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Background Autism spectrum disorder (ASD) patients often show altered responses to sensory stimuli as well as motor deficits, including an impairment of delay eyeblink conditioning (EBC), which involves integration of sensory signals in the cerebellum. Here, we identify abnormalities in parallel fiber (PF) and climbing fiber (CF) signaling in mouse cerebellar cortex that may contribute to these pathologies. Methods We use a mouse model for the human 15q11-13 duplication (patDp/+) and studied responses to sensory stimuli in Purkinje cells (PCs) 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. Results We find that spontaneous and sensory-evoked CF-calcium transients are enhanced in patDp/+ PCs and aversive movements are more severe across sensory modalities. We observe increased expression of the synaptic organizer neurexin 1 (Nrxn1) at CF synapses, and ectopic spread of these synapses to fine dendrites. CF-EPSCs recorded from PCs 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 (LTD), one of several plasticity types required for EBC learning. LTD is impaired in patDp/+ mice, but is rescued upon pharmacological enhancement of calcium signaling. Conclusions Our 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.

OBJECTIVE Transcranial magnetic stimulation (TMS) delivered over the cerebellum 5-7 ms prior to a stimulus over the contralateral primary motor cortex (M1) reduces the excitability of M1 output, a phenomenon termed cerebellar brain inhibition (CBI). The cerebellum receives sensory information for adaptive motor coordination and motor planning. Here, we explored through TMS whether a peripheral electrical stimulus modulates CBI. METHODS We studied the effect of right median nerve electrical stimulation (ES) on CBI from right cerebellum (conditioning stimulus, CS) to left M1 (test stimulus, TS) in 12 healthy subjects. The following ES-CS inter-stimulus intervals (ISIs) were tested: 25, 30 and 35 ms. CS-TS ISI was set at 5 ms. RESULTS We found significantly weaker CBI when the ES was delivered 25 ms (p

Neurons store information by changing synaptic input weights. In addition, they can adjust their membrane excitability to alter spike output. Here, we demonstrate a role of such “intrinsic plasticity” in behavioral learning in a mouse model that allows us to detect specific consequences of absent excitability modulation. Mice with a Purkinje-cell–specific knockout (KO) of the calcium-activated K+ channel SK2 (L7-SK2) show intact vestibulo-ocular reflex (VOR) gain adaptation but impaired eyeblink conditioning (EBC), which relies on the ability to establish associations between stimuli, with the eyelid closure itself depending on a transient suppression of spike firing. In these mice, the intrinsic plasticity of Purkinje cells is prevented without affecting long-term depression or potentiation at their parallel fiber (PF) input. In contrast to the typical spike pattern of EBC-supporting zebrin-negative Purkinje cells, L7-SK2 neurons show reduced background spiking but enhanced excitability. Thus, SK2 plasticity and excitability modulation are essential for specific forms of motor learning., This study shows that cerebellum-dependent motor learning involves an adjustment of membrane excitability in Purkinje cells via modulation of SK2-type K+ channels; this "intrinsic plasticity" requirement can be separated from changes in synaptic strength.

The development of multiple sclerosis, a major neurodegenerative disease, is due to both genetic and environmental factors that might trigger aberrant epigenetic changes of the genome. In this study, we analysed global DNA methylation in the brain of mice upon induction of experimental autoimmune encephalomyelitis (EAE), and the effect of environmental enrichment (EE). We demonstrate that global DNA methylation decreased in the striatum, but not in the cortex, of EAE mice compared to healthy controls, in particular in neuronal nitric oxide synthase (nNOS)-positive interneurons of this brain area. Also, in the striatum but again not in the cortex, decreased DNA methylation of the nNOS downstream effector, dexamethasone-induced Ras protein 1 (Dexras 1), was observed in EAE mice, and was paralleled by an increase in its mRNA. Interestingly, EE was able to revert EAE effects on mRNA expression and DNA methylation levels of Dexras 1 and reduced gene expression of nNOS and 5-lipoxygenase (Alox5). Conversely, interleukin-1β (IL-1β) gene expression was found up-regulated in EAE mice compared to controls and was not affected by EE. Taken together, these data demonstrate an unprecedented epigenetic modulation of nNOS-signaling in the pathogenesis of multiple sclerosis, and show that EE can specifically revert EAE effects on Dexras 1 along this pathway.

