Climbing Fiber Research Articles

Deficits Associated With Loss of STIM1 in Purkinje Neurons Including Motor Coordination Can Be Rescued by Loss of Septin 7.

Septins are cytoskeletal proteins that can assemble to form heteromeric filamentous complexes and regulate a range of membrane-associated cellular functions. SEPT7, a member of the septin family, functions as a negative regulator of the plasma membrane–localized store-operated Ca2+ entry (SOCE) channel, Orai in Drosophila neurons, and in human neural progenitor cells. Knockdown of STIM, a Ca2+ sensor in the endoplasmic reticulum (ER) and an integral component of SOCE, leads to flight deficits in Drosophila that can be rescued by partial loss of SEPT7 in neurons. Here, we tested the effect of reducing and removing SEPT7 in mouse Purkinje neurons (PNs) with the loss of STIM1. Mice with the complete knockout of STIM1 in PNs exhibit several age-dependent changes. These include altered gene expression in PNs, which correlates with increased synapses between climbing fiber (CF) axons and Purkinje neuron (PN) dendrites and a reduced ability to learn a motor coordination task. Removal of either one or two copies of the SEPT7 gene in STIM1 KO PNs restored the expression of a subset of genes, including several in the category of neuron projection development. Importantly, the rescue of gene expression in these animals is accompanied by normal CF-PN innervation and an improved ability to learn a motor coordination task in aging mice. Thus, the loss of SEPT7 in PNs further modulates cerebellar circuit function in STIM1 KO animals. Our findings are relevant in the context of identifying SEPT7 as a putative therapeutic target for various neurodegenerative diseases caused by reduced intracellular Ca2+ signaling.

Sensory Over-responsivity and Aberrant Plasticity in Cerebellar Cortex in a Mouse Model of Syndromic Autism

BackgroundPatients with autism spectrum disorder often show altered responses to sensory stimuli as well as motor deficits, including an impairment of delay eyeblink conditioning, which involves integration of sensory signals in the cerebellum. Here, we identify abnormalities in parallel fiber (PF) and climbing fiber (CF) signaling in the mouse cerebellar cortex that may contribute to these pathologies. MethodsWe used a mouse model for the human 15q11-13 duplication (patDp/+) and studied responses to sensory stimuli in Purkinje cells from awake mice using two-photon imaging of GCaMP6f signals. Moreover, we examined synaptic transmission and plasticity using in vitro electrophysiological, immunohistochemical, and confocal microscopic techniques. ResultsWe found that spontaneous and sensory-evoked CF-calcium transients are enhanced in patDp/+ Purkinje cells, and aversive movements are more severe across sensory modalities. We observed increased expression of the synaptic organizer NRXN1 at CF synapses and ectopic spread of these synapses to fine dendrites. CF–excitatory postsynaptic currents recorded from Purkinje cells are enlarged in patDp/+ mice, while responses to PF stimulation are reduced. Confocal measurements show reduced PF+CF-evoked spine calcium transients, a key trigger for PF long-term depression, one of several plasticity types required for eyeblink conditioning learning. Long-term depression is impaired in patDp/+ mice but is rescued on pharmacological enhancement of calcium signaling. ConclusionsOur findings suggest that this genetic abnormality causes a pathological inflation of CF signaling, possibly resulting from enhanced NRXN1 expression, with consequences for the representation of sensory stimuli by the CF input and for PF synaptic organization and plasticity.

Introduction of synaptotagmin 7 promotes facilitation at the climbing fiber to Purkinje cell synapse.

SUMMARYSynaptotagmin 7 (Syt7) is a high-affinity calcium sensor that is implicated in multiple aspects of synaptic transmission. Here, we study the influence of Syt7 on the climbing fiber (CF) to Purkinje cell (PC) synapse. We find that small facilitation and prominent calcium-dependent recovery from depression at this synapse do not rely on Syt7 and that Syt7 is not normally present in CFs. We expressed Syt7 in CFs to assess the consequences of introducing Syt7 to a synapse that normally lacks Syt7. Syt7 expression does not promote asynchronous release or accelerate recovery from depression. Syt7 decreases the excitatory postsynaptic current (EPSC) magnitude, consistent with a decrease in the initial probability of release (PR). Syt7 also increases synaptic facilitation to such a large extent that it could not arise solely as an indirect consequence of decreased PR. Thus, the primary consequence of Syt7 expression in CFs, which normally lack Syt7, is to promote synaptic facilitation.

