Mario Carta

Genetic ablation of the t-SNARE SNAP-25 distinguishes mechanisms of neuroexocytosis.

Axon outgrowth during development and neurotransmitter release depends on exocytotic mechanisms, although what protein machinery is common to or differentiates these processes remains unclear. Here we show that the neural t-SNARE (target-membrane-associated–soluble N-ethylmaleimide fusion protein attachment protein (SNAP) receptor) SNAP-25 is not required for nerve growth or stimulus-independent neurotransmitter release, but is essential for evoked synaptic transmission at neuromuscular junctions and central synapses. These results demonstrate that the development of neurotransmission requires the recruitment of a specialized SNARE core complex to meet the demands of regulated exocytosis.

Alcohol Enhances GABAergic Transmission to Cerebellar Granule Cells via an Increase in Golgi Cell Excitability

Alcohol intoxication alters coordination and motor skills, and this is responsible for a significant number of traffic accident-related deaths around the world. Although the precise mechanism of action of ethanol (EtOH) is presently unknown, studies suggest that it acts, in part, by interfering with normal cerebellar functioning. An important component of cerebellar circuits is the granule cell. The excitability of these abundantly expressed neurons is controlled by the Golgi cell, a subtype of GABAergic interneuron. Granule cells receive GABAergic input in the form of phasic and tonic currents that are mediated by synaptic and extrasynaptic receptors, respectively. Using the acute cerebellar slice preparation and patch-clamp electrophysiological techniques, we found that ethanol induces a parallel increase in both the frequency of spontaneous IPSCs and the magnitude of the tonic current. EtOH (50 mm) did not produce this effect when spontaneous action potentials were blocked with tetrodotoxin. Recordings in the loose-patch cell-attached configuration demonstrated that ethanol increases the frequency of spontaneous action potentials in Golgi cells. Taken together, these findings indicate that ethanol enhances GABAergic inhibition of granule cells via a presynaptic mechanism that involves an increase in action potential-dependent GABA release from Golgi cells. This effect is likely to have an impact on the flow of information through the cerebellar cortex and may contribute to the mechanism by which acute ingestion of alcoholic beverages induces motor impairment.

Neurosteroids enhance spontaneous glutamate release in hippocampal neurons. Possible role of metabotropic sigma1-like receptors.

Pregnenolone sulfate (PREGS), one of the most abundantly produced neurosteroids in the mammalian brain, improves cognitive performance in rodents. The mechanism of this effect has been attributed to its allosteric modulatory actions on glutamate- and γ-aminobutyric acid-gated ion channels. Here we report a novel effect of PREGS that could also mediate some of its actions in the nervous system. We found that PREGS induces a robust potentiation of the frequency but not the amplitude of miniature excitatory postsynaptic currents (mEPSCs) mediated by α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors in cultured hippocampal neurons. PREGS also decreased paired pulse facilitation of autaptic EPSCs evoked by depolarization, indicating that it modulates glutamate release probability presynaptically. PREGS potentiation of mEPSCs was mimicked by dehydroepiandrosterone sulfate and (+)-pentazocine but not by (−)-pentazocine, the synthetic (−)-enantiomer of PREGS or the inactive steroid isopregnanolone. The ς receptor antagonists, haloperidol and BD-1063, blocked the effect of PREGS on mEPSCs, as did pertussis toxin and the membrane-permeable Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (acetoxymethyl) ester. These results suggest that PREGS increases spontaneous glutamate release via activation of a presynaptic Gi/o-coupled ς receptor and an elevation in intracellular Ca2+ levels. We postulate that presynaptic actions of neurosteroids have a role in the maturation and/or maintenance of synaptic networks and the processing of information in the central nervous system.

Neurosteroid-Induced Plasticity of Immature Synapses via Retrograde Modulation of Presynaptic NMDA Receptors

Neurosteroids are produced de novo in neuronal and glial cells, which begin to express steroidogenic enzymes early in development. Studies suggest that neurosteroids may play important roles in neuronal circuit maturation via autocrine and/or paracrine actions. However, the mechanism of action of these agents is not fully understood. We report here that the excitatory neurosteroid pregnenolone sulfate induces a long-lasting strengthening of AMPA receptor-mediated synaptic transmission in rat hippocampal neurons during a restricted developmental period. Using the acute hippocampal slice preparation and patch-clamp electrophysiological techniques, we found that pregnenolone sulfate increases the frequency of AMPA-mediated miniature excitatory postsynaptic currents in CA1 pyramidal neurons. This effect could not be observed in slices from rats older than postnatal day 5. The mechanism of action of pregnenolone sulfate involved a short-term increase in the probability of glutamate release, and this effect is likely mediated by presynaptic NMDA receptors containing the NR2D subunit, which is transiently expressed in the hippocampus. The increase in glutamate release triggered a long-term enhancement of AMPA receptor function that requires activation of postsynaptic NMDA receptors containing NR2B subunits. Importantly, synaptic strengthening could also be triggered by postsynaptic neuron depolarization, and an anti-pregnenolone sulfate antibody scavenger blocked this effect. This finding indicates that a pregnenolone sulfate-like neurosteroid is a previously unrecognized retrograde messenger that is released in an activity-dependent manner during development.

