Cerebellar Synaptic Transmission Research Articles

An activator of voltage-gated K+ channels Kv1.1 as a therapeutic candidate for episodic ataxia type 1

Loss-of-function mutations in the KCNA1(Kv1.1) gene cause episodic ataxia type 1 (EA1), a neurological disease characterized by cerebellar dysfunction, ataxic attacks, persistent myokymia with painful cramps in skeletal muscles, and epilepsy. Precision medicine for EA1 treatment is currently unfeasible, as no drug that can enhance the activity of Kv1.1-containing channels and offset the functional defects caused by KCNA1 mutations has been clinically approved. Here, we uncovered that niflumic acid (NFA), a currently prescribed analgesic and anti-inflammatory drug with an excellent safety profile in the clinic, potentiates the activity of Kv1.1 channels. NFA increased Kv1.1 current amplitudes by enhancing the channel open probability, causing a hyperpolarizing shift in the voltage dependence of both channel opening and gating charge movement, slowing the OFF-gating current decay. NFA exerted similar actions on both homomeric Kv1.2 and heteromeric Kv1.1/Kv1.2 channels, which are formed in most brain structures. We show that through its potentiating action, NFA mitigated the EA1 mutation-induced functional defects in Kv1.1 and restored cerebellar synaptic transmission, Purkinje cell availability, and precision of firing. In addition, NFA ameliorated the motor performance of a knock-in mouse model of EA1 and restored the neuromuscular transmission and climbing ability in Shaker (Kv1.1) mutant Drosophila melanogaster flies (Sh5). By virtue of its multiple actions, NFA has strong potential as an efficacious single-molecule-based therapeutic agent for EA1 and serves as a valuable model for drug discovery.

Role of β3-adrenergic receptor in the modulation of synaptic transmission and plasticity in mouse cerebellar cortex.

Convergent lines of evidence have recently highlighted β3-adrenoreceptors (ARs) as a potentially critical target in the regulation of nervous and behavioral functions, including memory consolidation, anxiety, and depression. Nevertheless, the role of β3-ARs in the cerebellum has been never investigated. To address this issue, we first examined the effects of pharmacological manipulation of β3-ARs on motor learning in mice. We found that blockade of β3-ARs by SR 59230A impaired the acquisition of the rotarod task with no effect on general locomotion. Since the parallel fiber-Purkinje cell (PF-PC) synapse is considered to be the main cerebellar locus of motor learning, we assessed β3-AR modulatory action on this synapse as well as its expression in cerebellar slices. We demonstrate, for the first time, a strong expression of β3-ARs on Purkinje cell soma and dendrites. In addition, whole-cell patch-clamp recordings revealed that bath application of β3-AR agonist CL316,243 depressed the PF-PC excitatory postsynaptic currents via a postsynaptic mechanism mediated by the PI3K signaling pathway. Application of CL316,243 also interfered with the expression of PF long-term potentiation, whereas SR 59230A prevented the induction of LTD at PF-PC synapse. These results underline the critical role of β3-AR on cerebellar synaptic transmission and plasticity and provide a new mechanism for adrenergic modulation of motor learning.

Different types of multiple-synapse boutons in the cerebellar cortex between physically enriched and ataxic mutant mice.

Experience-dependent synapse remodeling is associated with information storage in the nervous system. Neuronal synapses show alteration in various neurological and cognitive disorders in their structure and function. At the ultrastructural level, parallel fiber boutons contacting multiple spines of Purkinje cells in the cerebellar cortex are commonly observed in physiologically enriched animals as well as pathological ataxic mutants. However, the dendritic origin of those spines on parallel fiber multiple-synapse boutons (MSBs) has been poorly understood. Here, we investigated this issue by 3-dimensional ultrastructural analysis to determine synaptic connectivity of MSBs in both mice housed in physically enriched environment and cerebellar ataxic mutants. Our results demonstrated that environmental enrichment selectively induced MSBs to contact spines from the same parent dendrite, indicating focal strengthening of synapse through the simultaneous activation of two adjacent spines. In contrast, ataxic mutants displaying impaired motor coordination had significantly more MSBs involving spines originating from different neighboring dendrites compared to both wild-type and environmentally enriched animals, suggesting that compromising multiple synapse formation may lead to abnormal motor behavior in the mutant mice. These findings propose that environmental stimulation in normal animals mainly involves the refinement of preexisting synaptic networks, whereas pathological ataxic conditions may results from less-selective but compromising multiple synaptic formation. This study underscores that different types of multiple synapse boutons may have disparate effects on cerebellar synaptic transmission.

