Synaptic responses evoked by tactile stimuli in Purkinje cells in mouse cerebellar cortex Crus II in vivo

Background

Sensory stimuli evoke responses in cerebellar Purkinje cells (PCs) via the mossy fiber-granule cell pathway. However, the properties of synaptic responses evoked by tactile stimulation in cerebellar PCs are unknown. The present study investigated the synaptic responses of PCs in response to an air-puff stimulation on the ipsilateral whisker pad in urethane-anesthetized mice.

Methods and Main Results

Thirty-three PCs were recorded from 48 urethane-anesthetized adult (6–8-week-old) HA/ICR mice by somatic or dendritic patch-clamp recording and pharmacological methods. Tactile stimulation to the ipsilateral whisker pad was delivered by an air-puff through a 12-gauge stainless steel tube connected with a pressurized injection system. Under current-clamp conditions (I = 0), the air-puff stimulation evoked strong inhibitory postsynaptic potentials (IPSPs) in the somata of PCs. Application of SR95531, a specific GABA_A receptor antagonist, blocked IPSPs and revealed stimulation-evoked simple spike firing. Under voltage-clamp conditions, tactile stimulation evoked a sequence of transient inward currents followed by strong outward currents in the somata and dendrites in PCs. Application of SR95531 blocked outward currents and revealed excitatory postsynaptic currents (EPSCs) in somata and a temporal summation of parallel fiber EPSCs in PC dendrites. We also demonstrated that PCs respond to both the onset and offset of the air-puff stimulation.

Conclusions

These findings indicated that tactile stimulation induced asynchronous parallel fiber excitatory inputs onto the dendrites of PCs, and failed to evoke strong EPSCs and spike firing in PCs, but induced the rapid activation of strong GABA_A receptor-mediated inhibitory postsynaptic currents in the somata and dendrites of PCs in the cerebellar cortex Crus II in urethane-anesthetized mice.     

Long-term depression of the cerebellar climbing fiber--Purkinje neuron synapse

In classic Marr-Albus-Ito models of cerebellar function, coactivation of the climbing fiber (CF) synapse, which provides massive, invariant excitation of Purkinje neurons (coding the unconditioned stimulus), together with a graded parallel fiber synaptic array (coding the conditioned stimulus) leads to long-term depression (LTD) of parallel fiber-Purkinje neuron synapses, underlying production of a conditioned response. Here, we show that the supposedly invariant CF synapse can also express LTD. Brief 5 Hz stimulation of the CF resulted in a sustained depression of CF EPSCs that did not spread to neighboring parallel fiber synapses. Like parallel fiber LTD, CF LTD required postsynaptic Ca2+ elevation, activation of group 1 mGluRs, and activation of PKC. CF LTD is potentially relevant for models of cerebellar motor control and learning and the developmental conversion from multiple to single CF innervation of Purkinje neurons.     

Interrelated modification of excitatory and inhibitory synapses in three-layer olivary-cerebellar neural network

The model of three-layer olivary-cerebellar neural network with modifiable excitatory and inhibitory connections between diverse elements is suggested. The same Hebbian modification rules are proposed for Purkinje cells, granule (input) cells, and deep cerebellar nuclei (output) cells. The inverse calcium-dependent modification rules for these cells and hippocampal/neocortical neurones or Golgi cells are conceivably the result of the involvement of cGMP and cAMP in postsynaptic processes. The sign of simultaneous modification of excitatory and inhibitory inputs to a cell is opposite and determined by the variations in pre- and/or postsynaptic cell activity. Modification of excitatory transmission between parallel fibers and Purkinje cells, mossy fibers and granule cells, and mossy fibers and deep cerebellar nuclei cells essentially depends on inhibition effected by stellate/basket cells, Golgi cells and Purkinje cells, respectively. The character of interrelated modifications of diverse synapses in all three layers of the network is influenced by olivary cell activity. In the absence (presence) of a signal from inferior olive, the long-term potentiation (depression) in the efficacy of a synapse between input mossy fiber and output cell can be induced. The results of the suggested model are in accordance with known experimental data.     

