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Objective: The frequency of inflammatory episodes in the early stages of multiple sclerosis (MS) has been correlated with late neurodegeneration, but the mechanism by which inflammation gives rise to delayed neuronal damage is unknown. Increased activity of the neurotransmitter glutamate is thought to play a role in the inflammation-driven neurodegenerative process of MS, and therefore we tested whether inflammatory cytokines released during acute MS attacks have the property of enhancing glutamate-mediated transmission and excitotoxicity in central neurons. Methods: We compared the effect of cerebrospinal fluid (CSF) from active and quiescent MS patients on glutamate-mediated excitatory postsynaptic currents (EPSCs) and excitotoxic damage in rodent brain slices. We also measured CSF concentrations of tumor necrosis factor-α, of interleukin-1β (IL-1β), and of IL-1 receptor antagonist (IL-1ra), and correlated cytokine levels with cortical excitability assessed in MS patients by means of paired-pulse transcranial magnetic stimulation (TMS). Results: CSF from MS patients with enhanced brain lesions at magnetic resonance imaging was able to increase spontaneous EPSC frequency and glutamate-mediated neuronal swelling in vitro, through a mechanism dependent on enhanced IL-1β signaling and increased glutamate α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor stimulation. Furthermore, IL-1β/IL-1ra ratio was significantly higher in the CSF of active MS subjects, and correlated with intracortical facilitation, an accredited TMS measure of glutamate transmission. Finally, we identified for the first time transient receptor potential vanilloid 1 channels as essential intermediates for the synaptic action of IL-1β on central glutamatergic synapses. Interpretation: Our results provide compelling evidence of the synaptic mechanism linking inflammation and excitotoxic neurodegeneration in MS. ANN NEUROL 2012;71:76–83

Abnormal dopamine (DA) transmission in the striatum plays a pivotal role in attention-deficit/hyperactivity disorder (ADHD). As striatal DA signalling modulates the endocannabinoid system (ECS), the present study was aimed at investigating cannabinoid CB1 receptor (CB1R) function in a model of ADHD obtained by triple point-mutation in the dopamine transporter (DAT) gene in mice, making them insensitive to cocaine [DAT cocaine-insensitive (DAT-CI) mice]. DAT-CI mice had a marked hyperactive phenotype, and neurophysiological recordings revealed that the sensitivity of CB1Rs controlling GABA-mediated synaptic currents [CB1Rs((GABA)) ] in the striatum was completely lost. In contrast, CB1Rs modulating glutamate transmission [CB1Rs((Glu)) ], and GABA(B) receptors were not affected in this model of ADHD. In DAT-CI mice, the blockade of CB1R((GABA)) function was complete even after cocaine or environmental manipulations activating the endogenous DA-dependent reward system, which are known to sensitize these receptors in control animals. Conversely, the hedonic property of sucrose was intact in DAT-CI mice, indicating normal sweet perception in these animals. Our results point to CB1Rs as novel molecular players in ADHD, and suggest that therapeutic strategies aimed at interfering with the ECS might prove effective in this disorder.

Background: Genetic and pharmacological inactivation of cannabinoid CB1 receptors (CB1Rs) exacerbates disease course in experimental autoimmune encephalomyelitis, suggesting that CB1Rs might play a role in the neurodegenerative damage associated with multiple sclerosis (MS). Objectives: To see whether CNR1 gene polymorphism could influence disease progression in relapsing–remitting MS. Methods: The genotype of 350 patients for the number of AAT repeats was characterized and correlation studies were performed with measures of disease severity and progression. Results: MS patients with the homozygous genotype for long AAT repeats in the CNR1 gene had more severe disease and higher risk of progression. These subjects had significantly higher scores on both the progression index and the MS severity scale. Furthermore, the percentage of patients with MS functional composite score progression or Bayesian Risk Estimate for MS (BREMS) score ≥2 (considered at very high risk of secondary progression) was significantly higher in the AAT long group than in the short group, while the frequency of patients with BREMS score ≤−0.63 (very likely to remain progression-free) was not significantly different between the two groups, although lower in the long group. Finally, the frequency of patients prescribed a second-line treatment was significantly higher among subjects of the AAT long group, providing a further, indirect indication of higher disease severity. Conclusions: The results of the present investigation point to CB1R as an important modulator of disease severity in relapsing MS subjects.

The endocannabinoid anandamide (AEA) plays a crucial role in emotional control, and inhibition of its degradation by the fatty acid amide hydrolase (FAAH) has a potent antianxiety effect. The mechanism by which the magnification of AEA activity reduces anxiety is still largely undetermined. By using FAAH mutant mice and both intraperitoneal and intracerebroventricular administration of the FAAH inhibitor (3'-(aminocarbonyl)[1,1'-biphenyl]-3-yl)-cyclohexylcarbamate (URB597), we found that enhanced AEA signaling reversed, via central cannabinoid CB1 receptors (CB1Rs), the anxious phenotype of mice exposed to social defeat stress. This behavioral effect was associated with preserved activity of CB1Rs regulating GABA transmission in the striatum, whereas these receptors were dramatically down-regulated by stress in control animals. The hypothalamic-pituitary-adrenal (HPA) axis was not involved in the antistress effects of FAAH inhibition, although the HPA axis is a biological target of endogenous AEA. We also provided some physiological indications that striatal CB1Rs regulating GABA synapses are not the receptor targets of FAAH inhibition, which rather resulted in the stimulation of striatal CB1Rs regulating glutamate transmission. Collectively, our findings suggest that preservation of cannabinoid CB1 receptor function within the striatum is a possible synaptic correlate of the antianxiety effects of FAAH inhibition.

