Parallel Fiber Stimulation Research Articles

Neuronal and Astrocytic Monoacylglycerol Lipase Limit the Spread of Endocannabinoid Signaling in the Cerebellum.

Endocannabinoids are diffusible lipophilic molecules that may spread to neighboring synapses. Monoacylglycerol lipase (MAGL) is the principal enzyme that degrades the endocannabinoid 2-arachidonoylglycerol (2-AG). Using knock-out mice in which MAGL is deleted globally or selectively in neurons and astrocytes, we investigated the extent to which neuronal and astrocytic MAGL limit the spread of 2-AG-mediated retrograde synaptic depression in cerebellar slices. A brief tetanic stimulation of parallel fibers in the molecular layer induced synaptically evoked suppression of excitation (SSE) in Purkinje cells, and both neuronal and astrocytic MAGL contribute to the termination of this form of endocannabinoid-mediated synaptic depression. The spread of SSE among Purkinje cells occurred only after global knock-out of MAGL or pharmacological blockade of either MAGL or glutamate uptake, but no spread was detected following neuron- or astrocyte-specific deletion of MAGL. The spread of endocannabinoid signaling was also influenced by the spatial pattern of synaptic stimulation, because it did not occur at spatially dispersed parallel fiber synapses induced by stimulating the granular layer. The tetanic stimulation of parallel fibers did not induce endocannabinoid-mediated synaptic suppression in Golgi cells even after disruption of MAGL and glutamate uptake, suggesting that heightened release of 2-AG by Purkinje cells does not spread the retrograde signal to parallel fibers that innervate Golgi cells. These results suggest that both neuronal and astrocytic MAGL limit the spatial diffusion of 2-AG and confer synapse-specificity of endocannabinoid signaling.

Counting Vesicular Release Events Reveals Binomial Release Statistics at Single Glutamatergic Synapses.

It is generally accepted that the output signal of individual central synapses saturates at high release probability, but it remains unclear whether the source of saturation is presynaptic, postsynaptic, or both presynaptic and postsynaptic. To clarify this and other issues concerning the function of synapses, we have developed new recording and analysis methods at single central glutamatergic synapses. We find that individual release events engage a high proportion of postsynaptic receptors (62%), revealing a larger component of postsynaptic saturation than anticipated. Conversely, we also find that the number of released synaptic vesicles is limited at each active zone. Altogether, our results argue for both presynaptic and postsynaptic contributions to signal saturation at single glutamatergic synapses.

Burst-Dependent Bidirectional Plasticity in the Cerebellum Is Driven by Presynaptic NMDA Receptors.

Numerous studies have shown that cerebellar function is related to the plasticity at the synapses between parallel fibers and Purkinje cells. How specific input patterns determine plasticity outcomes, as well as the biophysics underlying plasticity of these synapses, remain unclear. Here, we characterize the patterns of activity that lead to postsynaptically expressed LTP using both in vivo and in vitro experiments. Similar to the requirements of LTD, we find that high-frequency bursts are necessary to trigger LTP and that this burst-dependent plasticity depends on presynaptic NMDA receptors and nitric oxide (NO) signaling. We provide direct evidence for calcium entry through presynaptic NMDA receptors in a subpopulation of parallel fiber varicosities. Finally, we develop and experimentally verify a mechanistic plasticity model based on NO and calcium signaling. The model reproduces plasticity outcomes from data and predicts the effect of arbitrary patterns of synaptic inputs on Purkinje cells, thereby providing a unified description of plasticity.

Visualization of Ca2+Filling Mechanisms upon Synaptic Inputs in the Endoplasmic Reticulum of Cerebellar Purkinje Cells