Loss of GPRC5B impairs synapse formation of Purkinje cells with cerebellar nuclear neurons and disrupts cerebellar synaptic plasticity and motor learning

GPRC5B is a membrane glycoprotein robustly expressed in mouse cerebellar Purkinje cells (PCs). Its function is unknown. In Gprc5b−/− mice that lack GPRC5B, PCs develop distal axonal swellings in deep cerebellar nuclei (DCN). Numerous misshapen mitochondria, which generated excessive amounts of reactive oxygen species (ROS), accumulated in these distal axonal swellings. In primary cell cultures of Gprc5b−/− PCs, pharmacological reduction of ROS prevented the appearance of such swellings. To examine the physiological role of GPRC5B in PCs, we analyzed cerebellar synaptic transmission and cerebellum-dependent motor learning in Gprc5b−/− mice. Patch-clamp recordings in cerebellum slices in vitro revealed that the induction of long-term depression (LTD) at parallel fiber-PC synapses was normal in adult Gprc5b−/− mice, whereas the induction of long-term potentiation (LTP) at mossy fiber-DCN neuron synapses was attenuated in juvenile Gprc5b−/− mice. In Gprc5b−/− mice, long-term motor learning was impaired in both the rotarod test and the horizontal optokinetic response eye movement (HOKR) test. These observations suggest that GPRC5B plays not only an important role in the development of distal axons of PCs and formation of synapses with DCN neurons, but also in the synaptic plasticity that underlies long-term motor learning.

PRRT2 deficiency induces paroxysmal kinesigenic dyskinesia by regulating synaptic transmission in cerebellum

Mutations in the proline-rich transmembrane protein 2 (PRRT2) are associated with paroxysmal kinesigenic dyskinesia (PKD) and several other paroxysmal neurological diseases, but the PRRT2 function and pathogenic mechanisms remain largely obscure. Here we show that PRRT2 is a presynaptic protein that interacts with components of the SNARE complex and downregulates its formation. Loss-of-function mutant mice showed PKD-like phenotypes triggered by generalized seizures, hyperthermia, or optogenetic stimulation of the cerebellum. Mutant mice with specific PRRT2 deletion in cerebellar granule cells (GCs) recapitulate the behavioral phenotypes seen in Prrt2-null mice. Furthermore, recording made in cerebellar slices showed that optogenetic stimulation of GCs results in transient elevation followed by suppression of Purkinje cell firing. The anticonvulsant drug carbamazepine used in PKD treatment also relieved PKD-like behaviors in mutant mice. Together, our findings identify PRRT2 as a novel regulator of the SNARE complex and provide a circuit mechanism underlying the PRRT2-related behaviors.

Sex differences in cerebellar synaptic transmission and sex-specific responses to autism-linked Gabrb3 mutations in mice.

Neurons of the cerebellar nuclei (CbN) transmit cerebellar signals to premotor areas. The cerebellum expresses several autism-linked genes, including GABRB3, which encodes GABAA receptor β3 subunits and is among the maternal alleles deleted in Angelman syndrome. We tested how this Gabrb3 m-/p+ mutation affects CbN physiology in mice, separating responses of males and females. Wild-type mice showed sex differences in synaptic excitation, inhibition, and intrinsic properties. Relative to females, CbN cells of males had smaller synaptically evoked mGluR1/5-dependent currents, slower Purkinje-mediated IPSCs, and lower spontaneous firing rates, but rotarod performances were indistinguishable. In mutant CbN cells, IPSC kinetics were unchanged, but mutant males, unlike females, showed enlarged mGluR1/5 responses and accelerated spontaneous firing. These changes appear compensatory, since mutant males but not females performed indistinguishably from wild-type siblings on the rotarod task. Thus, sex differences in cerebellar physiology produce similar behavioral output, but provide distinct baselines for responses to mutations.