Neuronal glutamate transporters control activation of postsynaptic metabotropic glutamate receptors and influence cerebellar long-term depression.

Neuronal and glial isoforms of glutamate transporters show distinct distributions on membranes surrounding excitatory synapses, but specific roles for transporter subtypes remain unidentified. At parallel fiber (PF) synapses in cerebellum, neuronal glutamate transporters and metabotropic glutamate receptors (mGluRs) have overlapping postsynaptic distributions suggesting that postsynaptic transporters selectively regulate mGluR activation. We examined interactions between transporters and mGluRs by evoking mGluR-mediated excitatory postsynaptic currents (mGluR EPSCs) in slices of rat cerebellum. Selective inhibition of postsynaptic transporters enhanced mGluR EPSCs greater than 3-fold. Moreover, impairing glutamate uptake facilitated mGluR-dependent long-term depression at PF synapses. Our results demonstrate that uniquely positioned glutamate transporters strongly influence mGluR activation at cerebellar PF synapses. Postsynaptic glutamate uptake may serve as a general mechanism for regulating mGluR-initiated synaptic depression.     

Rapamycin suppresses the recurrent excitatory circuits of dentate gyrus in a mouse model of temporal lobe epilepsy

Recurrent excitatory circuits and abnormal recurrent excitatory inputs are essential in epileptogenesis. Studies in temporal lobe epilepsy have shown that mossy fiber sprouting, which represents synaptic reorganization, renders the formation of abnormal recurrent excitatory circuits and inputs. The mammalian target of rapamycin (mTOR) pathway has recently been proved important in mossy fiber sprouting. In the present study, rapamycin, a mTOR inhibiter, was injected into the mouse of temporal lobe epilepsy. Electrophysiological and histological properties of the hippocampus were investigated by whole cell patch clamp, extracellular recording and Timm staining. Following the development of epilepsy, frequency of spontaneous excitatory postsynaptic currents (EPSCs) and amplitude of antidromically evoked EPSCs in granule cells were remarkably increased, as well as the epileptiform activity and mossy fiber sprouting were detected, which indicated the formation of abnormal recurrent excitatory circuits. By the use of rapamycin, frequency of spontaneous EPSCs, amplitude of antidromically evoked EPSCs, the epileptiform activity and mossy fiber sprouting were all remarkably suppressed. Our findings suggested an anti-epileptogenic role of rapamycin by suppressing the recurrent excitatory circuits of dentate gyrus.     

Long-term potentiation of neuronal glutamate transporters

Persistent, use-dependent modulation of synaptic strength has been demonstrated for fast synaptic transmission mediated by glutamate and has been hypothesized to underlie persistent behavioral changes ranging from memory to addiction. Glutamate released at synapses is sequestered by the action of excitatory amino acid transporters (EAATs) in glia and postsynaptic neurons. So, the efficacy of glutamate transporter function is crucial for regulating glutamate spillover to adjacent presynaptic and postsynaptic receptors and the consequent induction of plastic or excitotoxic processes. Here, we report that tetanic stimulation of cerebellar climbing fiber-Purkinje cell synapses results in long-term potentiation (LTP) of a climbing fiber-evoked glutamate transporter current recorded in Purkinje cells. This LTP is postsynaptically expressed and requires activation of an mGluR1/PKC cascade. Together with a simultaneously induced long-term depression (LTD) of postsynaptic AMPA receptors, this might reflect an integrated antiexcitotoxic cellular response to strong climbing fiber synaptic activation, as occurs following an ischemic episode.     