W246G Mutant ELOVL4 Impairs Synaptic Plasticity in Parallel and Climbing Fibers and Causes Motor Defects in a Rat Model of SCA34

Spinocerebellar ataxia (SCA) is a neurodegenerative disorder characterized by ataxia and cerebellar atrophy. A number of different mutations gives rise to different types of SCA with characteristic ages of onset, symptomatology, and rates of progression. SCA type 34 (SCA34) is caused by mutations in ELOVL4 (ELOngation of Very Long-chain fatty acids 4), a fatty acid elongase essential for biosynthesis of Very Long Chain Saturated and Polyunsaturated Fatty Acids (VLC-SFA and VLC-PUFA, resp., ≥28 carbons), which have important functions in the brain, skin, retina, Meibomian glands, testes, and sperm. We generated a rat model of SCA34 by knock-in of the SCA34-causing 736T>G (p.W246G) ELOVL4 mutation. Rats carrying the mutation developed impaired motor deficits by 2 months of age. To understand the mechanism of these motor deficits, we performed electrophysiological studies using cerebellar slices from rats homozygous for W246G mutant ELOVL4 and found marked reduction of long-term potentiation at parallel fiber synapses and long-term depression at climbing fiber synapses onto Purkinje cells. Neuroanatomical analysis of the cerebellum showed normal cytoarchitectural organization with no evidence of degeneration out to 6 months of age. These results point to ELOVL4 as essential for motor function and cerebellar synaptic plasticity. The results further suggest that ataxia in SCA34 patients may arise from a primary impairment of synaptic plasticity and cerebellar network desynchronization before onset of neurodegeneration and progression of the disease at a later age.

An Autism-Associated Neuroligin-3 Mutation Affects Developmental Synapse Elimination in the Cerebellum.

Neuroligin is a postsynaptic cell-adhesion molecule that is involved in synapse formation and maturation by interacting with presynaptic neurexin. Mutations in neuroligin genes, including the arginine to cystein substitution at the 451st amino acid residue (R451C) of neuroligin-3 (NLGN3), have been identified in patients with autism spectrum disorder (ASD). Functional magnetic resonance imaging and examination of post-mortem brain in ASD patients implicate alteration of cerebellar morphology and Purkinje cell (PC) loss. In the present study, we examined possible association between the R451C mutation in NLGN3 and synaptic development and function in the mouse cerebellum. In NLGN3-R451C mutant mice, the expression of NLGN3 protein in the cerebellum was reduced to about 10% of the level of wild-type mice. Elimination of redundant climbing fiber (CF) to PC synapses was impaired from postnatal day 10–15 (P10–15) in NLGN3-R451C mutant mice, but majority of PCs became mono-innervated as in wild-type mice after P16. In NLGN3-R451C mutant mice, selective strengthening of a single CF relative to the other CFs in each PC was impaired from P16, which persisted into juvenile stage. Furthermore, the inhibition to excitation (I/E) balance of synaptic inputs to PCs was elevated, and calcium transients in the soma induced by strong and weak CF inputs were reduced in NLGN3-R451C mutant mice. These results suggest that a single point mutation in NLGN3 significantly influences the synapse development and refinement in cerebellar circuitry, which might be related to the pathogenesis of ASD.

Stochastic reaction-diffusion modeling of calcium dynamics in 3D dendritic spines of Purkinje cells

