Methods and kits for achieving synthetic nervous system states

Abstract

The present disclosure describes methods and compositions for inducing predetermined states in subjects through selective activation of neural populations. Various implementations relate more specifically to the use of synthetic receptors to capture and activate neurons associated with a particular state (e.g., a state of consciousness, a physiological state, a behavioral state, or the like).

Description

METHODS AND KITS FOR ACHIEVING SYNTHETIC NERVOUS SYSTEM STATESSTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This invention was made with government support under Grant No. R35G 146751, awarded by the National Institute of General Medical Sciences. The government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATION(S)

[0002] This application claims the priority of U.S. Provisional Patent Application No. 63 / 746,456, filed on January 17, 2025, and which is incorporated by reference herein in its entirety.TECHNICAL FIELD

[0003] This application relates to methods and kits for inducing predetermined states in subjects through selective activation of neural populations. It relates more specifically to the use of synthetic receptors to capture and activate neurons associated with a particular state (e.g. , a state of consciousness, a physiological state, a behavioral state, or the like).BACKGROUND

[0004] Many spatially segregated neural circuits are shown to regulate consciousness or recapitulate distinct components of an unconscious state. The dissection of these individual cell- types and circuits has yielded valuable insights into the regulation of sleep, arousal, and anesthesia with limited translatability for precise conscious state transitions that could replace current pharmacologic approaches.BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Some of the drawings submitted herein may be better understood in color. Applicant considers the color versions of the drawings as part of the original submission and reserves the right to present color images of the drawings in later proceedings.

[0006] FIG. 1 illustrates an environment in which a predetermined state is induced in a subject.

[0007] FIG. 2 illustrates an example process for inducing, in a subject, a predetermined state.

[0008] FIGs. 3A-3F illustrate Designer Receptor Exclusively Activated by a Designer Drug (DREADD) immunolabeling after synthetic activity-dependent capture.

[0009] FIGs. 4A-4J illustrate that Fos activity mapping reveals subcortical isoflurane-activated hotspots.

[0010] FIGs. 5A-5H illustrate intact whole brain activity mapping of isoflurane unconsciousness at single cell resolution.

[0011] FIGs. 6A-6E illustrate single unit temporal dynamics of isoflurane unconsciousness.

[0012] FIGs. 7A-7C illustrate response variability during isoflurane.

[0013] FIGs. 8A-8C illustrate differential dynamics of synthetic activity-dependent capture of unconsciousness vs. isoflurane.

[0014] FIGs. 9A-9J illustrate the effects of chemogenetic inhibition after synthetic activity-dependent capture of altered consciousness.

[0015] FIGs. 10A-10L illustrate synthetic activity-dependent capture of altered consciousness.

[0016] FIGs. 11A and 11B illustrate electrocorticography recordings of the synthetic unconscious state.

[0017] FIGs. 12A-12D illustrate identification of isoflurane-activated clusters from UNRAVEL analysis.

[0018] FIGs. 13A-13E illustrate network analysis that reveals hubs that functionally differentiate an isoflurane unconscious state.

[0019] FIGs. 14A-14F illustrate representative Fos immunolabeling from cleared intact brains.

[0020] FIGs. 15A-15K illustrate that isoflurane networks are denser across a variety of weight thresholds.DETAILED DESCRIPTION

[0021] There exists a limited understanding of mechanisms of unconsciousness and no tools to safely induce and reverse the unconscious state without medical oversight. Many spatially segregated neural circuits are shown to regulate consciousness or recapitulate distinct components of an unconscious state (Mashour, G. A. Neuron 112, 1553-1567 (2024)). The dissection of these individual cell types and circuits has yielded valuable insights into the regulation of sleep, arousal, and anesthesia with limited translatability for precise conscious state transitions that could replace current pharmacologic approaches (Ma, C. et al. Neuron 103, 323-334.e7 (2019); Ma, C. et al. Nat Neurosci 27, 249-258 (2024); Zhang, Z. et al. Cell 177, 1293-13O7.e16 (2019); Lu, J. et al. J. Neurosci. 20, 3830-3842 (2000); Silverman, D. et al. Sci Adv 11, eadq0651 (2025); Taylor, N. E. et al. Proc Natl Acad Sci U S A 113, 12826-12831 (2016); Castro, D. C. et al. Nature 598, 646-651 (2021); Rodriguez-Romaguera, J. et al. Cell Rep 33, 108362 (2020); Ramadasan-Nair, R et al. Anesthesiology 130, 423-434 (2019); Mashour, G. A. et al. Trends Neurosci 45, 722-732 (2022); Mashour, G. A. et al. eLife 10, e59525 (2021); Brown, E. N., et al. N Engl J Med 363, 2638-2650 (2010); Zhang, Z. et al. Nat Neurosci 18, 553-561 (2015); Nelson, L. E. et al. Anesthesiology 98, 428- 436 (2003); Zhou, W. et al. Proc Natl Acad Sci U S A 115, E10740-E10747 (2018); Solovey, G. et al. J. Neurosci. 35, 10866-10877 (2015)). A single, isolated neural correlate of consciousness has not been identified.

[0022] An integrated approach to produce unconsciousness via global central nervous system activation, in the absence of anesthetic agents, may pave the way for safe transitions in consciousness that can be applied in resourcescarce settings. Toward this goal, various implementations of the present disclosure leverage whole brain activitydependent chemogenetic capture of isoflurane-activated circuitry to achieve a synthetic state of anti-nociception and altered consciousness induced in the absence of anesthetic agents.

[0023] Various implementations described herein relate, more generally, to inducing predetermined states in subjects. A predetermined state, for instance, is characterized by activation of at least a portion of neurons within a subject’s body. Examples of predetermined states not only include states of consciousness, such as a state of unconsciousness, but also other states of satiety, states of euphoria, states of peripheral numbness, states of alertness (e.g., a state of stimulation), states of fear, sleep states, nondepressive states, nonpsychotic states, states of relaxation,exercise-induced states, thermoregulated states, or pharmacologically-induced states. A predetermined state, for instance, may include activation of a pattern of neurons in the central nervous system and / or the peripheral nervous system.

[0024] Various implementations described herein relate to methods and kits for selectively capturing and activating neurons associated with a predetermined state. The term "capturing” and its equivalents, as used herein, may refer to specifically identifying, such as by binding to or expressing a construct within, a population of cells (e.g., neurons). Various implementations include administration of a genetic construct that encodes a synthetic receptor. A subject may be induced into a first instance of the predetermined state. In various implementations, an expression agent is administered to the subject in order to cause expression of the synthetic receptor in neurons activated in the predetermined state. In order to induce a second instance of the predetermined state, a ligand configured to bind to the synthetic receptor may be administered to the subject.

[0025] Various implementations of the present disclosure enable subjects to be induced into various states safely and efficiently. For instance, conventional methods for pain management (e.g., oral or intravenous administration of one or more analgesics) may be associated with various off-target effects, such as gastrointestinal, cardiovascular, and renal effects. These off-target effects can be caused by interactions with non-pain-related physiological pathways. Use of the methods and kits described herein can mitigate these off-target effects through the use of synthetic ligands and receptors that facilitate selective activation of neuronal populations in order to achieve a particular state. In some examples, various methods described herein can include patient-administered treatments, thereby improving accessibility and efficacy of medical care for patients.

[0026] Various implementation described herein can be utilized for research and development purposes In some cases, brainwide mapping of neuronal populations associated with particular states can be identified using the disclosed methods and kits. Novel compounds or methods can be evaluated in order to identify, for instance, undesired activation of certain neurons. Accordingly, various implementations may improve the safety and efficacy of new therapeutic agents.

[0027] Particular examples are now described, with reference to the accompanying figures. The scope of this disclosure includes individual examples described herein as well as any combination of the examples and elements therein, unless otherwise specified.

[0028] FIG. 1 illustrates an environment 100 in which a predetermined state is induced in a subject 102. The subject 102 may be a human, a mammal, a non-human primate, a rodent, a mouse, a rat, or another animal. According to various examples, the subject 102 may be a patient in a clinical setting. In some implementations, the subject 102 may be in a non-clinical environment, such as a home or other location.

[0029] In some implementations, it may be beneficial to induce a state in the subject 102. The term “state” and its equivalents, as used herein, may refer to a condition of a subject as characterized by physical properties (e.g., heart rate, body temperature, etc.), mental properties (e.g., mood, awareness, etc.), physiological properties (e.g., neuralactivity, hormone levels, etc.), or behavioral properties (e.g., activity level, reaction time, etc.). Examples of states may include states of consciousness, such as an alert and conscious state, an unconscious state, a minimally conscious state, a state of altered self-awareness (e.g., meditative state, daydreaming state, etc.), a chemically-induced state (e.g., a sedated state, an anesthetized state, an alcohol-induced state, a psychedelic-induced state, etc.), or the like. Examples of states may include states of analgesia, states of satiety, states of euphoria, states of peripheral numbness, states of alertness (e.g., states of stimulation), states of fear, sleep states, nondepressive states, nonpsychotic states, states of relaxation, exercise-induced states, thermoregulated states, or pharmacologically-induced states. A state of analgesia may be characterized by a level of pain reduction (e.g., complete absence of pain perception, a partial reduction in pain sensitivity, etc.), a localized analgesic state, a centrally mediated analgesic state, or a pharmacologically induced analgesic state (e.g., opioid-induced or non-opioid analgesia), or the like. States of satiety can include a fully satiated state, a partially satiated state, a state of appetite suppression, a hormonally mediated satiety state, or the like. A state of euphoria may be characterized by a level of positive affect (e.g., a mildly positive affective state, a strongly positive affective state, a heightened mood state, etc.). States of euphoria can include a reward-associated euphoria, a chemically induced euphoric state, an exercise-induced euphoric state, a transient cognitive-emotional elevation, or the like. States of peripheral numbness can include localized sensory loss, partial tactile attenuation, paresthetic states (e.g., feeling tingling, burning, numbness, etc. in one or more areas of the body), pharmacologically induced peripheral sensory suppression, or the like. States of alertness can include a baseline wakeful state, a heightened alert state, a stimulated state, a stress-induced alert state, a pharmacologically enhanced alert state, a task-focused attentional state, or the like. States of fear can include an acute fear state (e.g., a fight-or-flight state), a sustained fear state (e.g., an anxiety-associated fear state), a physical fear response (e.g., a reflexive fear state), a context-dependent fear state, a cognitively mediated fear state, or the like. Sleep states can include light sleep states, deep sleep states, rapid eye movement (REM) sleep states, non-REM sleep states, transitional sleepwake states, or the like. Nondepressive states can include emotionally neutral states, stable mood states, adaptive affective states (e.g., a resilient mood state), or the like. Nonpsychotic states can include coherent perceptual states, organized-thought states, states of cognitive functioning without psychotic features, or the like. States of relaxation can include physical relaxation (e.g , muscular relaxation), mental calmness, meditative relaxation states, stress-recovery states, passive rest states, or the like. Exercise-induced states can include acute exertional states, post-exercise recovery states, elevated cardiovascular activation states, endorphin-associated states, fatigue-adaptive states, exercise-induced physiological states, or the like. Thermoregulated states can include normothermic states (e.g., a homeostatic thermoregulated state), hypothermic states (e.g., mild hypothermia, severe hypothermia, etc.), hyperthermic states (e.g., mild hyperthermia, severe hyperthermia, etc.), actively regulated thermal balance states, environmentally adapted thermal states, or the like Pharmacologically induced states can include drug-mediated physiological or cognitive states, such as stimulant-induced states, sedative-induced states, analgesic-induced states, anxiolytic-induced states, psychoactive substance-induced states, or the like.

[0030] In some examples, a state is associated with a behavior, such as a state of increased endorphins, adrenaline, dopamine, and other hormones in response to exercising or a state of increased motor neuron pathway and synaptic plasticity-related activity in response to physical therapy. A state, for instance, is associated with activation of a pattern of neurons in the central nervous system and / or the peripheral nervous system. When practicing methods disclosed herein, thresholds of physiological and / or behavioral measures can be set to define the absence or the presence of a state. The thresholds may be based on a population’s average physiological and / or behavioral measures when deemed to be within a state or may be individualized to a particular subject's predefined physiological and / or behavioral measures.

[0031] In various examples, inducing the state in the subject 102 may involve the risk of the various side effects. For instance, the subject 102 may benefit from analgesia, but it may be beneficial to avoid side effects associated with opioid medications, such as constipation, gastrointestinal irritation, respiratory depression, or cardiovascular effects. In some examples, the subject 102 may benefit from an antidepressant medication, but it may be beneficial to avoid peripheral toxicity, such as liver or kidney damage, that may be associated with the medication.

[0032] In various implementations, these issues can be addressed by specifically causing activation of neurons associated with the state in the subject 102 in order to induce the state of the subject 102. Various implementations of the present disclosure may enable individuals to enter the state in a safer, more effective, and more efficient manner. In some examples, the disclosed methods and kits include a synthetic ligand that specifically binds to a synthetic receptor in order to activate a particular pattern of neurons. Through the use of non-endogenous agents, various implementations can mitigate at least some of the off-target effects associated with conventional methods of administering a medication in order to induce the state in the subject 102. According to some examples, individuals can be induced into a state in situations where they conventionally were not able to be. For instance, an individual undergoing physical rehabilitation in order to restore motor function and improve neural pathways associated with movement can, using the implementations described herein, activate those neural pathways even when they are unable to perform rehabilitation exercises (e.g., when they are at home and / or recovering from exercising).

[0033] In various implementations, a therapeutically effective dose of a construct 104 encoding a synthetic receptor 106 is administered to the subject 102. The construct 104 may be delivered into the subject 102 using one or more delivery approaches configured to facilitate cellular update and / or biological activity of the construct 104. For instance, the construct 104 may be provided within (e.g., encapsulated or packaged within) a viral delivery system, such as a vector 108. The term “vector” may refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule, such as an expression construct. The transferred nucleic acid is generally linked to (e.g., inserted into) the vector nucleic acid molecule. The vector 108 may include sequences that direct autonomous replication in a cell or may include sequences that permit integration into host cell DNA. The vector 108 may include, for example, a plasmid (e.g., a DNA plasmid or RNA plasmid), a transposon, a cosmid, a bacterial artificial chromosome, or a viral vector.

[0034] The term “viral vector” is widely used to refer to a nucleic acid molecule that includes virus-derived components that facilitate transfer and expression of non-native nucleic acid molecules within a cell. The term "adeno-associated viral vector” may refer to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, that are primarily derived from AAV. The term "retroviral vector" may refer to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus. The term "lentiviral vector" may refer to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, that are primarily derived from a lentivirus, and so on. The term "hybrid vector" refers to a vector including structural and / or functional genetic elements from more than one virus type.

[0035] In some implementations, vectors (e.g, AAV) with capsids that cross the blood-brain barrier (BBB) are selected. In various cases, the vector 108 is modified to include a capsid that crosses the BBB. Examples of AAV with viral capsids that cross the blood brain barrier include AAV9 (Gombash et al., Front Mol Neurosci. 2014; 7:81), AAVrh.10 (Yang, et al., Mol Ther. 2014; 22(7): 1299-1309), AAV1R6, AAV1R7 (Albright et al. Mol Ther. 2018; 26(2): 510), rAAVrh.8 (Yang, et al, supra), AAV-BR1 (Marchio et al, EMBO Mol Med. 2016; 8(6): 592), AAV-PHP.S (Chan et al, Nat Neurosci. 2017; 20(8): 1172), AAV-PHP B (Deverman et al, Nat Biotechnol. 2016; 34(2): 204), AAV-PPS (Chen et al, Nat Med. 2009; 15: 1215), and PHP.eB.

