Vector set used for targeting specific neuron population in neural circuit, pharmaceutical composition containing same, and method for targeting specific neuron population in neural circuit

Abstract

The purpose of the present invention is to provide a novel method for targeting a neuron population that is a neural circuit element which has more limited functional localization. The purpose of the present invention is also to provide a method for treating a disease or the like by targeting a specific neuron population involved in a disease or the like by said novel method and controlling the neural activity thereof.　The present invention is a vector set used for targeting a specific neuron population in a neural circuit, said vector set being characterized by comprising first to third vectors, wherein: the first vector is an antegrade trans-synaptic virus vector that contains a base sequence which codes for a split-Cre recombinase consisting of a C-terminal-side sequence of a Cre recombinase; the second vector is a retrograde trans-synaptic virus vector that contains a base sequence which codes for a split-Cre recombinase consisting of an N-terminal-side sequence of a Cre recombinase; the third vector is a non-trans-synaptic virus vector that contains an inverted sequence of a gene sequence which codes for a desired protein and that has a loxP sequence and a loxP mutation sequence which face opposite to each other and which are at both ends of the inverted sequence; and a functional Cre recombinase is formed in a cell that has been infected with the first vector and the second vector.

Description

Vector set used for targeting specific neuronal populations in neural circuits, pharmaceutical composition containing the same, and method for targeting specific neuronal populations in neural circuits

[0001] The present invention relates to a vector set used for targeting a specific neuronal population in a neural circuit, a pharmaceutical composition containing the same, and a method for targeting a specific neuronal population in a neural circuit.

[0002] Mental disorders, neurodegenerative disorders, epilepsy, neurovascular disorders, migraines, and other disorders in the nervous system cause various disruptions in patients' lives, posing a major social problem. The cognitive functions of the nervous system, such as thinking, understanding, and judgment, are generated by the interaction of various functions, including sensory information processing, motor control, learning and memory, and emotional functions. These various functions are thought to exist with polarity in each brain region and in each neuron within that brain region. Recent neuroscience research has attempted to clarify the localization of various functions in each brain region and in each neuron within that brain region (see Non-Patent Documents 1 to 3).

[0003] In recent years, pharmaceutical companies and brain tech companies have been researching and developing technologies that enable artificial intervention in the information processing and communication of the nervous system, primarily for the treatment of neurological disorders (Non-Patent Document 4). Pharmaceutical companies have developed and marketed drugs targeting the information processing and communication between nervous system cells (synapses, the connections between neurons, and the surrounding glial cells), as well as between molecules within each cell, as treatments for psychiatric disorders such as depression, neurodegenerative disorders such as Parkinson's disease, and migraines. However, most of these therapeutic drugs act nonspecifically throughout the brain, rather than on brain regions, the long-distance interneuronal neural circuits connecting them, or the local neural circuits within brain regions. In light of recent neuroscience findings that suggest that various functions are localized in each brain region and each neuron within that brain region, it is believed that these therapeutic drugs' nonspecific, global effects on the brain may result in ineffective symptom improvement and significant side effects.

[0004] Neurons transmit information across neural circuits. It has become clear that the function of each neuron in these circuits is polarized and assigned based on both the afferent inputs it receives from other brain regions and the efferent outputs it sends to other brain regions. To study the neural activity and function of specific neuronal populations, anterograde and retrograde viral techniques have been widely used to identify neural circuit elements based on their inputs and outputs, respectively. The development of such techniques has contributed to significant advances in neuroscience research in recent years (see non-patent literature 2, 5-7).

[0005] Larry R Squire, Memory systems of the brain: a brief history and current perspective, Neurobiol Learn Mem., 2004 Nov;82(3):171-7Talia N Lerner, et al., Communication in Neural Circuits: Tools, Opportunities, and Challenges, Cell vol.164, March 10, 2016, 1136-1150Silvia Arber, Motor Circuits in Action:Specification, Connectivity, and Function, Neuron, vol.74, June 21, 2012, 975-989Optogenetics. Nat Biotechnol 37, 1420 (2019). https: / / doi.org / 10.1038 / s41587-019-0343-4Zingg, B.et.al, (2017). AAV-Mediated Anterograde Transsynaptic Tagging: Mapping Corticocollicular Input-Defined Neural Pathways for Defense Behaviors. Neuron, 93(1), 33-47Tervo, D. G. R.et.al., (2016). A Designer AAV Variant Permits Efficient Retrograde Access to Projection Neurons. Neuron, 92(2), 372-382Luo, L. (2021). Architectures of neuronal circuits. Science, 373(6559), eabg7285.

[0006] Under these circumstances, the present invention aims to provide a novel method for targeting neuronal populations, which are neural circuit elements with more restricted functional localization, and a method for treating diseases by targeting specific neuronal populations involved in diseases and controlling their neural activity using this method.

[0007] The present inventors have conducted research to study the neural activity and function of specific neuronal populations. We have used anterograde and retrograde viruses to identify neural circuit elements based on their inputs and outputs, respectively, and have applied this approach to a technology that enables recording and artificial intervention (manipulation) of neural information processing communication. This would enable artificial intervention in the information processing of neuronal populations, which are neural circuit elements identified based on both inputs and outputs with more precisely defined functional localizations, thereby enabling more effective suppression of side effects and specific elicitation of the desired therapeutic effect. We have successfully developed a technology that utilizes a combination of multiple recombinase enzymes and anterograde / retrograde viruses to target specific neuronal populations in neural circuits and investigate the functional input / output structure of neural circuits. The gist of the present invention is as follows.

[0008] [1] A vector set used to target a specific neuron population in a neural circuit, comprising first to third vectors, wherein the first vector is an anterograde transsynaptic viral vector comprising a base sequence encoding a split Cre recombinase consisting of the C-terminal sequence of Cre recombinase, the second vector is a retrograde transsynaptic viral vector comprising a base sequence encoding a split Cre recombinase consisting of the N-terminal sequence of Cre recombinase, and the third vector is a non-transsynaptic viral vector comprising an inverted sequence of a gene sequence encoding a desired protein, with a loxP sequence and a loxP mutant sequence in opposite directions at both ends of the inverted sequence, and wherein functional Cre recombinase is formed in cells infected with the first and second vectors. [2] A vector set used to target a specific neuron population in a neural circuit, comprising first to third vectors, wherein the first vector is a retrograde transsynaptic viral vector comprising a base sequence encoding a split Cre recombinase consisting of the C-terminal sequence of Cre recombinase, the second vector is an anterograde transsynaptic viral vector comprising a base sequence encoding a split Cre recombinase consisting of the N-terminal sequence of Cre recombinase, and the third vector is a non-transsynaptic viral vector comprising an inverted sequence of a gene sequence encoding a desired protein, with a loxP sequence and a loxP mutant sequence in opposite directions at both ends of the inverted sequence, and wherein functional Cre recombinase is formed in cells infected with the first and second vectors.[3] A vector set used to target a specific neuron population in a neural circuit, comprising first to third vectors, wherein the first vector is an anterograde transsynaptic viral vector comprising a base sequence encoding Cre recombinase, the second vector is a retrograde transsynaptic viral vector comprising an inverted sequence of a base sequence encoding flippase (Flp) and having a loxP sequence and a loxP mutant sequence in opposite directions at both ends of the inverted sequence, and the third vector is a non-transsynaptic viral vector comprising an inverted sequence of a gene sequence encoding a desired protein and having an FRT sequence and an FRT mutant sequence in opposite directions at both ends of the inverted sequence, wherein functional flippase is expressed in cells infected with the first and second vectors. [4] A vector set used for targeting a specific neuron population in a neural circuit, comprising first to third vectors, wherein the first vector is a retrograde transsynaptic viral vector containing a nucleotide sequence encoding Cre recombinase, the second vector is an anterograde transsynaptic viral vector containing an inverted sequence of a nucleotide sequence encoding flippase (Flp) and having a loxP sequence and a loxP mutant sequence in opposite orientations at both ends of the inverted sequence, and the third vector is a non-transsynaptic viral vector containing an inverted sequence of a gene sequence encoding a desired protein and having an FRT sequence and an FRT mutant sequence in opposite orientations at both ends of the inverted sequence, wherein functional flippase is expressed in cells infected with the first and second vectors. [5] The vector set according to any of [1] to [4], wherein the anterograde transsynaptic viral vector, the retrograde transsynaptic viral vector, and the non-transsynaptic viral vector are adeno-associated viral vectors. [6] The vector set according to any one of [1] to [5], wherein the desired protein is a protein involved in the control of neural activity and is used for controlling neural activity. [7] The vector set according to [6], wherein the protein involved in the control of neural activity is a protein involved in the inhibition of neural activity and is used for inhibiting neural activity.[8] The vector set according to [7], wherein the protein having the function of inhibiting neural activity is an inhibitory opsin and is used for inhibiting neural activity. [9] The vector set according to any one of [1] to [5], wherein the desired protein is a calcium indicator or a voltage indicator and is used to obtain a readout of neural activity.

[10] A pharmaceutical composition comprising the vector set according to any one of [1] to [8].

[11] A method for targeting a specific neuronal population in a neural circuit, comprising using the vector set according to any one of claims 1 to 5, and introducing an anterograde transsynaptic viral vector into an input region, a retrograde transsynaptic viral vector into an output region, and a non-transsynaptic viral vector into an intermediate target region.

[12] The method of targeting a specific neuron population in a neural circuit according to

[11] , wherein the input region is the cuneiform nucleus (CnF), the output region is the central nucleus of the amygdala (CeA), and the intermediate target region is the lateral and basal nuclei of the amygdala (LA / B).

[13] The method of

[11] or

[12] , which is used on animals other than humans.

[0009] The present invention identifies neuronal populations based on both input and output, enabling targeting of neural circuit elements with more specific neural activity dynamics and functions than identification based solely on input or output. This allows for the identification of new neuronal subclasses based on the anatomical connectivity between neurons resulting from the combination of input and output. Furthermore, the present invention provides tools for more detailed investigation and manipulation of the localization of functional properties of specific neural circuit elements based on the structure of the neural circuit's input and output. Applying the technology of the present invention to target neural circuit elements identified based on both input and output may potentially achieve desired therapeutic effects with minimal side effects, thereby broadening the scope of its applications and potentially leading to the development of new treatments for various nervous system disorders.

[0010] Figures 1(a-j) are diagrams illustrating the technical content of the present invention, i.e., the advantages of identifying neural circuit elements from both input and output and targeting anatomically defined neuronal populations in functionally localized neural circuits. Figures 2(a-h) are diagrams illustrating the anatomical and molecular characteristics of neurons in the lateral amygdala and basal ganglia (LA / B) labeled by input only, both input and output, and only output, respectively. Figures 3(a-k) are diagrams illustrating population-specific calcium activity dynamics of different neuronal populations in the lateral amygdala and basal ganglia (LA / B) identified by input only, both input and output, and only output, respectively, during fear conditioning. Figures 4(a-n) are diagrams illustrating population-specific calcium activity dynamics of different neuronal populations in the lateral amygdala and basal ganglia (LA / B) identified by input only, both input and output, and only output, respectively, during salience information processing. Figures 5(a-n) are diagrams illustrating the results of analyses of population-specific behavioral functions during fear conditioning using optogenetic inactivation of different neuronal populations in the lateral amygdala and basal ganglia (LA / B) identified from input only, both input and output, and output only. Figures 6(a-j) are diagrams illustrating the results of analyses of population-specific behavioral functions in a behavioral model of salience information processing using optogenetic inactivation of different neuronal populations in the LA / B identified from input only, both input and output, and output only. Figures 7(a-d) are diagrams illustrating the results of analyses of behavioral functions on freezing responses during fear memory retrieval after fear conditioning using optogenetic inactivation of neuronal populations in the LA / B identified from both input and output. Figure 8 is a diagram illustrating possible combinations of input regions, intermediate target regions, and output regions in the method of targeting specific neuronal populations of the present invention.

[0011] The present invention will be described in detail below. In this specification, unless otherwise specified, molecular biological techniques can be performed by methods described in general experimental manuals known to those skilled in the art or methods similar thereto. Furthermore, unless otherwise specified, the terms used in this specification should be interpreted as having the meanings commonly used in the technical field.