Presynaptic dysfunction caused by cerebrospinal fluid from a patient with the ataxic form of Hashimoto's encephalopathy

AbstractBackgroundHashimoto's encephalopathy is an autoimmune disease associated with Hashimoto's thyroiditis. The mechanism underlying the development of ataxia is unclear.AimWe examined whether impairment in synaptic transmission underlies the pathomechanism of Hashimoto's encephalopathy.MethodsWe tested the effects of cerebrospinal fluid from Hashimoto's encephalopathy patients on cerebellar synaptic transmission in rat cerebellar slices. The effects of cerebrospinal fluid from six patients with the ataxic form of Hashimoto's encephalopathy were examined using patch‐clamp recording in rat cerebellar slices.ResultsThe cerebrospinal fluid of one patient, but not those of the other five patients, impaired presynaptic short‐term plasticity between parallel fiber‐Purkinje cell transmission in the cerebellum. The patient whose cerebrospinal fluid induced presynaptic impairment had hypothyroidism with a high titer of anti‐thyroid antibodies, and was positive for anti‐NH2‐terminal of α‐enolase antibodies in the cerebrospinal fluid.ConclusionsThese results suggest that defective glutamate release is a potential pathomechanism in some patients with Hashimoto's encephalopathy.

Retrograde cPLA2α/Arachidonic Acid/2-AG Signaling Is Essential for Cerebellar Depolarization-Induced Suppression of Excitation and Long-Term Potentiation

Cytosolic phospholipase A2 alpha (cPLA2α) responds to micromolar intracellular Ca(2+) and produces arachidonic acid, which regulates cellular homeostasis, neurotoxicity, and inflammation. Endocannabinoids are the derivates of arachidonic acid and widely distributed in the cerebellum. However, the role of cPLA2α/arachidonic acid pathway in cerebellar synaptic transmission and plasticity is unknown. We utilized cPLA2α knockout mice and slice whole-cell patch clamp to study the action of cPLA2α/arachidonic acid signaling on the depolarization-induced suppression of excitation (DSE) and long-term potentiation at parallel fiber-Purkinje cell synapses. Our data showed that DSE was significantly inhibited but rescued by arachidonic acid in cPLA2α knockout mice. The degradation enzyme of 2-arachidonoylglycerol (2-AG), monoacylglycerol lipase, blocked DSE, while another catabolism enzyme for N-arachidonoylethanolamine, fatty acid amide hydrolase, did not, suggesting that 2-AG is responsible for DSE in Purkinje cells. Co-application of paxilline reversed the blockade of DSE by internal K(+), indicating that large-conductance Ca(2+)-activated potassium channel is sufficient to inhibit cPLA2α/arachidonic acid-mediated DSE. On the other hand, we found that 1Hz parallel fiber stimuli-triggered long-term potentiation (LTP) was deficient in cPLA2α knockout mice. LTP was also inhibited when AACOCF3, an inhibitor of cPLA2α, was given. Arachidonic acid was necessary for the LTP induction. Therefore, these data showed that cPLA2α/arachidonic acid/2-AG signaling pathway mediates DSE and LTP at parallel fiber-Purkinje cell synapse.

Is Anti-Gliadin Antibody Pathogenic in Gluten Ataxia? Analysis using Rat Cerebellar Slices and Patch-Clamp Recording

The significance of autoantibodies associated withneurologicalsymptoms has been the focus of interest. Recentstudies emphasized the pathogenic role of anti-gliadin antibody in gluten ataxia, an important disease of autoimmune cerebellar ataxia. To examine whether autoantibodies, including anti-gliadin antibody, play a pathogenic role, we analyzed the effects of CSF samples obtained from a Japanese patient with gluten ataxia on cerebellar synaptic transmission. Patch-clamp recordings were prepared from cerebellar Purkinje cells, the output cells from the cerebellar cortex, in mice cerebellar slices. The CSF (diluted 1:100) had no effects on the excitatory postsynaptic currents, and did not affect the release mechanisms of glutamate. These results do not support an idea that CSF autoantibodies, including anti-gliadin antibody, interfere cerebellar synaptic function so as to develop ataxia

Anti-Ca 2+ channel antibody attenuates Ca 2+ currents and mimics cerebellar ataxia in vivo

Voltage-gated Ca(2+) channels (VGCCs) are membrane proteins that determine the activity and survival of neurons, and mutations in the P/Q-type VGCCs are known to cause cerebellar ataxia. VGCC dysfunction may also underlie acquired peripheral and central nervous system diseases associated with small-cell lung cancer, including Lambert-Eaton myasthenic syndrome (LEMS) and paraneoplastic cerebellar ataxia (PCA). The pathogenic role of anti-VGCC antibody in LEMS is well established. Although anti-VGCC antibody is also found in a significant fraction of PCA patients, its contribution to PCA is unclear. Using a polyclonal peptide antibody against a major immunogenic region in P/Q-type VGCCs (the extracellular Domain-III S5-S6 loop), we demonstrated that such antibody was sufficient to inhibit VGCC function in neuronal and recombinant VGCCs, alter cerebellar synaptic transmission, and confer the phenotype of cerebellar ataxia. Our data support the hypothesis that anti-VGCC antibody may play a significant role in the pathogenesis of cerebellar dysfunction in PCA.

