The Spinocerebellar Ataxia 34-Causing W246G ELOVL4 Mutation Does Not Alter Cerebellar Neuron Populations in a Rat Model.

Spinocerebellar ataxia 34 (SCA34) is an autosomal dominant disease that arises from point mutations in the fatty acid elongase, Elongation of Very Long Chain Fatty Acids 4 (ELOVL4), which is essential for the synthesis of Very Long Chain-Saturated Fatty Acids (VLC-SFA) and Very Long Chain-Polyunsaturated Fatty Acids (VLC-PUFA) (28-34 carbons long). SCA34 is considered a neurodegenerative disease. However, a novel rat model of SCA34 (SCA34-KI rat) with knock-in of the W246G ELOVL4 mutation that causes human SCA34 shows early motor impairment and aberrant synaptic transmission and plasticity without overt neurodegeneration. ELOVL4 is expressed in neurogenic regions of the developing brain, is implicated in cell cycle regulation, and ELOVL4 mutations that cause neuroichthyosis lead to developmental brain malformation, suggesting that aberrant neuron generation due to ELOVL4 mutations might contribute to SCA34. To test whether W246G ELOVL4 altered neuronal generation or survival in the cerebellum, we compared the numbers of Purkinje cells, unipolar brush cells, molecular layer interneurons, granule and displaced granule cells in the cerebellum of wildtype, heterozygous, and homozygous SCA34-KI rats at four months of age, when motor impairment is already present. An unbiased, semi-automated method based on Cellpose 2.0 and ImageJ was used to quantify neuronal populations in cerebellar sections immunolabeled for known neuron-specific markers. Neuronal populations and cortical structure were unaffected by the W246G ELOVL4 mutation by four months of age, a time when synaptic and motor dysfunction are already present, suggesting that SCA34 pathology originates from synaptic dysfunction due to VLC-SFA deficiency, rather than aberrant neuronal production or neurodegeneration.

Opposing actions of CRF-R1 and CB1 receptor on facial stimulation-induced MLI-PC plasticity in mouse cerebellar cortex.

BACKGROUND: Corticotropin-releasing factor (CRF) is the major neuromodulator orchestrating the stress response, and is secreted by neurons in various regions of the brain. Cerebellar CRF is released by afferents from inferior olivary neurons and other brainstem nuclei in response to stressful challenges, and contributes to modulation of synaptic plasticity and motor learning behavior via its receptors. We recently found that CRF modulates facial stimulation-evoked molecular layer interneuron-Purkinje cell (MLI-PC) synaptic transmission via CRF type 1 receptor (CRF-R1) in vivo in mice, suggesting that CRF modulates sensory stimulation-evoked MLI-PC synaptic plasticity. However, the mechanism of how CRF modulates MLI-PC synaptic plasticity is unclear. We investigated the effect of CRF on facial stimulation-evoked MLI-PC long-term depression (LTD) in urethane-anesthetized mice by cell-attached recording technique and pharmacological methods. RESULTS: Facial stimulation at 1 Hz induced LTD of MLI-PC synaptic transmission under control conditions, but not in the presence of CRF (100 nM). The CRF-abolished MLI-PC LTD was restored by application of a selective CRF-R1 antagonist, BMS-763,534 (200 nM), but it was not restored by application of a selective CRF-R2 antagonist, antisauvagine-30 (200 nM). Blocking cannabinoid type 1 (CB1) receptor abolished the facial stimulation-induced MLI-PC LTD, and revealed a CRF-triggered MLI-PC long-term potentiation (LTP) via CRF-R1. Notably, either inhibition of protein kinase C (PKC) with chelerythrine (5 µM) or depletion of intracellular Ca2+ with cyclopiazonic acid (100 µM), completely prevented CRF-triggered MLI-PC LTP in mouse cerebellar cortex in vivo. CONCLUSIONS: The present results indicated that CRF blocked sensory stimulation-induced opioid-dependent MLI-PC LTD by triggering MLI-PC LTP through CRF-R1/PKC and intracellular Ca2+ signaling pathway in mouse cerebellar cortex. These results suggest that activation of CRF-R1 opposes opioid-mediated cerebellar MLI-PC plasticity in vivo in mice.

