N,N-dimethyl-thioamphetamine and methyl-thioamphetamine, two non-neurotoxic substrates of 5-HT transporters, have scant in vitro efficacy for the induction of transporter-mediated 5-HT release and currents.

We studied two non-neurotoxic amphetamine derivatives (methyl-thioamphetamine, MTA and N,N-dimethylMTA, DMMTA) interacting with serotonin (5-HT) transporters (SERTs) with affinities comparable to that of p-Cl-amphetamine (pCA). The rank order for their maximal effects in inducing both [(3)H]5-HT release from rat brain synaptosomes or hSERT-expressing HEK-293 cells, and currents in hSERT-expressing oocytes, was pCA >> MTA > or = DMMTA. A correlation between drug-induced release and currents is also strengthened by the similar bell shape of the dose-response curves. Release experiments indicated that MTA and DMMTA are SERT substrates although MTA is taken up by HEK-293 cells with a V(max) 40% lower than pCA. The weak effects of MTA and DMMTA in vitro might therefore be due to their properties as 'partial substrates' on the mechanisms, other than translocation, responsible for currents and/or release. After either local or systemic in vivo administration, MTA and DMMTA release 5-HT in a manner comparable to pCA. These findings confirm that the neurotoxic properties of some amphetamine derivatives are independent of their 5-HT-releasing activity in vivo. It is worth noting that only those amphetamine derivatives with high efficiency in inducing 5-HT release and currents in vitro have neurotoxic properties.

Functional expression of release-regulating glycine transporters GLYT1 on GABAergic neurons and GLYT2 on astrocytes in mouse spinal cord.

It is widely accepted that glycine transporters of the GLYT1 type are situated on astrocytes whereas GLYT2 are present on glycinergic neuronal terminals where they mediate glycine uptake. We here used purified preparations of mouse spinal cord nerve terminals (synaptosomes) and of astrocyte-derived subcellular particles (gliosomes) to characterize functionally and morphologically the glial versus neuronal distribution of GLYT1 and GLYT2. Both gliosomes and synaptosomes accumulated [3H]GABA through GAT1 transporters and, when exposed to glycine in superfusion conditions, they released the radioactive amino acid not in a receptor-dependent manner, but as a consequence of glycine penetration through selective transporters. The glycine-evoked release of [3H]GABA was exocytotic from synaptosomes but GAT1 carrier-mediated from gliosomes. Based on the sensitivity of the glycine effects to selective GLYT1 and GLYT2 blockers, the two transporters contributed equally to evoke [3H]GABA release from GABAergic synaptosomes; even more surprising, the 'neuronal' GLYT2 contributed more efficiently than the 'glial' GLYT1 to mediate the glycine effect in [3H]GABA releasing gliosomes. These functional results were largely confirmed by confocal microscopy analysis showing co-expression of GAT1 and GLYT2 in GFAP-positive gliosomes and of GAT1 and GLYT1 in MAP2-positive synaptosomes. To conclude, functional GLYT1 are present on neuronal axon terminals and functional GLYT2 are expressed on astrocytes, indicating not complete selectivity of glycine transporters in their glial versus neuronal localization in the spinal cord.

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 micromol/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.

The GABAergic-like system in the marine demosponge Chondrilla nucula.

Gamma-amino butyric acid (GABA) is believed to be the principal inhibitory neurotransmitter in the mammalian central nervous system, a function that has been extended to a number of invertebrate systems. The presence of GABA in the marine demosponge Chondrilla nucula was verified using immunofluorescence detection and high-pressure liquid chromatography. A strong GABA-like immunoreactivity (IR) was found associated with choanocytes, exopinacocytes, endopinacocytes lining inhalant, and exhalant canals, as well as in archaeocytes scattered in the mesohyl. The capacity to synthesize GABA from glutamate and to transport it into the vesicles was confirmed by the presence in C. nucula of glutamate decarboxylase (GAD) and vesicular GABA transporters (vGATs), respectively. GAD-like and vGAT-like IR show the same distribution as GABA-like IR. Supporting the similarity between sponge and mammalian proteins, bands with an apparent molecular weight of about 65-67 kDa and 57 kDa were detected using antibodies raised against mammalian GAD and vGAT, respectively. A functional metabotropic GABA(B)-like receptor is also present in C. nucula. Indeed, both GABA(B) R1 and R2 isoforms were detected by immunoblot and immunofluorescence. Also in this case, IR was found in choanocytes, exopinacocytes, and endopinacocytes. The content of GABA in C. nucula amounts to 1225.75 +/- 79 pmol/mg proteins and GABA is released into the medium when sponge cells are depolarized. In conclusion, this study is the first indication of the existence of the GABA biosynthetic enzyme GAD and of the GABA transporter vGAT in sponges, as well as the first demonstration that the neurotransmitter GABA is released extracellularly.