At glutamatergic synapses, both long-term potentiation (LTP) and long-term depression (LTD) can be induced at the same synaptic activation frequency. Instructive signals determine whether LTP or LTD is induced, by modulating local calcium transients. Synapses maintain the ability to potentiate or depress over a wide frequency range, but it remains unknown how calcium-controlled plasticity operates when frequency variations alone cause differences in calcium amplitudes. We addressed this problem at cerebellar parallel fiber-Purkinje cell synapses, which can undergo LTD or LTP in response to 1-Hz and 100-Hz stimulation. We observed that high-frequency activation elicits larger spine calcium transients than low-frequency stimulation under all stimulus conditions, but, regardless of activation frequency, climbing fiber (CF) coactivation provides an instructive signal that further enhances calcium transients and promotes LTD. At both frequencies, buffering calcium prevents LTD induction and LTP results instead, identifying the enhanced calcium signal amplitude as the critical parameter contributed by the instructive CF signal. These observations show that it is not absolute calcium amplitudes that determine whether LTD or LTP is evoked but, instead, the LTD threshold slides, thus preserving the requirement for relatively larger calcium transients for LTD than for LTP induction at any given stimulus frequency. Cerebellar LTD depends on the activation of calcium/calmodulin-dependent kinase II (CaMKII). Using genetically modified (TT305/6VA and T305D) mice, we identified α-CaMKII inhibition upon autophosphorylation at Thr305/306 as a molecular event underlying the threshold shift. This mechanism enables frequency-independent plasticity control by the instructive CF signal based on relative, not absolute, calcium thresholds.

Summary Postnatal cerebellar development is a precisely regulated process involving well-orchestrated expression of neural genes. Neurological phenotypes associated with CACNA1A gene defects have been increasingly recognized, yet the molecular principles underlying this association remain elusive. By characterizing a dose-dependent CACNA1A gene deficiency mouse model, we discovered that α1ACT, as a transcription factor and secondary protein of CACNA1A mRNA, drives dynamic gene expression networks within cerebellar Purkinje cells and is indispensable for neonatal survival. Perinatal loss of α1ACT leads to motor dysfunction through disruption of neurogenesis and synaptic regulatory networks. However, its elimination in adulthood has minimal effect on the cerebellum. These findings shed light on the critical role of α1ACT in facilitating neuronal development in both mice and humans and support a rationale for gene therapies for calcium-channel-associated cerebellar disorders. Finally, we show that bicistronic expression may be common to the voltage-gated calcium channel (VGCC) gene family and may help explain complex genetic syndromes.

A significant proportion of multiple sclerosis (MS) patients have functionally relevant cerebellar deficits, which significantly contribute to disability. Although clinical and experimental studies have been conducted to understand the pathophysiology of cerebellar dysfunction in MS, no electrophysiological and morphological studies have investigated potential alterations of synaptic connections of cerebellar Purkinje cells (PC). For this reason we analyzed cerebellar PC GABAergic connectivity in mice with MOG((35-55))-induced experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. We observed a strong reduction in the frequency of the spontaneous inhibitory post-synaptic currents (IPSCs) recorded from PCs during the symptomatic phase of the disease, and in presence of prominent microglia activation not only in the white matter (WM) but also in the molecular layer (ML). The massive GABAergic innervation on PCs from basket and stellate cells was reduced and associated to a decrease of the number of these inhibitory interneurons. On the contrary no significant loss of the PCs could be detected. Incubation of interleukin-1beta (IL-1β) was sufficient to mimic the electrophysiological alterations observed in EAE mice. We thus suggest that microglia and pro-inflammatory cytokines, together with a degeneration of basket and stellate cells and their synaptic terminals, contribute to impair GABAergic transmission on PCs during EAE. Our results support a growing body of evidence that GABAergic signaling is compromised in EAE and in MS, and show a selective susceptibility to neuronal and synaptic degeneration of cerebellar inhibitory interneurons.