Movement Rate Is Encoded and Influenced by Widespread, Coherent Activity of Cerebellar Molecular Layer Interneurons.

Inhibition from molecular layer interneurons (MLIs) is thought to play an important role in cerebellar function by sharpening the precision of Purkinje cell spike output. Yet the coding features of MLIs during behavior are poorly understood. To study MLI activity, we used in vivo Ca2+ imaging in head-fixed mice during the performance of a rhythmic motor behavior, licking during water consumption. MLIs were robustly active during lick-related movement across a lobule-specific region of the cerebellum showing high temporal correspondence within their population. Average MLI Ca2+ activity strongly correlated with movement rate but not to the intentional, or unexpected, adjustment of lick position or to sensory feedback that varied with task condition. Chemogenetic suppression of MLI output reduced lick rate and altered tongue movements, indicating that activity of these interneurons not only encodes temporal aspects of movement kinematics but also influences motor outcome pointing to an integral role in online control of rhythmic behavior.SIGNIFICANCE STATEMENT The cerebellum helps fine-tune coordinated motor actions via signaling from projection neurons called Purkinje cells. Molecular layer interneurons (MLIs) provide powerful inhibition onto Purkinje cells, but little is understood about how this inhibitory circuit is engaged during behavior or what type of information is transmitted through these neurons. Our work establishes that MLIs in the lateral cerebellum are broadly activated during movement with calcium activity corresponding to movement rate. We also show that suppression of MLI output slows and disorganizes the precise movement pattern. Therefore, MLIs are an important circuit element in the cerebellum allowing for accurate motor control.

Long Pauses in Cerebellar Interneurons in Anesthetized Animals.

Are long pauses in the firing of cerebellar interneurons (CINs) related to Purkinje cell (PC) pauses? If PC pauses affect the larger network, then we should find a close relationship between CIN pauses and those in PCs. We recorded activity of 241 cerebellar cortical neurons (206 CINs and 35 PCs) in three anesthetized cats. One fifth of the CINs and more than half of the PCs were identified as pausing. Pauses in CINs and PCs showed some differences: CIN mean pause length was shorter, and, after pauses, only CINs had sustained reduction in their firing rate (FR). Almost all pausing CINs fell into same cluster when we used different methods of clustering CINs by their spontaneous activity. The mean spontaneous firing rate of that cluster was approximately 53 Hz. We also examined cross-correlations in simultaneously recorded neurons. Of 39 cell pairs examined, 14 (35 %) had cross-correlations significantly different from those expected by chance. Almost half of the pairs with two CINs showed statistically significant negative correlations. In contrast, PC/CIN pairs did not often show significant effects in the cross-correlation (12/15 pairs). However, for both CIN/CIN and PC/CIN pairs, pauses in one unit tended to correspond to a reduction in the firing rate of the adjacent unit. In our view, our results support the possibility that previously reported PC bistability is part of a larger network response and not merely a biophysical property of PCs. Any functional role for PC bistability should probably be sought in the context of the broader network.

Synaptic Multivesicular Release in the Cerebellar Cortex: Its Mechanism and Role in Neural Encoding and Processing.

The number of synaptic vesicles released during fast release plays a major role in determining the strength of postsynaptic response. However, it remains unresolved how the number of vesicles released in response to action potentials is controlled at a single synapse. Recent findings suggest that the Cav2.1 subtype (P/Q-type) of voltage-gated calcium channels is responsible for inducing presynaptic multivesicular release (MVR) at rat cerebellar glutamatergic synapses from granule cells to molecular layer interneurons. The topographical distance from Cav2.1 channels to exocytotic Ca(2+) sensors is a critical determinant of MVR. In physiological trains of presynaptic neurons, MVR significantly impacts the excitability of postsynaptic neurons, not only by increasing peak amplitude but also by prolonging decay time of the postsynaptic currents. Therefore, MVR contributes additional complexity to neural encoding and processing in the cerebellar cortex.

