Luca Raiteri

Chronic Antidepressants Reduce Depolarization-Evoked Glutamate Release and Protein Interactions Favoring Formation of SNARE Complex in Hippocampus

Glutamate neurotransmission was recently implicated in the action of stress and in antidepressant mechanisms. We report that chronic (not acute) treatment with three antidepressants with different primary mechanisms (fluoxetine, reboxetine, and desipramine) markedly reduced depolarization-evoked release of glutamate, stimulated by 15 or 25 mm KCl, but not release of GABA. Endogenous glutamate and GABA release was measured in superfused synaptosomes, freshly prepared from hippocampus of drug-treated rats. Interestingly, treatment with the three drugs only barely changed the release of glutamate (and of GABA) induced by ionomycin. In synaptic membranes of chronically treated rats we found a marked reduction in the protein-protein interaction between syntaxin 1 and Thr286-phosphorylated αCaM kinase II (α-calcium/calmodulin-dependent protein kinase II) (an interaction previously proposed to promote neurotransmitter release) and a marked increase in the interaction between syntaxin 1 and Munc-18 (an interaction proposed to reduce neurotransmitter release). Furthermore, we found a selective reduction in the expression level of the three proteins forming the core SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex. These findings suggest that antidepressants work by stabilizing glutamate neurotransmission in the hippocampus and that they may represent a useful tool for the study of relationship between functional and molecular processes in nerve terminals.

Synaptosomes still viable after 25 years of superfusion.

Superfused synaptosomes have been utilized in studies of neurotransmitter release during 25 years. This review summarizes the aspects of neurotransmission that have been and could be successfully investigated with this technique. The major aim of the article is to draw attention on the versatility of superfused synaptosomes and to suggest how the system could be exploited in clarifying several aspects of synaptic neurochemistry including neurotransmitter transport, receptor localization, receptor-receptor interactions, functional aspects of multi-sited receptor complexes, receptor heterogeneity and mechanisms of neurotransmitter exocytosis-endocytosis.

Glia re-sealed particles freshly prepared from adult rat brain are competent for exocytotic release of glutamate

Glial subcellular re‐sealed particles (referred to as gliosomes here) were purified from rat cerebral cortex and investigated for their ability to release glutamate. Confocal microscopy showed that the glia‐specific proteins glial fibrillary acidic protein (GFAP) and S‐100, but not the neuronal proteins 95‐kDa postsynaptic density protein (PSD‐95), microtubule‐associated protein 2 (MAP‐2) and β‐tubulin III, were enriched in purified gliosomes. Furthermore, gliosomes exhibited labelling neither for integrin‐αM nor for myelin basic protein, which are specific for microglia and oligodendrocytes respectively. The Ca2+ ionophore ionomycin (0.1–5 µm) efficiently stimulated the release of tritium from gliosomes pre‐labelled with [3H]d‐aspartate and of endogenous glutamate in a Ca2+‐dependent and bafilomycin A1‐sensitive manner, suggesting the involvement of an exocytotic process. Accordingly, ionomycin was found to induce a Ca2+‐dependent increase in the vesicular fusion rate, when exocytosis was monitored with acridine orange. ATP stimulated [3H]d‐aspartate release in a concentration‐ (0.1–3 mm) and Ca2+‐dependent manner. The gliosomal fraction contained proteins of the exocytotic machinery [syntaxin‐1, vesicular‐associated membrane protein type 2 (VAMP‐2), 23‐kDa synaptosome‐associated protein (SNAP‐23) and 25‐kDa synaptosome‐associated protein (SNAP‐25)] co‐existing with GFAP immunoreactivity. Moreover, GFAP or VAMP‐2 co‐expressed with the vesicular glutamate transporter type 1. Consistent with ultrastructural analysis, several ∼30‐nm non‐clustered vesicles were present in the gliosome cytoplasm. It is concluded that gliosomes purified from adult brain contain glutamate‐accumulating vesicles and can release the amino acid by a process resembling neuronal exocytosis.

Effects of adenosine A1 and A2A receptor activation on the evoked release of glutamate from rat cerebrocortical synaptosomes

