José Sánchez-Prieto

Occurrence of a Large Ca2+‐Independent Release of Glutamate During Anoxia in Isolated Nerve Terminals (Synaptosomes)

Abstract: Isolated rat cerebral cortical synaptosomes made anoxic by addition of cyanide developed an inhibition of the Ca2+‐dependent release of glutamate 2 min after the addition of the metabolic inhibitor when the intrasynaptosomal ATP/ADP ratio decreased below 1.7. In contrast, cyanide induced a continuous efflux of glutamate through a Ca2+‐independent pathway that accounted for the release of 25% of total intrasynaptosomal glutamate in 5 min. The results suggest that a Ca2+‐independent release of glutamate could be implicated in the neurotoxic action of this amino acid during anoxia.

Group-I metabotropic glutamate receptors: hypotheses to explain their dual role in neurotoxicity and neuroprotection

The role of group-I metabotropic glutamate receptors (mGlu1 and 5) in neurodegeneration is still controversial. While antagonists of these receptors are consistently neuroprotective, agonists have been found to either amplify or attenuate excitotoxic neuronal death. At least three variables affect responses to agonists: (i) the presence of the NR2C subunit in the NMDA receptor complex; (ii) the existence of an activity-dependent functional switch of group-I mGlu receptors, similar to that described for the regulation of glutamate release; and (iii) the presence of astrocytes expressing mGlu5 receptors. Thus, a number of factors, including the heteromeric composition of NMDA receptors, the exposure time to drugs or to ambient glutamate, and the function of astrocytes clearing extracellular glutamate and producing neurotoxic or neuroprotective factors, must be taken into account when examining the role of group-I mGlu receptors in neurodegeneration/neuroprotection.

An Ion Channel Locus for the Protein Kinase C Potentiation of Transmitter Glutamate Release from Guinea Pig Cerebrocortical Synaptosomes

The mechanism by which protein kinase C (PKC) activates transmitter release from guinea pig cerebrocortical synaptosomes was investigated by employing parallel fluorescent assays of glutamate release, cytoplasmic free Ca2+, and plasma membrane potential. 4β‐Phorbol dibutyrate (4β‐ PDBu) enhances the Ca2+‐dependent, 4‐aminopyridine (4AP)‐evoked release of glutamate from synaptosomes, the 4AP‐evoked elevation of cytoplasmic free Ca2+, and the 4AP‐ evoked depolarization of the plasma membrane. 4β‐PDBu itself causes a slow depolarization, which may underlie the small effect of 4β‐PDBu on spontaneous, KCI‐evoked, and Ca2+‐independent/4AP‐evoked glutamate release. Because 4AP (but not KCI) generates spontaneous, tetrodotoxin‐sensitive action potentials in synaptosomes, a major locus of presynaptic PKC action is to enhance these action potentials, perhaps by inhibiting delayed rectifier K+ channels.

Presynaptic receptors and the control of glutamate exocytosis

When a typical glutamate-containing neurone fires, an action potential is propagated down the branching axon through more than a thousand varicosities. At each of these release sites the probability that a synaptic vesicle will be exocytosed into the synaptic cleft is individually controlled by means of presynaptic receptors: autoreceptors responding by positive or negative feedback to previously released transmitter, or heteroreceptors under the influence of other neurotransmitters or modulators. The simplest system in which to investigate presynaptic modulation is the isolated nerve terminal or synaptosome; studies with this preparation have revealed a complex interplay of signal-transduction pathways.

Switch from Facilitation to Inhibition of Excitatory Synaptic Transmission by Group I mGluR Desensitization

We have explored whether the desensitization of metabotropic glutamate receptors (mGluRs) coupled to phosphoinositide hydrolysis affects the role that they play in modulating glutamate release. In hippocampal nerve terminals, the agonist 3,5-dihydroxyphenylglycine (DHPG) facilitated evoked glutamate release, but a second stimulation 5 min later reduced rather than facilitated release. After a 30 min interval between stimulations, DHPG again facilitated glutamate release. In hippocampal slices, DHPG caused an inhibition of excitatory postsynaptic currents (EPSCs) recorded from CA1 neurons. However, when the effects of ambient glutamate were prevented, mGluR activation initially induced a facilitation of synaptic transmission, followed by an inhibition. We conclude that group I mGluRs have a dual action on glutamate release, switching from facilitatory to inhibitory upon receptor desensitization triggered by low concentrations of glutamate.

