fiber-Purkinje Cell Research Articles

α2δ-2 regulates synaptic GluK1 kainate receptors in Purkinje cells and motor coordination.

Gabapentin and pregabalin are inhibitory ligands of both α2δ-1 and α2δ-2 proteins (also known as subunits of voltage-activated Ca2+ channels) and are commonly prescribed for the treatment of neuropathic pain and epilepsy. However, these drugs can cause gait disorders and ataxia through unknown mechanisms. α2δ-2 and GluK1, a glutamate-gated kainate receptor subtype, are coexpressed in cerebellar Purkinje cells. In this study, we used a heterologous expression system and Purkinje cells to investigate the potential role of α2δ-2 in regulating GluK1-containing kainate receptor activity. Whole-cell patch clamp recordings showed that α2δ-2 coexpression augmented GluK1, but not GluK2, currents in HEK293 cells, and pregabalin abolished this augmentation. Pregabalin lost its inhibitory effect on GluK1 currents in HEK293 cells expressing both GluK1 and the α2δ-2(R282A) mutant. Blocking GluK1-containing receptors with UBP310 substantially reduced the amplitude of excitatory postsynaptic currents at parallel fiber-Purkinje cell synapses in mice. Also, pregabalin markedly attenuated the amplitude of excitatory postsynaptic currents and currents elicited by ATPA, a selective GluK1 receptor agonist, in Purkinje cells in Cacna2d1 knockout mice. Coimmunoprecipitation assays indicated that α2δ-2, but not α2δ-1, formed a protein complex with GluK1 in cerebellar tissues and HEK293 cells through its C terminus. Furthermore, α2δ-2 coexpression potentiated surface expression of GluK1 proteins in HEK293 cells, whereas pregabalin reduced GluK1 proteins in cerebellar synaptosomes. Disrupting α2δ-2-GluK1 interactions using α2δ-2 C-terminus peptide abrogated the potentiating effect of α2δ-2 on GluK1 currents and attenuated the amplitude of GluK1-mediated excitatory postsynaptic currents in Purkinje cells. However, neither pregabalin nor α2δ-2 C-terminus peptide had significant effect on P/Q-type currents in HEK293 cells. Additionally, CRISPR/Cas9-induced conditional knockdown of Cacna2d2 or Grik1 in Purkinje cells, as well as microinjection of α2δ-2 C-terminus peptide or UBP310 into the cerebellum, substantially impaired beam walking and rotarod performance in mice. Our study reveals that α2δ-2 directly interacts with GluK1 independently of its conventional role as a voltage-activated Ca2+ channel subunit. α2δ-2 regulates motor coordination by promoting synaptic expression and activity in GluK1-containing kainate receptors in Purkinje cells.

The effects of corticotropin-releasing factor on motor learning

Corticotropin-releasing factor (CRF) is mainly secreted from the hypothalamic paraventricular nuclei and plays a crucial role in stress-related responses. Recent studies have reported that CRF is a neuromodulator in the central nervous system. In the cerebellum, CRF is essential for the induction of long-term depression (LTD) at the parallel fiber-Purkinje cell synapses. Given that LTD is thought to be one of the fundamental mechanisms of motor learning, CRF may affect motor learning. However, the role of CRF in motor learning in vivo remains unclear. In this study, we aimed to examine the role of CRF in motor learning. This was achieved through a series of behavioral experiments involving the in vivo administration of CRF and its antagonists. Rats injected with CRF directly into the cerebellum exhibited superior performance on the rotarod test, especially during initial training phases, compared to control subjects. Conversely, rats receiving a CRF receptor antagonist demonstrated reduced endurance on the rotating rod compared to controls. Notably, CRF mRNA expression levels in the cerebellum did not show significant variance between the CRF-injected and control groups. These findings imply a critical role of endogenous CRF in cerebellar motor learning and suggest that exogenous CRF can augment this process. (199 words)

Systemic pharmacological suppression of neural activity reverses learning impairment in a mouse model of Fragile X syndrome