ERK-Dependent Modulation of Cerebellar Synaptic Plasticity after Chronic Δ9-Tetrahydrocannabinol Exposure

Chronic exposure to Delta9-tetrahydrocannabinol (THC) induces tolerance to cannabinoid-induced locomotor effects, which are mediated by cannabinoid receptors (CB1Rs) located in motor control regions, including the cerebellum. There is substantial evidence of cerebellar CB1R molecular adaptation and modifications in receptor signaling after prolonged cannabinoid exposure. However, very little is known about the effects of chronic cannabinoid administration on cerebellar synaptic plasticity, which may contribute to the development of cannabinoid behavioral tolerance. In the cerebellar cortex, activation of CB1R inhibits excitatory synaptic transmission at parallel fiber (PF)-Purkinje cell (PC) synapses by decreasing neurotransmitter release. Our study aimed to investigate the neurophysiological adaptive responses occurring at cerebellar PF-PC cell synapses after repeated THC exposure. In THC-tolerant mice, an increase of the basal release probability was found at PF-PC synapses, in parallel with a facilitation of slow mGluR1 (metabotropic glutamate receptor type 1)-mediated excitatory postsynaptic currents and a reduced sensitivity to the inhibitory effects of the CB1R agonist CP55,940 [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol]. Additionally, after repeated THC exposures, presynaptic PF-PC long-term potentiation was blocked by A1R (adenosine receptor-1) activation. Inhibition of the extracellular signal regulated kinase (ERK) pathway prevented these alterations of cerebellar synaptic transmission and plasticity. In summary, we provide evidence for ERK-dependent modulatory mechanisms at PF-PC synapses after chronic THC administration. This contributes to generation of forms of pathological synaptic plasticity that might play a role in cannabinoid dependence.

A role for zinc in cerebellar synaptic transmission?

There is considerable evidence that the transition metal zinc plays an important role in mammalian neural development, physiology and pathology. The most compelling evidence for a synaptic role for zinc comes from hippocampal studies: zinc is concentrated in the synaptic vesicles of some glutamatergic neurons, zinc can be released during neural activity and zinc can modulate postsynaptic GABA and glutamate receptors. The possibility that zinc is involved in cerebellar synaptic transmission is supported by the expression of specific zinc transporters (ZnT, including the synaptic vesicle transporter ZnT-3) in the cerebellar cortex. Furthermore, some subtypes of neurotransmitter receptors, expressed by cerebellar neurones, are highly sensitive to low concentrations of exogenous zinc. However there appears to be little chelatable (synaptic) zinc in the cerebellum, deletion of the ZnT-3 gene has no effect on motor phenotype and there is currently no evidence that zinc is released from cerebellar neurones to have physiological actions. Thus it is possible that the different types of zinc transporter found in the cerebellum play a neuroprotective rather than a signalling role.

Preferential localization of the hyperpolarization‐activated cyclic nucleotide‐gated cation channel subunit HCN1 in basket cell terminals of the rat cerebellum

Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels are involved in the control of neuronal excitability and plasticity. In this study, we used immunoblotting and immunohistochemical techniques to reveal the developmental expression and subcellular distribution of the HCN1 subunit in the cerebellar cortex. During postnatal development, the spatio-temporal expression of HCN1 correlated well with the morphological events occurring during the ontogenesis of cerebellar interneurons. Using immunoblotting techniques, HCN1 was weakly detected during the first postnatal week and continued to increase throughout postnatal development, peaking at postnatal day (P)15. At the light-microscopic level, HCN1 immunoreactivity was very weak until P7 whereas from P10-12 to adulthood it was strongly detected in the lower third of the molecular layer and in the Purkinje cell layer. HCN1 was present in axons running through the molecular layer and in the pericellular basket around Purkinje cells at P12, but in the periaxonal plexus (the pinceau) surrounding their initial segment only after P15. Using immunofluorescence, HCN1 colocalized with GAD65 and synaptophysin, demonstrating that the subunit was present in inhibitory axons and axon terminals. At the electron-microscopic level, in adulthood, HCN1 immunoparticles were detected at postsynaptic sites in basket and Purkinje cells but most immunoparticles were found at presynaptic sites in basket cell axons and in terminals. In the axon terminals, the distribution of HCN1 was relatively uniform along the extrasynaptic plasma membrane; this was confirmed using quantitative techniques. The present findings suggest that HCN1 channels may provide a significant route for modulating co-ordinated cerebellar synaptic transmission through basket cells.

