Optogenetic control of motor coordination by Gi/o protein-coupled vertebrate rhodopsin in cerebellar Purkinje cells

Abstract

G protein-coupled receptors are involved in the modulation of complex neuronal networks in the brain. To investigate the impact of a cell-specific G(i/o) protein-mediated signaling pathway on brain function, we created a new optogenetic mouse model in which the G(i/o) protein-coupled receptor vertebrate rhodopsin can be cell-specifically expressed with the aid of Cre recombinase. Here we use this mouse model to study the functional impact of G(i/o) modulation in cerebellar Purkinje cells (PCs). We show that in vivo light activation of vertebrate rhodopsin specifically expressed in PCs reduces simple spike firing that is comparable with the reduction in firing observed for the activation of cerebellar G(i/o)-coupled GABA(B) receptors. Notably, the light exposure of the cerebellar vermis in freely moving mice changes the motor behavior. Thus, our studies directly demonstrate that spike modulation via G(i/o)-mediated signaling in cerebellar PCs affects motor coordination and show a new promising approach for studying the physiological function of G protein-coupled receptor-mediated signaling in a cell type-specific manner.

FIGURE 1.

Cell type-specific Cre recombinase-mediated expression of vertebrate rhodopsin in cerebellar Purkinje cells. A, shown is a schematic description of the construct used to create the transgenic animals expressing floxed vRh. vRh was cloned into the pCZW vector, which contains a CMV enhancer and β-actin promoter and a lacZ expression cassette flanked by two loxP sequences. X-gal staining of sagittal brain slices from the vRh-GFP(Tg^flvRh-GFP) mouse line shows β-galactosidase expression throughout the brain with robust expression localized in the cerebellum (left) and the hippocampus (middle), and caudate putamen (right). B, a diagram revealing the results of Cre-mediated recombination events indicates an excision of the lacZ expression cassette and cell type-specific expression of vRh-GFP driven in PCs. Cre recombinase-mediated induction of vRh-GFP expression in PCs was accomplished by crossing vRh-GFP(Tg^flvRh-GFP) mice with Purkinje cell-specific CRE (Tg^Pcp2-cre) mice. PC-specific expression of vRh was verified by immunohistochemical staining for GFP (middle) and calbindin (a calcium-binding protein associated with Purkinje cells; left). Right, three-dimensional reconstruction of confocal z-stack images reveals colocalization of vRh-GFP and calbindin in the soma and proximal dendrites of PCs. Scale bars: left and middle, 25 μm; right,10 μm.

FIGURE 2.

In vivo photostimulation of the cerebellar vermis of vRh-GFP^PC induces a reduction in the firing rate of Purkinje cells. A, a Nissl stain of sagittal cerebellum slices after electrolytic lesions indicates that the in vivo recordings and the application of baclofen (see Fig. 3) were directed to the Purkinje cell layer. B, PC firing rate was recorded and calculated as the percent change in firing before and after light application. The vRh-GFP^PC transgenic line demonstrated a significant reduction in firing during the light pulse that persisted after 30 s once light was switched off. C, representative firing rates (Hz) of individual PCs from control and vRh-GFP^PC mice with and without illumination reveal that only neurons from the vRh-GFP^PC line reveal a decrease in the firing rate after light treatment. D, analysis of the CV after light application indicates no significant difference between wild type littermates and vRh-GFP^PC mice. E, raw traces of control littermates and vRh-GFP^PC mice before and during the 473-nm light pulse reveal a reduction in the PC firing rate only in vRh-GFP-positive transgenic mice. The presence of both simple and complex spikes as well as a comparative analysis of depth with a standard mouse brain atlas confirmed that recordings were taken from Purkinje cells. Statistical significance was evaluated with ANOVA (**, p < 0.01). Given values are the mean ± S.E.

FIGURE 3.

In vivo and in vitro application of baclofen decreases the firing rate of cerebellar Purkinje cells. A–E, in vivo recordings of cerebellar PCs before and after baclofen application are shown. A, comparative immunohistochemical staining of GABA_B1R (red) and vRh-GFP^PC (green) reveals the expression of GABA_B1R in both PCs and cerebellar cortical neurons. vRh-GFP^PC expression is restricted to PCs and colocalized partly (yellow) with GABA_B1Rs. Scale bar, 25 μm. B, percentage change in the number of spikes for a 10-s time interval during (t = 16–26 s) and after (t = 20–30 s) 1 mm baclofen application are compared with control (saline application). Bar graphs indicate a significant decrease in the firing of Purkinje cells with baclofen application. C, spike frequency (Hz) before and after saline or baclofen application for each recorded PC demonstrates an overall reduction in firing that corresponds to baclofen administration. D, calculated values for the CV between PCs treated with saline or baclofen reveals no significant difference in data dispersion between the two treatment groups. E, raw traces of PCs before and during either saline or baclofen treatment indicates reduced firing in the present of 1 mm baclofen. The presence of both simple and complex spikes as well as a comparative analysis of depth with a standard mouse brain atlas confirmed that recordings were taken from Purkinje cells. F–I, cerebellar slice recordings of PCs before and after baclofen application are shown. F, percentage change in spike number during the 10 mm baclofen bath application displays reduced firing. G, modifications in the overall firing rate (Hz) for the recorded PCs during baclofen application are shown. H, calculated CV values for extracellular PC recordings indicate no significant difference before and during baclofen treatment are shown. I, raw data traces demonstrate a decrease in PC firing with baclofen. Statistical significance was evaluated with ANOVA (*, p < 0.05). All values are the mean ± S.E.

FIGURE 4.

Light activation of vertebrate rhodopsin expressed in Purkinje cells of the cerebellum induces changes in motor behavior. A, shown is a photograph demonstrating the permanent placement of the cannula light guide used for behavioral testing. B, shown is pole test performance of control and vRh-GFP^PC mice (n = 10) before and after a 26-s light pulse. The light activation of vRh in vRh-GFP^PC mice results in either a fall (scored as 120 s) or an increase in the time required to descend from the pole. Light application to control littermates did not initiate any significant difference in the time required to descend the pole. C, rotarod performance of wild type littermates (n = 10) and vRh-GFP^PC mice before and after a 26-s light pulse is shown. Light activation of vRh in vRh-GFP^PC mice produces a significant decrease in rotarod performance when compared with wild type littermates. Performance between the two groups when no light pulse was applied reveals no significant difference. D, beam walk analysis demonstrates an increase in the time required to successfully cross the length of the beam after vRh activation in vRh-GFP^PC mice. Conversely, the time needed to cross the beam decreases in control littermates regardless of the light pulse. Falls were assigned a value of 120 s. Additionally, a measurement of the number of paw slips reveals a significant increase after light application for the left side of the vRh-GFP^PC mice, whereas control littermates experienced no significant increase in slips post-light application. E, grip strength assessment of wild type and vRh-GFP^PC mice before and after a 26-s light illumination is shown. No significant differences were observed for the grip strength of the front and hind paws between wild type littermates and vRh-GFP^PC mice before and after light application. Statistical significance in all behavior experiments was evaluated with ANOVA (*, p < 0.05; **, p < 0.01). Shown values are the mean ± S.E.