Maria Teresa Viscomi

N -Arachidonoyl-Dopamine Tunes Synaptic Transmission onto Dopaminergic Neurons by Activating both Cannabinoid and Vanilloid Receptors

In the present study, we used electrophysiological, biochemical, and confocal microscopy techniques, to investigate the functional role of transient receptor potential vanilloid type 1 (TRPV1) and cannabinoid type 1 receptors (CB1-R) in the substantia nigra pars compacta (SNpc) and their stimulation by the endocannabinoid N-arachidonoyl-dopamine (NADA). Liquid chromatography–mass spectrometry analyses revealed that a NADA-like compound is produced in substantia nigra slices, in conditions of hyperactivity. Moreover, the functional role of both TRPV1 and CB1-R in modulating synaptic transmission in this area was suggested by confocal microscopy data, showing TRPV1 and CB1-R immunoreactivity in punctate structures, probably representing synaptic contacts on cell bodies of the SNpc. In patch-clamp recordings from dopamine (DA) neurons of the SNpc, we found that NADA increases or reduces glutamatergic transmission onto DA neurons by activating TRPV1 and CB1 receptors, respectively, whereas it decreases GABAergic transmission via CB1 stimulation. Facilitation of glutamate release through TRPV1 was blocked in the presence of a selective blocker of the putative endocannabinoid membrane transporter (EMT), indicating that NADA needs to be taken up by cells to interact with this receptor. In line with these data, biochemical results demonstrated that NADA selectively acted at CB1-R when its re-uptake was blocked. Altogether these data demonstrate a significant role exerted by the endocannabinoid/endovanilloid NADA in the regulation of synaptic transmission to DA neurons of the SNpc. Moreover, they highlight a key function of the EMT transporter in promoting the stimulation of TRPV1 or CB1-R, thus favoring facilitation or inhibition of glutamate synaptic release.

Selective CB2 Receptor Agonism Protects Central Neurons from Remote Axotomy-Induced Apoptosis through the PI3K/Akt Pathway

Endocannabinoids are neuroprotective in vivo and in vitro, but the mechanisms by which they act are largely unknown. The present study addressed the role of cannabinoid receptors during remote cell death of central neurons in a model that is based on cerebellar lesions. A lesion in one cerebellar hemisphere induced remote cell death and type 2 cannabinoid receptor (CB2R) expression in contralateral precerebellar neurons. Of the selective agonists and antagonists that modulated cannabinoid receptor activity, we found that the CB2R agonist JWH-015 reduced neuronal loss and cytochrome-c release, leading to neurological recovery; these effects were reversed by the selective CB2R antagonist SR144528. Analysis of CB2R-triggered signal transduction demonstrated that in axotomized neurons, CB2R regulated Akt and JNK phosphorylation through a PI3K-dependent pathway, whereas other major signaling routes that are dependent on CB2R, such as ERK1/2 and p38, were not involved. This result was corroborated by the observation that the selective PI3K inhibitor LY294002 blocked the CB2R stimulation effects on neuronal survival as well as Akt and JNK phosphorylation levels. Together, these data demonstrate that axonal damage induces CB2R expression in central neurons and that stimulation of this receptor has a neuroprotective effect that is achieved through PI3K/Akt signaling.

Distinct Levels of Dopamine Denervation Differentially Alter Striatal Synaptic Plasticity and NMDA Receptor Subunit Composition

A correct interplay between dopamine (DA) and glutamate is essential for corticostriatal synaptic plasticity and motor activity. In an experimental model of Parkinsons disease (PD) obtained in rats, the complete depletion of striatal DA, mimicking advanced stages of the disease, results in the loss of both forms of striatal plasticity: long-term potentiation (LTP) and long-term depression (LTD). However, early PD stages are characterized by an incomplete reduction in striatal DA levels. The mechanism by which this incomplete reduction in DA level affects striatal synaptic plasticity and glutamatergic synapses is unknown. Here we present a model of early PD in which a partial denervation, causing mild motor deficits, selectively affects NMDA-dependent LTP but not LTD and dramatically alters NMDA receptor composition in the postsynaptic density. Our findings show that DA decrease influences corticostriatal synaptic plasticity depending on the level of depletion. The use of the TAT2A cell-permeable peptide, as an innovative therapeutic strategy in early PD, rescues physiological NMDA receptor composition, synaptic plasticity, and motor behavior.

