Silvia Coco

SNAP-25 Modulation of Calcium Dynamics Underlies Differences in GABAergic and Glutamatergic Responsiveness to Depolarization

SNAP-25 is a component of the SNARE complex implicated in synaptic vesicle exocytosis. In this study, we demonstrate that hippocampal GABAergic synapses, both in culture and in brain, lack SNAP-25 and are resistant to the action of botulinum toxins type A and E, which cleave this SNARE protein. Relative to glutamatergic neurons, which express SNAP-25, GABAergic cells were characterized by a higher calcium responsiveness to depolarization. Exogenous expression of SNAP-25 in GABAergic interneurons lowered calcium responsiveness, and SNAP-25 silencing in glutamatergic neurons increased calcium elevations evoked by depolarization. Expression of SNAP-25(1-197) but not of SNAP-25(1-180) inhibited calcium responsiveness, pointing to the involvement of the 180-197 residues in the observed function. These data indicate that SNAP-25 is crucial for the regulation of intracellular calcium dynamics and, possibly, of network excitability. SNAP-25 is therefore a multifunctional protein that participates in exocytotic function both at the mechanistic and at the regulatory level.

A Regulated Secretory Pathway in Cultured Hippocampal Astrocytes

Glial cells have been reported to express molecules originally discovered in neuronal and neuroendocrine cells, such as neuropeptides, neuropeptide processing enzymes, and ionic channels. To verify whether astrocytes may have regulated secretory vesicles, the primary cultures prepared from hippocampi of embryonic and neonatal rats were used to investigate the subcellular localization and secretory pathway followed by secretogranin II, a well known marker for dense-core granules. By indirect immunofluorescence, SgII was detected in a large number of cultured hippocampal astrocytes. Immunoreactivity for the granin was detected in the Golgi complex and in a population of dense-core vesicles stored in the cells. Subcellular fractionation experiments revealed that SgII was stored in a vesicle population with a density identical to that of the dense-core secretory granules present in rat pheochromocytoma cells. In line with these data, biochemical results indicated that 40–50% of secretogranin II synthesized during 18-h labeling was retained intracellularly over a 4-h chase period and released after treatment with different secretagogues. The most effective stimulus appeared to be phorbol ester in combination with ionomycin in the presence of extracellular Ca2+, a treatment that was found to produce a large and sustained increase in intracellular calcium [Ca2+] i transients. Our findings indicate that a regulated secretory pathway characterized by (i) the expression and stimulated exocytosis of a typical marker for regulated secretory granules, (ii) the presence of dense-core vesicles, and (iii) the ability to undergo [Ca2+] i increase upon specific stimuli is present in cultured hippocampal astrocytes.

A Common Exocytotic Mechanism Mediates Axonal and Dendritic Outgrowth

Outgrowth of the dendrites and the axon is the basis of the establishment of the neuronal shape, and it requires addition of new membrane to both growing processes. It is not yet clear whether one or two exocytotic pathways are responsible for the respective outgrowth of axons and dendrites. We have previously shown that tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) defines a novel network of tubulovesicular structures present both at the leading edge of elongating dendrites and axons of immature hippocampal neurons developing in primary culture and that TI-VAMP is an essential protein for neurite outgrowth in PC12 cells. Here we show that the expression of the N-terminal domain of TI-VAMP inhibits the outgrowth of both dendrites and axons in neurons in primary culture. This effect is more prominent at the earliest stages of the development of neurons in vitro. Expression of the N-terminal domain deleted form of TI-VAMP has the opposite effect. This constitutively active form of TI-VAMP localizes as the endogenous protein, particularly concentrating at the leading edge of growing axons. Our results suggest that a common exocytotic mechanism that relies on TI-VAMP mediates both axonal and dendritic outgrowth in developing neurons.

Non-synaptic localization of the glutamate transporter EAAC1 in cultured hippocampal neurons

It has been postulated for several years that the high affinity neuronal glutamate uptake system plays a role in clearing glutamate from the synaptic cleft. Four different glutamate transporter subtypes are now identified, the major neuronal one being EAAC1. To be a good candidate for the reuptake of glutamate at the synaptic cleft, EAAC1 should be properly located at synapses, either at pre‐ or postsynaptic sites. We have investigated the distribution of EAAC1 in primary cultures of hippocampal neurons, which represent an advantageous model for the study of synaptogenesis and synaptic specializations. We have demonstrated that EAAC1 immunoreactivity is segregated in the somatodendritic compartment of fully differentiated hippocampal neurons, where it is localized in the dendritic shaft and in the spine neck, outside the area facing the active zone. No co‐localization of EAAC1 immunoreactivity with the stainings produced by typical presynaptic and postsynaptic markers was ever observed, indicating that EAAC1 is not to be considered a synaptic protein. Accordingly, the developmental pattern of expression of EAAC1 was found to be different from that of typical synaptic markers. Moreover, EAAC1 was expressed in the somatodendritic compartment of hippocampal neurons already at stages preceding the formation of synaptic contacts, and was also expressed in GABAergic interneurons with identical subcellular distribution. Taken together, these data rule against a possible role for EAAC1 in the clearance of glutamate from within the cleft and in the regulation of its time in the synapse. They suggest an unconventional non‐synaptic function of this high‐affinity glutamate carrier, not restricted to glutamatergic fibres.

Traffic of botulinum toxins A and E in excitatory and inhibitory neurons.

