A modular tool for transsynaptic labeling based antibody mimetics

Abstract

Provided herein are methods and compositions for anterograde postsynaptic tracing which preferably do not affect synaptic transmission. The methods and compositions may be used to deliver polypeptide payloads to postsynaptic neurons in a manner that is exclusively transsynaptic, monosynaptic, nontoxic, and without retrograde spread.

Description

[0001]A MODULAR TOOL FOR TRANSSYNAPTIC LABELING BASED ANTIBODY MIMETICS This application claims the benefit of United States Provisional Patent Application No. 63 / 536,359, filed September 1, 2023, and United States Provisional Patent Application No.63 / 643,309, filed May 6, 2024, the entirety of which is incorporated herein by reference. This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on August 30, 2024, is named USCAP0002WO.xml and is 47,195 bytes in size. STATEMENT OF GOVERNMENT SUPPORT This invention was made with government support under Grant No. R01MH116989, awarded by the (NIH) National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE DISCLOSURE Although rabies virus has been widely used to trace circuits transsynaptically in a retrograde direction, tracing in an anterograde direction has proven more challenging. Several methods for anterograde tracing have been proposed; however, none has been shown to trace monosynaptically, from genetically determined cells, efficiently, in an anterograde- only direction, and without toxicity. Furthermore, none has been shown to be purely transsynaptic. This disclosure addresses these limitations of the current state of the art and provides related advantages as well. SUMMARY OF THE DISCLOSURE Using high titers of adeno-associated virus serotype one encoding Cre (AAV1- Cre) it is possible to infect source neurons in a particular brain region resulting in the labeling 14871-7173-5771, v. 3 of neurons in areas that receive synaptic input from those source cells17. Infecting the postsynaptic neurons with a second Cre-dependent AAV (AAV-DiO-GeneX) results in the expression of GeneX in these cells. Similarly, injections of AAV1-Cre into source neurons in transgenic mice expressing floxed reporters leads to the expression of reporters in downstream neurons. This system has the advantage of low toxicity, absence of polysynaptic labeling, and marking of projections throughout the brain when used with transgenic animals. However, this method cannot be applied to genetically specified starter neurons. Furthermore, AAV1 particles can transport both anterogradely and retrogradely, confounding results. Finally, there is no direct evidence for purely transsynaptic labeling. More recently, a transneuronal tracing system was developed based on an inactivated variant of yellow fever virus (YFV) used in vaccinations (Li et al., 2021). The original YFV variant has three limitations: It is toxic, moves retrogradely, and can jump multiple synapses. However, eliminating genes encoding the structural proteins C, M, and E, and NS1, which is necessary for replication, can mitigate these problems. The lack of CME can prevent the virus from transmitting beyond the initial cell, and controlling the amount of NS1 present with a Dox system can reduce toxicity and retrograde transmission. The resulting system requires four separate genes to be supplemented in starter cells and the level of NS1 to be carefully controlled to suppress the undesirable properties of YFV. Also, it has not been demonstrated that YFV can trace circuits from Cre-positive neurons in a transgenic animal. Finally, the mechanism of transneuronal transfer of YFV is unknown and it has not been shown to be strictly transsynaptic. Herpes Simplex Virus (HSV) and Vesicular Stomatitis Virus (VSV) have also been used to trace neuronal circuits in the anterograde direction. However, as with YFV, these viruses are toxic, travel retrogradely, and label neurons that are not immediately downstream (Lo et al., 2011; Beier et al., 2011). Trans Seq uses wheat germ agglutinin, a lectin, fused to mCherry (WGA-mCherry) to label cells transneuronally, allowing them to be sorted and subjected to single-cell transcriptome analysis (Tsai et al., 2022). This method does not allow for genetic access to postsynaptic cells and thus is not directly comparable to the techniques above or the one described in this paper. Furthermore, the mechanism by which transneuronal 24871-7173-5771, v. 3 labeling occurs in all three of the above methods is unknown and thus there is no direct evidence that any of the three is strictly transsynaptic. ATLAS is a method for anterograde postsynaptic tracing provided herein. ATLAS can utilize an antibody-like protein to direct postsynaptic binding (e.g., AMPA.FingR, which binds to GluA1, but does not affect synaptic transmission), and in this way select subpopulations of neurons can be selectively targeted, e.g., due to binding of specific neurotransmitter receptor(s). Applicant provides evidence that ATLAS mediates transsynaptic labeling through release of the AMPA.FingR from presynaptic neurons, binding to perisynaptic AMPA receptors, and uptake by the postsynaptic cell through endocytosis. Applicants show that this system can carry Cre to postsynaptic cells in cultures and label those cells that express a floxed reporter. Furthermore, Applicant shows that (Anterograde Transsynaptic Labeling using Antibody-like Sensors) ATLAS can mediate transsynaptic labeling from Cre-expressing cells in transgenic animals and, conversely, to cells expressing floxed reporters in transgenic animals. In these studies as shown in the examples, ATLAS did not label neurons downstream of inhibitory neurons, providing additional evidence against nonspecific spread. Tracing circuits in the cerebellum with postsynaptic neurons in close proximity provides further evidence for transsynaptic labeling. It was further observed that transsynaptic tracing by ATLAS is activity-dependent in vivo using designer receptors exclusively activated by designer drugs (“DREADDs”). Additional studies showed that including a nanobody to a postsynaptic neurotransmitter receptor (e.g., GABA receptor, nicotinic acetylcholine receptor, serotonin receptor, dopamine receptor, or metabotropic glutamate receptor) can be used to selectively deliver the polypeptide payload (e.g., Cre) from the presynaptic neuron to neuronal subtypes that express the postsynaptic neurotransmitter receptor (e.g., as observed based on the expression of a floxed label expressed after anterograde delivery of the Cre). The study of neural circuits has been revolutionized by the advent of monosynaptic tracing from genetically determined cells in the retrograde direction using rabies virus. However, mapping circuits in the anterograde direction, which is equally important, has proven to be challenging. Although anterograde tracing methods have been developed, each tool has weaknesses, including the lack of ability to trace from genetically determined neurons, retrograde labeling, multi-synaptic labeling, and toxicity. Applicant provides herein ATLAS 34871-7173-5771, v. 3 (Anterograde Transsynaptic Labeling using Antibody-like Sensors), a new method for anterograde tracing. ATLAS uses a rationally designed anterograde tracer that efficiently and specifically maps connections from genetically determined starter cells, monosynaptically and was observed without retrograde labeling or toxicity. The AMPA FingR, a recombinant antibody-like protein that binds specifically to the N-terminal domain of the AMPA receptor GLuA1, can be used. The AMPA FingR is released from presynaptic termini, diffuses across the synapse, and then binds to a subset of AMPA receptors undergoing endocytosis. Without wishing to be bound by any theory, data provided supports the idea that ATLAS works by a well-defined mechanism and is composed of independent components that can be optimized or exchanged to give specific functionality. In these ways, it offers advantages over traditional viral tracers. A variety of nanobodies or intrabodies can be used to cause postsynaptic receptor binding domain of the polypeptide-payload fusion to target specific neuronal circuits (e.g., GABA nanobody domains, Nicotinic Acetylcholine Receptor intrabody domain, serotonin Receptor Nanobody domain, Dopamine Receptor Fab fragment domain, or metabotropic glutamate receptor nanobody domain). Thus, constructs provided herein can be used to selectively target a variety of excitatory and / or inhibitory neuronal subtypes including, e.g., those expressing GABA receptors, acetylcholine receptors, dopamine receptors, serotonin receptors, and / or metabotropic glutamate receptors. An aspect of the present disclosure relates to a method for delivering a polypeptide payload transsynaptically in an anterograde direction to a postsynaptic neuron connected via synapses to a presynaptic neuron or neurons, comprising contacting the presynaptic neuron with a polynucleotide encoding a polypeptide-payload fusion and expressing a polypeptide of the polypeptide-payload fusion in the presynaptic neuron. The polypeptide-payload fusion may be capable of i) targeting the payload peptide and the receptor binder peptide to synaptic vesicles, ii) allowing the release of the payload peptide and receptor binder peptide into the synaptic cleft, iii) binding to a postsynaptic receptor, and iv) mediating the endocytic internalization of the peptide payload into the postsynaptic cell resulting in transport to the cytoplasm and / or nucleus of the postsynaptic neuron. The polypeptide-payload fusion may comprise: i) a synaptic vesicle targeting protein; ii) a protease cleavage domain; iii) a postsynaptic receptor binding domain, and iv) a payload. The postsynaptic receptor binding domain of the polypeptide-payload fusion may be selected from the group of: 44871-7173-5771, v. 3 AMPA.FingR domain, antibody fragment, single domain antibody, scFv, nanobody, and any antibody-like protein that can preferably selectively bind (preferably at high affinity and specificity) a post-synaptic receptor (preferably at an extracellular domain) in the synaptic cleft or in a perisynaptic area. The postsynaptic receptor binding domain of the polypeptide-payload fusion may be an antibody fragment, single domain antibody, F(ab), scFv, or nanobody that can selectively bind a neurotransmitter receptor, such as for example a GABA nanobody domain, Nicotinic Acetylcholine Receptor intrabody domain, serotonin Receptor Nanobody domain, Dopamine Receptor Fab fragment, Beta-Adrenergic receptor Nanobody domain, or metabotropic glutamate receptor nanobody domain. The AMPA.FingR domain may comprise a polypeptide having at least 85% sequence identity to SEQ ID NO: 14 across the full length of the polypeptide. In some preferred aspects, the protease cleavage domain of the polypeptide- payload fusion is a β-Secretase Cleavage (BACEcs) domain. The synaptic vesicle targeting protein domain may be: VAMP2, (VAMP2Δ (SEQ ID NO:2) and Synaptotagmin nanobody), or Synaptophysin. Data is provided showing that including both VAMP2Δ and Synaptotagmin in the same fusion protein can function similarly to including either VAMP2 or Synaptophysin in the fusion protein. The payload may be selected from a group of:Cre, Flp or other recombinase, CRISPR Cas9, NPAS4 or other transcription factor affecting neuronal excitability, and BDNF. The polypeptide-payload fusion may further comprise a detectable label. The detectable label may be selected from the group of: ALFA-tag peptide, FLAG peptide, HA peptide, halotag peptide, snaptag peptide, V5, and DHFR peptide. The polypeptide-payload fusion may further comprise a domain that binds capsid proteins of AAV. In some preferred aspects, the AAV capsid binding domain is a 4x Heptamer. The polypeptide- payload fusion may further comprise a nuclear localization peptide. The nuclear localization peptide may be a CCR5 zinc finger transcription factor DNA binding domain. The contacting may be in vitro or in vivo. The presynaptic neuron may be a mammalian presynaptic neuron. The payload may be a recombinase (e.g., a Cre recombinase, FLP recombinase, or Dre recombinase); and the recombinase may cause expression of a second polypeptide in the postsynaptic neuron. The second polypeptide may be a floxed gene or floxed reporter gene, wherein the reporter gene is preferably a fluorescent protein. The second polypeptide may be transgenically expressed by a non-human animal. The polynucleotide encoding the polypeptide-payload fusion is delivered to the presynaptic neuron via a viral vector, liposomes, 54871-7173-5771, v. 3 virus-like particles (VLPs), or nanoparticles. The viral vector may be an AAV (e.g., AAV8). The polynucleotide encoding the polypeptide-payload fusion may be operatively linked to a promoter that is active in neurons (e.g., a neuron specific promoter). The promoter may be a hSyn1 promotor, CAG promoter, GAD1 promoter, or Camkii promoter. Another aspect of the present disclosure relates to a polypeptide, wherein the polypeptide comprises: i) a synaptic vesicle targeting protein; ii) a protease cleavage domain; iii) a postsynaptic receptor binding domain, and iv) a payload. The postsynaptic receptor binding domain of the polypeptide may be selected from the group of: AMPA.FingR domain, GABA nanobody domain, Nicotinic Acetylcholine Receptor intrabody domain, serotonin Receptor Nanobody domain, Dopamine Receptor Fab fragment domain, Beta-Adrenergic receptor Nanobody domain, and metabotropic glutamate receptor nanobody domain. The AMPA.FingR domain may comprise a polypeptide having at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 14 across the full length of the polypeptide. The protease cleavage domain of the polypeptide may be a β -Secretase Cleavage (BACEcs) domain. The synaptic vesicle targeting protein domain may comprise 1, 2, or 3, of: VAMP2, VAMP2delta (SEQ ID NO:4), Synaptotagmin nanobody, or Synaptophysin. The synaptic vesicle targeting protein domain may comprise (i) VAMP2 (SEQ ID NO:1), (ii) VAMP2delta (SEQ ID NO:2) and synaptotagmin nanobody (SEQ ID NO:4), (iii) VAMP2delta (SEQ ID NO:2) and Synaptotagmin (SEQ ID NO:3), or (iv) synaptophysin. The synaptic vesicle targeting protein domain may comprise a fusion protein comprising VAMP2delta (SEQ ID NO:2), (ii) a synaptotagmin nanobody (SEQ ID NO:4), and (iii) Synaptotagmin (SEQ ID NO:3). The synaptic vesicle targeting protein domain may comprise SEQ ID NO:5, or a sequence having at least 85%, preferably at least 90%, more preferably at least 95% sequence identity. The synaptic vesicle targeting protein domain may be selected from the group of: VAMP2, Synaptotagmin nanobody, and Synaptophysin. The payload may be selected from a group of: Cre recombinase, FLP recombinase, CRISPR Cas9, NPAS4 or other transcription factor affecting neuronal excitability, or BDNF. The polypeptide may further comprise a detectable label. The detectable label may be selected from the group of: ALFA-tag peptide, FLAG peptide, HA peptide, halotag peptide, snaptag peptide, and DHFR peptide. The polypeptide may further comprise a domain that binds capsid proteins of AAV. The AAV capsid binding domain may be a 4x Heptamer. The polypeptide may further comprise a nuclear 64871-7173-5771, v. 3 localization peptide. The nuclear localization peptide may be a CCR5 zinc finger transcription factor. Yet another aspect of the present disclosure relates to an isolated polypeptide comprising an AMPA.FingR domain having at least 85%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO:13 across the full length of the polypeptide. Another aspect of the present disclosure relates to an isolated polynucleotide encoding the polypeptide provided above or herein, or its compliment. The polynucleotide may comprise a promotor operably linked to the polynucleotide. The promotor may be selected from a group of: hSyn1 promotor, CAG promoter, GAD1 promoter, Camkii promoter, the promoters for the Serotonin Transporter, Noradrenaline transporter, and the Dopamine transporter or other cell-type specific promoters. The polynucleotide may comprise or consist of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30, or a sequence having at least 85%, preferably at least 90%, more preferably at least 95% sequence identity thereto. Yet another aspect of the present disclosure relates to an isolated polynucleotide encoding a polypeptide selected from: a. hSyn1-VAMP2-ALFA-BACEcs-AMPA FingR- 4xHeptamer-Cre-CCR5 fusion comprising SEQ ID NO: 3, that is optionally detectably labeled; or b. hSyn1-VAMP2-BACEcsE-AMPA FingR-Cre fusion comprising SEQ ID NO: 4, that is optionally detectably labeled. Another aspect of the present disclosure relates to a vector comprising the isolated DNA polynucleotide provided above or herein, that is optionally detectably labeled. The vector may further comprise one or more regulatory polynucleotides operably linked to the isolated polynucleotides, that is / are optionally detectably labeled. The vector may be selected from a plasmid, a viral vector, a micelle, a liposome, or a lipid nanoparticle, that is optionally detectably labeled. The vector may be a viral vector, that is optionally detectably labeled. The vector may be an adenoviral vector or an adenoviral associated vector (AAV) or lentiviral vector, that is optionally detectably labeled. 74871-7173-5771, v. 3 Yet another aspect of the present disclosure relates to an isolated host cell comprising one or more of the polypeptides provided above or herein, the polynucleotide provided above or herein, and / or the vector provided above or herein. Another aspect of the present disclosure relates to the isolated host cell provided above or provided herein, wherein the cell is a neuron. The host cell may express a recombinase. The recombinase may be selected from the group: Cre recombinase, FLP recombinase, Dre recombinase. Yet another aspect of the present disclosure relates to a composition comprising a carrier and one or more of the fusions above or provided herein, the isolated polynucleotide above or provided herein, the vector above or provided herein and / or the isolated host cell above or provided herein. As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one. The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” may mean at least a second or more. Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the inherent variation in the method being employed to determine the value, the variation that exists among the study subjects, or a value that is within 10% of a stated value. As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. 84871-7173-5771, v. 3 Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. FIGS. 1A – 1F: Characterization of AMPA.FingR. (FIG. 1A) Western blot showing that AMPA.FingR, an antibody-like protein labels the same band as a GluA1 antibody. (FIG.1B) AMPA.FingR expressed in dissociated culture localizes perisynaptically. Postsynaptic sites labeled with PSD95.FingR. (FIG.1C) Expressed AMPA.FingR colocalizes with endogenous clathrin. (FIG.1D) Following expression of AMPA.FingR in CA1 neurons in cultured hippocampal slices whole cell recordings of spontaneous EPSCs showed no difference between control and transfected cells for EPSC and NMDA amplitudes or in prepulse facilitation. (FIG. 1E) Release of SEP from presynaptic terminal following transfection of VAMP2-BACEcs-AMPA.FingR-SEP. Each image was taken in succession at 1 sec. intervals. The bright spot in third panel indicates release of SEP, which is disbursed in approximately 1 sec. (FIG. 1F) Release of AMPA.FingR-SEP (green) from presynaptic terminal is visible beginning at the third panel and stays visible through the sixth panel, consistent with AMPA.FingR-SEP binding to perisynaptic sites after presynaptic release. Scale bar 5 µm. 94871-7173-5771, v. 3 FIGS. 2A – 2F: ATLAS can mediate transsynaptic labeling in culture. (FIG. 2A) Schematic of ATLAS. AMPA.FingR-Cre is targeted to synaptic vesicles by VAMP2. Once in presynaptic vesicles, endogenous β-secretase (BACE) cleaves the VAMP2- AMPA.FingR-Cre fusion at the BACE cleavage site (BACEcs), allowing AMPA.FingR-Cre to be released from synaptic vesicles into the synaptic cleft. Following diffusion across the synaptic cleft, AMPA.FingR binds to AMPA receptors, which are internalized through endocytosis. Following endocytosis AMPA.FingR-Cre moves from endosomes to the nucleus by an unknown mechanism. (FIG. 2B) Neuron from dissociated culture transfected with ATLASCre(VAMP2-BACEcs-AMPA.FingR-Cre) and infected with AAV-DiO-GFP. Arrow points to presynaptic neuron expressing VAMP2 (ALFA-tag, blue) and Cre (red), but not GFP (green). Arrowheads point to postsynaptic neurons expressing GFP and Cre, but not VAMP2. (FIGS.2C – 2E) Same cells as in a. showing labeling for GFP (green), Cre (red), and ALFA- tag (cyan). Scale bar 100 µm. (FIG.2F) Neuronal culture transfected Synaptophysin-RFP to mark presynaptic cell somas and their terminals, ATLASCre, and infected with DiO-GFP. Postsynaptic cell marked by GFP and dim Cre labeling. Terminals of presynaptic cells marked by Synaptophysin and Cre partially co-localize with dendritic spines on postsynaptic cells, consistent with transsynaptic labeling. Scale bar 100 µm. FIGS.3A – 3G: ATLAS can mediate transsynaptic labeling in vivo. (FIG.3A) Schematic of circuit with presynaptic cells in the prefrontal cortex (PFC) injected with AAV- ATLASCre(cyan) and postsynaptic cells in the striatum injected with AAV-DiO-mCherry (red). (FIG. 3B) Presynaptic cells labeled with At in the prefrontal cortex (cyan). (FIG. 3C) Abundant postsynaptic cells labeled with mCherry in the striatum indicate efficient anterograde tracing (red). (FIG.3D) Control section from brain injected with AAV-DiO-mCherry without AAV-ATLASCreinjection showing minimal staining. (FIG. 3E) Schematic of circuit with presynaptic cells in the primary visual cortex (V1, cyan) and postsynaptic cells in the Superior Colliculus (red). Lenti-Cre and AAV-ATLASFLPwere injected into V1 and AAV-DiO- mCherry was injected into the superior colliculus. (FIG.3F) Presynaptic cells labeled with At in V1 (cyan). (FIG. 3G) Postsynaptic cells labeled with mCherry in the Superior Colliculus (red). Scale bar 100 µm. 104871-7173-5771, v. 3 FIGS. 4A – 4D: ATLAS mediates transsynaptic tracing from genetically determined cells. (FIG. 4A) Schematic of circuit from dorsal Lateral Geniculate Nucleus (dLGN) to primary visual cortex (V1) (FIG. 4B) Neurons expressing At in dLGN in VIPR2- Cre mice injected with DiO-AAV-ATLASFLP (Cyan). (FIG.4C) Staining of postsynaptic cells in V1 expressing mCherry. (FIG.4D) Staining of mCherry in a control mouse without ATLAS injection. Scale bar 100 µm. FIGS. 5A – 5E: ATLAS does not mediate transneuronal labeling in the retrograde direction. (FIG. 5A) Schematic showing strategy for testing whether ATLAS can mediate retrograde tracing from the dLGN to retinal ganglion cells. dLGN neurons were labeled through an injection of AAV-retro-Cre in the cortex (green). A second injection AAV- DiO-ATLASCrewas made into Cre-expressing cells in the dLGN (cyan) and a third injection of fDiO into the retina. (FIG.5B) dLGN neurons expressing At (cyan) indicating successful retrograde labeling from V1 (cyan). (FIG. 5C) Complete absence of labeling in the retina consistent with ATLAS being unable to mediate retrograde transneuronal labeling. (FIG.5D) Schematic of control experiment where DiO-retro-FLP was injected into the dLGN. (FIG.5E) Abundant labeling of retinal ganglion cells expressing mCherry (red) confirms the strategy outlined in a. Scale bar 100 µm. FIGS. 6A – 6F: ATLAS can mediate transsynaptic tracing. (FIG. 6A) Schematic showing the circuit from the inferior olive (cyan) to Purkinje cells of the cerebellum (red). (FIG.6B) Labeling of presynaptic neurons in the inferior olive labeled with At following the injection of ATLASCre(cyan). (FIG.6C) Labeling of mCherry-expressing neurons in the molecular and Purkinje layers, but not the granule layer (red). (FIG. 6D) Same as c. with calbindin counterstain (green) showing that postsynaptic labeling was confined to the molecular and Purkinje layers consistent with high fidelity labeling. (FIG. 6E) Section from brain of SST-Cre mouse injected with DiO-ATLASFLPand fDiO-mCherry in the cortex. VAMP2-expressing inhibitory presynaptic cells (cyan). mCherry expressing postsynaptic cells (red). Merge (white) showing 100% co-labeling of At and mCherry labeling indicating that no nonsynaptic transneuronal labeling was mediated by ATLAS. (FIG. 6F) Mouse brain co- infected with lenti-Cre, DiO-ATLASFLP, and fDiO-mCherry shows relatively few presynaptic cells labeled with At (cyan), but abundant labeling of postsynaptic neurons with mCherry (red), 114871-7173-5771, v. 3 indicating very efficient postsynaptic labeling from excitatory neurons. Scale bar 25 µm in b.