The effects of adenosine A2A and A1 receptor activation on the release of glutamate were studied in rat cerebral cortex synaptosomes exposed in superfusion to adenosine receptor ligands. Adenosine (0.1 μM) produced a significant potentiation of the Ca2+‐dependent K+(15 mM)‐evoked [3H]‐D‐aspartate overflow (20.4±3.5%), which was blocked by A2A blocker SCH58261 (0.1 μM). At higher concentrations (10 – 1000 μM) adenosine inhibited in a DPCPX‐sensitive manner the Ca2+‐dependent K+‐evoked [3H]‐D‐aspartate overflow. The inhibitory effect of adenosine at 1000 μM was significantly increased by SCH58261. This inhibition was antagonized by 1 μM DPCPX. Adenosine did not produce any effect on basal release. The A2A receptor agonist CGS 21680 was ineffective on basal release, but stimulated the Ca2+‐dependent K+‐evoked overflow of [3H]‐D‐aspartate (EC50 ≃ 1 pM). The effect of 0.01 nM CGS 21680 was totally sensitive to the A2A receptor antagonist SCH58261 (IC50 ≃5 nM). The A1 receptor agonist CCPA inhibited the Ca2+‐dependent K+‐evoked [3H]‐D‐aspartate overflow (EC50 ≃ 20 nM). The effect of 100 nM CCPA was abolished by 100 nM of the A1 receptor antagonist DPCPX. The K+(15 mM)‐evoked overflow of endogenous glutamate was enhanced by CGS 21680 (0.01 nM) and inhibited by CCPA (0.1 μM). The effect of CGS 21680 was abolished by SCH58261 (0.1 μM) and that of CCPA by DPCPX (0.1 μM). It is concluded that adenosine and adenosine receptor agonists modulate glutamate release by activating inhibitory A1 and excitatory A2A receptors present on glutamatergic terminals of the rat cerebral cortex.

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.

Stimulation of excitatory amino acid release from adult mouse brain glia subcellular particles by high mobility group box 1 protein

The multifunctional protein high mobility group box 1 (HMGB1) is expressed in hippocampus and cerebellum of adult mouse brain. Our aim was to determine whether HMGB1 affects glutamatergic transmission by monitoring neurotransmitter release from glial (gliosomes) and neuronal (synaptosomes) re‐sealed subcellular particles isolated from cerebellum and hippocampus. HMGB1 induced release of the glutamate analogue [3H]d‐aspartate form gliosomes in a concentration‐dependent manner, whereas nerve terminals were insensitive to the protein. The HMGB1‐evoked release of [3H]d‐aspartate was independent of modifications of cytosolic Ca2+ , but it was blocked by dl‐threo‐β‐benzyloxyaspartate (dl‐TBOA), an inhibitor of glutamate transporters. HMGB1 also stimulated the release of endogenous glutamate in a Ca2+‐independent and dl‐TBOA‐sensitive manner. These findings suggest the involvement of carrier‐mediated release. Moreover, dihydrokainic acid, a selective inhibitor of glutamate transporter 1 (GLT1), does not block the effect of HMGB1, indicating a role for the glial glutamate‐aspartate transporter (GLAST) subtype in this response. We also demonstrate that HMGB1/glial particles association is promoted by Ca2+. Furthermore, although HMGB1 can physically interact with GLAST and the receptor for advanced glycation end products (RAGE), only its binding with RAGE is promoted by Ca2+. These results suggest that the HMGB1 cytokine could act as a modulator of glutamate homeostasis in adult mammal brain.

Sensitivity to selective adenosine A1 and A2A receptor antagonists of the release of glutamate induced by ischemia in rat cerebrocortical slices.

Adenosine released during cerebral ischemia is considered to act as a neuroprotectant, possibly through the inhibition of glutamate release. The involvement of A(1) and A(2A) receptors in the control of the rise of extracellular glutamate during ischemia was investigated by monitoring the effects of selective A(1) and A(2A) receptor antagonists on ischemia-evoked glutamate release in rat cerebrocortical slices.Slices were superfused with oxygen- and glucose-deprived medium and [(3)H]D-aspartate or endogenous glutamate was measured in the superfusate fractions. Withdrawal of Ca(2+) ions or addition of tetrodotoxin more than halved the ischemia-evoked efflux of [(3)H]D-aspartate or glutamate, compatible with a vesicular-like release. The glutamate transporter inhibitor DL-TBOA prevented the ischemia-evoked efflux of [(3)H]D-aspartate by about 40%, indicating a carrier-mediated efflux. The ischemia-evoked efflux of [(3)H]D-aspartate or glutamate was increased by the A(1) receptor antagonist DPCPX. The A(2A) antagonist SCH 58261 decreased [(3)H]D-aspartate or endogenous glutamate efflux (50 and 55% maximal inhibitions; EC(50): 14.9 and 7.6 nM, respectively); the drug was effective also if added during ischemia. No effect of either the A(1) or the A(2A) receptor antagonist was found on the ischemia-evoked efflux of [(3)H]D-aspartate in Ca(2+)-free medium. Our data suggest that adenosine released during cerebral ischemia can activate inhibitory A(1) and stimulatory A(2A) receptors that down- or up-regulate the vesicular-like component of glutamate release.

