Neurogenin 2 regulates progenitor cell-cycle progression and Purkinje cell dendritogenesis in cerebellar development

By serving as the sole output of the cerebellar cortex, integrating a myriad of afferent stimuli, Purkinje cells (PCs) constitute the principal neuron in cerebellar circuits. Several neurodegenerative cerebellar ataxias feature a selective cell-autonomous loss of PCs, warranting the development of regenerative strategies. To date, very little is known as to the regulatory cascades controlling PC development. During central nervous system development, the proneural gene neurogenin 2 (Neurog2) contributes to many distinct neuronal types by specifying their fate and/or dictating development of their morphological features. By analyzing a mouse knock-in line expressing Cre recombinase under the control of Neurog2 cis-acting sequences we show that, in the cerebellar primordium, Neurog2 is expressed by cycling progenitors cell-autonomously fated to become PCs, even when transplanted heterochronically. During cerebellar development, Neurog2 is expressed in G1 phase by progenitors poised to exit the cell cycle. We demonstrate that, in the absence of Neurog2, both cell-cycle progression and neuronal output are significantly affected, leading to an overall reduction of the mature cerebellar volume. Although PC fate identity is correctly specified, the maturation of their dendritic arbor is severely affected in the absence of Neurog2, as null PCs develop stunted and poorly branched dendrites, a defect evident from the early stages of dendritogenesis. Thus, Neurog2 represents a key regulator of PC development and maturation.     

Morphometric analysis of dendritic tree of cerebellar Purkinje-cells

Basic neural processes of sensorimotor adaptation can be observed by cellular-level studies on cerebellar cortex, both by electrophysiological and morphological means. In earlier studies we demonstrated double (sometimes triple or quadruple) rhythmic prespike activity patterns in dendritic microelectrode records taken from cerebellar Pc. By their active and passive interactions, the actual input pattern will turn into an arrhythmic output spiking, realizing a nonlinear and phase-sensitive integration. This curious complex spike-generating process can give rise to novel cerebellar functional models. The acting membrane dynamics rely not just upon the ionic current machinery but also upon the specific micromorphology of the dendritic tree, especially that of the branching areas. Although, statistical analyses on Pc dendrites generally followed the graph-theory, elaborated abstract parameters for complexity and hierarchy, and denied realistic geometry. Thus a novel specific morphometric study should be carried out, first applied to the main branching sites.     

Electrophysiological analysis of the circuitry and of the corticonuclear relationships in the agranular cerebellum of irradiated rats.

The cerebellar circuitry and the corticonuclear relationships were studied in the cerebellum of adult rats rendered agranular through 7 successive exposures to X-ray radiations during infancy. Data were obtained through examination of electrical responses induced in Purkinje cells (PC) and in neurons of the lateral vestibular nucleus (LVN) by cerebellar and spinal stimulations. In irradiated rats, PC exhibited antidromic activation with a high axonal threshold and 70% of them also presented typical climbing fiber responses (CFRs). By contrast, they exceptionnally exhibited responses via the mossy fiber (MF)-granule cell pathway, but two other classes of responses were identified: i) short latency single spike responses attributed to a direct excitatory impingement of MF onto PC; ii) atypical CFRs formed of high frequency bursts of simple spikes which were seen in 76% of PC tested. Furthermore, 53% of these cells also presented typical CFRs, strongly suggesting these PC were innervated by more than one CF, thus confirming previous data on the same type of agranular cerebellum. In the LVN neurons of control and irradiated rats, spinal and cerebellar stimulations evoked clear cut IPSPs. On the basis of their shape, latency, and occurrence in animals with or without cerebellum and with or without lesion of the CF pathway, they were interpreted as mediated through direct or synaptic activation of PC or through an extracerebellar pathway. In irradiated rats, the quantitative study of these IPSPs gave further arguments in favor of a multiinnervation of PC by CF and of an important reafferentation of MF onto PC. However, the functional efficiency of this reafferentation appeared very low, as tested by activation of MF originating in the spinal cord. Finally, the intracellular recording of LVN neurons showed that a large majority of PC axons retained normal synaptic connections with nuclear cells in treated animals, indicating that corticonuclear relationships do not markedly depend upon granule cells and normal CF input.     

