Stabilized step function opsin proteins and methods of using the same

Abstract

Provided herein are compositions comprising non-human animals comprising neurons expressing stabilized step function opsin proteins on neural plasma membranes and methods of using the same to selectively depolarize neurons residing in microcircuits of the pre-frontal cortex to affect one or more social behaviors, communications, and/or conditioned behaviors in the non-human animal.

What is claimed is: 
     
         1 . A non-human animal comprising a first light-activated cation channel protein expressed in neurons of the pre-frontal cortex of the animal, wherein the protein is capable of inducing depolarizing current in the neurons by light and exhibits rapid step-like activation in response to a single pulse of light having a first wavelength and deactivation in response to a pulse of light having a second wavelength, wherein the depolarizing current in the neurons is maintained for at least about ten minutes; and wherein the activation of the protein in the pre-frontal cortex neurons induces changes in social behaviors, communications, and/or conditioned behaviors in the animal. 
     
     
         2 . The animal of  claim 1 , wherein the protein comprises the amino acid sequence of ChR2, ChR1, VChR1, or VChR2 with an amino acid substitution at a residue corresponding to C128 or D156 of the amino acid sequence of ChR2. 
     
     
         3 . The animal of  claim 1 , wherein the protein comprises the amino acid sequence of ChR2, ChM, VChR1, or VChR2 with amino acid substitutions at a residues corresponding to C128 and D156 of the amino acid sequence of ChR2. 
     
     
         4 . The animal of  claim 2  or  3 , wherein the residue corresponding to C128 of the amino acid sequence of ChR2 is substituted to serine. 
     
     
         5 . The animal of  claim 2  or  3 , wherein the residue corresponding to D156 of the amino acid sequence of ChR2 is substituted to a non-acidic amino acid. 
     
     
         6 . The animal of  claim 2  or  3 , wherein the residue corresponding to D156 of the amino acid sequence of ChR2 is substituted to alanine. 
     
     
         7 . The animal of  claim 1 , wherein the protein comprises an amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO:1, and wherein C128 is substituted with serine and D156 is substituted with alanine. 
     
     
         8 . The animal of any one of  claims 1 - 7 , wherein the protein is expressed by delivering the polynucleotide sequence encoding the protein using a viral vector. 
     
     
         9 . The animal of  claim 8 , wherein the viral vector is an adeno-associated viral vector, a retroviral vector, a lentiviral, an adenoviral vector, or a HSV vector. 
     
     
         10 . The animal of any one of  claims 1 - 9 , wherein said social behaviors are selected from the group consisting of allogrooming, resident-intruder aggression, isolation-induced fighting, sexual behavior, parental behavior, social recognition, and auditory communication. 
     
     
         11 . The animal of  claim 11 , wherein the neurons are in the infralimbic or prelimbic subregions of the medial prefrontal cortex. 
     
     
         12 . The animal of any one of  claims 1 - 11 , wherein the protein is expressed in either inhibitory neurons or excitatory neurons of the pre-frontal cortex. 
     
     
         13 . The animal of  claim 12 , wherein the excitatory neurons are pyramidal neurons. 
     
     
         14 . The animal of  claim 12 , wherein the inhibitory neurons are GABAergic parvalbumin neurons. 
     
     
         15 . The animal of  claim 14 , wherein the animal further comprises a second light-activated protein expressed in said inhibitory neurons or said excitatory neurons of the pre-frontal cortex and wherein the second light activated protein is not expressed in the same neurons as the first light-activated protein. 
     
     
         16 . The animal of  claim 15 , wherein the second light activated protein is C1V1. 
     
     
         17 . A brain slice comprising neurons of the pre-frontal cortex, wherein a light-activated protein is expressed in the neurons of the pre-frontal cortex, wherein the protein is capable of inducing depolarizing current in the neurons by light and exhibits rapid step-like activation in response to a single pulse of light having a first wavelength and deactivation in response to a pulse of light having a second wavelength; wherein the depolarizing current in the neurons is maintained for at least about ten minutes. 
     
