Pharmacology of the dendritic action of acetylcholine and further observations on the somatic disinhibition in the rat hippocampus in situ.

In rats under urethane anaesthesia various cholinergic agonists and antagonists were microiontophorctically applied in the pyramidal layer of area CA1 and in the apical dendrites. using a twin set of multibarreled micropipettes. Thus, the somatic population spike and the dendritic negative field (field excitarorypostsynaptic potential) evoked by commissural stimulation and the effect of iontophoretic agents at either site were recorded simultaneously. Using this double recording system, several observations were made which confirmed the previous conclusions that acetylcholine has an important disinhibitory action at the level of the pyramidal cell bodies. Both muscarinic and nicotinic agents enhanced the somatic population spikes without altering the dendritic field excitatory postsynaptic potential. Local ejection of atropine, or scopolamine readily depressed or abolished the action of muscarinic agents (such as muscarine, metacholine or acetylcholine) without reducing the effects of nicotinic agents. Conversely, on several occasions, brief applications of curare or dihydro-/$-erythroidine selectively reduced the effects of dimethylphenylpiperazinium or tetramethyl~~mm~~niurn. Local appii~tions of acetyicholine in the apical dendrites reduced the field excitatory postsynaptic potential and the somatic population spike recorded simultaneously. This effect was reproduced by muscarinic agents (notably muscarine or metacho~ine)~ in contrast nicotinic agents (notably dimethylphenylpiper~tzinium or tetramethyiammonium) increased the fiefd excitatory postsynaptic potential and the simultaneously recorded somatic spike. This effect had a slow onset and a maximal increase was observed toward the end of a 20 s application. These actions were pharmacologically specific since local ejection of scopolamine or atropine completely blocked the action of muscarinic agents whereas curare or dihydro-fi-erythroidine selectively reduced the enhancement of the field excitatory postsynaptic potential produced by dimethylphenylpiperazinium or tetramethylammonium. In summary. both muscarinic and nicotinic receptors are involved in the facilitatory action of cholinomimetics at the cell soma, whereas muscarinic and nicotinic agents have an opposite actions on the dendritic tield excitatory postsynaptic potentials. Extensive ~ln~itornic~~l and biochemical studies have demonstrated the existence of a cholinergic afferent pathway which originates in the medial septum and diagonal band complex and projects both to the immediate vicinity of the pyramidal and granular layers and to various dendritic segments.29,3’,3’,40 Recent intracellular observations made irk citro and in aira have provided strong evidence in favour of a postsynaptic site of action of ACh.7.A.1”.” Hounsgaard’” first reported that local applications of ACh in the apical dendrites of CAI in the hippocampai slice * To whom reprint requests should be addressed. f PWWN c~&ir~.s~ Dep;irtment of nutrition and Food Sciences. M.I.T. Cambridge. MA 01139. U.S.A. + PW.WI~ &~WSS: McIntyre Bldg. McGill University, 365 Drummond Street. Montreal. Quebec H3G IYb Canada. /thh~~~intio~~~: ACh. acctylcholinc: GABA, y-aminobutpte: IPSP. inhibitory postsynaptic potential; EPSP. excitatory postsynaptic potential: DMPP. diphenylmethylpipera7inium: MeCh. metacholine: DHE. dihydro-/l-erythroidine: TMA. tetramethylammonium. Yi preparation reduce the excitatory postsynaptic potentials (EPSP) and provided evidence that this action was best explained by a presynaptic reduction of the release of excitatory transmitter. This was confirmed and extended in the same preparation more recently. AZ In the intact animal, Ben-Ari ef (11.~ have shown that microiontophoretic application of ACh in the pyramidal layer reduces the probably GABA-ergic inhibitory postsynaptic potential (IPSP) and its associated conductance change without reducing the response elicited by local application of GABA. Therefore ACh appears to modulate ptesyn~pticaily the excitatory and inhibitory drives which impinge upon the dendritic and somatic elements of the hippocampus. In the present report. we have studied the effects of dendritic and somatic applications of muscarinic and nicotinic agents in situ. To gain a better understanding of the dual modulatory action of ACh. we have used a method which was recently developed in this laboratory and which makes it possible to record simultaneous in situ somatic and dendritic potentials and to apply m~croionto~horetically various agents at either site.” This study has been reported else~hcre in brief.f*3” EXPERIMENTAL PROCEDURES Forty adult male Wistar rats (20@3OOg) were studied. They had access to food and water ad lib and were housed in cages under diurnal lighting conditions with lights on from 08.00 h. Experimental procedures were carried out under general anaesthesia with urethane (2~.3g/kg i.p.); The core temperature was maintained at 37”.-38°C and the heart rate was continuously monitored. The animal was placed in a conventional stereotaxic frame and bipolar electrodes were introduced to stimulate the ventral hippocampal commissure and fimbria (details in ref. 6). Seven barrelled micropipettes (4-7 pm tip diameter) were introduced through the overlying cortex to the regio superior of Ammon’s horn (CAI, ref. 27). The surface was then covered with agar (4% w/v in saline). In 20 experiments, the dendritic and somatic potentials were recorded simultaneously using a twin set of multibarrelled pipettes. This method has been described in detail elsewhere.’ ’ In brief. conventional multibarrelled electrodes were first prepared, then another multibarrelled pipette was bent with a microforge (approsimately 20-30” from vertical) and glued with dental cement to a (straight) muttibarrelled assembly. The vertical distance between the tips varied from 10&600 blrn depending on the dendritic segment under investi~tio~l: however, routinely the distance was 220-25O~im. so that one elcctrode was placed in the pyramidal layer, the other (deeper) one was situated optimally to record the negative field (EPSP) generated in the stratum radiatum by activation of Schaffer collaterals. The central barrel of each set contained NaCl (3 M) or pontamine sky blue (2:, w!v in 0.5 M Na acetate) for the determination of the electrode position at the end of the experiment. I5 The remaining barrels contained, in various permutations, the following drugs: acetylcholine f I M. ACh), muscarine Cl (0.05 M, MUSC); acetyl-/?-methylcholine C1 (0.1 M, MeCh); atropine sulphate (1 mM in 165 mM NaCl); scopolamine hydrobromide (I mM in 165 NaCli: hexamethonium Br (10 mM); gailamine trieth~od~dc (0.5 mM); mecamytamine (1 M); dihydro-~-ery~hro~d~nc (5 mM in 165 mM NaCL DHE, kindly donated by A. Ttautman); d-tubocurarine Cl (10 mM in 165 mM NaCII; tetramethylammonium hydroxide (1 M, TMA); diphenylmethylpiperazinium iodide (0.1 M in 165 mM NaCL DMPP); eserine sulphate, 0.1 M); Benzylpenicillin (G) Na (0.1 M in 165 mM NaCl); bicucullinc methiodide (IOmM in 165 mM NaCl, Pierce); picrotoxin (5 mM in 165 mM NaCl); GABA (1 M). Unless otherwise indicated all drugs were purchased from Sigma. At the end of the experiment, the rat was intracardiailj perfused with saline, foltowed by a 10% formalin sotution and the brains processed for acetylcholinesterase staining. using the method adapted by Lewis,” and Nissi or Klijver-Barrera stains to verify the location of stimulating and recording electrodes.