Studies on the asynchronous synaptic responses and endogenous potentiating substances of neurotransmission in the hippocampus
Creators: name: Chirwa, Sanika Samuel
Publisher: name: University of British Columbia
Year: 2010

description: In the hippocampus, transient tetanic stimulations of inputs, or brief simultaneous pairings of conditioning intracellular postsynaptic depolarizations with activated presynaptic afferents at low stimulation frequencies, result in input specific long-term potentiation (LTP) of synaptic transmission. LTP lasts for hours in vitro, or weeks in vivo, and it is thought to be involved in memory and learning. Experimental evidence in the literature suggests that postsynaptic mechanisms mediate LTP induction, whereas presynaptic mechanisms are involved in its maintenance. Since LTP is thought to be generated by postsynaptic mechanisms and to be subsequently maintained by presynaptic processes, this suggests the presence of feedback interactions during LTP development, however, the experimental evidence for such interactions is presently not available. Consequently, the present studies were conducted to examine possible feedback interactions between postsynaptic and presynaptic elements in the hippocampus. Furthermore, the experiments tested the hypothesis that substances released during tetanic stimulations caused the release of endogenous substances that interacted with activated afferents resulting in alterations in presynaptic functions and LTP production. Experiments were conducted using transversely sectioned guinea pig hippocampal slices. Briefly, physiological medium containing 3.5 mNi Ba++ and 0.5 mM Ca (denoted as Ba medium) was used to induce the asynchronous release of transmitters, observed as evoked miniature EPSPs (minEPSPs) in CA1b neurons after stimulation of the stratum radiatum. During transient Ba++ applications, short bursts of evoked minEPSPs were observed following stimulations of the stratum radiatum or conditioning depolarizing current injections into CA1b neurons. Moreover, the frequencies of minEPSPs were significantly increased immediately after simultaneous stimulations of the stratum radiatum and conditioning depolarizing current injections into CA1b neurons. Significant increases in the frequencies of evoked minEPSPs were also observed during LTP induced by tetanic stimulations. The above increases in the frequencies of evoked minEPSPs were attributed, in part, to presynaptic changes resulting in increases in transmitters released. However, a thorough quanta! analysis is requirea to substantiate this conclusion. In order to determine whether any substances released during tetanic stimulations were involved in the mooulation of presynaptic functions and induction of LTP, samples were collected from guinea pig hippocampus and rabbit neocortex. It was found that samples that were collected during tetanic stimulations of the guinea pig hippocampus in vivo or rabbit neocortex in vivo produced LTP in the guinea pig hippocampal slice in vitro. Applications of these samples after heating and cooling failed to induce LTP. Subsequent studies demonstrated that PC-12 cells incubated in growth medium treated with samples collected during tetanic stimulations of the rabbit neocortex developed extensive neurite growths. In contrast, PC-12 cell cultures incubated in (1) heated and cooled samples, (2) samples collected in the absence of tetanic stimulations of the rabbit neocortex, or (3) plain growth medium, failed to develop neurite growths. In addition, PC-12 cell cultures that were incubatea in growth medium containing samples collected during tetanic stimulations plus saccharin (10 mM), a substance known to inhibit N6F-dependent neurite growth, failed to develop neurites. In separate experiments it was found that saccharin could block (1) the synaptic potentiating effects of the above collected and applied endogenous substances, and (2) LTP induced with tetanic stimulations, in the guinea pig hippocampus in vitro. The concentrations of saccharin used in these studies had insignificant effects on resting membrane potentials, input resistances, spontaneous or evoked responses of CA1b neurons. Furthermore, CA1b neuronal depolarizations induced by N-methyl-DL-aspartate (NMDA) or with tetanic stimulations of the stratum radiatum, were not altered by saccharin applications. In addition, saccharin had insignificant effects on paired-pulse facilitation, post-tetanic potentiations, minEPSP frequencies in CA1b neurons, and Schaffer collaterals terminal excitability. These results suggest that saccharin blocked LTP through mechanisms different from either non-specific alterations in CA1b cell properties or NMDA receptor activation. Perhaps the agent antagonized LTP at a step beyond NMDA receptor activation. That saccharin blocked LTP caused by the applied neocortical sample as well as by tetanic stimulation of the stratum radiatum, and that saccharin also blocked neurite growth in PC-12 cells induced by the neocortical samples, raises the prospect that growth related substances are involved in LTP generation. In other control experiments, it was found that the potentiating effects of the collected endogenous substances were not antagonised by atropine or dihydro-e-erythroidine. Heated and then cooled solutions of glutamate (a putative transmitter at the Schaffer col laterals-CA1b synapses) still maintained their actions on the CA1b population spike. While brief applications of 2.5 μg/ml exogenous NGF (from Vipera lebetina) during low frequency stimulations of the stratum radiatum did not consistently induce LTP, this peptide significantly facilitated the development of LTP when applied in association with tetanic stimulations of weak inputs in the CA1b area. These weak inputs could not support LTP if tetanized in the absence of the exogenous NGF. The results of the studies in this thesis suggested that postsynaptic depolarizations modulated presynaptic functions in the hippocampus. Tetanic stimulations in hippocampus and neocortex caused the release of diffusible substances, which were probably growth related macromolecules, that interacted with activated presynaptic afferents and/or subsynaptic dendritic elements resulting in LTP development. The precise locus of actions of these agents awaits further investigations.