The endoplasmic reticulum (ER) plays crucial roles in intracellular Ca(2+) signaling, serving as both a source and sink of Ca(2+), and regulating a variety of physiological and pathophysiological events in neurons in the brain. However, spatiotemporal Ca(2+) dynamics within the ER in central neurons remain to be characterized. In this study, we visualized synaptic activity-dependent ER Ca(2+) dynamics in mouse cerebellar Purkinje cells (PCs) using an ER-targeted genetically encoded Ca(2+) indicator, G-CEPIA1er. We used brief parallel fiber stimulation to induce a local decrease in the ER luminal Ca(2+) concentration ([Ca(2+)]ER) in dendrites and spines. In this experimental system, the recovery of [Ca(2+)]ER takes several seconds, and recovery half-time depends on the extent of ER Ca(2+) depletion. By combining imaging analysis and numerical simulation, we show that the intraluminal diffusion of Ca(2+), rather than Ca(2+) reuptake, is the dominant mechanism for the replenishment of the local [Ca(2+)]ER depletion immediately following the stimulation. In spines, the ER filled almost simultaneously with parent dendrites, suggesting that the ER within the spine neck does not represent a significant barrier to Ca(2+) diffusion. Furthermore, we found that repetitive climbing fiber stimulation, which induces cytosolic Ca(2+) spikes in PCs, cumulatively increased [Ca(2+)]ER. These results indicate that the neuronal ER functions both as an intracellular tunnel to redistribute stored Ca(2+) within the neurons, and as a leaky integrator of Ca(2+) spike-inducing synaptic inputs.

The 5-HT7 receptor triggers cerebellar long-term synaptic depression via PKC-MAPK

The 5-HT7 receptor (5-HT7R) mediates important physiological effects of serotonin, such as memory and emotion, and is emerging as a therapeutic target for the treatment of cognitive disorders and depression. Although previous studies have revealed an expression of 5-HT7R in cerebellum, particularly at Purkinje cells, its functional role and signaling mechanisms have never been described. Using patch-clamp recordings in cerebellar slices of adult mice, we investigated the effects of a selective 5-HT7R agonist, LP-211, on the main plastic site of the cerebellar cortex, the parallel fiber-Purkinje cell synapse. Here we show that 5-HT7R activation induces long-term depression of parallel fiber-Purkinje cell synapse via a postsynaptic mechanism that involves the PKC-MAPK signaling pathway. Moreover, a 5-HT7R antagonist abolished the expression of PF-LTD, produced by pairing parallel fiber stimulation with Purkinje cell depolarization; whereas, application of a 5-HT7R agonist impaired LTP induced by 1 Hz parallel fiber stimulation. Our results indicate for the first time that 5-HT7R exerts a fine regulation of cerebellar bidirectional synaptic plasticity that might be involved in cognitive processes and neuropsychiatric disorders involving the cerebellum.

An excitatory GABA loop operating in vivo.

While it has been proposed that the conventional inhibitory neurotransmitter GABA can be excitatory in the mammalian brain, much remains to be learned concerning the circumstances and the cellular mechanisms governing potential excitatory GABA action. Using a combination of optogenetics and two-photon calcium imaging in vivo, we find that activation of chloride-permeable GABAA receptors in parallel fibers (PFs) of the cerebellar molecular layer of adult mice causes parallel fiber excitation. Stimulation of PFs at submaximal stimulus intensities leads to GABA release from molecular layer interneurons (MLIs), thus creating a positive feedback loop that enhances excitation near the center of an activated PF bundle. Our results imply that elevated chloride concentration can occur in specific intracellular compartments of mature mammalian neurons and suggest an excitatory role for GABAA receptors in the cerebellar cortex of adult mice.

Parallel fiber to Purkinje cell synaptic impairment in a mouse model of spinocerebellar ataxia type 27

Genetically inherited mutations in the fibroblast growth factor 14 (FGF14) gene lead to spinocerebellar ataxia type 27 (SCA27), an autosomal dominant disorder characterized by heterogeneous motor and cognitive impairments. Consistently, genetic deletion of Fgf14 in Fgf14−/− mice recapitulates salient features of the SCA27 human disease. In vitro molecular studies in cultured neurons indicate that the FGF14F145S SCA27 allele acts as a dominant negative mutant suppressing the FGF14 wild type function and resulting in inhibition of voltage-gated Na+ and Ca2+ channels. To gain insights in the cerebellar deficits in the animal model of the human disease, we applied whole-cell voltage-clamp in the acute cerebellar slice preparation to examine the properties of parallel fibers (PF) to Purkinje neuron synapses in Fgf14−/− mice and wild type littermates. We found that the AMPA receptor-mediated excitatory postsynaptic currents evoked by PF stimulation (PF-EPSCs) were significantly reduced in Fgf14−/− animals, while short-term plasticity, measured as paired-pulse facilitation (PPF), was enhanced. Measuring Sr2+-induced release of quanta from stimulated synapses, we found that the size of the PF-EPSCs was unchanged, ruling out a postsynaptic deficit. This phenotype was corroborated by decreased expression of VGLUT1, a specific presynaptic marker at PF-Purkinje neuron synapses. We next examined the mGluR1 receptor-induced response (mGluR1-EPSC) that under normal conditions requires a gradual build-up of glutamate concentration in the synaptic cleft, and found no changes in these responses in Fgf14−/− mice. These results provide evidence of a critical role of FGF14 in maintaining presynaptic function at PF-Purkinje neuron synapses highlighting critical target mechanisms to recapitulate the complexity of the SCA27 disease.