P2X2R purinergic receptor subunit mRNA and protein are expressed by all hypothalamic hypocretin/orexin neurons

Neurophysiologic data suggest that orexin neurons are directly excited by ATP through purinergic receptors (P2XR). Anatomical studies, though reporting P2XR in the hypothalamus, did not describe it in the perifornical hypothalamic area, where orexinergic neurons are located. Here we report the presence of the P2X2R subunit in the rat perifornical hypothalamus and demonstrate that hypothalamic orexin neurons express the P2X2R. Double immunohistochemistry showed that virtually all orexin‐immunoreactive neurons are also P2X2R immunoreactive, whereas 80% of P2X2R‐immunoreactive neurons are also orexin positive. Triple‐labeling experiments, combining fluorescence in situ hybridization for P2X2R mRNA and P2X2R/orexin double immunofluorescence, confirmed these findings. In addition, in situ hybridization demonstrated that P2X2R mRNA is localized in cellular processes of orexinergic neurons. The present data support neurophysiologic findings on ATP modulation of orexinergic function and provide direct evidence that the entire population of orexin neurons expresses a P2XR subtype, namely, P2X2R. Thus, purinergic transmission might intervene in modulating key functions known to be controlled by the orexinergic system, such as feeding behavior and arousal. J. Comp. Neurol. 498:58–67, 2006.

Impaired striatal D2 receptor function leads to enhanced GABA transmission in a mouse model of DYT1 dystonia.

DYT1 dystonia is caused by a deletion in a glutamic acid residue in the C-terminus of the protein torsinA, whose function is still largely unknown. Alterations in GABAergic signaling have been involved in the pathogenesis of dystonia. We recorded GABA- and glutamate-mediated synaptic currents from a striatal slice preparation obtained from a mouse model of DYT1 dystonia. In medium spiny neurons (MSNs) from mice expressing human mutant torsinA (hMT), we observed a significantly higher frequency, but not amplitude, of GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature currents (mIPSCs), whereas glutamate-dependent spontaneous excitatory synaptic currents (sEPSCs) were normal. No alterations were found in mice overexpressing normal human torsinA (hWT). To identify the possible sources of the increased GABAergic tone, we recorded GABAergic Fast-Spiking (FS) interneurons that exert a feed-forward inhibition on MSNs. However, both sEPSC and sIPSC recorded from hMT FS interneurons were comparable to hWT and non-transgenic (NT) mice. In physiological conditions, dopamine (DA) D2 receptor act presynaptically to reduce striatal GABA release. Of note, application of the D2-like receptor agonist quinpirole failed to reduce the frequency of sIPSCs in MSNs from hMT as compared to hWT and NT mice. Likewise, the inhibitory effect of quinpirole was lost on evoked IPSCs both in MSNs and FS interneurons from hMT mice. Our findings demonstrate a disinhibition of striatal GABAergic synaptic activity, that can be at least partially attributed to a D2 DA receptor dysfunction.

Metabotropic Glutamate Receptor 1 Mediates the Electrophysiological and Toxic Actions of the Cycad Derivative β-N-Methylamino-l-Alanine on Substantia Nigra Pars Compacta DAergic Neurons

Amyotrophic lateral sclerosis–Parkinson dementia complex (ALS-PDC) is a neurodegenerative disease with ALS, parkinsonism, and Alzheimers symptoms that is prevalent in the Guam population. β-N-Methylamino alanine (BMAA) has been proposed as the toxic agent damaging several neuronal types in ALS-PDC, including substantia nigra pars compacta dopaminergic (SNpc DAergic) neurons. BMAA is a mixed glutamate receptor agonist, but the specific pathways activated in DAergic neurons are not yet known. We combined electrophysiology, microfluorometry, and confocal microscopy analysis to monitor membrane potential/current, cytosolic calcium concentration ([Ca2+]i) changes, cytochrome-c (cyt-c) immunoreactivity, and reactive oxygen species (ROS) production induced by BMAA. Rapid toxin applications caused reversible membrane depolarization/inward current and increase of firing rate and [Ca2+]i in DAergic neurons. The inward current (IBMAA) was mainly mediated by activation of metabotropic glutamate receptor 1 (mGluR1), coupled to transient receptor potential (TRP) channels, and to a lesser extent, AMPA receptors. Indeed, mGluR1 (CPCCOEt) and TRP channels (SKF 96365; Ruthenium Red) antagonists reduced IBMAA, and a small component of IBMAA was reduced by the AMPA receptor antagonist CNQX. Calcium accumulation was mediated by mGluR1 but not by AMPA receptors. Application of a low concentration of NMDA potentiated the BMAA-mediated calcium increase. Prolonged exposure to BMAA caused significant modifications of membrane properties, calcium overload, cell shrinkage, massive cyt-c release into the cytosol and ROS production. In SNpc GABAergic neurons, BMAA activated only AMPA receptors. Our study identifies the mGluR1-activated mechanism induced by BMAA that may cause the neuronal degeneration and parkinsonian symptoms seen in ALS-PDC. Moreover, environmental exposure to BMAA might possibly also contribute to idiopathic PD.

