AU2015203097B2 - Light-activated chimeric opsins and methods of using the same - Google Patents

Abstract

Provided herein are compositions comprising light-activated chimeric proteins expressed on plasma membranes and methods of using the same to selectively depolarize excitatory or inhibitory neurons. WO 2012/061679 PCT/US2011/059276 a b VChR1-YFP C1V1-YFP x C1V1-ts-YFP 911 11 12= 0R VChR1 ClV1

Description

2015203097 10 Jun2015

AUSTRALIA

Patents Act 1990

THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY

COMPLETE SPECIFICATION STANDARD PATENT

Invention Title:

Light-activated chimeric opsins and methods of using the same

The following statement is a full description of this invention including the best method of performing it known to us :-

LIGHT-ACTIVATED CHIMERIC OPSINS AND 2015203097 10 Jun2015

METHODS OF USING THE SAME CROSS-REFERENCE TO RELATED APPLICATIONS This application is a divisional of Australia» Patent Application No, 201023220 which 5 eosTsspomls to International Application No. PCT/US2i l 1/059270 filed on November 4, 201 i which claims priority to LIS. Provisional Patent Application Nos, 01/41(),730 Sled on November 5, 2010:; §1/410,744 Sled on November 5, 2010; and b I /311,912 filed, on Inly 2b, 2011, toe disclosures of each of which are incorporated herei n by reference in their entireties.

10 TECHNICAL FIELD

This application pertains to compositions comprising animal cells expressing light-activated chimeric proteins on their plasma membranes and methods of using the same to selectively depolarize excitatory or inhibitory neurons residing in the same microcircuit in the pre-frontal cortex.

IS BACKGROUND

The neurophysiological substrates of most psychiatric disorders are poorly understood, despite rapidly emerging information on genetic factors that are associated with complex behavioral phenotypes such as those observed in autism and schizophrenia (Cichonet al., The American Journal of Psychiatry 166(5):540 (2009); O'Donovan et al., 20 Human Genetics 126(1): 3 (2009)). One remarkable emerging principle is that a very broad range of seemingly unrelated genetic abnormalities can give rise to the same class of psychiatric phenotype (such as social behavior dysfunction; Folstein & Rosen-Sheidley, Nature Reviews 2(12):943 (2001)). This surprising pattern has pointed to the need to identify simplifying circuit-level insights that could unify diverse genetic factors under a 25 common pathophysiological principle.

One such circuit-level hypothesis is that elevation in the ratio of cortical cellular excitation and inhibition (cellular E/I balance) could give rise to the social and cognitive deficits of autism (Rubenstein, Current Opinion in Neurology 23(2):118; Rubenstein & Merzenich, Genes, Brain, and Behavior 2(5):255 (2003)). This hypothesis could 30 potentially unify diverse streams of pathophysiological evidence, including the observation that many autism-related genes are linked to gain-of-function phenotypes in ion channels and synaptic proteins (Bourgeron, Current Opinion in Neurobiology 19 (2), 231 (2009)) and that -30% of autistic patients also show clinically apparent seizures (Gillberg & Billstedt, Acta Psychiatrica Scandinavica, 102(5):321 (2000)). However, it has not been clear if such an imbalance (to be relevant to disease symptoms) would be operative on the chronic (e.g. during development) or the acute timescale. Furthermore, this hypothesis is by no means universally accepted, in part because it has not yet been susceptible to direct testing. Pharmacological and electrical interventions lack the 5 necessary specificity to selectively favor activity (in a manner fundamentally distinct from receptor modulation) of neocortical excitatory cells over inhibitory cells, whether in the clinical setting or in freely behaving experimental mammals during social and cognitive tasks. It is perhaps related to challenges such as this that the social and cognitive deficits of autism and schizophrenia have proven largely unresponsive to conventional 10 psychopharmacology treatments in the clinic. 2015203097 10 Jun2015

Optogenetics is the combination of genetic and optical methods used to control specific events in targeted cells of living tissue, even within freely moving mammals and other animals, with the temporal precision (millisecond-timescale) needed to keep pace with functioning intact biological systems. The hallmark of optogenetics is the introduction of 15 fast light-activated channel proteins to the plasma membranes of target neuronal cells that allow temporally precise manipulation of neuronal membrane potential while maintaining cell-type resolution through the use of specific targeting mechanisms. Among the microbial opsins which can be used to investigate the function of neural systems are the channelrhodopsins (ChR2, ChRl, VChRl, and SFOs) used to promote depolarization in 20 response to light. In just a few short years, the field of optogenetics has furthered the fundamental scientific understanding of how specific cell types contribute to the function of biological tissues such as neural circuits in vivo. Moreover, on the clinical side, optogenetics-driven research has led to insights into Parkinson's disease and other neurological and psychiatric disorders. 25 However, there are limitations to existing optogenetic tools for exploring the hypothesis that elevation in the ratio of cortical E/I balance might be associated with the social and cognitive deficits of autism and other disorders such as schizophrenia. Conventional channelrhodopsin photocurrents display significant desensitization which precludes the generation of step-like changes in E/I balance (instead requiring ramping 30 or pulsing, which would not be suitable for investigation of stable changes in cellular E/I balance); moreover, both SFOs and conventional ChRs are driven by blue light, which precludes, within-preparation comparison of the effects of driving different populations of circuit elements (such as excitatory and inhibitory neurons). Therefore, what is needed is a tool that would allow the manipulation of cortical E/I balances and the monitoring of 2015203097 29 Aug 2017 gamma oscillations in cortical slices to permit the investigation of how these manipulations affect downstream neurons residing in the same microcircuit in the prefrontal cortex.

BRIEF SUMMARY OF THE INVENTION 5 Provided herein are compositions comprising chimeric light-activated protein cation channels which are capable of mediating a depolarizing current in the cell when the cell is illuminated with light.

Provided herein are animal cells comprising a light-activated protein expressed on the cell membrane, wherein the protein is (a) a chimeric protein derived from 10 VChRl from Volvox carteri and ChRl from Chlamydomonas reinhardti, wherein the protein comprises the amino acid sequence of VChRl having at least the first and second transmembrane helices replaced by the first and second transmembrane helices of ChRl; (b) is responsive to light; and (c) is capable of mediating a depolarizing current in the cell when the cell is illuminated with light. In some embodiments, the 15 cells are isolated or in a cell culture medium.

Provided herein in one embodiment is an isolated light-responsive chimeric polypeptide comprising an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:l.

Also provided herein is a population of cells comprising the cell expressing the 20 chimeric protein described herein on the cell membrane. Also provided herein are nonhuman animals and brain tissue slices comprising a cell expressing the chimeric protein described herein on the cell membrane.

Provided herein are polynucleotide comprising a nucleotide sequence encoding a light activated protein expressed on the cell membrane, wherein the protein is a 25 chimeric protein derived from VChRl from Volvox carteri and ChRl from

Chlamydomonas reinhardti, wherein the protein comprises the amino acid sequence of VChRl having at least the first and second transmembrane helices replaced by the first and second transmembrane helices of ChRl; is responsive to light; and is capable of mediating a depolarizing current in the cell when the cell is illuminated with light. 30 Vectors (such as expressing vectors) comprising the polynucleotides are also provided. In some embodiments, the expression vector is a viral vector (e.g., an AAV vector, a retroviral vector, an adenoviral vector, a HSV vector, or a lentiviral vector).

Also provided herein are methods of using the animal cells expressing the chimeric protein described herein on the cell membrane, the methods comprise 35 activating the chimeric protein with light. 2015203097 29 Aug 2017

Also provided herein are methods of selectively depolarizing excitatory or inhibitory neurons residing in the same microcircuit, the methods comprising: selectively depolarizing an excitatory neuron comprising a first light-activated protein, wherein the first light activated protein is depolarized when exposed to light having a 5 first wavelength; or selectively depolarizing an inhibitory neuron comprising a second light-activated protein, wherein the second light activated protein is depolarized when exposed to light having a second wavelength.

In some embodiments, the first or the second light activated protein is a chimeric protein derived from VChRl from Volvox carteri and ChRl from Chlamydomonas reinhardti, wherein the protein comprises the amino acid sequence of VChRl having at least the first and second transmembrane helices replaced by the first and second transmembrane helices of ChRl. In 5 some embodiments, wherein the first light-activated protein comprises an amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO: 1, 3, 5, or 7, and wherein the second light-activated protein comprises an amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO:l 1, 12, 13, or 14. 2015203097 10 Jun2015 A method of selectively depolarizing excitatory or inhibitory neurons residing in the 10 same microcircuit, the method comprising: expressing a first light-activated protein in an excitatory neuron; and expressing a second light activated protein in an inhibitory neuron, wherein the first light activated protein is independently depolarized when exposed to light having a first wavelength and wherein the second light activated protein is independently depolarized when exposed to light having a second wavelength. In some embodiments, the first 15 or the second light activated protein is a chimeric protein derived from VChRl from Volvox carteri and ChRl from Chlamydomonas reinhardti, wherein the protein comprises the amino acid sequence of VChRl having at least the first and second transmembrane helices replaced by the first and second transmembrane helices of ChRl. In some embodiments, the first light-activated protein comprises an amino acid sequence at least 95% identical to the sequence 20 shown in SEQ ID NO: 1,3,5, or 7, and wherein the second light-activated protein comprises an amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO:l 1, 12, 13, or 14.

Also provided herein are methods for identifying a chemical compound that selectively inhibits the depolarization of excitatory or inhibitory neurons residing in the same microcircuit, 25 the method comprising:(a) selectively depolarizing an excitatory neuron comprising a first light-activated protein with light having a first wavelength or selectively depolarizing an inhibitory neuron comprising a second light-activated protein with light having a second wavelength; (b) measuring an excitatory post synaptic potential (EPSP) in response to selectively depolarizing the excitatory neuron comprising a first light-activated protein or measuring an inhibitory post 30 synaptic current (IPSC) in response to selectively depolarizing an inhibitory neuron comprising a second light-activated protein; (c) contacting the excitatory neuron or the inhibitory neuron with a chemical compound; (d) measuring the excitatory post synaptic potential (EPSP) or measuring the inhibitory post synaptic current (IPSC) to determine if contacting either the excitatory neuron or the inhibitory neuron with the chemical compound selectively inhibits the 35 depolarization of either neuron. In some embodiments, the first or the second light activated protein is a chimeric protein derived from VChRl from Volvox carteri and ChRl from 2015203097 29 Aug 2017

Chlamydomonas reinhardti, wherein the protein comprises the amino acid sequence of VChRl having at least the first and second transmembrane helices replaced by the first and second transmembrane helices of ChRl. In some embodiments, the first light-activated protein comprises an amino acid sequence at least 95% identical to the 5 sequence shown in SEQ ID NO: 1, 3, 5, or 7, and wherein the second light-activated protein comprises an amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO:l 1, 12, 13, or 14.

It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the 10 present invention. These and other aspects of the invention will become apparent to one of skill in the art.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of 15 any other element, integer or step, or group of elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority 20 date of each of the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 depicts engineering of an improved red-shifted channelrhodopsin for combinatorial optogenetics. (a) Confocal images of cultured hippocampal neurons transfected with VChR 1-eYFP or CIVl-eYFP under the control of the CaMKIIa 25 promoter. Box denotes region expanded in the last panel, showing dendritic membrane localization of CIVl-tsYFP. Scale bars: 20 pm (left), 4 pm {right), (b) Peak photocurrents from whole-cell patch clamp recordings in-cultured hippocampal neurons expressing indicated opsins, (c) Sequence alignment of ChRl, ChR2 and VChRl. Splice sites for two ClVI variants are indicated. Putative transmembrane 30 helices 1-7 are indicated with bars (TM1-7); mutated amino acids indicated in grey, (d) Photocurrent amplitudes recorded in HEK cells expressing ClVI splice variants 1 and 2. (e) Single confocal plane images of cultured hippocampal neurons transfected with indicated opsins, fused to EYFP. DNA concentration was matched across constructs, (f) Action spectra of ChR2, VChRl, C1V1 wt, CIV (E122T), C1V1 (E162T), and 35 Cl VI (E122T/E162T). Photocurrents were collected with 2ms light pulses in HEK293 2015203097 29 Aug 2017 cells, (g) Ion permeance of Cl VI splice variant 1 as measured by photocurrent magnitude at -40 mV in HEK cells by whole cell patch clamp using cation-isolating external solutions. Data were normalized to the maximum peak Na current, (h) Schematic of the Cl VI chimera with point mutation positions indicated in white. ChRl 5 sequence indicated with black; VChRl sequence with grey. FIG. 2 depicts testing an improved red-shifted channelrhodopsin for combinatorial optogenetics. (a) Representative traces and summary plot of channel closure time constant (toff) in cultured neurons expressing the indicated opsins; traces are normalized to peak current, (b) C1V1-E122T inactivation compared to deactivation 10 of ChR2. (c) Inactivation of current in Cl VI double mutant E122T/E162T versus other Cl VI variants, (d) Mean peak photocurrents recorded in cultured neurons expressing the indicated opsins in response to a 2ms 542nm light pulse. FIG. 3 depicts photocurrents from acute slice recordings in prefrontal pyramidal neurons, (a) Peak photocurrents show consistent correlation with integrated fluorescence intensity, (b) Fluorescence-photocurrent relationship in ChR2(H134R) and C1V1(E122T/E162T). Black lines are linear fits to the data, (c) Acute slice recordings in 5 prefrontal pyramidal neurons stimulated with 560 nm light pulse trains or current injections at the indicated frequencies. Summary graph shows population data (n = 6). (d) Fraction of successful spikes to current injections (200 pA, 10 ms pulses; top left) or 2 ms light pulses at the indicated wavelengths and light power densities. All pulse trains consisted of 20x2 ms pulses delivered through the microscope objective using a Sutter DG-4 light source, 2015203097 10 Jun2015 10 filtered using 20 nm bandpass filters and additional neutral density filters to attenuate light power (n = 6 cells in 2 slices), (e) Voltage-clamp responses to 542 nm and 630 nm light pulses in cells expressing C1V1-E122T or C1V1-E 122ΊΥΕ 162T (top). Current-clamp recording in a Cl VI-E 122T expressing cell shows spiking in response to a 5 FIz train of 50 ms 630 nm light at 3.2 mW mm'2 (bottom), (f) Kinetics of red light response in 15 C1V1(E122T). Activation time constants (τοη) of photocurrents recorded from cultured neurons expressing C1V1(E122T) at 540 nm and 630 nm. Note that light powers were 3.2 mW mm-2 at 630 nm and 7.7 mW mm'2 at 540·nm (n = 5 cells, p = 0.0006 paired t-test), (g) Voltage clamp traces show responses in a neuron expressing Cl V1(E122T) to 630 nm light pulses. Pulse lengths are indicated above traces. xon calculated from the 150 ms trace is 20 67ms. (h) Current clamp recording from a neuron expressing C1VI(E122T) showing spikes elicited by 50ms pulses at 630 nm (power density 3.2 mW mm'2). FIG. 4 depicts independent activation of excitatory pyramidal neurons and inhibitory parvalbumin-expressing cells, (a) Current clamp recordings from cultured hippocampal neurons expressing Cl VI(El 22T/E162T) or ChR2(F1134R) in response to 25 2ms light pulses at 560nm or 405nm (5 Hz; 7.6 mW/mm2 at both wavelengths), (b)

Recording configuration in double-injected animals expressing Cl VI in cortical pyramidal neurons and ChR2 (H134R) in inhibitory parvalbumin-positive intemeurons. To independently express opsins, PV::Cre mice were injected with a two-virus mix containing Lenti-CaMKIla-C 1VI(E122T/E162T) and AAV5-EF la-DIO-Ch R2 (H 134R). (c) Voltage 3 0 clamp recordings from a non-expressing PYR neuron receiving synaptic input from C1V1 - expressing PYR-cells and ChR2-expressing PV-cells. Clamped at OmV, 405nm light pulses trigger short-latency IPSCs while 560nm pulses evoke only small, long-latency inhibitory synaptic responses, (d) Voltage clamp recording from the same cell shown in c. Clamped at -65mV, 560nm light pulses trigger EPSCs but 405nm pulses do not evoke detectable synaptic currents. Gray lines show individual events; black lines show light pulse-triggered averages, (e) mPFC optrode recording in an anesthetized PV::Cre mouse injected with CaMKIIa::ClVl(E162T)-ts-eYFP and Efla-DIO::ChR2-eYFP (diagram illustrates experimental setup). Violet (405 nm) light pulses are presented with variable 5 delay (At) relative to green light pulses (example traces), (f) Summary graph shows 2015203097 10 Jun2015 probability of green light-evoked spikes with violet pulses preceding the green light pulses by the indicated delays. Individual points are from single recordings. Black line shows average for all recordings (> 3 recording sites per bin), (g) Optrode recording from a mouse injected with viruses showing one presumed pyramidal unit and one presumed PV unit, 10 firing in response to 561 nm stimulation (right, upper waveform) and 405nm stimulation {right, lower waveform), respectively. FIG. 5 depicts combinatorial optogenetic excitation in distinct intact-system preparations, (a) Combinatorial projection control with C1V1-E122T/E 162T and ChR2-H134R in vitro. Experimental paradigm showing expression of Cl VI and ChR2 in cortico-15 thalamic (CT) and ventrobasal (VB) thalamo-cortical cells (TC), respectively, (b) Voltage-clamp recording from an nRT cell receiving projections both from CT and TC cells. Simultaneous stimulation (At = O ms) leads to a linear summation of evoked EPSCs from both projections, (c) Individual subthreshold inputs from TC and CT fibers lead to spiking in an nRT neuron only when inputs are precisely co-incident, (d) Delays between CT and 20 TC inputs are indicated on the left. Horizontal dashed lines indicate truncated spikes.

