Präsynaptische a2-Adrenozeptoren in hippokampalen Neuronen

Research project P 13920 Mechanisms of presynaptic inhibition exemplified by alpha2-adrenoceptors in hippocampal neurons Stefan BÖHM 28.06.1999 Presynaptic receptors are believed to shape neurotransmission at every synapse-in the central as well as peripheral nervous system. One archetypic presynaptic receptor is the (alpha2-adrenoceptor the function of which has been charachterized in greatest detail in sympathetic neurons where alpha2-autoreceptors mediate a feedback inhibition of noradrenaline release. The hippocampus receives a dense noradrenergic innervation. However, contrasting results have been obtained with noradrenaline in various in vitro preparations of hippocampal neurons, where the amine was found to elicit excitatory as well as inhibitory actions. In vivo, the amine appears to be primarily inhibitory, since the noradrenergic ascending system in the brain provides antiepileptogenic activity. The present project investigates mechanisms potentially underlying the antiepileptogenic activity of noradrenaline, and the preliminary data indicate that glutamatergic, but not gabaergic, hippocampal neurons are equipped with presynaptic adrenoceptors. Previous results had suggested that presynaptic alpha1-adrenoceptors may reduce glutamatergic transmission (Scanziani et al, 1993), whereas our results indicate that glutamatergic neurons are equipped with presynaptic alpha2-adrenoceptors. In the course of this project we will characterise the presynaptic adrenoceptors of glutamatergic nerve terminals by pharmacological as well as molecular techniques. In addition, this project will focus on the underlying signalling mechanisms which according to our preliminary results include an inhibition of voltage-gated Ca2+ channels. Furthermore, we will investigate as to how activation of G protein- coupled receptors may lead to a reduction of spontaneous (Ca2+-independent) glutamate release. Our preliminary data suggest that activation of GTP binding proteins is sufficient to hinder the interaction between SNARE proteins which have been implicated in vesicle exocytosis, (Söllner et al, 1993). We will test for direct interactions between G protein sub-units and SNARE components and for phosphorylation and/or dephosphorylation reactions posssibly involved in the inhibition of spontaneous glutamate release. Enhancement of glutamate release is a pathological key event in, for instance, cerebral ischemia and epilepsy. Therefore, reduction of glutamate release by agonists at presynaptic receptors, such as the (alpha2-adrenoceptors investigated here, or activation of downstream signalling cascades may provide potential strategies for neuroprotective pharmacotherapy.

Presynaptic receptors are believed to shape neurotransmission at every synapse in the central as well as peripheral nervous system. Initially, the focus of this project were presynaptic alpha2 -adrenoceptors in hippocampal neurons which were found to inhibit glutamate, but not GABA, release, thus providing the basis for the antiepileptogenic activity of the noradrenergic ascending system in the brain. A second and in the end predominant focus of the project were presynaptic autoreceptors of glutamatergic neurons in the hippocampus. Metabotropic glutamate receptors of the group III were revealed to require Ca2+-bound calmodulin to activate signalling cascades via G protein beta-gamma subunits. As a consequence, the autoinhibition of glutamate release via these receptors is activity-dependent as it is based not only on the release of glutamate, but also on increases in intracellular Ca2+. Thus, the more Ca2+ enters the glutamatergic nerve terminal, the more calmodulin will be available to support the signalling via presynaptic group III mGluRs. Increases in neuronal firing and enhancement of glutamate release are pathological key events in, for instance, cerebral ischemia and epilepsy. Therefore, reduction of glutamate release by agonists at any presynaptic receptor (such as alpha2-adrenoceptors) or activation of downstream signalling cascades may provide potential strategies for neuroprotective pharmacotherapy. In this respect, the presynaptic group III mGluRs appear to be particularly interesting targets, as their activation will not influence neuronal signalling unless the glutamatergic nerve terminals are depolarized by invading action potentials. Additional experiments supported by the project dealt with the role of SNARE complexes during vesicle exocytosis. In PC12 cells, a high molecular weight complex consisting of the SNARE proteins synaptobrevin, syntaxin and SNAP-25 was found to dissociate and reassociate under release promoting conditions. This reveals that such complexes are not only biochemical characteristics of proteins involved in membrane fusion, but also correlates of neuronal functions.