Increased Noise Level of Purkinje Cell Activities Minimizes Impact of Their Modulation during Sensorimotor Control

While firing rate is well established as a relevant parameter for encoding information exchanged between neurons, the significance of other parameters is more conjectural. Here, we show that regularity of neuronal spike activities affects sensorimotor processing in tottering mutants, which suffer from a mutation in P/Q-type voltage-gated calcium channels. While the modulation amplitude of the simple spike firing rate of their floccular Purkinje cells during optokinetic stimulation is indistinguishable from that of wild-types, the regularity of their firing is markedly disrupted. The gain and phase values of tottering's compensatory eye movements are indistinguishable from those of flocculectomized wild-types or from totterings with the flocculus treated with P/Q-type calcium channel blockers. Moreover, normal eye movements can be evoked in tottering when the flocculus is electrically stimulated with regular spike trains mimicking the firing pattern of normal simple spikes. This study demonstrates the importance of regularity of firing in Purkinje cells for neuronal information processing.

A biplanar slice preparation for studying cerebellar synaptic transmission

Unlike certain other brain areas, notably the hippocampal formation, synaptic transmission in the cerebellum, a 3-dimensional anatomical structure, is not ideally studied in a 2-dimensional slice preparation. We describe a method for cutting a cerebellar slice that incorporates two planes of section: one sagittal (in the plane of the Purkinje cell dendrites and of afferent and efferent fibres running in the white matter), and the other in the plane of the parallel fibres (granule cell axons). Details are given of how neurotransmission in the parallel fibre-to-Purkinje cell pathway and in the mossy fibre-to-granule cell pathway can be studied by applying a specially designed grease-gap recording technique to the biplanar slice. We further demonstrate the utility of the slice for intracellular recording.

Optokinetic stimulation increases corticotropin-releasing factor mRNA in inferior olivary neurons of rabbits

Stimulus-specific changes in levels of corticotropin-releasing factor (CRF) mRNA in inferior olivary neurons were studied in unanesthetized rabbits. The possible functional importance of CRF, a neuropeptide that is expressed in all inferior olivary neurons, in cerebellar synaptic transmission was investigated in a subpopulation of inferior olivary neurons, the caudal dorsal cap. Previous studies have shown that unidirectional, binocular optokinetic stimulation increases the level of discharge in neurons located in one of the caudal dorsal caps of the inferior olive and decreases the level of discharge in neurons in the opposite dorsal cap. We investigated the influence of prolonged (1-144 hr), unidirectional, binocular, optokinetic stimulus on the levels of CRF mRNA in dorsal cap neurons, measured with the technique of hybridization histochemistry. Rabbits were placed at the center of a cylindrical optokinetic drum that rotated at a constant velocity of 5 deg/sec, stimulating one eye in the posterior-to-anterior direction and the contralateral eye in the anterior-to-posterior direction. Posterior-to-anterior stimulation of the left eye evoked increased activity of inferior olivary neurons located in the right caudal dorsal cap. Conversely, anterior-to-posterior stimulation of the right eye evoked decreased activity of neurons in the left caudal dorsal cap. The levels of CRF mRNA in dorsal cap neurons that were activated by prolonged optokinetic stimulation were increased by a factor of 4 to 7 after 48 hr of stimulation and by more than a factor of 10 after 144 hr of optokinetic stimulation. These optokinetically induced increases in CRF mRNA decayed to background levels after 30 hr. Our observations implicate CRF in visual olivocerebellar function and suggest that CRF may play a role in the plastic control of eye movement mediated by the visual olivocerebellar system.

Selective antagonism of frog cerebellar synaptic transmission by manganese and cobalt ions

Parallel fiber-Purkinje cell synaptic transmission of the frog cerebellum can be selectively blocked by Mn 2+ and Co 2+. The interaction of these cations with Ca 2+ indicates that they act, as has been found for other chemical synapses, at the presynaptic terminals where Ca 2+ is required for release of neurotransmitters. Climbing fiber-Purkinje cell synaptic transmission was similarly blocked by Mg 2+, Mn 2+ and Co 2+.