Caffeine is the most commonly self-administered psychoactive substance worldwide. At usual doses, the effects of caffeine on vigilance, attention, mood and arousal largely depend on the modulation of central adenosine receptors. The present review article describes the action of caffeine within the striatum, to provide a possible molecular mechanism at the basis of the psychomotor and reinforcing properties of this pharmacological agent. The striatum is in fact a subcortical area involved in sensorimotor, cognitive, and emotional processes, and recent experimental findings showed that chronic caffeine consumption enhances the sensitivity of striatal GABAergic synapses to the stimulation of cannabinoid CB1 receptors. The endocannabinoid system is involved in the psychoactive effects of many compounds, and adenosine A2A receptors (the main receptor target of caffeine) elicit a permissive effect towards CB1 receptors, thus suggesting that A2A-CB1 receptor interaction plays a major role in the generation and maintenance of caffeine reinforcing behavior. Aim of this review is to describe the effects of caffeine on striatal neurotransmission with special reference to the modulation of the endocannabinoid system.

Several G protein-associated receptors and synaptic proteins function within lipid rafts, which are subdomains of the plasma membranes that contain high concentrations of cholesterol. In this study we addressed the possible role of lipid rafts in the control of endocannabinoid system in striatal slices. Disruption of lipid rafts following cholesterol depletion with methyl-beta-cyclodestrin (MCD) failed to affect synthesis and degradation of anandamide, while it caused a marked increase in the synthesis and concentration of 2-arachidonoylglycerol (2-AG), as well as in the binding activity of cannabinoid CB1 receptors. Surprisingly, endogenous 2-AG-mediated control of GABA transmission was not potentiated by MCD treatment and, in contrast, neither basal nor 3,5-Dihydroxyphenylglycine-stimulated 2-AG altered GABA synapses in cholesterol-depleted slices. Synaptic response to the cannabinoid CB1 receptor agonist HU210 was however intact in MCD-treated slices, indicating that reduced sensitivity of cannabinoid CB1 receptors does not explain why endogenous 2-AG is ineffective in inhibiting striatal GABA transmission after cholesterol depletion. Confocal microscopy analysis suggested that disruption of raft integrity by MCD might uncouple metabotropic glutamate 5-CB1 receptor interaction by altering the correct localization of both receptors in striatal neuron elements. In conclusion, our data indicate that disruption of raft integrity causes a complex alteration of the endocannabinoid signalling in the striatum.

Neurodegeneration is the irremediable pathological event occurring during chronic inflammatory diseases of the CNS. Here we show that, in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, inflammation is capable in enhancing glutamate transmission in the striatum and in promoting synaptic degeneration and dendritic spine loss. These alterations occur early in the disease course, are independent of demyelination, and are strongly associated with massive release of tumor necrosis factor-α from activated microglia. CNS invasion by myelin-specific blood-borne immune cells is the triggering event, and the downregulation of the early geneArc/Arg3.1, leading to the abnormal expression and phosphorylation of AMPA receptors, represents a culminating step in this cascade of neurodegenerative events. Accordingly, EAE-induced synaptopathy subsided during pharmacological blockade of AMPA receptors. Our data establish a link between neuroinflammation and synaptic degeneration and calls for early neuroprotective therapies in chronic inflammatory diseases of the CNS.

The endocannabinoid system (ECS) plays a fundamental role in the regulation of synaptic transmission. Exposure to stressful events triggers synaptic adaptations in many brain areas. The activity of the ECS in stress-responsive neural circuits suggests that it may be involved in the behavioral responses and synaptic effects typical of stress. In this review, we discuss evidence demonstrating that striatal ECS is modulated by stress. Chronic stress exposure alters endocannabinoid levels, cannabinoid CB1 receptor binding and cannabinoid CB1 receptor-mediated control of inhibitory synaptic transmission in the striatum. Recent studies have shown that impairment of endocannabinoid signalling is associated with inability to adapt to chronic stress and to the development of maladaptive behaviors. The ECS represents a novel potential pharmacological target to treat stress-associated neuropsychiatric conditions.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that primarily affects motor neurons. However, additional neuronal systems are also involved, and the aim of this study was to investigate the involvement of the nucleus striatum. By means of neurophysiological recordings in slices, we have investigated both excitatory and inhibitory synaptic transmission in the striatum of G93A-SOD1 ALS mice, along with the sensitivity of these synapses to cannabinoid CB1 receptor stimulation. We have observed reduced frequency of glutamate-mediated spontaneous excitatory postsynaptic currents (EPSCs) and increased frequency of GABA-mediated spontaneous inhibitory postsynaptic currents (IPSCs) recorded from striatal neurons of ALS mice, possibly due to presynaptic defects in transmitter release. The sensitivity of cannabinoid CB1 receptors controlling both glutamate and GABA transmission was remarkably potentiated in ALS mice, indicating that adaptations of the endocannabinoid system might be involved in the pathophysiology of ALS. In conclusion, our data identify possible physiological correlates of striatal dysfunction in ALS mice, and suggest that cannabinoid CB1 receptors might be potential therapeutic targets for this dramatic disease.

Of the endocannabinoids (eCBs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG) have received the most study. A functional interaction between these molecules has never been described. Using mouse brain slices, we found that stimulation of metabotropic glutamate 5 receptors by 3,5-dihydroxyphenylglycine (DHPG) depressed inhibitory transmission in the striatum through selective involvement of 2-AG metabolism and stimulation of presynaptic CB1 receptors. Elevation of AEA concentrations by pharmacological or genetic inhibition of AEA degradation reduced the levels, metabolism and physiological effects of 2-AG. Exogenous AEA and the stable AEA analog methanandamide inhibited basal and DHPG-stimulated 2-AG production, confirming that AEA is responsible for the downregulation of the other eCB. AEA is an endovanilloid substance, and the stimulation of transient receptor potential vanilloid 1 (TRPV1) channels mimicked the effects of endogenous AEA on 2-AG metabolism through a previously unknown glutathione-dependent pathway. Consistently, the interaction between AEA and 2-AG was lost after pharmacological and genetic inactivation of TRPV1 channels.

