Acute induction of epileptiform discharges by pilocarpine in the in vitro isolated guinea-pig brain requires enhancement of blood-brain barrier permeability

Abstract

Systemic application of the muscarinic agonist, pilocarpine, is commonly utilized to induce an acute status epilepticus that evolves into a chronic epileptic condition characterized by spontaneous seizures. Recent findings suggest that the status epilepticus induced by pilocarpine may be triggered by changes in the blood-brain barrier (BBB) permeability. We tested the role of the BBB in an acute pilocarpine model by using the in vitro model brain preparation and compared our finding with in vivo data. Arterial perfusion of the in vitro isolated guinea-pig brain with <1 mM pilocarpine did not cause epileptiform activity, but rather reduced synaptic transmission and induced steady fast (20-25 Hz) oscillatory activity in limbic cortices. These effects were reversibly blocked by co-perfusion of the muscarinic antagonist atropine sulfate (5 microM). Brain pilocarpine measurements in vivo and in vitro suggested modest BBB penetration. Pilocarpine induced epileptiform discharges only when perfused with compounds that enhance BBB permeability, such as bradykinin (n=2) or histamine (n=10). This pro-epileptic effect was abolished when the BBB-impermeable muscarinic antagonist atropine methyl bromide (5 microM) was co-perfused with histamine and pilocarpine. In the absence of BBB permeability enhancing drugs, pilocarpine induced epileptiform activity only after arterial perfusion at concentrations >10 mM. Ictal discharges correlated with a high intracerebral pilocarpine concentration measured by high pressure liquid chromatography. We propose that acute epileptiform discharges induced by pilocarpine treatment in the in vitro isolated brain preparation are mediated by a dose-dependent, atropine-sensitive muscarinic effect promoted by an increase in BBB permeability. Pilocarpine accumulation secondary to BBB permeability changes may contribute to in vivo ictogenesis in the pilocarpine epilepsy model.

Fig. 1

Cerebral and vascular levels of pilocarpine measured in vivo in the guinea-pig brain during SE. Mean pilocarpine values and brain/ blood ratio after 220 mg/kg i.p. injection in the guinea pig revealed poor penetration of pilocarpine into the CNS.

Fig. 2

Submillimolar concentrations of pilocarpine induce muscarine-receptor dependent fast activity in the hippocampus of the in vitro isolated guinea-pig brain. In the upper panel, the positions of the recording electrodes in the isolated brain are illustrated: PC, EC and hippocampus (CA1 area). (A) Reversible effect of arterial perfusion of pilocarpine 100 μM in PC, EC and CA1. (B) Fast activity in CA1 region induced by different concentrations of pilocarpine (100 and 800 μM) diluted in the arterial perfusate (middle traces). Field activity before and 20 min after pilocarpine perfusion is illustrated in the upper and lower traces. (C) The effect of pilocarpine observed in CA1 is abolished by co-perfusion of the isolated brain with pilocarpine and atropine sulfate (5 μM). (D) Mean frequency power of CA1 fast activity recorded during application of 800 μM pilocarpine (n=8; continuous thick line), co-application of 800 μM pilocarpine and atropine 5 μM (n=4; thin continuous line) and after drug washout (n=8; dotted line).

Fig. 3

Pilocarpine induces depression of both paired pulse disynaptic responses and polysynaptic propagation of activity. (A) Stimulation of the LOT induces in the PC a response characterized by a monosynaptic component (arrow) and a disynaptic component (arrowhead). Paired-pulse depression of the dpc is dramatically enhanced in the conditioned response to LOT stimulation (40 ms pulse interval) after perfusion with 800 μM pilocarpine (middle trace). (B) Quantitative results obtained in five experiments demonstrate that pilocarpine does not change the ratio between the conditioned (mpc2) and the conditioning (mpc1) monosynaptic PC component evoked by LOT pairing at 40 ms inter-pulse interval (upper panel), while it reversibly reduces significantly (two-tailed t-test, P<0.05) the ratio between the conditioned (dpc2) and the conditioning (dpc1) disynaptic component (lower panel). Values expressed as mean±S.D. (C) Recordings from EC and CA1 during paired LOT stimuli (100 ms interpulse interval). A reduction of the disynaptic component is observed also in the EC (upper traces). In CA1 the polysynaptic response to the conditioned LOT stimulus (asterisk) is reversibly abolished.

Fig. 4

Pro-epileptic effect of histamine and pilocarpine mediated by enhancement of BBB permeability. (A) Arterial co-perfusion of 100 μM histamine and 800 μM pilocarpine in the isolated guinea-pig brain induces a sustained ictal epileptiform discharge in area CA1 of the hippocampus. (B) The BBB-impermeable atropine methyl bromide (5 μM) reduces fast activity induced by 800 μM pilocarpine, when co-perfused for 10 min with the BBB-opener histamine (100 μM).

Fig. 5

Pilocarpine levels from in vitro isolated guinea-pig brains. HPLC pilocarpine levels were measured in frozen isolated brains in which pilocarpine was perfused in vitro for 10 min at 800 μM in the presence (open circles) or in the absence (filled squares) of histamine, an agent known to increase the permeability of the BBB. The data points for pilocarpine levels are plotted against the time of washout of the drug. In these brains electrophysiological recordings were performed to verify the presence of seizure-like activity during histamine–pilocarpine co-application. The initial cerebral concentrations of the drug were obtained by extrapolating the time-dependent decay and by using y axis (mM) intercepts.

Fig. 6

Pilocarpine/histamine-induced seizure-like activity is not determined by potassium enhancement. (A) Perfusion of 800 μM pilocarpine is followed by an increase of [K⁺]_o that correlates with the development of fast oscillatory activity in the field potential (FP) of CA1. In the bottom panel, details of the fast activity at time points a, b and c are illustrated. The duration of the pilocarpine perfusion is shown by the gray bar. (B) Comparable increase of [K⁺]_o is observed at the onset pilocarpine 800 μM and histamine 100 μM co-perfusion (arrowhead); a subsequent large increase in [K⁺]_o is associated to the onset of ictal discharges (arrow). The duration of the histamine–pilocarpine co-perfusion is shown by the gray bar.

Fig. 7

Epileptiform activity can be induced by supra-millimolar concentration of pilocarpine. (A) Arterial perfusion of the isolated guinea-pig brain preparation with supra-millimolar concentration of pilocarpine (13 mM) induces seizure-like activity in the CA1–EC region. (B) Epileptiform discharges induced by arterial application of a micromolar solution of kainic acid (4 μM).