Normalized number of action potentials (from 6 nRT cells) evoked by CT and TC fibers activated with variable latencies (At) indicates that CT and TC inputs lead to effective integration only if coincident within 5 ms. Summary data represent mean ± SEM. FIG. 6 depicts spectrotemporal separation and combinatorial control: circuit 25 modulation and emergent patterns in altered E/I states under ongoing synaptic activity, (a) Experimental paradigm for SSFO activation of PV neurons and Cl VI activation in pyramidal neurons, (b) Voltage clamp recording at 0 mV from a pyramidal neuron in an acute slice preparation from a PV::Cre mouse expressing CaMKIla::ClVl(E162T) and DIO-SSFO. SSFO and Cl VI are activated by a blue light pulse (2) and IPSC frequency is 30 increased by sustained SSFO activity (3; compare upper and lower traces in inset for pre-and post-activation IPSC activity). A sustained yellow light pulse deactivates SSFO and activates Cl VI and transiently increases IPSC frequency (4). Population power spectra (right) show gamma frequency activity during optical excitatory neuron activation (590 nm pulse) that is increased during coactivation of excitatory and PV neurons (470 nm pulse).

Diagrams below traces show predicted activity of Cl VI and SSFO during the experiment, (c) The observed gamma frequency peak was not dependent on prior PV neuron stimulation via SSFO. (d) Summary IPSC frequencies from (b) and (c) at baseline and after the initial blue or orange pulse. Diagrams below traces show predicted activity of Cl VI and SSFO 5 during the experiment. 2015203097 10 Jun2015

DETAILED DESCRIPTION

This invention provides, inter alia, compositions comprising animal cells expressing light-activated chimeric proteins on their plasma membranes and methods of using the same to selectively depolarize excitatory or inhibitory neurons residing in the same microcircuit 10 in the pre-frontal cortex. The inventors have developed chimeric proteins possessing unique physiochemical properties which for the first time permit experimental manipulation of cortical Efl elevations and the ability to monitor gamma oscillations in cortical slices. These unique light-sensitive chimeric proteins can be expressed in either excitatory or inhibitory neural circuits in the prefrontal cortex of nonhuman animals which can then be depolarized in 15 response to light having particular wavelengths. Furthermore, brain slices from non-human animals containing cortical excitatory or inhibitory neurons expressing the chimeric light-sensitive proteins disclosed herein can be used to search for chemical compounds which can selectively inhibit the depolarization of either excitatory or inhibitory neurons residing within the same neural circuit. 20 General Techniques

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A 25 Laboratory Manual, second edition (Sambrook et al., 1989) and Molecular Cloning: A

Laboratory Manual, third edition (Sambrook and Russel, 2001), (jointly referred to herein as “Sambrook”); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987, including supplements through 2001); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring 30 Harbor Publications, New York; Harlow and Lane (1999) Using Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (jointly referred to herein as “Harlow and Lane”), Beaucage et al. eds., Current Protocols in Nucleic Acid Chemistry John Wiley & Sons, Inc., New York, 2000), Handbook of Experimental Immunology, 4th edition (D. M. Weir & C. C. Blackwell, eds., Blackwell Science Inc., 1987); and Gene Transfer Vectors for Mammalian Cells (J. M. Miller & Μ. P. Calos, eds., 1987). 2015203097 10 Jun2015

Definitions

As used herein, the singular form “a”, “an”, and “the” includes plural references 5 unless indicated otherwise.

An “animal” can be a vertebrate, such as any common laboratory model organism, or a mammal. Mammals include, but are not limited to, humans, farm animals, sport animals, pets, primates, mice, rats, and other rodents.

An “amino acid substitution” or “mutation” as used herein means that at least one 10 amino acid component of a defined amino acid sequence is altered or substituted with another amino acid leading to the protein encoded by that amino acid sequence having altered activity or expression levels within a cell. A “chimeric protein” is a protein comprising one or more portions derived from one or more different proteins. Chimeric proteins may be produced by culturing a recombinant 15 cell transfected with a nucleic acid that encodes the chimeric protein.

It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such 20 higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. V1C1 Chimeric Proteins and Cells Expressing the Same 25 In some aspects, the animal cells disclosed herein comprise a chimeric light- sensitive protein, known as “Cl VI,” which is derived from the VChRl cation channel from Volvox carteri and the ChRl cation channel from Chlamydomonas Reinhardti. The protein may be comprised of the amino acid sequence of VChRl, but additionally can have at least the first and second transmembrane helices of the VChRl polypeptide replaced by the 30 corresponding first and second transmembrane helices of ChRl. Cl VI chimeric opsin proteins are assembled from pieces of other opsin proteins that do not express well alone in neurons and which are potent, redshifted, and stable channelrhodopsins. In some embodiments, the animal cell may express a second light-activated protein on the plasma membrane of the cell. The second light-activated protein can be capable of mediating a hyperpolarization of the cell plasma membrane in response to activation by light. Examples of light-activated proteins capable of mediating a hyperpolarization of the cell plasma membrane can be found, for example, in International Patent Application No: 2015203097 10 Jun2015 PCT/US2011/028893, the disclosure of which is incorporated by reference herein in its 5 entirety.

Embodiments of the present disclosure may also be directed toward modified or mutated versions of Cl VI. These proteins can be used alone or in combination with a variety of other opsins to assert optical control over neurons. In particular, the use of modified Cl VI, in connection with other opsins, is believed to be useful for optical control 10 over nervous system disorders. Specific uses of Cl VI relate to optogenetic systems or methods that correlate temporal, spatial and/or cell type-specific control over a neural circuit with measurable metrics. VI Cl chimeric proteins

Provided herein are light-activated chimeric proteins expressed on an animal cell 15 plasma membrane. In some aspects the light-activated protein is a chimeric protein derived from VChRl from Volvox carteri and ChRl from Chlamydomonas reinhardti. In some embodiments, the chimeric protein comprises the amino acid sequence of VChRl having at least the first and second transmembrane helices replaced by the corresponding first and second transmembrane helices of ChRl. In other embodiments, the chimeric protein 20 comprises the amino acid sequence of VChRl having the first and second transmembrane helices replaced by the corresponding first and second transmembrane helices of ChRl and further comprises at least a portion of the intracellular loop domain located between the second and third transmembrane helices replaced by the corresponding portion from ChRl. In some embodiments, the entire intracellular loop domain between the second and third 25 transmembrane helices of the chimeric light-activated protein can be replaced with the corresponding intracellular loop domain from ChRl. In other embodiments, the portion of the intercellular loop domain located between the second and third transmembrane helices that is replaced with the corresponding portion of ChRl can extend to A145 of SEQ ID NO:l. In other embodiments, the chimeric protein comprises the amino acid sequence of 30 VChRl having the first and second transmembrane helices and the intracellular loop domain replaced by the corresponding first and second transmembrane helices and intracellular loop domain of ChRl and further comprises at least a portion of the third transmembrane helix replaced by the corresponding portion of ChRl. In another embodiment, the portion of the third transmembrane helix replaced by the corresponding portion from ChRl can extend to W163 of SEQ ID NO:l. In some embodiments, the light-activated chimeric protein comprises the amino acids 1-145 of ChRl and amino acids 102316 of VChRl. In some embodiments, the light-activated chimeric protein comprises the amino acids 1-162 of ChRl and amino acids 119-316 of VChRl. In some embodiments, 2015203097 10 Jun2015 5 the light-activated chimeric protein can comprise an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 1 without the signal peptide sequence. In some embodiments, the light-activated chimeric protein can comprise an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in 10 SEQ ID NO: 1

In other embodiments, the light activated chimeric protein is capable of mediating a depolarizing current in the cell when the cell is illuminated with light. In some embodiments the light can be green light. In other embodiments, the light can have a wavelength of between about 540 nm to about 560 nm. In some embodiments, the light can 15 have a wavelength of about 542 nm. In some embodiments, the chimeric protein may not be capable of mediating a depolarizing current in the cell when the cell is illuminated with violet light. In some embodiments, the chimeric protein may not be capable of mediating a depolarizing current in the cell when the cell is illuminated with light having a wavelength of 405 nm. 20 In some embodiments, the protein can further comprise a C-terminal fluorescent protein. In some specific embodiments, the C-terminal fluorescent protein can be enhanced yellow fluorescent protein (EYFP), green fluorescent protein (GFP), cyan fluorescent protein (CFP), or red fluorescent protein (RFP). In some embodiments, the light-activated chimeric protein is modified by the addition of a trafficking signal (ts) which enhances 25 transport of the protein to the cell plasma membrane. In some embodiments, the trafficking signal is derived from the amino acid sequence of the human inward rectifier potassium channel Kir2.1. In some embodiments, the trafficking signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV. In some embodiments, the signal peptide sequence in the protein may be replaced with a different signal peptide sequence. 30 In some embodiments, the animal cell can be a neuronal cell, a muscle cell, or a stem cell. In one embodiment, the animal cell is a neuronal cell. In some embodiments the neuronal cell can be an excitatory neuron located in the pre-frontal cortex of a non-human animal. In other embodiments, the excitatory neuron can be a pyramidal neuron. In still other embodiments, the inhibitory neuron can be a parvalbumin neuron. In some embodiments the neuronal cell can be an inhibitory neuron located in the pre-frontal cortex of a non-human animal. In some embodiments the neuronal cell can be an inhibitory neuron located in the pre-frontal cortex of a non-human animal. 2015203097 10 Jun2015

In some embodiments, the animal cells can further comprise a second light-5 activated protein expressed on the cells’ plasma membrane. In some embodiments, the second light-activated protein can be capable of mediating a hyperpolarizing current in the cell when the cell is illuminated with light. In some embodiments the second light-activated protein can be NpHr, eNpHr2.0, eNpHr3.0, eNpHr3.1 or GtR3. Additional information regarding other light-activated cation channels, anion pumps, and proton pumps can be 10 found in U.S. Patent Application Publication Nos: 2009/0093403; and International Patent Application No: PCT/US2011/028893, the disclosures of which are hereby incorporated by reference herein in their entirety. In some embodiments, the light-activated chimeric protein can have enhanced photocurrents in neural cells exposed to light relative to cells expressing other light-activated cation channel proteins. In some embodiments, the 15 enhancement in photocurrent provided by the light-activated chimeric protein can be any of 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, or 15 fold, greater than cells expressing other light-activated cation channel proteins, inclusive.

Also provided herein is one or more light-activated proteins expressed on an animal 20 cell plasma membrane, wherein said one or more light activated proteins comprises a core amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 12, 13, or 14 and further comprising a trafficking signal (e.g., which enhances transport to the plasma membrane). The trafficking signal may be fused to the C-terminus of the core amino acid 25 sequence or may be fused to the N-terminus of the core amino acid sequence. In some embodiments, the trafficking signal can be linked to the core amino acid sequence by a linker. The linker can comprise any of 5, 10, 20, 30, 40, 50, 75, 100,125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length. The linker may further comprise a fluorescent protein, for example, but not limited to, an enhanced yellow fluorescent protein, 30 a red fluorescent protein, a green fluorescent protein, or a cyan fluorescent protein. In some embodiments, the trafficking signal can be derived from the amino acid sequence of the human inward rectifier potassium channel Kir2.1. In some embodiments, the trafficking signal can comprise the amino acid sequence KSRITSEGEYIPLDQIDINY. VI Cl chimeric mutant variants 2015203097 10 Jun2015

In some aspects, the invention includes polypeptides comprising substituted or mutated amino acid sequences, wherein the mutant polypeptide retains the characteristic light-activatable nature of the precursor Cl VI chimeric polypeptide but may also possess 5 altered properties in some specific aspects. For example the mutant light-activated chimeric proteins described herein may exhibit an increased level of expression both within an animal cell or on the animal cell plasma membrane; an altered responsiveness when exposed to different wavelengths of light, particularly red light; and/or a combination of traits whereby the chimeric Cl VI polypeptide possess the properties of low desensitization, 10 fast deactivation, low violet-light activation for minimal cross-activation with other light-activated cation channels, and/or strong expression in animal cells.

Light-activated chimeric proteins comprising amino acid substitutions or mutations include those in which one or more amino acid residues have undergone an amino acid substitution while retaining the ability to respond to light and the ability to control the 15 polarization state of a plasma membrane. For example, light-activated proteins comprising amino acid substitutions or mutations can be made by substituting one or more amino acids into the amino acid sequence corresponding to SEQ ID NO: 1. In some embodiments, the invention includes proteins comprising altered amino acid sequences in comparison with the amino acid sequence in SEQ ID NO:l, wherein the altered light-activated chimeric protein 20 retains the characteristic light-activated nature and/or the ability to regulate ion flow across plasma membranes of the protein with the amino acid sequence represented in SEQ ID NO:l but may have altered properties in some specific aspects.

Amino acid substitutions in a native protein sequence may be conservative or nonconservative and such substituted amino acid residues may or may not be one encoded by 25 the genetic code. The standard twenty amino acid “alphabet” is divided into chemical families based on chemical properties of their side chains. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, 30 leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and side chains having aromatic groups (e.g., tyrosine, phenylalanine, tryptophan, histidine). A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a chemically similar side chain (/. e., replacing an amino acid possessing a basic side chain with another amino acid with a basic side chain). A “non-conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a chemically different side chain (/. e., replacing an amino acid having a basic side chain with an amino acid having an aromatic side chain). The amino acid substitutions may 5 be conservative or non-conservative. Additionally, the amino acid substitutions may be located in the Cl VI retinal binding pocket, in one or more of the Cl VI intracellular loop domains, and/or in both the retinal binding pocket or the intracellular loop domains. 2015203097 10 Jun2015

Accordingly, provided herein are Cl VI chimeric light-activated proteins that may have specific amino acid substitutions at key positions throughout the retinal binding pocket 10 of the VChRl portion of the chimeric polypeptide. In some embodiments, the Cl VI protein can have a mutation at amino acid residue El22 of SEQ ID NO: 1. In some embodiments, the Cl VI protein can have a mutation at amino acid residue El62 of SEQ ID NO: 1. In other embodiments, the Cl VI protein can have a mutation at both amino acid residues E162 and E122 of SEQ ID NO:l. In some embodiments, each of the disclosed 15 mutant Cl VI chimeric proteins can have specific properties and characteristics for use in depolarizing the membrane of an animal cell in response to light. C1V1-E122 mutant polypeptides

Provided herein are the light-activated Cl VI chimeric proteins disclosed herein expressed on an animal cell plasma membrane, wherein one or more amino acid residues 20 have undergone an amino acid substitution while retaining Cl VI activity (i.e., the ability to catalyze the depolarization of an animal cell in response to light activation), and wherein the mutation can be at a glutamic acid residue corresponding to El22 of SEQ ID NO:1 (Cl VI-E122). In some embodiments, the C1V1-E122 mutant chimeric light-activated protein comprises substitutions introduced into the amino acid sequence shown in SEQ ID NO :1 at 25 amino acid El22 that can result in the chimeric protein having increased sensitivity to light, increased sensitivity to particular wavelengths of light, and/or increased ability to regulate the polarization state of the plasma membrane of the cell relative to Cl VI chimeric light-activated proteins that do not have a mutation at El22. In some embodiments, the mutation can be a conservative amino acid substitution. In some embodiments, the mutation can be a 30 non-conservative amino acid substitution. In some embodiments, the mutation at amino acid residue E122 can be to threonine (C1V1-E122T). In other embodiments, the light-activated chimeric protein can comprise an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 3 without the signal peptide sequence. In other embodiments, the light-activated chimeric protein can comprise an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 3. In other embodiments, the Cl VI-El22 mutant chimeric light-activated protein may be fused to a C-terminal trafficking signal. In some embodiments, the trafficking signal can be linked 5 to the Cl VI-E122 mutant chimeric light-activated protein by a linker. The linker can 2015203097 10 Jun2015 comprise any of 5, 10, 20, 30, 40, 50, 75, 100,125, 150,175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length. The linker may further comprise a fluorescent protein, for example, but not limited to, an enhanced yellow fluorescent protein, a red fluorescent protein, a green fluorescent protein, or a cyan fluorescent protein. In some embodiments, the 10 trafficking signal can be derived from the amino acid sequence of the human inward rectifier potassium channel Kir2.1. In some embodiments, the trafficking signal can comprise the amino acid sequence KSRITSEGEYIPLDQIDINV.