Sensory integration difficulties have been reported in autism, but their underlying brain-circuit mechanisms are underexplored. Using five autism-related mouse models, Shank3+/ΔC, Mecp2R308/Y, Cntnap2−/−, L7-Tsc1 (L7/Pcp2Cre::Tsc1flox/+), and patDp(15q11-13)/+, we report specific perturbations in delay eyeblink conditioning, a form of associative sensory learning requiring cerebellar plasticity. By distinguishing perturbations in the probability and characteristics of learned responses, we found that probability was reduced in Cntnap2−/−, patDp(15q11-13)/+, and L7/Pcp2Cre::Tsc1flox/+, which are associated with Purkinje-cell/deep-nuclear gene expression, along with Shank3+/ΔC. Amplitudes were smaller in L7/Pcp2Cre::Tsc1flox/+ as well as Shank3+/ΔC and Mecp2R308/Y, which are associated with granule cell pathway expression. Shank3+/ΔC and Mecp2R308/Y also showed aberrant response timing and reduced Purkinje-cell dendritic spine density. Overall, our observations are potentially accounted for by defects in instructed learning in the olivocerebellar loop and response representation in the granule cell pathway. Our findings indicate that defects in associative temporal binding of sensory events are widespread in autism mouse models. DOI: http://dx.doi.org/10.7554/eLife.06085.001, eLife digest On a windy day, hearing the sound of wind makes many individuals squint in anticipation in order to protect their eyes. Linking two sensations that arrive within a split second of one another, such as sound and the feeling of wind, is a type of learning that requires the cerebellum, a region found at the base of the brain. When done in a laboratory setting, this particular form of learning has been dubbed eyeblink conditioning. Individuals with autism tend to have difficulties with appropriate matching of different senses. For example, they have trouble identifying a video that goes with a spoken soundtrack. They also do not learn eyeblink conditioning the same way that other individuals do. However, it is not known which circuits in the brain are responsible for their difficulty. Kloth et al. now investigate this issue by asking whether versions of genes that increase the risk of autism in humans also disrupt eyeblink conditioning in mice. They tested five types of mouse model, each with a different genetic mutation that has previously been linked to autism. All five of these mutations cause defects in different cell types of the cerebellum, and all mice have abnormal social and habitual behaviors, similar to autistic people. The tests involved shining a bright light at the mice, which was followed, a split second later, by a puff of air that always causes the mice to blink. After this had occurred dozens of times, the mice started to blink earlier, as soon as the light appeared, in anticipation of the puff of air. To test whether the mice had successfully learned to respond to just the bright light, the light was also occasionally flashed without a puff of air. Kloth et al. found that the mice generally performed poorly in eyeblink conditioning, although in different ways depending on which cell types of the cerebellum were affected by the genetic mutations. Some mice blinked too soon or too late after the light appeared; others blinked weakly or less frequently; and some did not blink at all. This suggests that autism can affect the processing of sensory information in the cerebellum in different ways. This work is important because it demonstrates that a form of split-second multisensory learning is generally disrupted by autism genes. If defects in cerebellar learning are present early in life, they could keep autistic children from learning about the world around them, and drive their developing brains off track. Hundreds of autism genes have been found. Linking these genes to a single brain region identifies the cerebellum as an important anatomical target for future diagnosis and intervention. DOI: http://dx.doi.org/10.7554/eLife.06085.002

Corrigendum: Cerebellar plasticity and motor learning deficits in a copy-number variation mouse model of autism

A common feature of autism spectrum disorder (ASD) is the impairment of motor control and learning, occurring in a majority of children with autism, consistent with perturbation in cerebellar function. Here we report alterations in motor behaviour and cerebellar synaptic plasticity in a mouse model (patDp/+) for the human 15q11-13 duplication, one of the most frequently observed genetic aberrations in autism. These mice show ASD-resembling social behaviour deficits. We find that in patDp/+ mice delay eyeblink conditioning--a form of cerebellum-dependent motor learning--is impaired, and observe deregulation of a putative cellular mechanism for motor learning, long-term depression (LTD) at parallel fibre-Purkinje cell synapses. Moreover, developmental elimination of surplus climbing fibres--a model for activity-dependent synaptic pruning--is impaired. These findings point to deficits in synaptic plasticity and pruning as potential causes for motor problems and abnormal circuit development in autism.