     
         18 . The brain slice of  claim 17 , wherein the protein is selected from the group consisting of ChR2, ChR1, VChR1, or VChR2. 
     
     
         19 . The brain slice  claim 18 , wherein the opsin comprises at least one amino acid substitution at amino acid residues corresponding to C128 and D156 of the amino acid sequence of ChR2. 
     
     
         20 . The brain slice of  claim 19 , wherein the substitution at the amino acid residue corresponding to C128 of the amino acid sequence of ChR2 is a substitution to serine. 
     
     
         21 . The brain slice of  claim 19 , wherein the substitution at the amino acid residue corresponding to D156 of the amino acid sequence of ChR2 is a substitution to a non-acidic amino acid. 
     
     
         22 . The brain slice of  claim 19 , wherein the substitution at the amino acid residue corresponding to D156 of the amino acid sequence of ChR2 is a substitution to alanine. 
     
     
         23 . The brain slice of  claim 19 , wherein the protein comprises an amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO:1, and wherein C128 is substituted with serine and D156 is substituted with alanine. 
     
     
         24 . The brain slice of any one of  claims 17 - 23 , wherein the protein is expressed in either inhibitory neurons or excitatory neurons of the pre-frontal cortex. 
     
     
         25 . A method for identifying a chemical compound that inhibits the depolarization of excitatory or inhibitory neurons in the prefrontal cortex of a non-human animal, the method comprising:
 (a) depolarizing excitatory or inhibitory neurons in the prefrontal cortex of a non-human animal comprising a first light-activated protein cation channel protein expressed on the cell membrane of the neurons of the pre-frontal cortex of the animal, wherein the protein is capable of mediating a depolarizing current in the neurons when the neurons are illuminated with light, wherein the protein exhibits rapid step-like activation in response to a single pulse of light having a first wavelength and deactivation in response to a pulse of light having a second wavelength; wherein the depolarizing current in the neurons is maintained for at least about ten minutes; wherein the protein comprises the amino acid sequence of ChR2, ChR1, VChR1, or VChR2 with amino acid substitutions at amino acid residues corresponding to C128 and D156 of the amino acid sequence of ChR2; wherein the activation of the protein in the pre-frontal cortex neurons induces changes in social behaviors, communications, and/or conditioned behaviors in the animal;   (b) measuring an excitatory post synaptic potential (EPSP) or an inhibitory post synaptic current (IPSC) in response to selectively depolarizing the excitatory neurons comprising the light-activated protein;   (c) contacting the excitatory or inhibitory neurons with a chemical compound; and   (d) measuring the excitatory post synaptic potential (EPSP) or the inhibitory post synaptic current (IPSC) to determine if contacting the excitatory neurons with the chemical compound inhibits the depolarization of the neurons.   
     
     
         26 . The method of  claim 25 , wherein the light activated protein comprises an amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO:1 and wherein the protein has a D to A mutation at amino acid 156. 
     
     
         27 . The method of  claim 25 , wherein the single pulse of light having a first wavelength is for 100 milliseconds or less. 
     
     
         28 . The method of  claim 25 , wherein the first wavelength is blue light and wherein the second wavelength is green or yellow light. 
     
     
         29 . A method for identifying a chemical compound that restores a social behavior, communication, and/or conditioned behavior in a non-human animal, the method comprising:
 (a) depolarizing excitatory neurons in the prefrontal cortex of a non-human animal comprising a light-activated protein cation channel protein expressed on the cell membrane of the neurons, wherein the protein is capable of inducing a depolarizing current in the neurons when the neurons are illuminated with light, wherein the protein exhibits rapid step-like activation in response to a single pulse of light having a first wavelength and deactivation in response to a pulse of light having a second wavelength; wherein the depolarizing current in the neurons is maintained for at least about ten minutes; and wherein the protein comprises the amino acid sequence of ChR2, ChR1, VChR1, or VChR2 with amino acid substitutions at amino acid residues corresponding to C128 and D156 of the amino acid sequence of ChR2, wherein depolarizing the excitatory neuron inhibits one or more social behaviors, communications, and/or conditioned behaviors in the non-human animal;   (b) administering a chemical compound to the non-human animal; and   (c) determining if the administration of the chemical compound to the non-human animal restores said one or more social behaviors, communications, and/or conditioned behaviors in the non-human animal.   
     