In other embodiments, the Cl VI-El 22 chimeric protein is capable of mediating a depolarizing current in the cell when the cell is illuminated with light. In some 15 embodiments the light can be green light. In other embodiments, the light can have a wavelength of between about 540 nm to about 560 nm. In some embodiments, the light can have a wavelength of about 546 nm. In other embodiments, the Cl VI-El22 chimeric protein can mediate a depolarizing current in the cell when the cell is illuminated with red light. In some embodiments, the red light can have a wavelength of about 630 nm. In 20 some embodiments, the Cl VI-El 22 chimeric protein may not be capable of mediating a depolarizing current in the cell when the cell is illuminated with violet light. In some embodiments, the chimeric protein may not be capable of mediating a depolarizing current in the cell when the cell is illuminated with light having a wavelength of 405 nm. In some embodiments, the animal cell can be a neuronal cell, a muscle cell, or a stem cell. In one 25 embodiment, the animal cell can be a neuronal cell. In some embodiments the neuronal cell can be an excitatory neuron located in the pre-frontal cortex of a non-human animal. In other embodiments, the excitatory neuron can be a pyramidal neuron. In some embodiments the neuronal cell can be an inhibitory neuron located in the pre-frontal cortex of a non-human animal. In other embodiments, the excitatory neuron can be a pyramidal 30 neuron. In still other embodiments, the inhibitory neuron can be a parvalbumin neuron. In some embodiments, the animal cells can further comprise a second light-activated protein expressed on the cells’ plasma membrane. In some embodiments, the second light-activated protein can be capable of mediating a hyperpolarizing current in the cell when the cell is illuminated with light. In some embodiments the second light-activated protein can be NpHr, eNpHr2.0, eNpHr3.0, eNpHr3.1 or GtR3. 2015203097 10 Jun2015 C1V1-E162 mutant polypeptides

Provided herein are the light-activated Cl VI chimeric proteins disclosed herein 5 expressed on an animal cell plasma membrane, wherein one or more amino acid residues have undergone an amino acid substitution while retaining Cl VI activity (i.e., the ability to catalyze the depolarization of an animal cell in response to light activation), wherein the mutation can be at a glutamic acid residue corresponding to El62 of SEQ IDNO:l (Cl VI-E162). In some embodiments, the C1V1-E162 mutant chimeric light-activated protein 10 comprises substitutions introduced into the amino acid sequence shown in SEQ ID NO :1 at amino acid El62 that can result in the chimeric protein having increased sensitivity to light, increased sensitivity to particular wavelengths of light, and/or increased ability to regulate the polarization state of the plasma membrane of the cell relative to Cl VI chimeric light-activated proteins that do not have a mutation at El62. In some embodiments, the mutation 15 can be a conservative amino acid substitution. In some embodiments, the mutation can be a non-conservative amino acid substitution. In some embodiments, the mutation at amino acid residue El62 can be to threonine (C1V1-E162T). In other embodiments, the light-activated chimeric protein can comprise an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown iii SEQ 20 ID NO: 5 without the signal peptide sequence. In other embodiments, the light-activated chimeric protein can comprise an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 5. In other embodiments, the Cl VI-El62 mutant chimeric light-activated protein may be fused to a C-terminal trafficking signal. In some embodiments, the trafficking signal can be linked 25 to the Cl VI-El62 mutant chimeric light-activated protein by a linker. The linker can comprise any of 5, 10, 20, 30, 40, 50, 75, 100,125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length. The linker may further comprise a fluorescent protein, for example, but not limited to, an enhanced yellow fluorescent protein, a red fluorescent protein, a green fluorescent protein, or a cyan fluorescent protein. In some embodiments, the 30 trafficking signal can be derived from the amino acid sequence of the human inward rectifier potassium channel Kir2.1. In some embodiments, the trafficking signal can comprise the amino acid sequence KSRITSEGEYIPLDQIDINV.

In other embodiments, the Cl VI-El 62 chimeric protein is capable of mediating a depolarizing current in the cell when the cell is illuminated with light. In some embodiments the light can be green light. In other embodiments, the light can have a wavelength of between about 540 nm to about 535 nm. In some embodiments, the light can have a wavelength of about 542 nm. In other embodiments, the light can have a wavelength of about 530 nm. In some embodiments, the Cl VI-El 62 chimeric protein may not be 5 capable of mediating a depolarizing current in the cell when the cell is illuminated with violet light. In some embodiments, the chimeric protein may not be capable of mediating a depolarizing current in the cell when the cell is illuminated with light having a wavelength of 405 nm. In some embodiments, the Cl VI-El62 chimeric protein can further comprise a C-terminal fluorescent protein. In some embodiments, the animal cell can be a neuronal 10 cell, a muscle cell, or a stem cell. In one embodiment, the animal cell can be a neuronal cell. In some embodiments the neuronal cell can be an excitatory neuron located in the prefrontal cortex of a non-human animal. In other embodiments, the excitatory neuron can be a pyramidal neuron. In some embodiments the neuronal cell can be an inhibitory neuron located in the pre-frontal cortex of a non-human animal. In other embodiments, the 15 excitatory neuron can be a pyramidal neuron. In still other embodiments, the inhibitory neuron can be a parvalbumin neuron. In some embodiments, the animal cells can further comprise a second light-activated protein expressed on the cells’ plasma membrane. In some embodiments, the second light-activated protein can be capable of mediating a hyperpolarizing current in the cell when the cell is illuminated with light. In some 20 embodiments the second light-activated protein can be NpHr, eNpHr2.0, eNpHr3.0, 2015203097 10 Jun2015 eNpHr3.1 or GtR3. In some embodiments, the Cl VI-El62 light-activated chimeric protein can have an accelerated photocycle relative Cl VI proteins lacking mutations at El62 or relative to other light-activated cation channel proteins. In some embodiments, the C1V1-E162 light-activated chimeric protein can have a photocycle more than 1 fold, 1.5 fold, 2 25 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, or 5 fold faster than Cl VI proteins lacking mutations at El62 or relative to other light-activated cation channel proteins, inclusive. C1V1-E122/E162 double mutant polypeptides

Provided herein are the light-activated Cl VI chimeric proteins disclosed herein expressed on an animal cell plasma membrane, wherein one or more amino acid residues 30 have undergone an amino acid substitution while retaining ClVI activity (i.e., the ability to catalyze the depolarization of an animal cell in response to light activation), wherein the mutations can be at glutamic acid residues corresponding to El22 and El62 of SEQ ID NO:l (C1V1-E122/E162). In some embodiments, the C1V1-E122/E162 mutant chimeric light-activated protein can comprise substitutions introduced into the amino acid sequence shown in SEQ ID NO:1 at amino acid El22 and El62 that can result in the chimeric protein having increased sensitivity to light, increased sensitivity to particular wavelengths of light, and/or increased ability to regulate the polarization state of the plasma membrane of the cell relative to Cl Y1 chimeric light-activated proteins that do not have a mutation at El22 and 5 El62. In some embodiments, the mutations can be conservative amino acid substitutions. 2015203097 10 Jun2015

In some embodiments, the mutations can be non-conservative amino acid substitutions. In some embodiments, the mutations can be both conservative and non-conservative amino acid substitutions. In some embodiments, the mutation at amino acid residue El22 and at E162 can both be to threonine (C1V1-E122T/E162T). In other embodiments, the light-10 activated chimeric protein can comprise an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 7 without the signal peptide sequence. In other embodiments, the light-activated chimeric protein can comprise an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 7. In 15 other embodiments, the C1V1 -E122/E162 mutant chimeric light-activated protein may be fused to a C-terminal trafficking signal. In some embodiments, the trafficking signal can be linked to the C1V1 -E122/E 162 mutant chimeric light-activated protein by a linker. The linker can comprise any of 5, 10,20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length. The linker may further comprise a fluorescent 20 protein, for example, but not limited to, an enhanced yellow fluorescent protein, a red fluorescent protein, a green fluorescent protein, or a cyan fluorescent protein. In some embodiments, the trafficking signal can be derived from the amino acid sequence of the human inward rectifier potassium channel Kir2.1. In some embodiments, the trafficking signal can comprise the amino acid sequence KSRITSEGEYIPLDQIDINV. 25 In other embodiments, the C1V1 -E122/E 162 chimeric protein is capable of mediating a depolarizing current in the cell when the cell is illuminated with light. In some embodiments the light can be green light. In other embodiments, the light can have a wavelength of between about 540 nm to about 560 ran. In some embodiments, the light can have a wavelength of about 546 nm. In some embodiments, the C1V1-E122/E162 chimeric 30 protein may not be capable of mediating a depolarizing current in the cell when the cell is illuminated with violet light. In some embodiments, the chimeric protein may not be capable of mediating a depolarizing current in the cell when the cell is illuminated with light having a wavelength of 405 nm. In some embodiments, the Cl V1-E122/E162 chimeric protein can exhibit less activation when exposed to violet light relative to Cl VI proteins lacking mutations at E122/E162 or relative to other light-activated cation channel proteins. 2015203097 10 Jun2015

In some embodiments, the animal cell can be a neuronal cell, a muscle cell, or a stem cell.

In one embodiment, the animal cell can be a neuronal cell. In some embodiments the neuronal cell can be an excitatory neuron located in the pre-frontal cortex of a non-human 5 animal. In other embodiments, the excitatory neuron can be a pyramidal neuron. In some embodiments the neuronal cell can be an inhibitory neuron located in the pre-frontal cortex of a non-human animal. In still other embodiments, the inhibitory neuron can be a parvalbumin neuron. In some embodiments, the animal cells can further comprise a second light-activated protein expressed on the cells’ plasma membrane. In some embodiments, 10 the second light-activated protein can be capable of mediating a hyperpolarizing current in the cell when the cell is illuminated with light. In some embodiments the second light-activated protein can be NpHr, eNpHr2.0, eNpHr3.0, eNpHr3.1 or GtR3. In some embodiments, the C1V1-E122/E162 mutant light-activated chimeric protein can have decreased inactivation relative to Cl VI proteins lacking mutations at E122/E162 or relative 15 to other light-activated cation channel proteins. In some embodiments, the Cl VI- E122/E162 mutant light-activated chimeric protein can inactivate by any of about 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26v, 27%, 28%, 29%, or 30% compared to Cl VI proteins lacking mutations at E122/E162 or relative to other light-activated cation channel proteins, inclusive. In some embodiments, the Cl VI- E122/E162 20 light-activated chimeric protein can have a photocycle more than 1 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 5.5 fold, 6 fold, 6.5 fold, 7 fold, 7.5 fold, 8 fold, 8.5 fold, 9 fold, 9.5 fold, or 10 fold faster than Cl VI proteins lacking mutations at E122/E162 or relative to other light-activated cation channel proteins, inclusive.

Enhanced intracellular transport amino acid motifs 25 The present disclosure provides for the modification of light-activated chimeric proteins expressed in a cell by the addition of one or more amino acid sequence motifs which enhance transport to the plasma membranes of mammalian cells. Light-activated chimeric proteins having components derived from evolutionarily simpler organisms may not be expressed or tolerated by mammalian cells or may exhibit impaired subcellular 30 localization when expressed at high levels in mammalian cells. Consequently, in some embodiments, the chimeric light-activated protein expressed in a cell is fused to one or more amino acid sequence motifs selected from the group consisting of a signal peptide, an endoplasmic reticulum (ER) export signal, a membrane trafficking signal, and an N-terminal golgi export signal. The one or more amino acid sequence motifs which enhance light-activated chimeric protein transport to the plasma membranes of mammalian cells can be fused to the N-terminus, the C-terminus, or to both the N- and C-terminal ends of the light-activated protein. Optionally, the light-activated protein and the one or more amino acid sequence motifs may be separated by a linker. In some embodiments, the light-5 activated chimeric protein is modified by the addition of a trafficking signal (ts) which enhances transport of the protein to the cell plasma membrane. In some embodiments, the trafficking signal is derived from the amino acid sequence of the human inward rectifier potassium channel Kir2.1. In some embodiments, the trafficking signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV. Additional protein motifs which can 10 enhance light-activated protein transport to the plasma membrane of a cell are described in 2015203097 10 Jun2015 U.S. Patent Application No. 12/041,628, which is incorporated herein by reference in its entirety. In some embodiments, the signal peptide sequence in the chimeric protein is deleted or substituted with a signal peptide sequence from a different protein.

Animal cells, non-human animals, and brain slices 15 Provided herein are cells comprising the light activated chimeric proteins disclosed herein. In some embodiments, the cells are animal cells. In some embodiments, the animal cells comprise the Cl VI protein corresponding to SEQ ID NO:l. In other embodiments, the animal cells comprise the mutant C1V1-E122T protein corresponding to SEQ ID NO:3. In other embodiments, the animal cells comprise the mutant C1V1-E162T protein 20 corresponding to SEQ ID NO:5. In other embodiments, the animal cells comprise the mutant C1V1-E122T/E162T protein corresponding to SEQ ID N07. In some embodiments, the animal cell can be a neuronal cell, a muscle cell, or a stem cell. In one embodiment, the animal cell can be a neuronal cell. In some embodiments the neuronal cell can be an excitatory neuron located in the pre-frontal cortex of a non-human animal. In 25 other embodiments, the excitatory neuron can be a pyramidal neuron. In some embodiments the neuronal cell can be an inhibitory neuron located in the pre-frontal cortex of a non-human animal. In still other embodiments, the inhibitory neuron can be a parvalbumin neuron.

Also provided herein, are non-human animals comprising the light activated 30 chimeric proteins disclosed herein expressed on the cell membrane of the cells in the animals. In some embodiments, the animal cells comprise the Cl VI protein corresponding to SEQ ID NO: 1. In other embodiments, the animal cells comprise the mutant Cl VI-E122T protein corresponding to SEQ ID NO:3. In other embodiments, the animal cells comprise the mutant C1V1-E162T protein corresponding to SEQ ID NO:5. In other embodiments, the animal cells comprise the mutant C1V1-E122T/E162T protein corresponding to SEQ ID N07. In some embodiments, the animals comprising the light-activated chimeric proteins described herein are transgenically expressing said light-activated chimeric proteins. In other embodiments, the animals comprising the light-5 activated chimeric proteins described herein have been virally transfected with a vector carrying the light-activated protein such as, but not limited to, an adenoviral vector. 2015203097 10 Jun2015

Provided herein are living brain slices from a non-human animal comprising the light-activated chimeric proteins described herein expressed on the cell membrane of the cells in the slices. In some embodiments, the brain slices are from non-human animals 10 transgenically expressing the light-activated chimeric proteins described herein. In other embodiments, the brain slices are from non-human animals that have been virally transfected with a vector carrying said light-activated protein such as, but not limited to, an adenoviral vector. In some embodiments, the brain slices are coronal brain slices. In some embodiments, the brain slices are any of about 100 pm, about 150 pm, about 200 pm, about 15 250 pm, about 300 pm, about 350 pm, about 400 pm, about 450 pm, or about 500 pm thick, inclusive, including any thicknesses in between these numbers.

Isolated polynucleotides

Provided herein are isolated ClVI polynucleotides that encode any chimeric polypeptides described herein that, for example, have at least one activity of a Cl VI 20 polypeptide. The disclosure provides isolated, synthetic, or recombinant polynucleotides comprising a nucleic acid sequence having at least about 70%, e.g., at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%; 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or complete (100%) sequence identity to the nucleic acid of SEQ ID NO :2, 4, 6 or 8 over a region of at 25 least about 10, e.g., at least about 15, 20, 25, 30, 35, 40, 45, 50, 75, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nucleotides.

The disclosure specifically provides a nucleic acid encoding Cl VI and/or a mutant variant thereof. For example, the disclosure provides an isolated nucleic acid molecule, wherein the nucleic acid molecule encodes: (1) a polypeptide comprising an amino acid 30 sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,-98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1; (2) a polypeptide comprising an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:3, (3) a polypeptide comprising an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:5; or (4) a polypeptide comprising an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence represented by SEQ ID NO:7. 2015203097 10 Jun2015 5 Promoters and vectors

The disclosure also provides expression cassettes and/or vectors comprising the above-described nucleic acids. Suitably, the nucleic acid encoding a chimeric protein of the disclosure is operably linked to a promoter. Promoters are well known in the art. Any promoter that functions in the host cell can be used for expression of Cl VI and/or any 10 variant thereof of the present disclosure. Initiation control regions or promoters, which are useful to drive expression of a Cl VI chimeric protein or variant thereof in a specific animal cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these nucleic acids can be used.