The enhancement of associative synaptic plasticity often results in impaired rather than enhanced learning. Previously, we proposed that such learning impairments can result from saturation of the plasticity mechanism (Nguyen-Vu et al., 2017), or, more generally, from a history-dependent change in the threshold for plasticity. This hypothesis was based on experimental results from mice lacking two class I major histocompatibility molecules, MHCI H2-Kb and H2-Db (MHCI KbDb−/−), which have enhanced associative long-term depression at the parallel fiber-Purkinje cell synapses in the cerebellum (PF-Purkinje cell LTD). Here, we extend this work by testing predictions of the threshold metaplasticity hypothesis in a second mouse line with enhanced PF-Purkinje cell LTD, the Fmr1 knockout mouse model of Fragile X syndrome (FXS). Mice lacking Fmr1 gene expression in cerebellar Purkinje cells (L7-Fmr1 KO) were selectively impaired on two oculomotor learning tasks in which PF-Purkinje cell LTD has been implicated, with no impairment on LTD-independent oculomotor learning tasks. Consistent with the threshold metaplasticity hypothesis, behavioral pre-training designed to reverse LTD at the PF-Purkinje cell synapses eliminated the oculomotor learning deficit in the L7-Fmr1 KO mice, as previously reported in MHCI KbDb−/−mice. In addition, diazepam treatment to suppress neural activity and thereby limit the induction of associative LTD during the pre-training period also eliminated the learning deficits in L7-Fmr1 KO mice. These results support the hypothesis that cerebellar LTD-dependent learning is governed by an experience-dependent sliding threshold for plasticity. An increased threshold for LTD in response to elevated neural activity would tend to oppose firing rate stability, but could serve to stabilize synaptic weights and recently acquired memories. The metaplasticity perspective could inform the development of new clinical approaches for addressing learning impairments in autism and other disorders of the nervous system.

Systemic pharmacological suppression of neural activity reverses learning impairment in a mouse model of Fragile X syndrome.

The enhancement of associative synaptic plasticity often results in impaired rather than enhanced learning. Previously, we proposed that such learning impairments can result from saturation of the plasticity mechanism (Nguyen-Vu et al., 2017), or, more generally, from a history-dependent change in the threshold for plasticity. This hypothesis was based on experimental results from mice lacking two class I major histocompatibility molecules, MHCI H2-Kb and H2-Db (MHCI KbDb-/-), which have enhanced associative long-term depression at the parallel fiber-Purkinje cell synapses in the cerebellum (PF-Purkinje cell LTD). Here, we extend this work by testing predictions of the threshold metaplasticity hypothesis in a second mouse line with enhanced PF-Purkinje cell LTD, the Fmr1 knockout mouse model of Fragile X syndrome (FXS). Mice lacking Fmr1 gene expression in cerebellar Purkinje cells (L7-Fmr1 KO) were selectively impaired on two oculomotor learning tasks in which PF-Purkinje cell LTD has been implicated, with no impairment on LTD-independent oculomotor learning tasks. Consistent with the threshold metaplasticity hypothesis, behavioral pre-training designed to reverse LTD at the PF-Purkinje cell synapses eliminated the oculomotor learning deficit in the L7-Fmr1 KO mice, as previously reported in MHCI KbDb-/-mice. In addition, diazepam treatment to suppress neural activity and thereby limit the induction of associative LTD during the pre-training period also eliminated the learning deficits in L7-Fmr1 KO mice. These results support the hypothesis that cerebellar LTD-dependent learning is governed by an experience-dependent sliding threshold for plasticity. An increased threshold for LTD in response to elevated neural activity would tend to oppose firing rate stability, but could serve to stabilize synaptic weights and recently acquired memories. The metaplasticity perspective could inform the development of new clinical approaches for addressing learning impairments in autism and other disorders of the nervous system.

A Model with Dopamine Depletion in Basal Ganglia and Cerebellum Predicts Changes in Thalamocortical Beta Oscillations.

