Silvia Marinelli

Capsaicin activation of glutamatergic synaptic transmission in the rat locus coeruleus in vitro

The vanilloid receptor protein (VR1) is a well‐characterised integrator of noxious stimuli in peripheral sensory neurones. There is evidence for the presence of VR1 in the central nervous system, but little information as to its role there. In this study we have examined the actions of agonists for VR1 receptors in the rat locus coeruleus (LC), using whole‐cell patch‐clamp recordings from acutely isolated neurones and neurones in slices. Superfusion with capsaicin resulted in a concentration‐dependent increase in the frequency of isolated miniature excitatory postsynaptic currents (mEPSCs) in LC neurones. The mean amplitude of the mEPSCs was not affected by capsaicin. The effects of capsaicin (1 μM) were abolished by the VR1 receptor antagonists capsazepine (10 μM) and iodoresiniferatoxin (300 nm). Removal of extracellular Ca2+ abolished the capsaicin‐induced increase in frequency of mEPSCs. Capsaicin superfusion had no consistent effects on evoked excitatory postsynaptic currents. Capsaicin superfusion also resulted in the release of an adrenoceptor agonist in the LC but did not affect the membrane currents of acutely isolated LC neurones. These data demonstrate that the VR1 receptor appears to be located presynaptically on afferents to the LC, and that activation of VR1 may serve to potentiate the release of glutamate and adrenaline/noradrenaline in this brain region.

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.

Activation of TRPV1 in the VTA excites dopaminergic neurons and increases chemical- and noxious-induced dopamine release in the nucleus accumbens.

Dopamine (DA)-containing neurons of the ventral tegmental area (VTA) provide dopaminergic input to the nucleus accumbens and to the prefrontal cortex within the mesolimbic pathway. In the present study, we combined electrophysiological recordings and microdialysis techniques to investigate the function of transient receptor potential vanilloid 1 (TRPV1) channel in the VTA. In brain slices, application of the TRPV1 receptor agonist capsaicin increased the firing rate of rat dopamine neurons and in a proportion of tested cells (44%) it also induced a bursting behavior. The effects of capsaicin were concentration dependent. The increase in neuronal firing was dependent on enhanced glutamatergic transmission since it was blocked by the superfusion of the ionotropic glutamate antagonists, CNQX and AP5. Interestingly, microinjection of capsaicin into the VTA and noxious tail stimulation transiently enhanced dopamine release into the nucleus accumbens. Both the in vitro and in vivo effects were mediated by TRPV1 activation in the VTA since they were reduced by co-perfusion of the selective TRPV1 receptor antagonist iodoresineferatoxin. Our data suggest a novel role for TRPV1 channels in the mesencephalon of rat, namely activation of the DA system following a peripheral noxious stimulation.

Self-modulation of neocortical pyramidal neurons by endocannabinoids

Control of pyramidal neuron excitability is vital for the functioning of the neocortex. Somatodendritic slow self-inhibition (SSI) allows inhibitory neurons to regulate their own activity, but the existence of similar mechanisms in excitatory cells has not been shown. We found that in rodents endocannabinoids mediated SSI and long-term modulation of inhibitory connections in layer 2/3 pyramidal neurons with a distinct dendritic morphology, suggesting that a glutamatergic network in cortical circuits is self-regulated.

The Endocannabinoid 2-Arachidonoylglycerol Is Responsible for the Slow Self-Inhibition in Neocortical Interneurons