Distinct functional domains of Dystroglycan regulate inhibitory synapse formation and maintenance in cerebellar Purkinje cells.

Dystroglycan is a cell adhesion molecule that localizes to synapses throughout the nervous system. While Dystroglycan is required to maintain inhibitory synapses from cerebellar molecular layer interneurons (MLIs) onto Purkinje cells (PCs) whether initial synaptogenesis during development is dependent on Dystroglycan has not been examined. We show that conditional deletion of Dystroglycan from Purkinje cells prior to synaptogenesis results in impaired MLI:PC synapse formation and function due to reduced presynaptic inputs and abnormal postsynaptic GABAA receptor clustering. Using genetic manipulations that disrupt glycosylation of Dystroglycan or truncate its cytoplasmic domain, we show that Dystroglycan's role in synapse function requires both extracellular and intracellular interactions, whereas synapse formation requires only extracellular interactions. Together, these findings provide molecular insight into the mechanism of inhibitory synapse formation and maintenance in cerebellar cortex.

Specialized connectivity of molecular layer interneuron subtypes leads to disinhibition and synchronous inhibition of cerebellar Purkinje cells.

Molecular layer interneurons (MLIs) account for approximately 80% of the inhibitory interneurons in the cerebellar cortex and are vital to cerebellar processing. MLIs are thought to primarily inhibit Purkinje cells (PCs) and suppress the plasticity of synapses onto PCs. MLIs also inhibit, and are electrically coupled to, other MLIs, but the functional significance of these connections is not known. Here, we find that two recently recognized MLI subtypes, MLI1 and MLI2, have a highly specialized connectivity that allows them to serve distinct functional roles. MLI1s primarily inhibit PCs, are electrically coupled to each other, fire synchronously with other MLI1s on the millisecond timescale in vivo, and synchronously pause PC firing. MLI2s are not electrically coupled, primarily inhibit MLI1s and disinhibit PCs, and are well suited to gating cerebellar-dependent behavior and learning. The synchronous firing of electrically coupled MLI1s and disinhibition provided by MLI2s require a major re-evaluation of cerebellar processing.

Nicotine depresses facial stimulation-evoked molecular layer interneuron-Purkinje cell synaptic transmission via α7 nicotinic acetylcholine receptors in mouse cerebellar cortex.

Nicotine modulates cerebellar physiology function by interacting with nicotinic acetylcholine receptors (nAChRs) and is involved in modulation of cerebellar cortical circuitry functions. Here, we investigated the effect of nicotine on sensory stimulation-evoked molecular layer interneuron-Purkinje cell (MLI-PC) synaptic transmission mouse cerebellar cortex using in vivo cell-attached recording technique and pharmacological methods. The results show that micro-application of nicotine to the cerebellar molecular layer significantly decreased sensory stimulation-evoked MLI-PC synaptic transmission in mouse cerebellar cortex. Nicotine-induced depression in sensory stimulation-evoked MLI-PC synaptic transmission was abolished by either a non-selective nAChR blocker, hexamethonium, or the α7-nAChR antagonist methyllycaconitine (MLA), but not the selective α4ß2-nAChR antagonist dihydro-ß-erythroidine. Notably, molecular layer micro-application of nicotine did not significantly affect the number of spontaneous or facial stimulation-evoked action potentials of MLIs. Moreover, nicotine produced significant increases in the amplitude and frequency of miniature inhibitory postsynaptic currents of PCs, which were abolished by MLA in cerebellar slices. These results indicate that micro-application of nicotine to the cerebellar molecular layer depresses facial stimulation-induced MLI-PC synaptic transmission by activating α7 nAChRs, suggesting that cholinergic inputs modulate MLI-PC synapses to process sensory information in the cerebellar cortex of mice in vivo.