The Spontaneous Ataxic Mouse Mutant Tippy is Characterized by a Novel Purkinje Cell Morphogenesis and Degeneration Phenotype.

This study represents the first detailed analysis of the spontaneous neurological mouse mutant, tippy, uncovering its unique cerebellar phenotype. Homozygous tippy mutant mice are small, ataxic, and die around weaning. Although the cerebellum shows grossly normal foliation, tippy mutants display a complex cerebellar Purkinje cell phenotype consisting of abnormal dendritic branching with immature spine features and patchy, non-apoptotic cell death that is associated with widespread dystrophy and degeneration of the Purkinje cell axons throughout the white matter, the cerebellar nuclei, and the vestibular nuclei. Moderate anatomical abnormalities of climbing fiber innervation of tippy mutant Purkinje cells were not associated with changes in climbing fiber-EPSC amplitudes. However, decreased ESPC amplitudes were observed in response to parallel fiber stimulation and correlated well with anatomical evidence for patchy dark cell degeneration of Purkinje cell dendrites in the molecular layer. The data suggest that the Purkinje neurons are a primary target of the tippy mutation. Furthermore, we hypothesize that the Purkinje cell axonal pathology together with disruptions in the balance of climbing fiber and parallel fiber-Purkinje cell input in the cerebellar cortex underlie the ataxic phenotype in these mice. The constellation of Purkinje cell dendritic malformation and degeneration phenotypes in tippy mutants is unique and has not been reported in any other neurologic mutant. Fine mapping of the tippy mutation to a 2.1 MB region of distal chromosome 9, which does not encompass any gene previously implicated in cerebellar development or neuronal degeneration, confirms that the tippy mutation identifies novel biology and gene function.

Noradrenergic modulation of the parallel fiber-Purkinje cell synapse in mouse cerebellum

The signals arriving to Purkinje cells via parallel fibers are essential for all tasks in which the cerebellum is involved, including motor control, learning new motor skills and calibration of reflexes. Since learning also requires the activation of adrenergic receptors, we investigated the effects of adrenergic receptor agonists on the main plastic site of the cerebellar cortex, the parallel fiber-Purkinje cell synapse. Here we show that noradrenaline serves as an endogenous ligand for both α1-and α2-adrenergic receptors to produce synaptic depression between parallel fibers and Purkinje cells. On the contrary, PF-EPSCs were potentiated by the β-adrenergic receptor agonist isoproterenol. This short-term potentiation was postsynaptically expressed, required protein kinase A, and was mimicked by the β2-adrenoceptor agonist clenbuterol, suggesting that the β2-adrenoceptors mediate the noradrenergic facilitation of synaptic transmission between parallel fibers and Purkinje cells. Moreover, β-adrenoceptor activation lowered the threshold for cerebellar long-term potentiation induced by 1 Hz parallel fiber stimulation. The presence of both α and β adrenergic receptors on Purkinje cells suggests the existence of bidirectional mechanisms of regulation allowing the noradrenergic afferents to refine the signals arriving to Purkinje cells at particular arousal states or during learning.

Stimulus Discrimination in Cerebellar Purkinje Neurons

Cerebellar Purkinje neurons fire spontaneously in the absence of synaptic input. Overlaid on this intrinsic activity, excitatory input from parallel fibres can add simple spikes to the output train, whereas inhibitory input from interneurons can introduce pauses. These and other influences lead to an irregular spike train output in Purkinje neurons in vitro and in vivo, supplying a variable inhibitory drive to deep cerebellar nuclear neurons. From a computational perspective, this variability raises some questions, as individual spikes induced by excitatory inputs are indistinguishable from intrinsic firing activity. Although bursts of high-frequency excitatory input could be discriminated unambiguously from background activity, granule neurons are known to fire in vivo over a wide range of frequencies. This would mean that much of the sensory information relayed through the cerebellar cortex would be lost within the random variation in background activity. We speculated that alternative mechanisms for signal discrimination may exist, and sought to identify characteristic motifs within the sequence of spikes that followed stimulation events. We found that under certain conditions, parallel fibre stimulation could reliably add a “couplet” of spikes with an unusually short interspike interval to the output train. Therefore, despite representing a small fraction of the total number of spikes, these signals can be reliably discriminated from background firing on a moment-to-moment basis, and could result in a differential downstream response.