Parkinsonism is presented as a motor syndrome characterized by rigidity, tremors, and bradykinesia, with Parkinson's disease (PD) being the predominant cause. The discovery that those motor symptoms result from the death of dopaminergic cells in the substantia nigra led to focus most of parkinsonism research on the basal ganglia (BG). However, recent findings point to an active involvement of the cerebellum in this motor syndrome. Here, we have developed a multiscale computational model of the rodent brain's BG-cerebellar network. Simulations showed that a direct effect of dopamine depletion on the cerebellum must be taken into account to reproduce the alterations of neural activity in parkinsonism, particularly the increased beta oscillations widely reported in PD patients. Moreover, dopamine depletion indirectly impacted spike-time-dependent plasticity at the parallel fiber-Purkinje cell synapses, degrading associative motor learning as observed in parkinsonism. Overall, these results suggest a relevant involvement of cerebellum in parkinsonism associative motor symptoms.

3,5-Dimethyladamantan-1-amine Restores Short-term Synaptic Plasticity by Changing Function of Excitatory Amino Acid Transporters in Mouse Model of Spinocerebellar Ataxia Type 1

Introduction. Memantine is an agent that used for treatment of Alzheimer's type dementia. Memantine considerably reduces the effects of neurodegeneration, may potentially slow down the neurodegenerative changes in the cerebellum and may act as treatment of choice for spinocerebellar ataxia type 1 (SCA 1).
 Our objective was to study molecular mechanisms of the short-term synaptic plasticity improvement associated with long-term memantine use in SCA 1 transgenic mice.
 Materials and methods. The experiments were performed on 12-week-old CD1 mice. We created a mouse model of cerebellar astrogliosis after expression of mutant ataxin-1 (ATXN1[Q85]) in the Bergmann glia (BG). To model the astrocyte-mediated neurodegeneration in the cerebellum, the mice were injected with LVV GFAP-Flag-ATXN1[Q85] lentiviral vector (LVV) constructs intracortically. Some of the mice received 0.35 mg/kg memantine dissolved in drink water once daily for 9 weeks. The control animals were administered LVV GFAP-ATXN1[Q2]-Flag. Changes of the excitatory postsynaptic currents amplitudes from Purkinje cells (PC) were recorded by patch clamp. Expression of anti-EAAT1 in the cerebellar cortex was assessed using immunohistochemistry.
 Results. The reactive glia of the cerebellar cortex in SCA1 mice is characterized by a decrease in the immunoreactivity of anti-EAAT1, while chronic memantine use restores this capacity. The decay time of the excitatory postsynaptic current amplitude in the parallel fiber-Purkinje cell (PF-PC) synapses of the SCA1 mice is considerably longer, which indicates the slowing of glutamate reuptake and EAAT1 dysfunction. The prolonged presence of increased neurotransmitter levels in the synaptic cleft facilitates activation of the mGluR1 signaling and restoration of mGluR1-dependent synaptic plasticity in Purkinje cells of the SCA1 mice.
 Conclusions. The slowing of neurotransmitter reuptake associated with long-term memantine treatment improves mGluR1-dependent short-term synaptic plasticity of the Purkinje cells in the SCA1 mice. Restoration of synaptic plasticity in these animals may underlie partial reduction of ataxic syndrome.

Etomidate enhances cerebellar CF-PC synaptic plasticity through CB1 receptor/PKA cascade in vitro in mice

Etomidate (ET) is a widely used intravenous imidazole general anesthetic, which depresses the cerebellar neuronal activity by modulating various receptors activity and synaptic transmission. In this study, we investigated the effects of ET on the cerebellar climbing fiber-Purkinje cells (CF-PC) plasticity in vitro in mice using whole-cell recording technique and pharmacological methods. Our results demonstrated that CF tetanic stimulation produced a mGluR1-dependent long-term depression (LTD) of CF-PC excitatory postsynaptic currents (EPSCs), which was enhanced by bath application of ET (10 µM). Blockade of mGluR1 receptor with JNJ16259685, ET triggered the tetanic stimulation to induce a CF-PC LTD accompanied with an increase in paired-pulse ratio (PPR). The ET-triggered CF-PC LTD was abolished by extracellular administration of an N-methyl-(D)-aspartate (NMDA) receptor antagonist, D-APV, as well as by intracellular blockade of NMDA receptors activity with MK801. Furthermore, blocking cannabinoids 1 (CB1) receptor with AM251 or chelating intracellular Ca2+ with BAPTA, ET failed to trigger the CF-PC LTD. Moreover, the ET-triggered CF-PC LTD was abolished by inhibition of protein kinase A (PKA), but not by inhibition of protein kinase C inhibiter. The present results suggest that ET acts on postsynaptic NMDA receptor resulting in an enhancement of the cerebellar CF-PC LTD through CB1 receptor/PKA cascade in vitro in mice. These results provide new evidence and possible mechanism for ET anesthesia to affect motor learning and motor coordination by regulating cerebellar CF-PC LTD.