Activity-dependent long-term plasticity of synaptic transmission, such as in long-term potentiation (LTP) and long-term depression (LTD), provides a cellular correlate of experience-driven learning. While at excitatory synapses in the hippocampus and neocortex LTP is seen as the primary learning mechanism, it has been widely assumed that cerebellar motor learning is mediated by LTD at parallel fiber (PF)-Purkinje cell synapses instead. However, recent work on mouse mutants with deficits in AMPA receptor internalization has demonstrated that motor learning can occur in the absence of LTD, suggesting that LTD is not essential. Another recent study has shifted attention toward LTP at PF synapses, showing that blockade of LTP severely affects motor learning. Here, we review the cellular and molecular events that are involved in LTP induction and discuss whether LTP might indeed play a more significant role in cerebellar learning than previously anticipated.

Activity-dependent long-term plasticity of synaptic transmission, such as in long-term potentiation (LTP) and long-term depression (LTD), provides a cellular correlate of experience-driven learning. While at excitatory synapses in the hippocampus and neocortex LTP is seen as the primary learning mechanism, it has been widely assumed that cerebellar motor learning is mediated by LTD at parallel fiber (PF)-Purkinje cell synapses instead. However, recent work on mouse mutants with deficits in AMPA receptor internalization has demonstrated that motor learning can occur in the absence of LTD, suggesting that LTD is not essential. Another recent study has shifted attention toward LTP at PF synapses, showing that blockade of LTP severely affects motor learning. Here, we review the cellular and molecular events that are involved in LTP induction and discuss whether LTP might indeed play a more significant role in cerebellar learning than previously anticipated.

Cerebellar deficit contributes significantly to disability in multiple sclerosis (MS). Several clinical and experimental studies have investigated the pathophysiology of cerebellar dysfunction in this neuroinflammatory disorder, but the cellular and molecular mechanisms are still unclear. In experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, proinflammatory cytokines, together with a degeneration of inhibitory neurons, contribute to impair GABAergic transmission at Purkinje cells (PCs). Here, we investigated glutamatergic transmission to gain insight into the pathophysiology of cerebellar dysfunction in EAE. Electrophysiological recordings from PCs showed increased duration of spontaneous excitatory postsynaptic currents (EPSCs) during the symptomatic phase of EAE, suggesting an alteration of glutamate uptake played by Bergmann glia. We indeed observed an impaired functioning of the glutamate-aspartate transporter/excitatory amino acid transporter 1 (GLAST/EAAT1) in EAE cerebellum caused by protein downregulation and in correlation with prominent astroglia activation. We have also demonstrated that the proinflammatory cytokine interleukin-1β (IL-1β), released by a subset of activated microglia/macrophages and infiltrating lymphocytes, was involved directly in such synaptic alteration. In fact, brief incubation of IL-1β in normal cerebellar slices replicated EAE modifications through a rapid GLAST/EAAT1 downregulation, whereas incubation of an IL-1 receptor antagonist (IL-1ra) in EAE slices reduced spontaneous EPSC alterations. Finally, EAE mice treated with intracerebroventricular IL-1ra showed normal glutamatergic and GABAergic transmissions, along with GLAST/EAAT1 normalization, milder inflammation, and reduced motor deficits. These results highlight the crucial role played by the proinflammatory IL-1β in triggering molecular and synaptic events involved in neurodegenerative processes that characterize neuroinflammatory diseases such as MS.

In the adult nervous system, different neuronal classes show different regenerative behavior. Although previous studies demonstrated that olivocerebellar fibers are capable of axonal regeneration in a suitable environment as a response to injury, we have hitherto no details about the real dynamics of fiber regeneration. We set up a model of singularly axotomized climbing fibers (CF) to investigate their reparative properties in the adult central nervous system (CNS) in vivo. Here we describe the approach followed to characterize the reactive plasticity after injury.

Plasticity in the central nervous system in response to injury is a complex process involving axonal remodeling regulated by specific molecular pathways. Here, we dissected the role of growth-associated protein 43 (GAP-43; also known as neuromodulin and B-50) in axonal structural plasticity by using, as a model, climbing fibers. Single axonal branches were dissected by laser axotomy, avoiding collateral damage to the adjacent dendrite and the formation of a persistent glial scar. Despite the very small denervated area, the injured axons consistently reshape the connectivity with surrounding neurons. At the same time, adult climbing fibers react by sprouting new branches through the intact surroundings. Newly formed branches presented varicosities, suggesting that new axons were more than just exploratory sprouts. Correlative light and electron microscopy reveals that the sprouted branch contains large numbers of vesicles, with varicosities in the close vicinity of Purkinje dendrites. By using an RNA interference approach, we found that downregulating GAP-43 causes a significant increase in the turnover of presynaptic boutons. In addition, silencing hampers the generation of reactive sprouts. Our findings show the requirement of GAP-43 in sustaining synaptic stability and promoting the initiation of axonal regrowth.