Cerebellar Interneurons use Dendritic Voltage and Calcium Signals to Differentially Extract Information from Synaptic Activity

Non-linear summation of synaptic inputs within a dendritic branch endows neurons with multiple computational subunits, favoring different types of pattern detection. To date, most neuronal types have been described to display a supralinear summation of synaptic inputs, due to the activation of NMDAR or voltage-gated calcium channels, with a concomitant supralinearity of local Ca2+ signaling. Recently, evidence showed that in dendrites of cerebellar stellate cells, synaptic summation is sublinear, likely resulting from a reduced driving-force for synaptic currents caused by large local depolarizations. We expect these large synaptic depolarizations to cause a smaller fractional change in the driving force for Ca2+, therefore a more linear summation of dendritic Ca2+.In order to characterize the local Ca2+ and voltage responses to synaptic stimulation in cerebellar stellate cells, we combined two-photon targeted stimulation of parallel fibers, glutamate uncaging, and fast two-photon imaging of dendritic Ca2+ and voltage. Using fast line-scan imaging and the two-component voltage sensor DiO/DPA , we observed that, in accordance with numerical simulations, the local depolarization in the dendrites of stellate cells in response to the activation of a few synapses is rapid (<2ms), of large amplitude (up to 50mV) and distance-dependent, and is widely spread in the dendrite (several tens of µms). The measurement of Ca2+ transients in stellate cells dendrites showed that, in contrast, Ca2+ transients are more localized (<10µm), summed linearly in response to paired stimulation of parallel fibers, and supra-linearly in response to synaptic activation by high-frequency trains.Therefore Ca2+ and voltage in dendrites can obey different computational rules, the sublinear summation of voltage contributing to sparse-input detection and shaping the activation of Purkinje cells by parallel fibers, while supralinear Ca2+ is likely to contribute to regulation of synaptic plasticity.

Parallel fiber and climbing fiber responses in rat cerebellar cortical neurons in vivo

Over the last few years we have seen a rapidly increasing interest in the functions of the inhibitory interneurons of the cerebellar cortex. However, we still have very limited knowledge about their physiological properties in vivo. The present study provides the first description of their spontaneous firing properties and their responses to synaptic inputs under non-anesthetized conditions in the decerebrated rat in vivo. We describe the spike responses of molecular layer interneurons (MLI) in the hemispheric crus1/crus2 region and compare them with those of Purkinje cells (PCs) and Golgi cells (GCs), both with respect to spontaneous activity and responses evoked by direct electrical stimulation of parallel fibers (PFs) and climbing fibers (CFs). In agreement with previous findings in the cat, we found that the CF responses in the interneurons consisted of relatively long lasting excitatory modulations of the spike firing. In contrast, activation of PFs induced rapid but short-lasting excitatory spike responses in all types of neurons. We also explored PF input plasticity in the short-term (10 min) using combinations of PF and CF stimulation. With regard to in vivo recordings from cerebellar cortical neurons in the rat, the data presented here provide the first demonstration that PF input to PC can be potentiated using PF burst stimulation and they suggest that PF burst stimulation combined with CF input may lead to potentiation of PF inputs in MLIs. We conclude that the basic responsive properties of the cerebellar cortical neurons in the rat in vivo are similar to those observed in the cat and also that it is likely that similar mechanisms of PF input plasticity apply.

Ethanol affects NMDA receptor signaling at climbing fiber-Purkinje cell synapses in mice and impairs cerebellar LTD