Activation of α 2A and α 2B -adrenergic receptors inhibits tactile stimulation-evoked parallel fiber-Purkinje cell synaptic transmission in mouse cerebellar cortex.

The mechanism by which α2-adrenergic receptors (ARs) modulate the cerebellar parallel fiber-Purkinje cell (PF-PC) synaptic transmission is unclear. We investigated this issue using electrophysiological and neuropharmacological methods. Six- to eight-week-old ICR mice were used in the study. Under in vivo conditions, PF-PC synaptic transmission was evoked by facial stimulation of ipsilateral whisker pad, and recorded using cell-attached patch from PCs. Under in-vitro conditions, PF-PC synaptic transmission was evoked by electrical stimulation of the molecular layer in cerebellar slices, and was recorded using whole-cell recording from PCs. SR95531 (20 µM) was added to the ACSF during all recordings to prevent GABAA receptor-mediated inhibition. Air-puff stimulation of the ipsilateral whisker pad in-vivo evoked simple spike (eSS) firing of cerebellar PCs. Microapplication of noradrenaline (15 µM) to the molecular layer significantly decreased the numbers and frequency of eSS, an effect abolished by the α2-AR antagonist. Microapplication of an α2-AR agonist, UK14304 (1 µM), significantly decreased the numbers of eSS in PCs, which was abolished by either α2A- or α2B-AR antagonist, but not by α2C-AR antagonist. Under in-vitro conditions, application of UK 14304 significantly decreased the amplitude of PF-PC EPSCs and increased the paired-pulse ratio, which were abolished by either α2A- or α2B-AR antagonist. The present results indicate that activation of presynaptic α2A- and α2B-AR downregulates PF-PC synaptic transmission in mouse cerebellar cortex.

Gait disorders induced by photothrombotic cerebellar stroke in mice

Patients with cerebellar stroke display relatively mild ataxic gaits. These motor deficits often improve dramatically; however, the neural mechanisms of this improvement have yet to be elucidated. Previous studies in mouse models of gait ataxia, such as ho15J mice and cbln1-null mice, have shown that they have a dysfunction of parallel fiber-Purkinje cell synapses in the cerebellum. However, the effects of cerebellar stroke on the locomotor kinematics of wild-type mice are currently unknown. Here, we performed a kinematic analysis of gait ataxia caused by a photothrombotic stroke in the medial, vermal, and intermediate regions of the cerebellum of wild-type mice. We used the data and observations from this analysis to develop a model that will allow locomotive prognosis and indicate potential treatment regimens following a cerebellar stroke. Our analysis showed that mice performed poorly in a ladder rung test after a stroke. During walking on a treadmill, the mice with induced cerebellar stroke had an increased duty ratio of the hindlimb caused by shortened duration of the swing phase. Overall, our findings suggest that photothrombotic cerebellar infarction and kinematic gait analyses will provide a useful model for quantification of different types of acute management of cerebellar stroke in rodents.

Synapse-Specific Defects in Synaptic Transmission in the Cerebellum of W246G Mutant ELOVL4 Rats-a Model of Human SCA34.