Glutamate-mediated excitotoxicity is supposed to induce neurodegeneration in multiple sclerosis (MS). Glatiramer acetate (GA) is an immunomodulatory agent used in MS treatment with potential neuroprotective action. Aim of the present study was to investigate whether GA has effects on glutamate transmission alterations occurring in experimental autoimmune encephalomyelitis (EAE), to disclose a possible mechanism of GA-induced neuroprotection in this mouse model of MS. Single neuron electrophysiological recordings and immunofluorescence analysis of microglia activation were performed in the striatum of EAE mice, treated or not with GA, at different stages of the disease. GA treatment was able to reverse the tumor necrosis factor-α (TNF-α)-induced alterations of striatal glutamate-mediated excitatory postsynaptic currents (EPSCs) of EAE mice. Incubation of striatal slices of control animals with lymphocytes taken from EAE mice treated with GA failed to replicate such an anti-glutamatergic effect, while activated microglial cells stimulated with GA in vitro mimicked the effect of GA treatment of EAE mice. Consistently, EAE mice treated with GA had less microglial activation and less TNF-α expression than untreated EAE animals. Furthermore, direct application of GA to EAE slices replicated the in vivo protective activity of GA. Our results show that GA is neuroprotective against glutamate toxicity independently of its peripheral immunodulatory action, and through direct modulation of microglial activation and TNF-α release in the grey matter of EAE and possibly of MS brains.

In the adult nervous system, different populations of neurons correspond to different regenerative behavior. Although previous works showed that olivocerebellar fibers are capable of axonal regeneration in a suitable environment as a response to injury 1 , we have hitherto no details about the real dynamics of fiber regeneration. We set up a model of singularly axotomized climbing fibers (CF) to investigate their reparative properties in the adult central nervous system (CNS) in vivo . Time lapse two-photon imaging has been combined to laser nanosurgery 2, 3 to define a temporal pattern of the degenerative event and to follow the structural rearrangement after injury. To characterize the damage and to elucidate the possible formation of new synaptic contacts on the sprouted branches of the lesioned CF, we combined two-photon in vivo imaging with block face scanning electron microscopy (FIB-SEM). Here we describe the approach followed to characterize the reactive plasticity after injury. Keywords: In vivo imaging, two-photon microscopy, laser nanosurgery, cerebellum

Structural plasticity occurs physiologically or after brain damage to adapt or re-establish proper synaptic connections. This capacity depends on several intrinsic and extrinsic determinants that differ between neuron types. We reviewed the significant endogenous regenerative potential of the neurons of the inferior olive in the adult rodent brain and the structural remodeling of the terminal arbor of their axons the climbing fiber under various experimental conditions, focusing on the growth-associated protein GAP-43. Climbing fibers undergo remarkable collateral sprouting in the presence of denervated Purkinje cells that are available for new innervation. In addition, severed olivo-cerebellar axons regenerate across the white matter through a graft of embryonic Schwann cells. In contrast, climbing fibers undergo a regressive modification when their target is deleted. In vivo knockdown of GAP-43 in olivary neurons, leads to the atrophy of their climbing fibers and a reduction in the ability to sprout toward surrounding denervated Purkinje cells. These findings demonstrate that GAP-43 is essential for promoting denervation-induced sprouting and maintaining normal climbing fiber architecture.

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.