Antidepressant treatments and function of glutamate ionotropic receptors mediating amine release in hippocampus

Previous evidences showed that, besides noradrenaline (NA) and 5-hydroxytryptamine (5-HT), glutamate transmission is involved in the mechanism of action of antidepressants (ADs), although the relations between aminergic and glutamatergic systems are poorly understood. The aims of this investigation were to evaluate changes in the function of glutamate AMPA and NMDA receptors produced by acute and chronic administration of the two ADs reboxetine and fluoxetine, selective inhibitors of NA and 5-HT uptake, respectively. Rats were treated acutely (intraperitoneal injection) or chronically (osmotic minipump infusion) with reboxetine or fluoxetine. Isolated hippocampal nerve endings (synaptosomes) prepared following acute/chronic treatments were labelled with [(3)H]NA or [(3)H]5-HT and [(3)H]amine release was monitored during exposure in superfusion to NMDA/glycine, AMPA or K(+)-depolarization. Acute and chronic reboxetine reduced the release of [(3)H]NA evoked by NMDA/glycine or by AMPA. The NMDA/glycine-evoked release of [(3)H]NA was also down-regulated by chronic fluoxetine. Only acute, but not chronic, fluoxetine inhibited the AMPA-evoked release of [(3)H]5-HT. The release of [(3)H]NA and [(3)H]5-HT elicited by K(+)-depolarization was almost abolished by acute reboxetine or fluoxetine, respectively, but recovered during chronic ADs administration. ADs reduced NMDA receptor-mediated releasing effects in noradrenergic terminals after acute and chronic administration, although by different mechanisms. Chronic treatments markedly reduced the expression level of NR1 subunit in synaptic membranes. The noradrenergic and serotonergic release systems seem to be partly functionally interconnected and interact with glutamatergic transmission to down-regulate its function. The results obtained support the view that glutamate plays a major role in AD activity.

Coexistence and function of different neurotransmitter transporters in the plasma membrane of CNS neurons

Transporters able to recapture released neurotransmitters into neurons can no longer be considered as cell-specific neuronal markers. In fact, colocalization on one nerve terminal of transporters able to selectively recapture the released endogenously synthesized transmitter (homotransporters) and of transporters that can selectively take up transmitters/modulators originating from neighboring structures (heterotransporters) has been demonstrated to occur on several families of nerve terminals. Activation of heterotransporters often increases the release of the transmitter stored in the terminals on which the heterotransporters are localized. The release caused by heterotransporter activation takes place through multiple mechanisms including exocytosis, either dependent on external Ca(2+) or on Ca(2+) mobilized from intraterminal stores, and homotransporter reversal. Homocarrier-mediated release elicited by heterocarrier activation represents a clear case of transporter-transporter interaction. Although the functional significance of transporter coexpression on one nerve terminal remains to be established, it may in some instances reflect cotransmission. In other cases, heterotransporters may mediate modulation of basal transmitter release in addition to the modulation of the evoked release brought about by presynaptic heteroreceptors. Heterotransporters are also increasingly reported to exist on neuronal soma/dendrites. With the exception of EAAT4, the glutamate transporter/chloride channel situated on GABAergic Purkinje cells in the cerebellum, the functions of somatodendritic heterocarriers is not understood.

Adult astroglia is competent for Na+/Ca2+ exchanger-operated exocytotic glutamate release triggered by mild depolarization.

Glutamate release induced by mild depolarization was studied in astroglial preparations from the adult rat cerebral cortex, that is acutely isolated glial sub‐cellular particles (gliosomes), cultured adult or neonatal astrocytes, and neuron‐conditioned astrocytes. K+ (15, 35 mmol/L), 4‐aminopyridine (0.1, 1 mmol/L) or veratrine (1, 10 μmol/L) increased endogenous glutamate or [3H]d‐aspartate release from gliosomes. Neurotransmitter release was partly dependent on external Ca2+, suggesting the involvement of exocytotic‐like processes, and partly because of the reversal of glutamate transporters. K+ increased gliosomal membrane potential, cytosolic Ca2+ concentration [Ca2+]i, and vesicle fusion rate. Ca2+ entry into gliosomes and glutamate release were independent from voltage‐sensitive Ca2+ channel opening; they were instead abolished by 2‐[2‐[4‐(4‐nitrobenzyloxy)phenyl]ethyl]isothiurea (KB‐R7943), suggesting a role for the Na+/Ca2+ exchanger working in reverse mode. K+ (15, 35 mmol/L) elicited increase of [Ca2+]i and Ca2+‐dependent endogenous glutamate release in adult, not in neonatal, astrocytes in culture. Glutamate release was even more marked in in vitro neuron‐conditioned adult astrocytes. As seen for gliosomes, K+‐induced Ca2+ influx and glutamate release were abolished by KB‐R7943 also in cultured adult astrocytes. To conclude, depolarization triggers in vitro glutamate exocytosis from in situ matured adult astrocytes; an aptitude grounding on Ca2+ influx driven by the Na+/Ca2+ exchanger working in the reverse mode.