Elongation of very long fatty acids-4 (ELOVL4) mediates biosynthesis of very long chain-fatty acids (VLC-FA; ≥28 carbons). Various mutations in this enzyme result in spinocerebellar ataxia-34 (SCA34). We generated a rat model of human SCA34 by knock-in of a naturally occurring c.736T>G, p.W246G mutation in the Elovl4 gene. Our previous analysis of homozygous W246G mutant ELOVL4 rats (MUT) revealed early-onset gait disturbance and impaired synaptic transmission and plasticity at parallel fiber-Purkinje cell (PF-PC) and climbing fiber-Purkinje cell (CF-PC) synapses. However, the underlying mechanisms that caused these defects remained unknown. Here, we report detailed patch-clamp recordings from Purkinje cells that identify impaired synaptic mechanisms. Our results show that miniature EPSC (mEPSC) frequency is reduced in MUT rats with no change in mEPSC amplitude, suggesting a presynaptic defect of excitatory synaptic transmission on Purkinje cells. We also find alterations in inhibitory synaptic transmission as miniature IPSC (mIPSC) frequency and amplitude are increased in MUT Purkinje cells. Paired-pulse ratio is reduced at PF-PC synapses but increased at CF-PC synapses in MUT rats, which along with results from high-frequency stimulation suggest opposite changes in the release probability at these two synapses. In contrast, we identify exaggerated persistence of EPSC amplitude at CF-PC and PF-PC synapses in MUT cerebellum, suggesting a larger readily releasable pool (RRP) at both synapses. Furthermore, the dendritic spine density is reduced in MUT Purkinje cells. Thus, our results uncover novel mechanisms of action of VLC-FA at cerebellar synapses, and elucidate the synaptic dysfunction underlying SCA34 pathology.SIGNIFICANCE STATEMENT Very long chain-fatty acids (VLC-FA) are an understudied class of fatty acids that are present in the brain. They are critical for brain function as their deficiency caused by mutations in elongation of very long fatty acids-4 (ELOVL4), the enzyme that mediates their biosynthesis, results in neurologic diseases including spinocerebellar ataxia-34 (SCA34), neuroichthyosis, and Stargardt-like macular dystrophy. In this study, we investigated the synaptic defects present in a rat model of SCA34 and identified defects in presynaptic neurotransmitter release and dendritic spine density at synapses in the cerebellum, a brain region involved in motor coordination. These results advance our understanding of the synaptic mechanisms regulated by VLC-FA and describe the synaptic dysfunction that leads to motor incoordination in SCA34.

Rapid eye movement sleep contributes to the formation of new axonal varicosities in mouse cerebellar parallel fibers after motor training

Synaptic structural plasticity is essential for the development, learning and memory. It is well established that sleep plays important roles in synaptic plasticity after motor learning. In cerebellar cortex, parallel fibers of granule cells make excitatory synapses to the dendrites of Purkinje cells. However, the synaptic structural dynamics between parallel and Purkinje cells after motor training and the function of sleep in cerebellar synaptic plasticity remain unclear. Here, we used two-photon microscopy to examine presynaptic axonal structural dynamics at parallel fiber-Purkinje cell synapses and investigated the effect of REM sleep in synaptic plasticity of mouse cerebellar cortex following motor training. We found that motor training induces higher formation of new axonal varicosities in cerebellar parallel fibers. Our results also indicate that calcium activities of granule cells significantly increase during REM sleep, and REM sleep deprivation prevents motor training-induced formation of axonal varicosities in parallel fibers, suggesting that higher calcium activity of granule cells was crucial for promoting newly formed axonal varicosities after motor training. Together, these findings reveal the effect of motor training on parallel fiber presynaptic structural modification and the important role of REM sleep in synaptic plasticity in cerebellar cortex.

Glucagon-like peptide-1 facilitates cerebellar parallel fiber glutamate release through PKA signaling in mice in vitro