BACKGROUND AND PURPOSE: Glutamate transmission is dysregulated in both multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), the animal model of MS. A characteristic of EAE is increased glutamate transmission associated with up-regulation of AMPA receptors. However, little is known about the role of NMDA receptors in the synaptic modifications induced by EAE. EXPERIMENTAL APPROACH: The contribution of NMDA receptors to the alterations of glutamate transmission and disease severity in EAE mice was assessed by means of neurophysiological, morphological, Western blot, metabolic and clinical score assessments. KEY RESULTS: In our EAE mice, there was an NMDA receptor-dependent increase of glutamate release, associated with marked activation of the astroglia. Presynaptic NMDA receptors became overactive during EAE, increasing synaptic glutamate release by a mechanism dependent on voltage-gated sodium channels. By means of NAD(P)H autofluorescence analysis, we also found that EAE has a glutamate and NMDA receptor-dependent dysfunction of mitochondrial activity, which is known to contribute to the neurodegenerative damage of MS and EAE. Furthermore, pharmacological blockade of NMDA receptors in vivo ameliorated both synaptic transmission defects and of the clinical disease course of EAE mice, while EAE induced in mice with a genetically enhanced NMDA receptor signalling had opposite effects. CONCLUSIONS AND IMPLICATIONS: Our data, showing both sensitization of NMDA receptors and their involvement in the progression of the EAE disease, supggest that pharmacological impairment of NMDA receptor signalling would be a component of a neuroprotection strategy in MS Background and Purpose Glutamate transmission is dysregulated in both multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), the animal model of MS. A characteristic of EAE is increased glutamate transmission associated with up-regulation of AMPA receptors. However, little is known about the role of NMDA receptors in the synaptic modifications induced by EAE. Experimental Approach The contribution of NMDA receptors to the alterations of glutamate transmission and disease severity in EAE mice was assessed by means of neurophysiological, morphological, Western blot, metabolic and clinical score assessments. Key Results In our EAE mice, there was an NMDA receptor-dependent increase of glutamate release, associated with marked activation of the astroglia. Presynaptic NMDA receptors became overactive during EAE, increasing synaptic glutamate release by a mechanism dependent on voltage-gated sodium channels. By means of NAD(P)H autofluorescence analysis, we also found that EAE has a glutamate and NMDA receptor-dependent dysfunction of mitochondrial activity, which is known to contribute to the neurodegenerative damage of MS and EAE. Furthermore, pharmacological blockade of NMDA receptors in vivo ameliorated both synaptic transmission defects and of the clinical disease course of EAE mice, while EAE induced in mice with a genetically enhanced NMDA receptor signalling had opposite effects. Conclusions and Implications Our data, showing both sensitization of NMDA receptors and their involvement in the progression of the EAE disease, supggest that pharmacological impairment of NMDA receptor signalling would be a component of a neuroprotection strategy in MS. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

In the adult nervous system, different population of neurons corresponds to different regenerative behavior. Although previous works show that olivocerebellar fibers are capable of axonal regeneration in a suitable environment as a response to injury [1], we have hitherto no details about the real dynamics of fiber regeneration. We coupled two photon imaging to laser-induced lesions to perform in vivo multiphoton nanosurgery in the CNS of living mice expressing fluorescent proteins to investigate the reparative properties of Climbing Fibers (CFs) in the adult CNS, following the time evolution of this plastic process in vivo. Here we show that sprouting may take place in a murine model in the days that follow a sub-micrometric lesion on the distal portion of the CF. Furthermore this unique model allows, through manipulation of the viral vector, to explore the biochemical mechanisms underlying the reparative process. The great potential of long-term two-photon imaging, coupled to genetic manipulation, opens great opportunities to further investigate the dynamic properties of neurons and their rearrangement following an injury.

The adult mammalian central nervous system has a limited ability to establish new connections and to recover from traumatic or degenerative events. The olivo-cerebellar network represents an excellent model to investigate neuroprotection and repair in the brain during adulthood, due to its high plasticity and ordered synaptic organization. To shed light on the molecular mechanisms involved in these events, we focused on the growth-associated protein GAP-43 (also known as B-50 or neuromodulin). During development, this protein plays a crucial role in growth and in branch formation of neurites, while in the adult it is only expressed in a few brain regions, including the inferior olive (IO) where climbing fibres (CFs) originate. Following axotomy GAP-43 is usually up-regulated in association with regeneration. Here we describe an in vivo lentiviral-mediated gene silencing approach, used for the first time in the olivo-cerebellar system, to efficiently and specifically downregulate GAP-43 in rodents CFs. We show that lack of GAP-43 causes an atrophy of the CF in non-traumatic conditions, consisting in a decrease of its length, branching and number of synaptic boutons. We also investigated CF regenerative ability by inducing a subtotal lesion of the IO. Noteworthy, surviving CFs lacking GAP-43 were largely unable to sprout on surrounding Purkinje cells. Collectively, our results demonstrate that GAP-43 is essential both to maintain CFs structure in non-traumatic condition and to promote sprouting after partial lesion of the IO.

