Fast noninvasive activation and inhibition of neural and network activity by vertebrate rhodopsin and green algae channelrhodopsin

Abstract

Techniques for fast noninvasive control of neuronal excitability will be of major importance for analyzing and understanding neuronal networks and animal behavior. To develop these tools we demonstrated that two light-activated signaling proteins, vertebrate rat rhodopsin 4 (RO4) and the green algae channelrhodospin 2 (ChR2), could be used to control neuronal excitability and modulate synaptic transmission. Vertebrate rhodopsin couples to the Gi/o, pertussis toxin-sensitive pathway to allow modulation of G protein-gated inward rectifying potassium channels and voltage-gated Ca2+ channels. Light-mediated activation of RO4 in cultured hippocampal neurons reduces neuronal firing within ms by hyperpolarization of the somato-dendritic membrane and when activated at presynaptic sites modulates synaptic transmission and paired-pulse facilitation. In contrast, somato-dendritic activation of ChR2 depolarizes neurons sufficiently to induce immediate action potentials, which precisely follow the ChR2 activation up to light stimulation frequencies of 20 Hz. To demonstrate that these constructs are useful for regulating network behavior in intact organisms, embryonic chick spinal cords were electroporated with either construct, allowing the frequency of episodes of spontaneous bursting activity, known to be important for motor circuit formation, to be precisely controlled. Thus light-activated vertebrate RO4 and green algae ChR2 allow the antagonistic control of neuronal function within ms to s in a precise, reversible, and noninvasive manner in cultured neurons and intact vertebrate spinal cords.

Fig. 1.

Vertebrate rhodopsin modulates GIRK and P/Q-type Ca²⁺ channels via Gi/o-PTX-sensitive pathways. (A) K⁺ current traces of GIRK1/2 channels coexpressed with RO4 or mAChR-M2 in HEK293 cells before, during, and after light stimulation (Left) or 10 μM Carb application (Right). Currents were elicited by 500-ms voltage ramps from -100 to +50 mV. (B) Comparison of the GPCR-induced current increase in the presence and absence of 5 nmol PTX. (C) Time course traces of GPCR-mediated activation of GIRK currents. GIRK currents were recorded at -60 mV. (D) Comparison of the time constants of the GPCR-induced GIRK current changes before and after GPCR activation. (E) Ba²⁺ current traces of P/Q-type Ca²⁺ channels (α₁2.1, β_1b, and α₂δ subunits) coexpressed with RO4 or mAChR-M2 in HEK293 cells before, during, and after light stimulation (Left) or 10 μM Carb application (Right). (F) GPCR-induced depolarizing shift in the voltage dependence of activation curve of P/Q-type Ca²⁺ currents. Currents were elicited from a holding potential of -60 mV by 5-ms-long, 5-mV voltage steps from -10 to +65 mV. Relative tail currents were plotted against the voltage pulses. (G) Time course traces of GPCR-mediated inhibition of P/Q-type Ca²⁺ currents. Ba²⁺ currents were elicited by voltage pulses from -60 to +20 mV and measured every s. (H) Comparison of the time constants of the GPCR-induced P/Q-type channel current changes before and after GPCR activation. Throughout all experiments number in parentheses indicate the number of experiments and statistical significance as indicated (^*, P < 0.05; ^**, P < 0.01, ANOVA).

Fig. 2.

Functional expression and characterization of vertebrate rhodopsin in cultured hippocampal neurons. (A) Colocalization of RO4 and synaptobrevin in cultured hippocampal neurons. (Left) Fluorescence patterns of neurons from low-density hippocampal cultures transfected with RO4 reveal a punctate staining. RO4 was detected with an anti-RO4 antibody and visualized with an Alexa 488-coupled secondary antibody. (Center) Hippocampal cells were stained with an antisynaptobrevin II antibody and visualized with an Alexa 568-coupled secondary antibody. (Right) Overlay of RO4 and synaptobrevin II staining. Yellow indicates colocalization. (B) RO4 induced voltage change during a long (Upper) and short (Lower) light pulse. (C) Average GPCR (RO4, GABA_B)-induced hyperpolarization of cultured hippocampal neurons. Throughout the experiments GABA_B receptors were activated by application of 50 μM baclofen (Bacl). (D) Time course of GPCR (RO4, GABA_B)-induced hyperpolarization and recovery from hyperpolarization after switching off the light or washing out baclofen. (E) Voltage traces of current-induced (30 pA) neuronal firing of cultured hippocampal neurons before and during light activation of RO4. (F) Comparison of the number of action potentials measured after current injection for a neuron before and during light activation of RO4. (G) Comparison of EPSC amplitude before, during, and after light application for EPSCs measured in autaptic hippocampal cultures expressing RO4. EPSCs in autaptic hippocampal neurons were elicited by 2-ms voltage pulses from -60 to +10 mV. (H) Comparison of GPCR (RO4, GABA_B)-induced EPSC inhibition measured in autaptic hippocampal neurons. (I) Time constants of GPCR (RO4, GABA_B)-induced EPSC inhibition and release from inhibition. EPSCs were elicited every 5 s as described in G. (J) Autaptic EPSC traces elicited by 2-ms voltage pulses from -60 to +10 mV separated by 50 ms (20-Hz stimulation) before and after light activation of RO4. (K) Comparison of paired-pulse facilitation before and after GPCR (RO4, GABA_B) activation for a 20-Hz stimulation protocol. The amplitude of the second EPSC was compared with the first EPSC.