[0036] In some examples, the construct 104 is delivered using non-viral carrier, such as a lipid-based particular, a liposome, a nanoparticle (e.g, a polymeric nanoparticle, etc.), or the like. In various cases, the construct 104 is administered using a chemical transfection agent (e.g, cationic lipids or polymers, etc.), a physical delivery method (e.g, microinjection, ultrasound-mediates delivery, etc.), or a cell-mediated delivery system (e.g, modified cells).

[0037] In various implementations, in response to administration of the construct 104, the subject 102 is administered a therapeutically effective amount of an expression agent 110 while the subject 102 is in a first instance of a predetermined state. The expression agent 110 is configured to induce activity-dependent expression of the synthetic receptor 106. For instance, the expression agent 110 may cause the expression of the synthetic receptor 106 in at least part of a population of activated neurons 112 while the subject 102 is in the first instance of the predetermined state. The activated neurons 112, in some cases, have cellular activity while the subject 102 is in the predetermined state. In some examples, the cellular activity of the activated neurons 112 is greater than a threshold (e.g, a resting level). Resting neurons 114 in the subject 102, in various cases, do notexpress the synthetic receptor 106 in response to the administration of the expression agent 110. For instance, cellular activity of the resting neurons 114 is less than a threshold. Cellular activity, in various cases, may be associated with metabolic activity, biosynthetic activity, electrical activity, enzymatic activity, or another parameter associated with one or more cellular processes.

[0038] In various cases, the activated neurons 112 and / or the resting neurons 114 include neurons in the central nervous system and / or the peripheral nervous system of the subject 102. For instance, the activated neurons 112 and / or the resting neurons 114 may include neurons in the brain and / or neurons in the spinal cord of the subject 102. In some examples, the activated neurons 112 include neurons in two or more spatial regions (e.g, two or more brainregions, a brain region and a spinal region, etc.). In some examples, the activated neurons 112 include neurons in one spatial region. In various cases, the activated neurons 112 include neurons of two or more cell types. In various cases, the activated neurons 112 include neurons of one cell types. Neuronal cell types include, for instance, sensory neurons, motor neurons, interneurons, excitatory neurons, inhibitory neurons, modulatory neurons, or the like, or subtypes thereof. In various cases, the activated neurons 112 include neurons of two or more neural circuits. In various cases, the activated neurons 112 include neurons of one neural circuit.

[0039] In various cases, the expression agent 110 is associated with one or more immediate early genes, such as c-Fos. The expression agent 110, for instance, may include 4-hydroxytamoxifen (4-OHT). In some examples, the expression agent 110 may be administered within a certain time period of the subject 102 being in the predetermined state. For instance, expression of an immediate early gene may remain upregulated for a certain time period after the subject 102 leaves the predetermined state.

[0040] The synthetic receptor 106 is, in various cases, any receptor suitable for expression in the subject 102. In some instances, the receptor 106 includes an engineered receptor, such as a chemogenetic receptor. The receptor 106 may be activated by a specific synthetic ligand 116, rather than, for instance, by endogenous molecules. Examples of chemogenetic receptors include Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), such as hM3Dq (an excitatory DREADD), hM4Di (an inhibitory DREADD), or other engineered G protein-coupled receptors. In some cases, the receptor 106 includes a Pharmacologically Selective Actuator Module (PSAM) that is configured to be selectively activated by a Pharmacologically Selective Effector Molecule (PSEM). An example of a PSAM / PSEM system is PSAM-5HT3 and PSEM89S.

[0041] In response to the expression of the synthetic receptor 106 in at least some of the activated neurons 112 of the subject 102, the subject 102 can be induced into a second instance of the predetermined state by administration of the synthetic receptor ligand 116 to the subject 102. The ligand 116, in various cases, is configured to selectively bind to the synthetic receptor 106. In some instances, the ligand 116 does not correspond to an endogenous compound of the subject 102. The ligand 116, for instance, may include a synthetic compound. For instance, the ligand 116 may include clozapine-n-oxide. In some instances, the ligand 116 includes deschloroclozapine, Compound 21, JHU37152, JHU37160, or the like.

[0042] Therapeutically effective amounts include those that provide at least a part of a therapeutic treatment (e.g., achievement of or removal from a particular state). A “therapeutic treatment” includes a treatment administered to a subject (e.g., the subject 102) who, for instance, may benefit from being induced into or removed from a state. An “effective amount” is the amount of a formulation used to result in a desired physiological change in the subject (e.g., the subject 102). For example, an effective amount can provide a change in a metric associated with a state (e.g., a state of analgesia, a state of consciousness, or the like). A metric associated with the state may be associated with, for instance, a physiological effect, a behavioral effect, or a change in a marker (e.g., expression of a synthetic receptor, expression of a biomarker associated with the state, etc.) associated with the state. Effective amounts are oftenadministered for research purposes. Effective amounts disclosed herein can cause a statistically significant effect in an animal model or in vitro assay relevant to the assessment of a state. Therapeutically effective amounts and effective amounts are not mutually exclusive.

[0043] For administration, therapeutically effective amounts (also referred to herein as doses) can be initially estimated based on results from in vitro assays and / or animal model studies. Such information can be used to more accurately determine useful doses in subjects of interest. The actual dose amount administered to a particular subject (e.g., the subject 102) can be determined by a physician, veterinarian, or researcher taking into account parameters such as physical and physiological factors including target, body weight, the intended state, pathological conditions of the subject 102, previous or concurrent therapeutic interventions, and route of administration.

[0044] Useful doses, in some examples, are reported as a mass of the active ingredient (e.g., the expression agent 110, the synthetic ligand 116, etc.) per weight of the subject. Useful doses can range from 0.1 g / kg to 2 mg / kg. In other examples, a dose can include 0.1 pg / kg, 0.2 pg / kg, 0.5 pg / kg, 1 pg / kg, 10 pg / kg, 20 pg / kg, 50 pg / kg, 100 pg / kg, 250 pg / kg, 500 pg / kg, 750 pg / kg, 1000 pg / kg, 0.1 to 5 mg / kg or from 0.5 to 1 mg / kg. In other examples, a dose can include 1 mg / kg, 1.25 mg / kg, 1.5 mg / kg, 1 75 mg / kg, 2 mg / kg, 5 mg / kg, or more.

[0045] In some examples, useful doses of nucleic acids (e.g., the construct 104) can be reported as infectious units (e.g., viral particles, viral genomes, plaque-forming units, transducing units, mass of the nucleic acid, or the like) per weight of the subject. Useful doses can range from 102to 1025infectious units (IU) / kg. In other examples, a dose can include 102lU / kg, 103lU / kg, 104lU / kg, 105lU / kg, 1061 U / kg, 107lU / kg, 108lU / kg, 109lU / kg, 1010lU / kg, 1011lU / kg, 1012lU / kg, 1013lU / kg, 10141 U / kg, 10151 U / kg, 1016lU / kg, 10171 U / kg, 10181 U / kg, 10191 U / kg, 1020lU / kg, 1021lU / kg, 1022lU / kg, 10231 U / kg, 1024lU / kg, 10251 U / kg, or greater than 10251 U / kg.

[0046] Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., five times a day, four times a day, three times a day, twice a day, daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, or yearly). In particular implementations, the treatment protocol may be dictated by a clinical trial protocol or a US FDA-approved treatment protocol.

[0047] The compositions described herein (e.g., the vector 108, the expression agent 110, the synthetic ligand 116, etc.) can be administered by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion. Routes of administration can include auricular, cutaneous, epidural, interstitial, intraabdominal, intracerebral, intravenous, intradural, nasal, parenteral, percutaneous, perineural, oral, subcutaneous, transdermal, intradermal, intraarterial, intranodal, intravesicular, intrathecal, intraperitoneal, intraparenteral, intralesional, intramuscular, or sublingual administration.

[0048] In various implementations of the present disclosure, a composition (e.g., the vector 108, the expression agent 110, the ligand 116, etc.) can be administered by, e.g., injection, infusion, perfusion, or lavage. Routes of administrationcan include bolus intravenous, intradermal, intraarterial, intraparenteral, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesical, and / or subcutaneous administration.

[0049] In some implementations, a composition is administered with a pharmaceutically acceptable carrier. Exemplary pharmaceutically acceptable carriers and formulations are disclosed in Remington’s Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Moreover, formulations can be prepared to meet sterility, pyrogenicity, general safety and purity standards as required by United States FDA Office of Biological Standards and / or other relevant foreign regulatory agencies.

[0050] Exemplary generally used pharmaceutically acceptable carriers include at least one of a bulking agent or filler, a solvents or co-solvent, a dispersion media, a coating, a surfactant, an antioxidant (e.g., ascorbic acid, methionine, vitamin E, or the like), a preservative, an isotonic agent, an absorption delaying agent, a salt, a stabilizer, a buffering agent, a chelating agent (e.g., EDTA), a gel, a binder, a disintegration agent, a lubricants, or another carrier known in the art.

[0051] Exemplary buffering agents include citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and trimethylamine salts.

[0052] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0053] Exemplary isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0054] Exemplary stabilizers include organic sugars, polyhydric sugar alcohols, polyethylene glycol, sulfur-containing reducing agents, amino acids, low molecular weight polypeptides, proteins, immunoglobulins, hydrophilic polymers or polysaccharides.

[0055] Particular implementations of the present disclosure include kits. For instance, a kit may include one or more of the construct 104, the vector 108, the expression agent 110, or the ligand 116. One or more components of the kit may be formulated as an oral formulation, a subcutaneous formulation, a transdermal formulation, or a formulation for another route of administration described herein. In some examples, the kit includes a second construct that encodes a second receptor and / or a second synthetic ligand configured to specifically bind to the second receptor. In various cases, the kit includes an agent configured to reverse (e.g., remove) the subject from the predetermined state. In various cases, the kit includes an inhibitor agent configured to reduce activity and / or expression of the receptor 106. The kit may include materials in order to generate and / or modify administrable formulations. The kit may include materials in order to administer a composition to the subject (e.g., syringes, an antiseptic agent, etc.). The kit, in some cases, includes instructions in order to guide an individual (e.g., a healthcare provider, the subject 102, etc.) throughadministration of a formulation. In various examples, the kit includes one or more sensors in order to detect the activated neurons 112.

[0056] FIG. 2 illustrates an example process 200 for inducing, in a subject (e.g., the subject 102), a predetermined state, such as a state of analgesia. The process 200 may be performed by is performed by an entity, which may include one or more of an individual (e.g., a healthcare provider, the subject, etc.), a sensor, a specialized piece of equipment, or the like. According to some implementations, any of the steps of process 200 may be omitted.

[0057] At 202, the entity administers, to the subject, a therapeutically effective dose of a construct (e.g., the construct 104) encoding a synthetic receptor (e.g., the receptor 106). The construct, in various examples, is packaged in a delivery agent, such as a viral vector (e.g., the vector 108; an AAV vector, an adenoviral vector, a lentiviral vector, etc.), a nanoparticle, a microsphere, a liposome, a polymer complex, a hydrogel, or another suitable agent. In particular examples, the construct may be administered by retroorbitally injecting the therapeutically effective dose of a viral vector that includes the construct.

[0058] At 204, the entity administers, to the subject, a therapeutically effective dose of an expression agent (e.g. , the expression agent 110) that induces activity-dependent expression of the synthetic receptor in at least a portion of activated neurons in the subject (e.g., the activated neuron 112). In various implementations, the entity administers the expression agent while the subject is in a first instance of the predetermined state or within a particular time period of the subject being in the first instance of the predetermined state. For instance, a population of neurons that are activated when the subject is unconscious may remain activated for a duration of time after the subject has regained consciousness. The entity may administer the expression agent during this duration of time in order to capture the activated neurons of the subject.

[0059] In various cases, the subject is administered at least one inducing agent in order to induce the subject into the first instance of the predetermined state. For instance, the at least one inducing agent may include an anesthetic agent, neuroactive agent, a psychoactive agent, or a food item. In some instances, the inducing agent includes isoflurane and / or propofol. In some instances, the first instance of the predetermined state may be induced by causing the subject to perform one or more behaviors (e.g., exercising, meditating, etc.).

[0060] At 206, the entity induces the subject into a second instance of the predetermined state by administering a therapeutically effective dose of a ligand (e.g., the ligand 116) that specifically binds to the receptor. The ligand, in various cases, is not endogenous to the subject (e.g., does not naturally occur in the subject). In some examples, the subject may self-administer the ligand. In some examples, the subject is induced into the second instance of the predetermined state by acoustically stimulating at least a portion of the activated neurons.

[0061] According to various examples, the entity may reverse the subject from the predetermined state by administering, to the subject, a second synthetic receptor. In some instances, the second synthetic receptor is encoded by the construct or by a second construct. Accordingly, in some implementations, the expression agent may induce activity-dependent expression of the second synthetic receptor in at least a portion of activated neurons in the subject.In various cases, a second expression agent induces activity-dependent expression of the second synthetic receptor in at least a portion of activated neurons in the subject. The entity may administer, to the subject, a second ligand that specifically binds the second synthetic receptor. In some instances, the entity removes the subject from the predetermined state by administering, to the subject, a second construct that is configured to inactivate the synthetic receptor. For example, the second construct may encode a CRISPR / Cas9 system or components of another system configured to modulate expression of the synthetic receptor. The second construct may be packaged within the same delivery agent as the construct encoding the synthetic receptor. In various cases, the second construct is packaged within a second delivery agent (e.g., a second viral vector).

[0062] In some cases, the entity administers an inhibitor agent configured to reduce or eliminate expression of the receptor in the neurons of the subject. For instance, the inhibitor agent may be configured to bind to the synthetic receptor in order to prevent binding of the synthetic ligand. In some cases, the inhibitor agent is configured to cause neurons of the subject to internalize the receptor. In some cases, the inhibitor agent is configured to cause downregulation of the production of the synthetic receptor.

[0063] According to various implementations, the entity may identify at least one location of the portion of activated neurons by detecting, from a sensor, activation of the portion of the activated neurons. The sensor, for instance, may include a wireless subcutaneous mechanoacoustic device and / or electrocorticography electrodes. In some instances, the entity may identify a spatial distribution of the receptor (e.g., expression of the receptor) using a localization system, such as a nuclear localization system. See, for instance, Szelenyi, et al. Proc. Natl. Acad. Sci. U.S.A. 121, 62320250121 (2024).