Rapid Desensitization of the Metabotropic Glutamate Receptor that Facilitates Glutamate Release in Rat Cerebrocortical Nerve Terminals

The metabotropic autoreceptor of glutamatergic nerve terminals from the cerebral cortex of adult rats has been characterized. Receptor activation involves a rapid and transient increase in diacylglycerol, which is sensitive to l‐2‐amino‐3‐phosphonopropionate (l‐AP3) and l‐2‐amino‐4‐phosphonobutanoic acid (l‐AP4) and is partially blocked by pertussis toxin. Protein kinase C (PKC) has a negative feedback control in this transduction pathway because the activation of the kinase, either by phorbol esters or by the endogenous diacylglycerol produced by the receptor, results in a reversible receptor desensitization, with loss of the ability to further facilitate glutamate release. It is concluded that the facilitatory metabotropic receptor located at the glutamatergic nerve endings belongs to the subclass coupled to phosphoinositide hydrolysis and that the rapid and use‐dependent desensitization of the facilitatory pathway may underlie a mechanism to prevent its permanent activation and thereby to avoid neurotoxicity.

Presynaptic Modulation of Glutamate Release Targets Different Calcium Channels in Rat Cerebrocortical Nerve Terminals

We have studied which type/s of Ca2+ ‐channel/s support glutamate exocytosis and its modulation by presynaptic receptors in cerebrocortical nerve terminals. Depolarization of nerve terminals with 30 mM KCI induced a Ca2+ ‐dependent release of 3.64 ± 0.25 nmol/mg of protein. The addition of either 2 μM ω‐conotoxin‐GVIA or 200 nM ω‐agatoxin‐IVA reduced the KCI‐evoked release by 47.7 ± 3.5% and 70.4 ± 8.9% respectively, and by 85.7 ± 4.1% when both toxins were co‐applied. The activation of adenosine A1 receptors with N6‐cyclohexyladenosine or the activation of rnetabotropìc glutamate receptors with L(+)‐2‐amino‐4‐phosphonobutyrate inhibited the KCI‐evoked release by 41.0 ± 5.9 and 54.3 ± 10% respectively. The extent of these inhibitions was not altered by the prior addition of 2 μM ω‐conotoxin‐GVIA but they were significantly enhanced when ω‐agatoxin‐IVA was added together with the adenosine A1 receptor agonist or the metabotropic glutamate receptor agonist, suggesting that ω‐conotoxin‐GVIA‐sensitive and not ω‐agatoxin‐IVA‐sensitive Ca2+‐channels are ínvolved in the action of these inhibitory receptors. By contrast, the facilitation of glutamate release that follows the activation of the protein kinase C, either with phorbol esters or with the stimulation of phospholipase C‐linked metabotropic receptors, was expressed by both ω‐conotoxin‐GVIA‐sensitive and ω‐agatoxin‐sensitive Ca2+‐channels. It is concluded that different Ca2+‐channels support the modulation of glutamate release by presynaptic receptors.

Functional switch from facilitation to inhibition in the control of glutamate release by metabotropic glutamate receptors.