[0012] <Definitions> As stated above, all scientific and technical terms used in this application shall be interpreted in the sense commonly used in the art unless otherwise specified. In addition, when used in this application, the following terms have the meanings specified:

[0013] As used herein, the term "base sequence" is used interchangeably with "nucleotide sequence" or "nucleic acid sequence," and is represented as a sequence of deoxyribonucleotides (abbreviated as A, G, C, and T). For example, in the present invention, a "polynucleotide comprising the nucleic acid sequence represented by SEQ ID NO: 1" refers to a polynucleotide comprising the sequence represented by each of the deoxynucleotides A, G, C, and / or T in SEQ ID NO: 1. Furthermore, as used herein, the term "polynucleotide" is used interchangeably with "nucleic acid," "gene," or "nucleic acid molecule," and refers to a polymer of nucleotides, and refers to a series of polymers of nucleotides, deoxyribonucleotides, or ribonucleotides in either a single-stranded or double-stranded form, and includes known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides, unless otherwise specified.

[0014] The term "vector" means a construct capable of delivering, and preferably expressing, one or more genes or nucleic acid sequences of interest in a host cell.

[0015] In this specification, "PINCER," an abbreviation for Projection-based Intersectional Circuit-tagging Enabled by Recombinases, is used as the term to refer to the technology of the present invention for targeting specific neuronal populations in neural circuits.

[0016] The present invention will be specifically described below.

[0017] <Vector Set> [Split Cre-PINCER] One embodiment of the present invention is a vector set used to target a specific neuron population in a neural circuit, comprising first to third vectors, wherein the first vector is an anterograde transsynaptic viral vector comprising a nucleotide sequence encoding a split Cre recombinase (CCre) consisting of the C-terminal sequence of Cre recombinase, the second vector is a retrograde transsynaptic viral vector comprising a nucleotide sequence encoding a split Cre recombinase (NCre) consisting of the N-terminal sequence of Cre recombinase, and the third vector is a non-transsynaptic viral vector comprising an inverted sequence of a gene sequence encoding a desired protein, with a loxp sequence and a loxP mutation sequence in opposite orientations at both ends of the inverted sequence, and wherein functional Cre recombinase is formed in cells infected with the first vector and the second vector.

[0018] Another embodiment of the present invention is a vector set used to target a specific neuron population in a neural circuit, comprising first to third vectors, wherein the first vector is a retrograde transsynaptic viral vector comprising a split Cre recombinase (CCre) sequence consisting of the C-terminal sequence of Cre recombinase; the second vector is an anterograde transsynaptic viral vector comprising a split Cre recombinase (NCre) sequence consisting of the N-terminal sequence of Cre recombinase; and the third vector is a non-transsynaptic viral vector comprising an inverted sequence of a gene sequence encoding a desired protein, with a loxP sequence and a loxP mutant sequence in opposite orientations at both ends of the inverted sequence; and wherein functional Cre recombinase is formed in cells infected with the first and second vectors.

[0019] The above-described embodiment of the present invention utilizes the Cre / lox system, a known genetic engineering technology. In the Cre / lox system, the recombinase Cre recombinase acts on a DNA sequence called a loxP sequence or a loxP mutation sequence, resulting in site-specific gene recombination. Cre recombinase is an enzyme derived from bacteriophage P1 and is widely used in genetic engineering (Meinke, G. et al., Chem. Rev. 116, 12785-12820 (2016)). In this Cre / lox system, a control technique is also known in which Cre recombinase activity is reconstituted in a light-dependent or drug-dependent manner using split Cre, which is obtained by splitting Cre recombinase into two polypeptides (Hirrlinger, J. et al., PLoS One 4, e4286 (2009); Kuwasaki, Y. et al., Nature Biotechnology, 40(11), 1672-1679 (2022); Morikawa, K. et al., Nature Communications, 11(1), 2141 (2020); Jullien, N. et al., Nucleic Acids Research, 31(21), e131.(2003)).

[0020] That is, in the present invention, in a cell into which both the first vector and the second vector have been introduced (infected), a functional Cre recombinase is formed, in which a split Cre recombinase (CCre) sequence consisting of the C-terminal sequence of Cre recombinase and a split Cre recombinase (NCre) sequence consisting of the N-terminal sequence of Cre recombinase are joined. The third vector is a non-synaptic viral vector that contains an inverted sequence of a gene sequence encoding a desired protein and has a loxP sequence and a loxP mutation sequence in opposite directions at both ends of the inverted sequence. Therefore, when this third vector is introduced (infected) into a region into which both the first vector and the second vector have been introduced (infected), the inverted sequence of the gene sequence encoding the desired protein is inverted by the action of the functional Cre recombinase, allowing the desired protein to be normally expressed. In the present invention, the phrase "having a loxP sequence and a loxP mutant sequence in opposite directions at both ends of the inverted sequence" refers to the fact that, as shown in the center diagram in the left diagram of Figure 1f, the inverted sequence of the gene sequence encoding the desired protein is sandwiched between the loxP sequences in opposite directions, and the inverted sequence of the gene sequence encoding the desired protein is also sandwiched between the loxP mutant sequences in opposite directions. Cre recombinase inverts all sequences sandwiched between a set of loxP sequences in opposite directions or a set of loxP mutant sequences in opposite directions. Therefore, if the sequence is sandwiched between only one set of recognition sequences, repeated inversions will occur. Therefore, sandwiching the sequence between two sets of recognition sequences results in one inversion and then removing the loxP sequences that are aligned in the same direction after inversion, thereby preventing repeated inversions. The order of the sequences is preferably as follows:

[0021] loxP sequence-loxP mutation sequence-inverted sequence of the gene sequence encoding the desired protein-loxP sequence reversed sequence-loxP mutation sequence reversed sequence; loxP mutation sequence-loxP sequence-inverted sequence of the gene sequence encoding the desired protein-loxP mutation sequence reversed sequence-loxP sequence; inverted sequence of the loxP sequence-reverted sequence of the gene sequence encoding the desired protein-loxP sequence-loxP mutation sequence; inverted sequence of the loxP mutation sequence-reverted sequence of the gene sequence encoding the desired protein-loxP sequence-loxP mutation sequence; inverted sequence of the loxP mutation sequence-reverted sequence of the gene sequence encoding the desired protein-loxP mutation sequence-loxP sequence; inverted sequence of the loxP sequence-loxP mutation sequence-inverted sequence of the gene sequence encoding the desired protein-loxP sequence-loxP mutation sequence; or reverse sequence of loxP mutation sequence - loxP sequence - reverse sequence of gene sequence encoding desired protein - reverse sequence of loxP sequence - loxP mutation sequence

[0022] The loxP sequence is that shown in SEQ ID NO: 1 (ATAACTTCGTATA ATGTATGC TATACGAAGTTAT). The loxP mutant sequence is a sequence in which a part of the loxP sequence shown in SEQ ID NO: 1 is mutated, and examples thereof include the lox2272 sequence (SEQ ID NO: 2: ATAACTTCGTATA GGATACTT TATACGAAGTTA).

[0023] In the split Cre (SplitCre) system, CCre and NCre bind to form a functional Cre only in cells in which both a vector containing a base sequence encoding the C-terminal half of Cre (CCre) and a vector containing a base sequence encoding the N-terminal half of Cre (NCre) are present. This binding is called dimerization, and to induce dimerization of CCre and NCre, a sequence encoding a dimerizing protein is inserted into each of the CCre and NCre sequences. For example, it is possible to insert the sequence of a dimerizing protein called GCN4 into the CCre and NCre sequences. When a vector containing CCre and a vector containing NCre are introduced and transcribed and translated, GCN4-CCre and GCN4-NCre linked proteins are produced, respectively, and when GCN4 dimerizes and binds to each other, CCre and NCre can also bind to form functional Cre.

[0024] In the SplitCre system, a method has long been known in which either one of the dimerizer proteins FKBP or FRB, which dimerize only in the presence of rapamycin, is inserted into the sequences of CCre and NCre, so that only upon rapamycin administration, FKBP-CCre and FKR-NCre (logically either combination is acceptable) are led to bind with FKBP and FRB, and CCre and NCre bind to form a functional Cre. Recently, methods have been developed based on a similar logic in which, instead of FKBP and FRB, which dimerize only in the presence of rapamycin, the above-mentioned dimerizer combinations GCN4 and DocS and Coh2, which dimerize even without the presence of certain molecules such as rapamycin, are incorporated into SplitCre. Furthermore, dimerizer proteins that dimerize only when exposed to light of a specific wavelength, such as nMag and pMag (dimerizer proteins that dimerize only under blue light) and MagRed (the name of a combination of dimerizer proteins that dimerize only under red light), have also been developed.

[0025] It is expected that in the future, CCre and NCre sequences for SplitCre will be developed that utilize more efficient combinations of dimerizer proteins or combinations of proteins that dimerize depending on environmental factors such as specific wavelengths of light, wavelengths of vibrations such as sound, temperature, pH, magnetic field, etc. In the SplitCre-PINCER method of the present invention, any SplitCre (SplitCre) system can be logically used.

[0026] (First Vector and Second Vector) An important feature of the technology according to the above-described embodiment of the present invention is that when the first vector is an anterograde transsynaptic viral vector, the second vector is a retrograde transsynaptic viral vector; and when the first vector is a retrograde transsynaptic viral vector, the second vector is an anterograde transsynaptic viral vector, thereby identifying a neuronal population through both input and output. By introducing a third vector into the region identified through both input and output, desired proteins can be expressed, enabling visualization of the identified neuronal population and intervention in nervous system function through drugs or brain tech targeting specific neural circuit elements. The technology of the present invention allows targeting only the required area, potentially reducing side effects and achieving the desired therapeutic effect, thereby expanding the potential for developing new treatments for various nervous system disorders.

[0027] The "base sequence encoding a split Cre recombinase consisting of the C-terminal sequence of Cre recombinase" may be any base sequence that can form a base sequence encoding a functional Cre recombinase by binding to the "base sequence encoding a split Cre recombinase consisting of the N-terminal sequence of Cre recombinase," and is not limited to any particular sequence. For example, when the "base sequence encoding a split Cre recombinase consisting of the C-terminal sequence of Cre recombinase" is a base sequence corresponding to the amino acid sequence from the 60th to the 343rd amino acid in the amino acid sequence constituting Cre recombinase, the "base sequence encoding a split Cre recombinase consisting of the N-terminal sequence of Cre recombinase" may be a base sequence corresponding to the amino acid sequence from the 19th to the 59th amino acid in the amino acid sequence constituting Cre recombinase.

[0028] In the present invention, the base sequence of a polynucleotide represented by a SEQ ID NO: includes all polynucleotides that have one or more (e.g., 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 1, etc.) nucleotide deletions, substitutions, insertions, and / or additions, as long as the polynucleotide exerts the intended effect in a host cell when introduced into a vector to transform the host cell. Furthermore, the polynucleotide may simultaneously contain a combination of two or more of these deletions, substitutions, insertions, and additions. Generally, the smaller the number of nucleotide deletions, substitutions, insertions, and / or additions, the better.

[0029] Transsynaptic virus refers to the virus that can move from one neuron to another connected neuron through synapse.Examples of such transsynaptic virus include rhabdovirus, such as rabies virus, and alpha herpesvirus, such as pseudorabies or herpes simplex virus.In this specification, transsynaptic virus also includes the viral subunit that has the ability to move from one neuron to another connected neuron through synapse, and the biological vector, such as the modified virus that incorporates such subunit and shows the ability to move from one neuron to another connected neuron through synapse.

[0030] An anterograde transsynaptic viral vector is a viral vector encapsulated in an anterograde transsynaptic virus that is taken up into the cell body, transported anterogradely along the axon, and transsynaptically infects postsynaptic neurons from the nerve terminal. On the other hand, a retrograde transsynaptic viral vector is a viral vector that has the ability to infect in the opposite direction to anterograde transsynaptic viral vectors, and is encapsulated in a retrograde transsynaptic virus that invades a neuron through the axon terminal or axon, self-replicates within the infected cell, or invades a neuron, self-replicates within the infected cell, and then retrogradely infects input cells with which it has a synaptic connection. The anterograde transsynaptic viral vectors and retrograde transsynaptic viral vectors used in the present invention are not particularly limited as long as they have the above-mentioned properties, and examples of anterograde transsynaptic viral vectors include rAAV1, HSV1-H129ΔTK, VSVΔG (LCMV-G) (VSVΔG backbone vector), YFV-17D, Rabies, etc. Furthermore, examples of retrograde transsynaptic viral vectors include retroAAV2, CAV2 (pCAV vector), SAD Rabies (pSAD vector), CVS Rabies (pCVS vector), HSV1, VSV, etc.

