Multiple actions of acetylcholine on hippocampal pyramidal cells in organotypic explant cultures

Hippocampal cultures were prepared from 7- to 10-day-old rats by means of the roller-type technique. The preservation of the characteristic hippocampal cytoarchitecture allowed, after many weeks in vitro, impalement of pyramidal cells by microelectrodes under visual control. Application of 10(-7) to 10(-5) M acetylcholine to the bath depolarized hippocampal pyramidal cells, strongly increased their rate of firing and induced paroxysmal depolarization shifts. This depolarizing action was accompanied by a reduction in the amplitude of evoked postsynaptic potentials. Whereas it was not clear whether the decrease in the amplitude of the excitatory postsynaptic potentials was only a result of membrane depolarization, acetylcholine clearly and reversibly reduced the potency of evoked inhibitory postsynaptic potentials. Iontophoresis of acetylcholine to the perisomatic region of pyramidal neurons, like acetylcholine applied to the bath, increased their firing rate and powerfully decreased the amplitude and duration of spontaneous and evoked inhibitory postsynaptic potentials. In contrast, iontophoresis of acetylcholine in the pyramidal cell layer at a distance from the recorded neuron generated a hyperpolarizing response associated with a reduction in firing rate. At high current strength, the initial hyperpolarization was (often) followed by a paroxysmal depolarization shift. High frequency electrical stimulation with electrodes located close to the acetylcholine pipette in the pyramidal cell layer (i.e. about 1 mm away from the recorded neuron) mimicked the acetylcholine effect. Resistance measurements indicated that membrane input resistance was decreased in the majority of cells during application of acetylcholine. This decrease in membrane resistance may result from a direct action of acetylcholine or from an increased synaptic activity. Synaptic alterations induced by acetylcholine were quick in onset and in recovery, while the increase in the rate of firing occurred somewhat later. Atropine (10(-5) M), which had no significant action by itself, completely abolished the action of acetylcholine applied to the bath or by iontophoresis. In contradistinction, naloxone did not influence the acetylcholine effects, although opiates and opioid peptides produce paroxysmal depolarization shifts in pyramidal cells which resemble those induced by acetylcholine. Addition of 8-16 mM magnesium to the bathing solution or exposure of the cultures to a calcium-free solution containing 1 mM cobalt abolished the effects of acetylcholine. In the presence of 10(-6) g/ml tetrodotoxin, 10(-5) M acetylcholine decreased the membrane input resistance of pyramidal cells, reduced their threshold for the generation of tetrodotoxin-resistant spikes and generated paroxysmal depolarization shifts in a proportion of pyramidal cells...