Ethanol profoundly influences cerebellar circuit function and motor control. It has recently been demonstrated that functional N-methyl-(D)-aspartate (NMDA) receptors are postsynaptically expressed at climbing fiber (CF) to Purkinje cell synapses in the adult cerebellum. Using whole cell patch-clamp recordings from mouse cerebellar slices, we examined whether ethanol can affect NMDA receptor signaling in mature Purkinje cells. NMDA receptor-mediated currents were isolated by bath application of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzol[f]quinoxaline (NBQX). The remaining (D)-2-amino-5-phosphonovaleric acid ((D)-APV)-sensitive current was reduced by ethanol at concentrations as low as 10 mM. At a concentration of 50 mM ethanol, the blockade of (D)-APV-sensitive CF-excitatory postsynaptic currents was significantly stronger. Ethanol also altered the waveform of CF-evoked complex spikes by reducing the afterdepolarization. This effect was not seen when NMDA receptors were blocked by (D)-APV before ethanol wash-in. In contrast to CF synaptic transmission, parallel fiber (PF) synaptic inputs were not affected by ethanol. Finally, ethanol (10 mM) impaired long-term depression (LTD) at PF to Purkinje cell synapses as induced under control conditions by paired PF and CF activity. However, LTD induced by pairing PF stimulation with depolarizing voltage steps (substituting for CF activation) was not blocked by ethanol. These observations suggest that the sensitivity of cerebellar circuit function and plasticity to low concentrations of ethanol may be caused by an ethanol-mediated impairment of NMDA receptor signaling at CF synapses onto cerebellar Purkinje cells.

SK2 Channel Modulation Contributes to Compartment-Specific Dendritic Plasticity in Cerebellar Purkinje Cells

Small-conductance Ca(2+)-activated K(+) channels (SK channels) modulate excitability and curtail excitatory postsynaptic potentials (EPSPs) in neuronal dendrites. Here, we demonstrate long-lasting plasticity of intrinsic excitability (IE) in dendrites that results from changes in the gain of this regulatory mechanism. Using dendritic patch-clamp recordings from rat cerebellar Purkinje cells, we find that somatic depolarization or parallel fiber (PF) burst stimulation induce long-term amplification of synaptic responses to climbing fiber (CF) or PF stimulation and enhance the amplitude of passively propagated sodium spikes. Dendritic plasticity is mimicked and occluded by the SK channel blocker apamin and is absent in Purkinje cells from SK2 null mice. Triple-patch recordings from two dendritic sites and the soma and confocal calcium imaging studies show that local stimulation limits dendritic plasticity to the activated compartment of the dendrite. This plasticity mechanism allows Purkinje cells to adjust the SK2-mediated control of dendritic excitability in an activity-dependent manner.

Cav3-KCa3.1 complex enhances detection of facilitating parallel fiber inputs in cerebellar Purkinje cells

The ability for a neuron to distinguish important input signals from background noise is essential for proper circuit function. Several mechanisms exist to enhance signal detection. For example, short-term facilitation enhances burst transmission, while information from single spikes is transmitted more effectively through synapses that exhibit depression [1]. However, in the case of cerebellar Purkinje cells, where thousands of parallel fiber (PF) synaptic inputs impinge on the dendritic tree, postsynaptic mechanisms must play a role in increasing signal-to-noise ratio. Specifically, mechanisms must exist in Purkinje cells that allow transmission of granule cell bursts carrying sensory information while filtering out background input. Recently, we demonstrated that low voltage activated Cav3 calcium channels form a complex with intermediate conductance calcium-activated potassium channels (KCa3.1) in Purkinje cells [2]. We proposed that the Cav3-KCa3.1 complex enhances signal detection capabilities by selectively suppressing non-facilitating background inputs while allowing facilitating parallel fiber inputs to generate Purkinje cell spike output. We used a combination of modeling and experimental methods to examine the role of the Cav3-KCa3.1 complex in simultaneously enhancing detection of parallel fiber bursts and suppressing background inputs. The Cav3-KCa3.1 complex was simulated using a calcium-diffusion model with 10 hemispherical compartments around the Cav3 channel and the KCa3.1 channel placed within 50 nm of the calcium source. The Cav3-KCa3.1 complex was included in a single-compartment membrane model with HCN and synaptic conductances to determine its effect on excitatory postsynaptic potential (EPSP) summation. Whole-cell current-clamp recordings of Purkinje cells were also made from acute slices of rat cerebellum (P18-P25). Parallel fibers were stimulated using a monopolar stimulating electrode in the cerebellar molecular layer. We simulated 5 pulse 50 Hz trains of facilitating and non-facilitating EPSPs in the model. The Cav3-KCa3.1 complex reduced the summation of both sets of inputs. However, the facilitating inputs achieved a higher degree of summation than non-facilitating inputs, even though charge transfer for both trains was identical. Examination of the KCa3.1 current showed that synaptic facilitation compensated for the increase in KCa3.1 activation during the input train, allowing EPSPs to summate to higher voltages. When the Cav3-KCa3.1 complex was not included in the model, facilitating and non-facilitating inputs reached similar degrees of summation. Our computational results were confirmed by our current clamp recordings. Facilitating inputs evoked by direct PF stimulation generated more depolarization and spikes in Purkinje cells than non-facilitating simulated EPSPs (simEPSPs). During trains of simEPSPs, a supralinear increase in the rate of EPSP decay was observed for consecutive inputs, indicating an increased recruitment of hyperpolarizing current during the train. Application of Ni2+ (100 μM) to block Cav3 current removed this supralinear increase in rate of decay, confirming that the Cav3-KCa3.1 complex dynamically changes temporal summation during repetitive input trains. Our results demonstrate that the Cav3-KCa3.1 complex significantly affects the temporal summation of synaptic inputs. Specifically, the Cav3-KCa3.1 complex allows facilitating inputs from single PF sources to summate and generate spike output in Purkinje cells, while background inputs are quickly suppressed, improving the ability to respond to important sensory information.