Specifically, where recombinant expression of Cl VI chimeric proteins in an 15 excitatory neural cell is desired, a human calmodulin-dependent protein kinase II alpha (CaMKIIa) promoter may be used. In other embodiments, an elongation factor la (EF-la) promoter in conjunction with a Cre-inducible recombinant AAV vector can be used with parvalbumin-Cre transgenic mice to target expression Cl VI chimeric proteins to inhibitory neurons. 20 Also provided herein are vectors comprising the polynucleotides disclosed herein encoding a Cl VI chimeric polypeptide or any variant thereof. The vectors that can be administered according to the present invention also include vectors comprising a polynucleotide which encodes an RNA (e.g., RNAi, ribozymes, miRNA, siRNA) that when transcribed from the polynucleotides of the vector will result in the accumulation of light-25 activated chimeric proteins on the plasma membranes of target animal cells. Vectors which may be used, include, without limitation, lentiviral, HSV, and adenoviral vectors. Lentiviruses include, but are not limited to HIV-1, HIV-2, SIV, FIV and EIAV. Lentiviruses may be pseudotyped with the envelope proteins of other viruses, including, but not limited to VSV, rabies, Mo-MLV, baculovirus and Ebola. Such vectors may be prepared using 30 standard methods in the art.

In some embodiments, the vector is a recombinant AAV vector. AAV vectors are DNA viruses of relatively small size that can integrate, in a stable and sitespecific manner, into the genome of the cells that they infect. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies. The AAV genome has been cloned, sequenced and characterized. It encompasses approximately 4700 bases and contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as an origin of replication for the virus. The remainder of the genome is divided into two 5 essential regions that carry the encapsidation functions: the left-hand part of the genome, that contains the rep gene involved in viral replication and expression of the viral genes; and the right-hand part of the genome, that contains the cap gene encoding the capsid proteins of the virus. 2015203097 10 Jun2015

The application of AAV as a vector for gene therapy has been rapidly developed in 10 recent years. Wild-type AAV could infect, with a comparatively high titer, dividing or nondividing cells, or tissues of mammal, including human, and also can integrate into in human cells at specific site (on the long arm of chromosome 19) (Kotin, R. M., et al, Proc. Natl. Acad. Sci. USA 87: 2211-2215, 1990) (Samulski, R. J, et al, EMBOJ. 10: 3941-3950,1991 the disclosures of which are hereby incorporated by reference herein in their entireties). 15 AAV vector without the rep and cap genes loses specificity of site-specific integration, but may still mediate long-term stable expression of exogenous genes. AAV vector exists in cells in two forms, wherein one is episomic outside of the chromosome; another is integrated into the chromosome, with the former as the major form. Moreover, AAV has not hitherto been found to be associated with any human disease, nor any change of biological 20 characteristics arising from the integration has been observed. There are sixteen serotypes of AAV reported in literature, respectively named AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAV13, AAV 14, AAV15, and AAV 16, wherein AAV5 is originally isolated from humans (Bantel-Schaal, and H. zur Hausen. 1984. Virology 134: 52-63), while AAV1-4 and AAV6 are all found in the study of 25 adenovirus (Ursula Bantel-Schaal, Hajo Delius and Harald zur Hausen. J. Virol. 1999, 73: 939-947). AAV vectors may be prepared using standard methods in the art. Adeno-associated viruses of any serotype are suitable {See, e.g., Blacklow, pp. 165-174 of “Parvoviruses and Human Disease" J. R. Pattison, ed. (1988); Rose, Comprehensive Virology 3:1,1974; P. 30 Tattersall “The Evolution of Parvovirus Taxonomy" In Parvoviruses (JR Kerr, SF Cotmore. ME Bloom, RM Linden, CR Parrish, Eds.) p5-14, Hudder Arnold, London, UK (2006); and DE Bowles, JE Rabinowitz, RJ Samulski “The Genus Dependovirus" (JR Kerr, SF Cotmore. ME Bloom, RM Linden, CR Parrish, Eds.) pi5-23, Hudder Arnold, London, UK (2006), the disclosures of which are hereby incorporated by reference herein in their entireties). Methods for purifying for vectors may be found in, for example, U.S. Pat. Nos. 6566118, 6989264, and 6995006 and WO/1999/Ol 1764 titled “Methods for Generating High Titer Helper-free Preparation of Recombinant AAV Vectors", the disclosures of which are herein incorporated by reference in their entirety. Preparation of hybrid vectors is 5 described in, for example, PCT Application No. PCT/US2005/027091, the disclosure of which is herein incorporated by reference in its entirety. The use of vectors derived from the AAVs for transferring genes in vitro and in vivo has been described (See e.g., International Patent Application Publication Nos: 91/18088 and WO 93/09239; U.S. Patent Nos: 4,797,368, 6,596,535, and 5,139,941; and European Patent No: 0488528, all of which 10 are herein incorporated by reference in their entirety). These publications describe various 2015203097 10 Jun2015 AAV-derived constructs in which the rep and/or cap genes are deleted and replaced by a gene of interest, and the use of these constructs for transferring the gene of interest in vitro (into cultured cells) or in vivo (directly into an organism). The replication defective recombinant AAVs according to the invention can be prepared by co-transfecting a plasmid 15 containing the nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions, and a plasmid carrying the AAV encapsidation genes {rep and cap genes), into a cell line that is infected with a human helper virus (for example an adenovirus). The AAV recombinants that are produced are then purified by standard techniques. 20 In some embodiments, the vector(s) for use in the methods of the invention are encapsidated into a virus particle (e.g. AAV virus particle including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV10, AAVI 1, AAV 12, AAV13, AAV 14, AAV15, and AAV 16). Accordingly, the invention includes a recombinant virus particle (recombinant because it contains a recombinant polynucleotide) 25 comprising any of the vectors described herein. Methods of producing such particles are known in the art and are described in US Patent No. 6,596,535.

For the animal cells described herein, it is understood that one or more vectors may be administered to neural cells, heart cells, or stem cells. If more than one vector is used, it is understood that they may be administered at the same or at different times to the animal 30 cell.

Methods of the Invention

Provided herein are methods for selectively depolarizing excitatory or inhibitory neurons residing in the same microcircuit by expressing in those neurons the light-activated chimeric proteins described herein. In some embodiments, a first light-activated protein, such as those disclosed herein, can be expressed in an excitatory neuron while a second light-activated protein can be expressed in an inhibitory neuron. In some embodiments, the first light-activated protein expressed in the excitatory neuron can be activated by a different wavelength of light than the second light-activated protein expressed in the inhibitory 5 neuron. In some embodiments, the first and second light-activated proteins can be 2015203097 10 Jun2015 expressed in a living non-human animal or in a living brain slice from a non-human animal.

In other embodiments, a method is provided for identifying a chemical compound that selectively inhibits the depolarization of excitatory or inhibitory neurons residing in the same neural circuit by expressing in those neurons the light-activated chimeric proteins 10 described herein. In some embodiments, a first light-activated protein can be expressed in an excitatory neuron while a second light-activated protein can be expressed in an inhibitory neuron. In some embodiments, the first light-activated protein expressed in the excitatory neuron can be activated by a different wavelength of light than the second light-activated protein expressed in the inhibitory neuron. In some embodiments, the first and second 15 light-activated proteins can be expressed in a living non-human animal or in a living brain slice from a non-human animal.

Methods for selectively altering the E/I balance in neurons residing in the same microcircuit

In some aspects, there is provided a method for selectively depolarizing excitatory or 20 inhibitory neurons residing in the same microcircuit, the method comprising: selectively depolarizing an excitatory neuron comprising a first light-activated protein, wherein the first light-activated protein is depolarized when exposed to light having a first wavelength or selectively depolarizing an inhibitory neuron comprising a second light-activated protein, wherein the second light-activated protein is depolarized when exposed to light having a 25 second wavelength. In some embodiments, the first light-activated protein can comprise a protein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 1. In other embodiments, the first light-activated protein can comprise a protein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 3. In 30 some embodiments, the first light-activated protein can comprise a protein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 5. In some embodiments, the second light-activated protein can comprise a protein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:l 1. In some embodiments, the second light-activated protein can comprise a protein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the second light-activated protein can comprise a protein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 5 100% identical to the amino acid sequence shown in SEQ ID NO: 13. In some 2015203097 10 Jun2015 embodiments, the second light-activated protein can comprise a protein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 14. More information regarding the disclosure of other light-activated cation channels can be found in U.S. Patent Application Publication No: 10 2007/0054319; U.S. Patent Application No: 61/410,704; and International Patent

Application Publication No: WO 2010/056970, the disclosures of each of which are hereby incorporated by reference in their entireties.

In other aspects, there is provided a method for selectively depolarizing excitatory or inhibitory neurons residing in the same microcircuit, the method comprising: expressing a 15 first light-activated protein in an excitatory neuron; and expressing a second light-activated protein in an inhibitory neuron, wherein the first light-activated protein is independently depolarized when exposed to light having a first wavelength and wherein the second light-activated protein is independently depolarized when exposed to light having a second wavelength. In some embodiments, the first light-activated protein can comprise a protein at 20 least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 1. In other embodiments, the first light-activated protein can comprise a protein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, the first light-activated protein can comprise a protein at least 90%, 25 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 5. In some embodiments, the second light-activated protein can comprise a protein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 11. In some embodiments, the second light-activated protein can comprise a protein at least 90%, 91%, 30 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the second light-activated protein can comprise a protein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the second light-activated protein can comprise a protein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 14. 2015203097 10 Jun2015

In some embodiments, the first light-activated protein can be activated by green light. In one embodiment, the first light-activated protein can be activated by light having a 5 wavelength of about 560 nm. In one embodiment, the first light-activated protein can be activated by red light. In another embodiment, the first light-activated protein can be activated by light having a wavelength of about 630 nm. In other embodiments, the second light-activated protein can be activated by violet light. In one embodiment, the second light-activated protein can be activated by light having a wavelength of about 405 nm. In 10 other embodiments, the second light activated protein can be activated by green light. In some embodiments, the light-activated proteins are activated by light pulses that can have a duration for any of about 1 millisecond (ms), about 2 ms, about 3, ms, about 4, ms, about 5 ms, about 6 ms, about 7 ms, about 8 ms, about 9 ms, about 10 ms, about 15 ms, about 20 ms, about 25 ms, about 30 ms, about 35 ms, about 40 ms, about 45 ms, about 50 ms, about 15 60 ms, about 70 ms, about 80 ms, about 90 ms, about 100 ms, about 200 ms, about 300 ms, about 400 ms, about 500 ms, about 600 ms, about 700 ms, about 800 ms, about 900 ms, about 1 sec, about 1.25 sec, about 1.5 sec, or about 2 sec, inclusive, including any times in between these numbers. In some embodiments, the light-activated proteins are activated by light pulses that can have a light power density of any of about 0.05 mW mm , about 0.1 20 mW mm"2, about 0.25 mW mm"2, about 0.5 mW mm"2, about 0.75 mW mm"2, about 1 mW mm"2, about 2 mW mm"2, about 3 mW mm'2, about 4 mW mm"2, about 5 mW mm 2, about 6 mW mm"2, about 7 mW mm"2, about 8 mW mm’2, about 9 mW mm"2, about 10 mW mm 2, about 11 mW mm"2, about 12 mW mm"2, about 13 mW mm'2, about 14 mW mm 2, about mW mm"2, about 16 mW mm"2, about 17 mW mm"2, about 18 mW mm 2, about 19 mW mm 25 2, about 20 mW mm"2, about 21 mW mm'2, about 22 mW mm'2, about 23 mW mm"2, about 24 mW mm"2, or about 25 mW mm"2, inclusive, including any values between these numbers. In some embodiments the neuronal cell can be an excitatory neuron located in the pre-frontal cortex of a non-human animal. In other embodiments, the excitatory neuron can be a pyramidal neuron. In some embodiments the neuronal cell can be an inhibitory neuron 30 located in the pre-frontal cortex of a non-human animal. In still other embodiments, the inhibitory neuron can be a parvalbumin neuron. In some embodiments, the inhibitory and excitatory neurons can be in a living non-human animal. In other embodiments, the inhibitory and excitatory neurons can be in a brain slice from a non-human animal.

Methods for identifying a chemical compound that selectively alters the E/I balance in neurons residing in the same microcircuit 2015203097 10 Jun2015

In some aspects, there is provided a method for identifying a chemical compound that selectively inhibits the depolarization of excitatory or inhibitory neurons residing in the 5 same microcircuit, the method comprising: (a) selectively depolarizing an excitatory neuron comprising a first light-activated protein with light having a first wavelength or selectively depolarizing an inhibitory neuron comprising a second light-activated protein with light having a second wavelength; (b) measuring an excitatory post synaptic potential (EPSP) in response to selectively depolarizing the excitatory neuron comprising a first light-activated 10 protein or measuring an inhibitory post synaptic current (IPSC) in response to selectively depolarizing an inhibitory neuron comprising a second light-activated protein; (c) contacting the excitatory neuron or the inhibitory neuron with a chemical compound; (d) measuring the excitatory post synaptic potential (EPSP) or measuring the inhibitory post synaptic current (IPSC) to determine if contacting either the excitatory neuron or the 15 inhibitory neuron with the chemical compound selectively inhibits the depolarization of either neuron. In some embodiments, the first light-activated protein can comprise a protein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 1. In other embodiments, the first light-activated protein can comprise a protein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 20 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, the first light-activated protein can comprise a protein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 5. In some aspects, the second light-activated protein can comprise a protein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 25 100% identical to the amino acid sequence shown in SEQ ID NO: 11. In some embodiments, the second light-activated protein can comprise a protein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the second light-activated protein can comprise a protein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 30 100% identical to the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the second light-activated protein can comprise a protein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the chemical compound can be a member of a combinatorial chemical library. In other embodiments, the method further comprises assaying the chemical compound to determine if it adversely affects the function of cardiac tissue or the cardiac action potential in mammals. 2015203097 10 Jun2015

In some embodiments, the first light-activated protein can be activated by green light. In one embodiment, the first light-activated protein can be activated by light having a 5 wavelength of about 560 nm. In one embodiment, the first light-activated protein can be activated by red light. In another embodiment, the first light-activated protein can be activated by light having a wavelength of about 630 nm. In other embodiments, the second light-activated protein can be activated by violet light. In one embodiment, the second light-activated protein can be activated by light having a wavelength of about 405 nm. In 10 some embodiments, the light-activated proteins can be activated by light pulses that can have a duration for any of about 1 millisecond (ms), about 2 ms, about 3, ms, about 4, ms, about 5 ms, about 6 ms, about 7 ms, about 8 ms, about 9 ms, about 10 ms, about 15 ms, about 20 ms, about 25 ms, about 30 ms, about 35 ms, about 40 ms, about 45 ms, about 50 ms, about 60 ms, about 70 ms, about 80 ms, about 90 ms, about 100 ms, about 200 ms, 15 about 300 ms, about 400 ms, about 500 ms, about 600 ms, about 700 ms, about 800 ms,

about 900 ms, about 1 sec, about 1.25 sec, about 1.5 sec, or about 2 sec, inclusive, including any times in between these numbers. In some embodiments, the light-activated proteins can be activated by light pulses that can have a light power density of any of about 0.05 mW mm", about 0.1 mW mm" , about 0.25 mW mm", about 0.5 mW mm", about 0.75 mW mm" 20 2, about 1 mW mm"2, about 2 mW mm"2, about 3 mW mm"2, about 4 mW mm"2, about 5 mW mm", about 6 mW mm", about 7 mW mm', about 8 mW mm", about 9 mW mm", about 10 mW mm"2, about 11 mW mm"2, about 12 mW mm"2, about 13 mW mm"2, about 14 mW mm" , about mW mm" , about 16 mW mm" , about 17 mW mm", about 18 mW mm" , about 19 mW mm"2, about 20 mW mm"2, about 21 mW mm'2, about 22 mW mm"2, about 23 mW 25 mm", about 24 mW mm", or about 25 mW mm", inclusive, including any values between these numbers. In some embodiments the neuronal cell can be an excitatory neuron located in the pre-frontal cortex of a non-human animal. In other embodiments, the excitatory neuron can be a pyramidal neuron. In some embodiments the neuronal cell can be an inhibitory neuron located in the pre-frontal cortex of a non-human animal. In still other 30 embodiments, the inhibitory neuron can be a parvalbumin neuron. In some embodiments, the inhibitory and excitatory neurons can be in a living non-human animal. In other embodiments, the inhibitory and excitatory neurons can be in a brain slice from a nonhuman animal.

