The homeostatic effects of the RE-1 silencing transcription factor on cortical networks are altered under ictogenic conditions in the mouse.

AIM: The Repressor Element-1 Silencing Transcription Factor (REST) is an epigenetic master regulator playing a crucial role in the nervous system. In early developmental stages, REST downregulation promotes neuronal differentiation and the acquisition of the neuronal phenotype. In addition, postnatal fluctuations in REST expression contribute to shaping neuronal networks and maintaining network homeostasis. Here we investigate the role of the early postnatal deletion of neuronal REST in the assembly and strength of excitatory and inhibitory synaptic connections. METHODS: We investigated excitatory and inhibitory synaptic transmission by patch-clamp recordings in acute neocortical slices in a conditional knockout mouse model (RestGTi) in which Rest was deleted by delivering PHP.eB adeno-associated viruses encoding CRE recombinase under the control of the human synapsin I promoter in the lateral ventricles of P0-P1 pups. RESULTS: We show that, under physiological conditions, Rest deletion increased the intrinsic excitability of principal cortical neurons in the primary visual cortex and the density and strength of excitatory synaptic connections impinging on them, without affecting inhibitory transmission. Conversely, in the presence of a pathological excitation/inhibition imbalance induced by pentylenetetrazol, Rest deletion prevented the increase in synaptic excitation and decreased seizure severity. CONCLUSION: The data indicate that REST exerts distinct effects on the excitability of cortical circuits depending on whether it acts under physiological conditions or in the presence of pathologic network hyperexcitability. In the former case, REST preserves a correct excitatory/inhibitory balance in cortical circuits, while in the latter REST loses its homeostatic activity and may become pro-epileptogenic.

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

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

Somatosensory inputs modulate the excitability of cerebellar-cortical interaction.

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 < 0.001) and 35 ms (p < 0.001) earlier the CS over the ipsilateral cerebellum and a trend for 30 ms ES-CS ISI (p = 0.07). CONCLUSIONS: We hypothesize that the activation of cerebellar interneurons together with intrinsic properties of Purkinje cells may be responsible of the decreased CBI when the peripheral stimulation preceded the cerebellar stimulation of 25 and 35 ms. SIGNIFICANCE: To test the interaction between somatosensory inputs and cerebello-cortical pathway may be important in a variety of motor tasks and neuropsychiatric disorders.

Neuronal firing modulation by a membrane-targeted photoswitch.

Optical technologies allowing modulation of neuronal activity at high spatio-temporal resolution are becoming paramount in neuroscience. In this respect, azobenzene-based photoswitches are promising nanoscale tools for neuronal photostimulation. Here we engineered a light-sensitive azobenzene compound (Ziapin2) that stably partitions into the plasma membrane and causes its thinning through trans-dimerization in the dark, resulting in an increased membrane capacitance at steady state. We demonstrated that in neurons loaded with the compound, millisecond pulses of visible light induce a transient hyperpolarization followed by a delayed depolarization that triggers action potential firing. These effects are persistent and can be evoked in vivo up to 7 days, proving the potential of Ziapin2 for the modulation of membrane capacitance in the millisecond timescale, without directly affecting ion channels or local temperature.

SK2 channels in cerebellar Purkinje cells contribute to excitability modulation in motor-learning-specific memory traces.

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.

α1ACT Is Essential for Survival and Early Cerebellar Programming in a Critical Neonatal Window.

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.

Calcium threshold shift enables frequency-independent control of plasticity by an instructive signal.

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.

Activity-Dependent Plasticity of Spike Pauses in Cerebellar Purkinje Cells.

The plasticity of intrinsic excitability has been described in several types of neurons, but the significance of non-synaptic mechanisms in brain plasticity and learning remains elusive. Cerebellar Purkinje cells are inhibitory neurons that spontaneously fire action potentials at high frequencies and regulate activity in their target cells in the cerebellar nuclei by generating a characteristic spike burst-pause sequence upon synaptic activation. Using patch-clamp recordings from mouse Purkinje cells, we find that depolarization-triggered intrinsic plasticity enhances spike firing and shortens the duration of spike pauses. Pause plasticity is absent from mice lacking SK2-type potassium channels (SK2(-/-) mice) and in occlusion experiments using the SK channel blocker apamin, while apamin wash-in mimics pause reduction. Our findings demonstrate that spike pauses can be regulated through an activity-dependent, exclusively non-synaptic, SK2 channel-dependent mechanism and suggest that pause plasticity-by altering the Purkinje cell output-may be crucial to cerebellar information storage and learning.

Cerebellar associative sensory learning defects in five mouse autism models.

Sensory integration difficulties have been reported in autism, but their underlying brain-circuit mechanisms are underexplored. Using five autism-related mouse models, Shank3+/ΔC, Mecp2(R308/Y), Cntnap2-/-, L7-Tsc1 (L7/Pcp2(Cre)::Tsc1(flox/+)), 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/Pcp2(Cre)::Tsc1(flox/+), which are associated with Purkinje-cell/deep-nuclear gene expression, along with Shank3+/ΔC. Amplitudes were smaller in L7/Pcp2(Cre)::Tsc1(flox/+) as well as Shank3+/ΔC and Mecp2(R308/Y), which are associated with granule cell pathway expression. Shank3+/ΔC and Mecp2(R308/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.

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.

Cerebellar long-term potentiation: cellular mechanisms and role in learning.

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.

Interleukin-1ß alters glutamate transmission at purkinje cell synapses in a mouse model of multiple sclerosis.

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-1ß (IL-1ß), released by a subset of activated microglia/macrophages and infiltrating lymphocytes, was involved directly in such synaptic alteration. In fact, brief incubation of IL-1ß 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-1ß in triggering molecular and synaptic events involved in neurodegenerative processes that characterize neuroinflammatory diseases such as MS.

In vivo single branch axotomy induces GAP-43-dependent sprouting and synaptic remodeling in cerebellar cortex.

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.

Glatiramer acetate protects against inflammatory synaptopathy in experimental autoimmune encephalomyelitis.

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.

Structural plasticity of climbing fibers and the growth-associated protein GAP-43.

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 (IO) in the adult rodent brain and the structural remodeling of the terminal arbor of their axons, the climbing fiber (CF), under various experimental conditions, focusing on the growth-associated protein GAP-43. CFs undergo remarkable collateral sprouting in the presence of denervated Purkinje cells (PCs) 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, CFs undergo a regressive modification when their target is deleted. In vivo knockdown of GAP-43 in olivary neurons, leads to the atrophy of their CFs and a reduction in the ability to sprout toward surrounding denervated PCs. These findings demonstrate that GAP-43 is essential for promoting denervation-induced sprouting and maintaining normal CF architecture.

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.

GABAergic signaling and connectivity on Purkinje cells are impaired in experimental autoimmune encephalomyelitis.

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-1ß) 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.

Impaired sprouting and axonal atrophy in cerebellar climbing fibres following in vivo silencing of the growth-associated protein GAP-43.

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 channels modulate the synaptic effects of TNF-α and of IL-1ß in experimental autoimmune encephalomyelitis.

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 1ß (IL-1ß) 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-1ß-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.