Dendritic Signals Command Firing Dynamics in a Mathematical Model of Cerebellar Purkinje Cells

Dendrites of cerebellar Purkinje cells (PCs) respond to brief excitations from parallel fibers with lasting plateau depolarizations. It is unknown whether these plateaus are local events that boost the synaptic signals or they propagate to the soma and directly take part in setting the cell firing dynamics. To address this issue, we analyzed a likely mechanism underlying plateaus in three representations of a reconstructed PC with increasing complexity. Analysis in an infinite cable suggests that Ca plateaus triggered by direct excitatory inputs from parallel fibers and their mirror signals, valleys (putatively triggered by the local feed forward inhibitory network), cannot propagate. However, simulations of the model in electrotonic equivalent cables prove that Ca plateaus (resp. valleys) are conducted over the entire cell with velocities typical of passive events once they are triggered by threshold synaptic inputs that turn the membrane current inward (resp. outward) over the whole cell surface. Bifurcation analysis of the model in equivalent cables, and simulations in a fully reconstructed PC both indicate that dendritic Ca plateaus and valleys, respectively, command epochs of firing and silencing of PCs.     

Purkinje cells originate from cerebellar ventricular zone progenitors positive for Neph3 and E-cadherin

GABAergic Purkinje cells (PCs) provide the primary output from the cerebellar cortex, which controls movement and posture. Although the mechanisms of PC differentiation have been well studied, the precise origin and initial specification mechanism of PCs remain to be clarified. Here, we identified a cerebellar and spinal cord GABAergic progenitor-selective cell surface marker, Neph3, which is a direct downstream target gene of Ptf1a, an essential regulator of GABAergic neuron development. Using FACS, Neph3⁺ GABAergic progenitors were sorted from the embryonic cerebellum, and the cell fate of this population was mapped by culturing in vitro. We found that most of the Neph3⁺ populations sorted from the mouse E12.5 cerebellum were fated to differentiate into PCs while the remaining small fraction of Neph3⁺ cells were progenitors for Pax2⁺ interneurons, which are likely to be deep cerebellar nuclei GABAergic neurons. These results were confirmed by short-term in vivo lineage-tracing experiments using transgenic mice expressing Neph3 promoter-driven GFP. In addition, we identified E-cadherin as a marker selectively expressed by a dorsally localized subset of cerebellar Neph3⁺ cells. Sorting experiments revealed that the Neph3⁺ E-cadherin^high population in the embryonic cerebellum defined PC progenitors while progenitors for Pax2⁺ interneurons were enriched in the Neph3⁺ E-cadherin^low population. Taken together, our results identify two spatially demarcated subregions that generate distinct cerebellar GABAergic subtypes and reveal the origin of PCs in the ventricular zone of the cerebellar primordium.     

An Aberrant Cerebellar Development in Mice Lacking Matrix Metalloproteinase-3

Cell–cell and cell–matrix interactions are necessary for neuronal patterning and brain wiring during development. Matrix metalloproteinases (MMPs) are proteolytic enzymes capable of remodelling the pericellular environment and regulating signaling pathways through cleavage of a large degradome. MMPs have been suggested to affect cerebellar development, but the specific role of different MMPs in cerebellar morphogenesis remains unclear. Here, we report a role for MMP-3 in the histogenesis of the mouse cerebellar cortex. MMP-3 expression peaks during the second week of postnatal cerebellar development and is most prominently observed in Purkinje cells (PCs). In MMP-3 deficient (MMP-3^−/−) mice, a protracted granule cell (GC) tangential migration and a delayed GC radial migration results in a thicker and persistent external granular layer, a retarded arrival of GCs in the inner granular layer, and a delayed GABAergic interneuron migration. Importantly, these neuronal migration anomalies, as well as the consequent disturbed synaptogenesis on PCs, seem to be caused by an abnormal PC dendritogenesis, which results in reduced PC dendritic trees in the adult cerebellum. Of note, these developmental and adult cerebellar defects might contribute to the aberrant motor phenotype observed in MMP-3^−/− mice and suggest an involvement of MMP-3 in mouse cerebellar development.     