Fig. 3.

Functional expression and characterization of green algae ChR2 in cultured hippocampal neurons. (A) Colocalization of ChR2 and synaptobrevin in cultured hippocampal neurons. (Left) Fluorescence patterns of neurons from low-density hippocampal cultures transfected with GFP-ChR2 reveal a punctate staining. (Center) Hippocampal cells were stained with an antisynaptobrevin II antibody and visualized with an Alexa 568-coupled secondary antibody. (Right) Overlay of GFP-ChR2 and synaptobrevin II staining. Yellow indicates colocalization. (B) Voltage traces of ChR2-induced neuronal firing of cultured hippocampal neurons for light stimuli with increasing duration. (C) Voltage traces of ChR2-induced neuronal firing of cultured hippocampal neurons for light stimuli with different frequencies. (D) Number of action potentials measured in neurons expressing ChR2. Action potentials were elicited by a train of 10 stimuli for different light stimulation frequencies with a light duration of 5 ms. (E) Light activation of ChR2 expressed in excitatory (Upper) or inhibitory (Lower) presynaptic neurons induce activation or inhibition in the paired postsynaptic neurons. (E1 and E4) EPSC (Upper) or IPSC (Lower) were elicited by a 2-ms voltage pulse from -60 to +10 mV in the postsynaptic autaptic neuron. (E2 and E5) Light activation of the excitatory and inhibitory presynaptic cells expressing ChR2 induced EPSC (Upper) or IPSC (Lower) on the postsynaptic, autaptic neurons. (E3) Presynaptically (excitatory) light induced spiking or subthreshold depolarization (Inset) of the postsynaptic neuron after a single 5-ms light pulse (Left) or a 10-Hz/5-ms light stimulation protocol (Right). Five light pulses were applied. (E6) Presynaptically (inhibitory) light induced hyperpolarization of the postsynaptic neurons after a single 5-ms light pulse. (E7) Schematic diagram of the neuronal circuit analyzed. Gray indicates the presynaptic neuron expressing ChR2. (F) Average amplitude of the light induced EPSCs or IPSCs. (G) Average amplitude of the light-induced hyperpolarization (IPSP) or depolarization (EPSP), when the depolarization was not sufficient to trigger an action potential.

Fig. 4.

RO4 and ChR2 can be used to regulate the frequency of spontaneous rhythmic activity in isolated embryonic chick spinal cords and living embryos. (A) Diagram of isolated chicken spinal cord preparation showing the position of the recording suction electrode; regions electroporated with either ChR2 or RO4 are shown in gray. (B) Electrical recording from motor nerve of ChR2 lumbar-electroporated embryo showing two control episodes in the absence of light (Upper) with an expanded time base trace of a single episode shown (Lower). Bursts of many motor axons firing synchronously and individual motor axons firing asynchronously are noted. (C) Plot of the intervals (in min) between bursting episodes from a lumbar electroporated ChR2 embryo subjected to a long interval of continuous light (circles) or 3-s pulses of light (triangles); filled symbols indicate episodes elicited in the presence of light, and open circles indicate episodes occurring in the absence of light. (D) Electrical recordings showing episodes (denoted by brackets) occurring during several minutes of continuous light (Upper) or elicited by a 3-s pulse of light at the position of the asterisk (Lower). (E) Comparison of unit activity preceding bursts that occurred spontaneously in a nonelectroporated embryo (Top) or were elicited by light when ChR2 was expressed selectively in the lumbar cord (Middle) or cervical cord (Bottom). Time of light exposure is indicated by dashed line. (F) Bar graph of the percent change in motor unit activity occurring in control embryo and one electroporated at cervical or lumbar level during a 3-s exposure to light. (G) The frequency of axial movements of stage 25-26 embryos in ovo, 3 days after ChR2 was electroporated into cervical cord segments, in the presence or absence of 475 nM light. (H) Plot of intervals between bursting episodes in embryos electroporated with RO4 at lumbar level when exposed to a long interval of continuous light (circles) or 3-s light pulses at different repetition rates (triangles); filled symbols indicate episodes occurring in the presence of light, open symbols indicate those that occurred in the absence of light. (I) Activation of RO4 by brief light pulses triggers bursting episodes. (Top) After a spontaneous episode (no. 1) a 2-s light pulse was able to trigger a premature bursting episode (no. 2); both are shown on expanded time bases in Middle and Bottom, respectively (see text for more detail). (J) Bar graph of change in motor unit activity in the period preceding the first burst of a spontaneous episode or one evoked by light activation of RO4. (K) Light activation of RO4 can synchronize the bursting behavior of spinal cord motoneurons. Right and left sides of a RO4 lumbar electroporated cord exhibit independent (asynchronous) rhythms when they are surgically separated at the midline (top pair of traces) However, the bursts triggered after the cessation of a light stimulus results in their synchronization (bottom pair of traces). LS3, lumbar segment 3; Sp.N., spinal nerve.