- d., 100 µm in FIG.6E, FIG.6F. FIGS. 7A – 7B: Transsynaptic tracing with ATLAS is activity-dependent. (FIG. 7A) Section from mouse injected with AAV-ATLASCreand AAV-hHM4D(Gi)-HA in the PFC, and DiO-mCherry in the striatum, but without CNO injection. Abundant labeling of postsynaptic neurons by mCherry seen in the striatum. (FIG. 7B) Mouse with the same viral injections but with CNO injected daily. Seventy (70) percent less labeling was seen compared with the mouse in a. consistent with ATLAS being activity-dependent. FIG.8: A map of hSyn1-VAMP2-ALFA-BACECS-AMPA FingR-Cre-CCR5 vector. SEQ ID NO:27 is shown. FIG.9: hSyn1-VAMP2-ALFA-BACEcs-AMPA FingR-4xHeptamer-Cre- CCR5. SEQ ID NO:28 is shown. FIGS.10A-10L: Cellular mechanisms of release and uptake by ATLAS. (FIG. 10A) Western blot of lysate from dissociated neuronal cultures labeled with an anti-GluA1 Ab, AF-HA, and anti-GluA1 Ab + AF-HA (left to right). AF co-labels the higher MW band of two recognized by anti-GluA1 Ab. (FIG. 10B) DNA constructs used in the testing of ATLAS. Arrow denotes promoter. Rectangles are peptide domains. (FIG.10C) Live cortical neurons in culture expressing ss-AF-YFP (green) and PSD-95.FingR-tagRFP (red). AMPA.FingR appears to partially co-localize with PSD-95.FingR, consistent with it localizing perisynaptically, adjacent to PSD-95. Scale bar 5 µm. (FIG.10D) Cortical neurons in culture expressing ss-AF- YFP fixed and co-labeled for endogenous Clathrin. Colocalization (yellow) is consistent with AF localization to endocytic zones. Scale bar 5 µm (FIG. 10E) Cortical neurons in culture exposed to AF-HA (green) for 1 hour and co-labeled for endogenous MAP2 (red). Scale bar 25 µm. (FIG. 10F) Same as d., but with the addition of 10 mg / ml chlorpromazine, a clathrin inhibitor, to the culture medium. Scale bar 25 µm. (FIG.10G) Release of SEP from presynaptic sites in cultured cortical neurons expressing VAMP2-At-BACEcs-SEP. PSD-95.FingR- TagRFP (red), SEP (green). Scale bar 1 µm. (FIG. 10H) Same as g., but with VAMP2-At- BACEcs-AF-SEP. Note the increased brightness and longer duration of SEP fluorescence from VAMP2-At-BACEcs-AF-SEP vs. VAMP2-At-BACEcs-SEP, consistent with AF binding to 124871-7173-5771, v. 3 the postsynaptic cell. Scale bar 1 µm. (FIG. 10I) τ of VAMP2-At-BACEcs-AF-SEP is significantly increased vs. VAMP2-At-BACEcs-SEP. (FIG.10J) Adding 100 mM KCl to the culture medium causes a dramatic increase in the frequency of release events of VAMP2-At- BACEcs-SEP. (FIGS.10K-10L) k., l. Graphs of the differential of SEP fluorescence (ΔF) for VAMP2-At-BACEcs-AF-SEP with control medium vs. with medium containing 100 mM KCl. Each spike represents a single release event and does not reflect the duration of visible SEP fluorescence during each event (see Methods). * p < 0.05, ** p < 0.01. All tests Mann Whitney U test. FIGS.11A-11E: Design of ATLAS and testing in vitro. (FIG.11A) ATLASCre protein (VAMP2-At-BACEcs-AF-Cre) is expressed in a presynaptic starter cell and (1) targeted to synaptic vesicles by a VAMP2 domain. (2) A BACE cleavage site within the ATLAS protein is cleaved before release. (3) A peptide domain consisting of AMPA.FingR fused to Cre is released into the synaptic cleft and (4) subsequently binds to the extracellular domain of postsynaptic GluA1. (5) It is internalized through endocytosis and transported to the endosome. (6) AMPA.FingR-Cre moves to the nucleus, where Cre can catalyze the expression of a floxed reporter gene. (FIG.11B) Cortical neuron in culture expressing ATLASCre protein (arrow) following transfection, stained with anti-ALFA-tag nanobody (At). Note that four other neurons shown (arrowheads) do not express At. (FIG.11C) Cre staining of the same neurons as in b. including a neuron expressing ATLASCre(arrow), and four not expressing ATLASCre(arrowheads) provides evidence that Cre was transferred to the latter four neurons transneuronally. (FIG.11D) GFP labeling of postsynaptic cells (arrowheads, green) expressing Cre due to infection of AAV8-DIO-GFP, indicating that transneuronal Cre is nuclear. (FIG. 11E) Combined labeling of At, GFP, and Cre showing presynaptic (arrow) and postsynaptic neurons (arrowheads). Scale bars 100 µm. FIGS. 12A-12L: ATLAS mediates transsynaptic tracing in vivo. (FIG. 12A) Schematic depicting injections of AAV8-ATLASCre into the medial Prefrontal Cortex (mPFC, cyan) and AAV8-DIO-mCherry into the striatum (Str, red) to trace the unidirectional projection from the mPFC to the Str. (FIG. 12B) ALFA-tag (At) staining of ATLASCre in presynaptic cells in mPFC in a wild-type mouse. (FIG.12C) mCherry staining of postsynaptic cells in Str in the same brain as b. (FIG. 12D) Control brain injected only with AAV8-DIO-mCherry in 134871-7173-5771, v. 3 Str showing minimal background labeling. (FIG. 12E) Schematic depicting injections of AAV8-DIO-ATLASFLP and Lenti-Camkii-Cre into the primary visual cortex (V1, cyan) and AAV8-fDIO-mCherry into the Superior Colliculus (SC, red) to trace the unidirectional projection from V1 to SC. (FIG.12F) At staining of ATLASFLP in presynaptic cells in V1 in a wild-type mouse. (FIG. 12G) mCherry staining of postsynaptic cells in SC in the same brain as f. (FIG. 12H) Control brain injected only with AAV8-fDIO-mCherry in SC, showing the extent of background labeling. (FIG. 12I) Schematic depicting injections of AAV8-DIO- ATLASFLP into the dorsal Lateral Geniculate Nucleus (dLGN, cyan) and AAV8-fDIO- mCherry into V1 (red) to trace the projection from dLGN to V1 in a VIPR2-Cre mouse. An advantage of this strategy is that ATLAS cannot be expressed in V1 even if the virus is transported retrogradely. (FIG. 12J) At staining of ATLASFLPin presynaptic cells in dLGN. (FIG.12K) mCherry staining of postsynaptic cells in the V1 in the same brain as j. (FIG.12L) Control brain injected with only AAV8-fDIO-mCherry in V1 showing the extent of background labeling. Scale bars 100 µm. FIGS.13A-13G: ATLAS does not mediate nonsynaptic transneuronal labeling and transsynaptic labeling is specific. (FIG. 13A) Schematic of possible non-synaptic pathways for transneuronal labeling from an SST+ neuron. (FIG. 13B) SST-Cre mouse with AAV8-DIO-ATLASFLP and AAV-fDIO-mCherry co-injected into V1. Labeling of inhibitory presynaptic neurons with At. (FIG.13C) mCherry-labeled neurons from the same brain as in a. (FIG. 13D) Merged mCherry and At labeling shows that all neurons labeled with mCherry are also labeled with At, indicating that they are starter cells. Thus, there is no transneuronal labeling. (FIG. 13E) Schematic of configuration for whole cell patch clamp recording from random pairs of mCherry- and tdTomato-labeled and unlabeled striatal spiny projection neurons following ATLAS- mediated tracing from V1 cortex. EPSCs were generated as a result of optical activation of inputs from the V1 cortex. (FIG. 13F) Comparison of average EPSC trace from all optically evoked EPSCs from mCherry + and mCherry – striatal spiny projection neurons. (FIG. 13G) The amplitudes of EPSCs from mCherry + neurons are significantly larger than those from paired mCherry – neurons. FIGS.14A-14L: Transsynaptic labeling by ATLAS is exclusively anterograde and monosynaptic. (FIG. 14A) Schematic of the projection from mPFC to Str showing 144871-7173-5771, v. 3 injections of AAV8-DIO-ATLASFLPand Lenti-hsyn-Cre (Str, cyan), and AAV8-fDIO- mCherry (mPFC, red). (FIG.14B) At labeling of cells in Str. (FIG.14C) mCherry labeling of cells in mPFC in the same brain as b. (FIG. 14D) mCherry labeling of cells in mPFC in a control mouse brain with an injection of AAV8-fDIO-mCherry into the mPFC only. (FIG. 14E) Schematic of the disynaptic pathway from V1 to SC to PBG showing injection of ATLASCre (V1) and AAV8-DIO-mCherry (SC and PBG) for f-h. (FIG. 14F) At labeling of presynaptic cells in V1. (FIG. 14G) mCherry labeling of postsynaptic cells in the superficial gray layer of the superior colliuculus (SCsg) from the same brain as f. (FIG. 14H) mCherry labeling from the same brain as f. and g. shows no disynaptically labeled cells in PBG. (FIG. 14I) Schematic of projection from SCsg to PBG showing injections of ATLASCre (SCsg) and AAV8-DIO-mCherry (PBG). (FIG.14J) At labeling of presynaptic cells in SCsg. (FIG.14K) mCherry labeling of postsynaptic cells in PBG from the same brain as j. (FIG.14L) mCherry labeling in PBG following injection of AAV8-DIO-mCherry into PBG only in a control mouse brain. Scale bars 100 µm. FIGS.15A-15L: Transsynaptic tracing with ATLASsn. (FIG.15A) Schematic of ATLASsn. 1. Synaptotagmin nanobody (SYTnb) fused to the transmembrane domain of VAMP2 (VAMP2Δ) targets ATLAS to synaptic vesicles.2. AMPA.FingR-Cre is cleaved from VAMP2Δ.3. The synaptic vesicle fuses with the plasma membrane at the presynaptic terminal. 4. AMPA.FingR-Cre is released into the synaptic cleft. 5. SYTnb-VAMP2Δ-At is recycled back into the terminal, labeling the presynaptic site. (FIG.15B) ATLAS tracing in dissociated cortical culture transfected with AAV8-ATLASsnCre (labeled with At, cyan) and AAV8- BACE-HA and infected with AAV8-DIO-mCherry. Neuron labeled with mCherry (red) but not At is postsynaptic. At-labeled puncta colocalized with spine heads belonging to the postsynaptic neuron represent terminals of the presynaptic neuron. Scale bar 10 µm. (FIG. 15C) mPFC of the mouse injected with AAV8-ATLASsnCre (cyan) and incubated for two weeks. (FIG. 15D) Striatum of the same mouse as in c, injected with AAV8-DIO-mCherry (red). Area labeled with At (cyan) corresponding to presynaptic termini overlaps with area defined by cells labeled with mCherry consistent with transsynaptic labeling. (FIG. 15E) Schematic showing projections from the M1 to Str and thalamus (Thal) in the mouse. (FIG. 15F) M1 of a mouse co-injected with AAV8-BACE-GFP (green) and AAV8-ATLASsnCre (not shown). (FIG. 15G) Striatum of the same mouse as in f injected with AAV8-DIO-mCherry 154871-7173-5771, v. 3 showing mCherry-labeled neurons (red). (FIG.15H) Thalamus of the same mouse as in f and g injected with AAV8-DIO-mCherry showing mCherry-labeled neurons (red). (FIG. 15I) Schematic showing pathway from the ventral CA1 to the nucleus accumbens (ACB) and then to the lateral hypothalamic area (LHA) in the rat. (FIG. 15J) Ventral CA1 region of a rat injected with AAV8-ATLASsnCreshowing cell labeled with At (cyan). (FIG. 15K) ACB of the same rat as in j. injected with AAV8-DIO-tdTomato, showing tdTomato-labeled neurons (red). (FIG. 15L) ACB of the same rat as in j and k showing nerve terminals labeled with tdTomato (red) in the LHA. Scale bars 100 µm in c., d., f., g., h., 500 µm in j; 200 µm in k, l. FIGS. 16A-J: Testing of the AMPA.FingR. FIG. 16A, COS7 cells cotransfected with ss-Fn9.3-MYC, Stargazin-V5, and GluA1-HA. Surface labeling of MYC indicates binding of Fn9.3 to GluA1. FIG. 16B, Permeabilized labeling of HA in same cells as in a. FIG.16C, COS7 cells cotransfected with Fn6.5-MYC, Stargazin-V5, and GluA1-HA. Absence of surface MYC labeling indicates a lack of binding of Fn6.5 to GluA1. FIG. 16D, Permeabilized labeling of HA in same cells as in c. FIG. 16E., FIG. 16F: MYC surface labeling following co-expression of ss-Fn9.3-MYC with GFP in cultured cortical neurons. FIG. 16G., FIG. 16H: Lack of MYC surface labeling following co-expression of ss-Fn9.1- MYC with GFP in cultured cortical neurons. FIG. 16I, Addition of chlorpromazine, an endocytosis inhibitor, reduces uptake of AF-HA protein. FIG.16J, Patch clamp recordings of CA1 neurons in hippocampal slices transfected biolistically with ss-AF-YFP indicate that there is not a significant effect of AF expression on the amplitude of evoked AMPA- or NMDA receptor-mediated EPSCs, or on paired pulse facilitation compared to untransfected controls. Scale bars 20 pA and 20 ms for AMPA; 25 pA and 50 ms for NMDA; 20 pA and 20 ms for PPF. Scale bars in a-d, 20 µm. FIGS. 17A-F: Improving the efficiency of ATLAS by co-expressing BACE. FIG.17A, mCherry (red) and endogenous NeuN (green) labeling in Str in mouse brain infected with AAV8-ATLASCrein mPFC and AAV8-DIO-mCherry in Str. FIG. 17B, Same as in a. with a 1:1 ratio of AAV8-ATLASCre:BACE-HA injected into mPFC and AAV8-DIO-mCherry in Str. FIG. 17C, Same as in a. with 1:5 ratio of AAV8-ATLASCre:BACE-HA injected into mPFC and AAV8-DIO-mCherry in the Str. FIG. 17D, Closeup of region in c. showing approximately 10% of all neurons labeled in Str. FIG. 17E, Control staining from an 164871-7173-5771, v. 3 experiment with an injection of AAV8-DIO-mCherry only in Str. FIG. 17F, Quantitation shows that expressing ATLAS + BACE-HA 1:5 in mPFC leads to significantly higher labeling density in Str than ATLAS alone or ATLAS + BACE 1:1. *** p < 0.001 Tukey's multiple comparisons test. Scale bars 100 µm. FIGS. 18A-H: Connectivity of V1 to Str and mPFC to Str. FIG. 18A, High magnification histological sections showing presynaptic expression patterns in V1 of BACE (upper left), Cre-dependent ChR2 (upper right), ATLASsnCre(At, lower left), and merged (lower right). FIG. 18B, High magnification histological sections showing postsynaptic expression patterns in the dorsomedial striatum of mCherry / tdTomato (upper left), Cre- dependent ChR2 (upper right), and ATLASsnCre (At, lower left), and merged (lower right). FIG.18C, Schematic of configuration for whole cell patch clamp recording from Str neurons following ATLAS- mediated tracing from the mPFC. FIG.18D, Example traces show similar time courses of light-evoked EPSCs from mCherry- and mCherry+ striatum neurons following optical activation of inputs from the mPFC. FIG. 18E, Statistics comparing onset latency of eEPSC between mCherry- (n=7 cells from 3 animals) and mCherry+ (n = 8 cells from 3 animals) striatal neurons. Two-tailed unpaired t-test, N.S., P = 0.2251. FIG. 18F, Statistics comparing amplitudes of eEPSCs measured in mCherry- (n=7 cells from 3 animals) and mCherry+ (n=8 cells from 3 animals) striatal neurons. Two-tailed unpaired t test, N.S., P = 0.2766. FIG. 18G, Statistics comparing time constants of eEPSCs measured in mCherry- (n=7 cells from 3 animals) and mCherry+ (n=8 cells from 3 animals) striatal neurons. Two- tailed unpaired t-test, N.S., P = 0.2342. FIG. 18H, Traces illustrate the monosynaptic nature of the excitatory input to mCherry-labeled striatal neurons. eEPSC was suppressed by TTX application and rescued by 4AP. Scale bars 100 µm. FIGS. 19A-G: In vivo transsynaptic labeling with ATLAS is activity- dependent. FIG. 19A, Str of mouse injected with AAV8-ATLASCre+ AAV8-hHM3Dq- mCherry in mPFC and AAV8-DIO-GFP in Str. FIG.19B, Same as a, but with CNO injected intraperitoneally. FIG. 19C, Closeup of marked region in a. FIG.19D, Closeup of marked region in b. FIG. 19E, FIG. 19F: Presynaptic staining of At in the mPFC showing similar expression of ATLASCre for brains shown in a., b., respectively. FIG. 19G, Significantly 174871-7173-5771, v. 3 higher GFP expression was measured in Str of mice injected with CNO than in control mice (* indicates p < 0.05, Mann Withney U test). All scale bars 100 µm. FIG. 20A-H: Transsynaptic labeling of ATLASsnCre from the cortex to the striatum and thalamus. FIG. 20A, Coronal section from striatum of mouse in FIGS. 15C-D injected with AAV8-ATLASsnCre in the mPFC and AAV8-DIO-mCherry in the striatum and incubated for two weeks showing At labeling of presynaptic termini. FIG. 20B, Coronal section from striatum of same mouse as in a. showing mCherry-labeled neurons. FIG. 20C, Coronal section at the level of the striatum of mouse brain injected with AAV8-ATLASsnCre (not shown) + AAV8-BACE-GFP in the S1 cortex and AAV8-DIO-mCherry in the striatum and thalamus. Sections were stained for GFP (green), mCherry (red), and fluorescent Nissl stain (blue). FIG. 20D, Closeup of area marked in c. showing overlap of projections from neurons expressing AAV8-BACE-GFP (green) and neurons expressing mCherry. FIG. 20E, Coronal section at the level of the thalamus of the same mouse brain as in c. and d. showing projections of neurons expressing BACE-GFP (green), neurons expressing mCherry (red) and Nissl stain (blue). FIG.20F, Closeup of area marked in e. showing overlap of projections from neurons expressing AAV8- BACE-GFP (green) and neurons expressing mCherry. FIG.20G, Coronal section at the level of the striatum of a control mouse injected with AAV8-DIO- mCherry in the striatum in an identical manner to the mice in FIGS.5C-F, but with no injections in the cortex. Negligible mcherry staining (red) is visible in the striatum. Sections were also stained with Nissl stain (blue). FIG. 20H, Closeup of area marked in g. showing negligible mCherry labeling. Scale bars in FIGS. 20A, B, D, F, and H = 100 µm; in FIGS. 20C, E, and G = 1 mm. FIG. 21: Injection of AAV-DIO--SYTnb-VAMP2Δ-At-bcs-GN -Flp and AAV-fDIO-mCherry in the V1 cortex of a PV-Cre transgenic mouse followed by two weeks of incubation. Note abundant labeling of pyramidal neurons in layers 2 / 3 and 5. At+ cells label with mCherry, consistent with autaptic inhibition. FIGS. 22A-D: Injection of AAV-VAMP2-SYTnb-BACEcs-5HT3aNb-Cre in the Dorsal Raphe (22A-B) and AAV-DIO-GFP in the Ventral Tegmental area followed by two weeks of incubation (22C-D). Note abundant labeling of dopaminergic VTA neurons with GFP. 184871-7173-5771, v. 3 FIGS. 23A-D: AAV-VAMP2Δ-SYTnb- BACEcs-α7AchRNb-Cre was injected into the ventral nucleus of the diagonal band (23A-B) and AAV-DIO-GFP into the hippocampus (23C-D). GFP labeling in the dentate gyrus indicates transsynaptic labeling. FIG.24: AAV-VAMP2Δ-SYTnb- BACEcs-Drd2Nb-Cre was injected into the Substantia Nigra (left) and AAV-DIO-GFP into the striatum (right). GFP labeling in the striatum indicates transsynaptic labeling. FIG. 25: AAV-VAMP2Δ-SYTnb- BACEcs-mGluR5Nb-Cre was injected into the mPFC (left) and AAV-DIO-GFP into the striatum (right). GFP labeling in the striatum indicates transsynaptic labeling by ATLAS. DETAILED DESCRIPTION OF THE DISCLOSURE Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All nucleotide sequences provided herein are presented in the 5′ to 3′ direction. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods, devices, and materials are now described. All technical and patent publications cited herein are incorporated herein by reference in their entirety. Nothing herein is to be construed as an admission that the disclosure is not entitled to antedate such disclosure by virtue of prior disclosure. The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rdedition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of 194871-7173-5771, v. 3 Animal Cells: A Manual of Basic Technique, 5thedition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology. All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 1.0 or 0.1, as appropriate or alternatively by a variation of + / - 15 %, or alternatively 10% or alternatively 5% or alternatively 2%. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about”. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art. As used in the specification and claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a polypeptide” includes a plurality of polypeptides, including mixtures thereof. As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but do not exclude others. “Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the intended use. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure. 204871-7173-5771, v. 3 A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. The term “isolated” or “recombinant” as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively that are present in the natural source of the macromolecule as well as polypeptides. The term “isolated or recombinant nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polynucleotides, polypeptides and proteins that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. In other embodiments, the term “isolated or recombinant” means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature. For example, an isolated cell is a cell that is separated from tissue or cells of dissimilar phenotype or genotype. An isolated polynucleotide is separated from the 3′ and 5′ contiguous nucleotides with which it is normally associated in its native or natural environment, e.g., on the chromosome. As is apparent to those of skill in the art, a non- naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart. It is to be inferred without explicit recitation and unless otherwise intended, that when the present disclosure relates to a polypeptide, protein, polynucleotide, or antibody, an equivalent or a biologically equivalent of such is intended within the scope of this disclosure. As used herein, the term “biological equivalent thereof” is intended to be synonymous with “equivalent thereof” when referring to a reference protein, antibody, fragment, polypeptide or nucleic acid, intends those having minimal homology while still maintaining desired structure or functionality. Unless specifically recited herein, it is contemplated that any polynucleotide, polypeptide or protein mentioned herein also includes equivalents thereof. In one aspect, an 214871-7173-5771, v. 3 equivalent polynucleotide is one that hybridizes under stringent conditions to the polynucleotide or complement of the polynucleotide as described herein for use in the described methods. In another aspect, an equivalent antibody or antigen binding polypeptide intends one that binds with at least 70% , or alternatively at least 75% , or alternatively at least 80% , or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% affinity or higher affinity to a reference antibody or antigen binding fragment. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (for example, 80%, 85%, 90%, or 95%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. The alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. In particular preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non- redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST. Sequence identity and percent identity were determined by incorporating them into clustalW (available at the web address: / / align.genome.jp / , last accessed on March 7, 2011. “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence that may be aligned for purposes of comparison. When a positiond in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure. 224871-7173-5771, v. 3 “Homology” or “identity” or “similarity” can also refer to two nucleic acid molecules that hybridize under stringent conditions. As used herein, “expression” refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. The term “encode” as it is applied to polynucleotides refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and / or translated to produce the mRNA for the polypeptide and / or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom. As used herein, the terms “treating,” “treatment” and the like are used herein to mean obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disorder, sign or symptom thereof, and / or may be therapeutic in terms of a partial or complete cure for a disorder and / or adverse effect attributable to the disorder. To prevent intends to prevent a disorder or effect in vitro or in vivo in a system or subject that is predisposed to the disorder or effect. A “composition” is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant. A “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo. “Pharmaceutically acceptable carriers” refers to any diluents, excipients, or carriers that may be used in the compositions of the disclosure. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as 234871-7173-5771, v. 3 human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Suitable pharmaceutical carriers are described in Remington’s Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They are preferably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices. “Administration” can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue. Non-limiting examples of route of administration include oral administration, nasal administration, injection, and topical application. An agent of the present disclosure can be administered for therapy by any suitable route of administration. It will also be appreciated that the preferred route will vary with the condition and age of the recipient, and the disease being treated. The term “effective amount” refers to a quantity sufficient to achieve a desired effect. In the context of therapeutic or prophylactic applications, the effective amount will depend on the type and severity of the condition at issue and the characteristics of the individual subject, such as general health, age, sex, body weight, and tolerance to pharmaceutical 244871-7173-5771, v. 3 compositions. In the context of an immunogenic composition, in some embodiments the effective amount is the amount sufficient to result in a protective response against a pathogen. In other embodiments, the effective amount of an immunogenic composition is the amount sufficient to result in antibody generation against the antigen. In some embodiments, the effective amount is the amount required to confer passive immunity on a subject in need thereof. With respect to immunogenic compositions, in some embodiments the effective amount will depend on the intended use, the degree of immunogenicity of a particular antigenic compound, and the health / responsiveness of the subject’s immune system, in addition to the factors described above. The skilled artisan will be able to determine appropriate amounts depending on these and other factors. In the case of an in vitro application, in some embodiments the effective amount will depend on the size and nature of the application in question. It will also depend on the nature and sensitivity of the in vitro target and the methods in use. The skilled artisan will be able to determine the effective amount based on these and other considerations. The effective amount may comprise one or more administrations of a composition depending on the embodiment. A “plasmid” is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances. “Plasmids” used in genetic engineering are called “plasmid vectors”. Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacterium 254871-7173-5771, v. 3 produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for. A “viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like. Infectious tobacco mosaic virus (TMV)-based vectors can be used to manufacturer proteins and have been reported to express Griffithsin in tobacco leaves (O’Keefe et al., 2009). Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, Schlesinger & Dubensky (1999), Ying et al. (1999). In aspects where gene transfer is mediated by a retroviral vector, a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and a therapeutic gene. As used herein, “retroviral mediated gene transfer” or “retroviral transduction” carries the same meaning and refers to the process by which a gene or nucleic acid sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome. The virus can enter the host cell via its normal mechanism of infection or be modified such that it binds to a different host cell surface receptor or ligand to enter the cell. As used herein, retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism. Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus. In aspects where gene transfer is mediated by a DNA viral vector, such as an adenovirus (Ad) or adeno-associated virus (AAV), a vector construct refers to the polynucleotide comprising the viral genome or part thereof, and a transgene. Adenoviruses (Ads) are a relatively well-characterized, homogenous group of viruses, including over 50 serotypes. See, e.g., International PCT Application No. WO 95 / 27071. Ads do not require 264871-7173-5771, v. 3 integration into the host cell genome. Recombinant Ad derived vectors, particularly those that reduce the potential for recombination and generation of wild-type virus, have also been constructed. See, International PCT Application Nos. WO 95 / 00655 and WO 95 / 11984. Wild- type AAV has high infectivity and specificity integrating into the host cell’s genome. See, Hermonat & Muzyczka (1984) and Lebkowski, et al. (1988). Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, WI). In order to optimize expression and / or in vitro transcription, it may be necessary to remove, add or alter 5′ and / or 3′ untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites can be inserted immediately 5′ of the start codon to enhance expression. As used herein, the term “label” intends a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to the composition to be detected, e.g., N-terminal histadine tags (N-His), magnetically active isotopes, e.g.,115Sn,117Sn and119Sn, a non-radioactive isotopes such as13C and15N, polynucleotide or protein such as an antibody so as to generate a “labeled” composition. The term also includes sequences conjugated to the polynucleotide that will provide a signal upon expression of the inserted sequences, such as green fluorescent protein (GFP) and the like. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable. The labels can be suitable for small scale detection or more suitable for high-throughput screening. As such, suitable labels include, but are not limited to magnetically active isotopes, non-radioactive isotopes, radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. The label may be simply detected, or it may be quantified. A response that is simply detected generally comprises a response whose existence merely is confirmed, whereas a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, and / or other 274871-7173-5771, v. 3 property. In luminescence or fluorescence assays, the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component. Examples of luminescent labels that produce signals include, but are not limited to, bioluminescence and chemiluminescence. Detectable luminescence response generally comprises a change in, or an occurrence of, a luminescence signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art and described for example in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6thed.). Examples of luminescent probes include, but are not limited to, aequorin and luciferases. In another aspect, the fluorescent label is functionalized to facilitate covalent attachment to a cellular component present in or on the surface of the cell or tissue such as a cell surface marker. Suitable functional groups, including, but not are limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to attach the fluorescent label to a second molecule. The choice of the functional group of the fluorescent label will depend on the site of attachment to either a linker, the agent, the marker, or the second labeling agent. “Eukaryotic cells” comprise all of the life kingdoms except monera. They can be easily distinguished through a membrane bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane bound structure is the nucleus. Unless specifically recited, the term “host” includes a eukaryotic host, including, for example, yeast, higher plant, insect, and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include simian, bovine, porcine, murine, rat, avian, reptilian, and human. “Prokaryotic cells” that usually lack a nucleus or any other membrane bound organelles and are divided into two domains: bacteria and archaea. In addition to chromosomal DNA, these cells can also contain genetic information in a circular loop called an episome. Bacterial cells are very small, roughly the size of an animal mitochondrion (about 1-2µm in diameter and 10 µm long). Prokaryotic cells feature three major shapes: rod shaped, spherical, 284871-7173-5771, v. 3 and spiral. Instead of going through elaborate replication processes like eukaryotes, bacterial cells divide by binary fission. Examples include but are not limited to Bacillus bacteria, E. coli bacterium, and Salmonella bacterium. As used herein, the term “monosynaptic tracing” intends the use of a mutant neurotropic virus to label neurons that are monosynaptically connected (a monosynaptic connection is the connection of two neurons through a single junction, or synapse). The tracing is often achieved using a virus that has been modified, preventing movement to cells which have not also been genetically modified. By carefully targeting which cells are modified, researchers can use monosynaptic tracing to identify connections between neurons. See Lavin et al. (2019). Generally, two neurons are connected at a synapse. The presynaptic neuron releases a chemical transmitter to activate the postsynaptic neuron. The retrograde direction involves movement in the opposite direction, where a chemical or virus travels from the postsynaptic cell to the presynaptic cell. See Alger, (2002). For the purpose of this disclosure, “genetically determined cells” indicates the cells which have been genetically modified to express an exogenous polynucleotide such as a recombinase such as Cre or FLP that, in turn, allow expression of recombinase-dependent genes, i.e. Cre-dependent (floxed) or FLP-dependent genes. “Anterograde Transport” is transport which is the opposite of retrograde movement, that is movement from the presynaptic neuron to the postsynaptic neuron. See Saleeba et al. (2019). The term “transsynaptic” intends occurring or taking place across nerve synapses. In this context, and the broader context of neural circuits, transsynaptic indicates a molecule or virus that moves between two neurons using the small gap between them (the synapse). Tracers are chemicals or viruses that can be used to visualize and map the connections between neurons. Historically, they have been fluorescent or radiolabeled molecules. More recently, viral tracers have been developed to aid in visualizing the 294871-7173-5771, v. 3 connection between neurons. An anterograde tracer is one which moves from the presynaptic neuron to the postsynaptic neuron. Each amino acid has two common features: an amino group (N) and a carboxyl group (C). The amino group of one amino acid can bind to the carboxyl group of another amino acid, creating a chain of amino acids which becomes a protein. One end of the chain will be an amino group (N-terminus), and one end of the chain will be a carboxyl group (C- terminus). Here, N-terminus is designating which end of the protein chain is being discussed. Extracellular indicates that the N-terminus is located outside of the cell (as opposed to an intracellular terminus). AMPA receptor GluA1: AMPA stands for α-amino-3-hydroxy-5-methyl-4- isoxazole propionic acid. The AMPA receptors are a protein which plays a role in altering synaptic transmission between neurons. The AMPA receptors are made up of a number of subunits, one of which is glutamate receptor 1 (GluR1, also called GluA1). GluA1 is specifically involved in activity-dependent synaptic transmission (as opposed to maintaining basal synaptic transmission). Overall, AMPA works as a pore, allowing ions to flow through the cell membranes, enabling chemical and electrical signals to be passed between neurons. GluA1 is in the long-tailed group of AMPA subunits and is expressed in most neurons. “Cre (AAV1-Cre)”, “Cre” or “Cre recombinase” is an enzyme which performs recombination functions. The Cre protein has a high affinity for loxp DNA sequences. When two loxp sequences associate with Cre proteins, the two sites will bind together to become one DNA strand due to the catalytic function of Cre. The DNA between the sites can be excised or inverted. Therefore, Cre is a useful tool for DNA modification. Here, AAV1-Cre indicates a virus containing DNA to encode and produce Cre in the host cell. Note that expression in genetically modified cells refers to cells that express a recombinase and therefore can allow recombinase-dependent expression of genes. Synaptic Transmission: Neurons send signals to each other in order to physically transmit information. Within a single neuron, this is achieved using electric currents. However, neurons typically do not pass this electric current to other neurons, because each cell is separated by a small gap, called a synapse. In order to pass a signal across this gap, 304871-7173-5771, v. 3 neurons use synaptic transmission. Synaptic transmission utilizes chemicals released by the presynaptic neuron, which then travel across the synapse to interact with the post-synaptic neuron, thereby conveying a signal. Perisynaptic AMPA receptors: Perisynaptic indicates the area around the synapses, most commonly defined as the cleft and region near the synapse. Perisynaptic AMPA receptors are AMPA receptors (described above) that are located in this space. Inhibitory Neurons: Inhibitory neurons transmit signals similarly to other neurons. However, the key difference is that when an inhibitory neuron is activated, it prevents a subsequent neuron from activating. This contrasts with an excitatory neuron, which activates a subsequent neuron when activated. Inhibitory neurons make up a much smaller percentage of total neurons (10-20%). The designer receptor portion of the DREADD system is often targeted to certain cell populations. This is usually accomplished using viral vectors. The receptor can then be activated using the designed drug. When the receptor is activated, it can trigger the functions of the cell which would normally be triggered by a natural stimulus. In essence, DREADDs allow researchers to target specific cells for activation using an otherwise inert chemical. As used in the manuscript, DREADDs is referring to the experiments related to the activity-dependence of ATLAS, where the researchers used AAV-hM4D(Gi)-HA (designer receptor) and CNO (designer drug). Endocytic Zones: Endocytosis is a mechanism by which a cell can absorb or uptake molecules from the surrounding environment. Endocytosis is typically facilitated by clathrins, a protein which coats the vesicle taking the molecules into the cell. The endocytic zone is a clathrin-coated structure located on the postsynaptic neuron, where uptake of various molecules occurs. The endocytic zone is particularly important for uptake of AMPA receptors. Secretase Cleavage Site: Secretase is an enzyme which can cleave proteins at specific sites in order to create a new, shorter protein. The secretase cleavage site is a specific amino acid which allows the secretase enzyme to bind to the protein and cleave it. For the secretase enzyme BACE1, this site is the GLU11 amino acid of Amyloid beta protein. 314871-7173-5771, v. 3 However, there are a number of secretase enzymes, and each would have its own specific cleavage site. A promotor is a region of DNA which allows proteins to bind and initiate transcription. Each promoter has a unique sequence that allows for binding of appropriate proteins. Commonly used prokaryotic promoters include CMV (human cytomegalovirus), EF1a (Human elongation factor 1 alpha), CAG (Hybrid mammalian), PGK (Phospholycerate kinase), TRE (tetracycline response element), U6 (Human U6 nuclear), and UAS (Drosophilia containing Gal4 binding sites). These are commercially available from vendros such as addgene. Other bacterial promoters include T7 (T7 bacteriophage), Sp6 (Sp6 bacteriophage), lac (lac operon), araBad (arabinose metabolic operon), trp (E. coli tryptophan operon), and Ptac (hybrid of lac and trp). These are commercially available from vendors such as addgene. The bacterial promoters are often controlled by other molecules. For example, the trp promoter is repressible by tryptophan, and the lac promoter is induced by lactose but can be repressed by lacl. An enhancer is a sequence of DNA that allows specific proteins to bind and increase transcription of the target gene. There are a wide range of enhancers available for scientific application. Each of the promotors listed above has an associated enhancer. For example, the CMV promoter can be paired with a CMV enhancer. Further examples of enhancers include: Pgk enhancer, Tet1 enhancer, and the CD68 enhancer. These are commercially available from vendors such as addgene. Adenoviral vectors are modified viral vectors which can be engineered to include various DNA sequences for delivery to cells. There are a wide range of adenoviral vectors specifically designed for many different scientific purposes. Adenoviral vectors designed for chemogenetics and DREADD application include: pAAV-hSyn1-DIO- hM3D(gq)-mCherry, pAAV-CD68-hM4(gi)-mCherry, and pAAV-S5E2-Gq-P2A-dTomato. There are similarly designed adenoviral vectors used for other purposes, such as creating biosensors and controlling production of proteins. Each adenoviral vector contains the appropriate promotor and enhancer for the desired product. 324871-7173-5771, v. 3 Adeno-associated virus (AAV) forms the basis of adenoviral vectors. There are a number of AAV serotypes available for use in scientific research, including: AAV1, AAV2, AAV5, AAV6.2, AAV8, AAV9, AAVrh10, Retrograde AAV, and PHPeB. Each of these serotypes can form the basis of the adenoviral vector mentioned above and is used in combination with the appropriate promoters and enhancers. AAV can be used to deliver nucleic acid constructs described herein, including ATLAS constructs to presynaptic neurons and postsynaptic neurons. As shown in the below examples, AAV8 can be used to deliver constructs to the brain. In some aspects, liposomes or nanoparticles can be used to deliver a polynucleotide of the present disclosure to presynaptic and / or post synaptic neurons. Polypeptides Provided herein is a polypeptide comprising, consisting essentially of, or consisting of: i) a synaptic vesicle targeting protein; ii) a protease cleavage domain; iii) a postsynaptic receptor binding domain, and iv) a payload. A variety of neuronal receptors in the synaptic cleft may be selectively bound by the postsynaptic receptor binding domain, and in some aspects the postsynaptic receptor binding domain is an antibody fragment, single domain antibody, F(ab), scFv, or nanobody. In one aspect, the postsynaptic receptor binding domain of the polypeptide may be selected from the group of: AMPA.FingR domain, GABA nanobody domain, Nicotinic Acetylcholine Receptor intrabody domain, serotonin Receptor Nanobody domain, Dopamine Receptor Fab fragment domain, Beta-Adrenergic receptor Nanobody domain, and metabotropic glutamate receptor nanobody domain. The AMPA.FingR domain may comprise a polypeptide having at least 85%, or at least 90%, or at least 95%, or at least 98% or 100% sequence identity to SEQ ID NO: 14 across the full length of the polypeptide. Also provided is a polypeptide having a sequence as disclosed herein, and in particular a polypeptide disclosed in FIGs.8 and 9 of this disclosure as well as polypeptides having at least 85%, or at least 90%, or at least 95%, or at least 98% or 100% sequence identity to the sequences in the FIGs.8 and 9, or a polypeptide encoded by a polynucleotide disclosed in FIGs.8 and 9 or a polypeptide encoded by a polynucleotide having at least 85%, or at least 90%, or at least 95%, or at least 98% or 100% sequence identity to the sequences in the FIGs. 8 and 9. 334871-7173-5771, v. 3 It is anticipated that a wide variety of nanobodies, intrabodies and / or Fab fragments can be included in the postsynaptic receptor binding domain of the polypeptide to target a variety of neuronal subtypes. For example, the nanobody may target GABAAreceptors (e.g., Laverty et al., 2019), serotonin receptors such as 5HT3a (e.g., Hassaine et al., 2014), acetylcholine receptors including α7Ach receptor (e.g., Prevost et al. 2023), dopamine receptors such as the Drd2 dopamine receptor (Im et al., 2020), glutamate receptors including metabotropic glutamate receptor (e.g., mGluR5; Koehl et al., 2019). Thus, it is anticipated that a an extremely wide variety of neuronal subtypes that utilize specific types of neurotransmission with different neurotransmitters (e.g., GABA, acetylcholine, dopamine, serotonin) can be selectively targeted by choosing nanobodies that bind a particular post- synaptic receptor. It is anticipated that ligand-gated ion channel receptors or g-protein coupled receptors can thus be targeted. Further, by selecting nanobodies that selectively preferentially bind receptor subtypes (e.g., that target a particular α- β- or γ- subunit of a GABA-A receptor, that selectively bind a serotonin receptor or dopamine receptor subtype, etc.) additional neuronal specificity can be achieved. Examples of nanobodies that can be used include, e.g., GABA nanobody domains, Nicotinic Acetylcholine Receptor intrabody domains, serotonin Receptor Nanobody domains, Dopamine Receptor Fab fragment domains, Beta-Adrenergic receptor Nanobody domains, and metabotropic glutamate receptor nanobody domains. In another aspect, the protease cleavage domain of the polypeptide is a β- Secretase Cleavage (BACEcs) domain. The synaptic vesicle targeting protein domain may comprise 1, 2, 3, or more of: VAMP2 (Synaptobrevin, A SNARE protein), VAMP2delta (the transmembrane domain of VAMP2), Synaptotagmin nanobody (an antibody-like protein that recognizes the synaptic vesicle protein Synaptotagmin), or Synaptophysin, a synaptic vesicle protein. The synaptic vesicle targeting protein domain may comprise only one vesicle targeting domain, such as for example comprise VAMP2 only. In other aspects, a fusion protein can be used as the synaptic vesicle targeting protein domain; for example, the synaptic vesicle targeting protein domain may comprise (i) VAMP2 (e.g., SEQ ID NO:1) or (ii) VAMP2delta (e.g., SEQ ID NO:2) and a synaptotagmin nanobody (e.g., SEQ ID NO:4). VAMP2delta and the synaptotagmin nanobody can function as an obligate pair, and these two components together can function 344871-7173-5771, v. 3 similar to VAMP2 or synaptophysin. In some preferred aspects, the VAMP2delta–SYTnb– Synaptotagmin fusion protein (SEQ ID NO:5) is used as the synaptic vesicle targeting protein domain. As used herein, the term “payload” intends a protein delivered to a postsynaptic cell by ATLAS that is not necessary to mediate transsynaptic transport. Non-limiting examples of payloads are selected from a group of: Cre recombinase, CRISPR Cas9, NPAS4 or other transcription factor affecting neuronal excitability, or brain-derivied neurotrophic factor (BDNF). In a further aspect, the polypeptide further comprises a detectable label. Non- limiting examples of such include, a detectable label is selected from the group of: ALFA-tag peptide, FLAG peptide, HA peptide, halotag peptide, snaptag peptide, and DHFR peptide, all of which bind to either an antibody, nanobody or chemical ligand, allowing them to be labeled. The polypeptide can further comprise a domain that binds capsid proteins of AAV, e.g., a 4x Heptamer. In another aspect, the polypeptide further comprises a nuclear localization peptide for example wherein the nuclear localization peptide is CCR5 zinc finger transcription factor. Polynucleotides, Vectors and Host Cells This disclosure also provides isolated or recombinant polynucleotides encoding one or more of the above-identified polypeptides and their respective complementary strands. The polynucleotide can be a DNA or an RNA molecule. In one aspect, the polynucleotide further comprises a promotor operably linked to the polynucleotide. Non-limiting examples of such promoters include hSyn1 promotor, CAG promoter, GAD1 promoter, Camkii promoter or other cell-type specific promoters. This disclosure also provides an isolated polynucleotide encoding a polypeptide selected from: hSyn1-VAMP2-ALFA-BACEcs-AMPA FingR-4xHeptamer-Cre-CCR5 fusion comprising SEQ ID NO:27 (FIG. 8), that is optionally detectably labeled; or hSyn1-VAMP2- 354871-7173-5771, v. 3 BACEcsE-AMPA FingR-Cre fusion comprising SEQ ID NO:28 (FIG. 9), that is optionally detectably labeled. Further provided are vectors comprising the isolated DNA polynucleotide as described above, that is optionally detectably labeled or further optionally comprising regulatory polynucleotides operably linked to the isolated polynucleotides, that is optionally detectably labeled. Non-limiting examples of vectors include a plasmid, a viral vector, a micelle, a liposome, or a lipid nanoparticle, that is optionally detectably labeled. In one aspect, the vector is a viral vector, that is optionally detectably labeled. Non-limiting examples of viral vectors include an adenoviral vector or an adenoviral associated vector (AAV) or lentiviral vector, that is optionally detectably labeled. As noted above, the disclosure further provides the isolated or recombinant polynucleotide operatively linked to a promoter of RNA transcription, as well as other regulatory sequences for replication and / or transient or stable expression of the DNA or RNA. As used herein, the term “operatively linked” means positioned in such a manner that the promoter will direct transcription of RNA off the DNA molecule. Examples of such promoters are described herein and known in the art. In certain embodiments, cell specific promoters are used for cell specific expression of the inserted polynucleotide. Vectors that contain a promoter or a promoter / enhancer, with termination codons and selectable marker sequences, as well as a cloning site into which an inserted piece of DNA can be operatively linked to that promoter are known in the art and commercially available. For general methodology and cloning strategies, see Gene Expression Technology (Goeddel ed., Academic Press, Inc. (1991)) and references cited therein and Vectors: Essential Data Series (Gacesa and Ramji, eds., John Wiley & Sons, N.Y. (1994)) which contains maps, functional properties, commercial suppliers and a reference to GenEMBL accession numbers for various suitable vectors. Expression vectors containing these nucleic acids are useful to obtain host vector systems to produce proteins and polypeptides. It is implied that these expression vectors must be replicable in the host organisms either as episomes or as an integral part of the chromosomal DNA. Non-limiting examples of suitable expression vectors include plasmids, 364871-7173-5771, v. 3 yeast vectors, viral vectors, and liposomes. Adenoviral vectors are particularly useful for introducing genes into tissues in vivo because of their high levels of expression and efficient transformation of cells both in vitro and in vivo. When a nucleic acid is inserted into a suitable host cell, e.g., a prokaryotic or a eukaryotic cell and the host cell replicates, the protein can be recombinantly produced. Suitable host cells will depend on the vector and can include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells constructed using known methods. See Sambrook, et al. (1989) supra. In addition to the use of viral vector for insertion of exogenous nucleic acid into cells, the nucleic acid can be inserted into the host cell by methods known in the art such as transformation for bacterial cells; transfection using calcium phosphate precipitation for mammalian cells; or DEAE dextran; electroporation; or microinjection. See, Sambrook et al. (1989) supra, for methodology. Thus, this disclosure also provides a host cell, e.g., a mammalian cell, an animal cell (rat or mouse), a human cell, or a prokaryotic cell, such as a bacterial cell, containing a polynucleotide encoding the protein or polypeptide of this disclosure. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double and single stranded molecules. Unless otherwise specified or required, any embodiment of this disclosure that is a polynucleotide encompasses both the double stranded form and each of two complementary single stranded forms known or predicted to make up the double stranded form. Also provided is an isolated host cell comprising one or more of a polypeptide, a polynucleotide, or a vector as described herein. The host cell can be a prokaryotic or eukaryotic cell, e.g., a neuron. The eukaryotic cell can be a mammalian cell, such as a canine cell, a feline cell, an equine cell, a bovine cell, a murine cell a rat cell, a simian cell, or a human cell. A plurality of cells is also provided, wherein the polypeptides, vectors or polynucleotides are the same or different from each other. 