A new signalling pathway for parallel fibre presynaptic type 4 metabotropic glutamate receptors (mGluR4) in the rat cerebellar cortex

In the rodent cerebellum, pharmacological activation of mGluR4 acutely depresses excitatory synaptic transmission at parallel fibre–Purkinje cell synapses. This depression involves the inhibition of presynaptic calcium (Ca2+) influx that ultimately controls glutamate release. In this study, we investigate the molecular basis of mGluR4-mediated inhibition of presynaptic Ca2+ transients. Our results demonstrate that the mGluR4 effect does not depend on selective inhibition of a specific type of presynaptic voltage-gated Ca2+ channel, but rather involves modulation of all classes of Ca2+ channels present in the presynaptic terminals. In addition, this inhibitory effect does not involve the activation of G protein-activated inwardly rectifying potassium channels, TEA-sensitive potassium channels or two-pore-domain potassium channels. Furthermore, this inhibition does not require pertussis toxin-sensitive G proteins, and is independent of any effect on adenylyl cyclases, protein kinase A, mitogen-activated protein kinases or phosphoinositol-3 kinase activity. Interestingly we found that mGluR4 inhibition of presynaptic Ca2+ influx employs a newly defined signalling pathway, notably that involving the activation of phospholipase C and ultimately protein kinase C.

Lack of Kinase Regulation of Canonical Transient Receptor Potential 3 (TRPC3) Channel-dependent Currents in Cerebellar Purkinje Cells

Canonical transient receptor potential (TRPC) channels are widely expressed in the brain and play several roles in development and normal neuronal function. In the cerebellum, Purkinje cell TRPC3 channels underlie the slow excitatory postsynaptic potential observed after parallel fiber stimulation. In these cells TRPC3 channel opening requires stimulation of metabotropic glutamate receptor 1, activation of which can also lead to the induction of long term depression (LTD), which underlies cerebellar motor learning. LTD induction requires protein kinase C (PKC) and protein kinase G (PKG) activation, and although PKC phosphorylation targets are well established, virtually nothing is known about PKG targets in LTD. Because TRPC3 channels are inhibited after phosphorylation by PKC and PKG in expression systems, we examined whether native TRPC3 channels in Purkinje cells are a target for PKG or PKC, thereby contributing to cerebellar LTD. We find that in Purkinje cells, activation of TRPC3-dependent currents is not inhibited by conventional PKC or PKG to any significant extent and that inhibition of these kinases does not significantly impact on TRPC3-mediated currents either. Based on these and previous findings, we propose that TRPC3-dependent currents may differ significantly in their regulation from those overexpressed in expression systems.

Cerebellar long‐term depression requires dephosphorylation of TARP in Purkinje cells