[0031] In the above-described embodiment of the present invention, the first vector and the second vector may contain regulatory sequences such as a promoter, an enhancer sequence that assists mRNA transcription and protein translation, a Kozak sequence, a ribosome binding sequence, a terminator, a polyadenylation site, a WPRE sequence, etc., so as to enable expression of a gene of interest. Furthermore, as necessary, they may contain a selection marker sequence such as a drug resistance gene (e.g., a kanamycin resistance gene, an ampicillin resistance gene, a puromycin resistance gene, etc.), a thymidine kinase gene, a diphtheria toxin gene, or a reporter gene sequence such as mCherry (red fluorescent protein), green fluorescent protein (GFP), β-glucuronidase (GUS), or FLAG.

[0032] In the above-described embodiment of the present invention, the base sequence encoding the split Cre recombinase consisting of the C-terminal sequence of Cre recombinase contained in the first vector and the second vector, or the base sequence encoding the split Cre recombinase consisting of the N-terminal sequence of Cre recombinase, is preferably operably linked to a promoter sequence. Such a promoter sequence can be a promoter sequence that functions in cells constituting the brain region or cells constituting the neural circuit targeted for infection by transsynaptic or non-transsynaptic viruses, and examples thereof include the hSyn promoter, CAG promoter, and CMV promoter.

[0033] As used herein, "operably linked" refers to a juxtaposition wherein the described components are in a relationship permitting them to function in their intended manner. Specifically, the term refers to a functional link between a nucleic acid expression control sequence (e.g., a promoter and / or enhancer) and a polynucleotide sequence of interest, either directly or indirectly (when another polynucleotide sequence is present between them). In the present invention, a promoter sequence directs transcription of the linked polynucleotide of interest.

[0034] (Third Vector) The third vector is a non-synaptic viral vector containing an inverted sequence of a gene sequence encoding a desired protein, with a loxP sequence and a loxP mutation sequence in opposite orientations at both ends of the inverted sequence. Site-specific gene recombination occurs when the recombinase Cre recombinase acts on the loxP sequence and the loxP mutation sequence. In this embodiment, the inverted sequence of the gene sequence encoding the desired protein contains a loxP sequence and a loxP mutation sequence in opposite orientations at both ends. When Cre recombinase acts on the loxP sequence and the loxP mutation sequence in opposite orientations, the gene sequence sandwiched between them can be inverted. In other words, in cells into which both the first and second vectors have been introduced (infected), a functional Cre recombinase is formed, in which a split Cre recombinase sequence consisting of the C-terminal sequence of Cre recombinase and a split Cre recombinase sequence consisting of the N-terminal sequence of Cre recombinase are combined. When the third vector is introduced into the infected cell, the inversion sequence of the gene sequence encoding the desired protein is inverted by the action of functional Cre recombinase, allowing the desired protein to be expressed normally. Note that "having a loxP sequence and a loxP mutant sequence in opposite directions at both ends of the inversion sequence" is as described above.

[0035] As used herein, the term "non-synaptic viral vector" refers to a viral vector that does not fall under the category of the above-mentioned transsynaptic viruses. For example, an adeno-associated viral vector (AAV) can be used.

[0036] The "desired protein" in the inverted sequence of the gene sequence encoding the desired protein contained in the third vector is not particularly limited, but examples include proteins involved in the control of neural activity, indicator proteins that can be used to obtain a readout of neural activity, and visualizeable proteins such as fluorescent proteins, and it is preferable that the desired protein is a protein involved in the control of neural activity or an indicator protein that can be used to obtain a readout of neural activity.

[0037] The protein involved in the control of neural activity may be a "genetically encoded protein involved in the function of controlling neural activity" that is originally genetically encoded in the host, or a modified protein thereof, or a foreign protein not originally possessed by the host. Furthermore, the protein involved in the control of neural activity may be a protein involved in the inhibition of neural activity, or a protein involved in promoting (exciting) neural activity, but from the viewpoint of achieving superior effects of the present invention, a protein involved in the inhibition of neural activity is more preferable. Specific examples of proteins involved in the inhibition of neural activity include inhibitory opsins.

[0038] Preferred examples of the indicator protein include calcium indicators and voltage indicators that can be used to obtain readouts of neural activity.

[0039] As the desired protein, various candidates can be mentioned from the viewpoints of optogenetics, chemogenetics, sonogenetics, and (electro)magnetogenetics.

[0040] From the perspective of optogenetics, many proteins are known to be involved in excitatory (manipulation of the active system of neural activity) and inhibitory (manipulation of the inhibitory system of neural activity), but since each protein (opsin) responds to different conditions, such as the wavelength of light, it is desirable to select one based on the purpose.

[0041] For example, channelrhodopsin is the foundational protein for optogenetics, typically through direct stimulation of ion channels, resulting in rapid depolarization of neurons upon light illumination. Naturally occurring channelrhodopsins are proteins discovered in the green algae Chlamydomonas reinhardtii. Channelrhodopsin-1 (ChR1) is excited by blue light and allows nonspecific cation influx into cells upon stimulation. Channelrhodopsin-2 (ChR2) is also a blue-light-activated cation channel. The proteins used in optogenetics have been expanded both through the identification of novel ChRs from other algal species and the development of synthetic mutants that enhance ChR function. Examples of ChRs from other species include CsChR (from Chloromonas subdivisa), CoChR (from Chloromonas oogama), and SdChR (from Scherffelia dubia). Synthetic mutants have been created by gene point mutation, codon optimization, or chimeric fusion of two different ChR domains. Neuronal inhibitory ChR mutants have also been created and identified in other species. These mutants act as light-gated chloride channels, resulting in neuronal hyperpolarization. Examples of anion channel variants from other species include GtACR1 and GtACR2.

[0042] Halorhodopsin is a light-gated inward chloride pump isolated from halobacteria. Wild-type halorhodopsin, known as NpHR (from Natronomonas pharaoni), inhibits neuronal function by hyperpolarizing cells when triggered with yellow light. Archaerhodopsin-3 (Arch) from Halorubrum sodomense is also commonly used to inhibit neurons in optogenetic experiments. Arch is a light-activated outward proton pump that hyperpolarizes cells when triggered with green-yellow light. Leptosphaeria rhodopsin (Mac) is a blue-green light-activated proton pump derived from the fungus Leptosphaeria maculans. Mac and its mutants can inhibit neurons using blue-green light.

[0043] Optogenetics is a technology that uses laser light to manipulate neural cell activity at precise times and in specific brain regions, so it requires the implantation of optical fibers to deliver the laser light to specific brain regions.

[0044] Chemical genetics involves the use of artificial protein receptors, engineered to bind and interact with previously unrecognized small molecules. Similar to opsins in optogenetics, these receptors are genetically encoded proteins involved in the control of neural activity, and can be delivered to specific neuronal populations via viruses. These receptors are designed to be resistant to endogenous ligands and not to affect endogenous signaling. By introducing previously unrecognized small molecule compounds into the body as drugs and using viruses designed to bind and interact with these small molecule compounds, the neural activity of specific neuronal populations can be controlled by acting on the artificial protein receptors delivered to the neuronal populations. There are two main types of artificial protein receptors: DREADDs and PSAMs.

[0045] The main DREADDs involved in excitatory neuronal activity are Gq-DREADDs (hM1Dq, hM3Dq, hM5Dq), while the main DREADDs involved in inhibitory neuronal activity are Gi-DREADDs (hM2Di, hM4Di, KORD), etc. Ligands used for DREADDs include CNO, C21, DCZ (Clozapine), Perlapine, Olanzapine, Salvinorin B, etc.

[0046] The main PSAMs involved in excitatory neuronal activity include PSAM-Gly (PSAM4, PSAMQ79G, Q139G, PSAML141F, Y115F), and the main PSAMs involved in inhibitory neuronal activity include PSAM-5HT3 (PSAM4, PSAMQ79G, Q139G, PSAML141F, Y115F). Ligands used against PSAMs include Varenicline, uPSEM792, uPSEM817, PSEM22s, and PSEM89s.

[0047] In the present invention, when a chemical genetics approach is used, after sufficiently expressing the artificial protein receptor in the target cells using a third vector, a small molecule compound can be injected into the entire body in a manner similar to conventional drug administration. However, if there is a concern that the drug may cause adverse effects such as toxicity in other organs or other nervous systems, it is preferable to adopt a method of administering the drug in a limited area through a guide cannula placed in a specific nervous system region where the target cells are located.

[0048] Otogenetics is a technological field that utilizes genetically encoded proteins to detect specific ultrasound waves and open and close ion channels in neuronal membranes, thereby controlling ion dynamics and manipulating neuronal activity. Specific examples of proteins that have already been used in models such as nematodes, rats, mice, Xenopus laevis, and cultured cells include TRP-4, MscL, Piezo1, MEC-4, DEG / ENaC / ASIC, mPrestin (N7T, N308s), TREK-1 / 2, TRAAK, TRPP1 / 2, TRPC1, TRPM4, and TRPV1. This technology requires an acoustic device, such as a helmet, to deliver specific ultrasound frequencies to specific brain coordinates.

[0049] Electromagnetogenetics is a technological field that utilizes genetically encoded proteins to sense specific radio waves or magnetic fields, thereby opening and closing ion channels in neuronal membranes and controlling ion dynamics, thereby manipulating neuronal activity. Specific examples of proteins include Magneto2.0 (cation channel TRPV4 fused to paramagnetic protein ferritin), which can activate and inhibit neuronal activity using magnetic fields, and Anti-GFP-TRPV1 / GFP-ferritin, which can activate and inhibit neuronal activity using magnetic fields. This technology requires a large, helmet-like device that can deliver specific radio waves and magnetic fields to specific brain coordinates.

[0050] In the present invention, the third vector may include regulatory sequences such as a promoter, an enhancer sequence that assists mRNA transcription and protein translation, a Kozak sequence, a ribosome binding sequence, a terminator, a polyadenylation site, and a WPRE sequence, so as to enable expression of a gene of interest. Furthermore, if necessary, the third vector may include a selection marker sequence such as a drug resistance gene (e.g., a kanamycin resistance gene, an ampicillin resistance gene, a puromycin resistance gene, etc.), a thymidine kinase gene, or a diphtheria toxin gene, or a reporter gene sequence such as mCherry (red fluorescent protein), green fluorescent protein (GFP), β-glucuronidase (GUS), or FLAG.

[0051] In the present invention, the inverted sequence of the gene sequence encoding the desired protein contained in the third vector is preferably operably linked to a promoter sequence. Such promoter sequences can be those that function in cells constituting the brain region targeted for infection by transsynaptic and non-transsynaptic viruses, or in cells constituting neural circuits, and examples thereof include the hSyn promoter, CAG promoter, and CMV promoter. Here, "operably linked" is as defined above.

[0052] [Cre / Flp-PINCER] Another embodiment of the present invention is a vector set used to target a specific neuron population in a neural circuit, comprising first to third vectors, wherein the first vector is an anterograde transsynaptic viral vector comprising a nucleotide sequence encoding Cre recombinase; the second vector is a retrograde transsynaptic viral vector comprising an inverted sequence of a nucleotide sequence encoding flippase (Flp) and having a loxP sequence and a loxP mutant sequence in opposite orientations at both ends of the inverted sequence; and the third vector is a non-transsynaptic viral vector comprising an inverted sequence of a gene sequence encoding a desired protein and having an FRT sequence and an FRT mutant sequence in opposite orientations at both ends of the inverted sequence, wherein functional flippase is expressed in cells infected with the first and second vectors.