     
         30 . The method of  claim 29 , wherein the social behavior is selected from the group consisting of: allogrooming, resident-intruder aggression, isolation-induced fighting, sexual behavior, parental behavior, social recognition, and auditory communication. 
     
     
         31 . The method of  claim 29 , wherein the administration of the chemical compound decreases or inhibits depolarization of excitatory neurons. 
     
     
         32 . The method of  claim 29 , wherein the administration of the chemical compound increases depolarization of inhibitory neurons. 
     
     
         33 . A method of modeling the symptoms of a central nervous system (CNS) disorder in a patient, the method comprising:
 (a) identifying a target neural circuit associated with the CNS disorder;   (b) expressing light-responsive opsins in the target neural circuit;   (c) activating the light-responsive opsins in the target neural circuit; and   (d) monitoring a symptom of the central nervous system (CNS) disorder.   
     
     
         34 . A method of  claim 33 , wherein activating the light responsive opsins includes elevating an excitation/inhibition (E/I) balance in the target neural circuit while preserving the responsiveness of the targeted neural circuit to intrinsic electrical activity, wherein symptoms of the CNS disorder are temporally increased. 
     
     
         35 . The method of  claim 33 , wherein the target neural circuit is located in the prefrontal cortex. 
     
     
         36 . The method of  claim 33 , wherein the step of activating includes increasing the excitation relative to the inhibition in the target neural circuit. 
     
     
         37 . The method of  claim 33 , wherein the step of expressing further includes targeting cell-types of the target neural circuit for increased expression of exogenous light-responsive ion channels relative to other cell-types of the target neural circuit. 
     
     
         38 . A method of modeling the symptoms of a central nervous system (CNS) disorder in a patient, the method comprising: modifying an excitation/inhibition (E/I) balance in a target neural circuit of the CNS while preserving the responsiveness of the targeted neural circuit to intrinsic electrical activity, wherein symptoms of the CNS disorder are temporally increased. 
     
     
         39 . The method of  claim 38 , wherein the step of modifying an E/I balance includes applying optical stimulus to exogenous light-responsive ion channels that are expressed in cells of the targeted neural circuit of the CNS. 
     
     
         40 . The method of  claim 38 , wherein the target neural circuit is located in the prefrontal cortex. 
     
     
         41 . The method of  claim 38 , wherein the step of modifying includes increasing the excitation relative to the inhibition in the target neural circuit. 
     
     
         42 . The method of  claim 38 , further including the step of targeting cell-types of the target neural circuit for increased expression of exogenous light-responsive ion channels relative to other cell-types of the target neural circuit. 
     
     
         43 . A method comprising: assessing the effects of a treatment for a central nervous system (CNS) disorder by expressing light-responsive opsins in a target neural circuit associated with the CNS disorder; activating the light-responsive opsins in the target neural circuit, wherein symptoms of the CNS disorder are increased; applying the treatment; and monitoring the symptoms of the CNS disorder. 
     
     
         44 . A method for assessing the effects of a treatment for substance dependence, the method comprising: artificially inducing a central nervous system (CNS) disorder in an animal by activating the light-responsive opsins in a target neural circuit associated with the CNS disorder, wherein symptoms of the CNS disorder are increased; applying the treatment; and monitoring the symptoms of the CNS disorder. 
     
     
         45 . A system comprising: a set of neurons associated with a CNS disorder; a drug delivery device for providing drugs to the set of neurons; and a monitoring device for assessing activity of the set of neurons in response to the drugs being provided to the set of neurons. 
     
