Abstracts

The neocortex, an area involved in the generation and the propagation of the epileptiform activity, has a dense cholinergic innervation, which develops early in postnatal life. We therefore investigated the cholinergic effects on epileptiform discharge generation in immature rat neocortical slices on a model of disinhibition.
Evoked and spontaneous field potentials were recorded during perfusion with the GABA[sub]A[/sub] receptor antagonist bicuculline methiodide (BMI, 50 [mu]M ) from the deep layers of immature (postnatal day 10-20) sensorimotor cortex slices, obtained from Sprague-Dawley rat pups.
The anticholinesterase eserine (10[micro]M) diminished evoked field potential amplitude and duration by ~40%, but induced or increased significantly the rates of occurrence of spontaneous discharges of both interictal and ictal type. The non hydrolysable ACh-analog carbachol (CCh, 25[micro]M) depressed evoked discharges similarly to eserine, but increased the frequency of spontaneous discharges significantly more. The muscarinic antagonist atropine reversed fully these cholinergic effects (2.5[micro]M, n= 20), however the nicotinic receptor antagonist hexamethonium was ineffective (50[micro]M, n=3). Subtype-selective muscarinic antagonists decreased the rates of occurence of spontaneous discharges (AFDX 116 for M2 receptors, 1[micro]M, n=4, by 40%; 4 DAMP, M3, 1[micro]M, by 80%; gallamine, M4, n=5 by 50%; tropicamide, M4, 1[micro]M, n=6, by 60%), and the M1 antagonist pirenzepine (1[micro]M) abolished them in 8/12 slices, thus suggesting a major role for this subtype. Combination of the glutamate receptor antagonists APV (10[micro]M) and CNQX (10[micro]M; n=11) blocked the CCh-induced spontaneous discharges. The gap junction blocker halothane (2.5%, n=12) abolished them, while carbenoxolone (100-500[mu]M, n=8) depressed their frequency by ~65%. By contrast, perfusion with high Ca²⁺ (7mM; n=6), a manipulation that blocks polysynaptic connections, had no effect on the rates of these spontaneous discharges. The inhibitors of intracellular calcium mobilization dantrolene (100 [mu]M, n=5), a ryanodine receptor blocker and thapsigargin (1[mu]M, n=5), an inhibitor of the sarco/endoplasmic reticulum calcium ATPase, both decreased significantly the frequency of CCh-induced spontaneous discharges.
These findings demonstrate that activation of muscarinic receptors, of the M1 type in particular, in immature rat neocortex during GABA[sub]A[/sub] receptor blockade generates epileptiform discharges of ictal and interictal type by facilitating glutamatergic transmission. These muscarinic effects are partly mediated by an increase in intracellular calcium release, while activation of gap-junctions appears to be a [italic]sine qua non[/italic] condition for their expression.
[Supported by: NSERC, Savoy Foundation for Epilepsy [amp] Ste-Justine Research Centre.]