Glucagon-like peptide-1 (GLP-1) is mainly secreted by preproglucagon neurons; it plays important roles in modulating neuronal activity and synaptic transmission through its receptors. In the present study, we investigated the effects of GLP-1 on parallel fiber–Purkinje cell (PF-PC) synaptic transmission in mouse cerebellar slices using whole-cell patch-clamp recording and pharmacology methods. In the presence of a γ-aminobutyric acid type A receptor antagonist, bath application of GLP-1 (100 nM) enhanced PF-PC synaptic transmission, with an increased amplitude of evoked excitatory postsynaptic synaptic currents (EPSCs) and a decreased paired-pulse ratio. The GLP-1-induced enhancement of evoked EPSCs was abolished by a selective GLP-1 receptor antagonist, exendin 9–39, as well as by the extracellular application of a specific protein kinase A (PKA) inhibitor, KT5720. In contrast, inhibiting postsynaptic PKA with a protein kinase inhibitor peptide-containing internal solution failed to block the GLP-1-induced enhancement of evoked EPSCs. In the presence of a mixture of gabazine (20 μM) and tetrodotoxin (1 μM), application GLP-1 significantly increased frequency, but not amplitude of miniature EPSCs via PKA signaling pathway. The GLP-1-induced increase in miniature EPSC frequency was blocked by both exendin 9–39 and KT5720. Together, our results indicate that GLP-1 receptor activation enhances glutamate release at PF-PC synapses via the PKA signaling pathway, resulting in enhanced PF-PC synaptic transmission in mice in vitro. These findings suggest that, in living animals, GLP-1 has a critical role in the modulation of cerebellar function by regulating excitatory synaptic transmission at PF-PC synapses.

Optogenetic activation of mGluR1 signaling in the cerebellum induces synaptic plasticity

Neuronal plasticity underlying cerebellar learning behavior is strongly associated with type 1 metabotropic glutamate receptor (mGluR1) signaling. Activation of mGluR1 leads to activation of the Gq/11 pathway, which is involved in inducing synaptic plasticity at the parallel fiber-Purkinje cell synapse (PF-PC) in form of long-term depression (LTD). To optogenetically modulate mGluR1 signaling we fused mouse melanopsin (OPN4) that activates the Gq/11 pathway to the C-termini of mGluR1 splice variants (OPN4-mGluR1a and OPN4-mGluR1b). Activation of both OPN4-mGluR1 variants showed robust Ca2+ increase in HEK cells and PCs of cerebellar slices. We provide the prove-of-concept approach to modulate synaptic plasticity via optogenetic activation of OPN4-mGluR1a inducing LTD at the PF-PC synapse invitro. Moreover, we demonstrate that light activation of mGluR1a signaling pathway by OPN4-mGluR1a in PCs leads to an increase in intrinsic activity of PCs invivo and improved cerebellum driven learning behavior.

Dual STDP processes at Purkinje cells contribute to distinct improvements in accuracy and speed of saccadic eye movements.

Saccadic eye-movements play a crucial role in visuo-motor control by allowing rapid foveation onto new targets. However, the neural processes governing saccades adaptation are not fully understood. Saccades, due to the short-time of execution (20-100 ms) and the absence of sensory information for online feedback control, must be controlled in a ballistic manner. Incomplete measurements of the movement trajectory, such as the visual endpoint error, are supposedly used to form internal predictions about the movement kinematics resulting in predictive control. In order to characterize the synaptic and neural circuit mechanisms underlying predictive saccadic control, we have reconstructed the saccadic system in a digital controller embedding a spiking neural network of the cerebellum with spike timing-dependent plasticity (STDP) rules driving parallel fiber-Purkinje cell long-term potentiation and depression (LTP and LTD). This model implements a control policy based on a dual plasticity mechanism, resulting in the identification of the roles of LTP and LTD in regulating the overall quality of saccade kinematics: it turns out that LTD increases the accuracy by decreasing visual error and LTP increases the peak speed. The control policy also required cerebellar PCs to be divided into two subpopulations, characterized by burst or pause responses. To our knowledge, this is the first model that explains in mechanistic terms the visual error and peak speed regulation of ballistic eye movements in forward mode exploiting spike-timing to regulate firing in different populations of the neuronal network. This elementary model of saccades could be extended and applied to other more complex cases in which single jerks are concatenated to compose articulated and coordinated movements.

Loss of 4E-BP converts cerebellar long-term depression to long-term potentiation.

Genetic perturbances in translational regulation result in defects in cerebellar motor learning; however, little is known about the role of translational mechanisms in the regulation of cerebellar plasticity. We show that genetic removal of 4E-BP, a translational suppressor and target of mammalian target of rapamycin complex 1, results in a striking change in cerebellar synaptic plasticity. We find that cerebellar long-term depression (LTD) at parallel fiber-Purkinje cell synapses is converted to long-term potentiation in 4E-BP knockout mice. Biochemical and pharmacological experiments suggest that increased phosphatase activity largely accounts for the defects in LTD. Our results point to a model in which translational regulation through the action of 4E-BP plays a critical role in establishing the appropriate kinase/phosphatase balance required for normal synaptic plasticity in the cerebellum.