Fentanyl Inhibits Air Puff-Evoked Sensory Information Processing in Mouse Cerebellar Neurons Recorded in vivo.

Aim: To examine the effects of fentanyl, a potent mu-opioid receptor (MOR) agonist, on-air puff-evoked responses in Purkinje cells (PCs), and molecular layer interneurons (MLIs) using in vivo patch-clamp recordings in anesthetized mice. Methods: Male mice 6-8 weeks-old were anesthetized and fixed on a custom-made stereotaxic frame. The cerebellar surface was exposed and perfused with oxygenated artificial cerebrospinal fluid (ACSF). Patch-clamp recordings in the cell-attached mode were obtained from PCs and MLIs. Facial stimulation by air-puff of the ipsilateral whisker pad was performed through a pressurized injection system. Fentanyl citrate, CTOP, and H-89 dissolved in ACSF were perfused onto the cerebellar surface. Results: Fentanyl significantly inhibited the amplitude and area under the curve (AUC) of sensory stimulation-evoked inhibitory responses in PCs. Although fentanyl did not influence the frequency of simple spikes (SSs), it decreased the pause of SS. The IC50 of the fentanyl-induced suppression of the P1 response amplitude was 5.53 µM. The selective MOR antagonist CTOP abolished fentanyl-induced inhibitory responses in PCs. However, the application of CTOP alone increased the amplitude, AUC of P1, and the pause of SS. Notably, fentanyl significantly inhibited the tactile-evoked response of MLIs but did not affect their spontaneous firing. The fentanyl-induced decrease of inhibitory responses in PCs was partially prevented by a PKA inhibitor, H-89. Conclusions: These results suggest that fentanyl binds to MORs in MLIs to reduce GABAergic neurotransmission in MLI-PC projections and one potential mechanism is via modulation of the cAMP-PKA pathway.

Chronic ethanol exposure facilitates facial-evoked MLI-PC synaptic transmission via nitric oxide signaling pathway in vivo in mice.

Ethanol (EtOH) exposure causes alterations of motor coordination, balance, behavior, speech, and certain cognitive functions are considered to be caused partly by impairment of cerebellar circuits function and modulation of synaptic transmission. The cerebellar cortical molecular layer interneuron-Purkinje cell (MLI-PC) synapses are critical for various information integration and transmission, which are sensitive to acute and chronic EtOH exposure. The aim of this study is to investigate the effect of chronic ethanol exposure on the facial stimulation-evoked MLI-PC synaptic transmission in urethane-anesthetized mice, by electrophysiological recording and pharmacological methods. Under current-clamp recording conditions, air-puff stimulation of ipsilateral whisker pad evoked MLI-PC synaptic transmission, which expressed an inhibitory component (P1) followed by a pause of simple spike (SS) firing in cerebellar PCs. Chronic ethanol exposure did not change the latency of the facial stimulation-evoked responses in cerebellar PCs, but induced significant enhancement of the stimulation-evoked MLI-PC synaptic transmission, which expressed increases in amplitude of P1 and pause of SS firing. The amplitude of P1 and pause of SS in ethanol exposure group were significant higher than that in control group. Cerebellar surface application of nitric oxide synthesis (NOS) inhibitor, L-NNA (5 mM) significantly decreased the amplitude of P1 and the pause of SS firing in EtOH exposure group, but did no effect on control group. In contrast, cerebellar surface application of NO donor, SNAP (100 µM) significantly increased the amplitude of P1 and the pause of SS firing in control group, but not in EtOH exposure group. These results indicated that chronic EtOH exposure significantly facilitated the sensory-evoked MLI-PC synaptic transmission via NO signaling pathway in mouse cerebellar cortex.

Corticotrophin-Releasing Factor Modulates the Facial Stimulation-Evoked Molecular Layer Interneuron-Purkinje Cell Synaptic Transmission in vivo in Mice.