Dendritic NMDA receptors activate axonal calcium channels

NMDA receptor (NMDAR) activation can alter synaptic strength by regulating transmitter release from a variety of neurons in the CNS. As NMDARs are permeable to Ca(2+) and monovalent cations, they could alter release directly by increasing presynaptic Ca(2+) or indirectly by axonal depolarization sufficient to activate voltage-sensitive Ca(2+) channels (VSCCs). Using two-photon microscopy to measure Ca(2+) excursions, we found that somatic depolarization or focal activation of dendritic NMDARs elicited small Ca(2+) transients in axon varicosities of cerebellar stellate cell interneurons. These axonal transients resulted from Ca(2+) entry through VSCCs that were opened by the electrotonic spread of the NMDAR-mediated depolarization elicited in the dendrites. In contrast, we were unable to detect direct activation of NMDARs on axons, indicating an exclusive somatodendritic expression of functional NMDARs. In cerebellar stellate cells, dendritic NMDAR activation masquerades as a presynaptic phenomenon and may influence Ca(2+) -dependent forms of presynaptic plasticity and release.     

Mossy fibre contact triggers the targeting of Kv4.2 potassium channels to dendrites and synapses in developing cerebellar granule neurons

Compartmentalization of neuronal function is achieved by highly localized clustering of ion channels in discrete subcellular membrane domains. Voltage-gated potassium (Kv) channels exhibit highly variable cellular and subcellular patterns of expression. Here, we describe novel activity-dependent synaptic targeting of Kv4.2, a dendritic Kv channel, in cerebellar granule cells (GCs). In vivo, Kv4.2 channels are highly expressed in cerebellar glomeruli, specializations of GC dendrites that form synapses with mossy fibres. In contrast, in cultured GCs, Kv4.2 was found localized, not to dendrites but to cell bodies. To investigate the role of synaptic contacts, we developed a co-culture system with cells from pontine grey nucleus, the origin of mossy fibres. In these co-cultures, synaptic structures formed, and Kv4.2 was now targeted to these synaptic sites in a manner dependent on synaptic activity. Activation of NMDA- and/or AMPA-type glutamate receptors was necessary for the targeting of Kv4.2 in co-cultures, and activation of these receptor systems in GC monocultures induced dendritic targeting of Kv4.2 in the absence of synapse formation. These results indicate that the proper targeting of Kv4.2 channels is dynamically regulated by synaptic activity. This activity-dependent regulation of Kv4.2 localization provides a crucial yet dynamic link between synaptic activity and dendritic excitability.     

Bidirectional parallel fiber plasticity in the cerebellum under climbing fiber control

Cerebellar parallel fiber (PF)-Purkinje cell (PC) synapses can undergo postsynaptically expressed long-term depression (LTD) or long-term potentiation (LTP) depending on whether or not the climbing fiber (CF) input is coactivated during tetanization. Here, we show that modifications of the postsynaptic calcium load using the calcium chelator BAPTA or photolytic calcium uncaging result in a reversal of the expected polarity of synaptic gain change. At higher concentrations, BAPTA blocks PF-LTP. These data indicate that PF-LTD requires a higher calcium threshold amplitude than PF-LTP induction and suggest that CF activity acts as a polarity switch by providing dendritic calcium transients. Moreover, previous CF-LTD induction changes the relative PF-LTD versus -LTP induction probability. These findings suggest that bidirectional cerebellar learning is governed by a calcium threshold rule operating "inverse" to the mechanism previously described at other glutamatergic synapses (BCM rule) and that the LTD/LTP induction probability is under heterosynaptic climbing fiber control.     