Synaptic rearrangements in the peri-infarct regions are believed to contribute to the partial recovery of function that takes place after stroke. Here, we performed neurophysiological recordings from single neurons of rats with permanent occlusion of the middle cerebral artery (pMCAO) during the resolution of their neurological deficits. Our results show that complex and dynamic changes of glutamate transmission in the peri-infarct area parallel the recovery from brain infarct. We have observed that frequency and duration of spontaneous glutamate-mediated synaptic events were markedly increased in striatal neurons during the early phase of the recovery (3 days after pMCAO), due to potentiation of both NMDA (N-methyl-d-aspartate) and non-NMDA receptor-mediated transmission. In the late phase of recovery (7 days after pMCAO), glutamate transmission was still enhanced because of a selective facilitation of non-NMDA receptor-mediated transmission. Spiny projection neurons but not aspiny interneurons underwent detectable changes of synaptic excitability in the striatum following pMCAO, indicating that the process of neuronal adaptation after focal brain ischemia is cell-type-specific. Our results provide a synaptic correlate of the long-lasting brain hyperexcitability mediating recovery described with noninvasive neurophysiological approaches.

The endogenous cannabinoid system is involved in the regulation of the central reward pathway. Running wheel and sucrose consumption have rewarding and reinforcing properties in rodents, and share many neurochemical and behavioral characteristics with drug addiction. In this study, we investigated whether running wheel or sucrose consumption altered the sensitivity of striatal synapses to the activation of cannabinoid CB1 receptors. We found that cannabinoid CB1 receptor-mediated presynaptic control of striatal inhibitory postsynaptic currents was remarkably potentiated after these environmental manipulations. In contrast, the sensitivity of glutamate synapses to CB1 receptor stimulation was unaltered, as well as that of GABA synapses to the stimulation of presynaptic GABAB receptors. The sensitization of cannabinoid CB1 receptor-mediated responses was slowly reversible after the discontinuation of running wheel or sucrose consumption, and was also detectable following the mobilization of endocannabinoids by metabotropic glutamate receptor 5 stimulation. Finally, we found that the upregulation of cannabinoid transmission induced by wheel running or sucrose had a crucial role in the protective effects of these environmental manipulations against the motor and synaptic consequences of stress. The endogenous cannabinoid system is involved in the regulation of the central reward pathway. Running wheel and sucrose consumption have rewarding and reinforcing properties in rodents, and share many neurochemical and behavioral characteristics with drug addiction. In this study, we investigated whether running wheel or sucrose consumption altered the sensitivity of striatal synapses to the activation of cannabinoid CB1 receptors. We found that cannabinoid CB1 receptor-mediated presynaptic control of striatal inhibitory postsynaptic currents was remarkably potentiated after these environmental manipulations. In contrast, the sensitivity of glutamate synapses to CB1 receptor stimulation was unaltered, as well as that of GABA synapses to the stimulation of presynaptic GABAB receptors. The sensitization of cannabinoid CB1 receptor-mediated responses was slowly reversible after the discontinuation of running wheel or sucrose consumption, and was also detectable following the mobilization of endocannabinoids by metabotropic glutamate receptor 5 stimulation. Finally, we found that the upregulation of cannabinoid transmission induced by wheel running or sucrose had a crucial role in the protective effects of these environmental manipulations against the motor and synaptic consequences of stress. Neuropsychopharmacology (2010) 35, 374-387; doi: 10.1038/npp.2009.141; published online 23 September 2009

Summary Aim Natalizumab (NTZ) discontinuation leads to multiple sclerosis (MS) recurrence, but represents the only known strategy to limit the risk of progressive multifocal leukoencephalopathy (PML) in JCV seropositive patients. Here, we compared the clinical and imaging features of three groups of patients who discontinued NTZ treatment. Methods We treated 25 patients with subcutaneous INFβ-1b (INF group), 40 patients with glatiramer acetate (GA group), and 40 patients with GA plus pulse steroid (GA+CS group). Results Six of 25 patients (24%) of the INF group were relapse-free 6 months after NTZ suspension. In GA group, a significant higher proportion of patients (26 of 40 patients, 65%) were relapse-free (P

Background Absence of clinical and radiological activity in relapsing–remitting multiple sclerosis (RRMS) is perceived as disease remission. We explored the role of persisting inflammation during remission in disease evolution. Methods Cerebrospinal fluid (CSF) levels of interleukin 1β (IL-1β), a major proinflammatory cytokine, were measured in 170 RRMS patients at the time of clinical and radiological remission. These patients were then followed up for at least 4 years, and clinical, magnetic resonance imaging (MRI) and optical coherence tomography (OCT) measures of disease progression were recorded. Results Median follow-up of RRMS patients was 5 years. Detection of CSF IL-1β levels at the time of remission did not predict earlier relapse or new MRI lesion formation. Detection of IL-1β in the CSF was instead associated with higher progression index (PI) and Multiple Sclerosis Severity Scale (MSSS) scores at follow-up, and the number of patients with sustained Expanded Disability Status Scale (EDSS) or Multiple Sclerosis Functional Composite worsening at follow-up was higher in individuals with detectable levels of IL-1β. Patients with undetectable IL-1β in the CSF had significantly lower PI and MSSS scores and a higher probability of having a benign MS phenotype. Furthermore, patients with undetectable CSF levels of IL-1β had less retinal nerve fiber layer thickness and macular volume alterations visualized by OCT compared to patients with detectable IL-1β. Conclusions Our results suggest that persistence of a proinflammatory environment in RRMS patients during clinical and radiological remission influences midterm disease progression. Detection of IL-1β in the CSF at the time of remission appears to be a potential negative prognostic factor in RRMS patients.