[0064] In a particular example, the subject may be seeking rehabilitation due to an injury or physiological condition. The neural circuits associated with, for instance, certain movements may be impaired if the subject is unable to move certain parts of their body properly. The construct encoding the synthetic receptor may be administered to the subject in order to capture the neurons associated with motor function of the affected areas. The subject can be induced into a first instance of a therapeutic state when they undergo physical therapy in order to restore motor function of the affected areas. During the first instance of the therapeutic state, the subject may be administered the expression agent. In various implementations, the subject can then be administered the synthetic ligand in order to further strengthen the affected neural circuits (e.g., even when the subject is not performing rehabilitation exercises, such as when they are recovering from exercising). Whole brain capture of neural activity associated with rehabilitation may also capture neural populations associated with neuroendocrine function, inducing release of rehabilitation factors without the need for physical rehabilitation movement.EXPERIMENTAL EXAMPLE

[0065] This Experimental Example describes a cell-targeted, global network approach to manipulating consciousness. Many spatially segregated neural circuits are shown to regulate consciousness or recapitulate distinct components of an unconscious state (Mashour, G. A. Neuron 112, 1553-1567 (2024)). The dissection of theseindividual cell- types and circuits has yielded valuable insights into the regulation of sleep, arousal, and anesthesia with limited translatability for precise conscious state transitions that could replace current pharmacologic approaches (Ma, C. et al. Neuron 103, 323-334.e7 (2019); Ma, C. et al. Nat Neurosci 27, 249-258 (2024); Zhang, Z. et al. Cell 177, 1293-1307. e16 (2019); Lu, J. et al. J. Neurosci. 20, 3830-3842 (2000); Silverman, D. et al. Sci Adv 11, eadq0651 (2025); Taylor, N. E. et al. Proc Natl Acad Sci U S A 113, 12826-12831 (2016); Castro, D. C. et al. Nature 598, 646-651 (2021); Rodriguez-Romaguera, J. et al. Cell Rep 33, 108362 (2020); Ramadasan-Nair, R. et al. Anesthesiology 130, 423-434 (2019); Mashour, G. A. et al. Trends Neurosci 45, 722-732 (2022); Mashour, G. A. et al. eLife 10, e59525 (2021); Brown, E. N , et al. N Engl J Med 363, 2638-2650 (2010); Zhang, Z. et al. Nat Neurosci 18, 553-561 (2015); Nelson, L. E. et al. Anesthesiology 98, 428- 436 (2003); Zhou, W. et al. Proc Natl Acad Sci U S A 115, E10740-E10747 (2018); Solovey, G. et al. J. Neurosci. 35, 10866-10877 (2015)). A single, isolated neural correlate of consciousness has not been identified (Koch, C. Nature 557, S8-S12 (2018)). This Example demonstrates that consciousness is subserved by distinct, globally distributed cells in targetable circuits (Mashour, G. A. et al. Neuron 105, 776-798 (2020); Dehaene, S. et al. Proc Natl Acad Sci U S A95, 14529-14534 (1998)). If this is true, a distributed neural correlate of consciousness exists that can be synthetically targeted to generate precise state transitions.

[0066] This Example discloses a preclinical platform to test this theory using the genetic capture of brainwide activity during the anesthesia-induced unconscious state in mice. It is then evaluated if synthetic re- activation of those defined global neural substrates could recapitulate or modulate consciousness. The intact brainwide pattern of activity in the synthetic state as compared to isoflurane-induced unconsciousness is evaluated using cellular resolution Fos immediate early gene immunolabeling and light sheet microscopy. Functional network analysis of the brainwide patterns of co-activation or co-suppression indicates 9 key communities form the unconsciousness network. The Fos-based analysis of activity is extended to temporal single unit neural signatures using brainwide Neuropixels probe recordings. Together, this Example provides a synthetic platform for manipulating and analyzing cell-specific activity within the global network along with technical resources that provide key neural substrates for future studies.

[0067] Methods.

[0068] Mice. All experiments were approved by the National Institutes of Health and the University of Washington Institutional Animal Care and Use Committee. 3- to 6-month-old male and female c57bl / 6j or homozygous Fos2A iCreER(FosTRAP2) transgenic mice are used in all studies. Animals are group-housed in a 12-hour reverse light / dark cycle facility with ad libitum access to food and water. Animals used in electrocorticography or Neuropixels studies are singly housed during the recording experiments.

[0069] Intact brain tissue collection after isoflurane exposure. Mice were singly housed in the home cage for 3 days prior to tissue collection for intact whole brain Fos immunolabeling. Habituation to the testing room was achieved by placing mice in a temperature controlled red-light room for at least 2-3 hours a day for 3 days prior to tissue collection. On the test day, mice in the control condition remained singly housed in their home cage inside the testing room where isoflurane was delivered. Mice in the isoflurane condition were individually induced with 2% isoflurane and once lossof toe pinch reflex was confirmed, each mouse was maintained under general anesthesia for 180 minutes with 1.2%-1.3% isoflurane in oxygen delivered via nose cone The concentration of isoflurane delivered at each nose cone was confirmed to be within range using a gas analyzer prior to placing the mouse in the nose cone. Mice were actively warmed on a heating pad and continuously monitored by the experimenter. After 180 minutes, all mice were more deeply anesthetized with isoflurane and transcardially perfused with PBS followed by 10% formalin. Brains were dissected and post-fixed in formalin for 24 hours at 4°C prior to intact whole brain immunolabeling and clearing.

[0070] Intact brain clearing and immunolabeling. A previously published, modified version of the IDisco+ protocol was used to immunolabel and clear intact brain samples (Renier, N. et al. Cell 165, 1789-1802 (2016); Madangopal, R. et al. Proc Natl Acad Sci U S A 119, e2209382119 (2022)). The following antibodies were used: Synaptic Systems Rat anti-cFos (1:2000) for Fos immunolabeling or Rockland Rabbit anti-RFP (1:2000) (Pottstown, PA) for mCherry DREADD immunolabeling followed by Donkey anti-Rat or Donkey anti-Rabbit Jackson ImmunoResearch AlexaFluor 647 Fab2-conjugated secondary antibody (1:500) (West Grove, PA).

[0071] Intact brain imaging. Cleared, intact whole brains were imaged with a uniform axial resolution light sheet microscope (SmartSPIM, LifeCanvas Technologies) using either the 1.6x (5 micron (pm) voxels) or3.6x (1.8 pm voxel) objectives for Fos samples, and 3.6x for TRAP samples. Samples were mounted horizontally using a custom sample holder immersed in dibenzyl ether (DBE). Images were acquired in two channels (488 nanometer (nm): autofluorescence; 639 nm: mCherry / AlexaFluor-647) and stitched using the SmartSPIM acquisition software. TRAP+ images were down-sampled (0.5x) prior to image processing with isotropic 3.6 pm voxels. 3-dimensional images were visualized using Arivis Pro software (Zeiss).

[0072] FIGs. 3A-3F illustrate DREADD immunolabeling after synthetic activity-dependent capture. FIG. 3A illustrates the experimental timeline: FosTRAP2 transgenic mice received retroorbital injection of an AAV- PHP.eB Cre-dependent Gq-DREADD followed by activity-dependent capture (TRAP) during isoflurane exposure with 4-hydroxytamoxifen (4-OHT). After experiments, intact brains were immunolabeled for mCherry-DREADD and cleared by iDisco+. ClearMap was used to register brains, detect Fos+ cells, and generate cell counts and density heat maps. FIG. 3B illustrates mean TRAP+ cell density calculated across larger brain subdivisions after atlas registration by ClearMap shows similar TRAP+ cell density across the brain, except for significant differences in the hindbrain and cerebellum (1-way ANOVA, *p<0.05). FIG. 3C illustrates 3-dimensional mean density heatmap (n=7 mice). FIG. 3D illustrates mean TRAP+ density across all samples shown as voxel-based 2D-projection heatmaps (calibration bar is arbitrary units). FIG. 3E illustrates representative whole brain light sheet microscopy images after IDisco clearing and mCherry-DREADD immunolabeling digitally sliced to the 2D coronal plane from one control mouse expressing retroorbital AAV-PHP.eB Cre-dependent Gq-DREADD with oil vehicle administered during isoflurane exposure (non-TRAP control, see Methods) and (FIG 3F) one Gq-DREADD-expressing mouse that received 4-OHT during isoflurane exposure for activity-dependent capture of synthetic unconscious state. Abbreviations: SSp= primary somatosensory cortex, HDB = horizontal limb of diagonal band, BMA = basomedial amygdala, VMH = ventromedial hypothalamus,MiTG = microcellular tegmental nucleus, PrCnF = precuneiform area, PNO = pontine reticular, MnR = median raphe nucleus. Scale bar for full coronal slices is 1mm, inset scale bar is 500 pm.

[0073] ClearMap brainwide TRAP+ analysis. Seven intact formalin-fixed brain samples from FosTRAP2 transgenic mice infected with AAV-PHP.eB-DIO-Gq-DREADD-mCherry virus (see Retroorbital virus injection and Activitydependent labeling procedures) were processed using a modified version of the ClearMap pipeline as previously published (Madangopal, supra] Szelenyi, E. R. et al. Proc Natl Acad Sci U S A 121, e2320250121 (2024)). A pixel classifier was trained in llastik to segment and quantify single cells based on somatic signal classification. This was achieved by selecting 21 image tiles (200 x 200 x 220 pm) from three separate samples, cropped from dorsal (2), central (3), and ventral (2) positions of the brain. To improve segmentation accuracy, an additional three tiles were used to correct for artifacts in ventricular and boundary regions. The fully trained classifier was then applied to all samples, generating segmented cell counts per brain region and voxelized heatmaps. TRAP+ cell counts were computed within two different levels of hierarchy within the Unified Brain Atlas: 1) 11 major anatomical divisions (FIG.3B) and 914 minor subregions within those high-level anatomical divisions that no longer split into further daughter regions.

[0074] FIGs. 4A-4J illustrate Fos activity mapping that reveals subcortical isoflurane-activated hotspots. FIG. 4A illustrates intact whole brains were imaged horizontally with light sheet microscopy to acquire cellular resolution Fos+ signal then digitally resliced in the coronal plane at the indicated AP positions. FIG. 4B illustrates representative mean intensity projection of the raw 3.6X Fos+ signal from the isoflurane condition virtually re-sliced to the coronal plane at the indicated AP positions. FIG. 4C illustrates mean Fos+ density in the control condition across all samples (n=7) shown as a heatmap. FIG. 4D illustrates mean Fos+ density in the isoflurane condition across all samples (n=9) shown as a heatmap in the indicated AP coronal plane. FIG. 4E illustrates pairwise comparison of significant Fos+ regions in control vs isoflurane condition with blue indicating higher Fos+ activity in the isoflurane condition and red indicating higher Fos+ activity in control condition. FIG. 4F illustrates 3D mean Fos+ density heatmaps from the isoflurane condition shown as sections in FIG. 4D. FIG. 4G illustrates voxel-wise significance map of regions significantly regulated in the isoflurane (blue) or control (red) conditions. FIG. 4H illustrates representative raw Fos+ immunolabeling at the indicated regions: CeL = centrolateral amygdala; BNST = bed nucleus of stria terminalis; PVH = paraventricular hypothalamus; VMH = ventromedial hypothalamus; PBN = parabrachial nucleus; LC = locus coeruleus. FIG. 4I illustrates mean Fos density calculated across larger brain subdivisions after atlas registration by ClearMap in control vs isoflurane conditions shows significant differences in isocortex, hypothalamus, midbrain and cerebellum, (unpaired t-test, *p<0.05). FIG. 4J illustrates regions significantly activated in isoflurane (blue) or control (red). Scale bars shown are 1mm except in FIG. 4H insets where scale bar is 250 pm.

[0075] FIGs. 5A-5H illustrate intact whole brain activity mapping of isoflurane unconsciousness at single cell resolution. FIG. 5A illustrates the experimental overview. Three complementary analytical pipelines were used to analyze whole brain Fos activity map data from the control vs. isoflurane test conditions: ClearMap, b-d; UNRAVEL, e-h; and SMARTTR network analysis (see FIGs. 13A-13E), ClearMap registered brains, detected Fos+ cells, generated cell density heat maps, and used them for voxel- wise analyses. UNRAVEL registered brains and preprocessed immunofluorescence images before warping them to atlas space, z-scoring them, averaging hemispheres together, and running intensity-based voxel-wise analyses. Clusters of significant voxels defined by FDR correction were warped to full-resolution tissue space for validation via cell density measurements. FIG. 5B illustrates mean Fos+ density shown as a heatmap across the intact brain in all isoflurane samples (n=9). FIG. 5C illustrates mean Fos+ density heatmap across all control (n=7, top row) and isoflurane samples (n=9, bottom row) virtually sliced in the coronal plane at major anatomical subdivisions (‘level 2' - see Methods). FIG. 5D illustrates ClearMap atlas registration followed by pairwise analysis of z-scored Fos density by every anatomical subregion (stop-level') in the control vs isoflurane condition after FDR correction (q < 0.01). Direction of effect in isoflurane relative to control is shown in the bottom bar with unmarked bars and yellow bars indicating regions of enhanced Fos in isoflurane. (AON: anterior olfactory nucleus, CPu: caudate-putamen, CeA: central amygdala; BNST: bed nucleus of stria terminals; PVT: paraventricular thalamus; PVH: paraventricular hypothalamus; VMH: ventromedial hypothalamus; SC: superior colliculus; PBN: parabrachial nucleus; LC: locus coeruleus). Where isoflurane either (FIG. 5E) suppressed Fos (Control > Isoflurane) or (FIG. 5F) enhanced Fos (Control < Isoflurane) was mapped via UNRAVEL (q < 0.05). e-f) Clusters were mirrored in 3D brains. Valid clusters for each condition are shown in 3D brains and bar graphs. All data is shown as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001 (unpaired two-tailed t-tests). FIGs. 5G-5H illustrate sunburst plots summarize volumes of regions comprising valid clusters (outer rings represent finer anatomical granularity in the CCFv3 hierarchy). Abbreviations: {‘a’: ‘anterior’, ‘c’: ‘central nucleus’, ‘d’: ‘dorsal’, T: ‘lateral’, ‘m’: ‘motor related or medial’, ‘mo’: ‘molecular layer’, ‘p’: ‘primary or posterior’, ‘pl’: ‘posterolateral’, ‘po’: ‘preoptic’, T’: 'rostral', ‘s’: ‘secondary or supplemental', ‘sg’: ‘superficial gray layer or granule cell layer', ‘v’: 'ventral'}. FIGs. 5E-5H illustrate voxel-wise analysis of Fos+density and validation of significant clusters using UNRAVEL mapped onto major anatomic regions. See FIGs. 4A-4J, 12A-12D, and 14A-14F for additional analysis and representative images of Fos immunolabeling.

[0076] ClearMap brainwide Fos analysis. Sixteen intact brain samples (n=7 controls and n = 9 isoflurane) were processed using a modified version of the ClearMap pipeline as previously published (Madangopal, supra). Fos-positive activated cell counts were computed within two different levels of hierarchy within the Unified Brain Atlas: 1) 11 major anatomical divisions (FIGs. 4A-4J) and 914 minor subregions within those high-level anatomical divisions that no longer split into further daughter regions (FIG. 5D). To ensure accuracy within the hierarchical relationships, it was ensured that there were no overlapping spatial footprints and no double counting of any regions, following each subregion branch until the end or “stop-level.” The raw Fos cell counts were transformed to z-scores, and the data was normalized to account for regional volume differences. The home cage control group (control) was used to normalize the z-scores for each region of interest using the formula:z — ( — ) / o,where x represents the Fos count of the treatment group, p represents the mean Fos count of the control group, and cr represents the SD of the control group. The z-score was then converted into - Iog10 for visualization. For the high-level analysis, an unpaired t-test was used, *p<0.05 (FIG. 4I). For the stop-level analysis, a t-test was used, followed by the false discovery rate (FDR) multiple comparison correction with an alpha significance level of 0.01 (q < 0.01), two-tailed (FIG. 5D).

[0077] UNRAVEL Fos analysis. Whole brain Fos immunolabeling was analyzed using UNRAVEL (UN-biased high-Resolution Analysis and Validation of Ensembles using Light sheet images), with refactored Python code (documentation: b-heifets.github.io / UNRAVEL / index.html) (Rijsketic, D. R. et al. Neuropsychopharmacol. 48, 1798- 1807 (2023)). Autofluorescence images were resampled to 50 pm resolution, and tissue was masked using llastik (v1.4.0; pixel classification; all features) for N4 bias field correction (ANTsPy; 0.4.2). The resampled images were padded (15% voxels on all sides) and smoothed with a Gaussian filter (sigma = 0.4) before being registered with an average template from iDisco and light-sheet microscopy (LSFM), which was aligned with the Allen Mouse Brain Common Coordinate Framework (CCFv3) (Perens, J. et al. Neuroinformatics 19, 433-446 (2021)). Registration was performed using ANTsPy: ants.affinejnitializer(fixedjmage=autofluorescence_image, moving Jmage=template, search_factor=1 , radian_fraction=1, local_search_iterations=500), followed by ants.registration(fixed=autofluorescence_image, moving=template_aligned_with_tissue, grad_step=0.1 , syn_metric-CC’, syn_sampling=2, reg_iterations=(100, 70, 50, 20)). Registration accuracy was visually confirmed using FSLeyes (v0.30.1; FMRIB), with the atlas warped to each fixed registration input image. Fos immunofluorescence was enhanced through 3x3x3 spatial averaging and rolling ball background subtraction (pixel radius = 4). The resulting images were warped to 25 pm atlas space using transforms from registration. Each hemisphere was z-scored using a warped tissue mask and a hemispheric mask ((image - mean intensity in the brain) / standard deviation of intensity in the brain). The images were smoothed with a 100 pm kernel and mirrored to average the left and right sides. A unilateral atlas mask, excluding ventricles, fiber tracts, and undefined regions, spatially restricted the voxel-wise analyses.