Calcium (Ca2+) is a second messenger assumed to control changes in synaptic strength in the form of both long-term depression and long-term potentiation at Purkinje cell dendritic spine synapses via inositol trisphosphate (IP3)-induced Ca2+ release. These Ca2+ transients happen in response to stimuli from parallel fibers (PFs) from granule cells and climbing fibers (CFs) from the inferior olivary nucleus. These events occur at low numbers of free Ca2+, requiring stochastic single-particle methods when modeling them. We use the stochastic particle simulation program MCell to simulate Ca2+ transients within a three-dimensional Purkinje cell dendritic spine. The model spine includes the endoplasmic reticulum, several Ca2+ transporters, and endogenous buffer molecules. Our simulations successfully reproduce properties of Ca2+ transients in different dynamical situations. We test two different models of the IP3 receptor (IP3R). The model with nonlinear concentration response of binding of activating Ca2+ reproduces experimental results better than the model with linear response because of the filtering of noise. Our results also suggest that Ca2+-dependent inhibition of the IP3R needs to be slow to reproduce experimental results. Simulations suggest the experimentally observed optimal timing window of CF stimuli arises from the relative timing of CF influx of Ca2+ and IP3 production sensitizing IP3R for Ca2+-induced Ca2+ release. We also model ataxia, a loss of fine motor control assumed to be the result of malfunctioning information transmission at the granule to Purkinje cell synapse, resulting in a decrease or loss of Ca2+ transients. Finally, we propose possible ways of recovering Ca2+ transients under ataxia.

Influence of various temporal recoding on pavlovian eyeblink conditioning in the cerebellum

We consider the Pavlovian eyeblink conditioning (EBC) via repeated presentation of paired conditioned stimulus (tone) and unconditioned stimulus (US; airpuff). In an effective cerebellar ring network, we change the connection probability from Golgi to granule (GR) cells, and make a dynamical classification of various firing patterns of the GR cells. Individual GR cells are thus found to show various well- and ill-matched firing patterns relative to the US timing signal. Then, these variously-recoded signals are fed into the Purkinje cells (PCs) through the parallel-fibers (PFs). Based on such unique dynamical classification of various firing patterns, we make intensive investigations on the influence of various temporal recoding (i.e., firing patterns) of the GR cells on the synaptic plasticity of the PF-PC synapses and the subsequent learning process for the EBC. We first note that the variously-recoded PF signals are effectively depressed by the (error-teaching) instructor climbing-fiber (CF) signals from the inferior olive neuron. In the case of well-matched PF signals, they are strongly depressed through strong long-term depression (LTD) by the instructor CF signals due to good association between the in-phase PF and the instructor CF signals. On the other hand, practically no LTD occurs for the ill-matched PF signals because most of them have no association with the instructor CF signals. This kind of "effective" depression at the PF-PC synapses coordinates firings of PCs effectively, which then makes effective inhibitory coordination on the cerebellar nucleus neuron [which elicits conditioned response (CR; eyeblink)]. When the learning trial passes a threshold, acquisition of CR begins. In this case, the timing degree of CR becomes good due to presence of the ill-matched firing group which plays a role of protection barrier for the timing. With further increase in the number of trials, strength of CR (corresponding to the amplitude of eyelid closure) increases due to strong LTD in the well-matched firing group, while its timing degree decreases. In this way, the well- and the ill-matched firing groups play their own roles for the strength and the timing of CR, respectively. Thus, with increasing the number of learning trials, the (overall) learning efficiency degree (taking into consideration both timing and strength of CR) for the CR is increased, and eventually it becomes saturated. Finally, we also discuss dependence of the variety degree for firing patterns of the GR cells and the saturated learning efficiency degree of the CR on and their relations.

Part I: The Complex Spikes as One of the Cerebellar Secrets

The olivocerebellar tract has unique morphological, physiological, and developmental properties. Olivocerebellar axons are the source of multiple climbing fibers (CFs). The synapse between CFs and the Purkinje neuron is one of the most powerful excitatory in the central nervous system. Complex spikes are composed of an initial large amplitude spike followed by spikelets. The spatiotemporal patterns of complex/simple spikes complement the rate coding to enhance the accuracy of motor and cognitive processing, and to improve predictions related to internal models. Understanding the role of complex spikes is essential in clarifying how the cerebellar cortex contributes to learning, motor control, cognitive tasks, and the processing of emotions. This Cerebellar Classic is devoted to the pioneering work of Eccles, Llinás, and Sasaki on complex spikes using intracellular recordings from Purkinje neurons.

Stochastic Reaction-Diffusion Modeling of Calcium Dynamics in 3D-Dendritic Spines of Purkinje Cells

Calcium (Ca2+) is a second messenger assumed to control changes in synaptic strength in the form of both long-term depression (LTD) and long-term potentiation (LTP) at Purkinje cell dendritic spine synapses via inositol trisphosphate (IP3) induced Ca2+ release. These Ca2+ transients happen in response to stimuli from parallel fibers (PF) from granule cells and climbing fibers (CF) from the inferior olivary nucleus. These events occur at low numbers of free Ca2+ requiring stochastic single particle methods when modeling them.