374871-7173-5771, v. 3 In a further aspect, the host cell also expresses a recombinase, e.g., a recombinase is selected from the group: Cre recombinase, FLP recombinase, Dre recombinase. The polynucleotides can be conjugated to a detectable marker, e.g., an enzymatic label or a radioisotope for detection of nucleic acid and / or expression of the gene in a cell. A wide variety of appropriate detectable markers are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin / biotin, which are capable of giving a detectable signal. In one aspect, one may desire employing a fluorescent label or an enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally undesirable reagents. In the case of enzyme tags, calorimetric indicator substrates can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid containing samples. Thus, this disclosure further provides a method for detecting a single stranded polynucleotide or its complement, by contacting target single stranded polynucleotide with a labeled, single stranded polynucleotide (a probe) which is a portion of the polynucleotide of this disclosure under conditions permitting hybridization (preferably moderately stringent hybridization conditions) of complementary single stranded polynucleotides, or more preferably, under highly stringent hybridization conditions. Hybridized polynucleotide pairs are separated from un hybridized, single stranded polynucleotides. The hybridized polynucleotide pairs are detected using methods known to those of skill in the art and set forth, for example, in Sambrook et al. (1989) supra. The polynucleotide embodied in this disclosure can be obtained using chemical synthesis, recombinant cloning methods, PCR, or any combination thereof. Methods of chemical polynucleotide synthesis are known in the art and need not be described in detail herein. One of skill in the art can use the sequence data provided herein to obtain a desired polynucleotide by employing a DNA synthesizer or ordering from a commercial service. The polynucleotides of this disclosure can be isolated or replicated using PCR. The PCR technology is the subject matter of U.S. Patent Nos.4,683,195; 4,800,159; 4,754,065; and 4,683,202 and described in PCR: The Polymerase Chain Reaction (Mullis et al. eds., Birkhauser Press, Boston (1994)) or MacPherson et al. (1991) and (1995) supra, and references cited therein. Alternatively, one of skill in the art can use the sequences provided herein and a 384871-7173-5771, v. 3 commercial DNA synthesizer to replicate the DNA. Accordingly, this disclosure also provides a process for obtaining the polynucleotides of this disclosure by providing the linear sequence of the polynucleotide, nucleotides, appropriate primer molecules, chemicals, such as enzymes, and instructions for their replication and chemically replicating or linking the nucleotides in the proper orientation to obtain the polynucleotides. In a separate embodiment, these polynucleotides are further isolated. Still further, one of skill in the art can insert the polynucleotide into a suitable replication vector and insert the vector into a suitable host cell (prokaryotic or eukaryotic) for replication and amplification. The DNA so amplified can be isolated from the cell by methods known to those of skill in the art. A process for obtaining polynucleotides by this method is further provided herein as well as the polynucleotides so obtained. RNA can be obtained by first inserting a DNA polynucleotide into a suitable host cell. The DNA can be delivered by any appropriate method, e.g., by the use of an appropriate gene delivery vehicle (e.g., liposome, plasmid or vector) or by electroporation. When the cell replicates and the DNA is transcribed into RNA; the RNA can then be isolated using methods known to those of skill in the art, for example, as set forth in Sambrook et al. (1989) supra. For instance, mRNA can be isolated using various lytic enzymes or chemical solutions according to the procedures set forth in Sambrook et al. (1989) supra or extracted by nucleic acid binding resins following the accompanying instructions provided by manufactures. Compositions Compositions are further provided. The compositions comprise a carrier and one or more of an isolated polypeptide of the disclosure, an isolated polynucleotide of the disclosure, a vector of the disclosure, or an isolated host cell of the disclosure. The carriers can be one or more of a solid support or a pharmaceutically acceptable carrier. The compositions can further comprise an adjuvant or other components suitable for administration as vaccines. In one aspect, the compositions are formulated with one or more pharmaceutically acceptable excipients, diluents, carriers and / or adjuvants. In addition, embodiments of the compositions of the present disclosure include one or more of an isolated polypeptide of the disclosure, an isolated polynucleotide of the disclosure, a vector of the disclosure, or an isolated host cell of 394871-7173-5771, v. 3 the disclosure, or an antibody of the disclosure, formulated with one or more pharmaceutically acceptable auxiliary substances. For oral preparations, any one or more of an isolated or recombinant polypeptide as described herein, an isolated or recombinant polynucleotide as described herein, a vector as described herein, an isolated host cell as described herein, a small molecule or an antibody as described herein can be used alone or in pharmaceutical formulations of the disclosure comprising, or consisting essentially of, the compound in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. Pharmaceutical formulations and unit dose forms suitable for oral administration are particularly useful in the treatment of chronic conditions, infections, and therapies in which the patient self-administers the drug. In one aspect, the formulation is specific for pediatric administration. The disclosure provides pharmaceutical formulations in which the one or more of an isolated polypeptide of the disclosure, an isolated polynucleotide of the disclosure, a vector of the disclosure, an isolated host cell of the disclosure, or an antibody of the disclosure can be formulated into preparations for injection in accordance with the disclosure by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic 404871-7173-5771, v. 3 acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives or other antimicrobial agents. A non-limiting example of such is a antimicrobial agent such as other vaccine components such as surface antigens, e.g., an OMP P5, rsPilA, OMP 26, OMP P2, or Type IV Pilin protein (see Jurcisek and Bakaletz (2007); Murphy et al. (2009)) and antibacterial agents. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, a composition for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists. Aerosol formulations provided by the disclosure can be administered via inhalation and can be propellant or non-propellant based. For example, embodiments of the pharmaceutical formulations of the disclosure comprise a compound of the disclosure formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like. For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressurized container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. A non-limiting example of a non- propellant is a pump spray that is ejected from a closed container by means of mechanical force (i.e., pushing down a piston with one’s finger or by compression of the container, such as by a compressive force applied to the container wall or an elastic force exerted by the wall itself (e.g., by an elastic bladder)). Suppositories of the disclosure can be prepared by mixing a compound of the disclosure with any of a variety of bases such as emulsifying bases or water-soluble bases. Embodiments of this pharmaceutical formulation of a compound of the disclosure can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature. Unit dosage forms for oral or rectal administration, such as syrups, elixirs, and suspensions, may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more compounds of the disclosure. Similarly, unit dosage forms for injection 414871-7173-5771, v. 3 or intravenous administration may comprise a compound of the disclosure in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier. Embodiments of the pharmaceutical formulations of the disclosure include those in which one or more of an isolated polypeptide of the disclosure, an isolated polynucleotide of the disclosure, a vector of the disclosure, a small molecule for use in the disclosure, an isolated host cell of the disclosure, or an antibody of the disclosure is formulated in an injectable composition. Injectable pharmaceutical formulations of the disclosure are prepared as liquid solutions or suspensions; or as solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection. The preparation may also be emulsified, or the active ingredient may be encapsulated in liposome vehicles, in accordance with other embodiments of the pharmaceutical formulations of the disclosure. In an embodiment, one or more of an isolated polypeptide of the disclosure, an isolated polynucleotide of the disclosure, a vector of the disclosure, an isolated host cell of the disclosure, or an antibody of the disclosure is formulated for delivery by a continuous delivery system. The term “continuous delivery system” is used interchangeably herein with “controlled delivery system” and encompasses continuous (e.g., controlled) delivery devices (e.g., pumps) in combination with catheters, injection devices, and the like, a wide variety of which are known in the art. Mechanical or electromechanical infusion pumps can also be suitable for use with the present disclosure. Examples of such devices include those described in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852; 5,820,589; 5,643,207; 6,198,966; and the like. In general, delivery of a compound of the disclosure can be accomplished using any of a variety of refillable, pump systems. Pumps provide consistent, controlled release over time. In some embodiments, a compound of the disclosure is in a liquid formulation in a drug-impermeable reservoir and is delivered in a continuous fashion to the individual. In one embodiment, the drug delivery system is an at least partially implantable device. The implantable device can be implanted at any suitable implantation site using methods and devices well known in the art. An implantation site is a site within the body of a 424871-7173-5771, v. 3 subject at which a drug delivery device is introduced and positioned. Implantation sites include, but are not necessarily limited to, a subdermal, subcutaneous, intramuscular, or other suitable site within a subject’s body. Subcutaneous implantation sites are used in some embodiments because of convenience in implantation and removal of the drug delivery device. Drug release devices suitable for use in the disclosure may be based on any of a variety of modes of operation. For example, the drug release device can be based upon a diffusive system, a convective system, or an erodible system (e.g., an erosion-based system). For example, the drug release device can be an electrochemical pump, osmotic pump, an electroosmotic pump, a vapor pressure pump, or osmotic bursting matrix, e.g., where the drug is incorporated into a polymer and the polymer provides for release of drug formulation concomitant with degradation of a drug-impregnated polymeric material (e.g., a biodegradable, drug-impregnated polymeric material). In other embodiments, the drug release device is based upon an electrodiffusion system, an electrolytic pump, an effervescent pump, a piezoelectric pump, a hydrolytic system, etc. Drug release devices based upon a mechanical or electromechanical infusion pump can also be suitable for use with the present disclosure. Examples of such devices include those described in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852, and the like. In general, a subject treatment method can be accomplished using any of a variety of refillable, non-exchangeable pump systems. Pumps and other convective systems are generally preferred due to their more consistent, controlled release over time. Osmotic pumps are used in some embodiments due to their combined advantages of more consistent controlled release and relatively small size (see, e.g., PCT published application no. WO 97 / 27840 and U.S. Pat. Nos. 5,985,305 and 5,728,396). Exemplary osmotically-driven devices suitable for use in the disclosure include, but are not necessarily limited to, those described in U.S. Pat. Nos.3,760,984; 3,845,770; 3,916,899; 3,923,426; 3,987,790; 3,995,631; 3,916,899; 4,016,880; 4,036,228; 4,111,202; 4,111,203; 4,203,440; 4,203,442; 4,210,139; 4,327,725; 4,627,850; 4,865,845; 5,057,318; 5,059,423; 5,112,614; 5,137,727; 5,234,692; 5,234,693; 5,728,396; and the like. A further exemplary device that can be adapted for the present disclosure is the Synchromed infusion pump (Medtronic). 434871-7173-5771, v. 3 In some embodiments, the drug delivery device is an implantable device. The drug delivery device can be implanted at any suitable implantation site using methods and devices well known in the art. As noted herein, an implantation site is a site within the body of a subject at which a drug delivery device is introduced and positioned. Implantation sites include, but are not necessarily limited to a subdermal, subcutaneous, intramuscular, or other suitable site within a subject’s body. Suitable excipient vehicles for a compound of the disclosure are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Methods of preparing such dosage forms are known or will be apparent upon consideration of this disclosure, to those skilled in the art. See, e.g., Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985. The composition or formulation to be administered will, in any event, contain a quantity of the compound adequate to achieve the desired state in the subject being treated. Compositions of the present disclosure include those that comprise a sustained- release or controlled release matrix. In addition, embodiments of the present disclosure can be used in conjunction with other treatments that use sustained-release formulations. As used herein, a sustained-release matrix is a matrix made of materials, usually polymers, which are degradable by enzymatic or acid-based hydrolysis or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids. A sustained-release matrix desirably is chosen from biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (copolymers of lactic acid and glycolic acid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxcylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone. Illustrative biodegradable matrices include a polylactide matrix, a polyglycolide matrix, and a polylactide co-glycolide (co-polymers of lactic acid and glycolic acid) matrix. In another embodiment, the compositions of the present disclosure (as well as combination compositions separately or together) include those formed by impregnation of an 444871-7173-5771, v. 3 inhibiting agent described herein into absorptive materials, such as sutures, bandages, and gauze, or coated onto the surface of solid phase materials, such as surgical staples, zippers and catheters to deliver the compositions. Other delivery systems of this type will be readily apparent to those skilled in the art in view of the instant disclosure. Methods This disclosure also provides a method for delivering a polypeptide payload transsynaptically in an anterograde direction to a postsynaptic neuron connected via synapses to a presynaptic neuron or neurons, comprising contacting the presynaptic neuron with a polynucleotide encoding a polypeptide-payload fusion and expressing a polypeptide of the polypeptide-payload fusion in the presynaptic neuron. In one aspect, the polypeptide-payload fusion is capable of: i) targeting the payload peptide and the receptor binder peptide to synaptic vesicles, ii) allowing the release of the payload peptide and receptor binder peptide into the synaptic cleft, iii) binding to a postsynaptic receptor, and iv) mediating the endocytic internalization of the peptide payload into the postsynaptic cell resulting in transport to the cytoplasm and / or nucleus of the postsynaptic neuron. In another embodiment, the polypeptide- payload fusion comprises: i) a synaptic vesicle targeting protein; ii) a protease cleavage domain; iii) a postsynaptic receptor binding domain, and iv) a payload. Non-limiting examples of postsynaptic receptor binding domains of the polypeptide-payload fusion include AMPA.FingR domains, GABA nanobody domains, Nicotinic Acetylcholine Receptor intrabody domains, serotonin Receptor Nanobody domains, Dopamine Receptor Fab fragment domains, Beta-Adrenergic receptor Nanobody domains, and metabotropic glutamate receptor nanobody domains. In one aspect or the method, the AMPA.FingR domain comprises a polypeptide having at least 85%, or at least 90%, or at least 95%, or at least 95%, or 100% sequence identity to SEQ ID NO: 14 across the full length of the polypeptide. In another aspect, the protease cleavage domain of the polypeptide-payload fusion is a β-Secretase Cleavage (BACEcs) domain. 454871-7173-5771, v. 3 In a further aspect, the synaptic vesicle targeting protein domain is selected from the group of: VAMP2, Synaptotagmin nanobody, and Synaptophysin. In a further aspect of the methods, the payload is selected from a group of: Cre recombinase, FLP recombinase, CRISPR Cas9 (a DNA editing enzyme), NPAS4 (a transcription factor) or other transcription factor affecting neuronal excitability, or BDNF (Brain derived neurotrophic factor, a growth factor involved in synaptic plasticity). In a further aspect, the polypeptide-payload fusion further comprises a detectable label, non-limiting examples of such include ALFA-tag peptide, FLAG peptide, HA peptide, halotag peptide, snaptag peptide, or DHFR peptide, which are all peptides that can be labeled with an antibody, nanobody, or chemical ligand. In another aspect of the method, the polypeptide-payload fusion further comprises a domain that binds capsid proteins of AAV, e.g., a 4x Heptamer. In another aspect of the method, the polypeptide-payload fusion further comprising a nuclear localization peptide, e.g., a CCR5 zinc finger transcription factor DNA binding domain. The contacting can be done in vitro or in vivo. In a further aspect of the methods of this disclosure, the presynaptic neuron is a mammalian presynaptic neuron. Kits Kits containing the agents and instructions necessary to perform the in vitro and in vivo methods as described herein also are claimed. Accordingly, the disclosure provides kits for performing these methods which may include an interfering of this disclosure as well as instructions for carrying out the methods of this disclosure such as collecting tissue and / or performing the screen, and / or analyzing the results, and / or administration of an effective amount of an interfering agent as defined herein. These can be used alone or in combination with other suitable antimicrobial agents. 464871-7173-5771, v. 3 Examples The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Example 1 - Experimental Methods Histology for in vivo experiments Mouse brains were perfused with 40mL of phosphate-buffered saline (PBS) followed by 40mL of 4% paraformaldehyde in PBS. The brains were then fixed overnight in 4% paraformaldehyde at 4 °C and dehydrated in 30% sucrose solution for at least 48 hours. The brains were embedded in tissue freezing medium at -45 °C and then sectioned to a thickness of 25um-35um using cryostat. For immunohistochemistry, the brain sections were washed in PBS and incubated overnight in primary antibodies diluted in PBS containing 4% goat normal serum and 0.5% triton X-100. The primary antibodies used are as the following: RFP antibody, Rockland 600-401-379, 1:1000; Anti-GFP antibody, AVES gfp-1020, 1:1000; Calbindin-D-28K antibody, Sigma C9848-2ML, 1:1000; Fluotag-X2 anti-ALFA, NanoTag N1502-ALFA647-L, 1:1000 The sections were then washed with PBS and incubated in corresponding secondary antibodies for 2 hours. After incubation, the sections were washed with PBS again and then mounted onto the glass slides with DAPI Flouromount-G, EMS. The secondary antibodies used in this study and their dilution are as the following: Alexa Flour 488-conjugated goat anti-chicken or anti-mouse IgG, Thermo Fisher, 1:1000; Alexa Flour 594- conjugated antirabbit IgG, Thermo Fisher, 1:1000; Alexa Flour 647-conjugated goat anti- mouse IgG, Thermo Fisher, 1:1000. Results 474871-7173-5771, v. 3 Properties of the AMPA.FingR We used mRNA display, an in vitro selection procedure, to identify binders to a polypeptide encoding the aa 1-394 of the N-terminus of Rat GluA1 produced in Sf9 cells (see methods). This bait was used to screen a library of mRNA-protein fusions encoding the 10FNIII Ig-like domain of human Fibronectin, with 17 random residues engineered into the BC and FG loops and with a diversity of ≈ 10^13 (Gross et al., 2013). After the ninth round of selection, one of the Fibronection clones that was PCR-ed out, which we refer to as AMPA.FingR, or AF, was found to bind specifically to a band co-labeled with a GluA1 antibody on a western blot of neuronal lysate (FIG. 1A). To further characterize AF, we co- expressed it in cortical neurons in culture fused to a signal sequence, which caused it to be secreted by the secretory pathway, with PSD-95.FingR-GFP, which binds to postsynaptic sites. We found that AF partially overlapped with PSD-95.FingR, consistent with a perisynaptic localization (FIG. 1B). AF also colocalized with endogenous clathrin, consistent with localization to endocytic zones (FIG.1C). We also tested whether expression of AMPA.FingR affected the amplitude of spontaneous EPSCs of NMDA (FIG.1D, left) and AMPA receptors (FIG.1D, middle), and paired-pulse facilitation (FIG.1D, right). We found all three measures of synaptic transmission were not significantly different for cells expressing AF-YFP vs. for control cells, consistent with expression of AF not affecting synaptic transmission. VAMP2-mediated release from presynaptic sites Given that AF binds to GluA1 receptors and is endocytosed from perisynaptic sites, we reasoned that we could make a trans-synaptic tracer if we induced AF secretion from presynaptic terminals by fusing it to VAMP2 via its luminal domain. We then inserted a β- Secretase Cleavage Site (BACEcs) (Vassar et al., 1999) between AF and VAMP2 (to give VAMP2-BACEcs-AF), enabling endogenous BACE to cleave AF from VAMP2 and leave it free to exit the lumen of the synaptic vesicle upon exocytosis. To test this strategy, we substituted superecliptic pHluorin (Miesenbock et al., 1998) for AF and co-expressed the resulting plasmid (VAMP2-BACEcs-SEP) with PSD-95.FingR in cortical neurons in culture. We found 1-2 bright flashes lasting approximately (images taken at 1 Hz) appeared at points opposite postsynaptic sites (FIG. 1E), consistent with VAMP2 delivering SEP to synaptic vesicles, BACE cleaving off SEP, and SEP being released into the synaptic cleft. We then 484871-7173-5771, v. 3 fused AF to the C-terminus and following expression of the resulting construct (VAMP2- BACEcs-AF-SEP) in culture, we visualized flashes that were much brighter and decayed with a slower time constant (FIG. 1F). These results are consistent AF-SEP being released presynaptically, moving across the synaptic cleft, and binding to the adjacent postsynaptic neuron perisynaptically. Transsynaptic tracing in vitro and in vivo To complete the presynaptic ATLAS vector, Cre was substituted for SEP in VAMP2-BACEcs-AF-SEP and introduced an ALFA-tag (At) (Gotzke et al., 2019) to label the presynaptic neuron in which it is expressed, resulting in VAMP2-BACEcs-AF-Cre (ATLASCre). Given the above results, it was expected that following expression of ATLASCrein the presynaptic cell, VAMP2 should carry AMPA.FingR-Cre to synaptic vesicles, where it is cleaved off and released into the synaptic cleft (FIG.2A). At the postsynaptic membrane it binds to AMPA receptors whereupon it is endocytosed and carried to the nucleus. To test the strategy, ATLASCre were transfected into dissociated cultures that were also infected with AAV8-DiO-GFP. It was found that, indeed, there were cells stained with GFP and Cre, that were not labeled with At, consistent with transsynaptic transport of Cre (FIGS. 2B – 2E). Furthermore, when the presynaptic marker Synaptophysin-RFP was co-transfected with ATLASCre, Cre colocalized at presynaptic terminals and was found immediately adjacent to dendritic spines of GFP-expressing cells (Fig 2f). Thus, the results are consistent with ATLASCre mediating transsynaptic tracing and providing genetic access to postsynaptic cells. To test our strategy in vivo, we infected AAV-ATLASCre into the prefrontal cortex (PFC) and AAV8-DiO-mCherry into the striatum of an adult mouse (FIG. 3A). Following one week, we found At staining in the PFC and abundant mCherry staining in the striatum (FIG.3B, FIG.3C). In contrast, there was negligible staining in a control experiment without AAV-ATLASpre(FIG.3D). Because the projection from the PFC to striatum is strictly anterograde (ref), this result is consistent with the combination of AAV-ATLASCre and AAV- dio-mCherry mediating transsynaptic tracing. Furthermore, the efficiency of ATLAS was approximately 30 cells / mm2, consistent with the efficiency achieved with YFV in similar experiments59. 