Synaptic transmission and plasticity mediated by NMDA receptors (NMDARs) could modulate the severity of multiple sclerosis (MS). Here the role of NMDARs in MS was first explored in 691 subjects carrying specific allelic variants of the NR1 subunit gene or of the NR2B subunit gene of this glutamate receptor. The analysis was replicated for significant SNPs in an independent sample of 1548 MS subjects. The C allele of rs4880213 was found to be associated with reduced NMDAR-mediated cortical excitability, and with increased probability of having more disability than the CT/TT MS subjects. MS severity was higher in the CC group among relapsing-remitting MS (RR-MS) patients, while primary progressive MS (PP-MS) subjects homozygous for the T allele had more pronounced clinical worsening. Mean time to first relapse, but not to an active MRI scan, was lower in the CC group of RR-MS patients, and the number of subjects with two or more clinical relapses in the first two years of the disease was higher in CC compared to CT/TT group. Furthermore, the percentage of relapses associated with residual disability was lower in subjects carrying the T allele. Lesion load at the MRI was conversely unaffected by the C or T allele of this SNP in RR-MS patients. Axonal and neuronal degeneration at the optical coherence tomography was more severe in the TT group of PP-MS patients, while reduced retinal nerve fiber thickness had less consequences on visual acuity in RR-MS patients bearing the T allele. Finally, the T allele was associated with preserved cognitive abilities at the Rao's brief repeatable neuropsychological battery in RR-MS. Signaling through glutamate NMDARs enhances both compensatory synaptic plasticity and excitotoxic neurodegeneration, impacting in opposite ways on RR-MS and PP-MS pathophysiological mechanisms.

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.

Background: Multiple sclerosis (MS) patients discontinuing natalizumab treatment are at risk of disease reactivation. No clinical or surrogate parameters exist to identify patients at risk of post-natalizumab MS reactivation. Objective: To determine the role of natalizumab-induced lymphocytosis and of Akt polymorphisms in disease reactivation after natalizumab discontinuation. Methods: Peripheral leukocyte count and composition were monitored in 93 MS patients during natalizumab treatment, and in 56 of these subjects who discontinued the treatment. Genetic variants of the anti-apoptotic protein Akt were determined in all subjects because natalizumab modulates the apoptotic pathway and lymphocyte survival is regulated by the apoptotic cascade. Results: Natalizumab-induced peripheral lymphocytosis protected from post-natalizumab MS reactivation. Subjects who relapsed or had magnetic resonance imaging (MRI) worsening after treatment cessation, in fact, had milder peripheral lymphocyte increases during the treatment, largely caused by less marked T cell increase. Furthermore, subjects carrying a variant of the gene coding for Akt associated with reduced anti-apoptotic efficiency (rs2498804T) had lower lymphocytosis and higher risk of disease reactivation. Conclusion: This study identified one functionally meaningful genetic variant within the Akt signaling pathway that is associated with both lymphocyte count and composition alterations during natalizumab treatment, and with the risk of disease reactivation after natalizumab discontinuation.

The functional significance of adult neural stem and progenitor cells in hippocampal-dependent learning and memory has been well documented. Although adult neural stem and progenitor cells in the subventricular zone are known to migrate to, maintain and reorganize the olfactory bulb, it is less clear whether they are functionally required for other processes. Using a conditional transgenic mouse model, selective ablation of adult neural stem and progenitor cells in the subventricular zone induced a dramatic increase in morbidity and mortality of central nervous system disorders characterized by excitotoxicity-induced cell death accompanied by reactive inflammation, such as 4-aminopyridine-induced epilepsy and ischaemic stroke. To test the role of subventricular zone adult neural stem and progenitor cells in protecting central nervous system tissue from glutamatergic excitotoxicity, neurophysiological recordings of spontaneous excitatory postsynaptic currents from single medium spiny striatal neurons were measured on acute brain slices. Indeed, lipopolysaccharide-stimulated, but not unstimulated, subventricular zone adult neural stem and progenitor cells reverted the increased frequency and duration of spontaneous excitatory postsynaptic currents by secreting the endocannabinod arachidonoyl ethanolamide, a molecule that regulates glutamatergic tone through type 1 cannabinoid receptor (CB(1)) binding. In vivo restoration of cannabinoid levels, either by administration of the type 1 cannabinoid receptor agonist HU210 or the inhibitor of the principal catabolic enzyme fatty acid amide hydrolase, URB597, completely reverted the increased morbidity and mortality of adult neural stem and progenitor cell-ablated mice suffering from epilepsy and ischaemic stroke. Our results provide the first evidence that adult neural stem and progenitor cells located within the subventricular zone exert an 'innate' homeostatic regulatory role by protecting striatal neurons from glutamate-mediated excitotoxicity.

Abnormal glutamate-dependent synaptic excitation contributes to neuronal damage in multiple sclerosis (MS). Little is known about the involvement of the GABA system in this disorder. Here we found that cerebrospinal fluid (CSF) from MS patients with enhanced brain lesions on magnetic resonance imaging inhibited GABA transmission in mouse brain slices. Enhanced IL-1β neuronal action was responsible for this effect, because IL-1β receptor antagonist blocked, and exogenous IL-1β mimicked the synaptic effect of inflamed CSF. Our results provide evidence that focal inflammation in MS perturbs the cytokine milieu within the circulating CSF, resulting in diffuse GABAergic alteration in neurons.

Multiple sclerosis (MS) causes a variety of motor and sensory deficits and it is also associated with mood disturbances. It is unclear if anxiety and depression in MS entirely reflect a subjective reaction to a chronic disease causing motor disability or rather depend on specific effects of neuroinflammation in neuronal circuits. To answer this question, behavioral, electrophysiological, and immunofluorescence experiments were performed in mice with experimental autoimmune encephalomyelitis (EAE), which models MS in mice. First, we observed high anxiety indexes in EAE mice, preceding the appearance of motor defects. Then, we demonstrated that tumor necrosis factor α (TNF-α) has a crucial role in anxiety associated with neuroinflammation. In fact, intracerebroventricular (icv) administration of etanercept, an inhibitor of TNF-α signaling, resulted in anxiolytic-like effects in EAE-mice. Accordingly, icv injection of TNF-α induced per se overt anxious behavior in control mice. Moreover, we propose the striatum as one of the brain regions potentially involved in EAE anxious behavior. We observed that before disease onset EAE striatum presents elevated TNF-α levels and strong activated microglia, early signs of inflammation associated with alterations of striatal excitatory postsynaptic currents (EPSCs). Interestingly, etanercept corrected the synaptic defects of pre-symptomatic EAE mice while icv injection of TNF-α in non-EAE mice altered EPSCs, thus mimicking the synaptic effects of EAE. In conclusion, anxiety characterizes EAE course since the very early phases of the disease. TNF-α released from activated microglia mediates this effect likely through the modulation of striatal excitatory synaptic transmission.