Increased Climbing Fiber Lateral Crossings on Purkinje Cell Dendrites in the Cerebellar Hemisphere in Essential Tremor.

Climbing fibers (CFs) innervate Purkinje cells (PCs) with 1:1 relationship to ensure proper cerebellar function. Although CFs abnormally extend into the parallel fiber domain of PC dendrites in essential tremor (ET), the architecture of CFs in relation to PCs has yet to be investigated in detail. The aim of this work was to study the architecture of CFs in relation to PCs in ET. The number of PC somas and PC dendrites that a single CF crossed was quantified in the postmortem cerebellum of 15 ET cases and 15 control cases. In ET, CFs crossed a greater number of PC somas and PC dendrites than in control cases, raising the possibility that there is abnormal CF wiring onto the PCs. Interestingly, the increase in CF-PC crossings positively correlated with tremor severity. Patients with ET have increased CF crossings on PC dendrites. This abnormal architectural arrangement may contribute to synchronous brain activity and tremor. © 2021 International Parkinson and Movement Disorder Society.

Cerebellar developmental deficits underlie neurodegenerative disorder spinocerebellar ataxia type 23.

Spinocerebellar ataxia type 23 (SCA23) is a late‐onset neurodegenerative disorder characterized by slowly progressive gait and limb ataxia, for which there is no therapy available. It is caused by pathogenic variants in PDYN, which encodes prodynorphin (PDYN). PDYN is processed into the opioid peptides α‐neoendorphin and dynorphins (Dyn) A and B; inhibitory neurotransmitters that function in pain signaling, stress‐induced responses and addiction. Variants causing SCA23 mostly affect Dyn A, leading to loss of secondary structure and increased peptide stability. PDYN R212W mice express human PDYN containing the SCA23 variant p.R212W. These mice show progressive motor deficits from 3 months of age, climbing fiber (CF) deficits from 3 months of age, and Purkinje cell (PC) loss from 12 months of age. A mouse model for SCA1 showed similar CF deficits, and a recent study found additional developmental abnormalities, namely increased GABAergic interneuron connectivity and non‐cell autonomous disruption of PC function. As SCA23 mice show a similar pathology to SCA1 mice in adulthood, we hypothesized that SCA23 may also follow SCA1 pathology during development. Examining PDYN R212W cerebella during development, we uncovered developmental deficits from 2 weeks of age, namely a reduced number of GABAergic synapses on PC soma, possibly leading to the observed delay in early phase CF elimination between 2 and 3 weeks of age. Furthermore, CFs did not reach terminal height, leaving proximal PC dendrites open to be occupied by parallel fibers (PFs). The observed increase in vGlut1 protein—a marker for PF‐PC synapses—indicates that PFs indeed take over CF territory and have increased connectivity with PCs. Additionally, we detected altered expression of several critical Ca2+ channel subunits, potentially contributing to altered Ca2+ transients in PDYN R212W cerebella. These findings indicate that developmental abnormalities contribute to the SCA23 pathology and uncover a developmental role for PDYN in the cerebellum.

Direct translation of climbing fiber burst-mediated sensory coding into post-synaptic Purkinje cell dendritic calcium.

Climbing fibers (CFs) generate complex spikes (CS) and Ca2+ transients in cerebellar Purkinje cells (PCs), serving as instructive signals. The so-called 'all-or-none' character of CSs has been questioned since the CF burst was described. Although recent studies have indicated a sensory-driven enhancement of PC Ca2+ signals, how CF responds to sensory events and contributes to PC dendritic Ca2+ and CS remains unexplored. Here, single or simultaneous Ca2+ imaging of CFs and PCs in awake mice revealed the presynaptic CF Ca2+ amplitude encoded the sensory input's strength and directly influenced post-synaptic PC dendritic Ca2+ amplitude. The sensory-driven variability in CF Ca2+ amplitude depended on the number of spikes in the CF burst. Finally, the spike number of the CF burst determined the PC Ca2+ influx and CS properties. These results reveal the direct translation of sensory information-coding CF inputs into PC Ca2+, suggesting the sophisticated role of CFs as error signals.