[0078] Voxel-wise comparisons between the isoflurane and control groups (t-test design) were performed using nonparametric permutation testing (18,000 permutations) with the general linear model (randomise_parallel; FMRIB; v6.0.2). P- value maps were corrected for multiple comparisons using the false discovery rate (FDR) method, applied across a series of q-values to identify clusters of significant voxels. Data in FIGs. 5A-5H are from the most stringent q-value thresholds (q < 0.005, p < 0.000056 for Control < Isoflurane; q < 0.05, p < 0.0012 for Control > Isoflurane).

[0079] Clusters smaller than 100 voxels were excluded. Clusters were warped back to full-resolution tissue space for cell density measurements (Fos+ cells per cluster volume). Fos+ cells were segmented using llastik. 3D counting was performed using connected components-3d (v3.16.0; connectivity = 6). Clusters were considered valid if unpaired t-tests confirmed that differences in Fos labeling reflected changes in Fos+ cell densities. Statistical maps and other related data are available upon request. Scripts from UNRAVEL (github.com / b-heifets / UNRAVEL) are available at zenodo.org / records / 13655988.

[0080] Correlational, Permutation, and Network Analysis. All associated network analyses and visualizations were conducted with the SMARTTR package, igraph, tidygraph package, and custom written functions (Jin, M. et al. eLife 13:RP101327 (2025)). Pearson correlations between normalized Fos+ activity per region were calculated and asymptotic p-values for each correlation were determined using a one-sample t-test. Significantly correlated regions are visualized in heatmaps after FDR (Benjamini-Hochberg) correction using a significance threshold of q < 0.05. A permutation analysis was conducted to identify the functional connections which differed most between experimental groups Correlations differences were calculated by subtracting correlations from the Control group from respective correlations in the Isoflurane group (riSOfiurane - rcontroi). Each correlation difference was used as a test statistic against a null distribution produce by shuffling the labels of Control and Isoflurane mice 10,000 times, with the correlation difference recomputed for each shuffle. Each test statistic was compared to its individual null distributions to determine the p-value. Significance correlation differences for all analyses were thresholded using an alpha value of 0.001 and visualized as constructed network. Edge thickness in the permuted network represented magnitude of correlation difference, with regions represented as nodes. An agglomerative fast greedy modularity optimization algorithm was applied using tidygraph for community detection (Madangopal, supra).

[0081] Individual functional networks for the Control and Isoflurane test conditions were constructed by representing regions as nodes and Pearson correlation values as edges, with correlation magnitude corresponding to edge thickness. Since pairwise correlations were calculated for all pairs, the initial functional network is complete, with all nodes fully interconnected. Thresholding is often applied to assess the topological features of the most salient connections, however, there is little consensus on the best approach, (i.e., thresholding by connection strength or connection proportion) or choice of threshold (Szelenyi, supra). Thus, in this Example, networks for Control and Isoflurane were constructed across a range of alpha thresholds (FDR uncorrected) from 0.0001 to 1, and across a range of edge proportion thresholds, from 0.001 to 0.50.

[0082] In both the permuted and individual functional networks, several topology metrics per node, such as degree, clustering coefficient, efficiency, and betweenness centrality4 are automatically calculated using the create_networks() or create_permutation_network() functions in SMARTTR. Summary statistics are calculated using the summarise_networks() function, which averages nodal metrics to calculate mean degree, mean efficiency, mean clustering coefficient, and mean betweenness for a given network. To assess for “small-world” properties of empirical Control and Isoflurane networks (edge proportion of 3.15%), the clustering coefficient (C) and characteristic path length (L) were compared with the corresponding values (Crand, Lrand) averaged from 100 random null networks (Perens, J. et al. Neuroinformatics 19, 433-446 (2021)). Null networks are generated by applying the degree sequence-preserving maslov-sneppen6 rewiring algorithm with the rewire_network() function, and averages metrics were calculated with summarize_null_networks(). Number of edge rewirings per random network are equivalent to number of edges x 100.The small-world index (s), a ratio summarizing the propensity for high clustering while maintaining efficiency in a network, is defined here as:C / CrarL(iValues above 1 are considered indicative of small-world properties. All analysis code is available on (https: / / mj i n 1812. gi th u b . io / S M ARTTR)

[0083] Head-fixed implantation and habituation. For high density silicon probe (Neuropixels) recordings, mice were implanted with a custom-made titanium headplate (H.E. Parmer Company, TN) for head-fixation of the animal, and 3D-printed "well” providing safe access for probe insertion and retention of saline solution on the skull surface during recordings (International Brain Laboratory. Behavior: Appendix 1: IBL protocol for headbar implant surgery in mice.10437384 Bytes (2020)). Following recovery (2 weeks), mice were habituated to head-fixation for at least 3 days, starting with 15- to 20-min periods and increasing daily by 20 min until they exhibited minimal distress with fixation for 1 hour (International Brain Laboratory. Behavior: Appendix 2: IBL protocol for mice training. 2735505 Bytes (2020)). For head- fixed recordings, mice were placed in a 3D-printed mouse holder adapted for isoflurane delivery via a 3D-printed stereotax anesthetic nose cone (International Brain Laboratory. Behavior: Appendix 3: IBL protocol for setting up the behavioral training rig. 33751033 Bytes (2021)). During recordings, estimated isoflurane gas concentration was measured with a gas analyzer (BC Biomedical, Saint Charles, MO).

[0084] FIGs. 6A-6E illustrate single unit temporal dynamics of isoflurane unconsciousness. FIG. 6A illustrates the experiment schematic. Prior to recording sessions, Neuropixels 1.0 probe shanks were coated in Dil for later histological placement. A 5-min awake baseline period was recorded before isoflurane induction. During induction, foot pinch stimuli were delivered to determine loss-of-reflex (LOR), at which point the concentration was reduced to 1.3% to begin steady-state maintenance phase. FIG. 6B illustrates (left) intact iDisco-cleared brains were imaged with light sheet microscopy and registered to the Allen mouse brain atlas to obtain probe tract placements and (right) tract visualizations for all recordings. Shared colors represent tracts from the same mouse. FIG. 6C illustrates z-scored firing rates for induction and maintenance phases averaged across units from each recorded subregion within isocortex, hippocampus, amygdala, thalamus and hypothalamus. Isoflurane induction and maintenance elicited widespread reductions in average activity across cortical and subcortical regions. Normalized mean Fos density associated with each subregion (from FIGs. 5A-5H) is shown to the right. FIG. 6D illustrates k-means clustering of activity across induction and maintenance phases yielded 6 heterogeneous clusters composed of cortical and subcortical regions, generally differentiated by their relative activity between each isoflurane phase. FIG. 6E illustrates mean z-scored firing traces for 3 structures found to have significantly elevated Fos expression: reticular nucleus of thalamus (RT), medial amygdalar nucleus (MEA) and perireunensis nucleus (PR). Traces show 2 min before to 14 min following start of induction (left dashed line: “induction onset”) and 2 min before to 10 min following onset of maintenance phase (right dashed line: “LOR / maintenance start”). Shaded error indicates 95% confidence interval. Each animal had 2 recordingsessions and one recording was left out due to poor signal quality for a total of 7 recording sessions from 4 mice, yielding N=1048 units (Isocortex, n=37; Hippocampus, n=53; Amygdala, n=16; Thalamus, n=883, Hypothalamus, n=59). D, dorsal; V, ventral; A, anterior; P, posterior. See FIGs. 7A-7C for representative traces from all major brain regions.

[0085] FIGs. 7A-7C illustrate response variability during isoflurane. FIG. 7A illustrates heatmaps showing mean normalized firing for each major region (isocortex, hippocampus, amygdala, thalamus, and hypothalamus) across anesthetic phases (induction, maintenance, washout) with the total unit counts (n) for each region. FIG. 7B illustrates representative traces from units in major regions with diverse responses under isoflurane. For each major region, traces show 5 units (k=5 from total unit count n for each region) with the highest (top) and lowest (bottom) mean normalized firing during the maintenance phase. Left traces show 1 min prior and 5 min following onset of induction at the dashed line ("ISO on”). Right traces show 1 min prior and 10 min following onset of maintenance phase (first dashed line) and the “ISO off” time (right dashed line). FIG. 7C illustrates mean z-scored firing from each experimental phase was clustered (Ward’s method) for all subregions obtained in recordings. Hierarchical clustering of subpopulation means revealed 3 heterogeneous clusters (CHI=11.12), each with subclusters (e.g., 1a: Cluster 1a) linking spatially distributed cortical and subcortical structures.

[0086] FIGs. 8A-8C illustrate differential dynamics of synthetic activity-dependent capture of unconsciousness vs. isoflurane. FIG. 8A illustrates the experiment schematic. The first phase (“isoflurane anesthesia”) is as described in FIG. 6A, including isoflurane (ISO) onset but with an extended 25-min drug-washout period. After a 5-min baseline, phase 2 (“chemogenetic activation”) begins with CNO injection (5mg / kg) to induce synthetic unconsciousness (“CNOI NJ”), with a projected full onset at 15 min post-injection (“CNOONSET”). Ten minutes later the vaporizer is turned on to a 0.5% target concentration (“CNOI SO”) at which point there is loss-of-reflex (LOR) and turned off (“washout”) once LOR was detected via foot pinch. Dark bars before each onset type indicate relative baseline period. FIG. 8B illustrates population activity traces from major subcortical regions (top, amygdala; middle, thalamus; bottom, hypothalamus) across 4 experimental onset timepoints: isoflurane induction (1-ISO), CNO injection time (2-CNOINJ), estimated CNO onset (3-CNOONSET), post-CNO isoflurane induction (4-CNOISO). For each onset type, cell firing rates were z-scored to the relative baseline period (-2 to 0 min) and averaged per region. In all 3 regions, isoflurane elicited a slow, pronounced decrease in population activity regardless of chemogenetic context, with no significant change directly following CNO injection. In hypothalamus, population activity showed similar but slower decreases, including following CNO onset as well. FIG. 8C illustrates relative single-unit response distributions: Response indices were obtained for each neuron by calculating a normalized difference between their response and baseline mean firing rates (“response index”), reflecting the magnitude of their individual response after each onset type, for each major region shown. Across regions, responses to ISO, CNOISO and CNOONSET were significantly shifted to the left compared to relative baselines (Wilcoxon) except for CNOI NJ, for which only Thalamus showed a significant difference. Filled-in circles denote cells that had responsessignificantly greater or lower than 0, while open circles denote no significance, compared to a hypothetical distribution of same scale with location (mean) at O. ***p < 0.0001; **p < 0.001; *p < 0.01; n.s., not significant

[0087] High-density in vivo Neuropixels recordings. Male and female mice were prepared for head-fixed recordings in a behavioral rig adapted for isoflurane delivery and gas analyzer sampling at the nose cone. At least 1 day prior to each recording, craniotomies were made targeting regions of interest, planned beforehand using a specialized tool to take into consideration optimal probe orientations and angles of entry (5°-15°), and finally sealed with Kwik-Cast (World Precision Instruments, Sarasota, FL) (Birman, D. et al. eLife 12: RP91662 (2023)). When not sealed, craniotomies were kept moist with continuous applications of saline into the "well.” On recording days, mice were first head-fixed and allowed to acclimate before probe insertion. Neuropixels 1.0 probes were mounted on a micromanipulator system (Sensapex, SC) and lowered via the planned trajectory to 100 pirn beyond the target depth, then retracted after a 10-minute delay and allowed to rest for an additional 10 minute before starting the baseline recording. After a 5- minute awake baseline, mice were induced with isoflurane to an initial target 1.5% concentration. Intermittent foot pinches were used to determine loss-of-reflex (LOR) time. Following LOR, mice were maintained at 1.3% isoflurane to begin the steady-state maintenance phase recording (FIGs. 6A-6E). After 10 min, the vaporizer was turned off for washout period of recording (FIGs. 7A-7C). Mice were actively warmed via external heating pads. Each animal (n=4) had two recording sessions at least 24 hours apart, and one recording was left out due to poor signal quality for a total of 7 recording sessions. Altogether, units totaling N=1048; Isocortex, n=37; Hippocampus, n=53; Amygdala, n=16; Thalamus, n=883, Hypothalamus, n=59 were obtained. All recordings were acquired using SpikeGLX (Janelia Research Campus, Lansdowne, VA).

[0088] Histological alignment of recording tracts. To determine the placement of isolated units in the brain, sorted units were aligned to the probe tracts after intact brain imaging. After recordings, intact formalin-fixed brains were cleared and imaged with light sheet fluorescent microscopy for probe tract tracing in the Allen Brain Atlas space and mapped onto the locations of isolated units to identify their placement in the brain (FIG. 6B and see Intact brain clearing and imaging). After registering each brain to the Allen CCF, probe tracts made by application of the lipophilic CM- Dil dye (Thermo Fisher, Bothell, WA) to the probe shank before each recording were traced in Lasagna to yield a fitted line of XYZ coordinates, which were visualized in Brainrender (Tyson, A. L. et al. Sci Rep 12, 867 (2022); Campbell, R. et al. SainsburyWellcomeCentre / lasagna: Stable IBL. Zenodo (2020)). A separate open- source tool was used to adjust the alignment of activity recorded along the shank to the regions identified along the tract (Faulkner, M. Ephys Atlas GUI. https: / / github.com / int-brain-lab / iblapps / tree / master / atlaselectrophysiology. (2020)).

[0089] Neuropixels data processing. Spike sorting was performed with Kilosort4, isolated units were discarded if they had fewer than 1000 spikes across the whole recording and filtered out further based on quality metrics assessment (Pachitariu, M. et al. Nat Methods 21, 914-921 (2024); International Brain Laboratory, et al. Spike sorting pipeline for the International Brain Laboratory. (2022)). To prevent false negatives due to widely-observed prolongedinhibitions from anesthetic induction, units were primarily filtered out if they concurrently exhibited low presence and low firing rate (<0.5 Hz), or a poor score on L-ratio or D-prime isolation metrics (Hill, D. N. et al. Journal of Neuroscience 31, 8699-8705 (2011); Schmitzer-Torbert & Redish. J Neurophysiol 91, 2259-2272 (2004)). Remaining units were inspected manually using Phy (Pachitariu, supra). Postprocessing and curation was performed using custom scripts integrating open-source Python packages (Buccino, A. P. et al. eLife 9, e61834 (2020)).

[0090] Neuropixels data analysis. Spike times were binned into 250-ms bins and averaged to yield each unit’s mean firing rates. For representative isoflurane activity traces, normalized firing rates were calculated by first subtracting the mean firing during the last 2 min of the baseline phase, then z- scored across the whole recording. For mean region and subregion activity heatmaps, firing rates were z-scored across the recording. Mean rates during "induction,” “maintenance” or “washout" phases were taken across all units in each major region or subregion (FIGs. 7A-7C). Cortical and subcortical subregion normalized firing rates were averaged and compared via one-way ANOVA. For k-means clustering, subregion normalized firing rates were used as features for the elbow method analysis, yielding an optimal grouping of 6 clusters with asymptotic inertia and silhouette scores. All analyses were conducted using custom Python scripts integrating open- source packages.