Cerebellar long-term depression (LTD) at parallel fiber (PF)-Purkinje cell synapses is thought to play an essential role in certain forms of motor learning. Like hippocampal LTD, cerebellar LTD is mediated by the endocytosis of AMPA (α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate) receptors at postsynaptic sites. However, similar sets of kinases and phosphatases have opposite regulatory effects on hippocampal and cerebellar LTD, although the mechanisms responsible for this difference remain largely unclear. Activity-dependent dephosphorylation of stargazin (an AMPA receptor auxiliary protein) by calcineurin regulates hippocampal LTD, but whether and how stargazin is involved in cerebellar LTD is unknown. In this study, we showed that stargazin is highly phosphorylated at basal states and is dephosphorylated by the application of high KCl plus glutamate (K-glu) or of a metabotropic glutamate receptor agonist, (S)-3,5-dihydroxyphenylglycine (DHPG), both of which chemically induced LTD in cerebellar slices. This chemically induced dephosphorylation of stargazin was specifically blocked by a calcineurin inhibitor. Indeed, inclusion of the calcineurin auto-inhibitory peptide in the patch pipette solution completely inhibited the LTD induced by the conjunctive stimulation of PFs and Purkinje cells. Furthermore, in Purkinje cells expressing stargazin-9D, in which all nine serine residues are mutated to aspartate, neither conjunctive stimulus nor DHPG treatment induced LTD. Finally, immunohistochemical analyses revealed that neither K-glu nor DHPG induced the endocytosis of AMPA receptors in Purkinje cells expressing stargazin-9D. Together, these results indicate that hippocampal and cerebellar LTD share a common pathway, namely dephosphorylation of stargazin by calcineurin.

Transient expression of functional serotonin 5‐HT3 receptors by glutamatergic granule cells in the early postnatal mouse cerebellum

The serotonin 5-HT(3) receptor is the only ligand-gated ion channel activated by serotonin and is expressed by GABAergic interneurons in many brain regions, including the cortex, amygdala and hippocampus. Furthermore, 5-HT(3) receptors are expressed by glutamatergic Cajal-Retzius cells in the cerebral cortex. We used 5-HT(3A)/enhanced green fluorescent protein (EGFP) transgenic mice to show that 5-HT(3) receptors are also ubiquitously expressed by glutamatergic granule cells in the cerebellum during the first three postnatal weeks. Using whole-cell patch clamp recordings, we show that local application of either serotonin or the selective 5-HT(3) receptor agonist SR57227A to granule cells results in a small inward current, demonstrating a post- and/or extrasynaptic localisation of the 5-HT(3) receptors. Functional 5-HT(3) receptors were also observed presynaptically at the parallel fibre-Purkinje cell synapse. Pharmacological block using the selective 5-HT(3) receptor antagonist tropisetron induced a reduction in the frequency of miniature synaptic events recorded from Purkinje cells. Paired-pulse stimulation of parallel fibres on whole-cell voltage clamped Purkinje cells from 1-week-old mice did not yet show synaptic plasticity. In the presence of tropisetron, the parallel fibre-Purkinje cell synapse showed paired-pulse depression. Taken together, these results show that functional 5-HT(3) receptors are present during early postnatal development in the cerebellum, where they modulate synaptic plasticity.

Abnormalities in the Climbing Fiber-Purkinje Cell Circuitry Contribute to Neuronal Dysfunction inATXN1[82Q] Mice

One fundamental unanswered question in the field of polyglutamine diseases concerns the pathophysiology of neuronal dysfunction. Is there dysfunction in a specific neuronal population or circuit initially that contributes the onset of behavioral abnormalities? This study used a systems-level approach to investigate the functional integrity of the excitatory cerebellar cortical circuitry in vivo from several transgenic ATXN1 mouse lines. We tested the hypotheses that there are functional climbing fiber (CF)-Purkinje cell (PC) and parallel fiber (PF)-PC circuit abnormalities using flavoprotein autofluorescence optical imaging and extracellular field potential recordings. In early-symptomatic and symptomatic animals expressing ATXN1[82Q], there is a marked reduction in PC responsiveness to CF activation. Immunostaining of vesicular glutamate transporter type 2 demonstrated a decrement in CF extension on PC dendrites in symptomatic ATXN1[82Q] mice. In contrast, responses to PF stimulation were relatively normal. Importantly, the deficits in CF-PC synaptic transmission required expression of pathogenic ataxin-1 (ATXN1[82Q]) and for its entrance into the nucleus of PCs. Loss of endogenous mouse Atxn1 had no discernible effects. Furthermore, the abnormalities in CF-PC synaptic transmission were ameliorated when mutant transgene expression was prevented during postnatal cerebellar development. The results demonstrate the preferential susceptibility of the CF-PC circuit to the effects of ATXN1[82Q]. Further, this deficit likely contributes to the abnormal motor phenotype of ATXN1[82Q] mice. For polyglutamine diseases generally, the findings support a model whereby specific neuronal circuits suffer insults that alter function before cell death.