[0053] Another embodiment of the present invention is a vector set used to target a specific neuron population in a neural circuit, comprising first to third vectors, wherein the first vector is a retrograde transsynaptic viral vector comprising a nucleotide sequence encoding Cre recombinase; the second vector is an anterograde transsynaptic viral vector comprising an inverted sequence of a base sequence encoding flippase (Flp) and having a loxP sequence and a loxP mutant sequence in opposite orientations at both ends of the inverted sequence; and the third vector is a non-transsynaptic viral vector comprising an inverted sequence of a gene sequence encoding a desired protein and having an FRT sequence and an FRT mutant sequence in opposite orientations at both ends of the inverted sequence, wherein functional flippase is expressed in cells infected with the first and second vectors.

[0054] The above-described embodiment of the present invention utilizes the FLP-FRT system, a known genetic engineering technology. Flippase (Flp), a recombinase enzyme derived from budding yeast, recognizes FRT sequences to cause recombination, and is therefore a technology that is frequently used for the same purpose as the Cre / lox system described above.

[0055] That is, in the present invention, in cells into which both the first and second vectors have been introduced (infected), the functional Cre recombinase introduced by the first or second vector is expressed. This Cre recombinase recognizes the loxP sequence and the loxP mutant sequence present in opposite orientations at both ends of the inversion sequence of the nucleotide sequence encoding flippase (Flp) introduced by the first or second vector, thereby inverting the inversion sequence of the nucleotide sequence encoding flippase (Flp), thereby enabling the expression of functional flippase (Flp). Meanwhile, the third vector is a non-synaptic viral vector containing an inversion sequence of a gene sequence encoding a desired protein and having an FRT sequence and an FRT mutant sequence in opposite orientations at both ends of the inversion sequence, so that the functional flippase (Flp) recognizes the FRT sequence and the FRT mutant sequence, inverts the inversion sequence of the gene sequence encoding the desired protein, and enables the normal expression of the desired protein. In the present invention, "having an FRT sequence and an FRT mutant sequence in opposite directions at both ends of the inverted sequence" means that, as shown in the right diagram of Figure 1f, the FRT sequences in opposite directions are positioned so as to sandwich the inverted sequence of the gene sequence encoding the desired protein, and further, the FRT mutant sequences in opposite directions are also positioned so as to sandwich the inverted sequence of the gene sequence encoding the desired protein. Since flippase inverts all sequences sandwiched between a set of FRT sequences in opposite directions or a set of FRT mutant sequences in opposite directions, sandwiching the sequence with only one set of recognition sequences will result in repeated inversions. Therefore, sandwiching the sequence with two sets of recognition sequences will result in one inversion and then remove the FRT-based sequences that are aligned in the same direction after inversion, thereby stopping the repeated inversions. The order of the sequences is preferably as follows:

[0056] FRT-FRT variant-inverted sequence of gene encoding desired protein-FRT reverse-FRT variant-reverse FRT variant; FRT variant-FRT variant-inverted sequence of gene encoding desired protein-FRT variant-reverse FRT variant; FRT variant-reverse FRT variant-inverted sequence of gene encoding desired protein-FRT-FRT variant; FRT variant-reverse FRT variant-inverted sequence of gene encoding desired protein-FRT variant-FRT variant; FRT variant-reverse FRT variant-inverted sequence of gene encoding desired protein-FRT variant-FRT variant; FRT variant-reverse FRT variant-inverted sequence of gene encoding desired protein-FRT variant-FRT variant; FRT variant-reverse FRT variant-inverted sequence of gene encoding desired protein-FRT variant-FRT variant; or reverse sequence of FRT mutant sequence - FRT sequence - reverse sequence of gene sequence encoding desired protein - reverse sequence of FRT sequence - FRT mutant sequence

[0057] The FRT sequence is that shown in SEQ ID NO: 3 (GAAGTTCCTATTC TCTAGAAA GTATAGGAACTTC). The FRT mutant sequence is a sequence in which a portion of the FRT sequence shown in SEQ ID NO: 3 is mutated, and examples thereof include the F5 sequence (SEQ ID NO: 4: GAAGTTCCTATTC TTCAAAAG GTATAGGAACTTC).

[0058] (First Vector and Second Vector) An important feature of the technology according to the above-described embodiment of the present invention is that when the first vector is an anterograde transsynaptic viral vector, the second vector is a retrograde transsynaptic viral vector; and when the first vector is a retrograde transsynaptic viral vector, the second vector is an anterograde transsynaptic viral vector, thereby identifying a neuronal population through both input and output. By introducing a third vector into the region identified through both input and output, desired proteins can be expressed, enabling visualization of the identified neuronal population and intervention in nervous system function through drugs or brain tech targeting specific neural circuit elements. The technology of the present invention allows targeting only the required area, potentially reducing side effects and achieving the desired therapeutic effect, thereby expanding the potential for developing new treatments for various nervous system disorders.

[0059] The first vector or the second vector is an anterograde transsynaptic viral vector or a retrograde transsynaptic viral vector containing a nucleotide sequence encoding a Cre recombinase. The Cre recombinase may be any vector that exhibits functional Cre recombinase activity, and the nucleotide sequence contained in the first vector or the second vector may be any vector that encodes a Cre recombinase that exhibits functional Cre recombinase activity.

[0060] The first or second vector is a retrograde transsynaptic viral vector containing an inverted sequence of a base sequence encoding flippase (Flp), with a loxP sequence and a loxP mutation sequence at both ends of the inverted sequence, in opposite orientations. The flippase (Flp) may be any vector that exhibits functional flippase (Flp) activity, and the base sequence of the first or second vector may be a sequence encoding a flippase (Flp) that exhibits functional flippase (Flp) activity. A functional Cre recombinase expressed in a cell into which the first or second vector has been introduced recognizes the loxP sequence and the loxP mutation sequence, which are present in opposite orientations at both ends of the inverted sequence of the base sequence encoding flippase (Flp), and can invert the inverted sequence of the base sequence encoding flippase (Flp), thereby expressing functional flippase (Flp).

[0061] The explanations in the section [Split Cre-PINCER] can be applied directly to the explanations of transsynaptic viruses, anterograde transsynaptic viral vectors, and retrograde transsynaptic viral vectors.

[0062] In the above-described embodiment of the present invention, the first vector and the second vector may include regulatory sequences such as a promoter, an enhancer sequence that assists in mRNA transcription and protein translation, a Kozak sequence, a ribosome binding sequence, a terminator, a polyadenylation site, and a WPRE sequence, so as to enable expression of the gene of interest. Furthermore, if necessary, the vector may include a selection marker sequence such as a drug resistance gene (e.g., a kanamycin resistance gene, an ampicillin resistance gene, a puromycin resistance gene, etc.), a thymidine kinase gene, or a diphtheria toxin gene, or a reporter gene sequence such as mCherry (red fluorescent protein), green fluorescent protein (GFP), β-glucuronidase (GUS), or FLAG.

[0063] In the present invention, the first and second vectors preferably contain an inverted sequence of a nucleotide sequence encoding Cre recombinase or a nucleotide sequence encoding flippase (Flp), and the loxP sequences and loxP mutant sequences in opposite orientations at both ends of the inverted sequence are operably linked to a promoter sequence. Examples of such promoter sequences include promoter sequences that function in cells constituting brain regions or neural circuits targeted for infection by transsynaptic and non-transsynaptic viruses, such as the hSyn promoter, CAG promoter, and CMV promoter. Here, "operably linked" refers to the same as described above.

[0064] (Third Vector) The third vector is a non-transsynaptic viral vector that includes an inverted sequence of a gene sequence encoding a desired protein, with an FRT sequence and an FRT mutant sequence in opposite orientations at both ends of the inverted sequence. Site-specific gene recombination occurs when flippase (Flp), a recombinase enzyme, acts on the FRT sequence and the FRT mutant sequence in opposite orientations at both ends of the inverted sequence of a gene sequence encoding a desired protein. In this embodiment, when flippase (Flp) acts on the FRT sequence and the FRT mutant sequence in opposite orientations, the gene sequence sandwiched between them can be inverted. As described above, functional flippase (Flp) is formed in cells into which both the first and second vectors have been introduced (infected), and when a third vector is introduced into these infected cells, the inversion sequence of the gene sequence encoding the desired protein is inverted by the action of the functional flippase (Flp), thereby enabling the desired protein to be expressed normally. Note that the phrase "having an FRT sequence and an FRT mutant sequence in opposite directions at both ends of the inversion sequence" is as described above.

[0065] The explanation for the "desired protein" in the inverted sequence of the gene sequence encoding the desired protein contained in the third vector can be applied as is to the explanation in the section [Split Cre-PINCER].

[0066] In the above-described embodiment of the present invention, the inverted sequence of the gene sequence encoding the desired protein contained in the third vector is preferably operably linked to a promoter sequence. Such promoter sequences can be those that function in cells constituting the brain region targeted for infection by transsynaptic and non-transsynaptic viruses, cells constituting neural circuits, etc., and examples thereof include the hSyn promoter, CAG promoter, and CMV promoter. Here, "operably linked" is as defined above.

[0067] <Pharmaceutical Compositions> The present invention also encompasses pharmaceutical compositions containing the vector set of the present invention described above. As described above, the vector set of the present invention identifies neuronal populations based on both input and output, enabling targeting of neural circuit elements with more specific neural activity dynamics and functions compared to identification based on input alone or output alone. Furthermore, by targeting neural circuit elements identified based on both input and output and utilizing various proteins that can be introduced by a third vector, intervention in nervous system function using drugs or brain technology can be achieved with fewer side effects, resulting in the desired therapeutic effect and potentially providing new treatment methods for various nervous system disorders. The specific description of the vector set contained in the pharmaceutical composition of the present invention is directly applicable to the description in the above section on vector sets.

[0068] The pharmaceutical composition of the present invention can be formulated for an appropriate route of administration into the brain. The pharmaceutical composition of the present invention may also contain other ingredients, etc., as long as the effects of the present invention are not impaired. For example, the composition may contain other pharmacologically active substances, such as small molecule compounds useful in the above-mentioned chemical genetics techniques, devices such as guide cannulas for systemically injecting them throughout the body, or additional substances useful for physically preparing various dosage forms of the pharmaceutical composition of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, thickeners, and stabilizers.

[0069] The amount of each vector, compound, etc. contained in the pharmaceutical composition of the present invention is not particularly limited, and an appropriate amount can be selected depending on various conditions such as the type of disease, the age and symptoms of the patient, the administration route, the purpose of treatment, and the presence or absence of concomitant drugs.

[0070] <Method for targeting a specific neuronal population in a neural circuit> The present invention includes a method for targeting a specific neuronal population in a neural circuit, characterized by using the vector set of the present invention described above to introduce an anterograde transsynaptic viral vector into an input region, a retrograde transsynaptic viral vector into an output region, and a non-transsynaptic viral vector into an intermediate target region. The description of the vector set described above can be applied as is. The method of the present invention can be used in humans and non-human animals.

[0071] The input region, intermediate target region, and output region may be the combinations shown in FIG.

[0072] Among these, a preferred example is the CNS→CNS→CNS combination in Figure 8, in which the input region is the cuneiform nucleus (CnF), the output region is the central nucleus of the amygdala (CeA), and the intermediate target region is the lateral nucleus of the amygdala and basal nuclei of the amygdala (LA / B).

[0073] The input region, intermediate target region, and output region can be identified as follows. At present, anterograde transsynaptic viruses are a new type of virus, so it is preferable to identify the input region and output region from retrograde viruses, which are more established in this technical field, and select the input-output circuit. Specifically, this is as follows.