EXEMPLARY EMBODIMENTS 2015203097 10 Jun2015

The present disclosure relates to a light-activated chimera opsin that modifies a membrane voltage when expressed therein. While the present disclosure is not necessarily limited in these contexts, various aspects of the disclosure may be appreciated through 5 a discussion of examples using these and other contexts.

Various embodiments of the present disclosure relate to a light-activated opsin modified for expression in cell membranes including mammalian cells. The opsin is derived from a combination of two different opsins, Volvox channelrhodopsin (VChRl) and Chlamydomonas reinhardtii channelrhodopsin (ChRl). The opsin can be useful for 10 expressing at levels of a higher rate than either of the individual opsins from which it is derived.

In certain more specific embodiments, the genetic sequence of ChRl/VChRl chimera (Cl VI) is primarily VChRl. Portions of the VChRl sequence associated with trafficking are replaced with homologous sequences from ChRl. 15 Various embodiments relate to modification directed toward the addition of a trafficking signal to improve expression in mammalian cells.

Certain aspects of the present disclosure are directed to further modified versions of C1V1. For example, certain embodiments include a mutation E162T to C1V1, which experiments suggest provides an accelerated photocycle (e.g., almost 3-fold). 20 Various embodiments of the present disclosure relate to an optogenetic system or method that correlates temporal, spatial and/or cell-type-specific control over a neural circuit with measurable metrics. The optogenetic system uses a variety of opsins, including Cl VI and/or Cl VI variants, to assert control over portions of neural circuits. For instance, various metrics or symptoms might be associated with a neurological 25 disorder. The optogenetic system targets a neural circuit within a patient for selective control thereof. The optogenetic system involves monitoring the patient for the metrics or symptoms associated with the neurological disorder. In this manner, the optogenetic system can provide detailed information about the neural circuit, its function and/or the neurological disorder. 30 Consistent with the embodiments discussed herein, particular embodiments relate to studying and probing disorders using a variety of opsins. Other embodiments relate to the identification and/or study of phenotypes and endophenotypes. Still other embodiments relate to the identification of treatment targets.

Aspects of the present disclosure are directed toward the artificial inducement of disorder/disease states on a fast-temporal time scale. The use of an opsin such as Cl VI can be particularly useful based on characteristics regarding an accelerated photocycle. Moreover, certain embodiments allow for reversible disease states, which can be 5 particularly useful for establishing baseline/control points for testing and/or for testing the 2015203097 10 Jun2015 effects of a treatment on the same animal when exhibiting the disease state and when not exhibiting the disease state. The use of opsins such as Cl VI allows for the control of a cell using a light source. The Cl VI reacts to light, causing a change in the membrane potential of the cell. The removal of the light and the subsequent cessation of the activation of C1V1 10 allows for the cell to return to its baseline state. Various other possibilities exist, some of which are discussed in more detail herein.

Various aspects of the present disclosure are directed to an E122T mutation of a Cl VI opsin. In certain embodiments of the present disclosure, the E122T mutation shifts maximum absorption of Cl VI or its variants toward the red light spectrum with respect to 15 the un-mutated opsin.

Various embodiments of the present disclosure relate to an opsin modified for expression in mammalian cells and shifted, with respect to ChR2, for maximum absorption in the green light spectrum. The Cl VI opsin is derived from a combination of opsins and expresses at a higher rate than either of the opsins from which it is derived. The opsin, 20 C1V1, is derived from Volvox channelrhodopsin (VChRl) and Chlamydomonas reinhardtii channelrhodopsin (ChRl). The resulting opsin, Cl VI and its variants, have a maximum absorption at wavelengths between 530 nm and 546 nm.

Certain aspects of the present disclosure are directed to further modified versions of Cl VI. For example, certain embodiments include a mutation E122T, which shifts the 25 maximum absorption of Cl VI towards the red light spectrum. Other modifications can include an additional mutation E162T, which experiments suggest provides an accelerated photocycle in addition to the red shift provided by the E122T mutation.

In some embodiments, there is provided a transmembrane molecule derived from VChRl and having the traffic sequences replaced with homologous sequences from ChRl. 30 In some embodiments, the molecule further includes a mutation El22T. In other embodiments, the molecule further includes mutations at E162T and E122T. In certain embodiments, the molecule activates an ion channel in response to green light. In one embodiment, the molecule has a maximum light absorption of approximately 546nm. In another embodiment, the molecule has a maximum light absorption of approximately 535nm. 2015203097 10 Jun2015

In some embodiments, there is provided an animal cell comprising: an integrated exogenous molecule which expresses an ion channel that is responsive to red light; the 5 exogenous molecule derived from VChRl and including transmembrane traffic sequences thereof replaced by homologous sequences from ChRl. In some embodiments, the exogenous molecule further includes E122T. In other embodiments, the cell has a neural firing ratio of about 14% to 94% in response to light having wavelengths of405nm and 560 nm, respectively. In other embodiments, the cell has a neural firing ratio of about 11% 10 to 72% in response to light having wavelengths of405nm and 560 nm, respectively.

Additional example embodiments of the present disclosure relate to the use of a hybrid ChRl/VChRl chimera that contains no ChR2 sequence at all, is derived from two opsins genes that do not express well individually, and is herein referred to as Cl VI. Embodiments of the present disclosure also relate to improvements of the membrane 15 targeting of VChRl through the addition of a membrane trafficking signal derived from the Kjr2.1 channel. Confocal images from cultured neurons expressing VChRl-EYFP revealed a large proportion of intracellular protein compared with ChR2; therefore, membrane trafficking signal (ts) derived from the Kjr2.1 channel was used to improve the membrane targeting of VChRl. Membrane targeting of this VChRl-ts-EYFP was slightly enhanced 20 compared with VChRl-EYFP; however, mean photocurrents recorded from cultured hippocampal neurons expressing VChRl-ts-EYFP were only slightly larger than those of VChRl-EYFP. Accordingly, embodiments of the present disclosure relate to VChRl, which has been modified by exchanging helices with corresponding helices from other ChRs. For example, robust improvement has been discovered in two chimeras where helices 1 and 2 25 were replaced with the homologous segments from ChRl. It was discovered that whether splice sites were in the intracellular loop between helices 2 and 3 (at ChRl residue Alai45) or within helix 3 (at ChRl residue Trpl63), the resulting chimeras were both robustly expressed and showed similarly enhanced photocurrent and spectral properties. This result was unexpected as ChRl is only weakly expressed and poorly integrated into membranes of 30 most mammalian host cells.

Specific aspects of the present disclosure relate to microbial opsin genes adapted for neuroscience, allowing transduction of light pulse trains into millisecond-timescale membrane potential changes in specific cell types within the intact mammalian brain (e.g., channelrhodopsin (ChR2), Volvox channelrhodopsin (VChRl) and halorhodopsin (NpHR)). ChR2 is a rhodopsin derived from the unicellular green algae Chlamydomonas reinhardtii. The term "rhodopsin" as used herein is a protein that comprises at least two building blocks, an opsin protein, and a covalently bound cofactor, usually retinal (retinaldehyde). The rhodopsin ChR2 is derived from the opsin Channelopsin-2 (Chop2), 2015203097 10 Jun2015 5 originally named Chlamyopsin-4 (Cop4) in the Chlamydomonas genome. The temporal properties of one depolarizing channelrhodopsin, ChR2, include fast kinetics of activation and deactivation, affording generation of precisely timed action potential trains. For applications seeking long timescale activation, it has been discovered that the normally fast off-kinetics of the channelrhodopsins can be slowed. For example, certain implementations 10 of channelrhodopsins apply ImW/mm2 light for virtually the entire time in which depolarization is desired, which can be less than desirable.

Much of the discussion herein is directed to ChR2. Unless otherwise stated, the disclosure includes a number of similar variants. Examples include, but are not limited to, Chop2, ChR2-310, Chop2-310, and Volvox channelrhodopsin (VChRl). For further details 15 on VChRl, reference can be made to "Red-shifted optogenetic excitation: a tool for fast neural control derived from Volvox carteri," Nat Neurosci. June 2008, 11(6):631-3. Epub 2008 Apr 23, the disclosure of which is fully incorporated herein by reference in its entirety. In other implementations, similar modifications can be made to other opsin molecules. For instance, modifications/mutations can be made to ChR2 or VChRl variants. 20 Moreover the modified variants can be used in combination with light-activated ion pumps.

Embodiments of the present disclosure include relatively minor amino acid variants of the naturally occurring sequences. In one instance, the variants are greater than about 75% homologous to the protein sequence of the naturally occurring sequences. In other variants, the homology is greater than about 80%. Yet other variants have homology 25 greater than about 85%, greater than 90%, or even as high as about 93% to about 95% or about 98%. Homology in this context means sequence similarity or identity, with identity being preferred. This homology can be determined using standard techniques known in sequence analysis. The compositions of embodiments of the present disclosure include the protein and nucleic acid sequences provided herein, including variants which are more than 30 about 50% homologous to the provided sequence, more than about 55% homologous to the provided sequence, more than about 60% homologous to the provided sequence, more than about 65% homologous to the provided sequence, more than about 70% homologous to the provided sequence, more than about 75% homologous to the provided sequence, more than about 80% homologous to the provided sequence, more than about 85% homologous to the 2015203097 10 Jun2015 provided sequence, more than about 90% homologous to the provided sequence, or more than about 95% homologous to the provided sequence.

As used herein, “stimulation of a target cell” is generally used to describe modification of the properties of the cell. For instance, the stimulus of a target cell may 5 result in a change in the properties of the cell membrane that can lead to the depolarization or polarization of the target cell. In a particular instance, the target cell is a neuron and the stimulus affects the transmission of impulses by facilitating or inhibiting the generation of impulses (action potentials) by the neuron.

For further details on light-activated opsins, reference can be made to PCT 10 publication No. WO 2010/056970, entitled "Optically-Based Stimulation of Target Cells and Modifications Thereto," to Deisseroth et al., which is fully incorporated herein by reference in its entirety.

EXAMPLES

Example 1: Development of chimeric channelrhodopsin variant Cl VI 15 In this example, a tool that would permit the driving of cortical E/I elevations and the monitoring of gamma oscillations in cortical slices, as well as in vivo in live animal experiments, was sought, with three key properties: 1) much higher potency to enable dose-response investigation; 2) low desensitization to allow for step-like changes in E/I balance; and 3) redshifted excitation to allow comparative drive of different populations within the 20 same preparation.

These experiments were initially attempted with VChRl, which displays both a redshift and reduced desensitization14, but previous investigation suggested that photocurrents in cells expressing VChRl were small (-100-150 pA14), and did not elicit robust synaptic activity in downstream cells (not shown). Indeed, when first attempting to 25 express VChRl in cells, only small photocurrents were observed, (FIG. 1A) consistent with previous findings. Adding a membrane trafficking signal derived from the Kir2.1 channel to generate VChRl-is-EYFP delivered only a modest trend toward enhanced photocurrents compared with VChRl-EYFP (FIG IB). However, noting that in ChR2, replacing transmembrane segments with the homologous region from ChRl increased membrane 30 targeting and enhanced photocurrents, it was hypothesized that a similar systematic exchange between the helices of VChRl with the corresponding helices from other ChRs, might similarly result in enhanced membrane expression in HEK cells.

Materials and Methods 2015203097 10 Jun2015

Chimeric channelrhodopsin variant Cl VI was generated by fusing either a wild-type or human codon-optimized channelrhodopsin-1 with a human codon-adapted VChRl (GenBankTM accession number ACD70142.1) by overlap extension PCR. 5 Cl VI splice variants were generated by overlap PCR. Variant one contained the first 145 amino acids of ChRl and amino acids 102 to 316 of VChRl. Variant two contained the first 162 amino acids of ChRl and amino acids 119 to 316 of VChRl. The resultant chimeric PCR fragments were cloned into pECFP-Nl (Clonetech, Mountain View, CA) and into lentiviral expression vectors under the CaMKIIa promoter. The membrane trafficking 10 signal was derived from the Kir2.1 channel. Mutations were confirmed by sequencing the coding sequence and splice sites. For AAV-mediated gene delivery, opsin-EYFP fusions along with the CaMKIIa promoter were subcloned into a modified version of the pAAV2-MCS vector. Cre-dependent opsin expression was achieved by cloning the opsin-EYFP cassette in the reverse orientation between pairs of incompatible lox sites (loxP and 15 1 ox2722) to generate a double floxed inverted open reading frame (D10) under the control of the elongation factor la (EF- la) promoter. All constructs are available from the Deisseroth Lab (www.optogenetics.org). HEK293 cells were cultured in Dulbecco’s minimal essential medium supplemented with 10% fetal bovine serum, 2mM glutamine (Biochrome, Berlin, Germany), and 1% 20 (w/w) penicillin/streptomycin. Cells were seeded onto coverslips at a concentration of 0.175 x 106 cells/ml and supplemented with 1 μΜ all-trans retinal. Transient transfection was performed with Fugene 6 (Roche, Mannheim, Germany) and recordings were done 2028 hours later. Photocurrents in transiently transfected HEK293 cells were recorded by conventional whole-cell patch-clamp. The external solution contained [mM]: 140 NaCl, 2 25 CaCh, 2 MgCl2, 2 KC1,10 HEPES (pH 7.2). The internal solution contained [mM]: 110 NaCl, 10 EGTA, 2 MgCl2, 1 CaCl2, 5 KC1, 10 HEPES (pH was adjusted to 7.2 either using CsOH or HC1). Patch pipettes were pulled with micropipette puller model P-97 (Sutter Instrument Co., Novato, CA) from microhaematocrit-tubes (Hecht-Assistant, Sondheim, Germany) with 1.5-2 ΜΩ resistance. HEK cell whole-cell patch-clamping was 30 performed with an EPC 7 (HEKA, Elektronik GmbH, Lambrecht, Germany) amplifier.

Analog data was sampled at 20kHz, digitized with Digidatal440 (Molecular Devices, Foster City, CA) and displayed using pClamplO.l Software (Molecular Devices, Foster City, CA). For recording wavelength dependence, a light guide from a Polychrome V unit (TILL Photonics, Planegg, Germany) was mounted on the epiluminescence port of an Olympus 1X70 microscope. For reflecting light into the objective a beam splitter (70% R / 30% T) was used resulting in a final photon density of ~ 1 x IO22 photons m' s' at 470 nm on the coverslip. For recording the action spectra only 50% of the light intensity was used. The polychrome Y Unit was controlled with Tillvision Software (TILL Photonics, Planegg, 2015203097 10 Jun2015 5 Germany) synchronized with the pClamp Software.

Results

Interestingly, the most robust improvement in chimeras was found where helix 1 and 2 were replaced with the homologs from ChRl (FIG. 1C-D). Two chimeric ChRl-VChRl channels for membrane targeting and photocurrent size were tested in cultured HEK293 10 cells. The first was joined in the second intracellular loop after Alal45 of ChRl, and the second was joined within helix three after Trpl63 of ChRl (FIG. 1C). Whereas both variants were nearly equally well expressed in HEK293 cells (FIG. ID), in cultured neurons the second variant expressed more robustly (FIG. IE) and showed greatly enhanced peak photocurrents (888 ± 128 pA, n = 11 cells; p < 0.0005) compared with VChRl-EYFP (FIG. 15 IB). The action spectrum peak remained robustly redshifted relative to ChR2 (Table 1; FIG. IF), and the ionic selectivity of the chimera was similar to that previously reported for ChR2 and VChRl (FIG. 1G) Adding the Kir2.1 trafficking sequence to this hybrid trended to further increased photocurrents (1104 ± 123 pA, n = 12 cells; p < 0.0005 compared with VChRl-EYFP, p = 0.23 compared with C1V1-EYFP; FIG. IB; Tables 1- 2). The resulting 20 hybrid ChRl/VChRl chimera contains no ChR2 sequence at all, is remarkably derived from two opsin genes that do not express well alone, and is here referred to as CIV 1 (FIG. 1 A, H).

Example 2: Optimization of photocurrent kinetics of Cl VI

Fast deactivation properties28 of this redshifted opsin would be required for maximal 25 temporal as well as spectral separation from other opsins that are activated by wavelengths located towards the blue end of the visible spectrum. However, it was found that the photocurrents displayed by C 1 Vl-ts-EYFP exhibited > 10-fold slower decay than ChR2, and even slower decay than the original VChRl (FIG. 2A; Toff 156 ± 12 ms and 132 ± 12 ms for CIVl-ts-EYFP (n = 4) and VChRl-EYFP (n = 5), respectively; Table 1), potentially 30 precluding the use of CIV 1 for applications requiring rapid firing rates. To correct the photocurrent kinetics of Cl VI, the chromophore region was searched using known structural models ' (FIG. 1H) for mutations with faster photocycle kinetics, reduced inactivation and reduced blue absorption. Next, glutamate-122 was mutated to threonine, based on 2015203097 10 Jun2015 studies of the glutamate-rich motif in helix 2 showing that this mutation reduces inactivation.3

Materials and Methods

All point mutations in Cl VI vectors were generated in the plasmids by site-directed 5 mutagenesis (Agilent Technologies, Palo Alto, CA). The membrane trafficking signal was derived from the Kir2.1 channel. Mutations were confirmed by sequencing the coding sequence and splice sites.