Physiological properties of afferents and synaptic reorganization in the rat cerebellum degranulated by postnatal x-irradiation

Elimination of most granule, basket, and stellate interneurons in the rat cerebellum was achieved by repeated doses of low level x-irradiation applied during the first two weeks of postnatal life. Electrical stimulation of the brain stem and peripheral limbs was employed to investigate the properties of afferent cerebellar pathways and the nature of the reorganized neuronal synaptic circuitry in the degranulated cerebellum of the adult. Direct contacts of mossy fibers on Purkinje cells were indicated by short latency, single spike responses: 1.9 msec from the lateral reticular nucleus of brain stem and 5.4 msec from ipsilateral forlimb. These were shorter than in normal rats by 0.9 and 2.1 msec, respectively. The topography of projections from peripheral stimulation was approximately normal. Mossy fiber responses followed stimulation at up to 20/sec, whereas climbing fiber pathways fatigued at 10/sec. The latency of climbing fiber input to peripheral limb stimulation in x-irradiated cerebellum was 23 ± 8 (SD) msec. In x-irradiated rats, the climbing fiber pathways evoked highly variable extracellular burst responses and intracellular EPSPs of different, discrete sizes. These variable responses suggest that multiple climbing fibers contact single Purkinje cells. We conclude that each type of afferent retains identifying characteristics of transmission. However, rules for synaptic specification appear to break down so that: (1) abnormal classes of neurons develop synaptic connections, i.e., mossy fibers to Purkinje cells; (2) incorrect numbers of neurons share postsynaptic targets, i.e., more than one climbing fiber to a Purkinje cell; and (3) inhibitory synaptic actions may be carried out in the absence of the major inhibitory interneurons, i.e., Purkinje cell collaterals may be effective in lieu of basket and stellate cells.     

Physiological properties of afferents and synaptic reorganization in the rat cerebellum degranulated by postnatal X-irradiation.

Elimination of most granule, basket, and stellate interneurons in the rat cerebellum was achieved by repeated doses of low level x-irradiation applied during the first two weeks of postnatal life. Electrical stimulation of the brain stem and peripheral limbs was employed to investigate the properties of afferent cerebellar pathways and the nature of the reorganized neuronal synaptic circuitry in the degranulated cerebellum of the adult. Direct contacts of mossy fibers on Purkinje cells were indicated by short latency, single spike responses: 1.9 msec from the lateral reticular nucleus of brain stem and 5.4 msec from ipsilpateral forelimb. These were shorter than in normal rats by 0.9 and 2.1 msec, respectively. The topography of projections from peripheral stimulation was approximately normal. Mossy fiber responses followed stimulation at up to 20/sec, whereas climbing fiber pathways fatigued at 10/sec. The latency of climbing fiber input to peripheral limb stimulation in x-irradiated cerebellum was 23 +/- 8 (SD) msec. In x-irradiated rats, the climbing fiber pathways evoked highly variable extracellular burst responses and intracellular EPSPs of different, discrete sizes. These variable responses suggest that multiple climbing fibers contact single Purkinje cells. We conclude that each type of afferent retains identifying characteristics of transmission. However, rules for synaptic specification appear to break down so that: (1) abnormal classes of neurons develop synaptic connections, i.e., mossy fibers to Purkinje cells; (2) incorrect numbers of neurons share postsynaptic targets, i.e., more than one climbing fiber to a Purkinje cell; and (3) inhibitory synaptic actions may be carried out in the absence of the major inhibitory interneurons, i.e., Purkinje cell collaterals may be effective in lieu of basket and stellate cells.     

Kainate receptors differentially regulate release at two parallel fiber synapses

Presynaptic kainate receptors (KARs) facilitate or depress transmitter release at several synapses in the CNS. Here, we report that synaptically activated KARs presynaptically facilitate and depress transmission at parallel fiber synapses in the cerebellar cortex. Low-frequency stimulation of parallel fibers facilitates synapses onto both stellate cells and Purkinje cells, whereas high-frequency stimulation depresses stellate cell synapses but continues to facilitate Purkinje cell synapses. These effects are mimicked by exogenous KAR agonists and eliminated by blocking KARs. This differential frequency-dependent sensitivity of these two synapses regulates the balance of excitatory and inhibitory input to Purkinje cells and therefore their excitability.     

