Ottorino Belluzzi

Riluzole inhibits the persistent sodium current in mammalian CNS neurons.

The effects of 0.1–100 μm riluzole, a neuroprotective agent with anticonvulsant properties, were studied on neurons from rat brain cortex. Patch‐clamp whole‐cell recordings in voltage‐clamp mode were performed on thin slices to examine the effects of the drug on a noninactivating (persistent) Na+ current (INa,p). INa,p was selected because it enhances neuronal excitability near firing threshold, which makes it a potential target for anticonvulsant drugs. When added to the external solution, riluzole dose‐dependently inhibited INa,p up to a complete blocking of the current (EC50 2 μm), showing a significant effect at therapeutic drug concentrations. A comparative dose‐effect study was carried out in the same cells for the other main known action of riluzole, the inhibitory effect on the fast transient sodium current. This effect was confirmed in our experiments, but we found that it was achieved at levels much higher than putative therapeutic concentrations. Only the effect on INa,p, and not that on fast sodium current, can account for the reduction in neuronal excitability observed in cortical neurons following riluzole treatment at therapeutic concentrations, and this might represent a novel mechanism accounting for the anticonvulsant and neuroprotective properties of riluzole.

Neuronal differentiation potential of human adipose-derived mesenchymal stem cells

Adult mesenchymal stem cells derived from adipose tissue (A-MSC) have the capacity to differentiate in vitro into mesenchymal as well as endodermal and ectodermal cell lineages. We investigated the neuronal differentiation potential of human A-MSC with a protocol which included sphere formation and sequential culture in brain-derived neurotrophic factor (BDNF) and retinoic acid (RA). After 30 days, about 57% A-MSC showed morphological, immunocytochemical and electrophysiological evidence of initial neuronal differentiation. In fact, A-MSC displayed elongated shape with protrusion of two or three cellular processes, selectively expressed nestin and neuronal molecules (including GABA receptor and tyroxine hydroxilase) in the absence of glial phenotypic markers. Differentiated cells showed negative membrane potential (-60 mV), delayed rectifier potassium currents and TTX-sensitive sodium currents. Such changes were stable for at least 7 days after removal of differentiation medium. In view of these results and the easy availability of adipose tissue, A-MSC may be a ready source of adult MSC with neuronal differentiation potential, an useful tool to treat neurodegenerative diseases.

Functional properties of dopaminergic neurones in the mouse olfactory bulb

The olfactory bulb of mammals contains a large population of dopaminergic interneurones within the glomerular layer. Dopamine has been shown both in vivo and in vitro to modulate several aspects of olfactory information processing, but the functional properties of dopaminergic neurones have never been described due to the inability to recognize these cells in living preparations. To overcome this difficulty, we used a transgenic mouse strain harbouring an eGFP (enhanced green fluorescent protein) reporter construct under the promoter of tyrosine hydroxylase, the rate‐limiting enzyme for cathecolamine synthesis. As a result, we were able to identify dopaminergic neurones (TH‐GFP cells) in living preparations and, for the first time, we could study the functional properties of such neurones in the olfactory bulb, in both slices and dissociated cells. The most prominent feature of these cells was the autorhythmicity. In these cells we identified five main voltage‐dependent conductances: the two having largest amplitude were a fast transient Na+ current and a delayed rectifier K+ current. In addition, we observed three smaller inward currents, sustained by Na+ ions (persistent type) and by Ca2+ ions (LVA and HVA). Using pharmacological tools and ion substitution methods we showed that the pacemaking process is supported by the interplay of the persistent Na+ current and of a T‐type Ca2+ current. We carried out a complete kinetical analysis of the five conductances present in these cells, and developed a Hodgkin‐Huxley model of TH‐GFP cells, capable of reproducing accurately the properties of living cells, including autorhytmicity, and allowing a precise understanding of the process.