In the CNS, endocannabinoids are identified mainly as two endogenous lipids: anandamide, the ethanolamide of arachidonic acid, and 2-arachidonoylglycerol (2-AG). Endocannabinoids are known to inhibit transmitter release from presynaptic terminals; however we have recently demonstrated that they are also involved in slow self-inhibition (SSI) of layer V low-threshold spiking (LTS) interneurons in rat somatosensory cortex. SSI is induced by repetitive firing in LTS cells, which can express either cholecystokinin or somatostatin. SSI is triggered by an endocannabinoid-dependent activation of a prolonged somatodendritic K+ conductance and associated hyperpolarization in the same cell. The synthesis of both endocannabinoids is dependent on elevated [Ca2+]i such as occurs during sustained neuronal activity. To establish whether 2-AG mediates autocrine LTS-SSI, we blocked its biosynthesis from phospholipase C (PLC) and diacylglycerol lipases (DAGLs). Current-clamp recordings from LTS interneurons in acute neocortical slices showed that inclusion of DAGL inhibitors in the whole-cell pipette prevented the long-lasting hyperpolarization triggered by LTS cell repetitive firing. Similarly, extracellular applications of a PLC inhibitor prevented SSI in LTS interneurons. Moreover, metabotropic glutamate receptor-dependent activation of PLC produced a long-lasting hyperpolarization which was prevented by the CB1 antagonist AM251, as well as by PLC and DAGL inhibitors. The loss of SSI in the presence of intracellular DAGL blockers confirms that endocannabinoid production occurs in the same interneuron undergoing the persistent hyperpolarization. Since DAGLs produce no endocannabinoid other than 2-AG, these results identify this compound as the autocrine mediator responsible for the postsynaptic slow self-inhibition of neocortical LTS interneurons.

Altered cortico-striatal synaptic plasticity and related behavioural impairments in reeler mice

Reelin‐deficient mice have been used to investigate the role of this extracellular protein in cortico‐striatal plasticity and striatum‐related behaviours. Here we show that a repetitive electrical stimulation of the cortico‐striatal pathway elicited long‐term potentiation (LTP) in homozygous reeler (rl/rl) mice, while causing long‐term depression in their wild‐type (+/+) littermates. The N‐methyl‐d‐aspartic acid (NMDA) receptor antagonist d‐(–)‐2 amino‐5‐phosphonopentanoic acid prevented the induction of LTP in (rl/rl) mice, thus confirming that this form of synaptic plasticity was NMDA receptor‐dependent. Interestingly, in the presence of tiagabine, a blocker of γ‐aminobutyric acid (GABA) re‐uptake system, the probability that (rl/rl) mice showed LTP decreased significantly, thus suggesting an impaired GABAergic transmission in reeler mutants. Consistent with this view, a decreased density of parvalbumin‐positive GABAergic striatal interneurons was found in (rl/rl) mice in comparison to (+/+) mice. Finally, compatible with their abnormal striatal function (rl/rl) mice exhibited procedural learning deficits. Our data, showing alterations in cortico‐striatal plasticity largely depending on a depressed GABAergic tone, delineate a mechanism whereby the lack of reelin may affect cognitive functions.

Actions of nociceptin/orphanin FQ and other prepronociceptin products on rat rostral ventromedial medulla neurons in vitro.

1 Whole‐cell patch clamp recordings were made from rat rostral ventromedial medulla (RVM) neurons in vitro to investigate the cellular actions of the opioid‐like receptor ORL1 (NOP), ligand nociceptin/orphanin FQ and other putative prepronociceptin products. 2 Primary and secondary RVM neurons were identified as responding to the κ‐opioid receptor agonist U‐69593 (300 nm to 1 μm) and the μ‐ and δ‐opioid receptor agonist met‐enkephalin (10 μm), respectively. Both primary and secondary RVM neurons responded to nociceptin (3 nm to 1 μm) with an outward current that reversed polarity at –115 mV in brain slices and with inhibition of Ca2+ channel currents in acutely isolated cells. 3 The putative ORL1 antagonist J‐113397 (1 μm) produced no change in membrane current and abolished the outward current produced by nociceptin (100 nm). In contrast, Phe1ψ(CH2‐NH)Gly2]‐nociceptin‐(1‐13)NH2 (300 nm to 1 μm) alone produced an outward current and partially reduced the outward current produced by nociceptin (300 nm) when co‐applied. 4 In brain slices nociceptin (300 nm) reduced the amplitude of evoked GABAA receptor‐mediated inhibitory postsynaptic currents (IPSCs) but not non‐NMDA receptor‐mediated excitatory postsynaptic currents (EPSCs). 5 Met‐enkephalin (10 μm), but not nociceptin (300 nm), reduced the rate of spontaneous miniature IPSCs in normal external potassium solution (K+ 2.5 mm). In high external potassium (K+ 17.5 mm), nociceptin reduced the rate of miniature IPSCs in the presence (Ca2+ 2.4 mm, Mg2+ 1.2 mm) but not in the absence of external calcium (Ca2+ 0 mm, Mg2+ 10 mm, Cd2+ 10 μm). Nociceptin and met‐enkephalin had no effect on the amplitude of miniature IPSCs. 6 The putative nociceptin precursor products nocistatin (rat prepronociceptin125–132) and rat prepronociceptin154–181 had no effect on membrane currents, evoked IPSCs and evoked EPSCs. 7 These results indicate that nociceptin acts via the ORL1 receptor to directly inhibit both primary and secondary RVM neurons by activating a potassium conductance and by inhibiting calcium conductances. In addition, nociceptin inhibits GABA release within the RVM via a presynaptic Ca2+‐dependent mechanism. Thus, nociceptin has the potential to exert both disinhibitory and inhibitory effects on neuronal action potential firing within the RVM.