Results

The ChETA-homologous mutation E162T28 markedly accelerated the photocycle 10 almost 3-fold Toff 58 ± 4.7 ms, n = 7 cells; FIG. 2A, Table 1). Surprisingly, whereas analogous mutations in ChR2 or other microbial opsins have caused a red-shift ’ , in Cl VI this mutation shifted the action spectrum in the undesired direction, hypsochromic to 530 nm (FIG. IF; Table 1). C1V1-E122T inactivated only by 26% compared to 46% deactivation of ChR2 (FIG. 2B, Table 1); in addition, the spectrum was further red-shifted 15 to 546 nm (FIG. IF, Table 1) and showed a striking loss of the problematic blue shoulder of the C1V1 action spectrum. Finally, in the double mutant E122T/E162T, inactivation of the current was even lower than in the E122T mutant and the photocycle was still faster compared to E162T Toff 34 ± 4.9 ms, n = 7 cells; FIG. 2A, FIG. 2C, Table 1), while preserving the redshift and absence of the blue shoulder of the action spectrum. Moreover, 20 while the El22 mutant severely reduced photocurrent amplitude (FIG. 2D, the double mutant restored the very high currents characteristic of the original CIVl-ts. Thus, multiple surprising and useful properties of the individual mutations were conserved in the double mutant, trafficking-enhanced CIV 1 chimera. 2015203097 10 Jun2015

Table 1: Spectral/kinetic properties of ChR2, VChRl and C1V1 variants.

Peak activation wavelength was recorded in HEK cells using 2 ms light pulses at the peak activation wavelength. Deactivation kinetics (x0ff) and peak photocurrents were recorded in cultured hippocampal neurons using 2 ms light pulses at the maximal activation wavelength. 5 To identify the optimal variants for combinatorial activation with ChR2, the percent response at 405 nm and 560 nm was recorded in HEK cells. Desensitization of the photocurrent was recorded using 300 ms light pulses, quantifying the decay of the peak photocurrent (Imax) to the steady state.

Absorption maximum ( nm ) Toff Kinetics pH7.2 ( ms ) Peak current (pA) at -60 Mv* Ratio 405/560 Desensitation % ChR2 460± 6 (N=5) 10 ±1(N = 5) 816± 181 (N=5) 60% : 8% 65±8(N=5) (N = 7) VChRl 543±7(N= 7) 85 ±11(N = 284±54(N= 5) 9%: 82% 53±10(N=18) 6) (N = 7) C1V1 539 ±4(N= 10) 60 ±6(N= 6) 1035± 158 (N =6) 28%: 86% 46±12(N=14) (N= 10) C1V1(E162T) 530 ±4(N= 6) 23 ±5(N= 4) 1183±53(N= 6) 20%: 71% 41±12(N=7) (N = 6) C1V1(E122T) 546 ±5(N= 4) 55 ±8(N= 5) 572±21(N= 5) 14%: 94% 26±6(N 4) (N = 4) C1V1(E122T, E162T) 535 ±5(N= 7) 12 ±1(N= 5) 1072±89(N= 9) 11% : 72% (N = 7) 11±9 (N =9)

Table 2: Summary of p-values from unpaired /-test comparisons for peak photocurrent amplitude across all opsins shown in Table 1. Photocurrents were recorded in cultured neurons using a 2 ms light pulse at 540 nm (VChRl and C1VI variants) or 470 nm 15 (ChR2(H134R)). C1V1 C1V1

VchRl- YFP VChRl-ts- YFP C1V1- YFP ClVl-ts- YFP (E162T) -ts-Y (E162T/E122T) -ts-YFP ChR2(H134R)- YFP 1.0000 0.5770 0.0188 0.0029 6.5E-06 1.1E-05 0.0448 1.0000 0.266 0.0039 1.1E-06 0.0015 0.0579 1.0000 0.3372 0.0399 0.0788 0.8175 1.0000 0.4099 0.8442 0.4222 1.0000 0.3254 0.1490 1.0000 0.3001 1.0000

VChrl-YFP

VChRl-ts-YFP

C1V1-YFP

CIVl-ts-YFP

ClVl(E162T)-ts-Y C1V1(E162T/E122T)-

ts-YFP

ChR2(H134R)-YFP

Thus, multiple useful properties of the individual mutations were conserved together in the double mutant. 2015203097 10 Jun2015

Example 3: Use of novel C1V1 chimeras in prefrontal cortex neurons

To test these novel Cl VI opsin genes in neurons, lentiviral vectors encoding Cl VI-5 ts-EYFP and the point mutation combinations above were generated. These opsins were then expressed in cultured hippocampal neurons and recorded whole-cell photocurrents under identical stimulation conditions (2ms pulses, 542nm light, 5.5 mW mm'2) to determine whether the improvement in photocurrent amplitude resulted directly from the increased expression of CIV 1 compared to VChRl. 10 Materials and Methods

Animals

Wild-type or transgenic Parvalbumin::Cre C57/BL6J male mice were group housed three to five to a cage and kept on a reverse 12 hour light/dark cycle with ad libitum food and water. Experimental protocols were approved by Stanford University IACUC and meet 15 guidelines of the National Institutes of Health guide for the Care and Use of Laboratory Animals.

Whole cell patch-clamp electrophvsiolosv in hippocampal and cortical neurons

Primary hippocampal cultures were isolated from PO Sprague-Dawley rats, plated on Matrigel (Invitrogen)-coated glass coverslips and treated with FUDR to inhibit glia 20 overgrowth. Endotoxin-free plasmid DNA was transfected in cultured neurons using a HEPES buffered Saline/CaP04 mix. Electrophysiological recordings from individual neurons identified by fluorescent protein expression were obtained in Tyrode media ([mM] 150 NaCl, 4 KC1,2 MgC12,2 MgC12,10 D-glucose, 10 HEPES, pH 7.35 withNaOH) using a standard internal solution ([mM] 130 KGluconate, 10 KC1, 10 HEPES, 10 EGTA, 2 25 MgC12, pH 7.3 with KOH) in 3-5 ΜΩ glass pipettes. For cortical slice physiology, acute 300 gm coronal slices from 8-9 week old wild-type C57BL/6J or PV::Cre mice previously injected with virus were obtained in ice-cold sucrose cutting solution ([mM] 11 D-glucose, 234 sucrose, 2.5 KC1, 1.25 NaH2P04,10 MgS04, 0.5 CaC12, 26 NaHC03) using a Vibratome (Leica). Slices were recovered in oxygenated Artificial Cerebrospinal Fluid 30 (ACSF; [mM] 124 NaCl, 3 KC1, 1.3 MgC12, 2.4 CaC12,1.25 NaH2P04, 26 NaHC03,10 D-glucose) at 32°C for one hour. Individual neuron patches were obtained after identifying fluorescent protein expression from indicated prefrontal cortical layer under constant ACSF perfusion. Filtered light from a broad-wavelength xenon lamp source (Sutter Instruments DG-4) was coupled to the fluorescence port of the microscope (Leica DM-LFS A). Band pass filters (Semrock) had 20 nm bandwidth, and were adjusted with additional neutral density filters (ThorLabs) to equalize light power output across the spectrum. 2015203097 10 Jun2015

Cultured cell images were acquired on the same microscope using a Retiga Exi CCD 5 camera (Qimaging, Inc.) at 100 ms exposure with 30 gain. Illumination power density was 12 mW mm'2 at 500 nm with a standard EYFP filter set. Quantification of fluorescence was performed with ImageJ software by marking a region containing the soma and proximal neurites and calculating for each cell the total integrated pixel intensity in that region, rather than average fluorescence, since photocurrents are likely to be related to the total number of 10 membrane-bound channels rather than average channel expression per area.

Virus prevaration and injection

Both Lentiviral- and AAV-mediated gene delivery were used for heterologous expression of opsins in mice. Indicated opsins were driven by either Fluman calmodulin-dependent protein kinase II alpha (CaMKIIa) promoter to target cortical excitatory neurons 15 or Elongation Factor 1 a (EF-1 a) in conjunction with a Cre-inducible cassette and followed by the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Cre-inducible recombinant AAV vector was produced by the University of North Carolina Vector Core (Chapel Hill, NC, USA) and used in conjunction with parvalbumin::Cre transgenic mice to target parvalbumin positive ntemeurons. Briefly, AAV constructs were 20 subcloned into a modified version of the pAAV2-MCS, serotyped with AAV5 coat proteins and packaged by the viral vector core at the University of North Carolina. The final viral concentration of AAV vectors as lx 1012 genome copies (gc)/mL. Lentiviral constructs were generated as reported. All constructs are available from the Deisseroth Lab (www.optogenetics.org). Stereotactic viral injections were carried out under protocols 25 approved by Stanford University. Juvenile (4-6 weeks) mice kept under isoflurane anesthesia were arranged in a stereotactic frame (Kopf Instruments) and leveled using bregma and lambda skull landmarks. Craniotomies were preformed so as to cause minimal damage to cortical tissue. Infralimbic prefrontal cortex (IL; from bregma: 1.8mm anterior, 0.35mm lateral, -2.85mm ventral) was targeted using a lOuL syringe and 35g beveled 30 needle (Word Precision Instruments). Virus was infused at a rate of O.lpL/min. Subjects injected with virus for behavioral studies were additionally implanted with a chronic fiber optic coupling device to facilitate light delivery either with or without an attached penetrating cerebral fiber for local delivery to target cortical region as noted (Doric Lenses, Canada). Penetrating fibers were stereotactically inserted to a depth of-2.5mm from the same anterior and lateral coordinates and affixed using adhesive luting cement (C&amp;B MetaBond) prior to adhesive closure of the scalp (Vetbond, 3M). Animals were administered analgesic relief following recovery from surgery. 2015203097 10 Jun2015

Results 5 Recordings from cultured hippocampal neurons expressing individual constructs and an integrated fluorescence reading were obtained from each individual cell. In individual cells, fluorescence levels closely correlated with the measured photocurrent amplitudes across constructs (FIG. 3A). It was therefore concluded that the potently increased photocurrent of Cl VI resulted chiefly from improved expression in neurons. Since the 10 double mutant C1V1-E122T/E162T showed superior performance along all dimensions tested (photocurrent size, inactivation kinetics, and action spectrum), performance to ChR2(H134R) was also directly compared by measuring integrated somatic YFP fluorescence and peak photocurrents. Surprisingly, C1V1-E122T/E162T cells showed stronger photocurrents than ChR2-H134R cells at equivalent fluorescence levels (FIG. 3B), 15 potentially suggestive of increased unitary conductance.

To examine whether Cl V1-E122T/E162T would be suitable for optically driving spiking in pyramidal neurons, adeno-associated viral vectors harboring the Cl VI-E122T/E162T-ts-EYFP gene under the CaMKIIa promoter (AAV5-CaMKIIa-ClVl-E122T/E162T-ts-EYFP) were generated and injected the virus into the prefrontal cortex of 20 mice. Responses were recorded from expressing neurons in acute slices with 2 ms light pulse trains and compared with responses to trains of current injection (10 ms, 200 pA) at identical frequencies. It was found that the frequency response of neurons to 560 nm pulse trains was indistinguishable from the response to current injections at the same frequencies (FIG. 3C; n = 6 cells in 2 slices), suggesting that intrinsic properties of the cell and not C1V1 25 kinetics limit spiking performance at high rates. Similar performance properties were seen across a range of green, yellow, and amber illumination conditions, with strong performance at the moderate light intensities (<10 mW/mm2) suitable for in vivo applications in mammals (FIG. 3D). Indeed, responses at 540 nm and 590 nm were similarly effective at evoking precisely timed action potentials, with lower fidelity at lower light powers as 30 expected (FIG. 3D).

With the prominently red-shifted action spectrum, the possibility that CIV 1 might even be used to drive spiking with red light, not previously reported with any opsin and potentially important for allowing improved spectral separation as well as control of neurons in deeper tissue, was considered. Whether any Cl VI variants could be used to drive spikes using far-red light was therefore examined. Although the kinetics of Cl VI-E122T were slower than those of C1V1-E122T/E162T, its action spectrum was the most red-shifted of all variants (FIG. IF), and indeed it was found that cells expressing C1V1-E122T responded more strongly to red light (630nm) than cells expressing the double mutant (FIG. 2015203097 10 Jun2015 5 3E, top). Although on-kinetics of the E122T mutant at 630nm were slower than at 545nm (FIG. 3F), photocurrents were recruited using longer pulses of 630 nm light at moderate intensity (FIG. 3G) that sufficed to elicit defined spike trains (FIG. 3H; FIG. 3E, bottom).

Example 4: Use of novel C1V1 chimeras in living brain slices from the prefrontal cortex neurons of mice 10 The study sought to determine whether inhibitory and excitatory neurons residing within the same microcircuit could be targeted with the introduction of Cl VI variants. Independent activation of two neuronal populations within living brain slices was explored; in this case CaMKIIa-ClVl-E122T/E162Tts eYFP and EFla-DIO-ChR2-H134R-EYFP were expressed in mPFC of PV::Cre mice. 15 Materials and Methods

Acute 300 pm coronal slices isolated from 8-9 week old wild-type C57BF/6J or PV::Cre mice previously injected with virus were obtained in ice-cold sucrose cutting solution ([mM] 11 D-glucose, 234 sucrose, 2.5 KC1, 1.25 ΝηΗ2Ρ04, 10 MgSO^ 0.5 CaCl2, 26 NaHCCF) using a Vibratome (Feica). Slices were recovered in oxygenated Artificial 20 Cerebrospinal Fluid (ACSF; [mM] 124 NaCl, 3 KC1, 1.3 MgCl2, 2.4 CaCl2, 1.25 NaH2P04, 26 NaFlCOa, 10 D-glucose) at 32°C for one hour. Individual neuron patches were obtained after identifying fluorescent protein expression from indicated prefrontal cortical layer under constant ACSF perfusion. Filtered light from a broad-wavelength xenon lamp source (Sutter Instruments DG-4) was coupled to the fluorescence port of the microscope (Feica 25 DM-FFSA). Slice physiology data were imported into Matlab and analyzed using custom- written software. Power spectra were calculated using the wavelet method as described by

Sohal et al.55 Briefly, for each frequency /, the recorded traces were first filtered with a bandpass filter between / ± 5 FIz. The filtered traces were then convolved with the wavelet function: W(fti) = s(t} . ,(-0(205 where * denotes convolution, σ = 5/(6/). The squared amplitude of W(f,t) over a 500 msec window was then used to measure the power at various frequencies. All power spectra from slice recordings were normalized to 1 If 2015203097 10 Jun2015

Cultured cell images were acquired on the same microscope using a Retiga Exi 5 CCD camera (Qimaging inc.) at 100 ms exposure with the 30 gain. Illumination power density was 12 mW mm"2 at 500 nm with a standard EYFP filter set. Quantification of fluorescence was done with ImageJ software by marking a region containing the soma and proximal neuritis and calculating for each cell the total integrated pixel intensity in that region, rather than average fluorescence, since photocurrents are likely to be related to the 10 total number of membrane-bound channels rather than average channel expression per area.

Using current clamp, a single pyramidal cell was stimulated with a train of simulated EPSC waveforms. Individual sEPSC events had peak current magnitudes of 200 pA and decayed with a time constant of 2 ms. Each experiment was divided into 10 sweeps, each 10 15 seconds long and separated by 5 seconds to minimize rundown. Each sweep was divided into 500 ms segments. The total number of sEPSCs in each 500 ms segment was randomly chosen from a uniform distribution between 0 and 250. Then, the times of the sEPSCs within the 500 ms segment were randomly selected from a uniform distribution extending across the entire segment, simulating excitatory input from a population of unsynchronized 20 neurons. Empirically, these stimulation parameters reliably drove pyramidal neurons at firing rates from 0-30 Hz. In conditions marked as baseline, a 10 sec pulse of 590 nm light was delivered to completely inactivate the opsin before running the sEPSC protocol. In conditions where the opsin was activated, a 1 sec pulse of 470 nm light preceded the sEPSC protocol. 25 To understand the net effect of altered E/I balance on information processing, the mutual information between each neuron’s input sEPSC rate and output spike rate, which captures relevant changes in the shape of the 1-0 curve and in the response variability was computed. First, the joint distribution of sEPSC rate and spike rate by binning in time, sEPSC rate, and spike rate were estimated and the building of a joint histogram. Time bins 30 were 125 ms wide, and sEPSC rate was divided into 10 equally spaced bins from 0 to 500 Hz, although the mutual information results were consistent across a wide range of binning parameters. Spike rate was binned using the smallest meaningful bin width given the time bin width (e.g. 8 Hz bin width for 125 ms time bins). From this joint histogram, compute mutual information, as previously described was computed equaling the difference between response entropy and noise entropy. 2015203097 10 Jun2015

Response entropy quantifies the total amount of uncertainty in the output spike rate of the neuron. Noise entropy quantifies the uncertainty that remains in the output spike rate 5 given the input rate. Note that the maximum information that neural responses can transmit about the input stimulus is the entropy of the stimulus set. For 10 equally spaced input sEPSC rate bins and a uniform distribution of input rate over these bins, the entropy of the input rate is log2(10) = 3.322 bits.