Approach or avoidance behaviors are accompanied by perceptual vigilance for, affective reactivity to and behavioral predisposition towards rewarding or punitive stimuli, respectively. We detected three subpopulations of C57BL/6J mice that responded with avoiding, balancing or approaching behaviors not induced by any experimental manipulation but spontaneously displayed in an approach/avoidance conflict task. Although the detailed neuronal mechanisms underlying the balancing between approach and avoidance are not fully clarified, there is growing evidence that endocannabinoid system (ECS) plays a critical role in the control of these balancing actions. The sensitivity of dorsal striatal synapses to the activation of cannabinoid CB1 receptors was investigated in the subpopulations of spontaneously avoiding, balancing or approaching mice. Avoiding animals displayed decreased control of CB1 receptors on GABAergic striatal transmission and in parallel increase of behavioral inhibition. Conversely, approaching animals exhibited increased control of CB1 receptors and in parallel increase of explorative behavior. Balancing animals reacted with balanced responses between approach and avoidance patterns. Treating avoiding animals with URB597 (fatty acid amide hydrolase inhibitor) or approaching animals with AM251 (CB1 receptor inverse agonist) reverted their respective behavioral and electrophysiological patterns. Therefore, enhanced or reduced CB1-mediated control on dorsal striatal transmission represents the synaptic hallmark of the approach or avoidance behavior, respectively. Thus, the opposite spontaneous responses to conflicting stimuli are modulated by a different involvement of endocannabinoid signaling of dorsal striatal neurons in the range of temperamental traits related to individual differences.

Interleukin-1β (IL-1β) is involved in mood alterations associated with inflammatory illnesses and with stress. The synaptic basis of IL-1β-induced emotional disturbances is still unknown. To address the possible involvement of the endocannabinoid system in IL-1β-induced anxiety, we performed behavioral and neurophysiological studies in mice exposed to stress or to intracerebroventricular injections of this inflammatory cytokine or of its antagonist. We found that a single intracerebroventricular injection of IL-1β caused anxiety in mice, and abrogated the sensitivity of cannabinoid CB1 receptors (CB1Rs) controlling GABA synapses in the striatum. Identical behavioral and synaptic results were obtained following social defeat stress, and intracerebroventricular injection of IL-1 receptor antagonist reverted both effects. IL-1β-mediated inhibition of CB1R function was secondary to altered cholesterol composition within membrane lipid rafts, and required intact function of the transient receptor potential vanilloid 1 (TRPV1) channel, another element of the endocannabinoid system. Membrane lipid raft disruption and inhibition of cholesterol synthesis, in fact, abrogated IL-1β–CB1R coupling, and TRPV1−/− mice were indeed insensitive to the synaptic and behavioral effects of both IL-1β and stress. On the other hand, cholesterol enrichment of striatal slices mimicked the synaptic effects of IL-1β on CB1Rs only in control mice, while the same treatment was ineffective in slices prepared from TRPV1−/− mice. The present investigation identifies a previously unrecognized interaction between a major proinflammatory cytokine and the endocannabinoid system in the pathophysiology of anxiety.

Background There are two generally accepted strategies for treating multiple sclerosis (MS), preventing central nervous system (CNS) damage indirectly through immunomodulatory interventions and/or repairing CNS damage by promoting remyelination. Both approaches also provide neuroprotection since they can prevent, indirectly or directly, axonal damage. Objective Recent experimental and clinical evidence indicates that the novel immunomodulatory drug laquinimod can exert a neuroprotective role in MS. Whether laquinimod-mediated neuroprotection is exerted directly on neuronal cells or indirectly via peripheral immunomodulation is still unclear. Methods C57Bl/6 experimental autoimmune encephalomyelitis (EAE) mice, immunised with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide, were treated for 26 days with subcutaneous daily injections of laquinimod (from 1 to 25 mg/kg). Patch clamp electrophysiology was performed on acute brain striatal slices from EAE mice treated with daily (25 mg/kg) laquinimod and on acute brain striatal slices from control mice bathed with laquinimod (1–30 µM). Results Both preventive and therapeutic laquinimod treatment fully prevented the alterations of GABAergic synapses induced by EAE, the first limiting also glutamatergic synaptic alterations. This dual effect might, in turn, have limited glutamatergic excitotoxicity, a phenomenon previously observed early during EAE and possibly correlated with later axonal damage. Furthermore, laquinimod treatment also preserved cannabinoid CB1 receptor sensitivity, normally lost during EAE. Finally, laquinimod per se was able to regulate synaptic transmission by increasing inhibitory post-synaptic currents and, at the same time, reducing excitatory post-synaptic currents. Conclusions Our data suggest a novel neuroprotective mechanism by which laquinimod might in vivo protect from neuronal damage occurring as a consequence of inflammatory immune-mediated demyelination.

Abnormal dopamine (DA) transmission in the striatum plays a pivotal role in attention-deficit/hyperactivity disorder (ADHD). As striatal DA signalling modulates the endocannabinoid system (ECS), the present study was aimed at investigating cannabinoid CB1 receptor (CB1R) function in a model of ADHD obtained by triple point-mutation in the dopamine transporter (DAT) gene in mice, making them insensitive to cocaine [DAT cocaine-insensitive (DAT-CI) mice]. DAT-CI mice had a marked hyperactive phenotype, and neurophysiological recordings revealed that the sensitivity of CB1Rs controlling GABA-mediated synaptic currents [CB1Rs((GABA)) ] in the striatum was completely lost. In contrast, CB1Rs modulating glutamate transmission [CB1Rs((Glu)) ], and GABA(B) receptors were not affected in this model of ADHD. In DAT-CI mice, the blockade of CB1R((GABA)) function was complete even after cocaine or environmental manipulations activating the endogenous DA-dependent reward system, which are known to sensitize these receptors in control animals. Conversely, the hedonic property of sucrose was intact in DAT-CI mice, indicating normal sweet perception in these animals. Our results point to CB1Rs as novel molecular players in ADHD, and suggest that therapeutic strategies aimed at interfering with the ECS might prove effective in this disorder.