Corticotropin-releasing factor (CRF) is an important neuromodulator in central nervous system that modulates neuronal activity via its receptors during stress responses. In cerebellar cortex, CRF modulates the simple spike (SS) firing activity of Purkinje cells (PCs) has been previously demonstrated, whereas the effect of CRF on the molecular layer interneuron (MLI)-PC synaptic transmission is still unknown. In this study, we examined the effect of CRF on the facial stimulation-evoked cerebellar cortical MLI-PC synaptic transmission in urethane-anesthetized mice by in vivo cell-attached recording, neurobiotin juxtacellular labeling, immunohistochemistry techniques, and pharmacological method. Cell-attached recordings from cerebellar PCs showed that air-puff stimulation of ipsilateral whisker pad evoked a sequence of tiny parallel fiber volley (N1) followed by MLI-PC synaptic transmission (P1). Microapplication of CRF in cerebellar cortical molecular layer induced increases in amplitude of P1 and pause of SS firing. The CRF decreases in amplitude of P1 waveform were in a dose-dependent manner with the EC50 of 241 nM. The effects of CRF on amplitude of P1 and pause of SS firing were abolished by either a non-selective CRF receptor antagonist, α-helical CRF-(9-14), or a selective CRF-R1 antagonist, BMS-763534 (BMS, 200 nM), but were not prevented by a selective CRF-R2 antagonist, antisauvagine-30 (200 nM). Notably, application CRF not only induced a significant increase in spontaneous spike firing rate, but also produced a significant increase in the number of the facial stimulation-evoked action potential in MLIs. The effect of CRF on the activity of MLIs was blocked by the selective CRF-R1 antagonist, and the MLIs expressed the CRF-R1 imunoreactivity. These results indicate that CRF increases excitability of MLIs via CRF-R1, resulting in an enhancement of the facial stimulation-evoked MLI-PC synaptic transmission in vivo in mice.

Revisiting a theory of cerebellar cortex.

Long-term depression at parallel fiber-Purkinje cell synapses plays a principal role in learning in the cerebellum, which acts as a supervised learning machine. Recent experiments demonstrate various forms of synaptic plasticity at different sites within the cerebellum. In this article, we take into consideration synaptic plasticity at parallel fiber-molecular layer interneuron synapses as well as at parallel fiber-Purkinje cell synapses, and propose that the cerebellar cortex performs reinforcement learning, another form of learning that is more capable than supervised learning. We posit that through the use of reinforcement learning, the need for explicit teacher signals for learning in the cerebellum is eliminated; instead, learning can occur via responses from evaluative feedback. We demonstrate the learning capacity of cerebellar reinforcement learning using simple computer simulations of delay eyeblink conditioning and the cart-pole balancing task.

Inputs from Sequentially Developed Parallel Fibers Are Required for Cerebellar Organization.

Neuronal activity is believed to be important for brain development; however, it remains unclear as to how spatiotemporal distributions of synaptic excitation contribute to neural network formation. Bifurcated axons of cerebellar granule cells, parallel fibers (PFs), are made in an orderly inside-out manner during postnatal development. In this study, we induced a blockade of neurotransmitter release from specific bundles of developing PFs and tested the effects of biased PF inputs on cerebellar development. The blockade of different layers of PFs at different developmental times results in varying degrees of abnormal cerebellar development. Furthermore, cerebellar network abnormalities are not restored when PF inputs are restored in adulthood and, hence, result in motor dysfunction. We thus conclude that spatiotemporally unbiased synaptic transmission from sequentially developed PFs is crucial for cerebellar network formation and motor function, supporting the idea that unbiased excitatory synaptic transmission is crucial for network formation.

Chronic Ethanol Consumption Impairs the Tactile-Evoked Long-Term Depression at Cerebellar Molecular Layer Interneuron-Purkinje Cell Synapses in vivo in Mice.