[0091] Retroorbital virus injection. FosTRAP2 mice were briefly anesthetized with isoflurane (5-10-min) and administered adeno-associated virus (MV) by retroorbital injection to one eye. MV- PHP.eB-hSyn-DIO-hM3D(Gq)-mCherry (44361-PHPeB, Addgene) was used for activating DREADD and MV-PHP.eB-hSyn-DIO-hM4D(Gi)-mCherry (44362-PHPeB, Addgene) was used for inhibitory DREADD. Virus was diluted in total 10O L sterile saline to ~ 2-3x1011GC / mL based on prior studies (Chan, K. Y. et al. Nat Neurosci 20, 1172-1179 (2017)).

[0092] Activity-dependent labeling (TRAP). 3-4 weeks following retroorbital virus injection, FosTRAP2 mice were induced with 2% isoflurane and then maintained unresponsive at 1.2-1.3% for a total time of 180 min. Mice were actively warmed on a heating pad and continuously monitored by an experimenter. A single 50 mg / kg intraperitoneal (i.p.) injection of 4-hydroxytamoxifen (4-OHT, Sigma Aldrich), was administered at the 90-min halfway point during the isoflurane exposure to induce Cre-recombination and activity-dependent labeling (DeNardo, L. A. et al. Nat. Neurosci.22, 460-469 (2019)). Non-TRAP control mice also received retroorbital virus injections and isoflurane exposure but then sesame oil vehicle (Sigma Aldrich) in the equivalent volume of 4-OHT (0.2mL) was given i.p. at the 90-min halfway point.

[0093] Clozapine-n-oxide administration. Clozapine-n-oxide (CNO, Enzo Life Sciences, Long Island, NY) was solubilized in water and stored as 5 milligrams per milliliter (mg / mL) aliquots. For testing, the aliquot was diluted in sterile saline to an end concentration of 0.5 mg / mL for 5 mg / kg behavioral studies. Mice received i.p. injections according to their body weight ranging from 0.2 mL-0.3 mL for a 20 gram (g)-30 g mouse. The equivalent volume i.p. sterile saline was delivered for saline trials.

[0094] Electrocorticography surgical procedure & neural signal data recording. Electrocorticography (ECoG) was recorded using a tethered implant as previously described (Hsu, Y.-W. A. et al. J Biol Rhythms 32, 444-455 (2017)).Briefly, using aseptic stereotaxic surgical techniques under isoflurane anesthesia, a midline incision was made above the skull, and ECoG-recording electrodes (Pinnacle Technology, Lawrence, KS; No 8209: 0.10-in.) were screwed through cranial holes at the following coordinates:• Channel 1 (left frontal cortex): 1.5 mm lateral and 2 mm anterior to bregma• Channel 2 (right parietal cortex): 1.5 mm lateral and 2 mm posterior to bregma• Ground (visual cortex): 1.5 mm lateral and 4 mm posterior to bregma• Reference (cerebellum): 1.5 mm lateral and 6 mm posterior to bregma

[0095] Mice were singly housed in standard cages under a 12:12 light-dark (LD) cycle following surgery. After a 14-day recovery period, they were moved to recording cages, fitted with a tether and preamplifier (100x signal gain) connected to the Pinnacle Technology recording system, and given at least 1 day to acclimate before recording.

[0096] Animals were injected with saline or CNO (5mg / kg) at the same time of day on consecutive days (days 1 and 2, respectively), with pre-experimental (day 0) and post-experimental (day 3) days serving as baseline comparisons. To assess neural dynamics during isoflurane, mice were placed in a specialized chamber and induced with 3% isoflurane until fully immobile (10 minutes). Isoflurane was then discontinued, and the mice were allowed to fully wake (20-30 minutes). In all recording days, electromyography (EMG) signals were obtained by inserting a pair of silver wires into the neck muscles. All screws and wires were connected to a common 6-pin connector compatible with the Pinnacle recording device using silver wire. Signals were continuously recorded at 400 Hz with low-pass filters at 100 Hz. All recordings were saved and exported for processing using the European Data Format (EDF). Simultaneously, animal activity was continuously recorded using webcams.

[0097] For each animal, one hour of data (time-matched across days) was extracted for each condition (pre, saline, CNO, post), along with the entirety of the isoflurane induction condition, and saved to individual .edf files. Spectrograms were generated by low-pass filtering each file at 12 Hz and log-transforming the signal data. Additionally, slow wave signal-to-noise ratio (SNR) was calculated using minute-long bins by dividing mean power in the delta frequency band (1-4 Hz, primarily capturing delta waves associated with sleep and anesthesiaa) by mean power in surrounding frequencies (0.5-1 Hz + 4-7 Hz) (Amzica & Steriade. Electroencephalogr Clin Neurophysiol 107, 69-83 (1998)). All analyses were conducted using the left frontal cortex electrode (i.e., channel 1).

[0098] To quantify the change in delta (1-4 Hz) over the course of drug onset (i.e., 1 minute prior to and 15 minutes following drug onset), a linear mixed model predicting delta activity ratio was run using the interaction term time x condition (using the 'Pre' condition as reference and controlling for the random effect of individual animal). The results show that the only significant differences in delta activity were the increases seen over time in the CNO and ISO conditions, with no other significant direct or interaction effects.

[0099] FIGs. 9A-9J illustrate effects of chemogenetic inhibition after synthetic activity-dependent capture of altered consciousness. FIG. 9A illustrates the concentration of isoflurane (%), as measured with a gas analyzer sampling from the chamber, at which mice display loss of righting reflex during saline or CNO (10 mg / kg) is significantly reduced inGq-DREADD but not Gi-DREADD or non-TRAP control (n = 5-6 mice). FIG. 9B illustrates that there is no change in rotarod (4-rpm) performance in Gi-DREADD mice after 5 mg / kg CNO (n = 5 mice). CNO induces dose-dependent reduction in maximum speed (m / s) in the open field only in (FIG. 9C) Gq-DREADD mice (n = 6 mice) and not (FIG. 9D) Gi-DREADD mice (n = 5 mice, 3-4 trials per drug condition). FIG. 9E illustrates representative raster plot from all 6 (3 saline, 3 CNO) of one Gi-DREADD animal’s trials after analysis of classified behavior. FIG. 9F illustrates bouts of classified behavior for the average of saline and CNO (5 mg / kg) trials per animal expressing inhibiting Gi-DREADD virus (n = 5 mice). There is no change in (FIG. 9G) heart rate (BPM, beats per minute) (FIG. 9H) respiratory rate (breaths per minute) and (FIG. 91) temperature (Celsius) as recorded from the wireless mechano-acoustic device during trials shown in FIGs. 9E and 9F (n = 6 mice, 2-3 trials per drug condition). (FIG. 9J) Respiratory rate in Gq-DREADD TRAP mice is also unchanged. Drug onset time is approximated by average onset of immobility in Gq-DREADD mice (approx. 10 minutes after CNO injection). See FIGs. 10A-10L and Methods for experimental details. Error bars show mean ± SEM. 2-way repeated measures ANOVA with Tukey’s post-hoc tests (FIGs. 9C and 9D) and paired t-tests, *p<0.05, **p<0.01, ***p<0.001.

[0100] Open field test. CNO dose-dependent changes in maximum velocity were assessed in a 30 x 30 cm open field chamber (FIGs. 9C, 9D). Mice were habituated to the chamber for at least 3 days. On alternating days, they received intraperitoneal injection of either 1 mg / kg CNO, 5mg / kg CNO, or the equivalent volume of saline, and were placed in the box for 20-min with concurrent webcam video recording. A total of 3-4 trials per drug condition from 6 Gq-DREADD-expressing mice and 5 Gi-DREADD expressing mice were analyzed for changes in movement velocity using Any-MAZE video tracking software. GraphPad Prism was used to analyze data with 2-way ANOVA and Tukey multiple comparison post-tests.

[0101] Loss of righting reflex. Latency to loss of righting reflex was assessed in a chamber equipped for isoflurane delivery and sampling of isoflurane concentration with a gas analyzer (FIG. 9A). Mice were individually injected with 10 mg / kg CNO or saline in alternating trials at least one day apart and placed in the chamber. Isoflurane was administered stepwise from 0.5 to 2% on the vaporizer in 0.5 increments and loss of righting reflex was assessed at each stepwise increment with the exact sampled concentration recorded.

[0102] Warm water tail withdrawal test. Mice were tested for the latency to remove their tail from a warm water bath (52.5°C). Mice are restrained within a paper towel, and 1 / 3 to1 / 2of the tail is immersed, and the time to remove the tail (tail flick latency) is recorded (English, A. et al. eLife 12, RP89867 (2024); Abraham, A. D. et al. J Neurosci 38, 8031 — 8043 (2018); Chavkin, C. et al. Front Pharmacol 10, 88 (2019); Schattauer, S. S. et al. Nat Commun 8, 743 (2017)). This is done at baseline after saline, and then in 30-min intervals after CNO injection.

[0103] Hot plate test. Mice are placed on a 53°C plate for 30 seconds, and behaviors are video recorded (Abraham, A. D et al. Neuropsychopharmacology 45, 1105-1114 (2020)). This occurs at baseline and after CNO at 2-time intervals (30-min and 90-min post-CNO) interleaved between tail withdrawal tests. Latency to jump, groom paws, and number of occurrences for these behaviors are quantified from videos by two independent researchers.

[0104] FIGs. 10A-10L illustrate synthetic activity-dependent capture of altered consciousness. FIG. 10A illustrates the experimental timeline: FosTRAP2 transgenic mice received retroorbital injection of an AAV-PHP.eB Cre-dependent Gq-DREADD followed by activity-dependent capture (TRAP) during isoflurane exposure (1.2-1.3%) with 4-hydroxytamoxifen (4-OHT). Mice were tested in the absence of anesthetic after either clozapine-n-oxide (CNO) or saline injection in a modified open field test with concurrent videorecording and peripheral physiology recording using wireless mechano- acoustic (MA) devices, or neural physiology with electrocorticography (ECoG). (DREADD = designer receptor engineered to be activated by a designer drug). FIG. 10B illustrates that Gq-DREADD expressing mice show increased latency to fall off the rotarod at 4-rpm after CNO (n = 7 mice). See FIGs. 9A-9J for Gi-DREADD data. FIG. 10C illustrates that 1-4 Hz delta slow wave activity is prominent after isoflurane induction and (FIG. 10D) after CNO injection as compared to saline in Gq-DREADD mice (n = 4, see FIGs. 11A and 11 B for spectrograms). FIG.10E illustrates (left) heart rate (beats per minute) decreases after CNO injection as compared to saline, shown time-aligned to drug onset and (right) mean heart rate decreases after CNO induction in Gq-DREADD mice. FIG. 10F illustrates (left) body temperature (Celsius) changes over duration of trial, time-aligned to drug onset and (right) mean temperature decreases with CNO. FIG. 10G illustrates representative raster plot from all 6 (3 saline, 3 CNO) of one animal's trials after analysis of classified behavior. FIG. 10H illustrates bouts of classified behavior for the average of saline and CNO trials per animal (n= 7 mice, total of 18 CNO and 20 saline trials). The average of all saline or CNO trials per animal is shown in FIG. 10B and 10E-10H. Paired t-tests: *p<0.05, **p<0.01. FIG. 101 illustrates the experimental timeline: FosTRAP2 mice received retroorbital injection of either Cre-dependent activating Gq-DREADD or inhibitory Gi-DREADD. They underwent isoflurane exposure, during which either 4-OHT or oil vehicle (if non-TRAP control) was administered. On test day, mice were tested for anti- nociception following saline injection (Pre) and after 5 mg / kg CNO. Warm water tail withdrawal (tail flick) latency was tested at 30-, 60-, and 90-minutes (min) post-CNO injection, and hotplate behavior was tested at 30- and 90-min post-CNO injection interleaved with tail flick tests. FIG.10J illustrates (left) representative light sheet image after intact brain iDisco+ clearing and mCherry-immunolabeling for DREADD expression and (right) mean TRAP Gq-DREADD expression density (n=7 mice). Scale bar is 1 mm and 200 m in inset. See FIGs. 3A-3F. FIG. 10K illustrates that Gq-DREADD mice (n=8, triangles) show increased latency to tail flick compared to Gi-DREADD (n=7, pentagons) or non-TRAP control mice (n=8, circles) at every time point tested. FIG. 10L illustrates that in the hot plate test, Gq-DREADD mice do not jump off the hotplate, have fewer paw withdrawals off the hot plate at 90-min, and show increased latency to first paw withdrawal response at 30 and 90-min post-CNO. 2-way repeated measures ANOVA with Tukey's multiple comparisons: *p<0.05, **p<0.01. Error bars are the mean ± SEM.

[0105] Rotarod test. Coordination post CNO injection was tested for each mouse on a rotarod after modified open field testing (FIGs 9B, 10B). Mice were habituated for two days, 10 minutes per day, at 4 rotations per minute (rpm) before testing. On test day, mice received a saline i.p. injection in the morning and were placed on the rotarod at 4rpm. Latency to fall was recorded, unless animals stayed on for the duration of the experiment (10 minutes). Mice were recorded again in the afternoon with a 5 mg / kg i.p. CNO injection.

[0106] Wireless mechano-acoustic device implant. Mice were implanted with wireless mechano-acoustic devices (MA device: nVital, NeuroLux, Inc.) subcutaneously on the ventral surface while under isoflurane anesthesia for a duration of less than 30 minutes. They were then group housed following surgery. See Ouyang et al. for surgical procedure and device details (Ouyang, W. et al. Neuron 223:2764-2777 (2024)).

[0107] Modified open field test and MA device data collection. For 3 days before the experiment, mice were habituated to saline intraperitoneal (i.p.) injections and explored a 15cm x 15cm smaller, modified open field box for 10 minutes. The sides of the box were wrapped in 14G copper wire attached to tuner and power distribution control (PDC) boxes (NeuroLux, Inc, Northfield, IL). Implanted wireless mechano-acoustic (MA) devices were also tested during this time along with power input from the PDC box (7 watts). During each experiment day, the animal explored the enclosure for 2-min before an i.p. injection of either saline or 5mg / kg clozapine-n oxide (CNO, Enzo Life Sciences).

[0108] Recordings were taken with a web-camera positioned directly above the animal for 20 minutes. During each CNO trial, the first observable loss of mobility was documented as "drug onset.” The average latency to immobility in all CNO trials was used to time-align “drug onset” in saline trials. Each mouse underwent 3 saline trials and 2-3 CNO trials over 6 days with no more than one trial per day. A total of 18 CNO trials and 20 saline trials was analyzed from 7 Gq+mice (3 females, 4 males) in FIGs, 10A-10L. A total of 17 CNO trials and 17 saline trials was analyzed from 6 Gi+ mice (2 females, 4 males) in FIGs. 9A-9J. Behaviors for a sub-selection of animals were manually scored to identify commonly observed behaviors, as well as determine criteria for behavioral classifiers. Video recordings collected during the experiment were used for pose estimation and supervised behavioral analysis (see Modified open field test behavioral analysis). Data collected from the MA devices were processed with custom Python scripts. GraphPad Prism was used to analyze group means and paired t-tests.

[0109] Perfusion for tissue processing. After all studies, mice were deeply anesthetized with isoflurane and underwent transcardial perfusion with phosphate-buffered saline (PBS) followed by 10% formalin and dissection of the brain for further analysis (see Intact brain clearing and immunolabeling).