[0074] (1) A retrograde transsynaptic virus expressing a label protein (such as a fluorescent protein) is injected into the target region to identify the input region. (2) A non-transsynaptic virus expressing the label protein is injected into the target region, and nerve terminals of neurons that have cell bodies in the target region and express the label protein are located in other regions to identify the output region. (3) An anterograde virus expressing recombinase A, such as Cre or Flp, is injected into the input region, and a non-transsynaptic virus that expresses the label protein in a recombinase A-dependent manner is injected into the target region, and expression of the label protein in a neuronal subpopulation in the target region is confirmed. (4) A retrograde virus expressing recombinase A, such as Cre or Flp, is injected into the output region, and a non-transsynaptic virus that expresses the label protein in a recombinase A-dependent manner is injected into the target region, and expression of the label protein in a neuronal subpopulation in the target region is confirmed. (5) An anterograde virus expressing recombinase A such as Cre or Flp is injected into the input region, a retrograde virus expressing recombinase B such as Flp or SplitCreC is injected into the output region, and a non-transsynaptic virus expressing a label protein in a recombinase A+B-dependent manner is injected into the target region, and expression of the label protein in a neuronal subpopulation in the target region is confirmed (PINCER method). (6) In the system described above in (5), a virus expressing a protein for manipulating neural activity such as opsin or a protein for reading out neural activity such as GCaMP is injected into the target region instead of the label protein, and functional function of the desired protein in the neuronal subpopulation in the target region is confirmed. (PINCER method) (7) As the anterograde non-transsynaptic virus used in the present invention, AAV1 (adeno-associated virus) is preferable because of its low toxicity, but one major disadvantage is that it has been reported to infect synaptically not only anterogradely but also retrogradely. Future improvements are expected to lead to the development of an AAV1 that infects synaptically only anterogradely, but no such reports have been published to date. Therefore, at present, the PINCER technique is not suitable for use when there is a mutual neural connection between the input region and the target region (i.e., when the output region from the target region overlaps with the input region).Therefore, at this stage, a step between steps (2) and (3) is required: "inject a retrograde virus that expresses a label protein into the input region and confirm that the label protein is not expressed in the neuronal subpopulation in the target region." If a low-toxicity virus that infects synaptically only anterogradely is developed or discovered in the future, this additional step will become unnecessary.

[0075] By injecting each vector contained in the vector set of the present invention into the input region, intermediate target region, and output region identified by the above steps, specific neuronal populations in neural circuits can be targeted. By administering drugs tailored to the neuronal populations identified by the method of the present invention or by intervening in nervous system function using brain tech, it may be possible to achieve desired therapeutic effects with fewer side effects, and the broader range of applications will open up the possibility of creating new treatments for various nervous system diseases.

[0076] As described above, split Cre-PINCER and Cre / Flp-PINCER have been described as PINCER techniques that target specific neuronal populations in neural circuits, but similar effects can be expected using recombinases other than the Cre recombinase and flippase (Flp) recombinase used in these techniques. For example, the following systems are also thought to be applicable to PINCER technology.・Dre dDIO system: Nucleic Acids Research, Volume 32, Issue 20, 15 October 2004, Pages 6086-6095 ・vCre vcDIO system: Nucleic Acids Research, Volume 39, Issue 8, 1 April 2011, Page e49 ・sCre scDIO system: Nucleic Acids Research, Volume 39, Issue 8, 1 April 2011, Page e49 ・ΦC31 pSIO system:Neuron,VOLUME 112, ISSUE 1, P56-72.E4, JANUARY 03, 2024

[0077] In view of the above, the present invention also includes vector sets of the following embodiments.

[0078] A vector set used to target a specific neuron population in a neural circuit, comprising first to third vectors, wherein the first vector is an anterograde transsynaptic viral vector comprising a base sequence encoding a split recombinase consisting of the C-terminal sequence of recombinase X, the second vector is a retrograde transsynaptic viral vector comprising a base sequence encoding a split recombinase consisting of the N-terminal sequence of recombinase X, and the third vector is a non-transsynaptic viral vector comprising an inverted sequence of a gene sequence encoding a desired protein, and having recombinase X recognition sequences and / or mutated recombinase X recognition sequence sequences in opposite orientations at both ends of the inverted sequence, and wherein functional recombinase X is formed in cells infected with the first and second vectors.

[0079] A vector set used to target a specific neuron population in a neural circuit, comprising first to third vectors, wherein the first vector is a retrograde transsynaptic viral vector comprising a base sequence encoding a split recombinase consisting of the C-terminal sequence of recombinase X, the second vector is an anterograde transsynaptic viral vector comprising a base sequence encoding a split recombinase consisting of the N-terminal sequence of recombinase X, and the third vector is a non-transsynaptic viral vector comprising an inverted sequence of a gene sequence encoding a desired protein, and having recombinase X recognition sequences and / or mutated recombinase X recognition sequence sequences in opposite orientations at both ends of the inverted sequence, and wherein functional recombinase X is formed in cells infected with the first and second vectors.

[0080] 1. A vector set used to target a specific neuron population in a neural circuit, comprising first to third vectors, wherein the first vector is an anterograde transsynaptic viral vector comprising a nucleotide sequence encoding recombinase X; the second vector is a retrograde transsynaptic viral vector comprising an inverted sequence of a nucleotide sequence encoding recombinase Y, with recombinase X recognition sequences and / or mutated recombinase X recognition sequences in opposite orientations at both ends of the inverted sequence; and the third vector is a non-transsynaptic viral vector comprising an inverted sequence of a gene sequence encoding a desired protein, with recombinase Y recognition sequences and / or mutated recombinase Y recognition sequences in opposite orientations at both ends of the inverted sequence; and wherein functional recombinase Y is expressed in cells infected with the first and second vectors.

[0081] 1. A vector set used to target a specific neuron population in a neural circuit, comprising first to third vectors, wherein the first vector is a retrograde transsynaptic viral vector comprising a nucleotide sequence encoding recombinase X; the second vector is an anterograde transsynaptic viral vector comprising an inverted sequence of a nucleotide sequence encoding recombinase Y, with recombinase X recognition sequences and / or mutated recombinase X recognition sequences in opposite orientations at both ends of the inverted sequence; and the third vector is a non-transsynaptic viral vector comprising an inverted sequence of a gene sequence encoding a desired protein, with recombinase Y recognition sequences and / or mutated recombinase Y recognition sequences in opposite orientations at both ends of the inverted sequence; and wherein functional recombinase Y is expressed in cells infected with the first and second vectors.

[0082] The present invention also includes a pharmaceutical composition comprising any of the above vector sets.

[0083] The present invention also includes a method for targeting a specific neuronal population in a neural circuit, which is characterized by using any of the above-mentioned vector sets to introduce an anterograde transsynaptic viral vector into an input region, a retrograde transsynaptic viral vector into an output region, and a non-transsynaptic viral vector into an intermediate target region.

[0084] The present invention will be specifically described in the following examples, but the present invention should not be construed as being limited to these examples.

[0085] 1. Establishment of the Projection-based Intersectional Circuit-tagging Enabled by Recombinases (PINCER) Method Neurons transmit information across neural circuits. It has become clear that the function of each neuron in these circuits is assigned polarity based on both the afferent inputs it receives from other brain regions and the efferent outputs it sends to other brain regions. To study the neural activity and function of specific neuronal populations, methods using anterograde and retrograde viruses to identify neural circuit elements based on their inputs or outputs, respectively, have been widely used. The development of such methods has contributed to significant advances in neuroscience research in recent years.

[0086] However, neuroscience research has yet to develop a method for studying neuronal function based on both input and output. Therefore, the present inventors developed a technique for targeting specific neuronal populations in neural circuits by combining multiple recombinase enzymes and anterograde / retrograde viruses to investigate the functional input / output structure of neural circuits. The advantages of the present technique, i.e., identifying neural circuit elements based on both input and output and targeting anatomically defined neuronal populations in functionally localized neural circuits, are explained using the diagrams in Figure 1.

[0087] The neuronal population targeted in this study was identified by unidirectional input from the cuneate nucleus (CnF), which conveys aversive information to the lateral and basal amygdala (LA / B), and output to the central nucleus of the amygdala (CeA), a region involved in regulating different aspects of emotional responses. In our laboratory, we generated vectors in which the N-terminal portion of the split-cre recombinase enzyme (pAAV-NCre) was packaged in an anterograde transsynaptic adeno-associated virus (AAV) and the C-terminal portion (pAAV-CCre) was packaged in a retrograde AAV (Fig. 1f). By administering anterograde AAV-NCre to the CnF and retrograde AAV-CCre to the CeA, we demonstrated the efficacy of this approach by delivering cre-dependent transgene expression to specific neuronal populations identified by both input and output in the LA / B. Details are described below.

[0088] Figure 1a shows a schematic diagram of a neural circuit showing the flow of information through a target region, receiving afferent input from two brain regions 1 and 2 and sending efferent output to two regions 1 and 2. Figure 1b shows a conventional output-based targeting method for neural circuit elements using retrograde viral injection. Neuronal populations targeted by this method project to a specific output region 1 and receive inputs from multiple input regions 1 and 2. Figure 1c shows an input-based targeting method for neural circuit elements using anterograde transsynaptic viral injection. Neuronal populations identified by this method receive inputs from a specific input region 1 and project to multiple output regions 1 and 2.

[0089] On the other hand, Figure 1d shows a combinatorial strategy developed in this study that applies both retrograde and anterograde transsynaptic viruses to the PINCER method. This method allows targeting neural circuit elements based on both afferent input and efferent output. The PINCER method can be achieved by the following approaches based on the combination of two recombinase enzymes: (i) a method using the split-cre recombinase enzymes NCre and CCre (left panel of Figure 1d: SplitCre-PINCER method); (ii) a method using the Cre recombinase enzyme and the Flp recombinase enzyme (right panel of Figure 1d: Cre / Flp-PINCER method).

[0090] Figure 1e shows the combination of SplitCre-PINCER and Cre / Flp-PINCER viruses injected into the lateral amygdala and basal ganglia (LA / B) circuit, which receives input from the cuneate nucleus (CnF) and sends output to the central amygdala (CeA).

[0091] Figure 1f shows the reagents (basic vector designs) used in the SplitCre-PINCER method (left) and the Cre / Flp-PINCER method (right). rAAV1-hSyn-Cre was purchased from Addgene (addgene 105553-AAV1). CAV2-CMV-FLExloxP-Flp was purchased from Plateforme de Vectorologie de Montpellier. pAAV-hSyn-NCre (detailed in Fig. 1j-1), pAAV-CAG-CCre (detailed in Fig. 1j-2), pAAV-CAG-FLExFRT-mCherry (detailed in Fig. 1j-3), pAAV-CAG-FLExloxP-Flp (detailed in Fig. 1j-4), pAAV-hSyn-CCre (detailed in Fig. 1j-5), and pAAV-CAG-NCre (detailed in Fig. 1j-7) were constructed in-house and packaged into selected AAV serotypes. Figure 1f also shows the core sequence design of the pAAV plasmids packaged into the AAV used in the SplitCre-PICNER (left) and Cre / Flp-PINCER (right) methods. Open and filled triangles indicate incompatible loxP sites, and open and filled semicircles indicate incompatible FRT sites. Negative control experiments in which injection of either rAAV1-hSyn-NCre or retroAAV2-CAG-CCre and rAAV1-hSyn-Cre or retroAAV2-CAG-FLExloxP-Flp and CAV2-CMV-FLExloxP-Flp was omitted showed no labeling in the lateral amygdala and nucleus basalis (LA / B) (data not shown). We also confirmed that similar input / output-based labeling of neurons in the LA / B was possible using rAAV1-hSyn-CCre and retroAAV2-CAG-NCre in SplitCre-PINCER (data not shown). Negative control experiments in which either injection was omitted showed no labeling in the LA / B (data not shown).

[0092] Figure 1g shows images of CeA injection (rAAV5-CAG-eGFP+) to monitor leakage into the LA / B, along with labeling of neural circuit elements (rAAV5-FLExloxP-tdTomato+) identified from both CnF input and CeA output neurons using the SplitCre-PINCER method. This method was used when leakage of CeA into the LA / B was important in the experiment. The scale bar represents 500 μm.

[0093] Figure 1h shows the results of a time course analysis of viral expression, revealing more efficient neuronal labeling in LA / B neurons identified by both CnF input and CeA output using the SplitCre-PINCER method compared to the Cre / Flp-PINCER method. The arrows in the figure indicate neurons in the LA / B identified by both SplitCre-PINCER and Cre / Flp-PINCER CnF input and CeA output at different time points after viral injection. The scale bar indicates 200 μm.