Ccdc134 deficiency impairs cerebellar development and motor coordination

Coiled-coil domain containing 134 (CCDC134) has been shown to serve as an immune cytokine to exert antitumor effects and to act as a novel regulator of hADA2a to affect PCAF acetyltransferase activity. While Ccdc134 loss causes abnormal brain development in mice, the significance of CCDC134 in neuronal development in vivo is controversial. Here, we report that CCDC134 is highly expressed in Purkinje cells (PCs) at all developmental stages and regulates mammalian cerebellar development in a cell type-specific manner. Selective deletion of Ccdc134 in mouse neural stem cells (NSCs) caused defects in cerebellar morphogenesis, including a decrease in the number of PCs and impairment of PC dendritic growth, as well as abnormal granule cell development. Moreover, loss of Ccdc134 caused progressive motor dysfunction with deficits in motor coordination and motor learning. Finally, Ccdc134 deficiency inhibited Wnt signaling but increased Ataxin1 levels. Our findings provide evidence that CCDC134 plays an important role in cerebellar development, possibly through regulating Wnt signaling and Ataxin1 expression levels, and in controlling cerebellar function for motor coordination and motor learning, ultimately making it a potential contributor to cerebellar pathogenesis.     

Transcranial direct current stimulation of cerebellum alters spiking precision in cerebellar cortex: A modeling study of cellular responses

Transcranial direct current stimulation (tDCS) of the cerebellum has rapidly raised interest but the effects of tDCS on cerebellar neurons remain unclear. Assessing the cellular response to tDCS is challenging because of the uneven, highly stratified cytoarchitecture of the cerebellum, within which cellular morphologies, physiological properties, and function vary largely across several types of neurons. In this study, we combine MRI-based segmentation of the cerebellum and a finite element model of the tDCS-induced electric field (EF) inside the cerebellum to determine the field imposed on the cerebellar neurons throughout the region. We then pair the EF with multicompartment models of the Purkinje cell (PC), deep cerebellar neuron (DCN), and granule cell (GrC) and quantify the acute response of these neurons under various orientations, physiological conditions, and sequences of presynaptic stimuli. We show that cerebellar tDCS significantly modulates the postsynaptic spiking precision of the PC, which is expressed as a change in the spike count and timing in response to presynaptic stimuli. tDCS has modest effects, instead, on the PC tonic firing at rest and on the postsynaptic activity of DCN and GrC. In Purkinje cells, anodal tDCS shortens the repolarization phase following complex spikes (-14.7 ± 6.5% of baseline value, mean ± S.D.; max: -22.7%) and promotes burstiness with longer bursts compared to resting conditions. Cathodal tDCS, instead, promotes irregular spiking by enhancing somatic excitability and significantly prolongs the repolarization after complex spikes compared to baseline (+37.0 ± 28.9%, mean ± S.D.; max: +84.3%). tDCS-induced changes to the repolarization phase and firing pattern exceed 10% of the baseline values in Purkinje cells covering up to 20% of the cerebellar cortex, with the effects being distributed along the EF direction and concentrated in the area under the electrode over the cerebellum. Altogether, the acute effects of tDCS on cerebellum mainly focus on Purkinje cells and modulate the precision of the response to synaptic stimuli, thus having the largest impact when the cerebellar cortex is active. Since the spatiotemporal precision of the PC spiking is critical to learning and coordination, our results suggest cerebellar tDCS as a viable therapeutic option for disorders involving cerebellar hyperactivity such as ataxia.     

Aftereffect phenomena in neurons of the cerebellar cortex

The reactions of Purkinje cells (PC) of the cerebellar cortex during electrocutaneous stimulation of one of the extremities with different frequencies (from one stimulus in 10 sec to one to five stimuli per sec) were studied in experiments on cats. It was shown that the reactions to the first and subsequent stimuli were different. This indicates the presence of an aftereffect from the first stimulus. It is assumed that the variability of the responses of PC to infrequent stimuli is connected with changes in their functional state which develop in response to "spontaneous" cerebellopetal impulses, as well as to circulation of excitation in intracerebellar circuits. With an increase in the frequency of the stimuli, the changes in excitability induced by previous peripheral stimuli, not only in the reacting PC, but also in the whole neuronal network of the corresponding cerebellopetal pathway, evidently acquire paramount importance. The absence of a direct relationship between strong peripheral stimulation and the degree of the reactions of the PC may be due to the involvement of intermediate neurons both of the exciting and inhibitory type in the transmission of impulses at the level of the cerebellar cortex.N. I. Pirogov Vinnitsa Medical Institute. Translated from Neirofiziologiya, Vol. 2, No. 6, pp. 573–580, November–December, 1970.     