Mutual information calculated from undersampled probability distributions can be 10 biased upwards. Consequently, all reported values of mutual information, response entropy and noise entropy were corrected for bias due to undersampling. This correction is done by computing values from smaller fractions (from one-half to one-eighth) of the full data and extrapolating to the limit of infinite data. Using 125 ms time windows, the correction factors were always less than 0.07 bits. 15 Vectors were created and injections were performed as above.

Results

Using this array of multiply engineered opsin genes, the possibilities for combinatorial control of cells and projections within intact mammalian systems was explored. First, it was asked whether excitatory and inhibitory neurons residing within the 20 same microcircuit could be separably targeted by the respective introduction of Cl VI variants and conventional ChRs into these two cell populations. It was found that cultured hippocampal neurons expressing C1V1-E122T/E162T spiked in response to 2 ms green light pulses (560nm) but not violet light pulses. In contrast, cells expressing ChR2-H134R spiked in response to 2 ms 405nm light pulses, but not in response to 2ms 561 nm light 25 pulses (FIG. 4A). This principle was therefore tested within living brain slices; in this case AAV5-CaMKIIa::ClVl-E122T/E 162T-ts-mCherry along with AAV5-EFla-DIO::ChR2-H134R-EYFP in was expressed in rnPFC of PV::Cre mice (FIG. 4B). In pyramidal neurons not expressing any opsin, 405 nm light pulses triggered robust and fast inhibitory postsynaptic currents due to direct activation of PV cells (FIG. 4C), while 561 nm light 30 pulses triggered both short-latency EPSCs (FIG. 4D) and the expected long-latency polysynaptic IPSCs arising from Cl VI-expressing pyramidal cell drive of local inhibitory neurons (FIG. 4C).

Excitation of these independent cellular elements in vivo with optrode recordings was then explored (FIG. 4E, left). To examine the inhibitory effect of PV cell activity on pyramidal neuron spiking, an experimental protocol in which 5Hz violet light pulses (to activate ChR2 in PV cells) preceded 5 Hz green light pulses (to activate Cl VI in excitatory pyramidal neurons) with varying inter-pulse intervals was designed. When violet and green light pulses were separated by 100 ms (FIG. 4E, top trace), responses to green light pulses 5 were not affected by the violet pulses. However, as delays between violet and green pulses were reduced, green light-induced events became more readily inhibited and were completely abolished when light pulses were presented with sufficient synchrony (FIG. 4E, bottom trace', summary data in FIG. 4F). These data demonstrate combinatorial optogenetic activation within an intact mammal (driving one population alone or in precise temporal 2015203097 10 Jun2015 10 combination with another), capitalizing on the speed of the opsins and the properties of the delivered light pulses.

Example 5: Effect of independent activation of corticothalamic (CT) and thalamocortical (TCI glutamatereic axons impinging upon neurons of the reticular thalamic nucleus 15 To validate the combinatorial control property for axonal projections instead of direct cellular somata stimulation, the effect of independent activation of corticothalamic (CT) and thalamocortical (TC) glutamatergic axons impinging upon neurons of the reticular thalamic nucleus (nRT) (FIG. 5A) was examined in thalamic slices.

Materials and Methods 20 C57BL/6J wild-type (postnatal days 90-120) were anesthetized with pentobarbital (100 mg/kg, i.p.) and decapitated. The thalamic slice preparation and whole-cell patch-clamp recordings were performed. Recordings were obtained from nRT (reticular thalamic) and TC (relay thalamocortical) neurons visually identified using differential contrast optics with a Zeiss (Oberkochen, Germany), Axioskop microscope, and an infrared video camera. 25 For EPSCs and current-clamp recordings, the internal solution contained (in mM): 120 K-gluconate, 11 KC1, 1 MgCl2,1 CaCl2,10 Hepes, 1 EGTA. pH was adjusted to 7.4 with KOH (290 mOsm). Ecf was estimated ~ -60 mV based on the Nemst equation. Potentials were corrected for -15 mV liquid junction potential. For voltage-clamp experiments neurons were clamped at -80 mV and EPSCs were pharmacologically isolated by bath 30 application of the GABAA receptor antagonist picrotoxin (50 μΜ, Tocris). In all recording conditions, access resistance was monitored and cells were included for analysis only if the access resistance was <18 ΜΩ and the change of resistance was <25% over the course of the experiment. 600 nL rAAV5/CamKIIa-hChR2(H134R)-EYFP or 900 nL rAAV5-CaMKIIaClVl(E122T/E162T)-TS-mCherry virus was injected stereotaxically into ventrobasal thalamus (VB) or barrel cortex, respectively, of C57BL/6J wild-type mice in vivo, between post-natal days 30-35. Intra-cortical and intra-thalamic (VB) injections were 5 performed in the same mice (n=6). Intra-cortical injections were preformed (from bregma) 1.3 mm posterior, 3 mm lateral, 1.15 mm below the cortical surface. Intra-thalamic injections were 1.7 mm posterior, 1.5 mm lateral, 3.5 mm below the cortical surface. Mice were sacrificed ~2-3 months following injections and horizontal brain thalamic slices were made for optical stimulation and in vitro recordings as described above. VB thalamus was 10 removed to avoid disynaptic activation of nRT neurons via the CT-TC-nRT pathway. Cutting VB thalamus from slices removed all photosensitive cell bodies from the preparation, enabled direct examination of CTnRT and TC-nRT projections, and did not affect the electrical membrane properties of nRT neurons (not shown). Optical activation of ChR2-expressing TC and Cl VI-expressing CT axons were performed with 405 nm and 560 15 nm laser stimuli, respectively (5 ms duration light pulses, 2-3 mW) (OEM Laser Systems, 2015203097 10 Jun2015 MI) delivered with optic fiber (BFL 37-300, ThorLabs) upstream along the CT and TC pathways projecting to nRT. Minimal stimulation intensity was used, defined as the light power that resulted in 50 to 70% failures (30 - 50% successes), fixed response kinetics and low response amplitude variability. Consequent minimal evoked EPSCs presumably 20 resulted from selective optical activation of single CT or TC axons presynaptic to the recorded cell. The stimulation light power was slightly increased (~5% above minimal stimulation) until the number of failures became 0. CT and TC inputs were (simultaneously) stimulated and minimal evoked EPSCs and EPSPs (each individually subthreshold for action potential firing) were recorded in nRT cells. 25 Statistical significance was calculated using paired or unpaired two-tailed /-tests, as applicable. Data were analyzed using Matlab Statistics toolbox or Microsoft Excel

Results

Minimal stimulation of TC axons evoked large and fast excitatory post-synaptic currents (EPSCs) in nRT neurons, whereas minimal stimulation of CT axons evoked small 30 and slow EPSCs in nRT neurons (FIG. 5B), both typical for these pathways.

Next the synaptic integration of CT and TC inputs under variable delay conditions between these two inputs was examined. Subthreshold EPSPs from each pathway became suprathreshold for action potential firing only when coincident within 5 ms (FIG. 5C-D). The temporal precision of Cl VI and ChR2 activation allowed a reliable control of the delay between the TC and CT inputs and thus allowed determination of a narrow window (-5ms) of effective synaptic integration in nRT cells, not previously observable with existing electrical, pharmacological, or optogenetic techniques due to the reciprocal connectivity of cortex and thalamus as well as the close approximation of CT and TC axons. These results 5 demonstrate for the first time, in the same intact preparation, independent activation of distinct axonal projections to examine their combinatorial effects on the same target cell. 2015203097 10 Jun2015

Example 6: Use of C1V1 and SSFO to achieve spectrotemporal separation of neural activation within the same circuit

In both of the above two preparations, visible-spectrum violet (405 nm) and green 10 (560 nm) lasers were used to achieve separable activation of the two opsins. While 405 nm lasers deliver safe non-UV light, for many applications it may be preferable to use 470 nm laser light for the blue-responsive opsin, since 470 nm light will penetrate tissue more deeply, scatter less, and be more easily and economically delivered from common blue light sources. While this may seem impossible since 470 nm light will partially activate C1V1 15 (FIG. 1G) as well as ChR2, combinatorial control could be achievable even with 470 nm light, capitalizing on both the temporal properties of SSFO and the redshifted nature of Cl VI to achieve "spectrotemporal separation" within intact mammalian tissue. To test this possibility, it was decided to directly compare, within the same preparation, the effects on rhythmic activity of stably potentiating either excitatory or inhibitory cells (FIG. 6A) 20 Materials and Methods

ChR2-D156A and SSFO were generated by inserting point mutations into the pLenti-CaMKIIa-ChR2-EYFP-WPRE vector using site-directed mutagenesis (Quikchange II XL; Stratagene). Viral gene delivery, coronal brain sectioning, and patch clamp recording were performed as above. Double virus injections to express CaMKIIa::ClVl 25 and DIO-SSFO in the mPFC of PV::Cre mice were performed.

While handling cells or tissues expressing SSFO, care was taken to minimize light exposure to prevent activation by ambient light. Before each experiment, a 20s pulse of 590 nm light was applied to convert all of the SSFO channels to the dark state and prevent rundown of photocurrents. For acquisition of SSFO activation and deactivation spectra, 30 recordings from cultured neurons were made in voltage clamp mode. For recording activation spectra, a 1 s pulse of varying wavelength was applied, followed by a 10 s 590 nm pulse. Deactivation spectra were acquired by first applying a 1 s 470 nm pulse to activate SSFO, followed by a 10 s pulse of varying wavelength. Net activation or deactivation was calculated by dividing the photocurrent change after the first or second pulse, respectively, by the maximum photocurrent change induced by the peak wavelength for that cell. Negative values in deactivation spectra resulted from traces in which, for example, a 10 s 470nm pulse led to a slight increase in photocurrent rather than deactivate the channels. This could be the result of the relatively wide (20 nm) band-pass filter width 5 used for these recordings with the Sutter DG-4. Intermediate wavelengths (between 470nm and 520nm) are expected to have a mixed effect on the channel population for the same reasons. 2015203097 10 Jun2015

Photon flux calculations for SSFO integration properties were conducted by calculating the photon flux through the microscope objective at each light power, and then 10 dividing to reach the photon flux across the cell surface, based on the diameter of the recorded cells and approximating cell shape as a spheroid.

Results SSFO is a novel multiply-engineered channelrhodopsin with a decay constant of 29 minutes that can be effectively deactivated at the same wavelengths that activate ClVI and 15 permits bistable excitation of neurons over many minutes with enhanced light sensitivity.

Information regarding SSFOs can be found in International Patent Application Publication No: WO 2010/056970 and United States Patent Application Nos: 61/410,704 and 61/410,711, the contents of which are hereby incorporated by reference herein in their entireties. Double virus injections to express CaMKIIa::ClVl and DIO::SSFO in acute 20 slices from the mPFC of PV::Cre mice were performed. Under these conditions, excitatory pyramidal cells should respond to redshifted light, and inhibitory PV cells to blue light. Indeed, in response to a 1 s 470 nm light pulse to activate SSFO in the PV cells, the rate of ongoing IPSCs was stably increased from 8.5 ± 1.2 Hz at baseline (period 3, FIG. 6B) to 16.8 ±2.1 Hz after the blue light pulse (period 2; n = 4 recordings, p < 0.005, paired t-test; 25 FIG. 6C), showing persistent activation of the SSFO-expressing inhibitory cells. Even though 470 nm light will also transiently activate C 1V1, this activation can only occur during the light pulse itself due to the very fast deactivation of Cl VI after light-off; the prolonged post-light period is characterized by SSFO activity only (FIG. 6B), illustrating temporal separation of optogenetic control modes. Interestingly, during this prolonged 30 period of elevated PV neuron activity, no significantly elevated peak in the IPSC power spectrum was elicited, suggesting that direct activation of PV neurons alone in this reduced preparation is insufficient to elicit gamma synchrony in the network. However, in marked contrast, during the 470 nm light pulse itself when the same level of PV neuron activation but also partial activation of C IV 1-expressing pyramidal cells is also expected, a pronounced gamma peak was consistently observed (peak frequency 39.2 ± 3.5 Hz; n = 4 recordings;) that extended into the high-gamma range (>60Hz). 2015203097 10 Jun2015

Moreover, in the same experiments (indeed, later in the same recorded sweeps), direct activation in this case of Cl Y1-pyramidal cells alone with 590 nm light(which 5 simultaneously activates Cl VI in PY cells and deactivates the SSFO in PV cells) led to robust gamma synchrony, with a lower frequency peak (26.6 ± 1 Hz, n = 6 recordings). Demonstrating that any residual PV neuron activity linked to the prior history of SSFO activation in PV cells was not necessary for this effect, otherwise-identical sweeps with only a history of Cl VI activation in the pyramidal cells and no prior history of elevated 10 IPSC rate elicited the same result). These results illustrate the integrated principle of spectrotemporal combinatorial control, and also suggest that elevating activity in pyramidal neurons can give rise through network properties to gamma oscillations . Interestingly, during the 470 nm light pulse, when activation of both PV and pyramidal cells was expected, gamma synchrony was consistently observed at higher frequencies than when 15 only excitatory neurons were activated, supporting and extending information on the coordinated importance of both PV and pyramidal cells in eliciting gamma oscillations.. '

Conclusion

In the course of this work, a family of new tools was generated that are referred to as Cl VI variants. Cl VI is a red-shifted opsin gene assembled, remarkably, from pieces 20 of other opsin genes that do not express well alone in neurons, but which were identified in earlier genomic searches (VChRl and ChRl). Cl VI contains no ChR2 sequence at all, yet its multiply- engineered variants reported here now represent the most potent, most redshifted, and most stable channelrhodopsins known. Mutagenesis in key amino acid positions throughout the retinal binding pocket led to the generation of (1) C1V1(E162T), a 25 high-expressing redshifted opsin gene generated as a fast homolog of the ChETA mutation; (2) C1V1(E122T) which displays the reddest action spectrum shoulder and can even be used to fire action potentials with red light (3) C1V1(E122T/E162T) - a combination mutant with the lowest desensitization, fastest deactivation, least violet-light activation for minimal cross-activation with ChR2, and strong expression. Indeed, Cl VI 30 variants may be selected for different applications based on considerations of current size, deactivation kinetics, and action spectrum (Table 1)— for example, in two-photon work, since 2P activation of ChR2 has been difficult due to current size and rapid kinetics of channel closure, Cl V1(E162T) is likely to be of interest. The Cl VI variants enabled 2015203097 10 Jun2015 direct testing of the hypothesis that increasing levels of elevated cellular E/I balance would give rise to increasing intensities of gamma rhythmicity, a phenomenon previously linked to both schizophrenia and autism. Of course, the different tools are also synergistic; using Cl VI variants together with ChR2 permitted reliable and separate 5 driving of spiking in the two distinct neuronal populations addressed in this study - the excitatory pyramidal neurons and the fast-spiking, parvalbumin-expressing inhibitory intemeurons, and confirm that steady elevated cellular E/I balance was effective at generating gamma-band circuit activity, capitalizing on both kinetic and spectral differences in the optogenetic tools. This type of combinatorial activation can be 10 extended beyond multiple cell types to multiple neural pathway types—for example, the separable activation of spiking, within a single brain region, in two converging axonal afferent pathways arising from distinct locations— a long-sought goal of systems neuroscience.