Mechanisms Underlying Mu Opioid Receptor Effects on Parallel Fiber-Purkinje Cell Synaptic Transmission in Mouse Cerebellar Cortex.

μ-opioid receptors (MOR) are widely expressed in the brain, varying in density in different areas. Activation of MORs underlies analgesia, euphoria, but may lead to tolerance, dependence, and ultimately opioid addiction. The Purkinje cell (PC) is the only efferent neuron in the cerebellar cortex and receives glutamatergic synaptic inputs from the parallel fibers formed by the axons of granule cells. Studies have shown that MORs are expressed during the development of cerebellar cells. However, the distribution of MOR and their effects on PF-PC synaptic transmission remain unclear. To examine these questions, we used whole-cell patch clamp recordings and pharmacological methods to determine the effects and mechanisms of MOR activation on synaptic transmission at PF-PC synapses. The MOR-selective agonist DAMGO significantly reduced the amplitude and area under the curve (AUC) of PF-PC evoked (e) EPSCs, and increased the paired-pulse ratio (PPR).DAMGO-induced inhibitory effects on PF-PC eEPSCs and PPR were abolished by MOR specific blocker CTOP. Further, DAMGO significantly reduced the frequency of PF-PC mEPSCs, but had no obvious effect on their amplitude, suggesting a presynaptic site of action. The DAMGO-induced reduction in the frequency of PF-PC mEPSCs also was blocked by CTOP. A protein kinase A (PKA) inhibitor PKI added in the pipette solution did not affect the inhibitory effects on PF-PC mEPSCs induced by DAMGO. Both the PKA inhibitor K5720 and MEK inhibitor U0126 in artificial cerebrospinal fluid (ACSF) prevented the inhibitory effects of DAMGO on PF-PC mEPSCs. These findings reveal that MORs are expressed in presynaptic PF axon terminals, where DAMGO can activate presynaptic MORs to inhibit PF-PC synaptic transmission by regulating the release of glutamate. G-protein-dependent cAMP-PKA signaling pathway may be involved in this process.

Coexpression of calcineurin A and B subunits in various subcellular and synaptic compartments of cerebellar neurons and glia with particular abundance at parallel fiber-Purkinje cell synapses

Calcineurin (CN) is a Ca2+/calmodulin-dependent serine/threonine protein phosphatase consisting of catalytic CNA and regulatory CNB subunits, and links activity-dependent Ca2+ signals to various neural functions. Here we studied CN expression in the mouse brain by producing subunit-specific probes and antibodies. Of five CN subunits. CNAα, CNAβ, and CNB1 mRNAs were predominantly expressed over the brain from early embryonic to adult stage, and all were high in the telencephalon and cerebellum. Protein localization was examined in the cerebellum by immunofluorescence with cellular and terminal markers and by preembedding silver-enhanced immunogold microscopy. CNB1 and CNAβ were co-distributed in subcellular and synaptic elements of various cerebellar neurons and glia, whereas CNAα was exclusive in granule cell elements, including parallel fiber terminals. The present study thus discloses that CNB1 subunit well coexists with one or two CNA subunits in various cerebellar compartments. Moreover, high CN contents are provided to parallel fiber-Purkinje cell synapses, i.e., CNAα, CNAβ, and CNB1 in their presynaptic side and CNAβ and CNB1 in their postsynaptic side. These findings will be the anatomical basis, at least partly, for the known regulatory roles of postsynaptic CNs in long-term depression and presynaptic CNs in transmitter release function.

Neural Circuit Repair by Low-Intensity rTMS.