Cerebellar localization and colocalization of GABA and calcium binding protein-D28K

Immunocytochemical studies using antibodies raised against the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA) and against the 28 Kd vitamin D dependent calcium binding protein (calbindin) in the cerebellum, are reviewed. The GABA immunoreactive neurones found in the cerebellar cortex were the Purkinje cell (PC), the three classes of intrinsic inhibitory interneurones, stellate, basket and Golgi cells and the cells of Lugaro. Some of the neurons of the cerebellar nuclei were also found to be GABA immunoreactive. A part of these could be identified as extrinsic neurones projecting either back to the cerebellar cortex, or to the inferior olive, both these pathways being topographically highly organized but arising from independent parent neurons. The presumed inhibitory function of these two pathways are discussed. Calbindin immunoreactivity in the cerebellum was confined to the PCs, staining concerned the whole cell including soma, branching dendrites, axons and axons terminals. The antibody, which appears to be tightly bound to the PC in vivo, failed to stain some of the PC when cerebellar slices maintained in vitro were studied. The stability of the antigen-antibody binding and the use of calbindin as a marker specific for the PC in the cerebellum, is discussed. Co-localization of GABA with calbindin as well as with other calcium binding proteins are reported to be found in the PCs. While these co-localizations have led to much speculation, conclusive functional roles for them have not been identified at present.     

Cerebellar metabolism of phosphatidylcholine and its hydrosoluble precursors during bicuculline-induced convulsive seizures

The cerebellar incorporation of labeled choline into phosphatidylcholine (PC) and its hydrosoluble choline-containing precursors has been examined during the course of bicuculline-induced convulsive seizures. The labeling of phosphocholine and of PC diminished in these conditions whereas that of cytidine-5-diphosphate choline (CDP-choline) was practically unaffected. Moreover, the cerebellar pools of phosphocholine and CDP-choline increased by 75–100% after 6 min of convulsions; these compounds were formed from lipid through the action of phospholipases or through the reverse action of choline phosphotransferase. From the data reported in this paper it should also be inferred that the cytidylyltransferase reaction was activated. It is therefore concluded that the cerebellar metabolism of PC and its precursors was affected in various ways by the bicuculline-induced convulsive seizures.     

Astrotactin: a novel neuronal cell surface antigen that mediates neuron- astroglial interactions in cerebellar microcultures

A microculture system for mouse cerebellar cells has been used to identify an immune activity, raised in rabbits against postnatal cerebellar cells, that blocks neuron-glial interactions in vitro. In the presence of blocking antibodies, stable neuron-glial contacts did not form and neuronal induction of glial process outgrowth did not occur. Subsequently, neurons were randomly arranged in the cultures rather than organized along the arms of astroglia. We have named the immune activity that blocks neuron-astroglial interactions anti-astrotactin. Partial purification of the anti-astrotactin blocking antibodies was obtained by cellular absorption with PC12 cells, a clonal cell line which expresses both the N-CAM and NILE (Ng-CAM, L1) glycoproteins. Subsequent absorption with purified cerebellar granule cells, but not with astroglial cells, removed the blocking activity, suggesting that the antigen(s) bound by blocking antibodies are neuronal. Immunoprecipitation of [35S]methionine- or [3H]fucose-radiolabeled Triton extracts of early postnatal cerebellar cells showed that the unabsorbed antiserum recognized a large number of proteins. Among these were bands with apparent molecular masses of N-CAM (180 and 140 kD) and NILE (230 kD). After absorption of the immune serum with PC12 cells, the number of bands recognized by the antiserum was reduced to a prominent band at 100 kD and a diffuse smear of material between 80 and 90 kD. The prominent band at 100 kD was removed by subsequent absorption of the immune serum with granule cells, a step which removed the blocking activity in the cerebellar microculture assay. Further evidence suggests that the astrotactin activity is missing or defective on granule cells from the neurological mutant mouse weaver, an animal that suffers a failure of glial-guided neuronal migration. When anti-astrotactin Fab fragments were pre-absorbed with weaver cerebellar neurons and then tested in the functional assay of neuron-glial interactions, the immune blocking activity was not removed. In contrast, wild-type cerebellar neurons removed the anti-astrotactin blocking activity under the same conditions. Subsequently, when [3H]fucose-radiolabeled Triton extracts of weaver and normal cells were immunoprecipitated with whole or PC12-absorbed anti-astrotactin antiserum, the intensity of the band at 100 kD was reduced by 95% in weaver cells.     