δ-Glutamate Receptors Are Differentially Distributed at Parallel and Climbing Fiber Synapses on Purkinje Cells

Abstract: Neurons containing multiple excitatory inputs may sort and target glutamate receptor subtypes to subsets of synapses. A good model for testing this hypothesis is the Purkinje cell, which expresses significant levels of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate, kainate, N -methyl- d -aspartate, δ-, and metabotropic glutamate receptors. Purkinje cells receive two excitatory inputs, the parallel and climbing fibers; the combined effect of stimulation of these two inputs is to produce long-term depression of parallel fiber/Purkinje cell neurotransmission. Distribution of glutamate receptors in these two synapse populations in rat cerebella was studied using preembedding immunocytochemistry with antibodies to GluR1, GluR2/3, GluR5-7, NR1, δ1/2, and mGluR1α. Moderate/dense postsynaptic staining was most frequent in postsynaptic densities and spines of both parallel and climbing fiber synapses with mGluR1α antibody, was intermediate in frequency with GluR2/3 and GluR5-7 antibodies, and was least frequent with GluR1 and NR1 antibodies. The most striking finding was the absence of significant postsynaptic staining with δ1/2 antibody in climbing fiber synapses in adult animals, even though postsynaptic staining was prevalent in parallel fiber synapses with this antibody. In contrast to adults, moderate/dense postsynaptic immunolabeling of climbing fiber synapses with δ1/2 antibody was common in rats at 10 days postnatal. This study provides direct morphological evidence that δ-glutamate receptors are differentially targeted to synapse populations. Our results support previous suggestions that δ2 is involved in development of parallel and climbing fiber synapses and in long-term depression of parallel fiber/Purkinje synaptic responses in adults.     

Intrasynaptic ephaptic feedback in central synapses]

A review is given of experiments performed in the author's laboratory on slices from the rat visual cortex and hippocampus. The aim was to test the existence of the positive feedback in central synapses according to a mechanism of electrical (ephatic) linking proposed by A. L. Byzow. The hypothesis predicts that, in a subset of central synapses, artificial postsynaptic membrane potential (MP) hyperpolarization should increase the amplitude of the excitatory postsynaptic current (EPSC) and potential (EPSP) not only due to a deviation from the equilibrium potential but also due to increased presynaptic transmitter release. In a part of the experiments, we found changes in several traditional parameters of transmitter release during hyperpolarization: number of response failures, coefficient of variation of response amplitude and quantal content of minimal EPSC/EPSP. The effects were especially prominent for the giant mossy fibre-CA3 synapses. For them, "supralinear" amplitude-voltage relations at hyperpolarized membrane potentials and voltage--dependent paired--pulse facilitation ratios were found. All these "non-classical" effects disappeared when composite, rather than minimal, EPSCs were evoked. These data were consistent with simulation experiments performed on the Byzov's synaptic model with the ephaptic feedback and therefore they strengthen the hypothesis. Independent of their interpretation, the data reveal a novel feedback mechanism. The mechanism provides a possibility for the central postsynaptic neurone to control the efficacy of a subset of synapses via postsynaptic MP modifications. The mechanism can essentially increase the efficacy of large ("perforated") synapses. It explains the significance of the increased number of such synapses following experimental challenges such as leading to induction of the long-term potentiation or to behavioural conditioning.     

Formation of new synaptic contacts by Purkinje axon collaterals in the granular layer of deafferented cerebellar cortex of adult rat

In addition to (i) mossy terminals, (ii) Golgi axons, (iii) granule cell dendrites and (iv), occasionally, Golgi cell dendrites, a third axonal profile identified by morphological criteria as the collateral of Purkinje axons, has been found in 2% of all cerebellar glomeruli. These infrequent components of a few glomeruli, however, were never seen in normal cerebellar cortex to establish specialized synaptic contact with glomerular dendrites. Two to four weeks after surgical isolation of the cerebellar cortex, i.e. following the destruction of both efferent and afferent fibres, the number of glomeruli containing (hypertrophic) axonal branches of Purkinje cells has increased to 13% of all surveyed glomeruli. In addition, the Purkinje axon terminals in the mossy fibre-deprived glomeruli were observed to establish numerous Gray II-type synaptic contacts with surrounding granule cell dendrites. It is suggested that the development of heterologous synapses between hypertrophic, or even intact, Purkinje axon collaterals on the one hand and the mossy fibre-vacated granule cell dendrites on the other, is a compensatory, reactive process to the synaptic "desaturation" of granule neurons, which demonstrate a dormant potential of Purkinje cells to form new synaptic contacts in the adult cerebellum.     