Transient receptor potential vanilloid 1 (TRPV1) channels are involved in several inflammatory diseases. However, their action is still controversial, and both pro-inflammatory and anti-inflammatory roles have been described. We used a strain of TRPV1-KO mice to characterize the role of these channels in experimental autoimmune encephalomyelitis (EAE), which models multiple sclerosis (MS) in mice. EAE mice showed higher lethality in the peak phase of the disease and a better recovery of the surviving animals in the chronic stages, compared to their wild-type (WT) counterparts. By means of whole-cell patch clamp experiments in corticostriatal brain slices, we found that the absence of TRPV1 channels exacerbated the defect of glutamate transmission occurring in the peak phase of EAE, and attenuated the alterations of GABA synapses in the chronic phase of EAE, thus paralleling the dual effects of TRPV1-KO on the motor deficits of EAE mice. Furthermore, in slices from non-EAE mice, we found that genetic or pharmacological blockade of TRPV1 channels enhanced the synaptic effects of tumor necrosis factor α (TNF-α) on glutamate-mediated excitatory postsynaptic currents, and prevented the action of interleukin 1β (IL-1β) on GABAergic inhibitory postsynaptic currents. Together, our results suggest that TRPV1 channels contrast TNF-α-mediated synaptic deficits in the peak phase of EAE and, in the chronic stages, enhance IL-1β-induced GABAergic defects. The opposing interplay with the synaptic actions of the two major pro-inflammatory cytokines might explain the bimodal effects of TRPV1 ablation on the motor deficits of EAE, and suggests that the inflammatory milieu determines whether TRPV1 channels exert preferentially aversive or protective effects on neurons during neuroinflammatory diseases.

Synaptic dysfunction triggers neuronal damage in experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). While excessive glutamate signaling has been reported in the striatum of EAE, it is still uncertain whether GABA synapses are altered. Electrophysiological recordings showed a reduction of spontaneous GABAergic synaptic currents (sIPSCs) recorded from striatal projection neurons of mice with MOG(35−55)-induced EAE. GABAergic sIPSC deficits started in the acute phase of the disease (20–25 days post immunization, dpi), and were exacerbated at later time-points (35, 50, 70 and 90 dpi). Of note, in slices they were independent of microglial activation and of release of TNF-α. Indeed, sIPSC inhibition likely involved synaptic inputs arising from GABAergic interneurons, because EAE preferentially reduced sIPSCs of high amplitude, and was associated with a selective loss of striatal parvalbumin (PV)-positive GABAergic interneurons, which contact striatal projection neurons in their somatic region, giving rise to more efficient synaptic inhibition. Furthermore, we found also that the chronic persistence of pro-inflammatory cytokines were able, per se, to produce profound alterations of electrophysiological network properties, that were reverted by GABA administration. The results of the present investigation indicate defective GABA transmission in MS models depending from alteration of PV cells number and, in part, deriving from the effects of a chronic inflammation, and suggest that pharmacological agents potentiating GABA signaling might be considered to limit neuronal damage in MS patients.

In the adult nervous system, different population of neurons corresponds to different regenerative behavior. Although previous works show that olivocerebellar fibers are capable of axonal regeneration in a suitable environment as a response to injury 1 , we have hitherto no details about the real dynamics of fiber regeneration. We coupled two photon imaging to laser-induced lesions to perform in vivo multiphoton nanosurgery in the CNS of living mice expressing fluorescent proteins to investigate the reparative properties of Climbing Fibers (CFs) in the adult CNS, following the time evolution of this plastic process in vivo. Here we show that a regenerative event may take place in a murine model in the days that follow a sub-micrometric lesion on the distal portion of the climbing fiber. Furthermore this unique model could allow, through manipulation of the viral vector, to explore in detail the biochemical mechanisms underlying the reparative process. The great potential of long-term two photon imaging, coupled to genetic manipulation, opens great opportunities to further investigate the dynamic properties of neurons and their rearrangement following an injury.