The cerebellum is sensitive to ethanol (EtOH) consumption. Chronic EtOH consumption impairs motor learning by modulating the cerebellar circuitry synaptic transmission and long-term plasticity. Under in vitro conditions, acute EtOH inhibits both parallel fiber (PF) and climbing fiber (CF) long-term depression (LTD). However, thus far it has not been investigated how chronic EtOH consumption affects sensory stimulation-evoked LTD at the molecular layer interneurons (MLIs) to the Purkinje cell (PC) synapses (MLI-PC LTD) in the cerebellar cortex of living animals. In this study, we investigated the effect of chronic EtOH consumption on facial stimulation-evoked MLI-PC LTD, using an electrophysiological technique as well as pharmacological methods, in urethane-anesthetized mice. Our results showed that facial stimulation induced MLI-PC LTD in the control mice, but it could not be induced in mice with chronic EtOH consumption (0.8 g/kg; 28 days). Blocking the cannabinoid type 1 (CB1) receptor activity with AM-251, prevented MLI-PC LTD in the control mice, but revealed a nitric oxide (NO)-dependent long-term potentiation (LTP) of MLI-PC synaptic transmission (MLI-PC LTP) in the EtOH consumption mice. Notably, with the application of a NO donor, S-nitroso-N-Acetyl-D, L-penicillamine (SNAP) alone prevented the induction of MLI-PC LTD, but a mixture of SNAP and AM-251 revealed an MLI-PC LTP in control mice. In contrast, inhibiting NO synthase (NOS) revealed the facial stimulation-induced MLI-PC LTD in EtOH consumption mice. These results indicate that long-term EtOH consumption can impair the sensory stimulation-induced MLI-PC LTD via the activation of a NO signaling pathway in the cerebellar cortex in vivo in mice. Our results suggest that the chronic EtOH exposure causes a deficit in the cerebellar motor learning function and may be involved in the impaired MLI-PC GABAergic synaptic plasticity.

Effects of norepinephrine on spontaneous firing activity of cerebellar Purkinje cells in vivo in mice.

Norepinephrine (NE), from the locus coeruleus (LC), has been supported to affect GABAergic system and parallel fiber (PF)-Purkinje cell (PC) synaptic transmission via adrenoceptor in cerebellum cortex. However, the effects of NE on the spontaneous spike activity of cerebellar PCs in living mouse have not yet been fully understood. We here examined the effects of NE on the spontaneous activity of PC in urethane-anesthetized mice by electrophysiological and pharmacological methods. Cerebellar surface application of NE (2.5-25µM) reduced the PC simple spike (SS) firing rate in a dose-dependent manner. The half-inhibitory concentration (IC50) was 5.97µM. In contrast, NE significantly increased the spontaneous firing rate of molecular layer interneuron (MLI). Application of GABAA receptor antagonist, gabazine (SR95531, 20µM) not only blocked the NE-induced inhibition of PC SS firing but also revealed NE-induced excitation of cerebellar PC. Blocking AMPA receptors activity enhanced NE-induced inhibition of PC spontaneous activity. Moreover, the effects of NE on PC spontaneous activity were abolished by simultaneously blocking GABAA and AMPA receptors activity. These results indicated that NE bidirectional modulated the spontaneous activity of PCs via enhancing both inhibitory inputs from MLIs and excitatory inputs of parallel fibers, but NE-induced enhance of inhibitory inputs overwhelmed the excitatory inputs under in vivo conditions.

Dynamic properties of sensory stimulation evoked responses in mouse cerebellar granule cell layer and molecular layer.

Sensory information coming from climbing fiber and mossy fiber-granule cell pathways, generates motor-related outputs according to internal rules of integration and computation in the cerebellar cortex. However, the dynamic properties of sensory information processing in mouse cerebellar cortex are less understood. Here, we studied the dynamic properties of sensory stimulation-evoked responses in the cerebellar granule cell layer (GCL) and molecular layer (ML) by electrophysiological recordings method. Our data showed that air-puff stimulation (5-10 ms in duration) of the ipsilateral whisker pad evoked single-peak responses in the GCL and ML; whereas a duration of stimulation ≥30 ms in GCL and ≥60 ms in ML, evoked double-peak responses that corresponded with stimulation-on and -off responses via mossy fiber pathway. The highest frequency of stimulation train for evoking GCL responses was 33 Hz. In contrast, the highest frequency of stimulation train for evoking ML responses was 4 Hz. These results indicate that the cerebellar granule cells transfer the high-fidelity sensory information from mossy fibers, which is cut-off by molecular layer interneurons (MLIs). Our results suggest that the MLIs network acts as a low-pass filter during the processing of high-frequency sensory information.