[0094] Figure 1i shows quantification of neuronal labeling by the Cre / Flp-PINCER or SplitCre-PINCER method at various infection periods. Four mice were tested at each incubation time point after viral injection using either the Cre / Flp-PINCER or SplitCre-PINCER method. At 4 weeks after viral injection, the SplitCre-PINCER method (blue) labeled more neurons in the LA / B than the CreFlp-PINCER method (red). At 8 weeks after viral injection, both methods reached similar maximum labeling levels. *: P<0.05 (two-way repeated measures ANOVA, Cre / Flp-PINCER vs. splitCre-PINCER from week 2 to week 12). #: P<0.05 (Sidack's multiple comparisons test (post-hoc)). Error bars indicate s.e.m.

[0095] 2. Anatomical application of labeling neuronal populations in the LA / B with specific input / output connections Using the technology established above, we clarified the anatomical characteristics (Fig. 2a-b) and molecular differences of neuronal populations identified by input only, output only, and both input and output (Fig. 2c-e). We also demonstrated an application of labeling neuronal populations in the LA / B with specific input / output connections to the molecular characteristics of neurons targeted at output destinations (Fig. 2f-h), as a more detailed anatomical tracing of neuronal populations identified by input. Details are as follows.

[0096] As shown in Figure 2a, we quantified the overlap between neurons labeled only by cuneate nucleus (CnF) input (green) and neurons labeled by both CnF input and central amygdala (CeA) output (red) within the LA / B. Using the Cre / Flp-PICNER method, the viral combinations injected to label neurons in the LA / B from input only and both input and output are shown in the left panel of Figure 2a. The right panel shows images of neurons expressing eGFP (green) only (neurons receiving CnF input but not CeA output; arrows) and neurons co-expressing eGFP (green) and tdTomato (red) (neurons identified by both CnF input and CeA output; arrowheads). This method allowed us to quantify the proportion of neurons identified by both input and output among neurons identified by input only. The scale bar (right) represents 30 μm.

[0097] As shown in Figure 2b, we quantified the overlap between neurons labeled only by CeA output (red) and neurons labeled by both CnF input and CeA output (green) within the LA / B. The viral combinations used to label neurons within the LA / B via output only and both input and output using the Cre / Flp-PINCER method are shown in the left diagram of Figure 2b. rAAV5-CAG-FLExFRT-eYFP was generated in-house, packaged into a selected AAV serotype, and purified. For detailed methods, see Luo, R. et al. A dopaminergic switch for fear to safety transitions. Nat. Commun. 9, 2483 (2018). The right panel shows images of neurons expressing only tdTomato (red) (neurons that have CeA output but do not receive CnF input; arrows) and neurons co-expressing eYFP (green) and tdTomato (red) (yellow) (neurons identified by both CnF input and CeA output; arrowheads). The scale bar (right) represents 50 μm. This method enabled us to quantify the proportion of neurons identified by both input and output among neurons identified by output alone. We are currently counting the number of neurons of each type. The scale bar (right) represents 30 μm.

[0098] As shown in Figure 2c, we analyzed the co-expression of glutamatergic excitatory neuron markers (vGluT) and GABAergic inhibitory neuron markers (vGAT) in neurons labeled only by CnF input, neurons labeled by both CnF input and CeA output, and neurons labeled only by CeA output within the LA / B. Figure 2c shows the combinations of injected viruses used to label neurons in the LA / B identified by CnF input only, both CnF input and CeA output, and CeA output only, respectively, and outlines the experimental setup for co-labeling with vGluT and vGAT mRNA using hybridization chain reaction-fluorescence in situ hybridization (HCR-FISH).

[0099] Figure 2d shows images of neurons in the LA / B labeled from input only (top left), both input and output (center left), and output only (bottom left), as well as vGluT-expressing neurons. Among neurons identified in each projection pattern, vGluT co-expressing neurons are indicated by arrowheads, while non-co-expressing neurons are indicated by arrows. The pie charts (right) show that 34.39% of neurons labeled from input only (top, a total of 58 neurons from n = 4 mice), 81.07% of neurons labeled from input and output (center, a total of 60 neurons from n = 4 mice), and 74.15% of neurons labeled from output only (bottom, a total of 552 neurons from n = 3 mice) co-express vGluT. The scale bar (left) indicates 30 μm.

[0100] Figure 2e shows images of neurons in the LA / B labeled by input only (top left), both input and output (center left), and output only (bottom left) and vGAT-expressing neurons. Among neurons identified by each projection pattern, vGAT co-expressing cells are indicated by arrowheads, while non-co-expressing cells are indicated by arrows. The pie charts (right) show that 44.04% of neurons labeled by input only (top, a total of 58 cells from n = 4 mice), 2.17% of neurons labeled by input and output (center, a total of 60 cells from n = 4 mice), and 19.29% of neurons labeled by output only (bottom, a total of 552 cells from n = 3 mice) co-express vGAT. The scale bar (left) indicates 30 μm.

[0101] Figure 2f shows the application of PINCER to target neurons in the LA / B subunit that receive input from the CnF and send output to CeA neurons expressing SST, a marker for inhibitory neuron subtypes. We used an anterograde rAAV1 carrying the flp recombinase enzyme sequence to target CnF neurons and a modified rabies virus that specifically infects the CeA subunit from SST-expressing neurons in a retrograde manner. In SST-Cre transgenic rats, the virus combinations used to label LA / B neurons receiving input from the CnF with mCherry (red) and LA / B neurons that send output to SST-expressing neurons in the CeA with eGFP (green) are shown.

[0102] Figure 2g shows an image of starter cells (co-expressing green and red; arrows) infected with modified rabies virus in CeA. The helper virus required for modified rabies virus infection (green) immunolabels HA bound to the G protein (red). The scale bar indicates 200 μm.

[0103] Figure 2h shows an image of a neuron (arrowhead) in the lateral amygdala (LA) co-expressing eGFP (green: neurons projecting to SST-expressing neurons in the CeA) and mCherry (red: neurons receiving input from the CnF) (n = 3 animals, reproducibility confirmed). The scale bar represents 500 μm.

[0104] 3. Auditory Fear Conditioning Test Using photometry combined with PINCER, we compared the neural activity dynamics (calcium activity dynamics) and function of neuronal populations in the LA / B identified through both CnF input and CeA output during auditory fear conditioning with those in the lateral amygdala and basal ganglia (LA / B) identified through CnF input only, CeA output only, and both CnF input and CeA output. Results showed that all neuronal populations identified through input only, output only, and both input and output increased neural activity evoked by the auditory conditioned stimulus (CS) after aversive associative learning. Details are as follows.

[0105] Figure 3a shows a schematic diagram of the experiment for recording neural activity from each neuronal population using fiber photometry.

[0106] Figure 3b shows boxplots of the freezing response (%) to the auditory conditioned stimulus (CS) during fear conditioning (left panel: Day 3) and after conditioning (right panel: Day 4). Data for each projection pattern are shown for n = 18 animals, including those for which neurons were identified in each of the three projection patterns.

[0107] Figure 3c shows the viral combination used to express GCaMP6s in LA / B neurons identified with only cuneate nucleus (CnF) input for fiber photometry. AAV5-CAG-FLExloxP-GCaMP6s was purchased from Addgene (addgene 100842-AAV5). Figure 3d shows the mean dynamics of calcium responses to the CS before (black) and after (blue) fear conditioning in LA / B neurons identified with only input (n = 6). All calcium responses were significantly higher than the baseline before the CS. *: P < 0.05 (Wilcoxon test for comparison with the corresponding baseline). Shading indicates sem. Figure 3e shows a bar graph comparing the area under the curve (AUC) of the auditory CS-evoked calcium response increase before and after fear conditioning in LA / B neurons identified with only input. The AUC of the CS response after fear conditioning (blue) was significantly increased compared to before fear conditioning (black). *: P<0.05 (Wilcoxon test). Error bars are sem (standard error).

[0108] Figure 3f shows the viral combinations used to express GCaMP6s in LA / B neurons identified by the SplitCre-PINCER method, both as CnF inputs and as CeA outputs. Figure 3g shows the mean calcium response dynamics (n = 6) of LA / B neurons identified by both input and output responses to the CS before (black) and after (blue) fear conditioning. Responses to the CS after fear conditioning were significantly higher than the corresponding baseline before CS onset, whereas responses to the CS before conditioning were significantly lower than the baseline. ns: P > 0.05, *: P < 0.01 (Wilcoxon test for comparison with the corresponding baseline). Shading indicates sem (standard error). Figure 3h shows a bar graph comparing the area under the curve (AUC) of the auditory CS-evoked calcium response of LA / B neurons identified by both input and output responses before and after fear conditioning. The AUC of the CS response after fear conditioning (blue) was significantly higher than that before fear conditioning (black). *: P<0.01 (Wilcoxon test). Error bars are sem (standard error).

[0109] Figure 3i shows the viral combinations used to express GCaMP6s in LA / B neurons identified only by CeA output. Figure 3j shows the mean calcium response dynamics (n = 6) of LA / B neurons identified only by output before (black) and after (blue) fear conditioning. All CS-evoked calcium responses were significantly higher than the corresponding baseline before CS onset. *: P<0.01 (Wilcoxon test for comparison with the corresponding baseline). Shading indicates sem. Figure 3k shows a bar graph comparing the area under the curve (AUC) of the auditory CS-evoked calcium response of LA / B neurons identified only by output before and after fear conditioning. The AUC of the CS response after fear conditioning (blue) showed a significant increase compared to before fear conditioning (black). *: P<0.05 (Wilcoxon test). Error bars indicate sem.

[0110] Next, we examined population-specific calcium activity dynamics during salience information processing in distinct neuronal populations in the lateral amygdala and basal ganglia (LA / B) identified from input only, both input and output, and output only. Only neuronal populations identified from input only and output only responded to the auditory CS before aversive associative learning, demonstrating habituation of the CS response through repeated exposure to the CS, a hallmark of salience coding in neurons. Details are as follows. However, such response dynamics were not observed in neuronal populations identified from both input and output.

[0111] Figure 4a shows a schematic diagram of the experimental setup for recording neural activity from each neuronal population using fiber photometry. Figure 4b shows boxplots of freezing responses (%) during the presentation of an auditory conditioned stimulus (CS) before fear conditioning (left panel: Days 1 and 2), during fear conditioning (center panel: Day 3), and after fear conditioning (right panel: Day 4). Data from a total of 18 animals, including those with neuronal identification for each of the three projection patterns, are shown for each projection pattern.

[0112] The left panel of Figure 4c shows the viral combinations used to express GCaMP6s in LA / B neurons identified with only cuneate nucleus (CnF) input. The right panel of Figure 4c shows the mean calcium response dynamics of LA / B neurons identified with only input to the CS before (black) and after (blue) pre-CS exposure before fear conditioning (right, n = 6 mice). Shading indicates standard error of the mean (SEM). Figure 4d shows a bar graph comparing the area under the curve (AUC) of calcium responses to the CS in LA / B neurons identified with only input. Responses to the CS before pre-CS exposure were significantly higher than the corresponding baseline before CS onset, whereas responses to the CS after pre-CS exposure were significantly lower than the baseline. ns: P > 0.05, *: P < 0.01 (Wilcoxon test for comparison with the corresponding baseline). Shading indicates SEM. A significant decrease in AUC was observed from before (black) to after (blue) pre-CS exposure before fear conditioning (Fig. 4d). *: P<0.01 (Wilcoxon test). Error bars are sem. Figure 4e shows the mean dynamics of CS-evoked calcium responses of LA / B neurons identified by input alone (n = 6) before (black) and after (blue) fear conditioning after pre-CS exposure. Shading is sem. All CS-evoked calcium responses were significantly lower than the corresponding baseline before CS onset. ns: P>0.05 (Wilcoxon test for comparison with the corresponding baseline). Shading is sem. Bar graphs comparing the AUC of CS responses of LA / B neurons identified by input alone (Fig. 4f) showed no significant differences between CS responses before (black) and after (blue) fear conditioning. These data suggest that latent inhibition during fear conditioning blocks the enhancement of CS responses from before to after fear conditioning. ns: P>0.05 (Wilcoxon test). Error bars are sem (standard error).