494871-7173-5771, v. 3 Tracing from genetically-determined cells The ability to trace from genetically determined cell types is critical given the heterogeneous distribution of different neuronal types in many brain regions. To mediate genetically-determined tracing, AAV-dio-ATLASFLPwere generated, where expression of the presynaptic protein is Cre-dependent and the payload is FLP (Flip recombinase). This virus was tested by co-injecting it with Lenti-CAMKii-Cre (a lentivirus expressing Cre specifically in excitatory neurons) into V1 and then AAV-fDiO-mCherry into the superior colliculus. In addition to ALFA-tag staining in V1, we saw abundant staining for mCherry in the superior colliculus (FIGS.3E – 3G). Note that an advantage of this strategy is that lentivirus does not have any intrinsic retrograde or anterograde transneuronal transport that could cloud interpretation of the results, unlike virtually every pseudotype of AAV. To test whether AAV-dio-ATLASFLP is capable of transsynaptic labeling from Cre-positive cells in a transgenic mouse, we injected AAV-dio-ATLASFLP into a VIPR2-Cre line expressing Cre in neurons in the dorsal Lateral Geniculate Nucleus (dLGN) (Zhuang et al., 2021) and fDio-mCherry in V1. Staining of At was found specifically in the dLGN, and mCherry staining was dense in layer IV and deep layer III, and less dense in layers II and V, consistent with previous results (FIGS.4A – 4D) (Zhuang et al., 2021). Lack of retrograde labeling A major limitation of many virally-based anterograde transneuronal labeling methods is that they also mediate labeling in the retrograde direction. Because AF binds specifically to the AMPA receptor and AF-Rec (AMPA.FingR-recombinase) is released from presynaptic terminals, it is unlikely to mediate retrograde transneuronal labeling (FIG.2A). To determine whether AAV8-Dio-ATLASFLP mediates retrograde labeling, we investigated the retinal ganglion cell to dLGN to V1 pathway. We identified neurons in dLGN that project to V1, and therefore receive input from the retina, by injecting AAV-retro-Cre into V1. We then injected AAV-Dio-ATLASFLP into the dLGN and AAV-fDIO-mCherry into the retina (FIG. 5A). We found abundant AT staining in the dLGN, but no staining of retinal ganglion cells, indicating that no retrograde labeling occurred (FIG.5B, FIG.5C). As a control, we repeated the experiment, but injected dio-retro-Flp instead of dio-ATLASFLP(FIG. 5D). In this case, 504871-7173-5771, v. 3 many cells in the retina were labeled with mCherry (FIG. 5E), indicating that we injected the correct cells in the dLGN, and confirming that ALTAs does not mediate retrograde tracing. Labeling is transsynaptic Given that the secretion of AF-Cre likely occurs from presynaptic sites (FIG. 1G) and its endocytosis occurs perisynaptically (FIG.1B, FIG.1G), it is highly likely that the labeling mediated by VAMP2-AT-Bcs-AF-Cre is synaptic. To confirm this, we examined two separate adjacent circuits in the cerebellum, inferior olive to inhibitory neurons in the molecular and Purkinje cell layers and pontine nucleus to granule cells, to determine if both can be traced with fidelity. When we expressed AAV-ATLASCre in the inferior olive (FIG. 6A, FIG. 6B) and DiO-mCherry in the cerebellar cortex, we found expression of mCherry in Purkinje cells, basket cells, and stellate cells, but not in granule cells (FIG. 6C). Counterstaining with calbindin (FIG.6D) confirms that the staining with mCherry is confined to the molecular layer consistent with transsynaptic labeling. To further test whether nonsynaptic transneuronal labeling is possible, we injected AAV-DIO-VAMP2-AT-Bcs-AF-FLP and fDiO-mCherry into the cortex of an SST-Cre mouse, where inhibitory interneurons express Cre. When we examined the labeling in the cortex after two weeks, we saw no cells expressing mCherry that were not also labeled with At, indicating that no transneuronal labeling had occurred (FIG.6E). In contrast, the control experiment where we injected Lenti-CAMKii-Cre, DiO-ATLASFLP, and fDiO-mCherry into the cortex, causing expression of the presynaptic virus in excitatory neurons, showed many transsynaptic mCherry-labeled neurons in the cortex (FIG. 6F). Because transneuronal labeling from inhibitory presynaptic neurons would have to be nonsynaptic, this provides evidence that our method is not capable of mediating nonsynaptic, transneuronal labeling. Activity-dependence of ATLAS Because ATLAS mediates transsynaptic labeling through the release of AF-Rec from synaptic vesicles (FIG. 1F), it is very likely that labeling is activity-dependent. To test for activity dependence of transsynaptic labeling in vivo, we co-injected AAV-ATLASCre and AAV-hM4D(Gi)-HA into the PFC of adult wild-type mice. In one mouse, we injected CNO in the peritoneum daily, but not in the second. We found that in the mouse with CNO inject, 514871-7173-5771, v. 3 the amount of mCherry was roughly 30% of the magnitude of staining found in the mouse without CNO (FIG.7A, FIG.7B), despite similar expression patterns of ATLASCre in the PFC. Note that this is in line with reductions in activity seen after in vivo expression of hHM4D(Gi)- HA (Rodriguez et al., 2020). In conclusion, we have shown that ATLAs can mediate exclusively transsynaptic labeling that is monosynaptic, strictly anterograde, nontoxic, and activity- dependent from genetically determined starting cells. Sequences hSyn1 promoter (human synapsin promoter) AGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGC CTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCA AATTGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCG CGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGG CGCGCGCCACCGCCGCCTCAGCACTGAAGGCGCGCTGACGTCACTCGCCGGTCC CCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCA GCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCT GCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGG AGTCGTGTCGTGCCTGAGAGCGCAG (SEQ ID NO:11) VAMP2 (synaptic vesicle targeting protein) ATGTCGGCTACCGCTGCCACCGTCCCGCCTGCCGCCCCGGCCGGCG AGGGTGGCCCCCCTGCACCTCCTCCAAATCTTACCAGTAACAGGAGACTGCAGC AGACCCAGGCCCAGGTGGATGAGGTGGTGGACATCATGAGGGTGAATGTGGACA AGGTCCTGGAGCGGGACCAGAAGCTATCGGAACTGGATGATCGCGCAGATGCCC TCCAGGCAGGGGCCTCCCAGTTTGAAACAAGTGCAGCCAAGCTCAAGCGCAAAT ACTGGTGGAAAAACCTCAAGATGATGATCATCTTGGGAGTGATTTGCGCCATCAT CCTCATCATCATCATCGTTTACTTCAGCACT (SEQ ID NO:6) 524871-7173-5771, v. 3 MSATAATVPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVVDIMR VNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLKMMIILGVI CAIILIIIIVYFST (SEQ ID NO:1) VAMP2delta (delta mutant of the VAMP2 synaptic vesicle targeting protein) ATGATGATCATCTTGGGAGTGATTTGCGCCATCATCCTCATCATCAT CATCGTTTACTTCAGCACT (SEQ ID NO:7) MMIILGVICAIILIIIIVYFST (SEQ ID NO:2) Synaptotagmin Atgtcggctaccgctgccaccgtcccgcctgccgccccggccggcgagggtggcccccctgcacctcctcc aaatcttaccagtaacaggagactgcagcagacccaggcccaggtggatgaggtggtggacatcatgagggtgaatgtggacaagg tcctggagcgggaccagaagctatcggaactggatgatcgcgcagatgccctccaggcaggggcctcccagtttgaaacaagtgca gccaagctcaagcgcaaatactggtggaaaaacctcaagatgatgatcatcttgggagtgatttgcgccatcatcctcatcatcatcatc gtttacttcagcact (SEQ ID NO:8) MSATAATVPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVVDIMRVNVDKVLER DQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLKMMIILGVICAIILIIIIVYFS T (SEQ ID NO:3) Synaptotagmin nanobody (SYTnb) Atggaggtgcagctggaggagtctgggggcggcttggtgcggcctggggggtctctaagactctcctgtgcagcctctggattcccc ttcagtaaatattttatgagctgggtccgccaggctccaggaaaggggctcgaatgggtctcaactattagtgctagtggcaattatgaa acctatacagagtccgtgaagggccgattcaccatcgccagagacaacgccaagaacacgctgtatctgcagatgaacagcctgaaa ccggaggacacggccgtctattactgtgcaaaagggagctgggcccgggatatgacgaggggtcaggggacccaggtcactgtct cctcagaacccaag (SEQ ID NO:9) MEVQLEESGGGLVRPGGSLRLSCAASGFPFSKYFMSWVRQAPGKGLEWVSTISASG NYETYTESVKGRFTIARDNAKNTLYLQMNSLKPEDTAVYYCAKGSWARDMTRGQG TQVTVSSEPK (SEQ ID NO:4) 534871-7173-5771, v. 3 VAMP2delta–SYTnb–Synaptotagmin fusion protein Atgatgatcatcttgggagtgatttgcgccatcatcctcatcatcatcatcgtttacttcagcact (SEQ ID NO:10) MMIILGVICAIILIIIIVYFST (SEQ ID NO:5) BACEcs GAAATTAGTGAAGTAAACCTAGACGCTGAATTTAGA (SEQ ID NO:33) EISEVNLDAEFR (SEQ ID NO:12) AMPA FingR ATGCTCGAGGTCAAGGAAGCATCACCAACCAGCATCCAGATCAGC TGGAGCAGGAGGGACCAGGTGATTCGCTACTACCGCATCACCTACGGCGAAACT GGTGGCAATAGCCCTGTCCAAAAATTCACCGTGCCTGGCAGCAAATCCACTGCTA CCATCAGCGGCCTGAAACCTGGTGTTGACTATACCATCACGGTGTACGCCGTCAC GCCCTTCGCCAACTTCTGGGACTGGCACGGCCCGATCTCCATCAACTACCGCACC (SEQ ID NO:13) MLEVKEASPTSIQISWSRRDQVIRYYRITYGETGGNSPVQKFTVPGSKS TATISGLKPGVDYTITVYAVTPFANFWDWHGPISINYRT (SEQ ID NO:14) FLP recombinase sequence MPQFGILCKTPPKVLVRQFVERFERPSGEKIALCAAELTYLCWMITHN GTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTIIPYY GQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILNSFEY TSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVIIQCLVTETKTSV SRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVRSYN KALKKNAPYSIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVAR 544871-7173-5771, v. 3 TTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIR YPAWNGIISQEVLDYLSSYINRRI (SEQ ID NO:15) Dre Recombinase sequence MSELIISGSSGGFLRNIGKEYQEAAENFMRFMNDQGAYAPNTLRDLRL VFHSWARWCHARQLAWFPISPEMAREYFLQLHDADLASTTIDKHYAMLNMLLSHC GLPPLSDDKSVSLAMRRIRREAATEKGERTGQAIPLRWDDLKLLDVLLSRSERLVDL RNRAFLFVAYNTLMRMSEISRIRVGDLDQTGDTVTLHISHTKTITTAAGLDKVLSRRT TAVLNDWLDVSGLREHPDAVLFPPIHRSNKARITTTPLTAPAMEKIFSDAWVLLNKR DATPNKGRYRTWTGHSARVGAAIDMAEKQVSMVEIMQEGTWKKPETLMRYLRRG GVSVGANSRLMDS (SEQ ID NO:16) CAG promoter GCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAA CGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATA GGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGG CAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGG TAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACT TGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCA CGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTAT TTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGC GCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGT GCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGG CGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG (SEQ ID NO:17) NPAS4 MYRSTKGASKARRDQINAEIRNLKELLPLAEADKVRLSYLHIMSLACI YTRKGVFFAGGTPLAGPTGLLSAQELEDIVAALPGFLLVFTAEGKLLYLSESVSEHLG HSMVDLVAQGDSIYDIIDPADHLTVRQQLTMPSALDADRLFRCRFNTSKSLRRQSAG NKLVLIRGRFHAHPPGAYWAGNPVFTAFCAPLEPRPRPGPGPGPGPGPASLFLAMFQ 554871-7173-5771, v. 3 SRHAKDLALLDISESVLIYLGFERSELLCKSWYGLLHPEDLAHASSQHYRLLAENGDI QAEMVVRLQAKHGGWTWIYCMLYSDGPEGPITANNYPISDTEAWSLRQQLNSENT QAAYVLGTPAVLPSFSENVFSQEHCSNPLFTPALGTPRSASFPRAPELGVISTSEELAQ PSKELDFSYLPFPARPEPSLQADLSKDLVCTPPYTPHQPGGCAFLFSLHEPFQTHLPPP SSSLQEQLTPSTVTFSEQLTPSSATFPDPLTSSLQGQLTESSARSFEEQLTPCTSTFPDQL LPSTATFPEPLGSPTHEQLTPPSTAFQAHLNSPSQTFPEQLSPNPTKTYFAQEGCSFLYE KLPPSPSSPGNGDCTLLALAQLRGPLSVDVPLVPEGLLTPEASPVKQSFFHYTEKEQN EIDRLIQQISQLAQGMDRPFSAEAGTGGLEPLGGLEPLNPNLSLSGAGPPVLSLDLKP WKCQELDFLVDPDNLFLEETPVEDIFMDLSTPDPNGEWGSGDPEAEVPGGTLSPCNN LSPEDHSFLEDLATYETAFETGVSTFPYEGFADELHQLQSQVQDSFHEDGSGGEPTF (SEQ ID NO:18) BDNF (brain derived neurotrophic factor) MTILFLTMVISYFGCMKAAPMKEANIRGQGGLAYPGVRTHGTLESVN GPKAGSRGLTSLADTFEHVIEELLDEDQKVRPNEENNKDADLYTSRVMLSSQVPLEP PLLFLLEEYKNYLDAANMSMRVRRHSDPARRGELSVCDSISEWVTAADKKTAVDM SGGTVTVLEKVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSY VRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGR (SEQ ID NO:19) Flag peptide DYKDDDDK (SEQ ID NO:20) HA peptide YPYDVPDYA (SEQ ID NO:21) Halotag peptide MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIH DWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLI IDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVA 564871-7173-5771, v. 3 LVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQED NPDLIGSEIARWLSTLEISG (SEQ ID NO:22) SNAP-tag peptide MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVEVPA PAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK VVKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGG LAVKEWLLAHEGHRLGKPGLG (SEQ ID NO:23) DHFR peptide MKLSLMVAISKNGVIGNGPDIPWSAKGEQLLFKAITYNQWLLVGRKT FESMGALPNRKYAVVTRSSFTSDNENVLIFPSIKDALTNLKKITDHVIVSGGGEIYKSL IDQVDTLHISTIDIEPEGDVYFPEIPSNFRPVFTQDFASNINYSYQIWQKG (SEQ ID NO:24) GAD1 promoter GGCTTTGCTTCCTGGGCACTACAGCCCCACCTGAAGCAGGTGCGTT AGGGAGGAGTTGGGCTCTGCAGCTTGCCAGATTTTAATTCTGGCTTCCAGCGGAG GACCCTGCACCCCACGGTCCTCGCAAGAGTGTCTGGGAAGGTTTATTCGGTTGCT GAGTAGAGACGCAACAGACAGATCTACCTCAGCCCCTGCACCTTGGCTTGATAA GGCTGGAAAAGGAAGATTGCAACGTCGTGGTTGTTTCCAAACCCTTTTTCTATCT AGCCTTTTTCAGTCTCTCAACATTTAAGCAGCAAAAGAACTCGAGAGAGAAAAC TTGTACTAAATATCCTTCTTTGACCGGCCCTCTCCTCTCCCCACTACGGGGATTTC TGGGTGGCTCACAGTGGACAAGGAATAGGTAATCCCACCAAACCTTGAGGTGGG CTAAACTGGAGATTTTGGTATGGGTCTCCTACTGTAAAGAGAAAGCCTTCCGGGC TAACGGTATTCCTTTCGCAGTCTACTGCCTCCTTTGTGAACTAATGACTGTAATTA TTACCTCCCAGAGCTCTTTTGTTATCAACAACCCGGAGCCAGAGGGGGGTGGGG GATGGGCCCTTTGGGAAAAGGGAAAAAAATTTACAAAGCAAATTGCTGTCTACG AAGGGTTGGTTTTTTAAAAAGACAAACCCAATTCCAGCCTGCATTCGAAGTGGG GAGTGGCAAAATTTTTTTTCAAAGTATGATAAGATTTTAAAAATACTCAGAGCTG TGGGATTCAATTGCATTTCCACAGTATCGCTTTAAGTTACTATTTTTGACGAAGTA 574871-7173-5771, v. 3 AAATTCAGATGTTGAAAATCTGTATCTCTTGTTTTCATCCATTTCTTCCTTGGAAG GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGT TTAAGAACCTTTTTTTTTATTTCCGACCACTCATCCATGGTAAAAAGCTTCAACCC CAGCCAGCTGGCTTGAAGAGAATTATTTTGCAGGATTTAGTATGGATTTAAACAA AGACTCAGGCTCTCCAAGTCTTGTTGGCAAGGCTGGGCGGAAGCGCCCTTTCCAG GAGCTGTTACCTCTGCCAGCTGAAGCCACCAGGCCTGGAGGACCCTAAAGACTT ACTAAGTTGAGGCCTAGGGTCAAGGTGACTGCCTTCCCTTAGAGACACGCCTTGG GTAGCACCTCAGCAGTGCCAATCTCACCGCCCTCCCCCTCCCCACCTCGGGCCAC TCACCGGACCTCAGTGGGCATCCTCTAGCTAGATCCTCCCTGGCCCCCCTGGATT CTGGAAACCTTGGGAAGAAGATAAAGCTTTGTGGAGAGAATGCACTTGGTCATC TGCAGTTATTCGAGGTCTCCGGATCCCTAACAAAATAAGTGATCCTGGTCACCAA GACTAAAGGTCCCAGGGTTTCTCCTCCGGCAACAGCTTTGGTGACTCATTTGAAA AACTCAAAGAAGGAATAAAGGACTATGGAGAGCAGGGAAGCTGCAGTCCCTCG CTCGGAGAGAAGGAAAGGAGATCGGGAACGGTTTAAAGCAGCACTTTCCTGCAA GTCTTTCTTGAGCTCTCTGTAAGCCTGGAACTATGGGTTTAGTATAGGCAAGAGG AAACGGAAGCGGAGCCAGAGGGCCCCAAATCTTCCTCAAGGCGCCGTCCCTGCA TTTAAGAGCCTTTGGGAAAAGGGAAACAGAAAGGATTTTTTCATTCCTTTATTAG AAAACACAAACGAAACTACTTCTTTCTGGGCTGGGAAATTGCCACTGTTGTGTTC CTAGGCACCTGCTGATGGCAAGAACTCCTCTTGGGAAGGGACGAGGTCAGTGGG TTGAGTATCCCTGAAAAGATGGGAGTGCCTTTTCCTGGGATTTGGAAAAACTGAT TCGCTTACTTTGAGACTCCCTGAATCCTGTGTATGTGTCAGCTCTCACCCACTCCA AGAAGTTTCTGTTCGTTTTATTTATTTAATTTTTCTCAGCCTGAGGTCCTCAGTGA TAGATTCCAGCGTGGATTTTAATTGCTTCAATCAGCAGCCTTTCTCTTCAGCCGTC AGTCAAAACCTGGACGATGGGTCTGGGGACCCAGGCATGACTGCACAGAAACCG TACGACCCCCACCCCCACCCCCCAGGCTGGCTCAGCTCAGCAGGCAGACATTAG GCTCTGCTAAGGAGATGAAATGAGTTCGTTGGTGTGGAAGCGGTCCACCTTCCTC CCTCCTTGGTTCCTTCCCCAAGCTTTGGAGCTCTTCCTGATTGAGGCCAGGTGGCC TGGGCTCTTGAGGTCACAGCGACCTCAGCACTGGTTTGGAGGAAGGCGGGCGCG AGAACCACTTTTGCTCGGCAGCAGCCAGAGACAGCCCTCAAACAATGAGCCAAT CGATGCACACGTGAAATTGCCCTCGGCCACCATTCCAATCTTGCCAAGGTCTCCC 584871-7173-5771, v. 3 TGTCCCTGGGGTCACCTAGGCCAGCGTGCTGAGGGCTGTGGTAGCACCCTGGAC AGCGCGTTCCGGCAGAAGCAGACCTGGAG (SEQ ID NO:25) Camkii promoter CATTATGGCCTTAGGTCACTTCATCTCCATGGGGTTCTTCTTCTGAT TTTCTAGAAAATGAGATGGGGGTGCAGAGAGCTTCCTCAGTGACCTGCCCAGGG TCACATCAGAAATGTCAGAGCTAGAACTTGAACTCAGATTACTAATCTTAAATTC CATGCCTTGGGGGCATGCAAGTACGATATACAGAAGGAGTGAACTCATTAGGGC AGATGACCAATGAGTTTAGGAAAGAAGAGTCCAGGGCAGGGTACATCTACACCA CCCGCCCAGCCCTGGGTGAGTCCAGCCACGTTCACCTCATTATAGTTGCCTCTCT CCAGTCCTACCTTGACGGGAAGCACAAGCAGAAACTGGGACAGGAGCCCCAGGA GACCAAATCTTCATGGTCCCTCTGGGAGGATGGGTGGGGAGAGCTGTGGCAGAG GCCTCAGGAGGGGCCCTGCTGCTCAGTGGTGACAGATAGGGGTGAGAAAGCAGA CAGAGTCATTCCGTCAGCATTCTGGGTCTGTTTGGTACTTCTTCTCACGCTAAGGT GGCGGTGTGATATGCACAATGGCTAAAAAGCAGGGAGAGCTGGAAAGAAACAA GGACAGAGACAGAGGCCAAGTCAACCAGACCAATTCCCAGAGGAAGCAAAGAA ACCATTACAGAGACTACAAGGGGGAAGGGAAGGAGAGATGAATTAGCTTCCCCT GTAAACCTTAGAACCCAGCTGTTGCCAGGGCAACGGGGCAATACCTGTCTCTTCA GAGGAGATGAAGTTGCCAGGGTAACTACATCCTGTCTTTCTCAAGGACCATCCCA GAATGTGGCACCCACTAGCCGTTACCATAGCAACTGCCTCTTTGCCCCACTTAAT CCCATCCCGTCTGTTAAAAGGGCCCTATAGTTGGAGGTGGGGGAGGTAGGAAGA GCGATGATCACTTGTGGACTAAGTTTGTTCGCATCCCCTTCTCCAACCCCCTCAGT ACATCACCCTGGGGGAACAGGGTCCACTTGCTCCTGGGCCCACACAGTCCTGCA GTATTGTGTATATAAGGCCAGGGCAAAGAGGAGCAGGTTTTAAAGTGAAAGGCA GGCAGGTGTTGGGGAGGCAGTTACCGGGGCAACGGGAACAGGGCGTTTCGGAG GTGGTTGCCATGGGGACCTGGATGCTGACGAAGGCTCGCGAGGCTGTGAGCAGC CACAGTGCCCTGCTCAGAAGCCCCAAGCTCGTCAGTCAAGCCGGTTCTCCGTTTG CACTCAGGAGCACGGGCAGGCGAGTGGCCCCTAGTTCTGGGGGCAGC (SEQ ID NO:26) VAMP2-At-BACEcs-SEP protein sequence 594871-7173-5771, v. 3 MSATAATVPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVVDIMRVNVDKVLER DQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLKMMIILGVICAIILIIIIVYFS TTSPSRLEEELRRRLTEPELASEISEVNLDAEFRASSKGEELFTGVVPILVELDGDVNG HKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKR HDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNIL GHKLEYNYNDHQVYIMADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPV LLPDNHYLFTTSTLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK (SEQ ID NO:29) VAMP2-At-BACEcs-SEP nucleotide sequence Atgtcggctaccgctgccaccgtcccgcctgccgccccggccggcgagggtggcccccctgcacctcctccaaatcttaccagtaa caggagactgcagcagacccaggcccaggtggatgaggtggtggacatcatgagggtgaatgtggacaaggtcctggagcggga ccagaagctatcggaactggatgatcgcgcagatgccctccaggcaggggcctcccagtttgaaacaagtgcagccaagctcaagc gcaaatactggtggaaaaacctcaagatgatgatcatcttgggagtgatttgcgccatcatcctcatcatcatcatcgtttacttcagcact actagtccgagccgcctggaagaagaactgcgccgccgcctgaccgaaccggagctcgctagtGAAATTAGTGAAGT AAACCTAGACGCTGAATTTAGAGCTAGCAGTAAAGGAGAAGAACTTTTCACTGG AGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTG TCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTA TTTGCACTACTGGAAAACTACCTGTTCCTTGGCCAACACTTGTCACTACTTTAACT TATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGGCATGACTTTT TCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAG ATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTG TTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGG ACACAAATTGGAATACAACTATAACGATCACCAGGTGTACATCATGGCAGACAA ACAAAAGAATGGAATCAAAGCTAACTTCAAAATTAGACACAACATTGAAGATGG AGGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGGCC CGTCCTTTTACCAGACAACCATTACCTGTTTACAACTTCTACTCTTTCGAAAGATC CCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGA TTACACATGGCATGGATGAACTATACAAA (SEQ ID NO:31) VAMP2-At-BACEcs-AF-SEP protein sequence: 604871-7173-5771, v. 3 MSATAATVPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVVDIMRVNVDKVLER DQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLKMMIILGVICAIILIIIIVYFS TTSPSRLEEELRRRLTEPELASEISEVNLDAEFRASMLEVKEASPTSIQISWSRRDQVIR YYRITYGETGGNSPVQKFTVPGSKSTATISGLKPGVDYTITVYAVTPFANFWDWHGP ISINYRTGSDYKDDDDKGSGSSRGSGSGYVSRHPGGGCGYVSRHPGGGCGYVSRHP GGGCGYVSRHPGGGCKLSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYG KLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERT IFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMA DKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDP NEKRDHMVLLEFVTAAGITHGMDELYK (SEQ ID NO:30) VAMP2-At-BACEcs-AF-SEP nucleotide sequence: atgtcggctaccgctgccaccgtcccgcctgccgccccggccggcgagggtggcccccctgcacctcctcca aatcttaccagtaacaggagactgcagcagacccaggcccaggtggatgaggtggtggacatcatgagggtgaatgtggacaaggt cctggagcgggaccagaagctatcggaactggatgatcgcgcagatgccctccaggcaggggcctcccagtttgaaacaagtgcag ccaagctcaagcgcaaatactggtggaaaaacctcaagatgatgatcatcttgggagtgatttgcgccatcatcctcatcatcatcatcgt ttacttcagcactactagtccgagccgcctggaagaagaactgcgccgccgcctgaccgaaccggagctcgctagtGAAATTA GTGAAGTAAACCTAGACGCTGAATTTAGAGCTAGCATGCTCGAGGTCAAGGAAG CATCACCAACCAGCATCCAGATCAGCTGGAGCAGGAGGGACCAGGTGATTCGCT ACTACCGCATCACCTACGGCGAAACTGGTGGCAATAGCCCTGTCCAAAAATTCA CCGTGCCTGGCAGCAAATCCACTGCTACCATCAGCGGCCTGAAACCTGGTGTTGA CTATACCATCACGGTGTACGCCGTCACGCCCTTCGCCAACTTCTGGGACTGGCAC GGCCCGATCTCCATCAACTACCGCACCGGATCCGACTACAAGGATGACGACGAT AAGGGTAGCGGCTCCTCTAGAGGAAGCGGAAGCGGATATGTAAGCAGGCATCCC GGAGGCGGGTGCGGTTACGTTTCACGGCACCCCGGCGGTGGTTGCGGTTATGTA AGTCGACATCCGGGAGGAGGCTGCGGTTATGTGAGCCGTCATCCCGGAGGGGGA TGCAAGCTTAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTG AATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAG GTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACT ACCTGTTCCTTGGCCAACACTTGTCACTACTTTAACTTATGGTGTTCAATGCTTTT CAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCG 614871-7173-5771, v. 3 AAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGA CACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAA AGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAA CTATAACGATCACCAGGTGTACATCATGGCAGACAAACAAAAGAATGGAATCAA AGCTAACTTCAAAATTAGACACAACATTGAAGATGGAGGCGTTCAACTAGCAGA CCATTATCAACAAAATACTCCAATTGGCGATGGGCCCGTCCTTTTACCAGACAAC CATTACCTGTTTACAACTTCTACTCTTTCGAAAGATCCCAACGAAAAGAGAGACC ACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGA ACTATACAAA (SEQ ID NO:32) Example 2 - Generating AMPA.FingR, a binder to the N-terminus of GluA1 mRNA display, an in vitro selection procedure19, was used to identify binders to a polypeptide encoding aa 1-394 of the extracellular N-terminus of Rat GluA1 produced in Sf9 cells (see methods). This bait was used to screen a library of mRNA-protein fusions encoding the 10FNIII Ig-like domain of human Fibronectin, with 17 random residues engineered into the BC and FG loops and a diversity of ~1013 20, 21. After the 9th selection round, 81 Fibronectin (Fn) clones were amplified and sequenced, of which 24 were unique and full-length. Then each Fn clone was put into an expression vector that placed a signal sequence from GluA1 at the N-terminus to target it to the secretory pathway and added a MYC tag for labeling. The resulting plasmids (ss-Fn-MYC) were co-transfected into COS7 cells with GluA1-HA and Stargazin-V5. Surface labeling of MYC showed that four Fn clones labeled the surface of COS7 cells co-labeled with HA (Figs. 16a-j). These four clones were expressed in dissociated cultures of cortical neurons. Of the four Fibronectins expressed in the cultures, one clone, Fn9.3, gave the most robust labeling (Figs.16a-j). This clone was named AMPA.FingR (AF; FingR stands for Fibronectin intrabody generated with mRNA display). AF-HA protein was then generated and found that it bound specifically to a band co-labeled with a GluA1 antibody on a western blot of neuronal lysate (Fig. 10a, 10b). To characterize AF further, it was co-expressed in dissociated cortical neurons with PSD- 95.FingR-tagRFP, which binds to and labels postsynaptic sites20. We found that AF labeling partially overlapped with PSD-95.FingR-tagRFP, suggesting a perisynaptic localization (Fig. 10c). AF also co-localized with endogenous Clathrin, consistent with localization to endocytic 624871-7173-5771, v. 3 zones (Fig. 10d). Given that AF co-localizes with Clathrin and binds to GluA1, it was tested whether AF-HA protein is endocytosed by adding AF-HA protein to the medium of cortical neurons in culture. It was found that considerable AF-HA was detected in virtually all neurons labeled with MAP2 (37 of 38, n = 5 independent cultures, Fig.10e) within 1 hour of its addition. Furthermore, the addition of 10 mg / ml chlorpromazine, a clathrin inhibitor, significantly reduced the accumulation of AF-HA (4.2 ± 0.3 vs.2.1 ± 0.5, p < 0.01, Mann-Whitney U test, Figs.10f, 16a-j), confirming that internalization was mediated by endocytosis. Given that AF likely binds to GluA1 receptors and is endocytosed postsynaptically, it was reasoned that a trans-synaptic tracer could be made if AF was targeted to synaptic vesicles by fusing it to the luminal domain of the presynaptic protein, VAMP222. We inserted a β-Secretase cleavage site (BACEcs)23between AF and VAMP2 (to give VAMP2-BACEcs-AF), enabling endogenous BACE to cleave AF from VAMP2, leaving it free to exit the lumen of the synaptic vesicle upon exocytosis. In addition, an ALFA-tag (At) was added to label VAMP224. To test the strategy, superecliptic pHluorin (SEP)22was substituted for AF and co-expressed the resulting plasmid (VAMP2-At-BACEcs-SEP, Fig. 10b) with PSD-95.FingR-tagRFP in cortical neurons in culture. The release from acidic synaptic vesicles into the neutral pH of the synaptic cleft is expected to cause a sudden increase in the fluorescence of SEP, allowing it to be visualized22. Following overnight expression of VAMP2-At-BACEcs-SEP in neuronal cultures, we imaged SEP using widefield epifluorescence microscopy. Flashes of fluorescence were seen lasting approximately 2-8 sec (images taken at 1 Hz) appearing at points opposite postsynaptic sites (Fig.10g). This result is consistent with VAMP2 delivering SEP to synaptic vesicles, BACE cleaving off SEP, and SEP being released into the synaptic cleft. AF was also fused to SEP and expressed the resulting protein (VAMP2-At-BACEcs-AF-SEP, Fig. 10b) in culture and observed flashes that were much brighter and decayed with a much longer time constant (6 ± 2 sec vs.147 ± 60 sec, n = 5 independent cultures, Figs. 10h, 10i), a significant difference (P < 0.01, Mann Whitney U test). This result is consistent with AF binding to the postsynaptic membrane following release and remaining until it is endocytosed. To further test whether AF-SEP is released from synaptic vesicles, it was examined whether depolarization increases the number of release events. The same cultures were imaged before and after adding 100 mM KCl and counted the number of bright flashes corresponding to release events. A ~15-fold increase was found in release events 634871-7173-5771, v. 3 with KCl (50 ± 6 vs. 3 ± 1 for control, P < 0.005, t-test, n = 5; Figs. 10j-l). Together, these results are consistent with AF-SEP being released from synaptic vesicles, moving across the synaptic cleft, binding to the adjacent postsynaptic neuron perisynaptically, and then being endocytosed. Given that AF likely binds to the extracellular N-terminal domain of GluA1, it was tested whether the expression of ss-AF-YFP affects synaptic transmission to hippocampal CA1 neurons. It was found that in cells in hippocampal slices transfected with ss-AF-YFP using biolistics, the amplitudes of spontaneous EPSCs of AMPA and NMDA receptors and paired-pulse facilitation were not significantly different from control cells, consistent with the expression of AF not affecting synaptic transmission (Figs.16a-j). Transsynaptic tracing in vitro and in vivo The results in Fig.10h are consistent with VAMP2 carrying AF-SEP to synaptic vesicles, where it is cleaved off and released into the synaptic cleft following expression of ATLAS in the presynaptic cell (Fig. 11a). To generate a protein capable of mediating transsynaptic tracing, Cre recombinase was introduced into the construct, replacing SEP, to generate VAMP2-At-BACEcs-AF-Cre (ATLASCre, Fig. 10b). Following expression of ATLASCre, AF-Cre should be released into the synaptic cleft and then bind to AMPA receptors at the postsynaptic membrane after which it needs to be endocytosed and carried to the nucleus to provide genetic access to the postsynaptic cell (Fig. 11a). To facilitate this final step, the DNA binding domain from CCR520, 25, which contains a robust nuclear localization signal, was added to the ATLAS protein. To test the strategy, ATLASCre was transfected into dissociated cultures infected with AAV encoding a floxed GFP reporter (AAV8-DIO-GFP). Cells with GFP and Cre were observed that were not labeled with the starter cell marker, At, consistent with the transneuronal transport of Cre and recombination of the floxed allele, implying transport of Cre to the nucleus (Fig. 11b-e). Thus, our results are consistent with