[0110] Modified open field test behavioral analysis. Pose estimation models were created to track 9 body-part keypoints using SLEAP (snout, left ear, right ear, left lateral, right lateral, centroid, tail base, tail center, and tail-tip) (Jin, supra). Missing pose-estimation data was interpolated using forward fill and the data was smoothed using a Savitsky-Golay filter across a 500ms sliding window. Grooming, rearing, and Straub tail were detected using random forest classifiers (Goodwin, N. L. et al. Nat Neurosci 27, 1411-1424 (2024)). Freezing and circling were detected using heuristic rules as previously described (Sabnis, G. et al. bioRxiv 2024.05.29.596520 (2024); Lopez, G. C. et al. Curr Biol 35, 2433-2433.e5 (2024)).

[0111] Annotation. Behavior was annotated in 20-minute video clips obtained from the modified open field test. For fitting the Straub tail classifier, a sampled annotated dataset was used, in which the behavior was present in 24244frames (808s) and behavior was absent in 627312 frames (20910s). For fitting the grooming classifier, a sampled annotated dataset was used, in which the behavior was present in 47005 frames (1566s) and behavior absent in 94010 frames (3133s). For fitting the rearing classifier, a sampled annotated dataset was used, in which the behavior was present 31596 frames (1053s) and behavior absent in 315960 frames (10530s).

[0112] Featurization. Video and pose-estimation data was featurized using image, movement, geometry, circular-and frequentist statistical methods (see below). For acceptable runtimes, GPU accelerated and multiprocessing methods available through the SimBA API were used (Goodwin, supra). Features were computed in metric space after calculating the pixel-to-metric conversion factors in the SimBA graphical interface. Classifications were smoothened to remove any detected bouts less than 200ms in length. Computations are primarily dependent on numba and shapely libraries and the featurization classes are available on the SimBA GitHub repository (, S. K. et al. Numba: a LLVM-based Python JIT compiler, in Proceedings of the Second Workshop on the LLVM Compiler Infrastructure in HPC (2015); Gillies, S. et al. Shapely. Zenodo (2024)).

[0113] Grooming and rearing behavioral classifiers. The same feature set was used to create classifiers for rearing and grooming. This feature set included movement and acceleration measures of individual body-parts as well as paired body-part movement Spearman correlations in sliding time windows (0.25-2s). Distribution descriptive statistics (mean, variance, sum, z- scores, skew, median absolute deviations, mean absolute change, root mean square) of the animal hull geometry and animal hull sub-geometries (animal hull width, length, and area, as well as head area, posterior hull area, anterior hull area, left lateral hull area, right lateral hull area) were computed in sliding windows of 0.25-2 seconds. Spearman correlations were computed between the hull sub-geometry areas in the same sized sliding time-windows. Using circular statistics, descriptive statistics of animal directions were calculated, including animal circular range, animal circular standard deviation, and directional vector lengths in sliding windows of 0.25-2 seconds.

[0114] Straub tail classifier. For accurate Straub tail segmentation, video background subtraction was performed first and the mice were centered and egocentrically aligned in the videos. Next, a buffered (1.75 cm in each direction) polygonal geometry was defined using the mouse tail key-points (tail-base, tail center, tail-tip) and sliced the tail geometry from each frame. Using the tail geometry coordinates, the tail geometry mean area, standard deviation, mean absolute deviation were computed in sliding time-windows of 1-2 seconds. For each frame, the tail geometry was compared to the tail geometry 0.5-2 seconds prior using Hausdorff distances. For the sliced images of the tails, the current tail image was compared to the tail image 0.5-2 seconds prior using mean squared error of the raw RGB pixel values. Descriptive statistics (average, variance, sum, mean absolute change) of the tail tip and tail center movements, and the tail length were also computed in sliding windows of 1-2 seconds.

[0115] Feature importance. Model Gini feature importance scores showed the following key feature determinants for each behavior: (1) Grooming was primarily influenced by features describing the distribution of the animal's head area, body length, and snout movement within sliding temporal windows. (2) Rearing was primarily driven by features capturing the distribution of the distance between the animal’s ears, head area, and body width in sliding temporalwindows. (3) Straub tail was mainly determined by features describing the distribution of the tail-tip movements, tail length and area, overall body area and right-side hull area, as well as correlations between the movements of the tail tip and tail base in sliding temporal windows.

[0116] Performance. Out-of-sample frame wise performance showed strong performance for grooming (precision: 0.92, recall: 0.99, f1: 0.96), rearing, (precision: 0.98, recall: 0.96, f1: 0.97) and Straub tail (precision: 0.99, recall: 0.96, f1: 0.98).

[0117] Heuristic models. Freezing and circling were detected using heuristic rules as previously described. Circling was scored as present when the directional circular range of the animal was above 320 degrees, and the animal movement (computed from the animal centroid key-point) was above 6 cm in the preceding 10 seconds time-window. Freezing was scored as present in frames where the velocity (computed from the mean movement of the nape, snout, and tail-base body-parts) fell below 5 mm / s in the preceding 3 seconds. Although the nape body-part was not pose-estimated, this body- part was inferred as halfway between the two ears.

[0118] Results.

[0119] Activity-dependent capture of a synthetic unconscious state. If unconsciousness is regulated by discrete brainwide-activated cells rather than a non- specific disruption of neural activity, unconsciousness should be recapitulated by those activated cells. To capture brainwide anesthesia-activated unconsciousness circuitry, Fos-TRAP2 (Fos2A- iCreER) mice received retroorbital injections of AAV-PHP.eB virus expressing Cre-dependent designer receptors engineered to be activated by designer drugs (DREADDs) (DeNardo, supra,' Chan, supra). Three to four weeks later, mice underwent 180-min of isoflurane exposure (1.2-1.3%) and halfway through the exposure, at 90-min, received an injection of 4-hydroxytamoxifen (4-OHT) to induce activity-dependent DREADD expression in brainwide isoflurane-activated cells (TRAP) (DeNardo, supra] DeNardo & Luo, supra). On separate subsequent days, mice were implanted with either a wireless mechano-acoustic device or electrodes for electrocorticography to characterize peripheral physiologic and neurophysiologic effects, respectively, as well as behavioral changes after chemogenetic manipulation (FIG. 10A) (Ouyang, supra).

[0120] It was first assayed if coordinated movement, as measured in the rotarod test, is affected by chemogenetic stimulation of brainwide anesthesia-activated circuitry. Mice underwent 3 days of training and then were placed on a slowly rotating rod (4 revolutions per minute, rpm) that poses minimal challenge to motor coordination under control conditions. After clozapine-n-oxide (CNO) injection (5 mg / kg), mice expressing activating Gq-DREADD fall off the rotarod when compared to their respective saline trials, indicating a loss of coordinated movement (FIG. 10B), which is not observed in mice expressing brainwide inhibitory Gi-DREADD (FIG. 9B).

[0121] Anesthesia-induced unconsciousness in rodents is commonly defined by loss of righting reflex (LORR, a postural reflex that allows rodents to regain the upright position. This behavioral definition is not used for other altered states of consciousness like after psychedelic treatment, and there are efforts to identify alternatives to righting reflex for studies of anesthesia-induced unconsciousness, such as machine-guided behavioral classification duringconsciousness state transitions (Dickinson, R. et al. Anesthesiology 93, 837-843 (2000); Aboharb, F. et al. ClasNat Commun 16, 1590 (2025); Heifets, B. D. et al. Science Translational Medicine 11, eaaw6435 (2019); Gao & Calderon. Sci Rep 10, 20280 (2020)). It was found that CNO (10 mg / kg) potentiates LORR with subanesthetic isoflurane administration (FIG. 9A). A significant shift toward LORR occurs after synthetic chemogenetic stimulation of isofl urane-activated brainwide circuitry, and not inhibition nor non-TRAP control conditions (FIG. 9A). 1 mg / kg and 5 mg / kg CNO produce dose-dependent reduction in movement velocity in activating Gq-DREADD-expressing mice (FIG. 9C) but not inhibitory Gi-DREADD-expressing mice (FIG. 9D). In behavioral tests, 5mg / kg CNO were used, as that dose is validated to saturate DREADD receptors with minimal off-target effects, does not modify behavior when administered to non-TRAP control mice, and does not fully immobilize Gq-DREADD-expressing mice, allowing analysis of behavioral repertoires of the altered state of consciousness (Manvich, D. F. et al. Sci Rep 8, 3840 (2018); Jendryka, M. et al. Sci Rep 9, 4522 (2019)).

[0122] FIGs. 11 A and 11 B illustrate electrocorticography recordings of the synthetic unconscious state. FIG. 11A illustrates the experimental timeline: FosTRAP2 transgenic mice received retroorbital AAV injection expressing Gq-DREADD and activity-dependent capture during isoflurane exposure, followed by neural electrocorticography (ECoG) recordings over five days (DREADD = designer receptor engineered to be activated by a designer drug, 4-OHT = 4-hydroxytamoxifen, ECoG = electrocorticography, CNO = clozapine n-oxide, ISO = isoflurane). FIG. 11B illustrates that Day 1 (labeled “1 - Pre") shows the average 40-minute baseline recording without manipulations. Recordings on Days 2-4 were time-aligned to the approximated onset of intraperitoneal CNO (10 min post-injection) indicated by the white dotted line. Day 5 (labeled “5 - Post”) was obtained without drug manipulation. The spectrograms shown are an average of 4 mice. See FIGs 10A-10L and Methods for further details.

[0123] Gq-DREADD-expressing mice underwent placement of electrocorticography (ECoG) and electromyography electrodes to assess changes in slow wave delta oscillations in the 1 to 4 Hz frequency range after CNO or saline, as compared to their isoflurane induction (FIGs. 10C, 10D, 11 A, and 11 B). An approximate “drug onset” dashed line is highlighted where the average onset of behavioral immobility occurs in CNO trials, typically 10 minutes following injection. Like with isoflurane induction, CNO induction promotes a significant increase in slow wave (1-4 Hz) signal to noise ratio (FIGs. 10C, 10D). Group-averaged spectrograms analyzed pre- and post-CNO exposure show a return to baseline occurs 24-hours following the CNO trial (FIG. 11 B).

[0124] It was next observed that synthetic stimulation of isoflurane-activated brainwide circuitry produces distinct behaviors, with differing temporal onset and distributions, when mice are tested and recorded in a modified open field chamber in the absence of anesthetic drug. An example raster plot of 6 cumulative saline and CNO trials collected from one Gq-DREADD-expressing mouse shows the representative distribution of 5 behaviors before and after drug onset (FIG. 10G). Using machine-guided supervised behavioral classification to quantify behavior relative to control saline injections, it was found that induction with CNO results in decreased rearing and grooming behavior, concomitant with an increase in stereotyped circling behavior, Straub tail, and ultimately immobility and freezing (FIG. 10H).

[0125] Since altered consciousness may be associated with widespread physiologic changes, peripheral physiologic data collected from wireless mechano-acoustic devices that was previously validated for use under isoflurane was analyzed (Ouyang, supra] Hsueh, B. etal. Nature 615, 292-299 (2023)). Behavioral and physiological data was aligned to the average latency offirst loss of immobility observed across CNO induction trials (approximately 10-min after CNO, “drug onset”). Consistent with an altered state of consciousness, significant decreases in body temperature and heart rate recorded after drug onset were observed (FIGs. 10E, 10F). Respiratory rate remains unchanged (FIG. 9J).

[0126] In contrast, significant behavioral changes are not observed, besides decreased rearing, in brainwide Gi-DREADD-expressing mice (FIGs. 9E, 9F). Mice that express Gi-DREADD never show the atypical behaviors, such as Straub tail, seen in the Gq-DREADD mice (FIGs. 9E, 9F). They also retain stable peripheral physiology after CNO (FIGs. 9G-9I).

[0127] Since anesthesia-induced unconsciousness can encompass analgesia, the anti-nociceptive function of the synthetic state was sought to be identified. Mice were tested for thermal anti-nociceptive responses in the warm water tail withdrawal (tail flick) test interleaved with hotplate testing after saline and at 30-min time points after CNO injection (FIG. 101). In the tail flick test, chemogenetic activation promoted significantly delayed tail withdrawal (FIG. 10K). In hotplate testing, chemogenetic activation reduced jumping off the hot plate, decreased paw withdrawals, and increased latency to first response after CNO (FIG. 10L). There was no effect of chemogenetic manipulation on thermal antinociceptive responding in non-TRAP control or TRAP Gi-DREADD mice at all time points tested (FIGs. 10K, 10L). Following all behavioral testing, intact brains were cleared and immunolabeled for DREADD expression using iDisco+ and light sheet microscopy to quantify brainwide DREADD distribution (FIGs. 10J and 3A-3F).

[0128] Together, the synthetic induction of an altered state of consciousness that mirrors patterns of behavioral, neural and physiologic activity observed in natural sleep, torpor, and anesthesia is described (Amzica & Steriade. Electroencephalogr Clin Neurophysiol 107, 69-83 (1998); Hrvatin, S. et al. Nature 583, 115-121 (2020); Huang, Y.-G. et al. Sleep 44, zsab093 (2021); Akeju, O. et al. Clinical Neurophysiology 127, 2472-2481 (2016)). The synthetic state is characterized by a significant increase in 1-4Hz slow wave oscillations, atypical freezing behavior, hypothermia, and loss of coordinated movement. The anti-nociceptive profile accompanying this state transition may be confounded by and reflect the synthetic altered state of consciousness, though it presents a potential platform for dissociating neural circuit components of analgesia and unconsciousness, if such a dissociation exists (Weinrich, J. A. et al. bioRxiv 2023.04.03.534475 (2023)).

[0129] Single cell resolution brainwide activity network of isoflurane unconsciousness. To map the neural architecture of synthetically altered consciousness at single cell resolution across the intact brain, iDisco+ tissue clearing with mCherry-DREADD immunolabeling was used, followed by light sheet fluorescent microscopy (FIG. 3A). Globally, consistent brainwide TRAP2 cell population density across gross cortical and subcortical divisions was observed, besides significant elevations in the hindbrain and decreases in the cerebellum (FIG. 3B). To better understand brain region-specific differences, a voxel-based analysis was used where 3D and 2D TRAP2 densityheatmaps exhibit clear patterns of region-specific TRAP2 enrichment (FIGs. 3C, 3D). These expression patterns are not seen in non-TRAP controls (FIGs. 3E, 3F).

[0130] While the TRAP2 approach is highly specific (>95% of activity labelled cells are Fos+), it is only moderately efficient (-65% of Fos+ cells are activity labeled) (Allen, W. E. et al. Science 357, 1149-1155 (2017)). Similarly, following retro- orbital injection at viral titers like the disclosed (2-3 x 1011), PHP.eB neuron infection efficiency is high in the cortex (70% of neurons are infected), but moderate in other brain areas like the striatum (50% of neurons) (Chan, K. Y. et al. Nat Neurosci 20, 1172-1179 (2017)). Though these transgenic and viral efficiencies are sufficient for inducing robust behavioral changes (FIGs. 10A-10L), they introduce technical bias for analyzing endogenous brainwide structural and functional mechanisms associated with isoflurane-induced unconsciousness.

[0131] To overcome this, an unbiased brainwide approach was applied using immunolabeling for the immediate early gene Fos to examine the isoflurane-activated unconsciousness network at cellular resolution. Recent studies using endogenous Fos mapping after isoflurane focused on isolated brain regions such as the hypothalamus using a subanesthetic dose or performed Fos immunolabeling in non-contiguous 2D slices rather than the intact brain (Wasilczuk, A. Z. et al. Proc Natl Acad Sci U S A 121, e2312913120 (2024); Rijsketic, supra). Here, applying an approach that more closely captures globally distributed neurons, the network-level mechanism of anesthesia-induced altered consciousness can be probed in relation to understanding the key cell- specific network nodes engaged in the synthetic altered state.