[0113] The left panel of Figure 4g shows the viral combinations used to express GCaMP6s in LA / B neurons identified by the SplitCre-PINCER method as both CnF inputs and central amygdala (CeA) outputs. The right panel of Figure 4g shows the mean calcium response dynamics (n = 6) of LA / B neurons identified by both input and output evoked by CS before (black) and after (blue) pre-CS exposure before fear conditioning. Shading indicates standard error of mean (SEM). The response to the CS before pre-CS exposure was significantly lower than baseline both before and after CS onset. ns: P > 0.05 (Wilcoxon test for comparison with the corresponding baseline). Shading indicates standard error of mean (SEM). A bar graph comparing the AUC of the CS response of LA / B neurons identified by both input and output (Figure 4h) showed no difference in AUC between before (black) and after (blue) pre-CS exposure before fear conditioning. These data suggest that LA / B neurons identified through both input and output do not respond to calcium CS before or after pre-CS exposure prior to fear conditioning. ns: P > 0.05 (Wilcoxon test). Error bars are sem. Figure 4i shows the mean dynamics of CS-evoked calcium responses (n = 6) of LA / B neurons identified through both input and output, before (black) and after (blue) fear conditioning after pre-CS exposure. Shading is sem. All CS-evoked calcium responses were significantly lower than the corresponding baseline before CS onset. ns: P > 0.05 (Wilcoxon test for comparison with the corresponding baseline). Shading is sem. Bar graphs comparing the AUCs of CS responses of LA / B neurons identified through both input and output (Fig. 4j) showed no significant differences in CS responses before (black) and after (blue) fear conditioning. These data suggest that latent inhibitory mechanisms underlying fear conditioning prevent the fear-conditioned enhancement of CS responses from occurring before and after fear conditioning. ns: P > 0.05 (Wilcoxon test), error bars sem (standard error).

[0114] The left panel of Figure 4k shows the viral combinations used to express GCaMP6s in LA / B neurons identified only by CeA output. The right panel of Figure 4k shows the mean dynamics of calcium responses to the CS before (black) and after (blue) pre-CS exposure before fear conditioning (right, n = 6 mice). Shading indicates standard error of mean (SEM). The pre-CS response was significantly lower than baseline both before and after CS onset. *: P < 0.01 (Wilcoxon test for comparison with corresponding baseline). Shading indicates SEM. A bar graph comparing the AUC of the CS response of LA / B neurons identified only by output (Figure 4l) showed a significant decrease in AUC from before (black) to after (blue) pre-CS exposure before fear conditioning. **: P < 0.01 (Wilcoxon test). Error bars indicate SEM. Figure 4m shows the mean dynamics of CS-evoked calcium responses of neurons in the LA / B identified from output alone (n = 6) before (black) and after (blue) fear conditioning following pre-CS exposure. Shading indicates standard error of the mean (SEM). All CS-evoked calcium responses were significantly lower than the corresponding baseline before CS onset. ns: P > 0.05 (Wilcoxon test for comparison with the corresponding baseline). Shading indicates standard error of the mean (SEM). A bar graph comparing the AUC of CS responses of neurons in the LA / B identified from output alone (Fig. 4n) showed no significant difference between CS responses before (black) and after (blue) fear conditioning. These data suggest that latent inhibitory mechanisms are at work in response to fear conditioning, preventing the enhancement of CS responses from before to after fear conditioning. ns: P > 0.05 (Wilcoxon test). Error bars indicate standard error of the mean (SEM).

[0115] Next, we investigated the population-specific behavioral functions of different neuronal populations in the lateral amygdala and basal ganglia (LA / B) identified by input-only, input / output, and output-only methods using optogenetic inactivation. Functionally, all of these neuronal populations were required for long-term aversive memory formation, whereas only the neuronal population identified by output-only was required for short-term aversive learning. Details are as follows.

[0116] Figure 5a shows a schematic diagram of the optogenetic manipulation experiment during fear conditioning. An orange laser (589 nm) was applied during the shock unconditioned stimulus (US) period to determine the timing of optogenetic manipulation. The left panel of Figure 5b shows the viral combinations used to express ArchT-tdTomato in neurons in the LA / B identified only from cuneus nucleus (CnF) inputs for optogenetic manipulation. rAAV5-CAG-FLExloxP-ArchT-tdTomato (addgene 28305-AAV5) and rAAV5-CAG-FLExloxP-tdTomato (addgene 28306-AAV5) were purchased from Addgene. The right panel of Figure 5b shows an image of a neuron in the LA / B identified only from inputs expressing ArchT-tdTomato. Fiber tracks are indicated by white dotted lines. The scale bar is 500 μm. Bar graphs of auditory conditioned stimulus (CS)-induced freezing responses during fear conditioning (Fig. 5c) showed no significant difference between the control group (n = 8) with tdTomato-only expression in LA / B neurons identified from input alone and the experimental group (n = 8) with ArchT-tdTomato expression. ns: P > 0.05 (Mann-Whitney test). Error bars are sem. Bar graphs of freezing responses during memory retrieval (Fig. 5d) showed significantly reduced freezing responses in the experimental group (n = 8) with ArchT-tdTomato expression in LA / B neurons identified from input alone compared with the control group (n = 8) with tdTomato-only expression. Optogenetic inactivation of neurons in the LA / B identified solely from input during US presentation inhibited long-term memory formation in aversive associative learning, indicating that neural circuit elements in the LA / B identified solely from input are essential for long-term memory formation in aversive associative learning. *: P<0.05 (Mann-Whitney test). Error bars are sem (standard error).

[0117] The left panel of Figure 5e shows the viral combinations used to express ArchT-tdTomato in LA / B neurons identified by SplitCre-PINCER from both CnF input and central amygdala (CeA) output. The right panel of Figure 5e shows images of ArchT-tdTomato-expressing neurons identified by both input and output. Fiber tracks are indicated by white dotted lines. The scale bar represents 500 μm. The bar graph of CS-induced freezing responses during fear conditioning (Figure 5f) shows no significant difference between the control group (n = 8) expressing tdTomato alone and the experimental group (n = 8) expressing ArchT-tdTomato in LA / B neurons identified by SplitCre-PINCER from both input and output. ns: P > 0.05 (Mann-Whitney test). Error bars represent sem (standard error). Furthermore, the bar graph of freezing responses during memory retrieval (Fig. 5g) showed significantly reduced freezing responses in the experimental group (n = 8) in which ArchT-tdTomato was expressed in LA / B neurons identified through both input and output by the SplitCre-PINCER method, compared with the control group (n = 8) in which only tdTomato was expressed. Optogenetic inactivation of LA / B neurons identified through both input and output during fear conditioning inhibited long-term memory formation in aversive associative learning, indicating that LA / B neural circuit elements identified through both input and output are essential for long-term memory formation in aversive associative learning. *: P<0.05 (Mann-Whitney test). Error bars are sem (standard error).

[0118] The left panel of Figure 5h shows the viral combination used to express ArchT-tdTomato in LA / B neurons identified only from the CeA output. The right panel of Figure 5h shows images of LA / B neurons identified only from the output that expressed ArchT-tdTomato. Fiber tracks are indicated by white dotted lines. The scale bar represents 500 μm. The bar graph of CS-induced freezing responses during fear conditioning (Figure 5i) shows that ArchT-tdTomato expression significantly dampened the increase in freezing responses during fear conditioning during CS presentation in the experimental group (n = 8) compared with the control group (n = 8) in LA / B neurons identified only from the output. **: P < 0.01 (Mann-Whitney test). Error bars are sem (standard error). Furthermore, the bar graph of freezing responses during memory retrieval (Fig. 5j) showed significantly reduced freezing responses in the experimental group (n = 8) with ArchT-tdTomato expression in LA / B neurons identified solely from output compared with the control group (n = 8) with tdTomato expression alone. Optogenetic inactivation of LA / B neurons identified solely from output during US presentation inhibited aversive associative learning and its long-term memory formation, indicating that LA / B neural circuit elements identified solely from output are essential for aversive associative learning and its long-term memory formation. *: P<0.05 (Mann-Whitney test). Error bars are sem (standard error).

[0119] Figure 5k shows the viral combinations used to express ArchT-tdTomato in neurons identified by Cre / Flp-PINCER from both CnF input and CeA output. For pAAV-CAG-FLExFRT-ArchT-mCherry, a plasmid generated in-house was packaged into a specific AAV serotype and purified. A bar graph of CS-induced freezing responses during fear conditioning (Figure 5l) showed no significant difference between the control group (n = 8) expressing tdTomato alone and the experimental group (n = 8) expressing ArchT-tdTomato in neurons in the LA / B identified by Cre / Flp-PINCER from both input and output. ns: P > 0.05 (Mann-Whitney test). Error bars are sem (standard error). Furthermore, the bar graph of freezing responses during memory retrieval (Fig. 5m) showed that freezing responses were significantly reduced in the experimental group (n = 8) in which ArchT-tdTomato was expressed in neurons in the LA / B identified by both input and output using the Cre / Flp-PINCER method, compared with the control group (n = 8) in which only tdTomato was expressed. Optogenetic inactivation of neurons in the LA / B identified by both input and output using the Cre / Flp-PINCER method during shock presentation during fear conditioning demonstrated a significant impairment of long-term memory formation in aversive associative learning, similar to the SplitCre-PINCER method. *: P<0.05 (Mann-Whitney test). Error bars are sem (standard error).

[0120] Figure 5n shows a map of the vector packaged in the virus used in the Cre / Flp-PINCER method, which was prepared in our laboratory.

[0121] Next, to investigate whether these neuronal populations within the LA / B are functionally involved in Salience information processing in different ways, we performed optogenetic experiments using a behavioral model of Salience, which allows us to examine latent inhibition. Optogenetic inhibition of neuronal populations identified only from inputs and only from outputs within the LA / B during CS exposure before aversive associative learning reduced the impairment of subsequent aversive associative learning due to latent inhibition. In contrast, inhibition of neuronal populations identified only from inputs / outputs that do not respond to the CS before aversive associative learning did not have a modulating effect on latent inhibition. Details are as follows.

[0122] Figure 6a shows a schematic diagram of the optogenetic manipulation experiment on latent inhibition of fear conditioning, a behavioral model of salience information processing. In this behavioral experiment, repeated exposure to an auditory conditioned stimulus (CS) before fear conditioning weakens aversive associative memory by activating latent inhibition of fear conditioning. An orange laser (589 nm), which determines the timing of the optogenetic manipulation, was irradiated onto the LA / B during the pre-CS exposure period before fear conditioning.

[0123] The left panel of Figure 6b shows the viral combinations used to express ArchT-tdTomato in neurons in the LA / B identified with only CnF input for optogenetic manipulation. The bar graph of freezing responses during pre-CS exposure before fear conditioning (right panel of Figure 6b) showed no statistical difference between the control group (n = 8) with tdTomato-only expression in neurons in the LA / B identified with only input (n = 8) and the experimental group (n = 8) with ArchT-tdTomato expression. ns: P > 0.05 (Mann-Whitney test). Error bars are sem. Furthermore, the bar graph of CS-induced freezing responses during fear conditioning (Fig. 6c) shows that the increase in freezing responses during fear conditioning during CS presentation was significantly enhanced in the experimental group (n = 8) expressing ArchT-tdTomato compared with the control group (n = 8) expressing tdTomato alone in neurons in the LA / B identified by input alone. **: P<0.01 (Mann-Whitney test; error bars are sem). Bar graphs of freezing responses during memory retrieval (Fig. 6d) showed that freezing responses were significantly increased in the experimental group (n = 8) with ArchT-tdTomato expression in LA / B neurons identified by input alone compared with the control group (n = 8) with tdTomato expression alone. Inactivation of neural activity of LA / B neural circuit elements identified by input alone during pre-CS exposure before fear conditioning reduced the effect of latent inhibitory function on aversive associative learning and its long-term memory formation, indicating that LA / B neural circuit elements identified by input alone are essential for latent inhibitory function on aversive associative learning and its long-term memory formation. *: P<0.05 (Mann-Whitney test; error bars are sem).