Botulinum neurotoxins (BoNTs), proteases specific for the SNARE proteins, are used to study the molecular machinery supporting exocytosis and are used to treat human diseases characterized by cholinergic hyperactivity. The recent extension of the use of BoNTs to central nervous system (CNS) pathologies prompted the study of their traffic in central neurons. We used fluorescent BoNT/A and BoNT/E to study the penetration, the translocation and the catalytic action of these toxins in excitatory and inhibitory neurons. We show that BoNT/A and BoNT/E, besides preferentially inhibiting synaptic vesicle recycling at glutamatergic relative to Gamma‐aminobutyric acid (GABA)‐ergic neurons, are more efficient in impairing the release of excitatory than inhibitory neurotransmitter from brain synaptosomes. This differential effect does not result from a defective penetration of the toxin in line with the presence of the BoNT/A receptor, synaptic vesicle protein 2 (SV2), in both types of neurons. Interestingly, exogenous expression of SNAP‐25 in GABAergic neurons confers sensitivity to BoNT/A. These results indicate that the expression of the toxin substrate, and not the toxin penetration, most likely accounts for the distinct effects of the two neurotoxins at the two types of terminals and support the use of BoNTs for the therapy of CNS diseases caused by the altered activity of selected neuronal populations.

Internalization and Proteolytic Action of Botulinum Toxins in CNS Neurons and Astrocytes

Abstract: Tetanus and botulinum toxins bind and are internalized at the neuromuscular junction. Botulinum neurotoxins (BoNTs) enter the cytosol at the motor nerve terminal; tetanus neurotoxin (TeNT) proceeds retroaxonally inside the motor axon to reach the spinal cord inhibitory interneurons. Although the major target of BoNTs is the peripheral cholinergic terminals, CNS neurons are susceptible to intoxication as well. We investigated the route of entry and the proteolytic activity of BoNT/B and BoNT/F in cultured hippocampal neurons and astrocytes. We show that, differently from TeNT, which enters hippocampal neurons via the process of synaptic vesicle (SV) recycling, BoNTs are internalized and cleave the substrate synaptobrevin/VAMP2 via a process independent of synaptic activity. Labeling of living neurons with Texas Red‐conjugated BoNTs and fluoresceinated dextran revealed that these toxins enter hippocampal neurons via endocytic processes not mediated by SV recycling. Botulinum toxins also exploit endocytosis to enter cultured astrocytes, where they partially cleave cellubrevin, a ubiquitous synaptobrevin/VAMP isoform. These results indicate that, in spite of their closely related protein structure, TeNT and BoNTs use different routes to penetrate hippocampal neurons. These findings bear important implications for the identification of the protein receptors of clostridial toxins.

Synaptogenesis in hippocampal cultures.

Hippocampal cultures offer unique advantages for the study of neuronal development and synaptogenesis. Studies performed on this model enabled dissection of the temporal sequence of events which lead to the differentiation of pre- and postsynaptic compartments.

Analysis of SNAP-25 immunoreactivity in hippocampal inhibitory neurons during development in culture and in situ

Synaptosomal associated protein of 25 kDa (SNAP-25) is a component of the soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) complex which plays a central role in synaptic vesicle exocytosis. We have previously demonstrated that adult rat hippocampal GABAergic synapses, both in culture and in brain, are virtually devoid of SNAP-25 immunoreactivity and are less sensitive to the action of botulinum toxin type A, which cleaves this SNARE protein [Neuron 41 (2004) 599]. In the present study, we extend our findings to the adult mouse hippocampus and we also provide demonstration that hippocampal inhibitory synapses lacking SNAP-25 labeling belong to parvalbumin-, calretinin- and cholecystokinin-positive interneurons. A partial colocalization between SNAP-25 and glutamic acid decarboxylase is instead detectable in developing mouse hippocampus at P0 and, at a lesser extent, at P5. In rat embryonic hippocampal cultures at early developmental stages, SNAP-25 immunoreactivity is detectable in a percentage of GABAergic neurons, which progressively reduces with time in culture. Consistent with the presence of the substrate, botulinum toxin type A is partially effective in inhibiting synaptic vesicle recycling in immature GABAergic neurons. Since SNAP-25, beside its role as a SNARE protein, is involved in additional processes, such as neurite outgrowth and regulation of calcium dynamics, the presence of higher levels of the protein at specific stages of neuronal differentiation may have implications for the construction and for the functional properties of brain circuits.

Astrocytes are required for the oscillatory activity in cultured hippocampal neurons

Synchronous oscillations of intracellular calcium concentration ([Ca2+]i) and of membrane potential occurred in a limited population of glutamatergic hippocampal neurons grown in primary cultures. The oscillatory activity occurred in synaptically connected cells only when they were in the presence of astrocytes. Microcultures containing only one or a few neurons also displayed oscillatory activity, provided that glial cells participated in the network. The glutamate‐transporter inhibitors L‐trans‐pyrrolidine‐2,4‐dicarboxylic acid (PDC) and dihydrokainate, which produce an accumulation of glutamate in the synaptic microenvironment, impaired the oscillatory activity. Moreover, in neurons not spontaneously oscillating, though in the presence of astrocytes, oscillations were induced by exogenous l‐glutamate, but not by the stereoisomer d‐glutamate, which is not taken up by glutamate transporters. These data demonstrate that astrocytes are essential for neuronal oscillatory activity and provide evidence that removal of glutamate from the synaptic environment is one of the major mechanisms by which glial cells allow the repetitive excitation of the postsynaptic cell.

Mechanisms of synaptogenesis in hippocampal neurons in primary culture

To improve our understanding of the mechanisms which regulate the formation and the functional maturation of synaptic contacts between neurons, we used hippocampal neurons maintained in primary cultures as experimental system. In this model, which offers several advantages for the study of neuronal development and synaptogenesis, we investigated some of the cellular mechanisms underlying the formation of presynaptic and postsynaptic compartments.