Cerebellar compensation is a reliable model of lesion-induced plasticity occurring through profound synaptic and neurochemical modifications in cortical and sub-cortical regions. As the recovery from cerebellar deficits progresses, the firstly enhanced glutamate striatal transmission is then normalized. The time course of cerebellar compensation and the concomitant striatal modifications might be influenced by protocols of environmental enrichment (EE) differently timed in respect to cerebellar lesion. In the present study, we analyzed the effects of different EE protocols on postural and locomotor behaviors (by means of a neurological rating scale), and on striatal synaptic activity (by means of recordings of spontaneous glutamate-mediated excitatory postsynaptic currents (sEPSCs)) and on morphological correlates (by means of density and dendritic length of Fast Spiking (FS) interneurons) following hemicerebellectomy (HCb) in rats. Cerebellar motor deficits were reduced faster in the enriched animals in comparison to standard housed HCbed rats. The beneficial influence of EE was higher in the animals enriched before the HCb than in rats enriched only after the lesion. In parallel, the HCb-induced increase in striatal sEPSCs was not observed in rats enriched before HCb and attenuated in rats enriched after HCb. Furthermore, the EE prevented the shrinkage of dendritic arborization of FS striatal interneurons. Also this effect was more marked in animals enriched before than after the HCb. The exposure to EE exerted either neuro-protective or therapeutic actions on the cerebellar deficits. The experience-dependent changes of the synaptic and neuronal connectivity observed in the striatal neurons may represent one of the mechanisms through which the enrichment facilitates functional compensation following the cerebellar damage.

Cannabinoid CB1 receptors (CB1Rs) regulate the neurodegenerative damage of experimental autoimmune encephalomyelitis (EAE) and of multiple sclerosis (MS). The mechanism by which CB1R stimulation exerts protective effects is still unclear. Here we show that pharmacological activation of CB1Rs dampens the tumor necrosis factor α (TNFα)-mediated potentiation of striatal spontaneous glutamate-mediated excitatory postsynaptic currents (EPSCs), which is believed to cogently contribute to the inflammation-induced neurodegenerative damage observed in EAE mice. Furthermore, mice lacking CB1Rs showed a more severe clinical course and, in parallel, exacerbated alterations of sEPSC duration after induction of EAE, indicating that endogenous cannabinoids activate CB1Rs and mitigate the synaptotoxic action of TNFα in EAE. Consistently, we found that mice lacking the fatty acid amide hydrolase (FAAH), and thus expressing abnormally high brain levels of the endocannabinoid anandamide, developed a less severe EAE associated with preserved TNFα-induced sEPSC alterations. CB1Rs are important modulators of EAE pathophysiology, and might play a mechanistic role in the neurodegenerative damage of MS patients.

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.

The role of brain-derived neurotrophic factor (BDNF) in emotional processes suggests an interaction with the endocannabinoid system. Here, we addressed the functional interplay between BDNF and cannabinoid CB(1) receptors (CB(1)Rs) in the striatum, a brain area in which both BDNF and CB(1)s play a role in the emotional consequences of stress and of rewarding experiences. BDNF potently inhibited CB(1)R function in the striatum, through a mechanism mediated by altered cholesterol metabolism and membrane lipid raft function. The effect of BDNF was restricted to CB(1)Rs controlling GABA-mediated IPSCs (CB(1)R((GABA))), whereas CB(1)Rs modulating glutamate transmission and GABA(B) receptors were not affected. The action of BDNF on CB(1)R((GABA)) function was tyrosine kinase dependent and was complete even after receptor sensitization with cocaine or environmental manipulations activating the dopamine (DA)-dependent reward system. In mice lacking one copy of the BDNF gene (BDNF(+/−)), CB(1)R((GABA)) responses were potentiated and were preserved from the action of haloperidol, a DA D(2) receptor (D(2)R) antagonist able to fully abolish CB(1)R((GABA)) function in rewarded animals. Haloperidol also enhanced BDNF levels in the striatum, suggesting that this neurotrophin may act as a downstream effector of D(2)Rs in the modulation of cannabinoid signaling. Accordingly, 5 d cocaine exposure both reduced striatal BDNF levels and increased CB(1)R((GABA)) activity, through a mechanism dependent on D(2)Rs. The present study identifies a novel mechanism of CB(1)R regulation mediated by BDNF and cholesterol metabolism and provides some evidence that DA D(2)R-dependent modulation of striatal CB(1)R activity is mediated by this neurotrophin.

Transcriptional silencing of the gene encoding the fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS). FMRP acts as a translational repressor at central synapses, and molecular and synaptic plasticity studies have shown that the absence of this protein alters metabotropic glutamate 5 receptors (mGlu5Rs)-mediated signaling. In the striatum of mice lacking FMRP, we found enhanced activity of diacylglycerol lipase (DAGL), the enzyme limiting 2-arachidonoylglicerol (2-AG) synthesis, associated with altered sensitivity of GABA synapses to the mobilization of this endocannabinoid by mGlu5R stimulation with DHPG. Mice lacking another repressor of synaptic protein synthesis, BC1 RNA, also showed potentiated mGlu5R-driven 2-AG responses, indicating that both FMRP and BC1 RNA act as physiological constraints of mGlu5R/endocannabinoid coupling at central synapses. The effects of FMRP ablation on DAGL activity and on DHPG-mediated inhibition of GABA synapses were enhanced by simultaneous genetic inactivation of FMRP and BC1 RNA. In double FMRP and BC1 RNA lacking mice, striatal levels of 2-AG were also enhanced compared with control animals and to single mutants. Our data indicate for the first time that mGlu5R-driven endocannabinoid signaling in the striatum is under the control of both FMRP and BC1 RNA. The abnormal mGlu5R/2-AG coupling found in FMRP-KO mice emphasizes the involvement of mGlu5Rs in the synaptic defects of FXS, and identifies the modulation of the endocannabinoid system as a novel target for the treatment of this severe neuropsychiatric disorder.

