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Serontonergic, purinergic, and calcium dependent mechanisms in rat hippocampal pyramidal cells

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Title: Serontonergic, purinergic, and calcium dependent mechanisms in rat hippocampal pyramidal cells
Author: Obenaus, Andre
Degree Doctor of Philosophy - PhD
Program Physiology
Copyright Date: 1989
Abstract: This series of investigations using the in vitro rat hippocampal slice, examines the role of two important neuromodulatory compounds, serotonin and adenosine. The agonists and antagonists of these compounds were examined for their effects on normal evoked synaptic activity and on epileptiform activity in a low calcium (Ca²⁺) bursting model. Perfusion of serotonin (5-HT) in the CA1 region and dentate gyrus (DG) reduced the evoked population spike amplitude. The largest reductions, followed the perfusion of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (a 5-HT[sub 1A] receptor agonist). While application of the 5-HT₂ receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), did not result any changes in the CA1 region, and only slight reductions in the DG. Application of adenosine elicited a reduction in the evoked population spike, with the specific agonist, 2-chloroadenosine, being much more effective. Perfusion of the degradative enzyme for adenosine, adenosine deaminase potentiated the amplitude of the population spike. Perfusion of the purine antagonist, theophylline, elicited a transient increase in the population spike amplitude, proving that serotonergic and purinergic compounds have the ability to exert inhibitory effects on evoked synaptic transmission in the hippocampus. Rhythmic synchronous bursting discharges are observed in the CA1 region of hippocampal slices when perfused with artificial cerebrospinal fluid (ACSF) containing low concentrations of calcium. Such bursts are characterized by shifts in the extracellular DC potential upon which population spikes are superimposed: this phenomenon provides a simple model to study cellular hyper-excitability which occurs in the absence of synaptic transmission. The introduction of 5-HT resulted in a reduction in burst rate only at high doses (e.g. 50μM), while 8-OH-DPAT depressed it significantly at all doses tested. Application of adenosine yielded significant reductions only if high concentrations were used. The catabolic enzyme, adenosine deaminase increased the burst rate 2-10 times above control burst rates. Likewise, addition of two methylxanthines, known adenosine antagonists, 3-isobutyl-l-methylxanthine (IBMX) and theophylline to the perfusate reversibly increased the burst rate, strongly indicating that large concentrations of purines are being released to control burst behavior. Acting together the serotonergic and purinergic systems modulate hippocampal activity. Convulsant-induced burst discharges in normal CA1 pyramidal neurons activate the N-methyl-D-aspartate (NMDA) receptor. Surprisingly the NMDA antagonist, D-2-amino-5-phosphonovaleric acid (D-APV), displayed a significant and reversible inhibitory effect on burst rates in the presence of low extracellular Ca²⁺. This reduction suggests that glutamate or aspartate may released under low Ca²⁺ conditions and that these may be exerting an excitatory effect leading to the bursting activity. Evidence is presented which demonstrates possible roles for serotonergic, purinergic and possibly excitatory amino acids in modulating the mechanisms underlying the generation of presumed non-synaptic bursting phenomena. Previous studies have suggested that these compounds may exert their effects via modulation of free [Ca²⁺][sub i], however, Fura-2 microspectrofluorimetry in cultured hippocampal neurons treated with serotonergic and purinergic agonists and antagonists found no such changes in [Ca²⁺][sub i]. Long-term potentiation (LTP) is characterized by a long lasting increase in the efficacy of neurotransmission. An essential function for calcium ions in the induction of LTP has been established and a particular emphasis has been placed on the role of NMDA receptor activation in gating a post-synaptic influx of calcium (Mayer and Westbrook 1987a). The present experiments used dantrolene to blockade intraneuronal calcium release and were able to completely block the induction of.both tetanic and Ca²⁺-induced LTP in the CA1 region of the rat hippocampal slice. This drug inhibits calcium release from the sarcoplasmic reticulum and also diminishes the rise in intraneuronal calcium ion concentrations elicited by NMDA receptor activation in cultured CA1 pyramidal cells. Dantrolene does not block NMDA gated membrane currents or voltage activated Ca²⁺currents in these cells (Mody et al 1989). In contrast to reported effects in the DG granule cell layer, NMDA receptors do not seem to be directly involved in Ca²⁺-induced LTP. In the CA1 pyramidal cell layer the NMDA antagonist D-APV did not block the induction of LTP by transient exposure of hippocampal slices to a high Ca²⁺-containing medium. To extend these findings, the role of intraneuronal Ca²⁺ was investigated on cultured hippocampal neurons using Fura-2 microspectrofluorimetry. In neurons perfused with a medium containing normal Ca²⁺, the resting [Ca²⁺][sub i] was approximately 100 nM. Perfusion of high Ca²⁺for 10 min revealed a rapid and sustained increase in [Ca²⁺][sub i] which persisted after the return to normal [Ca²⁺][sub o]. Application of dantrolene in this experimental paradigm prevented the rise in [Ca²⁺][sub i]. Interestingly, D-APV applied prior to and during the high [Ca²⁺][sub o] perfusion, did not effect the changes in [Ca²⁺][sub i]. The combined results from both the hippocampal slice preparation and neuronal cultures suggest that while not excluding pre-synaptic involvement in the maintenance of LTP, the release of calcium from intraneuronal stores, rather than a transmembrane calcium influx may be the critical post-synaptic feature underlying the induction of both tetanic and Ca²⁺ -induced LTP.
URI: http://hdl.handle.net/2429/29256
Series/Report no. UBC Retrospective Theses Digitization Project [http://www.library.ubc.ca/archives/retro_theses/]
Scholarly Level: Graduate

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