Selective neuroinhibitory effects of taurine in slices of rat main olfactory bulb

Taurine is abundant in the main olfactory bulb, exceeding glutamate and GABA in concentration. In whole-cell patch-clamp recordings in rat olfactory bulb slices, taurine inhibited principal neurons, mitral and tufted cells. In these cells, taurine decreased the input resistance and caused a shift of the membrane potential toward the chloride equilibrium potential. The taurine actions were sustained under the blockade of transmitter release and were reversible and dose-dependent. At a concentration of 5 mM, typically used in this study, taurine showed 90% of its maximal effect. GABA(A) antagonists, bicuculline and picrotoxin, blocked the taurine actions, whereas the glycine receptor antagonist strychnine and GABA(B) antagonists, CGP 55845A and CGP 35348, were ineffective. These findings are consistent with taurine directly activating GABA(A) receptors and inducing chloride conductance. Taurine had no effect on periglomerular and granule interneurons. The subunit composition of GABA(A) receptors in these cells, differing from those in mitral and tufted cells, may account for taurine insensitivity of the interneurons. Taurine suppressed olfactory nerve-evoked monosynaptic responses of mitral and tufted cells while chloride conductance was blocked. This action was mimicked by the GABA(B) agonist baclofen and abolished by CGP 55845A; CGP 35348, which primarily blocks postsynaptic GABA(B) receptors, was ineffective. The taurine effect most likely was due to GABA(B) receptor-mediated inhibition of presynaptic glutamate release. Neither taurine nor baclofen affected responses of periglomerular cells. The lack of a baclofen effect implies that functional GABA(B) receptors are absent from olfactory nerve terminals that contact periglomerular cells. These results indicate that taurine decreases the excitability of mitral and tufted cells and their responses to olfactory nerve stimulation without influencing periglomerular and granule cells. Selective effects of taurine in the olfactory bulb may represent a physiologic mechanism that is involved in the inhibitory shaping of the activation pattern of principal neurons.

Functional heterogeneity of periglomerular cells in the rat olfactory bulb

The periglomerular cells of the rat olfactory bulb, a virtually unknown population of interneurons, have been studied applying the whole‐cell patch‐clamp technique to thin slices. A prominent result, obtained under current‐clamp conditions, is that these cells appear to be functionally heterogeneous, and show distinct excitability profiles. Voltage‐clamp analysis allows the identification of the ionic basis of these differences and suggests a division into at least two classes, based on the characteristics of the K+ conductances. The first group displays two K+ conductances (delayed rectifier, gKV, and fast transient, gA) of similar amplitude, and under current‐clamp conditions shows the usual outward rectifying behaviour at depolarized potentials. The second group has a large gA, and a small or absent gKV. Consequently, following sustained depolarizations under current‐clamp conditions leading to inactivation of gA, these neurons do not show any sign of outward rectification and behave as ohmic elements, as normally observed only at hyperpolarized potentials. The transition ion zinc (10–300 μm) affects gA but not gKV. The inactivation process (steady‐state curve and rate constant) is strongly altered by Zn2+, the activation process less so; open‐channel conductance is not affected. The Zn2+ effect is unlikely to be due to surface charge screening or to a mechanism involving channel block. In view of the substantial presence of zinc ions in the olfactory nerve terminals, its actions on the A‐current could be of some relevance for physiological function.

NMDA-dependent, network-driven oscillatory activity induced by bicuculline or removal of Mg2+ in rat olfactory bulb neurons

Spontaneous, low‐frequency voltage oscillations (LFOs) were observed in the neurons of rat olfactory bulb upon disinhibition with GABAA antagonists and/or removal of Mg2+ from external saline. Ordinarily, LFOs presented a highly organized temporal structure, with bursts recurring regularly at about 0.05 Hz. Slow depolarizing shifts with similar frequencies were observed in all types of bulbar neurons. Simultaneous recordings from mutually independent neurons showed that LFOs were highly synchronized in distinct cells. The occurrence of LFOs was prevented by NMDA, but not AMPA/kainate, receptor antagonists. The oscillations were also halted by Ca2+ antagonists and tetrodotoxin. The pace of the oscillations was reset by stimulation of the olfactory nerve but not by direct injection of depolarizing current into the oscillating cell. Removal of the outer portion of the slice with a cut along the external plexiform layer provided crucial evidence that the bursting activity first initiated in the glomerular region and propagated synaptically downstream towards the inner layers, suggesting an organizing role for olfactory glomeruli.