The role of the cerebellum in modulating voluntary limb movement commands

We recorded the activity of cerebellar Purkinje cells (PCs), primary motor cortical (M1) neurons, and limb EMG signals while monkeys executed a sequential reaching and button pressing task. PC simple spike discharge generally correlated well with the activity of one or more forelimb muscles. Surprisingly, given the inhibitory projection of PCs, only about one quarter of the correlations were negative. The largest group of neurons burst during movement and were positively correlated with EMG signals, while another significant group burst and were negatively correlated. Among the PCs that paused during movement most were negatively correlated with EMG. The strength of these various correlations was somewhat weaker, on average, than equivalent correlations between M1 neurons and EMG signals. On the other hand, there were no significant differences in the timing of the onset of movement related discharge among these groups of PCs, or between the PCs and M1 neurons. PC discharge was modulated largely in phase, or directly out of phase, with muscle activity. The nearly synchronous activation of PCs and muscles yielded positive correlations, despite the fact that the synaptic effect of the PC discharge is inhibitory. The apparent function of this inhibition is to restrain activity in the limb premotor network, shaping it into a spatiotemporal pattern that is appropriate for controlling the many muscles that participate in this task. The observed timing suggests that the cerebellar cortex learns to modulate PC discharge predictively. Through the cerebellar nucleus, this PC signal is combined with an underlying cerebral cortical signal. In this manner the cerebellum refines the descending command as compared with the relatively crude version generated when the cerebellum is damaged.     

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents ³. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells ¹¹ are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period ⁴. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.     

Optogenetic manipulation of cerebellar Purkinje cell activity in vivo

Purkinje cells (PCs) are the sole output neurons of the cerebellar cortex. Although their anatomical connections and physiological response properties have been extensively studied, the causal role of their activity in behavioral, cognitive and autonomic functions is still unclear because PC activity cannot be selectively controlled. Here we developed a novel technique using optogenetics for selective and rapidly reversible manipulation of PC activity in vivo. We injected into rat cerebellar cortex lentiviruses expressing either the light-activated cationic channel channelrhodopsin-2 (ChR2) or light-driven chloride pump halorhodopsin (eNpHR) under the control of the PC-specific L7 promoter. Transgene expression was observed in most PCs (ChR2, 92.6%; eNpHR, 95.3%), as determined by immunohistochemical analysis. In vivo electrophysiological recordings showed that all light-responsive PCs in ChR2-transduced rats increased frequency of simple spike in response to blue laser illumination. Similarly, most light-responsive PCs (93.8%) in eNpHR-transduced rats decreased frequency of simple spike in response to orange laser illumination. We then applied these techniques to characterize the roles of rat cerebellar uvula, one of the cardiovascular regulatory regions in the cerebellum, in resting blood pressure (BP) regulation in anesthetized rats. ChR2-mediated photostimulation and eNpHR-mediated photoinhibition of the uvula had opposite effects on resting BP, inducing depressor and pressor responses, respectively. In contrast, manipulation of PC activity within the neighboring lobule VIII had no effect on BP. Blue and orange laser illumination onto PBS-injected lobule IX didn't affect BP, indicating the observed effects on BP were actually due to PC activation and inhibition. These results clearly demonstrate that the optogenetic method we developed here will provide a powerful way to elucidate a causal relationship between local PC activity and functions of the cerebellum.