It is increasingly accepted that excessive glutamate release plays a key role in the pathophysiology of grey matter damage in multiple sclerosis (MS). The mechanisms causing abnormal glutamate transmission in this disorder are however largely unexplored. By means of electrophysiological recordings from single striatal neurons in slices, we found that the presymptomatic and acute phases of experimental autoimmune encephalomyelitis (EAE), a preclinical model of MS, are associated with enhanced synaptic release of glutamate. The reverse mode of action of axonal Na(+)/Ca(++) exchanger, secondary to abnormal functioning of voltage-dependent Na(+) channels, was identified as a major cause of this alteration. In fact, inhibition of the Na(+)/Ca(++) exchanger with bepridil or with KB-R7943, which selectively blocks the reverse mode of the exchanger, reduced the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) recorded from striatal neurons in EAE mice but not in control animals. In the presence of tetrodotoxin (TTX), a blocker of voltage-dependent Na(+) channels, the effect of bepridil was normalized in acute (25 days post-immunization) EAE mice, indicating that axonal accumulation of Na(+) ions flowing through voltage-dependent Na(+) channels plays a role in the abnormal activity of the Na(+)/Ca(++) exchanger in EAE. Our data reveal an important role of Na(+)/Ca(++) exchanger and of voltage-dependent Na(+) channels in the pathological process of EAE, and provide a rationale for the use of neuroprotective strategies since the very early stages of MS.

Caffeine, the psychoactive ingredient of coffee and of many soft drinks, is frequently abused by humans especially during stressful live events. The endocannabinoid system is involved in the central effects of many psychoactive compounds and of stress. Whether caffeine alters the cannabinoid system and interferes with stress-induced synaptic alterations is however unknown. We have studied electrophysiologically the sensitivity of cannabinoid receptors modulating synaptic transmission in the striatum of mice exposed to caffeine in their drinking solution. Chronic caffeine assumption sensitized GABAergic synapses to the presynaptic effect of cannabinoid CB1 receptor stimulation by exo- and endocannabinoids. Caffeine was conversely unable to affect the sensitivity of cannabinoid receptors modulating glutamate transmission. The synaptic effects of caffeine were slowly reversible after its removal from the drinking solution. Furthermore, although exposure to caffeine for only 24h did not produce measurable changes of the sensitivity of cannabinoid CB1 receptors, it was able to contrast the down-regulation of CB1 receptor-mediated responses after social defeat stress. Our data suggest that the cannabinoid system is implicated in the psychoactive properties of caffeine and in the ability of caffeine to reduce the pathological consequences of stress.

Transient receptor potential vanilloid 1 (TRPV1) channels participate in the modulation of synaptic transmission in the periphery and in central structures. Here, we investigated the role of TRPV1 channels in the control of both excitatory and inhibitory transmission in the striatum. Pharmacological stimulation of TRPV1 channels with capsaicin (10 nM) selectively enhanced the frequency of glutamate-mediated spontaneous (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs) recorded from putative striatal medium spiny neurons. Capsaicin-mediated response underwent a rapid rundown, and was no longer detected in the majority of the neurons when the concentration of the drug was in the micromolar range, possibly due to receptor desensitization. Consistently, the totality of striatal neurons responded to capsaicin (10 nM or 10 microM) after prevention of desensitization of TRPV1 channels with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA). PMA was able per se to increase sEPSC frequency. The effects of capsaicin and of PMA were absent after pharmacological or genetic inactivation of TRPV1 channels. Finally, we provided evidence for anandamide as an endovanilloid substance in the striatum, since genetic inhibition of anandamide degradation resulted in a tonic activation of TRPV1 channels modulating glutamate but not GABA release. TRPV1-mediated regulation of excitatory transmission in the striatum might be important for the final output to other basal ganglia structures, and might play a role in several physiological and pathological processes.

Recent findings proposed that the cerebellum and the striatum, key structures in motor control, are more interconnected than commonly believed, and that the cerebellum may influence striatal activity. In the present study, the possible changes of synaptic transmission in the striatum of hemicerebellectomized rats have been investigated. Neurophysiological recordings showed a significant facilitation of glutamate transmission in the contralateral striatum occurring early following hemicerebellectomy. This process of synaptic adaptation appears to be relevant for the compensation of cerebellar deficits. Accordingly, pharmacological blockade of glutamate N-methyl-d-aspartate (NMDA) receptors with MK-801 prevented the rearrangement of excitatory synapses in the striatum and interfered with the recovery from motor disturbances in rats with cerebellar lesions. Hemicerebellectomy also perturbed gamma-aminobutyric acid (GABA) transmission in contralateral but not ipsilateral striatum. The present findings advance the role of striatal excitatory transmission in the compensation of cerebellar deficits, providing support to the notion that adaptations of striatal function exert a role in the recovery of cerebellar symptoms.

Rhes is a small GTP-binding protein prominently localized in the striatum. Previous findings obtained in cell culture systems demonstrated an involvement of Rhes in cAMP/PKA signalling pathway, at a level proximal to the activation of heterotrimeric G-protein complex. However, its role in the striatum has been, so far, only supposed. Here we studied the involvement of Rhes in dopaminergic signalling, by employing mice with a null mutation in the Rhes gene. We demonstrated that the absence of Rhes modulates cAMP/PKA signalling in both striatopallidal and striatonigral projection neurons by increasing Golf. protein levels and, in turn, influencing motor responses challenged by dopaminergic agonist/antagonist. Interestingly, we also show that Rhes is required for a correct dopamine-mediated GTP binding, a function mainly associated to stimulation of dopamine D2 receptors. Altogether, our results indicate that Rhes is an important modulator of dopaminergic transmission in the striatum. (C) 2007 Elsevier Inc. All rights reserved.