ATLASCre mediating transsynaptic tracing and providing genetic access to postsynaptic cells. To test the strategy in vivo, AAV8-ATLAScre were infected into the medial prefrontal cortex (mPFC) and AAV8-DIO-mCherry into the striatum (Str) of an adult mouse (Fig. 12a). Following one week, At staining was found in mPFC, and abundant mCherry 644871-7173-5771, v. 3 staining in the Str (Fig.12b, c). In contrast, there was negligible mCherry staining in a control experiment without AAV8-ATLAScre (Fig.12d). Because the projection from mPFC to Str is strictly anterograde26, this result is consistent with AAV8-ATLAScremediating the transsynaptic transport of Cre into the nucleus of the postsynaptic cell, causing mCherry labeling. Tracing from genetically-determined cells The ability to trace from genetically determined cell types is critical, given the heterogeneous distribution of different neuronal types in many brain regions. To mediate genetically determined tracing, AAV8-DIO-ATLASFLP were generated (Fig. 10b), where expression of ATLAS is Cre-dependent and the payload is FLP (Flip recombinase). This construct was tested by tracing the projection from the primary visual cortex (V1) to the superior colliculus (SC), which is strictly anterograde27. Lenti-Camkii-Cre (a lentivirus expressing Cre specifically in excitatory neurons) and AAV8-DIO-ATLASFLP were co-injected into V1 and AAV8-fDIO-mCherry into SC. In addition to At staining in V1, abundant staining was seen for mCherry in SC that was not observed in the control (Fig. 12e-h). Note that an advantage of this strategy is that lentivirus does not have any intrinsic retrograde or anterograde transneuronal transport that could cloud the interpretation of the results, unlike virtually every pseudotype of AAV17. To test the ability of ATLAS to trace from labeled cells in transgenic Cre mouse lines, the projection was examined from the dorsal Lateral Geniculate Nucleus (dLGN) to V1 in a VIPR2-Cre line expressing Cre in neurons in the dLGN28. AAV8-DIO-ATLASFLP were injected into dLGN and AAV8-fDIO-mCherry into V1. Staining of At was found specifically in the dLGN, and mCherry staining in V1 was dense in layer IV and less dense in layers II / III and V (Fig.12i-l), consistent with known anatomy29. These results are consistent with ATLAS tracing anterogradely from Cre-expressing neurons in wild-type and transgenic mice. Increasing efficiency of transsynaptic labeling through co-infection of BACE To measure the efficiency of transsynaptic tracing by ATLAS, AAV8- ATLASCre were injected in mPFC and AAV8-DIO-mCherry in Str, and the total number of 654871-7173-5771, v. 3 labeled cells were counted in four consecutive 25 µm thick sections after one week of incubation, and the density per mm2was calculated. Initially, 160 + / - 14 labeled cells / mm2were observed (n = 9 from 3 mice). One possible inefficiency in this system is that there may be insufficient endogenous BACE in presynaptic cells to cleave AF-Cre from VAMP2. To test this hypothesis, exogenous BACE were co-expressed with ATLASCreand the resulting efficiency was measured. When AAV8-BACE-HA and AAV8-ATLASCre were co-injected into mPFC in equal amounts, it did not significantly affect the number of postsynaptic cells labeled with mCherry (150 ± 20 cells / mm2, n = 6 from 3 mice, p = 0.95, Tukey's multiple comparisons, Figs. 17a-f). However, when AAV8-BACE-HA and AAV8-ATLASCrewere injected in a 5:1 ratio, a significant increase was seen in the number of cells in Str labeled with mCherry, compared to both AAV8-ATLAS + BACE 1:1 and AAV-ATLAS alone (260 ± 10 / mm2, n = 12 from 4 mice; p < 0.001, Tukey's multiple comparisons test, Figs.17a-f). With this ratio of BACE to ATLAS, approximately 10% of postsynaptic neurons labeled were seen (Figs.17a-f). Thus, when supplemented with exogenous BACE, ATLAS labels cells in the Str at a similar density to YFV in a similar experiment16. Labeling is transsynaptic It has been shown that AF-Cre is secreted from presynaptic sites and endocytosed, likely through interaction with postsynaptic GluA1 (Figs.10a-l). Given the lack of other plausible mechanisms by which ATLAS can leave the presynaptic cell and enter the postsynaptic cell, it is likely that transneuronal labeling mediated by ATLAS occurs exclusively through synapses. To test this hypothesis in vivo, it was asked whether transneuronal labeling could happen from inhibitory neurons. Given that uptake of ATLAS is mediated by GluA1 receptors, which are not found at inhibitory synapses, if ATLAS were expressed in inhibitory neurons, any transneuronal labeling would have to be nonsynaptic (Fig. 13a). Accordingly, AAV8-DIO-ATLASFLPand AAV8-fDIO-mCherry were injected into V1 of an SST-Cre mouse, where somatostatin-positive inhibitory interneurons express Cre30. When the labeling was examined in the cortex after two weeks, no cells expressing mCherry were seen that were not also labeled with At (Fig. 13b-d), indicating that no transneuronal labeling had occurred. Thus, the results of the above experiments, in combination with experiments in culture, led to a conclusion that ATLAS labels postsynaptic neurons exclusively 664871-7173-5771, v. 3 through the transsynaptic transfer of proteins. Such an observation has not been made for any other transneuronal labeling method. Example 3 - Electrophysiology experiments confirm the specificity of ATLAS tracing To test for the specificity of ATLAS tracing, optogenetically evoked currents we measured in the circuit from the primary visual cortex to the striatum. AAV8-ATLASsnCre (Figs. 10b, 15a) and AAV8-BACE-HA were co-infected in V1, and AAV8-DIO- mCherry / tdTomato in Str. Two weeks later, AAV8-DIO-ChR2-GFP were injected in the same area in V1 as the first injection, to manipulate ATLAS-labeled presynaptic inputs while avoiding expression competition between ATLAS and ChR2. Histology two weeks after the second injection revealed efficient expression of At, HA, ChR2 in V1 and Cre-dependent mCherry / tdTomato in striatum, consistent with the projection pattern of Str-projecting V1 neurons (Figs. 18a-h). To compare V1 innervation between ATLAS-labeled vs unlabeled postsynaptic neurons, optically evoked EPSCs (oEPSCs) were measured following a 2 ms flash of 473 nm blue light in acute brain slices prepared from mice similarly injected with virus four weeks earlier (Fig.13e). Specifically, sequential, paired whole-cell recordings were performed where oEPSCs were measured of an ATLAS-labeled (mCherry / tdTomato+) and an unlabeled (mCherry / tdTomato-) neuron in the same field of view (100 x 100 µm; Fig.13e, f).9 pairs of cells (from four mice) were recorded, in which 9 of 9 labeled cells carried a current, compared with 7 of 9 unlabeled cells (Fig.13f, g). The existence of oEPSCs in unlabeled cells can be due to inefficiencies of ATLAS labeling or ChR2 infection of other V1 neurons that receive inputs from ATLAS-labeled presynaptic cells. Importantly, oEPSCs recorded from labeled cells were significantly larger in amplitude and total charge compared with those of unlabeled cells (amplitude: -139 ± 37 pA vs. -59 ± 19 pA, p < 0.005, Wilcoxon Signed-Rank Test, total charge: -0.137 ± 0.03 pC vs. -0.045 ± 0.01 pC, p < 0.005, Wilcoxon Signed-Rank Test, plotted as absolute values in Fig. 13g). These results suggest that when ATLASCreand a Cre-dependent reporter are simultaneously expressed in different brain regions, cells exhibiting Cre-dependent labeling are very likely to receive synaptic inputs from cells expressing ATLASCre. ATLAS tracing does not affect synaptic transmission 674871-7173-5771, v. 3 To test whether ATLAS disrupts synaptic transmission, AAV8-ATLASsnCre(Figs. 10b, 15a), AAV8-BACE-HA, and AAV8-DIO-ChR2-GFP, were co-infected into the mPFC, and AAV8-DIO-mCherry in the striatum of wild-type mice. The mPFC to Str projection was chosen because connectivity of this circuit is particularly dense. Since it was found that ATLAS labels 10-15% of target neurons in this circuit (Figs. 17a-f), it was reasoned that it should be possible to find both labeled and non-labeled cells that are synaptically connected to ChR2 expressing neurons, so it could be compared whether ATLAS labeling affects the cell's physiological properties. Four weeks after virus injection, coronal slices were cut of striatum approximately 350 µm thick and incubated them in artificial cerebral spinal fluid for in vitro slice recording. Whole-cell patch clamp recording was performed to measure photo- stimulation (470nm) evoked excitatory postsynaptic current (eEPSC) from striatal cells (Figs. 18a-h). To evaluate the potential effect of ATLAS tracing on synaptic transmission, we compared the striatum cells with and without mCherry expression (Figs.18a-h). Robust evoked EPSCs were recorded in mCherry-expressing cells with an onset latency of < 5 ms following the light flash in 8 of 8 cells (from 3 mice), indicating that the presynaptic ChR2-expressing cells were synaptically connected with mCherry-labeled cells (Figs.18a-h). Furthermore, when non-labeled postsynaptic cells were recorded from, ChR2-evoked EPSCs were not significantly different from those of labeled cells in terms of onset latency, amplitude, and time constant (Figs. 18a-h, n = 7 cells from 3 mice). This result is consistent with transsynaptic labeling with ATLAS not affecting synaptic transmission, and thus being nontoxic. Moreover, it was further verified that the evoked currents are monosynaptic through sequential application of TTX, which blocks action potentials, and thus polysynaptic transmission, and 4AP, which blocks K+channels and facilitates direct activation of presynaptic terminals (Figs.18a-h). ATLAS is exclusively anterograde and monosynaptic A significant limitation of many virally-based anterograde transneuronal labeling methods is that they also mediate labeling in the retrograde direction16, 17. Because AF binds specifically to the AMPA receptor, and AF-Rec (AF-recombinase) is released from presynaptic terminals, it is unlikely to mediate retrograde transneuronal labeling (Fig.11a). To determine whether ATLAS mediates retrograde labeling in vivo, AAV8-DIO-ATLASFLP and Lenti-hsyn-Cre were injected into Str and AAV8-fDIO-mCherry into mPFC (Fig. 14a). 684871-7173-5771, v. 3 Because the mPFC to Str projection is unidirectional and lentivirus does not spread retrogradely, any labeling in mPFC would be due to retrograde transneuronal labeling mediated by ATLAS. However, despite abundant At labeling in Str, indicating that ATLAS was expressed at a high level, no labeling was found above background in mPFC (Fig. 14b-d). These data are consistent with ATLAS not mediating retrograde transport. To test whether ATLAS can mediate transsynaptic labeling that is monosynaptic, a critical property for transsynaptic tracing results to be interpretable, it was tried to trace the polysynaptic pathway from V1 to SC to the Parabigeminal nucleus (PBG)16. AAV8-ATLASCreinto V1 and AAV8-DIO-mCherry were injected into SC and PBG (Fig.14e). Abundant transsynaptic mCherry labeling was found in SC but none in PBG (Fig. 14f-h). Furthermore, when AAV8-ATLASCrewere injected into SC, abundant labeling was seen in PBG, demonstrating that ATLAS is capable of tracing the SC to PBG pathway (Fig. 14i-l). Thus, transsynaptic labeling mediated by ATLAS is exclusively anterograde and monosynaptic. Activity-dependence of ATLAS Because ATLAS mediates transsynaptic labeling through the release of AF- Recombinase from synaptic vesicles and release is dramatically increased in high concentrations of KCl (Fig. 10j-l), transsynaptic labeling is likely activity-dependent. To test for activity dependence of transsynaptic labeling in vivo, AAV8-ATLASCre + AAV8- hHM3Dq-mCherry, an excitatory Dreadd31, were injected into mPFC and AAV8-DIO-GFP into Str (Figs. 19a-g). We injected one group of mice with CNO intraperitoneally but not the other and compared GFP expression in Str (Figs. 19a-g). It was found that GFP fluorescence intensity increased by 95% (37 ± 5 a.u., n = 4 vs. 19 ± 2 a.u., n = 4) in the mice injected with CNO vs. those without, a significant difference (p < 0.05, Mann Whitney U test), despite having roughly equal At expression in the mPFC (Figs.19a-g). Thus, our experiments confirm that ATLAS mediates activity-dependent transsynaptic labeling. An alternate synaptic vesicle targeting domain ATLAS is comprised of multiple independent components, each of which can thus, in principle, be modified or upgraded to improve efficiency or alter function, which is not 694871-7173-5771, v. 3 possible with conventional viral tracers. This property was utilized to optimize the VAMP2 component of ATLAS responsible for presynaptic targeting. Although VAMP2 is widely used as a presynaptic marker, it can produce toxicity when expressed at a high level, which could affect the efficiency of ATLAS tracing. Accordingly, the cytoplasmic domain of VAMP2 was switched out for a nanobody against Synaptotagmin 1 (SYTnb)32to give SYTnb-VAMP2Δ- At-BACEcs-AMPA.FingR-Cre (ATLASsnCre, Figs.10b, 15a), where VAMP2Δ consists of the extracellular and transmembrane domains of VAMP2. To test ATLASsn-Cre, it was transfected into dissociated cultures of cortical neurons while infecting them with AAV8-DIO- mCherry. Abundant cells were seen that were labeled with mCherry, but not with AT, indicating that they were labeled transsynaptically (Fig. 15b). A closeup image of such a postsynaptic cell revealed At labeling opposite its dendritic spines, consistent with presynaptic labeling (Fig.15b, inset). To test AAV8-ATLASsnCrein vivo, it and AAV8-BACE-HA were injected into the mPFC and AAV8-DIO-mCherry into the striatum. After 2 weeks of incubation, expression of At was seen in the mPFC and abundant mCherry labeling in Str (Figs.15c, 15d, 20a-h). The dorsal region of postsynaptic mCherry labeling colocalized almost perfectly with At labeling representing presynaptic termini (Figs. 15d, 20a-h), consistent with ATLASsn mediating transsynaptic labeling. To further test ATLASsn in vivo, AAV8-ATLASsnCre and AAV8- BACE-GFP were co-infected in M1 cortex and AAV8-DIO-mCherry in the Str and thalamus (Thal, Fig. 15e). After two weeks incubation, staining of GFP and mCherry revealed labeled presynaptic cells in M1 (Fig. 6f) and postsynaptic cells in Str (Fig. 15g) and Thal (Fig. 15h). Overlap of GFP and mCherry labeling in Str and Thal (Figs. 20a-h) is consistent with transsynaptic labeling. Furthermore, minimal background was observed in the Str when AAV8-DIO-mCherry alone was injected (Figs.20a-h). ATLAS mediates transsynaptic labeling in the rat It is highly desirable that transsynaptic labeling techniques work in systems other than the mouse. Given the high sequence similarity between AMPA receptors in mice and rats, it was tested whether ATLAS would work in the rat. Accordingly, AAV8- ATLASsnCreand AAV8-BACE-HA were injected into the CA1 region of the ventral hippocampus of a rat, and AAV8-DIO-tdTomato into its nucleus accumbens (ACB, Fig.15i), 704871-7173-5771, v. 3 to which the CA1 neurons project. At staining was observed in CA1 (Fig. 15j), and dense tdTomato labeling in ACB (Fig.15k), consistent with transsynaptic labeling. Finally, tdTomato labeling of axons within the lateral hypothalamic area (LHA, Fig.15l), which receives axonal projections from ACB33, confirms the postsynaptic labeling of neurons within ACB. Example 4 – Additional Methods Target preparation, mRNA display, and screening. The amino terminal domain of GluA1 (1-394 a.a.) fused to a biotin acceptor tag (AviTag, Avidity) grown in suspension cultures of Sf9 cells was used as a target for the mRNA display selection. mRNA display procedure was carried out as described in49. Screening for binding FingRs was performed in Cos7 cells expressing ss-GluA1-HA (where ss is the signal sequence of GluA1) and Stargazin-V5 with the candidate FingRs fused to a Myc tag. Cos7 cells were initially stained live with anti-MYC antibody and then fixed, permeabilized, and stained for HA and V5. Fns labeling the surface of cells expressing GluA1 and Stargazin were then expressed in dissociated cultures and tested for the ability to label endogenous GluA1. Western blot. Lysates from cultured neurons were collected in NP40 lysis buffer (150 mM NaCl, 100 mM Tris-HCl, 1% NP40 in PBS) containing 1X protease inhibitors (cOmplete ULTRA tablets, Roche, cat# 05892970001). Samples were run on mini-protean TGX gels (Biorad, cat# 4569034) and transferred to LF PVDF membrane (Biorad, cat# 1620264). The membrane was blocked in 5% dry milk in PBS. The membrane was probed with anti-GluR1 (EMD Millipore ABN241; anti-HA, Cell Signaling 3724S; anti-tubulin, Sigma T6168; and purified AF-HA). Membranes were imaged with the LI-COR Odyssey IR imaging system. Dissociated cultures. Pups from timed pregnant Sprague Dawley rats (E19) (Charles River) were used for dissociated cultured cortical neurons as described in50. Briefly, cortices from E19 pups were trypsinized in Neuronal Isolation Enzyme with papain (ThermoScientific 88285) and triturated into a single cell suspension. Cells were plated at a density of 1.5 X 10^5 per well on 6-well plates in Neurobasal media (Gibco 21103-049) containing B27 supplement (Gibco 17504-044), Fetal Bovine Serum Characterized (Cytiva SH30071.03), glutaMAX-I (Gibco 2523105) and gentamicin (Sigma G1397). Media was 714871-7173-5771, v. 3 changed after 4 hours, and after 7 days, it was replaced with neurobasal media without Fetal Bovine Serum. Transfection. Cultured neurons were transfected using the CalPhos Mammalian Transfection Kit (Takara Cat# 631312). Briefly, 8 µg of plasmid DNA was incubated with 100 ml of 2M CaCl2 in sterile H2O; this mixture was added to 100 ml of 2X HEPES-buffered saline and incubated at room temperature for 20 minutes. The mixture was then added to the cultured neurons dropwise and incubated for 1-3 hours for crystal formation. After incubation, the media was removed, and neurons were washed with HEPES-NaCl buffer (140mM NaCl, 5mM KCl, 24mM D-glucose, and 10 mM HEPES, pH 7.2). After washing, transfected neurons were maintained in conditioned neurobasal media. Immunocytochemistry. Dissociated cultured neurons were fixed in 4% Paraformaldehyde (EMS 15714) for five minutes, followed by 3 X 5 minute washes in 1x phosphate buffered saline (PBS). After washes, neurons were blocked for 30 minutes in blocking buffer (5% Normal goat serum, 1% Bovine serum albumin, 0.1% Triton-100 in PBS). Following blocking, neurons were incubated with primary antibodies for 1 hour at room temperature (anti-GFP, Aves GFP-1020; anti-Map2, Abcam ab5392; anti-clathrin, GeneTex GTX80218; anti-mCherry, Rockland M11217; anti-RFP, Invitrogen; anti-GluA1, EMD Millipore ABN241; anti-PSD-95, NeuroMab 75-348; anti-HA, cell signaling 3724S; anti-Cre, EMD Millipore MAB3120), followed by 3 X 5 minute washes in PBS. Neurons were incubated in secondary antibodies in blocking buffer for 1 hour at room temperature in the dark (Alexa Fluor 488 goat anti-chicken, Invitrogen A-11006; Alexa Fluor 594 goat anti-rabbit; Invitrogen A-11012; Alexa Fluor 594 goat anti-mouse, Invitrogen A-11005; Alexa Fluor 647 goat anti- mouse, Invitrogen A-21235; Alexa Fluor 647 goat anti-rabbit, Invitrogen A-21244; JF549 halotag ligand, Janelia Farm; JF647 halotag ligand, Janelia Farm; FluoTag-X2 anti-alfa 647, NanoTag biotechnologies N1502 AF647). Neurons were mounted using Dapi-fluoromount-G (Electron microscopy services 17984-24). Microscopy. Fixed dissociated neurons were imaged using Olympus IX-71 and IX-81 microscopes. Confocal images were acquired using a Zeiss 880 microscope. 724871-7173-5771, v. 3 Protein purification. AF-HA protein was purified from the media following secretion by HEK293T cells. Briefly, HEK293T cells were transfected with the plasmid pCAG-ss-AF-3XHA-TEVcs-HALOTag using Lipofectamine 2000 (Invitrogen, Cat# 11668- 019) following the manufacturer's protocol and medium containing the secreted protein AF- HA-TEVcs-HALOTag was collected. The medium was incubated with HaloLink resin (Promega, Cat# G1914) overnight at 4oC. The resin was washed 3 times with wash buffer (100 mM Tris, pH7.6, 150 mM NaCl, 0.5% Triton-X), and the resin was then incubated with TEV protease (New England Biolabs, Cat# P8112S) overnight at 4oC. Next, the supernatant was collected and incubated with NEBExpress Ni Resin (New England Biolabs, Cat# S1428S) overnight at 4oC to remove the TEV protease from the purified protein. Purified protein was then dialyzed into 20mM HEPES, 250mM NaCl pH 7.4, and concentrated using the Amicon Ultra centrifugal filters (cat# UFC501096). Endocytosis experiments. Neuronal cultures were incubated with 1 µM purified AF-HA protein for 1hr at 37oC. Next, cultures were washed 3 times with 1X Hanks Balanced Salt Solution containing 10 mM HEPES, fixed with 4% paraformaldehyde, and stained for anti-HA (Cell signaling 3724S) and anti-Map2 (Abcam ab5392). Presynaptic release experiments. Plasmids encoding VAMP2-BACEcs-SEP (1ug) or VAMP2-BACEcs-AF-SEP (1 µg) with HA-BACE (2 µg) were transfected into 15-18 DIV cultured neurons using the calcium phosphate method (see below). Two days before transfection, the cultures were infected with AAV8-Cre and AAV8-PSD-95.FingR-TdTomato to label postsynaptic sites. Neurons were imaged 3 to 5 days later with or without 100 mM KCl. Time-lapse imaging was performed for 200 frames at an interval of 1000 ms and an exposure of 500 ms. Time constants were measured using curve fitting functions in GraphPad Prism. Videos of ΔF of SEP during release events were generated using ImageJ by taking the difference of adjacent image frames to eliminate background and SEP fluorescence beyond the initial frame after release. Electrophysiology (testing of AMPA.FingR). All experiments were performed in accordance with NIH Guidelines for the Care and Use of Laboratory Animals, and all procedures were approved by the Institutional Animal Care and Use Committee of the University of Southern California. As previously described, 400 µm thick rat organotypic 734871-7173-5771, v. 3 hippocampal slice cultures were prepared from male and female P6 to P8 Sprague Dawley rats 51. Tissue was isolated, and an MX-TS tissue slicer (Siskiyou) was used to make 400 µm transverse sections. Tissue slices were placed on squares of Biopore Membrane Filter Roll (Millipore) and placed on Millicell Cell Culture inserts (Millipore) in 35 mm dishes. Slices were fed 1mL of culture media containing MEM + HEPES (Gibco Cat#12360-038), horse serum (25%), HBSS (25%) and L-glutamine (1 mM). Media was exchanged every other day. Whole-cell recordings were performed on DIV7-8. During recordings, slices were maintained in room-temperature artificial cerebrospinal fluid (aCSF) containing 119 mM NaCl, 2.5 mM KCl, 1 mM NaH2PO4, 26.2 mM NaHCO311 mM glucose, 4 mM CaCl2, and 4 mM MgSO4. 