Lipid rafts regulate 2-arachidonoylglycerol metabolism and physiological activity in the striatum

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‐β‐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.

Dopamine neuronal loss contributes to memory and reward dysfunction in a model of Alzheimer’s disease

Alterations of the dopaminergic (DAergic) system are frequently reported in Alzheimers disease (AD) patients and are commonly linked to cognitive and non-cognitive symptoms. However, the cause of DAergic system dysfunction in AD remains to be elucidated. We investigated alterations of the midbrain DAergic system in the Tg2576 mouse model of AD, overexpressing a mutated human amyloid precursor protein (APPswe). Here, we found an age-dependent DAergic neuron loss in the ventral tegmental area (VTA) at pre-plaque stages, although substantia nigra pars compacta (SNpc) DAergic neurons were intact. The selective VTA DAergic neuron degeneration results in lower DA outflow in the hippocampus and nucleus accumbens (NAc) shell. The progression of DAergic cell death correlates with impairments in CA1 synaptic plasticity, memory performance and food reward processing. We conclude that in this mouse model of AD, degeneration of VTA DAergic neurons at pre-plaque stages contributes to memory deficits and dysfunction of reward processing.

Remote Neurodegeneration: Multiple Actors for One Play

Remote neurodegeneration significantly influences the clinical outcome in many central nervous system (CNS) pathologies, such as stroke, multiple sclerosis, and traumatic brain and spinal cord injuries. Because these processes develop days or months after injury, they are accompanied by a therapeutic window of opportunity. The complexity and clinical significance of remote damage is prompting many groups to examine the factors of remote degeneration. This research is providing insights into key unanswered questions, opening new avenues for innovative neuroprotective therapies. In this review, we evaluate data from various remote degeneration models to describe the complexity of the systems that are involved and the importance of their interactions in reducing damage and promoting recovery after brain lesions. Specifically, we recapitulate the current data on remote neuronal degeneration, focusing on molecular and cellular events, as studied in stroke and brain and spinal cord injury models. Remote damage is a multifactorial phenomenon in which many components become active in specific time frames. Days, weeks, or months after injury onset, the interplay between key effectors differentially affects neuronal survival and functional outcomes. In particular, we discuss apoptosis, inflammation, oxidative damage, and autophagy—all of which mediate remote degeneration at specific times. We also review current findings on the pharmacological manipulation of remote degeneration mechanisms in reducing damage and sustaining outcomes. These novel treatments differ from those that have been proposed to limit primary lesion site damage, representing new perspectives on neuroprotection.

Increased levels of p70S6 phosphorylation in the G93A mouse model of Amyotrophic Lateral Sclerosis and in valine-exposed cortical neurons in culture

The higher risk factor for Amyotrophic Lateral Sclerosis (ALS) among Italian soccer players is a question that is still debated. One of the hypotheses that have been formulated to explain a possible link between ALS and soccer players is related to the abuse of dietary supplements and drugs for enhancing sporting performance. In particular, it has been reported that branched-chain amino acids (BCAAs) are widely used among athletes as nutritional supplements. To observe the possible effect of BCAAs on neuronal electrical properties, we performed electrophysiological experiments on Control cultured cortical neurons and on neurons after BCAA treatment. BCAA-treated neurons showed hyperexcitability and rapamycin was able to suppress it and significantly reduce the level of mTOR, Akt and p70S6 phosphorylation. Interestingly, the hyperexcitability previously reported in cortical neurons from a genetic mouse model of ALS (G93A) was also reversed by rapamycin treatment. Moreover, both G93A and valine-treated neurons presented significantly higher levels of Pp70S6 when compared to control neurons, strongly indicating the involvement of this substrate in ALS pathology. Finally, we performed electrophysiological experiments on motor cortex slices from Control and G93A mice and those fed with a BCAA-enriched diet. We observed that neuron excitability was comparable between G93A and BCAA-enriched diet mice, but was significantly higher than in Control mice. These findings, besides strongly indicating that BCAAs specifically induce hyperexcitability, seem to suggest the involvement of p70S6 substrate in ALS pathology.