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.

Co-existence of GABA and Glu transporters in the central nervous system.

Co-localization of transporters able to recapture the released or endogenously synthesized transmitter (homotransporters) and of transporters that can selectively take up transmitters/modulators originating from neighbouring structures (heterotransporters) has been demonstrated to occur within the same axon terminal of several neuronal phenotypes. Activation of terminal heterotransporters invariably leads to the release of the transmitter specific to the terminal. Heterotransporters are also increasingly reported to exist on neuronal soma/dendrites and nerve terminals, on the basis of morphological experiments. The functions of somatodendritic heterotransporters has been investigated only in a very limited number of cases. Release-regulating GABA heterotransporters of the GAT-1 type exist on Glu nerve terminals in different rodent brain regions including spinal cord. Activation of GABA heterotransporters provokes release of Glu, which takes place by reversal of the Glu homotransporter and by anion channel opening. Interestingly, the release of Glu induced by GABA in spinal cord is dramatically enhanced in a transgenic mouse model of amyotrophic lateral sclerosis and this effect seems to represent the most precocious mechanism that increases extracellular Glu concentration, reported to occur in the pathomechanism.

Glutamate release induced by activation of glycine and GABA transporters in spinal cord is enhanced in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a progressive and fatal neurodegenerative disease, involving both upper and lower motor neurons, the cause of which is obscure, although glutamate (GLU)-induced excitotoxicity has been suggested to play a major role. We studied the release of [3H]d-aspartate ([3H]d-ASP) and endogenous glutamate evoked by glycine (GLY) or GABA from spinal cord synaptosomes in mice expressing a mutant form of human SOD1 with a Gly93Ala substitution ([SOD1-G93A(+)]), a transgenic model of amyotrophic lateral sclerosis, in mice expressing the non-mutated form of human SOD1 [SOD1+], and in non-transgenic littermates [SOD1(-)/G93A(-)]. In parallel experiments, we also studied the release of [3H]GABA evoked by GLY and that of [3H]GLY evoked by GABA. Mutant mice were killed at advanced phase of pathology or during the pre-symptomatic period. In SOD1(-)/G93A(-) or SOD1(+) mice GLY evoked [3H]d-ASP and [3H]GABA release, while GABA caused [3H]d-ASP, but not [3H]GLY, release. The GLY-evoked release of [3H]d-ASP, but not that of [3H]GABA, and the GABA-evoked [3H]d-ASP release, but not that of [3H]GLY, were more pronounced in SOD1-G93A(+) than in SOD1(+) or SOD1(-)/G93A(-) mice. Furthermore, the excessive potentiation of [3H]d-ASP by GLY or GABA was already present in asymptomatic 30-40 day-old SOD1-G93A(+) mice. The releases of endogenous glutamate and GABA also were enhanced by GLY and the GLY-evoked release of endogenous glutamate, but not of endogenous GABA, was higher in SOD1-G93A(+) than in control animals. Potentiation of the spontaneous amino acid release is likely to be mediated by activation of a GLY or a GABA transporter, since the effect of GLY was counteracted by the GLY transporter blocker glycyldodecylamide but not by the GLY receptor antagonists strychnine and 5,7-dichlorokynurenate while the effect of GABA was diminished by the GABA transporter blocker SKF89976-A but not by the GABA receptor antagonists SR9531 and CGP52432. It is concluded that the glutamate release machinery seems excessively functional in SOD1-G93A(+) animals.

Redox-dependent modulation of the carrot SV channel by cytosolic pH.

Currents mediated by a slow vacuolar (SV) channel were recorded and characterized in vacuoles from cultured carrot cells. The carrot channel shows the typical functional characteristics reported for channels of the SV category previously identified in other plants, i.e., slow voltage-dependent activation kinetics, current activation favoured by cytosolic calcium and permeability to different monovalent cations. The carrot channel is strongly activated by cytosolic reducing agents (such as dithiothreitol, DTT, and glutathione, GSH) and has a peculiar dependence on cytosolic pH, which, in turn, is affected by the concentration of cytosolic reducing agents. Specifically, in 1 mM DTT or GSH the channel displayed a maximum conductance at neutral pH. The normalized conductance did not depend significantly on DTT concentration at acidic pH, while at alkaline pH the attenuation of the normalized conductance declines with increasing DTT concentration. Our results suggest two pH-titratable groups within the carrot SV channel, one of these depending on cysteine residues exposed to the cytosolic side of the vacuole.