Desynchronization of Neocortical Networks by Asynchronous Release of GABA at Autaptic and Synaptic Contacts from Fast-Spiking Interneurons

An activity-dependent long-lasting asynchronous release of GABA from identified fast-spiking inhibitory neurons in the neocortex can impair the reliability and temporal precision of activity in a cortical network.

Electrophysiology of the neuroprotective agent riluzole on striatal spiny neurons

Striatal spiny neurons are selectively vulnerable in Huntingtons disease (HD). No effective treatment is available to limit neuronal death in this pathological condition. In an experimental model of HD, a beneficial effect has recently been reported by the neuroprotective agent riluzole. We performed intracellular recordings in order to characterize the electrophysiological effects of this compound on striatal spiny neurons. Riluzole (0.1-100 microM) affected neither the resting membrane potential nor the input resistance/membrane conductance of the recorded cells. Bath application of this pharmacological agent produced a dose-dependent reduction of the number of spikes evoked by long-lasting depolarizing pulses. The EC50 value for this effect was 0.5 microM. Low doses of riluzole selectively reduced the firing frequency in the last part of the depolarizing pulse suggesting a use-dependent action at low concentrations of this compound. Riluzole produced a dose-dependent reduction of the amplitude of the corticostriatal glutamatergic excitatory post-synaptic potentials (EPSPs) with an extrapolated EC50 value of 6 microM. This effect was reversible and maximal at a concentration of 100 microM. Paired-pulse facilitation (PPF) was not affected by riluzole suggesting that the reduction of excitatory transmission was not only caused by a decrease of presynaptic release. Accordingly, riluzole also reduced the amplitude of membrane depolarization induced by exogenous glutamate. The modulatory action of riluzole on the activity of striatal spiny neurons might support the use of this drug in experimental models of excitotoxicity and in the neurodegenerative disorders involving the striatum.

ProNGF\NGF imbalance triggers learning and memory deficits, neurodegeneration and spontaneous epileptic-like discharges in transgenic mice

ProNGF, the precursor of mature nerve growth factor (NGF), is the most abundant form of NGF in the brain. ProNGF and mature NGF differ significantly in their receptor interaction properties and in their bioactivity. ProNGF increases markedly in the cortex of Alzheimer’s disease (AD) brains and proNGF\NGF imbalance has been postulated to play a role in neurodegeneration. However, a direct proof for a causal link between increased proNGF and AD neurodegeneration is lacking. In order to evaluate the consequences of increased levels of proNGF in the postnatal brain, transgenic mice expressing a furin cleavage-resistant form of proNGF, under the control of the neuron-specific mouse Thy1.2 promoter, were derived and characterized. Different transgenic lines displayed a phenotypic gradient of neurodegenerative severity features. We focused the analysis on the two lines TgproNGF#3 and TgproNGF#72, which shared learning and memory impairments in behavioral tests, cholinergic deficit and increased Aβ-peptide immunoreactivity. In addition, TgproNGF#3 mice developed Aβ oligomer immunoreactivity, as well as late diffuse astrocytosis. Both TgproNGF lines also display electrophysiological alterations related to spontaneous epileptic-like events. The results provide direct evidence that alterations in the proNGF/NGF balance in the adult brain can be an upstream driver of neurodegeneration, contributing to a circular loop linking alterations of proNGF/NGF equilibrium to excitatory/inhibitory synaptic imbalance and amyloid precursor protein (APP) dysmetabolism.