Climbing fiber synapses rapidly and transiently inhibit neighboring Purkinje cells via ephaptic coupling

Climbing fibers (CFs) from the inferior olive (IO) make strong excitatory synapses onto cerebellar Purkinje cell (PC) dendrites, and trigger distinctive responses known as complex spikes (CSs). We find that in awake mice, a CS in one PC suppresses conventional simple spikes (SSs) in neighboring PCs for several milliseconds. This involves a novel ephaptic coupling, in which an excitatory synapse generates large negative extracellular signals that nonsynaptically inhibit neighboring PCs. The distance dependence of CS-SS ephaptic signaling, combined with the known CF divergence, allows a single IO neuron to influence the output of the cerebellum by synchronously suppressing the firing of potentially over one hundred PCs. Optogenetic studies in vivo, and dynamic clamp studies in slice, indicate that such brief PC suppression, either as a result of ephaptic signaling or other mechanisms, can effectively promote firing in neurons in the deep cerebellar nuclei with remarkable speed and precision.

Propofol facilitates climbing fiber-Purkinje cell synaptic transmission via NMDA receptor in vitro in mice

Propofol is generally used for the induction and maintenance of anesthesia in clinical procedures via activation of γ -aminobutyric acid A (GABAA) receptors. When administered at the clinical dose, propofol use is associated with movement disorders, including dystonia and ataxia, suggesting that propofol administration impacts the function of cerebellar neuronal circuitry. In this study, we investigated the effect of propofol on climbing fiber (CF)-Purkinje cell (PC) synaptic transmission in mouse cerebellar slices in the absence of GABAergic inhibition using a whole-cell recording technique and pharmacological methods. Our results showed that bath application of propofol enhanced CF-PC synaptic transmission, which was demonstrated by an increased amplitude and area under the curve (AUC) of the excitatory postsynaptic currents (EPSCs) accompanied by a decrease in the paired-pulse ratio (PPR). The propofol-induced increase in the amplitude of P1 was concentration-dependent with a half effective concentration (EC50) of 20.9 μM. The propofol-induced increases in the amplitude and AUC of CF-PC EPSCs were abolished by an N-Methyl-D-aspartate (NMDA) receptor blocker. Furthermore, the application of NMDA enhanced CF-PC EPSCs and overwhelmed the effect of propofol on CF-PC EPSCs. Moreover, intracellular blockade of NMDA receptors attenuated the propofol-induced enhancement of CF-PC synaptic transmission but strengthened the propofol-induced change in the PPR. These results indicate that propofol enhances CF-PC synaptic transmission by activation of NMDA receptors in the mouse cerebellar cortex, suggesting that propofol administration might be involved in propofol-induced dysfunction of the cerebellum via NMDA receptors.

Is Purkinje Neuron Hyperpolarisation Important for Cerebellar Synaptic Plasticity? A Retrospective and Prospective Analysis

Two recent studies have demonstrated that the dendritic Ca2+ signal associated with a climbing fibre (CF) input to the cerebellar Purkinje neuron (PN) depends on the membrane potential (Vm). Specifically, when the cell is hyperpolarised, this signal is mediated by T-type voltage-gated Ca2+ channels; in contrast, when the cell is firing, the CF-PN signal is mediated by P/Q-type voltage-gated Ca2+ channels. When the CF input is paired with parallel fibre (PF) activity, the signal is locally amplified at the sites of PF-activated synapses according to the Vm at the time of the CF input, suggesting that the standing Vm is a critical parameter for the induction of PF synaptic plasticity. In this review, I analyse how the Vm can potentially play a role in cerebellar learning focussing, in particular, on the hyperpolarised state that appears to occur episodically, since PNs are mostly firing under physiological conditions. By revisiting the recent literature reporting in vivo recordings and synaptic plasticity studies, I speculate on how a putative role of the PN Vm can provide an interpretation for the results of these studies.