[0132] FIGs. 13A-13E illustrate network analysis that reveals hubs that functionally differentiate an isoflurane unconscious state. Regional correlation heatmaps for (FIG. 13A) Control and (FIG. 13B) Isoflurane condition based on FIGs. 5A-5H (ClearMap atlas-registered mean Fos density data). Groupings of subregions by their larger parent regions are shown as sub-facets. FIG. 13C illustrates a combined network (bottom) showing the overlapping functional connectivity patterns of the individual control (top, red) and isoflurane (middle, blue) functional networks with matching network density (edge proportion = 3.15%) and the combined network below (see FIGs. 15A-15K for proportional thresholding). FIG. 13D illustrates a functional difference network generated by plotting significant permuted regional correlation differences as edges (lines), and the regions they connect as nodes (circles). The uniquely shaded convex hull polygons denote regions classified as members of the same community. Key community nodes are abbreviated C1: LM = lateral mamillary nucleus ; C2: MnPO = median preoptic nucleus ; C3: LPB = lateral parabrachial nucleus, C4: LA = lateroanterior hypothalamic nucleus; C5: MITg = microcellular tegmental nucleus; C6: Bar = Barrington nucleus; C7: VTM = ventral tuberomammillary nucleus ; C8: CeA = central amygdalar nucleus; C9: PrEW = pre-Edinger- Westphal nucleus. FIG. 13E illustrates anatomical distribution of key community nodes identified in FIG. 13D. See FIGs. 15A-15K for additional details.

[0133] FIGs. 12A-12D illustrate identification of isoflurane-activated clusters from UNRAVEL analysis. Voxel-wise data from FIGs. 5E-5H were warped to the Allen Mouse Brain Common Coordinate Framework (CCFv3) for display in coronal brain slices, overlaid with a colored wireframe using FSLeyes. Input images for voxel-wise comparisons wereaveraged for each group to show background-subtracted and z-scored Fos immunofluorescence. FDR-corrected p-value maps show increases (warm colors) and decreases (cool colors) in Fos expression resulting from isoflurane treatment. For each effect direction, the most stringent q-value threshold is fully opaque (100%), the next most stringent is semi-opaque (66%), and the least stringent is less opaque (33%). Valid clusters with significant differences in Fos+ cell density were randomly colored and labeled with their IDs. Abbreviations: Adj. = adjusted; Avg. = average; SS = somatosensory; OT = olfactory tubercle; SI = substantia innominata; BNST = bed nucleus of the stria terminalis; MA = magnocellular nucleus; PVH = paraventricular hypothalamic nucleus; Hypothal. = hypothalamus; CeA = central amygdala; Thai. = thalamus; Zl = zona incerta; COA = cortical amygdalar area; MB = midbrain; Sup. = superior; PAG = periaqueductal gray; Inf. = inferior.

[0134] First, an intact brainwide functional 'connectome' of isoflurane-induced unconsciousness was created. Mice were anesthetized with isoflurane at 1.2-1.3% for 180-min prior to fixation and intact brain processing (FIG. 5A). Control mice were singly housed in the home cage (see Methods). Two orthogonal approaches based on different pipelines (ClearMap and UNRAVEL) were used to analyze the whole brain imaging datasets via cell segmentation, counting, atlas registration, and brain region cell count annotation (FIGs. 5A-5H, 4A-4J, and 12A-12D). This was done in order to increase analytical rigor through the convergence of key brain regions across analyses. This analysis was followed with network and community analysis in SMARTTR (FIGs. 13A-13E) (Jin, supra). Together, these data provide a comprehensive technical resource describing brainwide cellular activation patterns after isoflurane exposure (FIGs.5A-5H) and an analysis of the brainwide networks to identify key functional communities associated with altered consciousness (FIGs. 13A-13E).

[0135] FIGs. 14A-14F illustrate representative Fos immunolabeling from cleared intact brains. Images from a representative control (left column) and an isoflurane mouse (right column) from FIGs. 5A-5H. Images were acquired with a light sheet microscope using a 3.6X objective in the horizontal plane and then re-sliced to the coronal plane to identify (FIG. 14A) BNST = bed nucleus of the stria terminalis, (FIG. 14B) PVH = paraventricular hypothalamus, (FIG.14C) CeA = central amygdala, (FIG. 14D) VMH = ventromedial hypothalamus, (FIG. 14E) PBN = parabrachial nucleus, (FIG. 14F) LC = locus coeruleus. Scale bars are 1mm for the full coronal slice and 500 urn for the inset image.

[0136] ClearMap and UNRAVEL provide complementary approaches to quantifying Fos density. While ClearMap uses the traditional approach of segmenting cellsand performing cell counts within annotated brain regions, UNRAVEL first quantifies bulk signal to identify clusters of significant voxel differences (agnostic to brain atlas region), counts Fos+ cells within these clusters, and summarizes predominant brain regions encompassed by each significant cluster (see Methods for a more detailed discussion). Using both approaches, overall differential whole brain activity patterns in the isoflurane state were found relative to control mice (FIGs. 5A-5H; see FIGs. 14A-14F for representative images). When analyzed at a gross hierarchical level, the overall total mean Fos density is consistent across analyzed samples, significantly decreases with isoflurane in the isocortex and midbrain, and increases in the olfactory areas, cortical subplate, hypothalamic areas and cerebellum (FIG. 4I). At the granular level, isoflurane increases activity withinsubregions of the striatum (caudoputamen and central amygdala), pallidum (bed nucleus of the stria terminalis), thalamus (paraventricular nucleus), hypothalamus (paraventricular and ventromedial nuclei), and hindbrain (parabrachial nucleus, locus coeruleus, and reticular nucleus) (FIGs. 5B-5D). UNRAVEL confirmed these increases in activity (FIGs. 5E-5H) and extended the findings (FIGs. 12A-12D). Together, a cortical to subcortical shift in activity in the isoflurane condition is described with key region-specific subcortical hotspots observed at cellular resolution in the intact brain.

[0137] FIGs. 15A-15K illustrate that isoflurane networks are denser across a variety of weight thresholds. FIG. 15A illustrates control (red) and isoflurane (blue) networks thresholded at various alpha values FIG. 15B illustrates mean degree, clustering, betweenness, and efficiency metrics calculated across a range of significance thresholds and (FIG.15C) edge proportion thresholds. FIG. 15D illustrates assessment of small-world properties of individual networks plotted in FIGs. 13A-13E. Small-world index (a) for both networks are above the threshold of 1 (black dotted horizontal line). The control network (FIG. 15E) clustering coefficient and (FIG. 15F) path length as well as the isoflurane (FIG.15G) clustering coefficient and (FIG. 15H) path length are higher than randomly rewired networks with matching degree sequences. FIG. 151 illustrates distributions of the Pearson correlation coefficients between all unique regional connections and their associated (FIG FIG. 15J) p-value for the control and isoflurane networks. FIG. 15K illustrates a volcano plot illustrating the regional correlations which most differ between the networks. The horizontal line marked with a triangle denotes a significance threshold of p < 0.001. Points in the upper right (control < isoflurane) and upper left quadrants (control > isoflurane), defined by vertical line lines, indicate regional correlation differences with a magnitude shift greater than 1 between groups. Two-tailed Students t-test: **p<0.01 , ****p<0.0001. Error bars represent mean ± SEM.

[0138] Network analysis reveals high interconnectivity of distributed communities. A given brain state is composed of dynamic interconnectivity between brain regions, so the brainwide Fos mapping datasets were leveraged to investigate how functional activity networks are changed in isoflurane-induced unconsciousness at the cellular resolution (FIGs. 13A-13E). Applying network analysis approaches to study the structure of connectivity (i.e., network topology), can generate insights about the functional patterns that differentiate behavioral states (Goodwin, supra; Pereira, T. D. et al. SLEAP: Nat Methods 19, 486-495 (2022); Renier, supra). SMARTTR, a newly developed accessible R package that supports the importation of externally mapped IEG datasets, was applied for network analysis and visualization (Jin, supra). Fos was cross-correlated and area-normalized across all mapped subregions (FIGs. 13A, 13B) and a higher number of strongly positive correlations in the isoflurane condition were found (FIGs.151, 15J). Since Fos is an activity-dependent marker, positive correlations between brain regions represent either coactivation or co- suppression, while negative correlations represent opposing activation.

[0139] To better investigate which individual connections are most functionally altered, each regional correlation coefficient in the control condition was subtracted from its corresponding value in the isoflurane condition, and each difference was compared to a permuted null distribution. Consistent with the global correlation analysis, there was agreater proportion of positively shifted connections in isoflurane FIGs. 13C and 15A-15K). Next, a functional difference network was constructed by visualizing the most significant correlation differences (p < 0.001) as connections or edges (lines), and the regions they connect as nodes (circles) (FIG. 13D). This network revealed a naturally high interconnectivity amongst regions with the most functionally different connections, visualized as a large, connected component (I. e., sub-network), rather than as collections of small, isolated networks or individual connections, as would be expected from chance.

[0140] Many complex real networks can be broken into subparts called communities, defined by nodes with higher interconnectivity or probability of interconnectivity to other members within their community versus without. An agglomerative community detection algorithm was used and nine distinct communities were discovered within the functional difference network (FIG. 13D, 13E) (Clauset, A. et al. Phys. Rev. E 70, 06611 (2004)). Metrics of nodal influence on a network, termed centrality, include measures such as degree (the number of direct connections to a node) and betweenness (how often a particular node lies on the shortest path between two other nodes). Examination of degree and betweenness revealed higher centrality of regions such as the median preoptic nucleus, microcellular tegmental nucleus, lateral mammillary nucleus, locus coeruleus, and lateral parabrachial nucleus. Qualitatively, these metrics align well with the central, influential positioning of these regions within distinct, spatially distributed communities.

[0141] Individual networks (edge proportion 3.15%) were constructed to examine more general topological properties (FIGs. 13C and 15A-15K). In the context of the brain, the property of small-worldness can be considered a balance between the capacity for functional specialization and integration (Watts & Strogatz. Nature 393, 440-442 (1998)). Both the control and isoflurane networks exhibited small-worldness, defined as a > 1 , although a was lower in isoflurane (3.61) compared to control (5.67) (FIG. 15D). Global network properties were examined across a range of alpha thresholds and proportional thresholds, finding the density of connections in the isoflurane network is consistently higher across a range of alpha thresholds, reflected in the higher trajectories of mean degree, clustering, and efficiency (FIG. 15B). This effect is mitigated when controlling for edge density (FIG. 15C). As a result, it was found that global topological properties are highly influenced by greater functional interconnectivity in the isoflurane unconsciousness network.

[0142] Heterogeneous brainwide single-unit activity in isoflurane unconsciousness. Since Fos is correlative and only a snapshot of activity, brainwide single unit activity (Neuropixels) was recorded from mice during isoflurane-induced unconsciousness to investigate the natural temporal dynamics of key structures in the network (FIGs. 6A, 6B). Neural responses to isoflurane induction and maintenance were summarized by comparing them to the mean Fos density difference observed for each structure between control and isoflurane conditions from the Fos activity map (FIGs. 6C, 6D).

[0143] Consistent with Fos immunolabeling, average population activity was significantly lower in cortical versus subcortical structures following isoflurane exposure (FIG. 6C). To examine shared dynamics among distributed neuralpopulations, k-means clustering was performed, and 6 clusters were identified that best described relative activity patterns between induction and maintenance phases (FIG. 6D). Clusters were heterogeneous, composed of both cortical and subcortical regions, though in general, clusters with greater relative firing during maintenance contained more subcortical regions compared to those with more cortical subregions. Fos density examined in parallel to each cluster subregion revealed that changes in neural activity generally trended with Fos differences.

[0144] Neural activity was highlighted in 3 subregions (reticular nucleus of thalamus, medial amygdalar nucleus and perireunensis nucleus) that showed significantly greater activation by isoflurane in the brainwide Fos analysis (FIGs.5A-5H). A temporal view of the mean normalized firing for these structures during isoflurane induction revealed timedependent shifts as well as maintained firing rates throughout induction (FIG. 6E). Over the recording session, isoflurane exposure led to broadly reduced firing rates, accounting for relatively low phase averages (FIGs. 6C, 6D). Maintenance activity was generally greater than the induction phase mean. When examining recordings across major brain regions, high individual variability was observed within each region, with some units showing greater and others showing lower firing than the average (FIGs. 7A-7C). As a result, spatially distributed brain regions demonstrate high heterogeneity in responding to isoflurane-induced unconsciousness, suggesting possible fluctuations of functional interconnectivity occur (Huang, Z. et al. Nat Commun 15, 7496 (2024); Vlisides, P. E. et al. Anesthesiology 130, 885-897 (2019); Claar, L. D. et al. eLife 12, RP84630 (2023)).

[0145] Discussion

[0146] This Example presents a genetic capture model for dissecting cellular contributions within the intact network. It was found that the synthetic state of altered consciousness recapitulates hallmarks of sedation and antinociception, driving slow wave oscillations and increasing subcortical activity while reducing cortical activity. The viral-genetic synthetic model provides a potential neuromodulatory platform for generating precise, desired consciousness state transitions, with applications for inducing sleep, sedation, analgesia or anesthesia when further refined for use outside of a transgenic mouse system. This Example defines the brainwide circuitry captured by the synthetic state compared to isoflurane-induced unconsciousness using intact brain activity mapping and functional network analysis, identifying 9 key communities in the unconsciousness network. Since Fos represents a static, indirect measure of neural activity, brainwide neural firing patterns of isoflurane-induced unconsciousnesswas further analyzed using Neuropixels high density silicone probes and their comparison to Fos. Overall increased subcortical activity was found with heterogeneous responding in any one subregion.