We have investigated the role of metabotropic glutamate receptors linked to phosphoinositide hydrolysis in the control of glutamate release in cerebrocortical nerve terminals. The activation of these receptors with the agonist 3,5-dihydroxyphenylglycine enhanced intrasynaptosomal diacylglycerol and facilitated both the depolarization-induced increase in the cytosolic free Ca2+ concentration and the release of glutamate. However, 5 min after receptor activation, a second stimulation of the pathway with the agonist failed to produce diacylglycerol and to facilitate glutamate release. Interestingly, during the period in which the diacylglycerol response was desensitized, a strong agonist-induced inhibition of Ca2+entry and glutamate release was observed. This change in the presynaptic effects of 3,5-dihydroxyphenylglycine is reversible since 30 min after the first stimulation, the agonist-induced inhibition of release disappeared, whereas both the production of diacylglycerol and the facilitation of glutamate release were recovered. The tonic elevation of the extracellular glutamate concentration from basal levels (0.8 μm) up to 5 μm also produced the switch from facilitation to inhibition in the receptor response. The existence of this activity-dependent switch in the presynaptic control of glutamate release suggests that release facilitation is limited to conditions under which an appropriate clearance of synaptic glutamate exists, probably to prevent the neurotoxic accumulation of glutamate in the synapse.

Neuroprotectant minocycline depresses glutamatergic neurotransmission and Ca2+ signalling in hippocampal neurons

The mechanism of the neuroprotective action of the tetracycline antibiotic minocycline against various neuron insults is controversial. In an attempt to clarify this mechanism, we have studied here its effects on various electrophysiological parameters, Ca2+ signalling, and glutamate release, in primary cultures of rat hippocampal neurons, and in synaptosomes. Spontaneous excitatory postsynaptic currents and action potential firing were drastically decreased by minocycline at concentrations known to afford neuroprotection. The drug also blocked whole‐cell inward Na+ currents (INa) by 20%, and the whole‐cell Ca2+ current (ICa) by about 30%. Minocycline inhibited glutamate‐evoked elevation of the cytosolic Ca2+ concentration ([Ca2+]c) by nearly 40%, and K+‐evoked glutamate release from synaptosomes by 63%. Minocycline also depressed the frequency and amplitude of spontaneous excitatory postsynaptic currents, but did not affect the whole‐cell inward current elicited by γ‐aminobutyric acid or glutamate. This pharmacological profile suggests that the neuroprotective effects of minocycline might be associated with the mitigation of neuronal excitability, glutamate release, and Ca2+ overloading.

A restricted population of CB1 cannabinoid receptors with neuroprotective activity

Significance Cannabinoids and their endogenous counterparts, the so-called endocannabinoids, promote neuroprotection in laboratory animals by engaging CB1 cannabinoid receptors, one of the most abundant types of receptors in the brain. However, the assessment of the physiological relevance and therapeutic potential of the CB1 receptor in neurological diseases is hampered, at least in part, by the lack of knowledge of the neuron-population specificity of CB1 receptor action. This study shows that a unique and well-defined population of CB1 receptors, namely that located on glutamatergic terminals, plays a key neuroprotective role in the mouse brain. This finding opens a new conceptual view on how the CB1 receptor evokes neuroprotection, and provides preclinical support for improving the development of cannabinoid-based neuroprotective therapies. The CB1 cannabinoid receptor, the main molecular target of endocannabinoids and cannabis active components, is the most abundant G protein-coupled receptor in the mammalian brain. Of note, CB1 receptors are expressed at the synapses of two opposing (i.e., GABAergic/inhibitory and glutamatergic/excitatory) neuronal populations, so the activation of one and/or another receptor population may conceivably evoke different effects. Despite the widely reported neuroprotective activity of the CB1 receptor in animal models, the precise pathophysiological relevance of those two CB1 receptor pools in neurodegenerative processes is unknown. Here, we first induced excitotoxic damage in the mouse brain by (i) administering quinolinic acid to conditional mutant animals lacking CB1 receptors selectively in GABAergic or glutamatergic neurons, and (ii) manipulating corticostriatal glutamatergic projections remotely with a designer receptor exclusively activated by designer drug pharmacogenetic approach. We next examined the alterations that occur in the R6/2 mouse, a well-established model of Huntington disease, upon (i) fully knocking out CB1 receptors, and (ii) deleting CB1 receptors selectively in corticostriatal glutamatergic or striatal GABAergic neurons. The data unequivocally identify the restricted population of CB1 receptors located on glutamatergic terminals as an indispensable player in the neuroprotective activity of (endo)cannabinoids, therefore suggesting that this precise receptor pool constitutes a promising target for neuroprotective therapeutic strategies.