     
         46 . The system of  claim 45 , wherein the set of neurons include light-responsive opsins, and wherein the system further includes an optical delivery system for exciting the neurons by activating the light-responsive opsins. 
     
     
         47 . The system of  claim 45 , wherein the monitoring device is further configured to assess the activity of the set of neurons by monitoring electrical activation of the set of neurons. 
     
     
         48 . The system of  claim 45 , wherein the monitoring device is further configured to assess the activity of the set of neurons by monitoring electrical activation of the set of neurons. 
     
     
         49 . A method of  claim 33 , wherein activating the light responsive opsins includes lowering an excitation/inhibition (E/I) balance in the target neural circuit while preserving the responsiveness of the targeted neural circuit to intrinsic electrical activity. 
     
     
         50 . A method comprising: elevating an excitation/inhibition (EA) balance in a targeted neural circuit in a prefrontal cortex of a subject/patient while preserving the responsiveness of the targeted neural circuit to intrinsic electrical activity, wherein symptoms of a disorder are temporally increased; introducing a stimulus to the subject; and monitoring the symptoms of the disorder. 
     
     
         51 . The method of  claim 50 , further including assessing the effect of the stimulus on the symptoms. 
     
     
         52 . The method of  claim 50 , wherein the symptoms of the disorder include symptoms of schizophrenia. 
     
     
         53 . The method of  claim 50 , wherein the symptoms of the disorder include symptoms of autism. 
     
     
         54 . The method of  claim 50 , wherein the stimulus is a drug-based treatment. 
     
     
         55 . The method of  claim 50 , wherein the stimulus is a psychological-based treatment. 
     
     
         56 . The method of  claim 54 , wherein pyramidal cells and parvalbumin cells in the prefrontal cortex are monitored for an effect of the drug-based treatment. 
     
     
         57 . The method of  claim 50 , further including determining the relative effect of excitatory pyramidal neurons and parvalbumin-expressing inhibitory intemeurons in the prefrontal cortex on the E/I balance in the targeted neural circuit. 
     
     
         58 . The method of  claim 57 , further including assessing the relative effect of a drug on the pyramidal neurons and the parvalbumin intemeurons. 
     
     
         59 . The method of  claim 50 , wherein the stimulus inhibits pyramidal neurons in the prefrontal cortex. 
     
     
         60 . The method of  claim 50 , wherein the stimulus excites pyramidal neurons in the prefrontal cortex. 
     
     
         61 . The method of  claim 50 , wherein the stimulus inhibits inhibitory or parvalbumin-expressing interneurons in the prefrontal cortex. 
     
     
         62 . The method of  claim 50 , wherein the stimulus excites inhibitory or parvalbumin-expressing interneurons in the prefrontal cortex. 
     
     
         63 . The method of  claim 50 , wherein the stimulus excites one of a set of pyramidal neurons and parvalbumin-expressing interneurons in the prefrontal cortex and inhibiting the other one of the set of pyramidal neurons and parvalbumin-expressing interneurons in the prefrontal cortex. 
     
     
         64 . A method comprising: providing a stimulus to a subject, the subject exhibiting symptoms of a neurological disease; and assessing the effect of the stimulus on the symptoms. 
     
     
         65 . The method of  claim 64 , wherein the stimulus is a potential treatment and assessing the effect of the stimulus includes determining the efficacy of the potential treatment. 
     
     
         66 . The method of  claim 16 , further including modifying the potential treatment based on the determined efficacy of the potential treatment, delivering the modified potential treatment to the subject, and accessing the efficacy of the modified potential treatment. 
     
     
         67 . Devices, reagents, tools, technologies, methods and approaches for using models of disorders to study the disorders, to identify phenotypes/endophenotypes, and/or to identify treatments. 
     
     
         68 . A method comprising: lowering an excitation/inhibition (E/I) balance in a targeted neural circuit in a prefrontal cortex of a subject while preserving the responsiveness of the targeted neural circuit to intrinsic electrical activity; introducing a stimulus to the subject; and monitoring the symptoms of the disorder.