Facial stimulation induces long-term depression at cerebellar molecular layer interneuron-Purkinje cell synapses in vivo in mice.

Cerebellar long-term synaptic plasticity has been proposed to provide a cellular mechanism for motor learning. Numerous studies have demonstrated the induction and mechanisms of synaptic plasticity at parallel fiber-Purkinje cell (PF-PC), parallel fiber-molecular layer interneurons (PF-MLI) and mossy fiber-granule cell (MF-GC) synapses, but no study has investigated sensory stimulation-evoked synaptic plasticity at MLI-PC synapses in the cerebellar cortex of living animals. We studied the expression and mechanism of MLI-PC GABAergic synaptic plasticity induced by a train of facial stimulation in urethane-anesthetized mice by cell-attached recordings and pharmacological methods. We found that 1 Hz, but not a 2 Hz or 4 Hz, facial stimulation induced a long-term depression (LTD) of GABAergic transmission at MLI-PC synapses, which was accompanied with a decrease in the stimulation-evoked pause of spike firing in PCs, but did not induce a significant change in the properties of the sensory-evoked spike events of MLIs. The MLI-PC GABAergic LTD could be prevented by blocking cannabinoid type 1 (CB1) receptors, and could be pharmacologically induced by a CB1 receptor agonist. Additionally, 1 Hz facial stimulation delivered in the presence of a metabotropic glutamate receptor 1 (mGluR1) antagonist, JNJ16259685, still induced the MLI-PC GABAergic LTD, whereas blocking N-methyl-D-aspartate (NMDA) receptors during 1 Hz facial stimulation abolished the expression of MLI-PC GABAergic LTD. These results indicate that sensory stimulation can induce an endocannabinoid (eCB)-dependent LTD of GABAergic transmission at MLI-PC synapses via activation of NMDA receptors in cerebellar cortical Crus II in vivo in mice. Our results suggest that the sensory stimulation-evoked MLI-PC GABAergic synaptic plasticity may play a critical role in motor learning in animals.

N-methyl-d-aspartate inhibits cerebellar Purkinje cell activity via the excitation of molecular layer interneurons under in vivo conditions in mice.

N-methyl-d-aspartate (NMDA) receptors play a key role in synaptic transmission, and are widely expressed on the membrane of granule cells, parallel fibers, and molecular layer interneurons (MLIs) in the cerebellar cortex of mammals. In cerebellar slices, activation of NMDA receptors increases inhibitory postsynaptic currents (IPSCs) of Purkinje cells (PCs). However, the effects of NMDA on the cerebellar network under in vivo conditions are currently unclear. In the present study, we examined the effects of NMDA on the spontaneous activity of PCs and MLIs in urethane-anesthetized mice by electrophysiological, pharmacological, and juxtacellular labeling methods. Our results revealed that cerebellar surface application of NMDA (5-200µM) reduced the PC simple spike (SS) firing rate in a dose-dependent manner. Application of GABAA receptor antagonist, SR95531 (20µM) abolished NMDA-induced inhibition of PCs spontaneous activity, and revealed NMDA-induced excitation of cerebellar PCs. NMDA receptor antagonist, DAP-V (250µM) did not affect the mean frequency of SS firing, but the SS firing rate of PCs became more regular than the control. In addition, NMDA increased the spike firing of both basket-type and stellate-type MLIs. Overall, these results indicated that NMDA-induced excitation of MLIs at the cerebellar surface may inhibit PC activity. Thus, NMDA receptors of MLIs may play a key role in regulating the spontaneous activity of PCs, and in information transmission and integration in cerebellar cortex.