5 μM 2-chloroadenosine and 0.1 mM picrotoxin were also added to the aCSF to dampen epileptiform activity and block GABAAreceptor activity, respectively. Osmolarity was adjusted to 310-315 mOsm. aCSF was saturated with 95% O2 / 5% CO2 throughout the recording. Borosilicate recording electrodes were filled with an internal whole-cell recording solution containing 135 mM CsMeSO4, 8 mM NaCl, 10 mM Hepes, 0.3 mM EGTA, 5 mM QX-314, 4 mM Mg-ATP, and 0.3 mM Na-GTP. Osmolarity was adjusted to 290–298 mOsm and pH-buffered at 7.3–7.4. CA1 pyramidal neurons were identified using differential interference phase contrast microscopy while GFP-expressing transfected neurons were identified using epifluorescence microscopy. Dual whole-cell recordings of CA1 pyramidal neurons were made through simultaneous recordings from a transfected neuron and a neighboring, non-transfected control neuron. Synaptic responses were evoked by stimulating with a monopolar glass electrode filled with aCSF in the stratum radiatum. Membrane holding current, pipette series resistance and input resistance were monitored throughout recording sessions. Data were acquired using a Multiclamp 700B amplifier (Molecular Devices), filtered at 2 kHz, and digitized at 10 kHz. AMPAR-evoked EPSCS (-eEPSCs) were measured at -70mV. NMDAR- eEPSCs were measured at +40 mV and were temporally isolated by measuring amplitudes 150 ms following the stimulus, at which point the AMPAR-eEPSC had completely decayed. For paired-pulse facilitation, paired pulses at 40ms inter-pulse interval were delivered, and the ratio of peak amplitudes was analyzed to generate barplots depicting mean ± SEM. Data analysis was performed using Igor Pro (Wavemetrics). In the scatter plots for simultaneous dual whole- cell recordings, each open circle represents one paired recording, and the closed circle 744871-7173-5771, v. 3 represents the average of all paired recordings. No more than one paired recording was performed on any given hippocampal slice. Sparse biolistic transfections were performed on day in vitro 1 (DIV1) as previously described52, 53.50 µg of mixed plasmid DNA encoding ss-AF-YFP was coated on 1μm-diameter gold particles in 0.5 mM spermidine, precipitated with 0.1 mM CaCl2, and washed four times in pure ethanol. These DNA-coated gold particles were then coated onto PVC tubing, dried briefly using ultra-pure N2gas, and stored at 4 °C in desiccant. Before use, the gold particles were brought to room temperature and delivered to slice cultures via a Helios Gene Gun (BioRad). Construct expression was confirmed by YFP epifluorescence. Transsynaptic tracing in culture. Neuronal cultures (14 DIV) were transfected with plasmids for ATLAS (1 µg) and HA-BACE (2 µg) using the calcium phosphate method. Transfection was followed by infection with AAV8-DIO-GFP (with human synapsin, hSyn, promoter) and incubated for 7 days. Neurons were fixed and probed as described. Animal preparation and stereotaxic surgery (mouse Figs.12a-l, 13b-g, 14a- 1, 15c, and 15d). All experiments were performed in accordance with NIH Guidelines for the Care and Use of Laboratory Animals. All procedures used in this study were approved by the Animal Care and Use Committee at the University of Southern California. Male and female Wild-type mice (C57BL / 6J, Jackson Laboratories, stain #000664), Vipr2-Cre (Cre expression in dLGN, Jackson Laboratories, strain #031332), and Sst-IRES-Cre (Cre expression in somatostatin interneurons, Jackson Laboratories strain #013044) mice aged 2–6 months were used in this study. Mice were housed in a light-controlled (12 h light / dark cycle) environment with ad libitum access to food and water. For stereotaxic viral injection, mice were anesthetized initially in an induction chamber containing 4% isoflurane mixed with oxygen and then transferred to a stereotaxic frame equipped with a heating pad. Anesthesia was maintained throughout the procedure using continuous delivery of 1.5% isoflurane through a nose cone at a rate of 0.5 L / min. The scalp was shaved, and a small incision was made along the midline to expose the skull. After leveling the head relative to the stereotaxic frame, injection coordinates based on Paxinos and Franklin's 4th Edition54were used to mark the location on the skull directly above the target area, and a small hole (0.5 mm diameter) was drilled. Viruses were 754871-7173-5771, v. 3 delivered through pulled glass micropipettes using pressure injection via a micropump. Total injection volumes ranged from 50 to 600 nl, at 20 to 60 nl / min. Following injection, the micropipette was left in place for 15 min to minimize virus diffusion into the pipette track. After withdrawing the micropipette, the scalp was sutured closed. Animals were recovered from anesthesia on a heating pad to minimize inflammation and discomfort and returned to their home cage. Animals were administered Ketofen (5mg / kg) at the beginning of the surgical procedure and again every 24 h for 2d following surgery. Bregma and Lambda coordinates are listed in millimeters as follows: AP is the distance from Lambda, ML is the distance from midline, and DV is the distance from Lambda except for V1 and SC, which use the distance from the surface of the cortex. Medial Prefrontal Cortex (mPFC) (6.3, 0.3, 1.7); Str (5.1, 1.8, 3.4); dorsal lateral geniculate nucleus (dLGN) (2.2, 2.2, 2.9); Primary Visual cortex (V1) (0.8, 2.4, 0.6); Superior colliculus (SC) (0.6, 0.8, 1.2); Parabigeminal nucleus (PBG) (0.5, 1.8, 3.3); For Figs.15g-I, the injections of AAV8-ATLASCre were made in the somatosensory cortex of wild-type mice and AAV8-Ef1a-fDIO-mCherry into the striatum and thalamus. Histology (mouse Figs.12a-l, 13b-d, 14a-l, 15c, and 15d). Mouse brains were perfused with 40 mL of phosphate-buffered saline (PBS) followed by 40 mL of 4% paraformaldehyde in PBS. The brains were then fixed overnight in 4% paraformaldehyde at 4oC and dehydrated in 30% sucrose solution for at least 48 hours. The brains were embedded in tissue freezing medium at -45oC and then sectioned to a thickness of 25µm – 35 µm using a cryostat. For immunohistochemistry, the brain sections were washed in PBS and incubated overnight in primary antibodies diluted in PBS containing 4% goat normal serum and 0.5% triton X-100. The primary antibodies used are: rabbit anti-RFP, Rockland (600-401-379), 1:1000; chicken anti-GFP antibody, AVES (gfp-1020), 1:1000; 1:1000; Fluotag-X2 anti- ALFA, NanoTag (N1502-ALFA647-L), 1:1000 The sections were then washed with PBS and incubated in corresponding secondary antibodies for 2 hours. After incubation, the sections were washed with PBS and mounted onto glass slides with DAPI Flouromount-G, EMS. The secondary antibodies used in this study and their dilution are as follows: Alexa Flour 488- conjugated goat anti-chicken or anti-mouse IgG, Thermo Fisher, 1:1000; Alexa Flour 594- conjugated anti-rabbit IgG, Thermo Fisher, 1:1000; Alexa Flour 647-conjugated goat anti- mouse IgG, Thermo Fisher, 1:1000. 764871-7173-5771, v. 3 Stereotaxic surgery and histology (mouse, Fig. 15 e-h). The ATLASsnCreviral vector AAV8- SYTnb-VAMP2Δ-At-BACEcs-AMPA.FingR-Cre and AAV8-BACE- GFP (1:1, 200 nl) were injected stereotaxically into the motor cortex of wild-type mice at (1.0, 2.0, 1.8). They also received an injection of a Cre-dependent reporter vector: AAV8-Ef1a- FLEX-tdTomato (addgene: 28306) into the striatum (0.5, 2.0, 4.0) and thalamus (-1.55, 1.5, 3.75). Following a survival time of 2 weeks animals were perfused transcardially with saline followed by 4% formaldehyde, brains were removed, post-fixed for 24 hours and transferred to 20% sucrose in PBS. Brains were coronally sectioned with a freezing microtome (50 µm) and sections processed with immunohistochemical techniques to visualize cortical neurons injected with AAV8-BACE-GFP (a marker for ATLAS), mCherry or tdTomato in neurons transsynaptically labeled in the striatum and thalamus with sequential incubation in rabbit anti- RFP (600-401-379, Rockland Immunochemicals) and chicken anti-GFP (A10262, Invitrogen), and the goat anti-chicken 488 (A32931, Invitrogen) and goat anti-rabbit 555 (A32732, Invitrogen.) For each brain every section was mounted sequentially onto slides, sections were counterstained with Neurotrace Blue 435 (N21479, Invitrogen), and coverslipped with Aqua- polymount (18606, Polysciences). Slides with 12-15 coronal brain sections were imaged on a Zeiss EpiFluorescent Microscope equipped with Xylis broad spectrum LED illumination (Excelitas Technologies), a Hammamatsu Orca Flash4.0 camera and Ludl stage drive controlled by Neurolucida Imaging software (MBF Bioscience, Williston VT). Imaged sections were reconstructed into a whole brain volume, labeled neurons marked and registered to the Allen common mouse atlas with NeuroInfo software (MBF Biosciences)55. Animal preparation and stereotaxic surgery (rat). Male Sprague-Dawley rats (Envigo, Indianapolis, IN) weighing 300–400 g were used in this experiment. Animals were individually housed in shoebox cages and given ad libitum access to chow (Rodent Diet 5001, LabDiet, St. Louis, MO) and water. Rats were housed in a 12 h:12 h reverse light / dark cycle (lights off at 11:00 a.m.). All experiments were performed in accordance with NIH Guidelines for the Care and Use of Laboratory Animals, and all procedures were approved by the Institutional Animal Care and Use Committee of the University of Southern California. Rats were first anesthetized and sedated via intramuscular injections of ketamine (90 mg / kg), xylazine (2.8 mg / kg), and acepromazine (0.72 mg / kg) cocktail, prepped for surgery, and placed in stereotaxic apparatus. Stereotaxic injections of viruses were delivered using a micro-infusion 774871-7173-5771, v. 3 pump (Harvard Apparatus, Cambridge, MA, USA) connected to a 33-gauge microsyringe injector attached to a PE20 catheter and Hamilton syringe. The flow rate was calibrated and set to 5 µl / min. Injectors were left in place for 3 min post-injection. Following viral injections, animals were surgically closed with sutures. Viruses were incubated for 21 days after injection to allow for viral transduction and expression. AAV8-ATLASsnCrewas unilaterally injected into the CA1v at the following coordinates: −4.9 mm AP, ±4.8 mm ML, −7.8 mm DV (zeroing at skull site). AAV-pCAG-FLEX-tdTomato-WPRE (Addgene; Catalog #: 51503) was unilaterally injected into the ACB at the following coordinates: +1.2 mm AP, ±1.0 mm ML, −6.75 mm DV (zeroing at dura). The injection volume was 100 nl / site. For control experiments animals were injected with AAV-pCAG-FLEX-tdTomato-WPRE unilaterally in the ACB, but no injection was made in the ventral CA1. Histology (rats). Rats were first anesthetized and sedated via intramuscular injections of ketamine (90 mg / kg), xylazine (2.8 mg / kg), and acepromazine (0.72 mg / kg) cocktail and then perfused using 0.9% sterile saline (pH 7.4), followed by 4% paraformaldehyde (PFA) in 0.1 M borate buffer (pH 9.5; PFA). Brains were extracted and post- fixed with 12% sucrose in PFA overnight, and then flash-frozen in methyl-butane cooled by dry ice. Brains were sectioned into 30-µm sections on a microtome cooled with dry ice and collected in an antifreeze solution, and stored in a −20 °C freezer. The following IHC fluorescence labeling procedures were adapted from previous work56. Rabbit anti-RFP (1:2000, Rockland Inc., Limerick, PA, USA; Catalog #:600- 401-379; Clonality: Polyclonal) and FluoTag®-X2 anti-ALFA (1:1000, NanoTag Biotechnologies; Catalog #: N1502) were used. Rabbit anti-RFP was prepared in 0.02 M potassium phosphate-buffered saline (KPBS) solution containing 0.2% sodium azide and 2.0% normal donkey serum and stored at 4 °C overnight. After many series of washing with 0.02 M KPBS, brain sections were incubated in a secondary antibody solution for RFP and the ALFA- tag nanobody. Sections were stored overnight at 4 °C (Jackson Immunoresearch; West Grove, PA), then mounted, airdried, and coverslipped using 50% glycerol in 0.02 M KPBS and clear nail polish to seal the coverslip onto the slide. Photomicrographs were acquired using a Nikon 80i (Nikon DS-QI1,1280 × 1024 resolution, 1.45 megapixel) microscope under epifluorescence. 784871-7173-5771, v. 3 AAV virus production and purification. Briefly, one day before transfection, Hek293T cells were plated at 1.5 x 107cells per 15 cm dish. Plasmids for pHelper, AAV2 / 8 (Gifts from Dr. Fan Wang), and gene of interest were transfected using PEI (1mg / ml). The cells were cultured for 72 hrs. Cells were scraped, and the pellet from 6 dishes was resuspended in 4 ml of cell lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8.5). The pellet was then frozen in a dry ice / ethanol bath and thawed in a 37oC water bath for three cycles. The cell lysate was thawed in a 37 °C water bath, and benzonase was added at a final concentration of 50 U / ml for 30 min. This was followed by centrifugation at 4,500 rpm for 30 min at 4 °C in a tabletop centrifuge. Subsequently, vector-containing supernatant was collected and loaded over an iodixanol density gradient, centrifuged at 50,000 rpm for 2 h at 18 °C, and concentrated using VIVASPIN 2010K MWCO filter tubes. Lentivirus production and purification. Briefly, one day before transfection, Hek293T cells were plated at 1.5 x 107cells per 15 cm dish. Plasmids for pMD2.G (Addgene #12259), psPAX2 (Addgene #12260), and gene of interest were transfected using PEI (1mg / ml) and cultured for 72 hrs. Cell supernatant was collected and centrifuged at 1,500g for 15 min. The virus-containing supernatant was filtered through a 0.45 µm filter flask, loaded into sterilized 50 ml centrifuge tubes with 7.5 ml of 10% sucrose solution, and centrifuged for 4 hours at 10,000g and 4oC. After centrifugation, the supernatant was 50 µl of ice-cold PBS added (without Ca2+and Mg2+) to cover the virus pellet and let sit at 4oC for 2 hours. The pellet was resuspended gently and spun at 7,000 rpm for 5 min. Finally, the supernatant was concentrated using VIVASPIN 2030K MWCO (SARTORIUS) filter units with PBS. Plasmid Constructs. The signal sequence from GluA1 (AA 1-18) was inserted upstream of the AMPA.FingR sequence and YFP was inserted downstream of the AF sequence to generate ss-AF-YFP. For all constructs containing VAMP2, the complete sequence of VAMP2 was inserted upstream on an ALFA-Tag (SRLEEELRRRLTE) followed by a BACE ( ^-site Amyloid Precursor Protein-cleaving enzyme 1) cut site (EISEVNLDAEFR). In the case of VAMP2-BACEcs-SEP, the BACE cleavage site (BACEcs) was followed by super ecliptic pHluorin (SEP). For VAMP2-BACEcs-AF-SEP, the BACEcs was followed by the AF sequence and SEP downstream of AF. For DIO-ATLASFLP, Cre was removed from the ATLAS construct and replaced with a Myc Tag (EQKLISEEDL) followed by the recombinase Flp. 794871-7173-5771, v. 3 Nested LoxP sites were then added on either side of the coding sequence. GFP-HA-BACE1 plasmid was purchased from addgene (#165032), and the GFP was removed to make HA- BACE. AAV constructs were generated by ligating the above-mentioned sequences into a pAAV plasmid containing the human synapsin (hsyn) promoter and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). The plasmid Lenti-hsyn-Cre was purchased from addgene (#86641), and the human synapsin promoter was replaced with the CamKII promoter to generate Lenti-CamKII-Cre. Viruses. The following viruses were purchased from addgene : AAV8-hsyn- hM3D(Gq)-mcherry #50474, AAVrg-EF1a-Cre #55636, AAVrg-Ef1a-DIO-Flp-WPRE-hGH #87306, AAV8.hSyn.DIO.mCherry #50459, AAV8-Ef1a-fDIO-mCherry #114471, AAV8.hsyn.DIO.eGFP.WPRE.bGH #50457, and AAV8.hSyn.hM3D.mCherry #50474. Quantification of activity-dependent transsynaptic labeling. For experiments to quantify transsynaptic labeling with and without BACE, mCherry-expressing cells were counted within an identical 1 mm2region over four consecutive 25 µm stained sections of Str. In all cases, the sections examined were roughly the same distance from the site of AAV injection. For experiments to quantify activity-dependent transsynaptic labeling, total GFP fluorescence minus background was measured identically in Str in sections that were equivalent distances from the injection site using ImageJ. Electrophysiology (Figs. 18a-h). To determine whether ATLAS labeling disrupted neurotransmission between mPFC axons and striatal neurons, wild type C57BL / 6J mice co-infected with AAV8-ATLASsnFLP, AAV8-BACE-HA, and AAV8-DIO-ChR2-GFP into the mPFC, and fDIO-mCherry in the striatum were used for slice recording. Note that details of the stereotaxic surgery are the same as used in Figs. 12a-d, and 15c, d. Four weeks following the injections, animals were decapitated following anesthesia, and the brain was rapidly removed and immersed in an ice-cold dissection buffer (composition: 60 mM NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 25 mM NaHCO3, 115 mM sucrose, 10 mM glucose, 7 mM MgCl2, 0.5 mM CaCl2; saturated with 95% O2and 5% CO2; pH = 7.4). Coronal slices of 350 µm thickness were sectioned by a vibrating microtome (Leica VT1000s) and recovered for 30 min in a submersion chamber filled with warmed (35°C) ACSF (composition:119 mM NaCl, 26.2 mM NaHCO3, 11 mM glucose, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, and 1.2 804871-7173-5771, v. 3 NaH2PO4, 2 mM Sodium Pyruvate, 0.5 mM VC). Striatum neurons surrounded by GFP+ fibers were visualized under a fluorescence microscope (Olympus BX51 WI). Patch pipettes (~4–5 MΩ resistance) filled with a cesium-based internal solution (composition: 125 mM cesium gluconate, 5 mM TEA-Cl, 2 mM NaCl, 2 mM CsCl, 10 mM HEPES, 10 mM EGTA, 4 mM ATP, 0.3 mM GTP, and 10 mM phosphocreatine; pH = 7.25; 290 mOsm) were used for whole- cell recordings. Signals were recorded with an Axopatch 700B amplifier (Molecular Devices) under voltage clamp mode at a holding voltage of -70 mV for excitatory currents, filtered at 2 kHz and sampled at 10 kHz. Tetrodotoxin (TTX, 1 μM) and 4-aminopyridine (4-AP, 1 mM) were added to the external solution for recording monosynaptic responses to blue light stimulation (5 ms pulse, 3 mW power, 5–10 trials). Surgery (Fig. 13e-g). Mice were anesthetized with 5% isoflurane and maintained under surgery with 1.5% isoflurane and 0.08% oxygen. All experiments were performed in accordance with NIH Guidelines for the Care and Use of Laboratory Animals, and all procedures were approved by the Institutional Animal Care and Use Committee of Harvard Medical School. Surgeries were performed under aseptic conditions within a stereotaxic frame (David Kopf Instruments). For all surgeries, the skull was exposed and small craniotomies were drilled. Injections were performed through a pulled glass pipette containing virus, driven by a syringe pump (Harvard Apparatus, #883015) and infused at a rate of 30 nl / min. After injections, pipettes were slowly withdrawn (less than 100 µm / s) at least 5 min after the end of the infusion. At the end of each surgery, the wound was sutured and mice were placed in a cage with a heating pad until their activity recovered. Afterwards, mice were returned to their homecage and were given pre- and post-operative oral carprofen (CPG, 5 mg / kg / day) and monitored daily for at least 5 days post-surgery. Viruses were injected in two rounds of surgeries to minimize expression competition between ATLAS viruses (i.e. ATLASsnCre, BACE-HA) and Cre-dependent ChR2. Details of each round of surgery are as follows. Wild type animals undergo the first round of surgery between 4-8 weeks of age. Before the start of surgery, presynaptic virus cocktail was prepared by mixing AAV8-hSyn- ATLASsnCre and AAV8-hSyn-BACE-HA in 3:1 ratio (titer is 2x1013gc / ml for both viruses). During surgery, presynaptic virus cocktail was injected at three separate sites in V1. In addition, 814871-7173-5771, v. 3 AAV8-hSyn-DIO-mCherry (2 mice, titer: 8x1012gc / ml) or AAV9-CAG-DIO-tdTomato (2 mice, titer: 7x1012gc / ml) was injected into two separate sites in striatum.14 days after the first surgery, AAV8-hSyn-DIO-ChR2-GFP (titer: 1x1013gc / ml) was injected at the same three V1 sites as the first surgery. Coordinates of V1 injection sites were as follows (coordinates are relative to bregma and D / V is relative to the brain surface, see below).300 nl of presynaptic virus cocktail (AAV8-ATLASsnCreand AAV8-BACE-HA) was injected per site for the first surgery.200 nl of AAV8-hSyn-DIO-ChR2 was injected per site for the second surgery. V1 site 1: -2.75 mm A / P, ±2.5 mm M / L, -0.65 mm D / V V1 site 2: -3.80 mm A / P, ±2.9 mm M / L, -0.65 mm D / V V1 site 3: -3.80 mm A / P, ±2.0 mm M / L, -0.65 mm D / V Coordinates of striatum injection sites were as follows (coordinates are relative to bregma and D / V is relative to the brain surface, see below). 500 nl of postsynaptic virus (Cre-dependent mCherry or tdTomato) was injected at striatum site 1 (anterior) while 400 nl of virus was injected at striatum site 2 (posterior). Striatum site 1: 0.70 mm A / P, ±1.60 mm M / L, -2.22 mm D / V Striatum site 2: -0.58 mm A / P, ±2.75 mm M / L, -2.75 mm D / V Electrophysiology (Fig.13e-g). Brain slices were obtained from two- to three- month-old mice (both male and female) using standard techniques. The experiments closely followed the procedures outlined in previous studies57, 58. Mice were anesthetized using isoflurane inhalation and subsequently subjected to transcardial perfusion with ice-cold artificial cerebrospinal fluid (ACSF) composed of the following (in mM): 125 NaCl, 2.5 KCl, 25 NaHCO3, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, and 11 glucose (final osmolarity of 300–305 mOsm / kg). The brain was removed from the skull, placed in ice-cold ACSF, and 250-300 μm coronal brain slices were obtained on a Leica VT1000 S vibratome. Slices were initially placed in a holding chamber for 10 minutes at 34 °C in a choline-based solution composed of (in mM): 110 choline chloride, 25 NaHCO3, 2.5 KCl, 7 MgCl2, 0.5 CaCl2, 1.25 NaH2PO4, 25 glucose, 11.6 ascorbic acid, and 3.1 pyruvic acid. Following the choline incubation, slices were transferred to a second chamber with ACSF also maintained at 34 °C for a minimum of 30 824871-7173-5771, v. 3 minutes. The chamber was subsequently shifted to room temperature for the duration of the experiment. All recordings were made within 4-5 hours of slicing. The temperature was maintained at 32 °C and carbogen-bubbled ACSF was perfused at a rate of 2–3 mL / min for all experiments. Striatal projection neurons were targeted for recordings using differential interference contrast (DIC) optics in wild-type mice on an upright microscope (Olympus BX51WI). Monosynaptically connected striatal neurons were identified by their mCherry- positive or tdTomato-positive somata. Fluorescence-negative neurons with GABAergic interneuron physiological properties (membrane tau decay < 0.8 ms for both fast-spiking and persistent low-threshold spiking subtypes; input resistance > 500 MΩ in persistent low- threshold spiking subtype) were excluded from the analysis. Striatal neurons were patched in whole-cell voltage-clamp configurations using borosilicate glass electrodes (3-5 MΩ) filled with cesium-based (voltage-clamp) internal solution to measure excitatory postsynaptic currents (EPSCs). The solution contained (in mM): 135 CsMeSO3, 10 HEPES, 1 EGTA, 3.3 QX-314 (Cl− salt), 4 Mg-ATP, 0.3 Na-GTP, 8 Na2- Phosphocreatine, with pH adjusted to 7.3 using CsOH, resulting in an osmolarity of 295 mOsm·kg−1. Whole-cell recordings were acquired with a MultiClamp 700B amplifier (Molecular Devices) and digitized at 10 kHz with a National Instruments data acquisition device (NI USB-6343). All recorded neurons exhibited electrophysiological characteristics of spiny projection neurons. All synaptic currents were recorded with a cesium-based internal and monitored at a holding potential of -70 mV. Series resistance and leak currents were monitored continuously. Fluorescent positive and negative neurons were patched as randomly ordered sequential pairs, strictly within 1 field of view of each other. ChR2-mediated synaptic currents from V1 were optically evoked using 2 ms pulses of 473 nm light at light powers of 2 mW whose power output was controlled through an acousto-optic modulator. Quality control criteria were a stable baseline membrane resistance (variation in Rm< 10%) and a stable baseline resting potential with median standard deviation of ≤ 0.5 mV across recordings. Optically evoked EPSCs were analyzed post-hoc using custom written scripts for Matlab. Example 5: ATLAS tracers for circuits mediated by GABA, Serotonin, Acetylcholine, Dopamine and Metabotropic Glutamate 834871-7173-5771, v. 3 The above ATLAS constructs that include AMPA.FingR target glutamatergic circuits because the AMPA.FingR binds exclusively to AMPA receptors, which mediate glutamatergic synaptic transmission. ATLAS was used to trace other types of neuronal circuits by replacing AMPA.FingR with a binder to the receptor for a different neurotransmitter. As described below, selectively targeting various neuronal populations was observed using ATLAS approaches that target different brain receptors that are modulated by different neurotransmitters. i. GABA: To make a tracer for GABAergic circuits, a GABA receptor nanobody (Nb38) that binds to the alpha1 subunit of the GABAA receptor (Laverty et al.2019) was included in constructs. To test this version of ATLAS we co-injected AAV-DIO-SYTnb- VAMP2∆-At-bcs-GN-Flp (where GB is a GABA receptor nanobody) and AAV-fDIO- mCherry into the V1 cortex of PV-Cre transgenic mice. We found abundant staining of mCherry, particularly in layer 5 and layer 2 / 3, consistent with efficient transsynaptic labeling (FIG. 21). Note that 100% of neurons expressing At were labeled by an anti-PV antibody (112 / 112). ii. Serotonin – to trace circuits mediated by serotonin, an ATLAS tracer incorporating a nanobody that binds to the serotonin receptor, 5HT3aRNb (Hassaine et al. 2014), was generated to produce: VAMP2D-SytNb-BACEcs-5HT3aRNb-Cre (FIGS.22A-D). AAV-VAMP2-SYTnb-BACEcs-5HT3aNb-Cre was then expressed in the dorsal raphe and AAV-DIO-GFP in the VTA, and incubated for two weeks. Bright and specific labeling of dopaminergic neurons in the VTA with GFP were observed, indicating that ATLAS successfully labeled this serotonergic circuit. iii. Acetylcholine – An ATLAS tracer incorporating a nanobody that binds to the α7Ach receptor, α7AchRNb, (Prevost et al., 2023) was generated to produce VAMP2∆- SytNb-BACEcs- α7AchRNb-Cre. AAV-VAMP2∆-SYTnb-BACEcs-α7AchRNb-Cre was expressed in the ventral nucleus of the diagonal band and AAV-DIO-GFP in the hippocampus. Prolific GFP labeling of dentate granule cells in the ipsilateral dentate gyrus consistent was observed, with labeling of cholinergic circuits mediated by the α7Ach Receptor. Results are shown in FIGS.23A-D. 844871-7173-5771, v. 3 iv. Dopamine – An ATLAS tracer incorporating a nanobody that binds to the Drd2 dopamine receptor (Im et al., 2020) generated to produce the construct (VAMP2∆- SytNb-BACEcs-Drd2RNb-Cre). VAMP2∆-SYTnb-BACEcs-Drd2RNb-Cre was expressed in the substantia nigra and DIO-GFP in the striatum. GFP labeling of ipsilateral medium spiny neurons was observed, consistent with labeling of dopamine circuits (FIG.24). v. Metabotropic Glutamate circuits – An ATLAS tracer incorporating a nanobody that binds to the metabotropic glutamate receptor mGluR5 (Koehl et al., 2019) was generated to produce the construct: (VAMP2∆-SytNb-BACEcs-mGluR5Nb-Cre). VAMP2∆- SYTnb-BACEcs-Drd2RNb-Cre was expressed in the mPFC and DIO-GFP in the striatum. Prolific GFP labeling of ipsilateral medium spiny neurons was observed, consistent with labeling of metabotropic glutamate circuits. Results are shown in FIG.25. 854871-7173-5771, v. 3 Equivalents It is to be understood that while the disclosure has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the disclosure. Other aspects, advantages, and modifications within the scope of the disclosure will be apparent to those skilled in the art to which the disclosure pertains. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All nucleotide sequences provided herein are presented in the 5′ to 3′ direction. The disclosures illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure claimed. Thus, it should be understood that although the present disclosure has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the disclosures embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this disclosure. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure. The disclosure has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the disclosure with a proviso or 864871-7173-5771, v. 3 negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control. 874871-7173-5771, v. 3 REFERENCES: Li, E., et al., Anterograde transneuronal tracing and genetic control with engineered yellow fever vaccine YFV-17D. 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Claims