The ability of cannabinoids to modulate both inflammatory and degenerative neuronal damage prompted investigations on the potential benefits of such compounds in multiple sclerosis (MS) and in animal models of this disorder. Here we measured endocannabinoid levels, metabolism and binding, and physiological activities in 26 patients with MS (17 females, aged 19-43 years), 25 healthy controls and in mice with experimental autoimmune encephalomyelitis (EAE), a preclinical model of MS. Our results show that MS and EAE are associated with significant alterations of the endocannabinoid system. We found that anandamide (AEA), but not 2-arachidonoylglycerol (2-AG), was increased in the CSF of relapsing MS patients. AEA concentrations were also higher in peripheral lymphocytes of these patients, an effect associated with increased synthesis and reduced degradation of this endocannabinoid. Increased synthesis, reduced degradation, and increased levels of AEA were also detected in the brains of EAE mice in the acute phase of the disease, possibly accounting for its anti-excitotoxic action in this disorder. Accordingly, neurophysiological recordings from single neurons confirmed that excitatory transmission in EAE slices is inhibited by CB1 receptor activation, while inhibitory transmission is not. Our study suggests that targeting the endocannabinoid system might be useful for the treatment of MS.

Genomic location of sequences encoding small nucleolar RNAs (snoRNAs) is peculiar in all eukaryotes from yeast to mammals: most of them are encoded within the introns of host genes. In Saccharomyces cerevisiae, seven snoRNAs show this location. In this work we demonstrate that the position of snoRNA-coding regions with respect to splicing consensus sequences is critical: yeast strains expressing mutant constructs containing shorter or longer spacers (the regions between snoRNA ends and intron splice sites) show a drop in accumulation of U24 and U18 snoRNAs. Further mutational analysis demonstrates that altering the distance between the 3′ end of the snoRNA and the branch point is the most important constraint for snoRNA biosynthesis, and that stable external stems, which are sometimes present in introns containing snoRNAs, can overcome the positional effect. Surprisingly enough, splicing of the host introns is clearly affected in most of these constructs indicating that, at least in S. cerevisiae, an incorrect location of snoRNA-coding sequences within the host intron is detrimental to the splicing process. This is different with respect to what was demonstrated in mammals, where the activity of the splicing machinery seems to be dominant with respect to the assembly of snoRNPs, and it is not affected by the location of snoRNA sequences. We also show that intronic box C/D snoRNA recognition and assembly of snoRNPs occur during transcription when splicing sequences are recognized.

Dopamine D2receptor (D2DR)-mediated transmission in the striatum is remarkably flexible, and changes in its efficacy have been heavily implicated in a variety of physiological and pathological conditions. Although receptor-associated proteins are clearly involved in specific forms of synaptic plasticity, the molecular mechanisms regulating the sensitivity of D2receptors in this brain area are essentially obscure. We have studied the physiological responses of the D2DR stimulations in mice lacking the brain cytoplasmic RNA BC1, a small noncoding dendritically localized RNA that is supposed to play a role in mRNA translation. We show that the efficiency of D2-mediated transmission regulating striatal GABA synapses is under the control of BC1 RNA, through a negative influence on D2receptor protein level affecting the functional pool of receptors. Ablation of the BC1 gene did not result in widespread dysregulation of synaptic transmission, because the sensitivity of cannabinoid CB1receptors was intact in the striatum of BC1 knock-out (KO) mice despite D2and CB1receptors mediated similar electrophysiological actions. Interestingly, the fragile X mental retardation protein FMRP, one of the multiple BC1 partners, is not involved in the BC1 effects on the D2-mediated transmission. Because D2DR mRNA is apparently equally translated in the BC1-KO and wild-type mice, whereas the protein level is higher in BC1-KO mice, we suggest that BC1 RNA controls D2DR indirectly, probably regulating translation of molecules involved in D2DR turnover and/or stability.

Synaptic transmission in the striatum is regulated by metabotropic glutamate (mGlu) receptors through pre- and postsynaptic mechanisms. We investigated the involvement of mGlu 1 and 5 receptors in the control of both excitatory and inhibitory transmission in the striatum. The mGlu 1 and 5 receptor agonist 3,5-DHPG failed to affect glutamate transmission, while it caused a biphasic effect on GABA transmission, characterized by early increase and late decrease in the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from striatal principal neurons. Both mGlu 1 and 5 receptors were involved in the early response to 3,5-DHPG, through membrane depolarization of striatal GABAergic interneurons and action potential generation. The 3,5-DHPG-mediated late depression of inhibitory inputs to striatal principal neurons was conversely secondary to mGlu 5 receptor activation and subsequent endocannabinoid release. In conclusion, we have identified an mGlu-dependent mechanism of GABA transmission regulation of potential relevance for physiological neuronal activity.

Decreased activity of mitochondrial complex I and II is implicated in the pathophysiology of progressive supranuclear palsy (PSP) and Huntington's disease (HD), respectively. Both disorders preferentially affect the nucleus striatum, a brain area particularly vulnerable to excitotoxic damage. To gain insights into the pathophysiology of neuronal degeneration during PSP and HD, here we studied the possible interplay between excitatory transmission and mitochondrial complex I and II inhibition in the development of striatal damage. By using in vitro neurophysiological recordings and cell swelling measures in corticostriatal slices, we found that stimulation of NMDA receptors significantly contributed to the neurotoxic effects of 3-nitropropionic acid (3-NP) but not of rotenone, selective inhibitors of mitochondrial complex II and I, respectively. We also found that blockade of a subset of NMDA receptors containing the NR2B subunit was sufficient to protect the striatum from the injurious effects of 3-NP, an effect unrelated to the prevention of membrane excitation by NMDA receptor stimulation. Pharmacological inhibition of dopamine receptors, conversely, failed to modulate both rotenone- and 3-NP-induced neuronal damage. Our results indicate that the cellular mechanisms leading to striatal neuronal death are different following inhibition of distinct mitochondrial complexes of the respiratory chain, implying that neuroprotective strategies in PSP and HD must significantly differ.