Developmentally Regulated Actions of Alcohol on Hippocampal Glutamatergic Transmission

Ethanol exposure during fetal development is a leading cause of learning disabilities. Studies suggest that it alters learning and memory by permanently damaging the hippocampus. It is generally assumed that this is mediated, in part, via alterations in glutamatergic transmission. Although NMDA receptors are presumed to be the most sensitive targets of ethanol in immature neurons, this issue has not been explored in the developing hippocampus. We performed whole-cell patch-clamp recordings in hippocampal slices from neonatal rats. Unexpectedly, we found that acute ethanol (10–50 mm) exposure depresses inward currents elicited by local application of exogenous AMPA, but not NMDA, in CA3 pyramidal neurons. These findings revealed a direct effect of ethanol on postsynaptic AMPA receptors. Ethanol significantly decreased the amplitude of both AMPA and NMDA receptor-mediated EPSCs evoked by electrical stimulation. This effect was associated with an increase in the paired-pulse ratio and a decrease in the frequency of miniature EPSCs driven by depolarization of axonal terminals. These findings demonstrate that ethanol also acts at the presynaptic level. ω-Conotoxin-GVIA occluded the effect of ethanol on NMDA EPSCs, indicating that ethanol decreases glutamate release via inhibition of N-type voltage-gated Ca2+ channels. In more mature rats, ethanol did not affect the probability of glutamate release or postsynaptic AMPA receptor-mediated currents, but it did inhibit NMDA-mediated currents. We conclude that the mechanism by which ethanol inhibits glutamatergic transmission is age dependent and challenge the view that postsynaptic NMDA receptors are the primary targets of ethanol early in development.

Changes in Expression and Function of Extrasynaptic GABAA Receptors in the Rat Hippocampus during Pregnancy and after Delivery

Pregnancy is associated with changes in mood and anxiety level as well as with marked hormonal fluctuations. Increases in the brain concentrations of neuroactive steroids during pregnancy in rats are accompanied by changes in expression of subunits of the GABA type A receptor (GABAA-R) in the brain. Granule cells of the dentate gyrus (DGGCs) exhibit two components of inhibitory GABAergic transmission: a phasic component mediated by synaptic GABAA-Rs, and a tonic component mediated by extrasynaptic GABAA-Rs. Recordings of GABAergic currents were obtained from hippocampal slices prepared from rats in estrus, at pregnancy day 15 (P15) or P19, or at 2 d after delivery. Exogenous GABA or 3α,5α-THP induced an increase in tonic current in DGGCs that was significantly greater at P19 than in estrus. Neither tonic nor phasic currents were affected by pregnancy in CA1 pyramidal cells. Immunohistochemical analysis revealed a marked increase in the abundance of the δ subunit of the GABAA-R and a concomitant decrease in that of the γ2 subunit in the hippocampus at P19. Expression of the α4 subunit did not change during pregnancy but was increased 2 d after delivery. Treatment of rats from P12 to P18 with the 5α-reductase inhibitor finasteride prevented the changes in tonic current and in δ and γ2 subunit expression normally apparent at P19. These data suggest that the number of extrasynaptic GABAA-Rs is increased in DGGCs during late pregnancy as a consequence of the associated marked fluctuations in the brain levels of neuroactive steroids.

Synaptic Localization and Activity of ADAM10 Regulate Excitatory Synapses through N-Cadherin Cleavage

N-Cadherin has an important role during dendrite arborization, axon guidance, and synaptogenesis. In particular, at synaptic sites, N-cadherin is involved in the regulation of cell–cell adhesion and in morphology and plasticity control. Recent studies have shown that N-cadherin can be cleaved by the metalloproteinase ADAM10. Here we demonstrate that impairing ADAM10 localization and activity at synaptic sites decreases its processing of N-cadherin. This leads to an accumulation of the full-length form of N-cadherin, to an increase in spine head width, and to modifications of the number and function of glutamate receptors of AMPA type, both in vitro and in vivo. Our results indicate a key role for ADAM10 in the complex sequence of events through which N-cadherin affects spine maturation and controls structure and function of glutamatergic synapses.