Cholinergic Modulation of Dopaminergic Neurons in the Mouse Olfactory Bulb

Considerable evidence exists for an extrinsic cholinergic influence in the maturation and function of the main olfactory bulb. In this study, we addressed the muscarinic modulation of dopaminergic neurons in this structure. We used different patch-clamp techniques to characterize the diverse roles of muscarinic agonists on identified dopaminergic neurons in a transgenic animal model expressing a reporter protein (green fluorescent protein) under the tyrosine hydroxylase promoter. Bath application of acetylcholine (1 mM) in slices and in enzymatically dissociated cells reduced the spontaneous firing of dopaminergic neurons recorded in cell-attached mode. In whole-cell configuration no effect of the agonist was observed, unless using the perforated patch technique, thus suggesting the involvement of a diffusible second messenger. The effect was mediated by metabotropic receptors as it was blocked by atropine and mimicked by the m2 agonist oxotremorine (10 muM). The reduction of periglomerular cell firing by muscarinic activation results from a membrane-potential hyperpolarization caused by activation of a potassium conductance. This modulation of dopaminergic interneurons may be important in the processing of sensory information and may be relevant to understand the mechanisms underlying the olfactory dysfunctions occurring in neurodegenerative diseases affecting the dopaminergic and/or cholinergic systems.

FGF-2 Overexpression Increases Excitability and Seizure Susceptibility but Decreases Seizure-Induced Cell Loss

Fibroblast growth factor 2 (FGF-2) has multiple, pleiotropic effects on the nervous system that include neurogenesis, neuroprotection and neuroplasticity. Thus, alteration in FGF-2 expression patterns may have a profound impact in brain function, both in normal physiology and in pathology. Here, we used FGF-2 transgenic mice (TgFGF2) to study the effects of endogenous FGF-2 overexpression on susceptibility to seizures and to the pathological consequences of seizures. TgFGF2 mice display increased FGF-2 expression in hippocampal pyramidal neurons and dentate granule cells. Increased density of glutamatergic synaptic vesicles was observed in the hippocampus of TgFGF2 mice, and electrophysiological data (input/output curves and patch-clamp recordings in CA1) confirmed an increase in excitatory inputs in CA1, suggesting the presence of a latent hyperexcitability. Indeed, TgFGF2 mice displayed increased susceptibility to kainate-induced seizures compared with wild-type (WT) littermates, in that latency to generalized seizure onset was reduced, whereas behavioral seizure scores and lethality were increased. Finally, WT and TgFGF2 mice with similar seizure scores were used for examining seizure-induced cellular consequences. Neurogenesis and mossy fiber sprouting were not significantly different between the two groups. In contrast, cell damage (assessed with Fluoro-Jade B, silver impregnation and anti-caspase 3 immunohistochemistry) was significantly lower in TgFGF2 mice, especially in the areas of overexpression (CA1 and CA3), indicating reduction of seizure-induced necrosis and apoptosis. These data suggest that FGF-2 may be implicated in seizure susceptibility and in seizure-induced plasticity, exerting different, and apparently contrasting effects: favoring ictogenesis but reducing seizure-induced cell death.

Excitatory synapses in the glomerular triad of frog olfactory bulb in vitro.

Whole-cell patch clamp recording techniques were applied to periglomerular (PG) cells in slices of the frog olfactory bulb (OB) to study the properties of the excitatory synapses in the triad formed by the olfactory nerve (ON) and the dendrites of mitral/tufted (MT) cells and PG cells. The postsynaptic response evoked by ON stimulation was glutamatergic and could be dissected into NMDA and non-NMDA components of equivalent amplitudes. The dendro-dendritic synapse between MT and PG cells could be activated following antidromic stimulation of the lateral and medial olfactory tract (LOT and MOT). In this case the postsynaptic potentials had amplitudes and durations comparable to those obtained by ON stimulation, the neurotransmitter was glutamate, but the synapse was largely dominated by the slow NMDA component.

Electrophysiological evidence for a PGE-mediated presynaptic control of acetylcholine output in the guinea-pig superior cervical ganglion

Abstract Intracellular recordings from single ganglion neurons show that 10 −8 −10 −7 M PGE 1 reversibly blocks synaptic transmission in isolated preparations of the guinea-pig superior cervical ganglion (SCG), when added to the superfusing medium. Neither resting potential nor membrane resistance of the impaled neurons appear to be affected by PGE 1 . Quantal analyses of transmitter release demonstrate that the number of quanta liberated per volley is sharply reduced by PGE 1 treatment whereas the amplitude of the elementary event does not appear to be significantly changed.