Systems, methods and compositions for optical stimulation of target cells

Abstract

Stimulation of target cells using light, e.g., in vivo or in vitro, is implemented using a variety of methods and devices. One example involves a vector for delivering a light-activated NpHR-based molecule comprising a nucleic acid sequence that codes for light-activated NpHR-based molecule and a promoter. Either a high expression of the molecule manifests a toxicity level that is less than about 75%, or the light-activated NpHR-based proteins are expressed using at least two NpHR-based molecular variants. Each of the variants characterized in being useful for expressing a light-activated NpHR-based molecule that responds to light by producing an inhibitory current to dissuade depolarization of the neuron. Other aspects and embodiments are directed to systems, methods, kits, compositions of matter and molecules for ion pumps or for controlling inhibitory currents in a cell (e.g., in in vivo and in vitro environments).

1 - 24 . (canceled) 
     
     
         25 . A method for controlling a voltage level across a cell membrane of a cell, the method comprising:
 a) expressing in a neuron a variant opsin polypeptide derived from Natronomonas pharaonis (NpHR), wherein the variant NpHR opsin polypeptide comprises:
 i) an NpHR opsin polypeptide encoded by a mammalian codon optimized nucleotide sequence; and 
 ii) a heterologous endoplasmic reticulum (ER) export signal comprising the amino acid sequence set forth in SEQ ID NO:  16 , wherein the variant NpHR opsin polypeptide exhibits reduced toxicity in the neuron compared to toxicity induced by wild-type NpHR; and 
   b) measuring the voltage level across the cell membrane; and   c) exposing the neuron to light of a wavelength that activates the polypeptide, wherein said exposing results in producing a current across the cell membrane of the cell that is responsive to the measured voltage level.   
     
     
         26 . The method of  claim 25 , wherein the variant NpHR polypeptide comprises a heterologous signal peptide. 
     
     
         27 . The method of  claim 26 , wherein the heterologous signal peptide is a nicotinic acetylcholine receptor signal peptide. 
     
     
         28 . The method of  claim 27 , wherein the heterologous signal peptide comprises the amino acid sequence set forth in SEQ ID NO:13. 
     
     
         29 . The method of  claim 25 , wherein the nucleotide sequence is present in a recombinant expression vector. 
     
     
         30 . The method of  claim 29 , wherein the nucleotide sequence is operably linked to a neuron-specific promoter. 
     
     
         31 . The method of  claim 30 , wherein the neuron-specific promoter is a CaMKIIα promoter. 
     
     
         32 . The method of  claim 30 , wherein the neuron-specific promoter is a cholinergic neuron-specific promoter. 
     
     
         33 . The method of  claim 29 , wherein the expression vector is a viral expression vector. 
     
     
         34 . The method of  claim 25 , wherein the variant NpHR polypeptide is encoded by nucleotides 1-873 of SEQ ID NO:1. 
     
     
         35 . The method of  claim 25 , wherein the light has a wavelength of from 573 nm to 613 nm. 
     
     
         36 . The method of  claim 25 , wherein the nucleic acid comprising the mammalian codon optimized nucleotide sequence that encodes the variant NpHR polypeptide further comprises a nucleic acid comprising a nucleotide sequence encoding a channelrhodopsin-2 polypeptide. 
     
     
         37 . A method of controlling behavior of an animal in vivo, the method comprising:
 a) expressing in the muscle or cholinergic motoneurons of the animal a variant opsin polypeptide derived from  Natronomonas pharaonis  (NpHR), wherein the variant NpHR opsin polypeptide comprises:
 i) an NpHR opsin polypeptide encoded by a mammalian codon optimized nucleotide sequence; and 
 ii) a heterologous endoplasmic reticulum (ER) export signal comprising the amino acid sequence set forth in SEQ ID NO: 16, wherein the variant NpHR opsin polypeptide exhibits reduced toxicity in the animal compared to toxicity induced by wild-type NpHR; and 
   b) exposing the animal to light of a wavelength that activates the polypeptide, wherein said exposing results in rapid inhibition of movement and relaxed bodies of the animal.   
     
     
         38 . The method of  claim 37 , wherein the light is exposed to the animal for 1 or 10 seconds. 
     
     
         39 . The method of  claim 38 , further comprising measuring the behavior of an animal after activating the polypeptide with light. 
     
     
         40 . The method of  claim 39 , wherein the behavior is a swimming behavior. 
     
     
         41 . The method of  claim 40 , wherein activation of the NpHR opsin polypeptide when the animal is exposed to light for 1 or 10 seconds results in reduced or arrested swimming behavior. 
     
     
         42 . The method of  claim 41 , wherein the arrested swimming behavior is reversed following light termination.