Electrically evoked GABA release in rat hippocampus CA1 region and its changes during kindling epileptogenesis.

Previous findings on changes in K+-induced GABA release from hippocampal slices during kindling epileptogenesis were reinvestigated using physiological electrical stimulation. For that purpose, a procedure was developed enabling neurochemical monitoring of GABA release locally in the CA1 region of rat hippocampal slices upon tetanic stimulation of Schaffer-collateral fibers. In the presence of a GABA reuptake blocker, subsequent application of short (3 s) pulses of 50-Hz stimuli induced a local transient increase in GABA release. In slices from fully kindled animals, 24 h after the last generalized seizure, tetanically stimulated GABA release was increased in comparison to control slices. In slices from long-term kindled animals, 4-5 weeks after the last seizure, tetanically stimulated GABA release had returned to control levels. Application of the broad low-affinity GABAB receptor antagonist saclofen increased the tetanically stimulated GABA release in control slices, but had no effect in fully kindled slices. In slices from long-term kindled animals, however, saclofen enhanced GABA release similarly as in control slices. We conclude that the transient increase in tetanus-induced GABA release during kindling epileptogenesis is seizure-related, and probably caused by temporarily impaired presynaptic GABAB receptors. The possible relevance of this finding for GABA transmission in epilepsy is discussed.

Differential signaling in presynaptic neurotransmitter release.

Neuronal communication is tightly regulated by presynaptic signaling, thereby temporarily and locally secreting one or more transmitters in order to exert propagation or modulation of network activity. In the last 2 decades our insight into the molecular regulation of presynaptic transmitter vesicle traffic and fusion has exponentionally grown due to the identification of specific functional interactions between presynaptic proteins involved in these processes. In addition, a plethora of extracellular and intracellular messengers regulate neurotransmitter release, occasionally leading to short- or long-term adaptations of the synapse to altered environmental signals. Important in this respect is the ability of various nerve terminals to diverge their output by differentiation in secretion of co-localized transmitters. This divergence in presynaptic signaling may converge in the postsynaptic target neuron or spread to neighbouring cells. In this review differential presynaptic signaling mechanisms will be related to their potential divergent roles in transmitter release.

Regulation of vesicle traffic and neurotransmitter release in isolated nerve terminals.

In this overview current insights in the regulation of presynaptic transmitter release, mainly acquired in studies using isolated CNS nerve terminals are highlighted. The following aspects are described. (i) The usefulness of pinched-off nerve terminals, so-called synaptosomes, for biochemical and ultrastructural studies of presynaptic stimulus-secretion coupling. (ii) The regulation of neurotransmitter release by multiple Ca2+ channels, with special emphasis on the specificity of different classes of these channels with respect to the release of distinct types of neurotransmitters, that are often co-localized, such as amino acids and neuropeptides. (iii) Possible molecular mechanisms involved in targeting synaptic vesicle (SV) traffic toward the active zone. (iv) The role of presynaptic receptors in regulating transmitter release, with special emphasis on different glutamate subtype receptors. Isolated nerve terminals are of great value as model system in order to obtain a better understanding of the regulation of the release of distinct classes of neurotransmitters in tiny CNS nerve endings.

Neurotransmitter release from tottering mice nerve terminals with reduced expression of mutated P- and Q-type Ca2+-channels.

Neurotransmitter release is triggered by Ca2+-influx through multiple sub-types of high voltage-activated Ca2+-channels. Tottering mice have a mutation in the alpha1A pore-forming subunit of P- and Q-type Ca2+-channels, two prominent sub-types that regulate transmitter release from central nerve terminals. Immunoblotting analysis of purified forebrain terminals from tottering mice revealed an 85% reduction in the protein expression level of the mutated alpha1A subunit compared to expression of the alpha1A subunit in wild-type terminals. In contrast, the expression of the alpha1B subunit of the N-type Ca2+-channels was unchanged. Release of the amino acids glutamate and GABA and of the neuropeptide cholecystokinin (CCK) induced by a short (100 ms) depolarization pulse was unchanged in the terminals of tottering mice. Studies using specific blockers of Ca2+-channels however, revealed a reduced contribution of P- and Q-type Ca2+-channels to glutamate and cholecystokinin release, whereas a greater reliance on N-type Ca2+-channels for release of these transmitters was observed. In contrast, the contribution of the P-, Q- and N-type Ca2+-channels to the release of GABA was not altered in tottering mice. These results indicate that the expression of the alpha1A subunit was decreased in terminals from tottering mice, and that a decreased contribution of P- and Q-type Ca2+-channels to the release of glutamate and cholecystokinin was functionally compensated by an increased contribution of N-type Ca2+-channels.

Glutamate transporters alterations in the reorganizing dentate gyrus are associated with progressive seizure activity in chronic epileptic rats.

The expression of glial and neuronal glutamate transporter proteins was investigated in the hippocampal region at different time points after electrically induced status epilepticus (SE) in the rat. This experimental rat model for mesial temporal lobe epilepsy is characterized by cell loss, gliosis, synaptic reorganization, and chronic seizures after a latent period. Despite extensive gliosis, immunocytochemistry revealed only an up-regulation of both glial transporters localized at the outer aspect of the inner molecular layer (iml) in chronic epileptic rats. The neuronal EAAC1 transporter was increased in many somata of individual CA1-3 neurons and granule cells that had survived after SE; this up-regulation was still present in the chronic epileptic phase. In contrast, a permanent decrease of EAAC1 immunoreactivity was observed in the iml of the dentate gyrus. This permanent decrease in EAAC1 expression, which was only observed in rats that experienced progressive spontaneous seizure activity, could lead to abnormal glutamate levels in the iml once new abnormal glutamatergic synaptic contacts are formed by means of sprouted mossy fibers. Considering the steady growth of reorganizing mossy fibers in the iml, the absence of a glutamate reuptake mechanism in this region could contribute to progression of spontaneous seizure activity, which occurs with a similar time course.

A biochemical approach to study sub-second endogenous release of diverse neurotransmitters from central nerve terminals.

Exocytosis in central nerve terminals is rapidly triggered by the influx of calcium through high voltage sensitive Ca2+ -channels. Mainly due to their small size, studies in which neurotransmitter release from these terminals was determined at the sub-second time-scale are still rather limited. Here we describe the use of a pneumatic rapid mixing device, allowing application of short (> or = 50 ms) K+ -depolarizing pulses to purified nerve terminals, synaptosomes, to trigger endogenous release of different transmitter types. A consistent, Ca2+ -dependent exocytotic release of the amino acid transmitters, glutamate and GABA, from synaptosomes purified from rat and mouse brain was observed after 100 ms depolarization. For determination of amino acid release after longer depolarizations (> 100 ms), transporter blockers had to be added to prevent clearance of the vesicularly released transmitters. Ca2+ -dependent release of the neuropeptide cholecystokinin occured only after 250 ms depolarization. In addition, the time-courses of amino acid and cholecystokinin release were clearly different. The fast Ca2+ -dependent release of all transmitters was selectively and strongly inhibited by the P/Q-type Ca2+ -channel blocker omega-Agatoxin IVA. In conclusion, this approach allows direct measurement of Ca2+ -dependent release of diverse endogenous neurotransmitters from central nerve terminals upon depolarization pulses at a physiologically relevant, sub-second, time scale.