Quantal release of glutamate generates pure kainate and mixed AMPA/kainate EPSCs in hippocampal neurons

The relative contribution of kainate receptors to ongoing glutamatergic activity is at present unknown. We report the presence of spontaneous, miniature, and minimal stimulation-evoked excitatory postsynaptic currents (EPSCs) that are mediated solely by kainate receptors (EPSC(kainate)) or by both AMPA and kainate receptors (EPSC(AMPA/kainate)). EPSC(kainate) and EPSC(AMPA/kainate) are selectively enriched in CA1 interneurons and mossy fibers synapses of CA3 pyramidal neurons, respectively. In CA1 interneurons, the decay time constant of EPSC(kainate) (circa 10 ms) is comparable to values obtained in heterologous expression systems. In both hippocampal neurons, the quantal release of glutamate generates kainate receptor-mediated EPSCs that provide as much as half of the total glutamatergic current. Kainate receptors are, therefore, key players of the ongoing glutamatergic transmission in the hippocampus.     

alphaCaMKII Is essential for cerebellar LTD and motor learning

Activation of postsynaptic alpha-calcium/calmodulin-dependent protein kinase II (alphaCaMKII) by calcium influx is a prerequisite for the induction of long-term potentiation (LTP) at most excitatory synapses in the hippocampus and cortex. Here we show that postsynaptic LTP is unaffected at parallel fiber-Purkinje cell synapses in the cerebellum of alphaCaMKII(-/-) mice. In contrast, a long-term depression (LTD) protocol resulted in only transient depression in juvenile alphaCaMKII(-/-) mutants and in robust potentiation in adult mutants. This suggests that the function of alphaCaMKII in parallel fiber-Purkinje cell plasticity is opposite to its function at excitatory hippocampal and cortical synapses. Furthermore, alphaCaMKII(-/-) mice showed impaired gain-increase adaptation of both the vestibular ocular reflex and optokinetic reflex. Since Purkinje cells are the only cells in the cerebellum that express alphaCaMKII, our data suggest that an impairment of parallel fiber LTD, while leaving LTP intact, is sufficient to disrupt this form of cerebellar learning.     

Interrelated modification of excitatory and inhibitory synaptic connections in the olivary-cerebellar neuronal network

The model of simultaneous interrelated modification in the efficacy of synaptic inputs to different neurons of the olivary-cerebellar network is developed. The model is based on the following features of the network: simultaneous activation of the input layer (granule) cells and the output layer (deep cerebellar nuclei) cells by mossy fibers; simultaneous activation of Purkinje cells and cerebellar cells of the input and output layers by climbing fibers and their collaterals; the existence of local feedback excitatory, inhibitory, and disinhibitory circuits. The rise (decrease) of posttetanic Ca2+ concentration in reference to the level produced by previous stimulation causes the decrease (increase) in cGMP-dependent protein kinase G activity, and increase (decrease) inprotein phosphatase 1 activity. Subsequent dephosphorylation (phosphorylation) of ionotropic receptors results in simultaneous LTD (LTP) of the excitatory input together with the LTP (LTD) of the inhibitory input to the same neuron. The character of interrelated modifications of synapses at different cerebellar levels strongly depends on the olivary cell activity. In the presence (absence) of the signal from the inferior olive LTD (LTP) of the output cerebellar signal can be induced.     