WHAT IS CLAIMED IS:

1. A method for delivering a polypeptide payload transsynaptically in an anterograde direction to a postsynaptic neuron connected via synapses to a presynaptic neuron or neurons, comprising contacting the presynaptic neuron with a polynucleotide encoding a polypeptide-payload fusion and expressing a polypeptide of the polypeptide- payload fusion in the presynaptic neuron.

2. The method of claim 1, wherein the polypeptide-payload fusion is capable of i) targeting the payload peptide and the receptor binder peptide to synaptic vesicles, ii) allowing the release of the payload peptide and receptor binder peptide into the synaptic cleft, iii) binding to a postsynaptic receptor, and iv) mediating the endocytic internalization of the peptide payload into the postsynaptic cell resulting in transport to the cytoplasm and / or nucleus of the postsynaptic neuron.

3. The method of claim 1 or 2, wherein the polypeptide-payload fusion comprises: i) a synaptic vesicle targeting protein; ii) a protease cleavage domain; iii) a postsynaptic receptor binding domain, and iv) a payload.

4. The method of claim 3, wherein the postsynaptic receptor binding domain of the polypeptide-payload fusion is selected from the group of: AMPA.FingR domain, a nanobody, scFv, single domain antibody, and Fab fragment.

5. The method of claim 4, wherein the postsynaptic receptor binding domain of the polypeptide-payload fusion is a GABA nanobody domain, Nicotinic Acetylcholine Receptor intrabody domain, serotonin Receptor Nanobody domain, Dopamine Receptor Fab fragment domain, Beta-Adrenergic receptor Nanobody domain, or a metabotropic glutamate receptor nanobody domains.

6. The method of claim 4, wherein the AMPA.FingR domain comprises a polypeptide having at least 85% sequence identity to SEQ ID NO: 14 across the full length of the polypeptide. 974871-7173-5771, v.

37. The method of any of claims 3-6, wherein the protease cleavage domain of the polypeptide-payload fusion is a β-Secretase Cleavage (BACEcs) domain.

8. The method of any of claims 3-7, wherein the synaptic vesicle targeting protein domain is selected from the group of: VAMP2, VAMP2Δ (SEQ ID NO:2), Synaptotagmin nanobody, and Synaptophysin.

9. The method of any of claims 3-8, wherein the payload is selected from a group of: Cre recombinase, CRISPR Cas9, NPAS4, a transcription factor affecting neuronal excitability, and BDNF.

10. The method of any of claims 1-9, wherein the polypeptide-payload fusion further comprises a detectable label.

11. The method of claim 10, wherein the detectable label is selected from the group of: ALFA-tag peptide, FLAG peptide, HA peptide, halotag peptide, snaptag peptide, V5, and DHFR peptide.

12. The method of any of claim 1-11, wherein the polypeptide-payload fusion further comprises a domain that binds capsid proteins of AAV.

13. The method of claim 12, wherein the AAV capsid binding domain is a 4x Heptamer.

14. The method of any of claims 1-13, wherein the polypeptide-payload fusion further comprising a nuclear localization peptide.

15. The method of claim 14, wherein the nuclear localization peptide is CCR5 zinc finger transcription factor DNA binding domain.

16. The method of any of claims 1-15, wherein the contacting is in vitro or in vivo.

17. The method of any of claims 1-16, wherein the presynaptic neuron is a mammalian presynaptic neuron. 984871-7173-5771, v.

318. The method of any one of claims 1-17, wherein the payload is a recombinase.

19. The method of claim 18, wherein the recombinase is Cre recombinase, FLP recombinase, or Dre recombinase; and wherein the recombinase causes expression of a second polypeptide in the postsynaptic neuron.

20. The method of claim 19, wherein the second polypeptide is a floxed gene or floxed reporter gene, wherein the reporter gene is preferably a fluorescent protein.

21. The method of any one of claims 19-20, wherein the second polypeptide is transgenically expressed by a non-human animal.

22. The method of any one of claims 1-20, wherein the polynucleotide encoding the polypeptide-payload fusion is delivered to the presynaptic neuron via a viral vector, liposomes, virus-like particles (VLPs), or nanoparticles.

23. The method of claim 22, wherein the viral vector is an AAV, preferably AAV8.

24. The method of any one of claims 1-23, wherein the polynucleotide encoding the polypeptide-payload fusion is operatively linked to a promoter that is active in neurons, preferably wherein the promoter is a neuron specific promoter.

25. The method of claim 24, wherein the promoter is hSyn1 promotor, CAG promoter, GAD1 promoter, or Camkii promoter.

26. A polypeptide, wherein the polypeptide comprises: i) a synaptic vesicle targeting protein; ii) a protease cleavage domain; iii) a postsynaptic receptor binding domain, and iv) a payload.

27. The polypeptide of claim 26, wherein the postsynaptic receptor binding domain of the polypeptide is selected from the group of: AMPA.FingR domain, GABA nanobody domain, Nicotinic Acetylcholine Receptor intrabody domain, serotonin Receptor Nanobody domain, Dopamine Receptor Fab fragment domain, Beta-Adrenergic receptor Nanobody domain, and metabotropic glutamate receptor nanobody domains. 994871-7173-5771, v.

328. The polypeptide of claim 27, wherein the AMPA.FingR domain comprises a polypeptide having at least 85% sequence identity to SEQ ID NO: 14 across the full length of the polypeptide.

29. The polypeptide of any of claims 26-28, wherein the protease cleavage domain of the polypeptide is a β -Secretase Cleavage (BACEcs) domain.

30. The polypeptide of any of claims 26-29, wherein the synaptic vesicle targeting protein domain comprises 1, 2, or 3, of: VAMP2, VAMP2delta (SEQ ID NO:2), Synaptotagmin nanobody, or Synaptophysin.

31. The polypeptide of claim 30, wherein the synaptic vesicle targeting protein domain comprises (i) VAMP2 (SEQ ID NO:1), (ii) VAMP2delta (SEQ ID NO:2) and synaptotagmin nanobody (SEQ ID NO:4), (iii) VAMP2delta (SEQ ID NO:2) and Synaptotagmin (SEQ ID NO:3), or (iv) synaptophysin.

32. The polypeptide of claim 31, wherein the synaptic vesicle targeting protein domain comprises a fusion protein comprising VAMP2delta (SEQ ID NO:2), (ii) a synaptotagmin nanobody (SEQ ID NO:4), and (iii) Synaptotagmin (SEQ ID NO:3).

33. The polypeptide of claim 32, wherein the synaptic vesicle targeting protein domain comprises SEQ ID NO:5, or a sequence having at least 85%, preferably at least 90%, more preferably at least 95% sequence identity.

34. The polypeptide of any of claims 26-29, wherein the synaptic vesicle targeting protein domain is: VAMP2, (VAMP2 delta and Synaptotagmin nanobody), or Synaptophysin.

35. The polypeptide of any of claims 26-34, wherein the payload is selected from a group of: Cre recombinase, FLP recombinase, CRISPR Cas9, NPAS4 or other transcription factor affecting neuronal excitability, or BDNF.

36. The polypeptide of any of claims 26-35, wherein the polypeptide further comprises a detectable label. 1004871-7173-5771, v.

337. The polypeptide of claim 36, wherein the detectable label is selected from the group of: ALFA-tag peptide, FLAG peptide, HA peptide, halotag peptide, snaptag peptide, and DHFR peptide.

38. The polypeptide of any of claims 26-37, wherein the polypeptide further comprises a domain that binds capsid proteins of AAV.

39. The polypeptide of claim 38, wherein the AAV capsid binding domain is a 4x Heptamer.

40. The polypeptide of any of claims 26-39, wherein the polypeptide further comprises a nuclear localization peptide.

41. The polypeptide of claim 40, wherein the nuclear localization peptide is CCR5 zinc finger transcription factor.

42. An isolated polypeptide comprising an AMPA.FingR domain having at least 85%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO:13 across the full length of the polypeptide.

43. An isolated polynucleotide encoding the polypeptide of any of claims 26-42, or its compliment.

44. The polynucleotide of claim 43, further comprising a promotor operably linked to the polynucleotide.

45. The polynucleotide of claim 44, wherein the promotor is selected from a group of: hSyn1 promotor, CAG promoter, GAD1 promoter, Camkii promoter, the promoters for the Serotonin Transporter, Noradrenaline transporter, and the Dopamine transporter or other cell-type specific promoters.

46. The polynucleotide of claim 43, wherein the polynucleotide comprises or consists of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30, or a sequence having at least 85%, preferably at least 90%, more preferably at least 95% sequence identity thereto. 1014871-7173-5771, v.

347. An isolated polynucleotide encoding a polypeptide selected from: a. hSyn1-VAMP2-ALFA-BACEcs-AMPA FingR-4xHeptamer-Cre- CCR5 fusion comprising SEQ ID NO: 3, that is optionally detectably labeled; or b. hSyn1-VAMP2-BACEcsE-AMPA FingR-Cre fusion comprising SEQ ID NO: 4, that is optionally detectably labeled.

48. A vector comprising the isolated DNA polynucleotide of any of claims 44-47, that is optionally detectably labeled.

49. The vector of claim 48, further comprising one or more regulatory polynucleotides operably linked to the isolated polynucleotides, that is optionally detectably labeled.

50. The vector of claim 48 or 49, wherein the vector is selected from a plasmid, a viral vector, a micelle, a liposome, or a lipid nanoparticle, that is optionally detectably labeled.

51. The vector of claim 48 or 49, wherein the vector is a viral vector, that is optionally detectably labeled.

52. The viral vector of claim 51, wherein the vector is an adenoviral vector or an adenoviral associated vector (AAV) or lentiviral vector, that is optionally detectably labeled.

53. An isolated host cell comprising one or more of the polypeptides of any of claims 26-43, the polynucleotide of claim 43-47, and / or the vector of any of claims 49-52.

54. The isolated host cell of claim 53, wherein the cell is a neuron.

55. The isolated host cell of claim 53 or 54, wherein the host cell expresses a recombinase.

56. The isolated host cell of claim 55, wherein the recombinase is selected from the group: Cre recombinase, FLP recombinase, Dre recombinase. 1024871-7173-5771, v.

357. A composition comprising a carrier and one or more of the fusions of any of claims 26-43, the isolated polynucleotide of claim 43-47, the vector of any of claims 48-52 and / or the isolated host cell of claim 53-56. 1034871-7173-5771, v. 3

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