[0147] Recent circuit dissection studies in rodents have focused on stimulating or inhibiting isolated brain nuclei to uncover the contributions of individual brain regions to unconsciousness and arousal. These studies provide insights into region-specific contributions in isolation of global brain circuit activity leading to several paradoxical findings. For example, isolated stimulation of the median preoptic nucleus contributes to arousal from anesthesia despite it having high activity during sleep and anesthesia maintenance. Selective stimulation of GABA or glutamate subpopulations in the median preoptic nucleus is insufficient for generating unconsciousness (Vanini, G. et al. Curr Biol 30, 779 (2020)).The disclosed whole brain connectome identifies the median preoptic nucleus as one key community node contributing to the unconscious state. This suggests that the median preoptic nucleus, while significantly regulated by isoflurane, is insufficient to produce unconsciousness in isolation of the brainwide network. Other key community nodes include the lateral parabrachial nucleus and the central amygdala, each found previously to modulate pain responding under anesthesia. Parabrachial nucleus stimulation also does not independently generate unconsciousness, rather accelerates the arousal from an unconscious state (Muindi, F. et al. Behav. Brain Res. 306, 20-25 (2016)). The disclosed findings highlight the complexity of integrating this heterogeneous functional interconnectivity and underline the importance of harnessing an integrated, cell-specific systems-level investigation to understand mechanisms of altered consciousness.EXAMPLE CLAUSES1. A method, including: administering, to a subject, a therapeutically effective dose of a construct encoding a synthetic receptor; administering, to the subject in a first instance of a predetermined state characterized by a population of activated neurons of the subject, a therapeutically effective dose of an agent that induces activitydependent expression of the synthetic receptor in at least a portion of the activated neurons; and inducing the subject into a second instance of the predetermined state by administering, to the subject, a therapeutically effective dose of a ligand that specifically binds the synthetic receptor.2. The method of clause 1 , wherein the construct is within a viral vector.3. The method of clause 2, wherein the viral vector includes an adeno-associated viral vector and / or a lentiviral vector.4. The method of clause 2 or 3, the construct being a first construct, the synthetic receptor being a first synthetic receptor, and the ligand being a first ligand, wherein the viral vector further includes a second construct encoding a second synthetic receptor, and wherein the method further includes: removing the subject from the predetermined state by administering, to the subject, a therapeutically effective dose of a second ligand that specifically binds the second synthetic receptor.5. The method of clause 4, wherein administering, to the subject, a therapeutically effective dose of the second ligand that specifically binds the second synthetic receptor includes administering the therapeutically effective dose of the second ligand auricularly, cutaneously, epidurally, interstitially, intraabdominally, intracerebrally, intravenously, intradurally, nasally, parenterally, percutaneously, perineurally, orally, subcutaneously, or transdermally.6. The method of any of clauses 1-5, wherein the synthetic receptor includes a chemogenetic receptor.7. The method of clause 6, wherein the chemogenetic receptor includes a Designer Receptor Exclusively Activated by a Designer Drug (DREADD).8. The method of any of clauses 1-7, wherein administering, to the subject, the therapeutically effective dose of the construct includes retroorbitally injecting the therapeutically effective dose of the construct.9. The method of any of clauses 1-8, wherein the predetermined state includes a predetermined state of consciousness.10. The method of any of clauses 1-9, wherein the predetermined state includes an unconscious state.11. The method of any of clauses 1-10, wherein the predetermined state includes at least one of a state of analgesia, a state of fear, a state of satiety, a state of euphoria, a state of peripheral numbness, a state of alertness, a sleep state, a nondepressive state, a nonpsychotic state, a state of relaxation, an exercise-induced state, a thermoregulated state, or a pharmacologically-induced state.12 The method of any of clauses 1-11, wherein the activated neurons include neurons in the brain and / or spinal cord of the subject.13. The method of any of clauses 1-12, wherein the activated neurons include neurons in the peripheral nervous system of the subject.14. The method of any of clauses 1-13, wherein the activated neurons include at least one of: neurons in two or more spatial regions, neurons of two or more types, or neurons of two or more neural circuits.15 The method of any of clauses 1-14, wherein the agent includes 4-hydroxytamoxifen16. The method of any of clauses 1-15, wherein administering, to the subject, the therapeutically effective dose of the ligand that specifically binds the synthetic receptor includes administering the therapeutically effective dose of the ligand auricularly, cutaneously, epidurally, interstitially, intraabdominally, intracerebrally, intravenously, intradurally, nasally, parenterally, percutaneously, perineurally, orally, subcutaneously, or transdermally.17. The method of any of clauses 1-16, wherein the ligand includes clozapine-n-oxide.18 The method of any of clauses 1-17, further including: inducing the subject into the first instance of the predetermined state by administering, to the subject, at least one inducing agent.19. The method of clause 18, wherein the at least one inducing agent includes at least one of an anesthetic agent, a neuroactive agent, a psychoactive agent, or a food item.20. The method of clause 19, wherein the anesthetic agent includes isoflurane.21. The method of clause 19 or 20, wherein the anesthetic agent includes propofol.22 The method of any of clauses 1-21, further including: reducing expression of the synthetic receptor in the portion of the activated neurons by administering, to the subject, an inhibitor.23. The method of clause 22, wherein the inhibitor is configured to downregulate production of the synthetic receptor in the portion of the activated neurons or to cause internalization of the synthetic receptor.24. A method of inducing a subject into a predetermined state by activating a portion of neurons of the subject.25. The method of clause 24, wherein activating the portion of the neurons of the subject includes: administering, to the subject, a therapeutically effective dose of a designer ligand that specifically binds a synthetic receptor expressed by the portion of neurons of the subject.26. The method of clause 25, further including: inducing expression of the synthetic receptor by the portion of neurons.27. The method of any of clauses 24-26, wherein activating the portion of the neurons of the subject includes: acoustically stimulating the portion of neurons.28 The method of any of clauses 24-27, further including: identifying at least one location of the portion of neurons in the subject by detecting, from a sensor, activation of the portion of neurons when the subject is in the predetermined state.29. The method of clause 28, wherein the sensor includes a wireless subcutaneous mechanoacoustic device and / or electrocorticography electrodes.30. A kit, including: a therapeutically effective dose of a construct encoding a synthetic receptor; a therapeutically effective dose of an agent that induces activity-dependent expression of the synthetic receptor in at least a portion of activated neurons of a subject in a predetermined state; and a therapeutically effective dose of a ligand that specifically binds the synthetic receptor.31. The kit of clause 30, wherein the construct is within a viral vector.32. The kit of clause 31 , wherein the viral vector includes an adeno-associated viral vector and / or a lentiviral vector.33 The kit of clause 31 or 32, the construct being a first construct, the synthetic receptor being a first synthetic receptor, and the ligand being a first ligand, wherein the viral vector further includes a second construct encoding a second synthetic receptor, and wherein the kit further includes a therapeutically effective dose of a second ligand that specifically binds the second synthetic receptor.34. The kit of any of clauses 30-33, wherein the synthetic receptor includes a chemogenetic receptor.35. The kit of clause 34, wherein the chemogenetic receptor includes a Designer Receptor Exclusively Activated by a Designer Drug (DREADD).36. The kit of any of clauses 30-35, wherein the predetermined state includes at least one of a state of analgesia, a state of fear, a state of unconsciousness, state of satiety, a state of euphoria, a state of peripheral numbness, a state of alertness, a sleep state, a nondepressive state, a nonpsychotic state, or a state of relaxation.37. The kit of any of clauses 30-36, wherein the activated neurons include neurons in the brain and / or spinal cord of the subject.38 The kit of any of clauses 30-37, wherein the activated neurons include neurons in the peripheral nervous system of the subject.39. The kit of any of clauses 30-38, wherein the agent includes 4-hydroxytamoxifen.40. The kit of any of clauses 30-39, wherein the therapeutically effective dose of the ligand includes an oral formulation, a subcutaneous formulation, or a transdermal formulation.41. The kit of any of clauses 30-40, wherein the ligand includes clozapine-n-oxide.42. The kit of any of clauses 30-41 , further including: an inhibitor configured to reduce expression of the synthetic receptor in the portion of activated neurons.

[0148] The features disclosed in the foregoing description, or the following claims, or the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for attaining the disclosed result, as appropriate, may, separately, or in any combination of such features, be used for realizing implementations of the disclosure in diverse forms thereof.

[0149] As will be understood by one of ordinary skill in the art, each implementation disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of' limits the scope of the implementation to the specified elements, steps, ingredients or components and to those that do not materially affect the implementation. As used herein, the term “based on" is equivalent to “based at least partly on,” unless otherwise specified.

[0150] Unless otherwise indicated, all numbers expressing quantities, properties, conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1 % of the stated value.

[0151] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0152] The terms “a,” “an,” “the” and similar referents used in the context of describing implementations (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate implementations of the disclosure and does not pose a limitation on the scope of the disclosure. No language in the specification should be construed as indicating any non-claimed element essential to the practice of implementations of the disclosure.

[0153] Groupings of alternative elements or implementations disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0154] "Specifically binds" refers to an association of a binding domain (of, for example, a CAR binding domain or a nanoparticle selected cell targeting ligand) to its cognate binding molecule with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1 / M) equal to or greater than 105M1, while not significantly associating with any other molecules or components in a relevant environment sample. “Specifically binds” is also referred to as “binds” herein. Binding domains may be classified as "high affinity" or "low affinity". In particular embodiments, "high affinity" binding domains refer to those binding domains with a Ka of at least 107M1, at least 108M , at least 109M1, at least 1010M1, at least 1011M1, at least 1012M1, or at least 1013M L In particular embodiments, "low affinity" binding domains refer to those binding domains with a Ka of up to 107M’1, up to 106M1, up to 105M’1. Alternatively, affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10-5M to 10-13M). In certain embodiments, a binding domain may have "enhanced affinity," which refers to a selected or engineered binding domains with stronger binding to a cognate binding molecule than a wild type (or parent) binding domain. For example, enhanced affinity may be due to a Ka (equilibrium association constant) for the cognate binding molecule that is higher than the reference binding domain or due to a Kd (dissociation constant) for the cognate binding molecule that is less than that of the reference binding domain, or due to an off-rate (Koff) for the cognate binding molecule that is less than that of the reference binding domain. A variety of assays are known for detecting binding domains that specifically bind a particular cognate binding molecule as well as determining binding affinities, such as Western blot, ELISA, and BIACORE® analysis (see also, e.g., Scatchard, et al., 1949, Ann. N.Y. Acad. Sci. 51:660; and US 5,283,173, US 5,468,614, or the equivalent).

[0155] Unless otherwise indicated, the practice of the present disclosure can employ conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA. These methods are described in the following publications. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4nd Edition (2012); F. M. Ausubel, et al. eds., Current Protocols in Molecular Biology, (2003); the series Methods In Enzymology (Academic Press, Inc.); Behlke, et al., Polymerase Chain Reaction: Theory and Technology (2019); Greenfield, ed. Antibodies, A Laboratory Manual, Second Edition (2014); and Capes-Davis and R. I. Freshney, eds. Freshney's Culture of Animal Cells 8th Edition (2021).

[0156] Certain implementations are described herein, including the best mode known to the inventors for carrying out implementations of the disclosure. Of course, variations on these described implementations will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for implementations to be practiced otherwise than specifically described herein. Accordingly, the scope of this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by implementations of the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIMSWhat is claimed is:

1. A method, comprising:administering, to a subject, a therapeutically effective dose of a construct encoding a synthetic receptor; administering, to the subject in a first instance of a predetermined state characterized by a population of activated neurons of the subject, a therapeutically effective dose of an agent that induces activity-dependent expression of the synthetic receptor in at least a portion of the activated neurons; andinducing the subject into a second instance of the predetermined state by administering, to the subject, a therapeutically effective dose of a ligand that specifically binds the synthetic receptor.

2. The method of claim 1 , wherein the construct is within a viral vector.

3. The method of claim 2, wherein the viral vector comprises an adeno-associated viral vector and / or a lentiviral vector.

4. The method of claim 2, the construct being a first construct, the synthetic receptor being a first synthetic receptor, and the ligand being a first ligand, wherein the viral vector further comprises a second construct encoding a second synthetic receptor, andwherein the method further comprises:removing the subject from the predetermined state by administering, to the subject, a therapeutically effective dose of a second ligand that specifically binds the second synthetic receptor.

5. The method of claim 4, wherein administering, to the subject, a therapeutically effective dose of the second ligand that specifically binds the second synthetic receptor comprises administering the therapeutically effective dose of the second ligand auricularly, cutaneously, epidurally, interstitially, intraabdominally, intracerebrally, intravenously, intradurally, nasally, parenterally, percutaneously, perineurally, orally, subcutaneously, or transdermally.

6. The method of claim 1 , wherein the synthetic receptor comprises a chemogenetic receptor.

7. The method of claim 6, wherein the chemogenetic receptor comprises a Designer Receptor Exclusively Activated by a Designer Drug (DREADD).

8. The method of claim 1, wherein administering, to the subject, the therapeutically effective dose of the construct comprises retroorbitally injecting the therapeutically effective dose of the construct.

9. The method of claim 1 , wherein the predetermined state comprises a predetermined state of consciousness.

10. The method of claim 1 , wherein the predetermined state comprises an unconscious state.

11. The method of claim 1 , wherein the predetermined state comprises at least one of a state of analgesia, a state of fear, a state of satiety, a state of euphoria, a state of peripheral numbness, a state of alertness, a sleep state, a nondepressive state, a nonpsychotic state, a state of relaxation, an exercise-induced state, a thermoregulated state, or a pharmacologically-induced state.

12. The method of claim 1, wherein the activated neurons comprise neurons in the brain and / or spinal cord of the subject.

13. The method of claim 1, wherein the activated neurons comprise neurons in the peripheral nervous system of the subject.

14. The method of claim 1, wherein the activated neurons comprise at least one of: neurons in two or more spatial regions, neurons of two or more types, or neurons of two or more neural circuits.

15. The method of claim 1, wherein the agent comprises 4-hydroxytamoxifen.16 The method of claim 1, wherein administering, to the subject, the therapeutically effective dose of the ligand that specifically binds the synthetic receptor comprises administering the therapeutically effective dose of the ligand auricularly, cutaneously, epidurally, interstitially, intraabdominally, intracerebrally, intravenously, intradurally, nasally, parenterally, percutaneously, perineurally, orally, subcutaneously, or transdermally.

17. The method of claim 1 , wherein the ligand comprises clozapine-n-oxide.

18. The method of claim 1, further comprising:inducing the subject into the first instance of the predetermined state by administering, to the subject, at least one inducing agent.

19. The method of claim 18, wherein the at least one inducing agent comprises at least one of an anesthetic agent, a neuroactive agent, a psychoactive agent, or a food item.

20. The method of claim 19, wherein the anesthetic agent comprises isoflurane.

21. The method of claim 19, wherein the anesthetic agent comprises propofol.22 The method of claim 1 , further comprising:reducing expression of the synthetic receptor in the portion of the activated neurons by administering, to the subject, an inhibitor.

23. The method of claim 22, wherein the inhibitor is configured to downregulate production of the synthetic receptor in the portion of the activated neurons or to cause internalization of the synthetic receptor.

24. A method of inducing a subject into a predetermined state by activating a portion of neurons of the subject.25 The method of claim 24, wherein activating the portion of the neurons of the subject comprises:administering, to the subject, a therapeutically effective dose of a designer ligand that specifically binds a synthetic receptor expressed by the portion of neurons of the subject.

26. The method of claim 25, further comprising:inducing expression of the synthetic receptor by the portion of neurons.

27. The method of claim 24, wherein activating the portion of the neurons of the subject comprises:acoustically stimulating the portion of neurons.

28. The method of claim 24, further comprising:identifying at least one location of the portion of neurons in the subject by detecting, from a sensor, activation of the portion of neurons when the subject is in the predetermined state29. The method of claim 28, wherein the sensor comprises a wireless subcutaneous mechanoacoustic device and / or electrocorticography electrodes.

30. A kit, comprising:a therapeutically effective dose of a construct encoding a synthetic receptor;a therapeutically effective dose of an agent that induces activity-dependent expression of the synthetic receptor in at least a portion of activated neurons of a subject in a predetermined state; anda therapeutically effective dose of a ligand that specifically binds the synthetic receptor.

31. The kit of claim 30, wherein the construct is within a viral vector.

32. The kit of claim 31 , wherein the viral vector comprises an adeno-associated viral vector and / or a I entivi ral vector.

33. The kit of claim 31 , the construct being a first construct, the synthetic receptor being a first synthetic receptor, and the ligand being a first ligand, wherein the viral vector further comprises a second construct encoding a second synthetic receptor, andwherein the kit further comprises a therapeutically effective dose of a second ligand that specifically binds the second synthetic receptor.

34. The kit of claim 30, wherein the synthetic receptor comprises a chemogenetic receptor.

35. The kit of claim 34, wherein the chemogenetic receptor comprises a Designer Receptor Exclusively Activated by a Designer Drug (DREADD).

36. The kit of claim 30, wherein the predetermined state comprises at least one of a state of analgesia, a state of fear, a state of unconsciousness, state of satiety, a state of euphoria, a state of peripheral numbness, a state of alertness, a sleep state, a nondepressive state, a nonpsychotic state, or a state of relaxation.

37. The kit of claim 30, wherein the activated neurons comprise neurons in the brain and / or spinal cord of the subject.38 The kit of claim 30, wherein the activated neurons comprise neurons in the peripheral nervous system of the subject.

39. The kit of claim 30, wherein the agent comprises 4-hydroxytamoxifen.

40. The kit of claim 30, wherein the therapeutically effective dose of the ligand comprises an oral formulation, a subcutaneous formulation, or a transdermal formulation.

41. The kit of claim 30, wherein the ligand comprises clozapine-n-oxide.42 The kit of claim 30, further comprising:an inhibitor configured to reduce expression of the synthetic receptor in the portion of activated neurons.

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