[3H]GABA binding in the cerebellum of the reeler murine mutant

In the cerebellum of the reeler mutant mouse, characterized morphologically by depletion of the granule cell population and abnormal synapse formation, increased GABA concentration and alterations in [³H]GABA binding have been observed. This study shows decreased affinity of the Na⁺-independent, high affinity GABA binding component of synaptosomal membranes and an increased affinity of the Na⁺-dependent, high affinity GABA binding component in reeler cerebellar homogenate and synaptic membranes. In contrast to the changes in affinity, the number of both Na⁺-dependent and Na⁺-independent binding sites was not significantly altered. The decreased affinity of the Na⁺-independent GABA binding and the increased affinity of the Na⁺-dependent binding, evidenced only in cerebellar tissue, were interpreted to indicate, respectively, hypo- and hypersensitivity of the postsynaptic and presynaptic elements of cerebellar GABAergic synapses, induced by the depressed excitatory granule cell input and/or the increased mossy fiber contact with the ectopic Purkinje cells.     

Synaptic destabilization by neuronal Nogo-A

Formation and maintenance of a neuronal network is based on a balance between plasticity and stability of synaptic connections. Several molecules have been found to regulate the maintenance of excitatory synapses but nothing is known about the molecular mechanisms involved in synaptic stabilization versus disassembly at inhibitory synapses. Here, we demonstrate that Nogo-A, which is well known to be present in myelin and inhibit growth in the adult CNS, is present in inhibitory presynaptic terminals in cerebellar Purkinje cells at the time of Purkinje cell-Deep Cerebellar Nuclei (DCN) inhibitory synapse formation and is then downregulated during synapse maturation. We addressed the role of neuronal Nogo-A in synapse maturation by generating several mouse lines overexpressing Nogo-A, starting at postnatal ages and throughout adult life, specifically in cerebellar Purkinje cells and their terminals. The overexpression of Nogo-A induced a progressive disassembly, retraction and loss of the inhibitory Purkinje cell terminals. This led to deficits in motor learning and coordination in the transgenic mice. Prior to synapse disassembly, the overexpression of neuronal Nogo-A led to the downregulation of the synaptic scaffold proteins spectrin, spectrin-E and β-catenin in the postsynaptic neurons. Our data suggest that neuronal Nogo-A might play a role in the maintenance of inhibitory synapses by modulating the expression of synaptic anchoring molecules. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

mGluR2 postsynaptically senses granule cell inputs at Golgi cell synapses

In the cerebellar circuit, Golgi cells are thought to contribute to information processing and integration via feedback mechanisms. In these mechanisms, dynamic modulation of Golgi cell excitability is necessary because GABA from Golgi cells causes tonic inhibition on granule cells. We studied the role and synaptic mechanisms of postsynaptic metabotropic glutamate receptor subtype 2 (mGluR2) at granule cell-Golgi cell synapses, using whole-cell recording of green fluorescent protein-positive Golgi cells of wild-type and mGluR2-deficient mice. Postsynaptic mGluR2 was activated by glutamate from granule cells and hyperpolarized Golgi cells via G protein-coupled inwardly rectifying K+ channels (GIRKs). This hyperpolarization conferred long-lasting silencing of Golgi cells, the duration and extents of which were dependent on stimulus strengths. Postsynaptic mGluR2 thus senses inputs from granule cells and is most likely important for spatiotemporal modulation of mossy fiber-granule cell transmission before distributing inputs to Purkinje cells.     

Retrograde inhibition of presynaptic calcium influx by endogenous cannabinoids at excitatory synapses onto Purkinje cells

Brief depolarization of cerebellar Purkinje cells was found to inhibit parallel fiber and climbing fiber EPSCs for tens of seconds. This depolarization-induced suppression of excitation (DSE) is accompanied by altered paired-pulse plasticity, suggesting a presynaptic locus. Fluorometric imaging revealed that postsynaptic depolarization also reduces presynaptic calcium influx. The inhibition of both presynaptic calcium influx and EPSCs is eliminated by buffering postsynaptic calcium with BAPTA. The cannabinoid CB1 receptor antagonist AM251 prevents DSE, and the agonist WIN 55,212-2 occludes DSE. These findings suggest that Purkinje cells release endogenous cannabinoids in response to elevated calcium, thereby inhibiting presynaptic calcium entry and suppressing transmitter release. DSE may provide a way for cells to use their firing rate to dynamically regulate synaptic inputs. Together with previous studies, these findings suggest a widespread role for endogenous cannabinoids in retrograde synaptic inhibition.