[0124] The left panel of Figure 6e shows the viral combinations used to express ArchT-tdTomato in neurons in the LA / B identified by the SplitCre-PINCER method from both CnF input and central amygdala (CeA) output. The bar graph of freezing responses during pre-CS exposure before fear conditioning (right panel of Figure 6e) showed no statistically significant differences between the control group (n = 8) expressing tdTomato alone and the experimental group (n = 8) expressing ArchT-tdTomato in neurons in the LA / B identified from both input and output. ns: P > 0.05 (Mann-Whitney test). Error bars are sem. The bar graph of CS-induced freezing during fear conditioning (Fig. 6f) shows no difference in freezing during CS presentation during fear conditioning between the experimental group (n = 8) expressing ArchT-tdTomato compared with the control group (n = 8) expressing only tdTomato in neurons in the LA / B identified from both input and output. ns: P > 0.05 (Mann-Whitney test). Error bars are sem. The bar graph of freezing during memory retrieval (Fig. 6g) also shows no difference in freezing between the experimental group (n = 8) expressing ArchT-tdTomato in neurons in the LA / B identified from both input and output (n = 8) and the control group (n = 8) expressing only tdTomato. Inactivation of neural activity of the LA / B neural circuit elements identified through both input and output during pre-CS exposure before fear conditioning did not affect the effect of latent inhibitory function on long-term memory formation of aversive associative learning, indicating that the LA / B neural circuit elements identified through both input and output do not have a latent inhibitory function on aversive associative learning and its long-term memory formation. ns: P>0.05 (Mann-Whitney test), error bars are sem (standard error).

[0125] The left panel of Figure 6h shows the viral combinations used to express ArchT-tdTomato in LA / B neurons identified only from the CeA output. The bar graph of freezing responses during pre-CS exposure before fear conditioning (right panel of Figure 6h) showed no statistical difference between the control group (n = 8) expressing tdTomato only in LA / B neurons identified only from the output and the experimental group (n = 8) expressing ArchT-tdTomato. ns: P > 0.05 (Mann-Whitney test). Error bars are sem. The bar graph of CS-induced freezing responses during fear conditioning (Figure 6i) showed no difference in freezing responses during CS presentation during fear conditioning between the experimental group (n = 8) expressing ArchT-tdTomato compared to the control group (n = 8) expressing tdTomato only in LA / B neurons identified only from the output. ns: P>0.05 (Mann-Whitney test). Error bars are sem. Furthermore, the bar graph of freezing responses during memory retrieval (Fig. 6j) showed significantly increased freezing responses in the ArchT-tdTomato experimental group (n = 8) compared with the tdTomato-only control group (n = 8). Inactivation of neural activity in LA / B neural circuit elements identified by output alone during pre-CS exposure before fear conditioning reduced the effect of latent inhibitory function on long-term memory formation of aversive associative learning, indicating that LA / B neural circuit elements identified by output alone are essential for latent inhibitory function on long-term memory formation of aversive associative learning. *: P<0.05 (Mann-Whitney test). Error bars are sem.

[0126] This indicates that neuronal populations in the LA / B identified by input only or output only perform more general aversive associative learning and memory and its salience processing functions, whereas neuronal populations identified by both input and output only function specifically in the formation of aversive long-term memories.

[0127] 4. Methods for alleviating PTSD symptoms It is known that a symptom of PTSD is an excessive aversion response to stimuli such as loud noises after a traumatic experience. When considering these excessive symptoms in the context of fear conditioning, the following explanations can be proposed. - Before the traumatic experience: The individual is not afraid of hearing loud noises (CS) such as fireworks in a safe situation. - During the traumatic experience: In a dangerous situation such as a battlefield during war or a major earthquake, the individual experiences and associates the loud noise (CS) with the fear of death (US), resulting in a fearful response (UCR). - After the traumatic experience: When the individual again hears a loud noise (CS) such as fireworks in a safe situation, the fearful response (UCR) to loud noises (CS) associated with the traumatic experience is recalled, resulting in a fear (CR) to the loud noise of fireworks (CS). (CS: conditioned stimulus, US: unconditioned stimulus, UCR: unconditioned response, CR: conditioned response)

[0128] This explanation for PTSD symptoms was also proposed in a review paper (YehudaLeDoux_PTSDReview_Neuron2007) in the history of research into the emotional learning mechanisms of the amygdala. Based on this explanation, when considering ways to alleviate PTSD symptoms by suppressing excessive aversion responses (CR) to stimuli such as loud noises (CS) in PTSD patients after a traumatic experience, one possible method would be to artificially suppress the neural activity of neurons localized to the function of controlling aversion responses (CR) to CS during fear memory recall.

[0129] Therefore, we investigated the behavioral function of optogenetic inactivation of neuronal activity in the LA / B, identified from both input and output, on the freezing response during fear memory retrieval after fear conditioning.

[0130] Figure 7a shows a schematic diagram of the optogenetic manipulation experiment during fear memory retrieval after fear conditioning. An orange laser (589 nm), which determined the timing of optogenetic manipulation, was irradiated during the conditioned stimulus (CS) presentation period during fear memory retrieval.

[0131] Figure 7b shows the viral combinations used to express ArchT-tdTomato in LA / B neurons identified from both the cuneate nucleus (CnF) input and the central amygdala (CeA) output using the Cre / Flp-PINCER method for optogenetic manipulation. A bar graph of CS-induced freezing responses during fear conditioning (Fig. 7c) showed no significant differences between the control group (n = 4) expressing tdTomato alone and the experimental group (n = 4) expressing ArchT-tdTomato in LA / B neurons identified from both the input and output. ns: P>0.05 (two-way repeated measures ANOVA, control group vs. experimental group for CS-US1 to CS-US3; error bars are sem (standard error). Furthermore, the bar graph of freezing responses during memory retrieval (Fig. 7d) showed that in the experimental group (n = 4) with ArchT-tdTomato expression in neurons in the LA / B identified from both input and output, freezing responses were significantly reduced when the laser was irradiated during CS presentation (CS+ON) compared with when it was not irradiated (CS+OFF). *: P<0.05 (Mann-Whitney test). On the other hand, in the experimental group (n = 4) with ArchT-tdTomato expression in neurons in the LA / B identified from both input and output, freezing responses were significantly reduced when the laser was irradiated during CS presentation (CS+ON) compared with when it was not irradiated (CS+OFF). In the control group (n = 4) expressing only o, there was no difference in freezing responses when the laser was not irradiated (CS+OFF) or when it was irradiated (CS+ON) during CS presentation. ns: P>0.05 (Mann-Whitney test). Inactivation of neural activity in the LA / B neural circuit elements identified through both input and output during CS presentation during optogenetic fear memory retrieval suppressed fear responses based on long-term memory in aversive associative learning, indicating that the LA / B neural circuit elements identified through both input and output are essential for fear responses based on long-term memory in aversive associative learning. Error bars are sem (standard error).

[0132] These results demonstrate that optogenetic suppression of neural activity in the LA / B neuronal population identified by both CnF input and CeA output using PICNER during tone (CS) presentation alone during fear memory retrieval after fear conditioning reduces fear-induced freezing (CR). This suggests that the neural activity of the LA / B neuronal population identified by both CnF input and CeA output is responsible for the fear-induced freezing (CR) in response to tone-only presentation (CS) during fear memory retrieval. Based on these data, if PICNER can be used to suppress the neural activity of the LA / B neuronal population identified by both CnF input and CeA output in PTSD patients during exposure to loud noises after a traumatic experience, as in this experiment, it may be possible to alleviate stress symptoms in PTSD patients.

[0133] The present invention identifies neuronal populations based on both input and output, enabling targeting of neural circuit elements with more specific neural activity dynamics and functions than identification based solely on input or output. This allows for the identification of new neuronal subclasses based on the anatomical connectivity between neurons resulting from the combination of input and output. Furthermore, the present invention provides tools for more detailed investigation and manipulation of the localization of functional properties of specific neural circuit elements based on the structure of the neural circuit's input and output. Applying the technology of the present invention to target neural circuit elements identified based on both input and output may potentially achieve desired therapeutic effects with minimal side effects, thereby broadening the scope of its applications and potentially leading to the development of new treatments for various nervous system disorders.

Claims

1. A vector set used to target a specific neuron population in a neural circuit, comprising first to third vectors, wherein the first vector is an anterograde transsynaptic viral vector comprising a base sequence encoding a split Cre recombinase consisting of the C-terminal sequence of Cre recombinase, the second vector is a retrograde transsynaptic viral vector comprising a base sequence encoding a split Cre recombinase consisting of the N-terminal sequence of Cre recombinase, and the third vector is a non-transsynaptic viral vector comprising an inverted sequence of a gene sequence encoding a desired protein, with a loxP sequence and a loxP mutant sequence in opposite orientations at both ends of the inverted sequence, and wherein functional Cre recombinase is formed in cells infected with the first and second vectors.

2. A vector set used to target a specific neuron population in a neural circuit, comprising first to third vectors, wherein the first vector is a retrograde transsynaptic viral vector comprising a base sequence encoding a split Cre recombinase consisting of the C-terminal sequence of Cre recombinase, the second vector is an anterograde transsynaptic viral vector comprising a base sequence encoding a split Cre recombinase consisting of the N-terminal sequence of Cre recombinase, and the third vector is a non-transsynaptic viral vector comprising an inverted sequence of a gene sequence encoding a desired protein, with a loxP sequence and a loxP mutant sequence in opposite orientations at both ends of the inverted sequence, and wherein functional Cre recombinase is formed in cells infected with the first and second vectors.

3. A vector set used to target a specific neuron population in a neural circuit, comprising first to third vectors, wherein the first vector is an anterograde transsynaptic viral vector comprising a nucleotide sequence encoding Cre recombinase, the second vector is a retrograde transsynaptic viral vector comprising an inverted sequence of a nucleotide sequence encoding flippase (Flp) and having a loxP sequence and a loxP mutant sequence in opposite orientations at both ends of the inverted sequence, and the third vector is a non-transsynaptic viral vector comprising an inverted sequence of a gene sequence encoding a desired protein and having an FRT sequence and an FRT mutant sequence in opposite orientations at both ends of the inverted sequence, wherein functional flippase is expressed in cells infected with the first and second vectors.

4. A vector set used to target a specific neuron population in a neural circuit, comprising first to third vectors, wherein the first vector is a retrograde transsynaptic viral vector comprising a base sequence encoding Cre recombinase, the second vector is an anterograde transsynaptic viral vector comprising an inverted sequence of a base sequence encoding flippase (Flp) and having a loxP sequence and a loxP mutant sequence in opposite orientations at both ends of the inverted sequence, and the third vector is a non-transsynaptic viral vector comprising an inverted sequence of a gene sequence encoding a desired protein and having an FRT sequence and an FRT mutant sequence in opposite orientations at both ends of the inverted sequence, wherein functional flippase is expressed in cells infected with the first and second vectors.

5. A vector set according to any one of claims 1 to 4, wherein the anterograde transsynaptic viral vector, the retrograde transsynaptic viral vector, and the non-transsynaptic viral vector are adeno-associated viral vectors.

6. A vector set according to any one of claims 1 to 5, wherein the desired protein is a protein involved in the control of neural activity and is used for the control of neural activity.

7. The vector set according to claim 6, wherein the protein involved in the control of neural activity is a protein involved in the inhibition of neural activity and is used for the inhibition of neural activity.

8. The vector set according to claim 7, wherein the protein having the function of inhibiting neural activity is an inhibitory opsin and is used for inhibiting neural activity.

9. A vector set according to any one of claims 1 to 5, wherein the desired protein is a calcium indicator or a voltage indicator and is used to obtain a readout of neural activity.

10. A pharmaceutical composition comprising the vector set according to any one of claims 1 to 8.

11. A method for targeting a specific neuronal population in a neural circuit, comprising using the vector set described in any one of claims 1 to 5, and introducing an anterograde transsynaptic viral vector into an input region, a retrograde transsynaptic viral vector into an output region, and a non-transsynaptic viral vector into an intermediate target region, said method comprising the steps of:

12. The method of targeting a specific population of neurons in a neural circuit of claim 11, wherein the input region is the cuneiform nucleus (CnF), the output region is the central nucleus of the amygdala (CeA), and the intermediate target region is the lateral and basal nuclei of the amygdala (LA / B).

13. The method of claim 11 or 12, which is used on animals other than humans.

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