Alcohol Impairs Long-Term Depression at the Cerebellar Parallel Fiber-Purkinje Cell Synapse

Acute alcohol consumption causes deficits in motor coordination and gait, suggesting an involvement of cerebellar circuits, which play a role in the fine adjustment of movements and in motor learning. It has previously been shown that ethanol modulates inhibitory transmission in the cerebellum and affects synaptic transmission and plasticity at excitatory climbing fiber (CF) to Purkinje cell synapses. However, it has not been examined thus far how acute ethanol application affects long-term depression (LTD) and long-term potentiation (LTP) at excitatory parallel fiber (PF) to Purkinje cell synapses, which are assumed to mediate forms of cerebellar motor learning. To examine ethanol effects on PF synaptic transmission and plasticity, we performed whole cell patch-clamp recordings from Purkinje cells in rat cerebellar slices. We found that ethanol (50 mM) selectively blocked PF-LTD induction, whereas it did not change the amplitude of excitatory postsynaptic currents at PF synapses. In contrast, ethanol application reduced voltage-gated calcium currents and type 1 metabotropic glutamate receptor (mGluR1)-dependent responses in Purkinje cells, both of which are involved in PF-LTD induction. The selectivity of these effects is emphasized by the observation that ethanol did not impair PF-LTP and that PF-LTP could readily be induced in the presence of the group I mGluR antagonist AIDA or the mGluR1a antagonist LY367385. Taken together, these findings identify calcium currents and mGluR1-dependent signaling pathways as potential ethanol targets and suggest that an ethanol-induced blockade of PF-LTD could contribute to the motor coordination deficits resulting from alcohol consumption.

Alcohol Potently Modulates Climbing Fiber→Purkinje Neuron Synapses: Role of Metabotropic Glutamate Receptors

Consumption of alcoholic beverages produces alterations in motor coordination and equilibrium that are responsible for millions of accidental deaths. Studies indicate that ethanol produces these alterations by affecting the cerebellum, a brain region involved in the control of motor systems. Purkinje neurons of the cerebellar cortex have been shown to be particularly important targets of ethanol. However, its mechanism of action at these neurons is poorly understood. We hypothesized that ethanol could modulate Purkinje neuron function by altering the excitatory input provided by the climbing fiber from the inferior olive, which evokes a powerful all-or-none response denoted as the complex spike. To test this hypothesis, we performed whole-cell patch-clamp electrophysiological and Ca2+ imaging experiments in acute slices from rat cerebella. We found that ethanol potently inhibits the late phase of the complex spike and that this effect is the result of inhibition of type-1 metabotropic glutamate receptor-dependent responses at the postsynaptic level. Moreover, ethanol inhibited climbing fiber long-term depression, a form of synaptic plasticity that also depends on activation of these metabotropic receptors. Our findings identify the climbing fiber→Purkinje neuron synapse as an important target of ethanol in the cerebellar cortex and indicate that ethanol significantly affects cerebellar circuits even at concentrations as low as 10 mm (legal blood alcohol level in the United States is below 0.08 g/dl = 17 mm).

Social isolation‐induced increase in α4 and δ subunit gene expression is associated with a greater efficacy of ethanol on steroidogenesis and GABAA receptor function

Previously we have demonstrated that social isolation of rats reduces both the cerebrocortical and plasma concentrations of 3α‐hydroxy‐5α‐pregnan‐20‐one (3α,5α‐TH PROG), and potentiates the positive effects of acute ethanol administration on the concentrations of this neurosteroid. We now show that the ethanol‐induced increase in 3α,5α‐TH PROG is more pronounced in the brain than in the plasma of isolated rats. The ability of ethanol to inhibit isoniazid‐induced convulsions is greater in isolated rats than in group‐housed animals and this effect is prevented by treatment with finasteride. Social isolation modified the effects of ethanol on the amounts of steroidogenic regulatory protein mRNA and protein in the brain. Moreover, ethanol increased the amplitude of GABAA receptor‐mediated miniature inhibitory postsynaptic currents recorded from CA1 pyramidal neurones with greater potency in hippocampal slices prepared from socially isolated rats than in those from group‐housed rats, an effect inhibited by finasteride. The amounts of the α4 and δ subunits of the GABAA receptor in the hippocampus were increased in isolated rats as were GABAA receptor‐mediated tonic inhibitory currents in granule cells of the dentate gyrus. These results suggest that social isolation results in changes in GABAA receptor expression in the brain, and in an enhancement of the stimulatory effect of ethanol on brain steroidogenesis, GABAA receptor function and associated behaviour.