Phospholipase C β3 is Required for Climbing Fiber Synapse Elimination in Aldolase C-positive Compartments of the Developing Mouse Cerebellum

In the cerebellum of neonatal mice, multiple climbing fibers (CFs) form excitatory synapses on each Purkinje cell (PC). Only one CF is strengthened in each PC from postnatal day 3 (P3) to P7, whereas the other weaker CFs are eliminated progressively from ∼P7 to ∼P11 (early phase of CF elimination) and from ∼P12 to ∼P17 (late phase of CF elimination). Type 1 metabotropic glutamate receptor (mGluR1) triggers a canonical pathway in PCs for the late phase of CF elimination. Among downstream signaling molecules of mGluR1, phospholipase C β3 (PLCβ3) and β4 (PLCβ4) are expressed complementarily in PCs of aldolase C (Aldoc)-positive (+) and Aldoc-negative (−) cerebellar compartments, respectively. PLCβ4 is reported to mediate the late phase of CF elimination in the anterior half of the cerebellar vermis which corresponds to the Aldoc (−) region. However, roles of PLCβ3 and Aldoc in CF synapse elimination are unknown. Here, we investigated CF innervation of PCs in Aldoc-tdTomato knock-in mice that underwent lentivirus-mediated knockdown (KD) of PLCβ3 in PCs during postnatal development. By recording CF-mediated excitatory postsynaptic currents from PCs and immunostaining CF synaptic terminals, we found that significantly higher percentage of PCs with PLCβ3-KD remained multiply innervated by CFs in Aldoc (+) compartments after P12, which was accompanied by impaired elimination of somatic CF synapses and reduced dendritic CF translocation. In contrast, deletion of Aldoc had no effect on CF synapse elimination. These results suggest that PLCβ3 is required for the late phase of CF elimination in Aldoc (+) PCs.

α2δ-2 Protein Controls Structure and Function at the Cerebellar Climbing Fiber Synapse.

α2δ proteins (Cacna2d1-4) are auxiliary subunits of voltage-dependent calcium channels that also drive synapse formation and maturation. Because cerebellar Purkinje cells (PCs) predominantly, if not exclusively, express one isoform of this family, α2δ-2 (Cacna2d2), we used PCs as a model system to examine roles of α2δ in excitatory synaptic function in male and female Cacna2d2 knock-out (KO) mice. Whole-cell recordings of PCs from acute cerebellar slices revealed altered climbing fiber (CF)-evoked complex spike generation, as well as increased amplitude and faster decay of CF-evoked EPSCs. CF terminals in the KO were localized more proximally on PC dendrites, as indicated by VGLUT2+ immunoreactive puncta, and computational modeling demonstrated that the increased EPSC amplitude can be partly attributed to the more proximal location of CF terminals. In addition, CFs in KO mice exhibited increased multivesicular transmission, corresponding to greater sustained responses during repetitive stimulation, despite a reduction in the measured probability of release. Electron microscopy demonstrated that mutant CF terminals had twice as many vesicle release sites, providing a morphologic explanation for the enhanced glutamate release. Though KO CFs evoked larger amplitude EPSCs, the charge transfer was the same as wild-type as a result of increased glutamate reuptake, producing faster decay kinetics. Together, the larger, faster EPSCs in the KO explain the altered complex spike responses, which degrade information transfer from PCs and likely contribute to ataxia in Cacna2d2 KO mice. Our results also illustrate the multidimensional synaptic roles of α2δ proteins.SIGNIFICANCE STATEMENT α2δ proteins (Cacna2d1-4) regulate synaptic transmission and synaptogenesis, but coexpression of multiple α2δ isoforms has obscured a clear understanding of how various α2δ proteins control synaptic function. We focused on roles of the α2δ-2 protein (Cacna2d2), the deletion of which causes cerebellar ataxia and epilepsy in mice and humans. Because cerebellar Purkinje cells (PCs) only express this single isoform, we studied excitatory climbing fiber synaptic function onto PCs in Cacna2d2 KO mice. Using optical and electrophysiological analysis, we provide a detailed description of the changes in PCs lacking α2δ-2, and provide a comprehensive mechanistic explanation for how functional synaptic phenotypes contribute to the altered cerebellar output.

The Origin of Physiological Local mGluR1 Supralinear Ca2+ Signals in Cerebellar Purkinje Neurons.

