STUDIES ON THE ASYNCHRONOUS SYNAPTIC RESPONSES AND ENDOGENOUS
POTENTIATING SUBSTANCES OF NEUROTRANSMISSION
IN THE HIPPOCAMPUS
By
SANIKA SAMUEL CHIRWA
B.Sc, (Pharmacy), The University of British Columbia, 1981 M.Sc, (Pharmarmacology & Therapeutics.). The University of British Columbia, 1985
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
in
THE FACULTY OF GRADUATE STUDIES
(Department of Pharmacology & Therapeutics,
Faculty of Medicine,
The University of British Columbia)
We accept this thesis as conforming
to the required standard
THE UNIVERSITY OF BRITISH COLUMBIA
1988 September
©Sanika Samuel Chirwa, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.
Department of Pharmacology and Therapeutics
The University of British Columbia Vancouver, Canada
Date 26 September 1988
DE-6 (2/88) CHIRWA ii
ABSTRACT
In the hippocampus, transient tetanic stimulations of inputs, or brief simultaneous pairings of conditioning intracellular postsynaptic depolariz• ations with activated presynaptic afferents at low stimulation frequencies, result in input specific long-term potentiation (LTP) of synaptic transmis• sion. 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 presyn• aptic 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 inter• actions 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 produc• tion.
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 asyn• chronous release of transmitters, observed as evoked miniature EPSPs
(minEPSPs) in CA^b neurons after stimulation of the stratum radiatum.
During transient Ba++ applications, short bursts of evoked minEPSPs were CHIKWA iii observed following stimulations of the stratum radiatum or conditioning
depolarizing current injections into CA^b neurons. Moreover, the frequen• cies of minEPSPs were significantly increased immediately after simultaneous stimulations of the stratum radiatum and conditioning depolarizing current
injections into CA^ neurons. Significant increases in the frequencies of evoked minEPSPs were also observed during LTP induced by tetanic stimula• tions. The above increases in the frequencies of evoked minEPSPs were
attributed, in part, to presynaptic changes resulting in increases in trans• mitters 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 neocor• tex 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. CHIRwA iv
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 CA^b neurons. Furthermore, CA^ 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
CA^ neurons, and Schaffer collaterals terminal excitability. These results suggest that saccharin blocked LTP through mechanisms different from either non-specific alterations in CA^ 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 radia• tum, 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-CA^ synapses) still maintained their actions on the
CAj^ population spike. While brief applications of 2.5 yg/ml exogenous
NGF (from Vipera lebetina) during low frequency stimulations of the stratum CHIKWA v radiatum did not consistently induce LTP, this peptide significantly facili• tated the development of LTP when applied in association with tetanic stimu•
lations of weak inputs in the CA^ 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 inter•
acted with activated presynaptic afferents and/or subsynaptic dendritic
elements resulting in LTP development. The precise locus of actions of
these agents awaits further investigations.
Research Supervisor CHIRWA vi
TABLE OF CONTENTS
Chapter Title Page No.
A. TITLE PAGE i
B. ABSTRACT