The Modulation of Epileptiform Activity in Rat Hippocampal Slices by Adenine Nucleotides

Ross, Fiona M (1997) The Modulation of Epileptiform Activity in Rat Hippocampal Slices by Adenine Nucleotides. PhD thesis, University of Glasgow.

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In this study the most consistent method of inducing epileptiform activity in hippocampal slices involved recording from the pyramidal cell layer of the CA3 region whilst bathing the slice in a medium containing no added magnesium and 4-aminopyridine (4AP) (50?M). The bursts produced were interictal in nature and inhibited by kynurenate (ImM) or 2-amino-5-phosphonopentanoic acid (20 and 40muM) suggesting that both NMDA and non-NMDA receptors were involved. The effect of ATP and other adenine nucleotides on the rate of spontaneous epileptiform activity was investigated. ATP and adenosine were equipotent at decreasing discharge rate at concentrations above 10muM. The depression produced by ATP was characterised by being inhibited by the A1 receptor antagonist 8-cyclopentyl, 1,3-dimethylxanthine (CPT) but resistant to adenosine deaminase (at a concentration which annulled the effect of adenosine). P2 receptor antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and suramin) were also unable to block ATP depression of bursting. AMP depressed epileptiform activity similarly to ATP. Its effect was inhibited by CPT and 5'-adenylic acid deaminase (AMPase). AMPase also blocked the depression in discharge rate elicited by ATP. 5'-nucleotidase is involved in the metabolism of AMP to adenosine. The combination of 5'-nucleotidase and adenosine deaminase with AMP or ATP resulted in no significant change in the frequency of activity. These later results, therefore, suggest that ATP acts through metabolism to AMP. The antagonism by CPT supports the involvement of A1 receptors. The ATP analogues alpha,beta-methyleneATP and 2-methylthioATP were also tested as well as uridine triphosphate. Only alpha,beta-methyleneATP (10muM) altered the discharge rate producing an increase in the frequency of spontaneous activity which was inhibited by suramin and PPADS. These results are indicative of an excitatory P2X receptor in the hippocampal CA3 region. Several have been cloned and found expressed in the hippocampus but none with the characteristics displayed in this study. BzATP, an agonist at the P2Z receptor, decreased the discharge rate during a 10 minute perfusion. This depression was not reversed upon washing but continued to progress. The combination of BzATP and CPT produced an increase in discharge rate which was smaller than that caused by CPT alone. This suggests that A1 receptors may not be involved in producing the effect of BzATP. The alpha, omega-adenine dinucleotides Ap4A and Ap5A produced a concentration-dependent depression in discharge rate. Adenosine deaminase inhibited the effect of 1muM but not the early stages of the depression produced by 10?M Ap4A and Ap5A. CPT but not AMPase antagonised the depression in rate produced by Ap4A. This suggests that in the CA3 region of the hippocampus AP4A and Ap5A act partly by stimulating xanthine sensitive receptors directly and partly through the formation of the metabolite, adenosine. AMPase initially elevated the discharge rate to a small extent but a subsequent fall in rate occurred during the wash period which continued until a plateau was reached at which the rate remained for up to 90 minutes. IMP was ineffective. Dialysis of AMPase neither affected the enzymatic activity of AMPase nor the following depression. Boiling the enzyme rendered it inactive in that it could no longer prevent the effect of AMP. The sustained depression in activity produced by AMPase was also inhibited by denaturisation. It was proposed, therefore, that AMPase induces a form of long term depression (LTD). The induction of this LTD, as measured by an effect on discharge rate, was not altered by antagonists of A1, opioid or GABAA receptors nor inhibitors of cyclo-oxygenase, nitric oxide synthase or protein kinase.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Trevor Stone
Keywords: Neurosciences
Date of Award: 1997
Depositing User: Enlighten Team
Unique ID: glathesis:1997-74733
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 27 Sep 2019 16:45
Last Modified: 27 Sep 2019 16:45

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