Electromagnetic brain stimulation is a promising treatment in neurology and psychiatry. However, clinical outcomes are variable and underlying mechanisms remain ill-defined, impeding the development of new effective stimulation protocols. There is increasing application of repetitive transcranial magnetic stimulation (rTMS) to the cerebellum to induce forebrain plasticity through its long-distance cerebello-cerebral circuits. To better understand what magnetic stimulation does within the cerebellum, we have developed tools to generate defined low-intensity (LI) magnetic fields and deliver them in vivo, in 3D organotypic culture and in primary cultures, over a range of stimulation parameters. Here we show that low-intensity rTMS (LI-rTMS) to the cerebellum induces axon growth and synapse formation providing olivocerebellar reinnervation. This repair depends on stimulation pattern, with complex biomimetic patterns being most effective, and this requires the presence of a cellular magnetoreceptor, cryptochrome. To explain these reparative changes, we found that repair-promoting LI-rTMS patterns, but not ineffective ones, increased c-fos expression in Purkinje neurons, consistent with the production of reactive oxygen species by activated cryptochrome. Rather than activating neurons via induced electric currents, we propose that weak magnetic fields act through cryptochrome, activating intracellular signals that induce climbing fibre-Purkinje cell reinnervation. This information opens new routes to optimize cerebellar magnetic stimulation and its potential role as an effective treatment for neurological diseases.

グルタミン酸受容体GluD1は高次脳機能領域に強く発現し、小脳では平行線維－プルキンエ細胞シナプスの形成に関与する

Of the two members of the δ subfamily of ionotropic glutamate receptors, GluD2 is exclusively expressed at parallel fiber-Purkinje cell (PF-PC) synapses in the cerebellum, and regulates their structural and functional connectivity. However, little is known to date regarding cellular and synaptic expression of GluD1 and its role in synaptic circuit formation. In the present study, we investigated this issue by producing specific and sensitive histochemical probes for GluD1 and analyzing cerebellar synaptic circuits in GluD1-knockout mice. GluD1 was widely expressed in the adult mouse brain, with high levels in higher brain regions, including the cerebral cortex, striatum, limbic regions (hippocampus, nucleus accumbens, lateral septum, bed nucleus stria terminalis, lateral habenula, and central nucleus of the amygdala), and cerebellar cortex. In the cerebellar cortex, GluD1 mRNA was expressed at the highest level in molecular layer interneurons, and its immunoreactivity was concentrated at PF synapses on interneuron somata. In GluD1-knockout mice, the density of PF synapses on interneuron somata was significantly reduced, and the size and number of interneurons were significantly diminished. Thus, GluD1 is common to GluD2 in expression at PF synapses, but distinct from GluD2 in neuronal expression in the cerebellar cortex, i,e., GluD1 in interneurons and GluD2 in PCs. Furthermore, GluD1 regulates the connectivity of PF-interneuron synapses, and promotes the differentiation and/or survival of molecular layer interneurons. These results suggest that GluD1 works in concert with GluD2 for the construction of cerebellar synaptic wiring through distinct neuronal and synaptic expressions and also their shared synapse-connecting function.

Associative anticipatory learning and control of the cerebellar cortex based on the spike-timing-dependent plasticity of the parallel fiber-Purkinje cell synapses

Time delays are inevitable in the neural processing of sensorimotor systems; small delays can cause severe damage to movement accuracy and stability. It is strongly suggested that the cerebellum compensates for delays in neural signal processing and performs predictive control. Neural computational theories have explored concepts of the internal models of control objects—believed to avoid delays by providing internal feedback information—although there has been no clear relevance to neural processing. The timing-dependent plasticity of parallel fiber-Purkinje cell synapses is well known. The long-term depression of the synapse is observed when parallel fiber activation precedes climbing fiber activation within −50–300 ms, and is the greatest within 50–200 ms. This paper presents a theory that this temporal difference of 50–200 ms is the basis for an associative anticipation of as many milliseconds. Associative learning can theoretically connect an input signal to a desired signal; therefore, a 50–200 ms earlier input signal can be connected to a desired output signal through temporary asymmetric plasticity. After learning is completed, an input signal generates a desired output signal that appears 50–200 ms later. For the associative learning of temporally continuous signals, this study integrates the universal function approximation capability of the cerebellar cortex model and temporally asymmetric synaptic plasticity to create the theory of associative anticipatory learning of the cerebellum. The effective motor control of this learning is demonstrated by adaptively stabilizing an inverted pendulum with a delay similar to that done by humans.