In combination with fluorescent protein (XFP) expression techniques, two-photon microscopy has become an indispensable tool to image cortical plasticity in living mice. In parallel to its application in imaging, multi-photon absorption has also been used as a tool for the dissection of single neurites with submicrometric precision without causing any visible collateral damage to the surrounding neuronal structures. In this work, multi-photon nanosurgery is applied to dissect single climbing fibers expressing GFP in the cerebellar cortex. The morphological consequences are then characterized with time lapse 3-dimensional two-photon imaging over a period of minutes to days after the procedure. Preliminary investigations show that the laser induced fiber dissection recalls a regenerative process in the fiber itself over a period of days. These results show the possibility of this innovative technique to investigate regenerative processes in adult brain. In parallel with imaging and manipulation technique, non-linear microscopy offers the opportunity to optically record electrical activity in intact neuronal networks. In this work, we combined the advantages of second-harmonic generation (SHG) with a random access (RA) excitation scheme to realize a new microscope (RASH) capable of optically recording fast membrane potential events occurring in a wide-field of view. The RASH microscope, in combination with bulk loading of tissue with FM4-64 dye, was used to simultaneously record electrical activity from clusters of Purkinje cells in acute cerebellar slices. Complex spikes, both synchronous and asynchronous, were optically recorded simultaneously across a given population of neurons. Spontaneous electrical activity was also monitored simultaneously in pairs of neurons, where action potentials were recorded without averaging across trials. These results show the strength of this technique in describing the temporal dynamics of neuronal assemblies, opening promising perspectives in understanding the computations of neuronal networks.

Multiple sclerosis (MS) is characterized by auto-reactive T cells that respond to central nervous system (CNS)-based antigens and affect motor, sensory as well as behavioral and cognitive functions. Cognitive deficits are now considered an early manifestation of the disease in MS patients. However, the pathophysiology responsible for the cognitive symptoms in MS remains unclear. Increasing evidence from a mouse model of MS, the experimental autoimmune encephalomyelitis (EAE), suggests a correlation between the synaptopathy induced by microglia activation in the early phase of the disease and cognitive dysfunction. In particular, EAE causes deficits in hippocampal-dependent learning and memory that are associated with early microglial activation, synaptic loss and neurodegeneration. Interestingly, inflammatory cytokines released from infiltrating lymphocytes or activated microglia are able to alter synaptic transmission. Increased glutamate-mediated transmission and loss of GABAergic inputs were observed in EAE. They may thus underlie cognitive dysfunction in this model and in MS.

Synaptosome-associated protein of 25 kDa (SNAP25) is a component of the fusion complex that mediates synaptic vesicle exocytosis, regulates calcium dynamics and neuronal plasticity. Despite its crucial role in vesicle release, SNAP25 is not distributed homogenously within the brain. It seems to be virtually absent in mature inhibitory terminals and is observed in a subtype of excitatory neurons defined by the expression of vesicular glutamate transporter 1 (VGluT1). Since a complementary distribution of VGluT1 and VGluT2 in excitatory synapses is correlated with different probabilities of release (Pr), we evaluated whether SNAP25 localization is associated with specific synaptic properties. In the cerebellum, climbing fiber (CF) and parallel fiber (PF) inputs, which impinge onto the same Purkinje cell (PC), have very different functional properties. In the cerebellum of adult rats, using confocal and electron microscopy, we observed that VGluT2-positive CFs, characterized by a high Pr, only weakly express SNAP25, while VGluT1-positive PFs that show a low Pr abundantly express SNAP25. Moreover, SNAP25 was less profuse in the VGluT2-positive rosettes of mossy fibers (MFs) and was almost absent in inhibitory terminals. We extended our analysis to the SNAP23 homolog; this is expressed at different levels in both gamma-aminobutyric acid-containing terminals (GABAergic) and glutamatergic terminals of the cerebellar cortex. In conclusion, the preferential localization of SNAP25 in specific synaptic boutons suggests a correlation between SNAP25 and the Pr. This evidence supports the hypothesis that SNAP25 has a modulatory role in shaping synaptic responses.