In mouse cerebellar Purkinje neurons (PNs), the climbing fiber (CF) input provides a signal to parallel fiber (PF) synapses, triggering PF synaptic plasticity. This signal is given by supralinear Ca2+ transients, associated with the CF synaptic potential and colocalized with the PF Ca2+ influx, occurring only when PF activity precedes the CF input. Here, we unravel the biophysical determinants of supralinear Ca2+ signals associated with paired PF-CF synaptic activity. We used membrane potential (Vm) and Ca2+ imaging to investigate the local CF-associated Ca2+ influx following a train of PF synaptic potentials in two cases: (1) when the dendritic Vm is hyperpolarized below the resting Vm, and (2) when the dendritic Vm is at rest. We found that supralinear Ca2+ signals are mediated by type-1 metabotropic glutamate receptors (mGluR1s) when the CF input is delayed by 100-150 ms from the first PF input in both cases. When the dendrite is hyperpolarized only, however, mGluR1s boost neighboring T-type channels, providing a mechanism for local coincident detection of PF-CF activity. The resulting Ca2+ elevation is locally amplified by saturation of endogenous Ca2+ buffers produced by the PF-associated Ca2+ influx via the mGluR1-mediated nonselective cation conductance. In contrast, when the dendritic Vm is at rest, mGluR1s increase dendritic excitability by inactivating A-type K+ channels, but this phenomenon is not restricted to the activated PF synapses. Thus, Vm is likely a crucial parameter in determining PF synaptic plasticity, and the occurrence of hyperpolarization episodes is expected to play an important role in motor learning.SIGNIFICANCE STATEMENT In Purkinje neurons, parallel fiber synaptic plasticity, determined by coincident activation of the climbing fiber input, underlies cerebellar learning. We unravel the biophysical mechanisms allowing the CF input to produce a local Ca2+ signal exclusively at the sites of activated parallel fibers. We show that when the membrane potential is hyperpolarized with respect to the resting membrane potential, type-1 metabotropic glutamate receptors locally enhance Ca2+ influx mediated by T-type Ca2+ channels, and that this signal is amplified by saturation of endogenous buffer also mediated by the same receptors. The combination of these two mechanisms is therefore capable of producing a Ca2+ signal at the activated parallel fiber sites, suggesting a role of Purkinje neuron membrane potential in cerebellar learning.

NPC1 deficiency impairs cerebellar postnatal development of microglia and climbing fiber refinement in a mouse model of Niemann-Pick disease type C.

ABSTRACTLittle is known about the effects of NPC1 deficiency in brain development and whether these effects contribute to neurodegeneration in Niemann–Pick disease type C (NPC). Degeneration of cerebellar Purkinje cells occurs at an earlier stage and to a greater extent in NPC; therefore, we analyzed the effect of NPC1 deficiency on microglia and on climbing fiber synaptic refinement during cerebellar postnatal development using the Npc1nmf164 mouse. Our analysis revealed that NPC1 deficiency leads to early phenotypic changes in microglia that are not associated with an innate immune response. However, the lack of NPC1 in Npc1nmf164 mice significantly affected the early development of microglia by delaying the radial migration, increasing the proliferation and impairing the differentiation of microglia precursor cells during postnatal development. Additionally, increased phagocytic activity of differentiating microglia was observed at the end of the second postnatal week in Npc1nmf164 mice. Moreover, significant climbing fiber synaptic refinement deficits along with an increased engulfment of climbing fiber synaptic elements by microglia were found in Npc1nmf164 mice, suggesting that profound developmental defects in microglia and synaptic connectivity might precede and predispose Purkinje cells to early neurodegeneration in NPC.

Developmental Rewiring between Cerebellar Climbing Fibers and Purkinje Cells Begins with Positive Feedback Synapse Addition

SUMMARYDuring postnatal development, cerebellar climbing fibers alter their innervation strengths onto supernumerary Purkinje cell targets, generating a one-to-few connectivity pattern in adulthood. To get insight about the processes responsible for this remapping, we reconstructed serial electron microscopy datasets from mice during the first postnatal week. Between days 3 and 7, individual climbing fibers selectively add many synapses onto a subset of Purkinje targets in a positive-feedback manner, without pruning synapses from other targets. Active zone sizes of synapses associated with powerful versus weak inputs are indistinguishable. Changes in synapse number are thus the predominant form of early developmental plasticity. Finally, the numbers of climbing fibers and Purkinje cells in a local region nearly match. Initial over-innervation of Purkinje cells by climbing fibers is therefore economical: the number of axons entering a region is enough to assure that each ultimately retains a postsynaptic target and that none branched there in vain.