The examples, which are intended to be purely exemplary of the invention and 15 should therefore not be considered to limit the invention in any way, also describe and detail aspects and embodiments of the invention discussed above. The foregoing examples and detailed description are offered by way of illustration and not by way of limitation. All publications, patent applications, and patents cited in this specification are herein incorporated by reference as if each individual publication, patent application, or patent 20 were specifically and individually indicated to be incorporated by reference. Various embodiments described above, and discussed in the attached Appendices may be implemented together and/or in other manners. One or more of the aspects in the present disclosure and in the Appendices can also be implemented in a more separated or integrated manner, as should be apparent and is useful in accordance with particular target applications. In particular, all 25 publications and appendices cited herein are expressly incorporated herein by reference for the purpose of describing and disclosing compositions and methodologies which might be used in connection with the invention. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the 30 teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. 2015203097 10 Jun2015

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SEQUENCES 2015203097 10 Jun2015 SEQ ID NO:I (Humanized C1V1 amino acid sequence)

MSRRPWLLALALAVALAAGSAGASTGSDATVPVATQDGPDYVFHRAHERMLFQTSYTLENNGSVI CIPNNGQCFCLAWLKSNGTNAEKLAANILQWITFALSALCLMFYGYQTWKSTCGWEEIYVATIEMI 5 KFIIEYFHEFDEPAVIYSSNGNKTVWLRYAEWLLTCPVLLIHLSNLTGLKDDYSKRTMGLLVSDVGC IVWGATSAMCTGWTKILFFLISLSYGMYTYFHAAKVYIEAFHTVPKGICRELVRVMAWTFFVAWG MFPVLFLLGTEGFGHISPYGSAIGHSILDLIAKNMWGVLGNYLRVKIHEHILLYGDIRKKQKITIAGQ EMEVETLVAEEED SEQ ED NO:2 (Humanized C1V1 nucleotide sequence) 10 atgtcgcgacgcccgtggctccttgctctcgcattggcggtggcgcttgcagcgggatcggcaggagcgtcaaccggaagcgatgcgaccgtccccgtggct acgcaagacggaccagattacgtgttccacagagcccacgagcggatgttgtttcagacatcatacacacttgaaaacaatggtagcgtcatttgcatccctaac aatgggcagtgtttttgcctggcctggttgaaatcgaacggtacgaacgccgagaagctggcggcgaacattctgcagtggatcacattcgcactctcggcgct ctgccttatgttctatggctaccagacttggaaatccacgtgtggttgggaagagatctacgtagcaaccattgaaatgatcaagtttatcattgagtatttccatgagt ttgacgaaccggccgtaatctactcatcgaatgggaataagacagtctggttgaggtatgcggagtggctcctcacctgcccggtccttctgatccatctgagcaa 15 cctcacaggcctgaaggacgattatagcaaaaggactatgggcctgttggtttctgatgtgggatgcatcgtgtggggcgcaaccagcgccatgtgtacggggt ggacgaagatcctgttcttcctcatctcattgagctatggtatgtatacctattttcatgctgctaaagtttatatcgaagcattccacacagttccaaaagggatttgtc gagaactggtccgagtgatggcctggacattctttgtggcttggggaatgtttccagtcctgtttctgctgggcacggaaggattcggtcatatcagcccttatggat ctgccattgggcactccatcctcgacctgattgcaaagaacatgtggggtgtgctggggaattacctgcgcgtcaaaatccacgagcacatcctgttgtatggcg acatcagaaagaagcagaaaattacgatcgccggccaagagatggaggttgagacactggtggctgaagaggaggactaa 20 SEQ ID NO:3 (Humanized C1V1 E122T amino acid sequence)

MSRRPWLLALALAVALAAGSAGASTGSDATVPVATQDGPDYVFHRAHERMLFQTSYTLENNGSVICIPNNGQCFC IAWLKSNGTTSIAEKLAAMIjQWnFALSAl£lJVlFYGYQTWiCSI03WEIIWATTE]VIIKFnEYFHEFEEPAVIYSSNGNKrV WLRYAEWLLTCPVLLIHLSNLTGLKDDYSKRTMGLLVSDVGCIVWGATSAMCTGWI’KILFFLISLSYGMYTYFHAAK WIEAFHTVPKGICRELVRVMAWTFFVAWGMFPVLFlXGTEGFGHISPYGSAIGHSrLDLIAKNMWGVLGNYI.RV 25 KIHEHILLYGDfRKKQKI'IlAGQEMEVETLVAFEED SEQ Π) NO:4 (Humanized C1V1 E122T nucleotide sequence) atgtcgcgacgcccgtggctccttgctctcgcattggcggtggcgcttgcagcgggatcggcaggagcgtcaaccggaagcgatgcgaccgtccccgtggc tacgcaagacggaccagattacgtgttccacagagcccacgagcggatgttgtttcagacatcatacacacttgaaaacaatggtagcgtcatttgcatccctaa 30 caatgggcagtgtttttgcctggcctggttgaaatcgaacggtacgaacgccgagaagctggcggcgaacattctgcagtggatcacattcgcactctcggcg ctctgccttatgttctatggctaccagacttggaaatccacgtgtggttgggaaaccatctacgtagcaaccattgaaatgatcaagtttatcattgagtatttccatg agtttgacgaaccggccgtaatctactcatcgaatgggaataagacagtctggttgaggtatgcggagtggctcctcacctgcccggtccttctgatccatctga gcaacctcacaggcctgaaggacgattatagcaaaaggactatgggcctgttggtttctgatgtgggatgcatcgtgtggggcgcaaccagcgccatgtgtac ggggtggacgaagatectgttetteeteatcteattgagGiaiggialglaiaeeiatttteaigeigetaaagtttaiategaageatteeaeaeagtteeaaaaggg 35 atttgtcgagaactggtccgagtgatggcctggacattctttgtggcttggggaatgtttccagtcctgtttctgctgggcacggaaggattcggtcatatcagccc ttatggatctgccattgggcactccatcctcgacctgattgcaaagaacatgtggggtgtgctggggaattacctgcgcgtcaaaatccacgagcacatcctgtt gtatggcgacatcagaaagaagcagaaaattacgatcgccggccaagagatggaggttgagacactggtggctgaagaggaggactaa SEQ ID NO:5 (Humanized C1V1 E162T amino acid sequence) 2015203097 10 Jun2015

MSRRPWLLALALAVALAAGSAGASTGSDATVPVATQDGPDYVFHRAHERMLFQTSYTLENNGSVI CIPNNGQCFCLAWLKSNGTNAEKLAANILQWITFALSALCLMFYGYQTWKSTCGWEEIYVATIEMI KFIIEYFHEFDEPAVIYSSNGNKTVWLRYATWLLTCPVLLIHLSNLTGLKDDYSKRTMGLLVSDVGC 5 IVWGATSAMCTGWTKILFFLISLSYGMYTYFHAAKVYIEAFHTVPKGICRELVRVMAWTFFVAWG MFP VLFLLGTEGF GHISPYGS AIGHSILDLIAKNM W G VLGN YLRVKIFIEHILLY GDIRKKQKITIAGQ EMEVETLVAEEED SEQ ID NO:6 (Humanized C1V1 E162T nucleotide sequence) atgtcgcgacgcccgtggctccttgctctcgcattggcggtggcgcttgcagcgggatcggcaggagcgtcaaccggaagcgatgcgaccgtccccgtggct 10 acgcaagacggaccagattacgtgttccacagagcccacgagcggatgttgtttcagacatcatacacacttgaaaacaatggtagcgtcatttgcatccctaac aatgggcagtgtttttgcctggcctggttgaaatcgaacggtacgaacgccgagaagctggcggcgaacattctgcagtggatcacattcgcactctcggcgct ctgccttatgttctatggctaccagacttggaaatccacgtgtggttgggaagagatctacgtagcaaccattgaaatgatcaagtttatcattgagtatttccatgagt ttgacgaaccggccgtaatctactcatcgaatgggaataagacagtctggttgaggtatgcgacgtggctcctcacctgcccggtccttctgatccatctgagcaa cctcacaggcctgaaggacgattatagcaaaaggactatgggcctgttggtttctgatgtgggatgcatcgtgtggggcgcaaccagcgccatgtgtacggggt 15 ggacgaagatcctgttcttcctcatctcattgagctatggtatgtatacctattttcatgctgctaaagtttatatcgaagcattccacacagttccaaaagggatttgtc gagaactggtccgagtgatggcctggacattctttgtggcttggggaatgtttccagtcctgtttctgctgggcacggaaggattcggtcatatcagcccttatggat ctgccattgggcactccatcctcgacctgattgcaaagaacatgtggggtgtgctggggaattacctgcgcgtcaaaatccacgagcacatcctgttgtatggcg acatcagaaagaagcagaaaattacgatcgccggccaagagatggaggttgagacactggtggctgaagaggaggactaa SEQ ID NO:7 (Humanized C1V1 E122T/E162T amino acid sequence)

20 MSRRPWLLALALAVALAAGSAGASTGSDATVPVATQDGPDYVFHRAHERMLFQTSYTLENNGSVI CIPNNGQCFCLAWLKSNGTNAEKLAANILQWITFALSALCLMFYGYQTWKSTCGWETIYVATIEMI KFIIEYFHEFDEPAVIYSSNGNKTVWLRYATWLLTCPVLLIHLSNLTGLKDDYSKRTMGLLVSDVGC IVWGATSAMCTGWTKILFFLISLSYGMYTYFHAAKVYIEAFHTVPKGICRELVRVMAWTFFVAWG MFP VLFLLGTEGFGHISP Y GS AIGHSILDLIAKNM W G VLGN YLRVKIFIEHILLY GDIRKKQKITIAGQ 25 EMEVETLVAEEED SEQ Π) NO:8 (Humanized C1V1 E122T/E162T nucleotide sequence) atgtcgcgacgcccgtggctccttgctctcgcattggcggtggcgcttgcagcgggatcggcaggagcgtcaaccggaagcgatgcgaccgtccccgtggct acgcaagacggaccagattacgtgttccacagagcccacgagcggatgttgtttcagacatcatacacacttgaaaacaatggtagcgtcatttgcatccctaac 30 aatgggcagtgtttttgcctggcctggttgaaatcgaacggtacgaacgccgagaagctggcggcgaacattctgcagtggatcacattcgcactctcggcgct ctgccttatgttctatggctaccagacttggaaatccacgtgtggttgggaaaccatctacgtagcaaccattgaaatgatcaagtttatcattgagtatttccatgagt ttgacgaaccggccgtaatctactcatcgaatgggaataagacagtctggttgaggtatgcgacgtggctcctcacctgcccggtccttctgatccatctgagcaa cctcacaggcctgaaggacgattatagcaaaaggactatgggcctgttggtttctgatgtgggatgcatcgtgtggggcgcaaccagcgccatgtgtacggggt ggacgaagatcctgttcttcctcatctcattgagctatggtatgtatacctattttcatgctgctaaagtttatatcgaagcattccacacagttccaaaagggatttgtc 35 gagaactggtccgagtgatggcctggacattctttgtggcttggggaatgtttccagtcctgtttctgctgggcacggaaggattcggtcatatcagcccttatggat 2015203097 10 Jun2015 ctgccattgggcactccatcctcgacctgattgcaaagaacatgtggggtgtgctggggaattacctgcgcgtcaaaatccacgagcacatcctgttgtatggcg acatcagaaagaagcagaaaattacgatcgccggccaagagatggaggttgagacactggtggctgaagaggaggactaa SEQ ID NO:9 (Alternative Humanized C1V1 amino acid sequence (C1V1_25))

MSRRPWLLALALAVALAAGSAGASTGSDAWPVATQDGPDYWHRAHERMLFQTSYTLENNGSVrCIPNNGQCF 5 CLAWLKSNGTNAEKLAAMLQWITFALSALCLMFYGYQTWKSTCGWEEIYVATIEMIKFIIEYFHEFDEPATLWLS SGNGVWMRYGEWLLTCPVLLIHLSNLTGLKDDYSKRTMGLLVSDVGCIVWGATSAMCTGWTKILFFLISLSYG MYTYFFiAAKVYIEAFHWPKGICRELVRVMAWTFFVAWGMFPVLFLLGTEGFGFIISPYGSATGHSILDLIAKNM WGVLGNYLRVKIHEHILLYGDIRKKQKITIAGQEMEVETLVAEEED SEQ Π) NO:10 (Alternative Humanized C1V1 nucleotide sequence (CIV1_25)) 10 Atgagcagacggccctggctgctggccctggctctcgctgtggccctggccgccggcagcgccggagccagcaccggcagcgacgccaccgtgcccgtt gccacacaggacggccccgactacgtgttccaccgggcccacgagcggatgctgttccagaccagctacacccttgaaaacaacggcagcgtgatctgcatc cccaacaacggccagtgcttctgcctggcctggctgaagtccaacggcaccaacgccgagaagctggccgccaacatcctgcagtggatcaccttcgccctg tctgccctgtgcctgatgttctacggctaccagacctggaagtccacctgcggctgggaggaaatctacgtggccaccatcgagatgatcaagttcatcatcgag tacttccacgagttcgacgagcccgccaccctgtggctgtccagcggaaacggcgtggtgtggatgagatacggcgagtggctgctgacctgccctgtgctgc 15 tgatccacctgagcaacctgaccggactgaaggatgactacagcaagagaaccatgggactgctggtgtccgatgtgggatgcatcgtgtggggagccacct ccgccatgtgcaccggatggaccaagatcctgttcttcctgatcagcctgagctacggaatgtacacctacttccacgccgccaaggtgtacattgaggcctttca caccgtgcctaagggaatctgcagagaactggtcagagtgatggcctggaccttcttcgtggcctggggaatgttccctgtgctgttcctgctgggaaccgagg gattcggacacatcagcccttacggaagcgccatcggacacagcatcctggatctgatcgccaagaacatgtggggagtgctgggaaactacctgagagtga agatccacgagcacatcctgctgtacggcgacatcagaaagaagcagaagatcaccatcgccggacaggaaatggaagtcgagaccctggtggccgagga 20 agaggat SEQ Π) NO:ll ChR2 amino acid sequence

MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGTNGAQTASNVLQWLAA GFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKNPSMLYLATGHRVQWLR YAEWLLTCPVILIHLSNLTGLSNDYSRRTMGLLVSDIGTIVWGATSAMATGYVKVIFFCL 25 GLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVSWGMFPILFILGPEGFGVL SVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDIRKTTKLNIGGTEIEVETLV EDEAEAGAVP SEQ Π) NO:12 ChR2(H134R)

3 0 MDYGGALS AVGRELLF VTNP WVNGS VLVPEDQCYC AG WIESRGTNGAQTASNVLQ WLAA

GFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKNPSMLYLATGHRVQWLR YAEWLLTCPVILIRLSNLTGLSNDYSRRTMGLLVSDIGTIVWGATSAMATGYVKVIFFCL GLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVSWGMFPILFILGPEGFGVL SVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDIRKTTKLNIGGTEIEVETLV 35 EDEAEAGAVP 2015203097 10 Jun2015

SEQ ID NO:13 SFO

MDYGGALSAVGRELLFVTNPVWNGSVLVPEDQCYCAGWIESRGTNGAQTASNVLQWLAAGFSILL LMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKNPSMLYLATGHRVQWLRYAEWLLTSPVILI HLSNLTGLSNDYSRRTMGLLVSDIGTIVWGATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGY 5 HTVPKGRCRQVVTGMAWLFFVSWGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYL RVLIHEHILIHGDIRKTTKLNIGGTEIEVETLVEDEAEAGAVP SEQ ID NO:14 (SSFO)

10 MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGTNGAQTASNVLQWLAAGFSILL LMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKNPSMLYLATGHRVQWLRYAEWLLTSPVILI HLSNLTGLSNDYSRRTMGLLVSAIGTIVWGATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGY HTVPKGRCRQVVTGMAWLFFVSWGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYL RVLIHEHILIHGDIRKTTKLNIGGTEIEVETLVEDEAEAGAVP 58

Claims (9)

1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOW:

   1. An isolated light-responsive chimeric polypeptide comprising an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.
2. 2. The chimeric polypeptide of claim 1, wherein the polypeptide is activated by light of a wavelength between about 540 nm to about 560 nm.
3. 3. The chimeric polypeptide of claim 1 or claim 2, further comprising a C-terminal trafficking signal.
4. 4. The chimeric polypeptide of claim 3, wherein the trafficking signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 15).
5. 5. The chimeric polypeptide of any one of claims 1 to 4, wherein the light-responsive chimeric polypeptide comprises an amino acid sequence having at least about 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:l.
6. 6. The chimeric polypeptide of any one of claims 1 to 5, wherein the polypeptide comprises a Glu to Thr amino acid substitution at position 122 relative to the amino acid sequence set forth in SEQ ID NO: 1.
7. 7. The chimeric polypeptide of any one of claims 1 to 6, wherein the polypeptide comprises a Glu to Thr amino acid substitution at position 162 relative to the amino acid sequence set forth in SEQ ID NO: 1.
8. 8. The chimeric polypeptide of any one of claims 1 to 7, wherein the polypeptide comprises a Glu to Thr amino acid substitution at position 122 and a Glu to Thr amino acid substitution at position 162 relative to the amino acid sequence set forth in SEQ ID NO:l.
9. 9. The chimeric polypeptide of any one of claims 1 to 8, wherein the light-responsive chimeric polypeptide comprises an amino acid sequence having at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:l.