Fig. 1. Antagonism of CB1 receptors by SR141716A causes SE-like activity in “epileptic” hippocampal neurons in cultures. Whole-cell current clamp recordings were obtained from hippocampal pyramidal neurons in cultures before and 1-day after a 3-h exposure to low Mg²⁺ solution. (A) Recording from a representative control neuron displaying intrinsic baseline activity consisting of intermittent spontaneous action potentials. (B) A continuous 30-min recording from a representative “epileptic” neuron 1-day following a 3-h exposure to low Mg²⁺ solution. Two SREDs or spontaneous seizure episodes lasting 60–80 s can be seen in this time frame. These SREDs occurred throughout the life of the cultures and are indicative of the pathophysiological “epileptic” phenotype. (C) Representative current-clamp recording showing transition of an epileptic neuron from SREDs to continuous epileptiform discharges (SE) after the addition of the CB1 receptor antagonist SR141716A (1 μM). After the appearance of first seizure event SR141716A was applied to neuron and within a minute after application the neuron manifested a response of essentially continuous epileptiform activity, a characteristic of SE. Also note the increased seizure frequency and duration. The patterns shown in “C” demonstrate the transitions from very rapid (10–15 Hz) to slower (3–5 Hz) spike discharges that occur during the continuous epileptiform discharges. (a): is an expansion of a segment from “C” showing the transition to SE-like activity on a faster time scale. (b and c): are further expansions of segments from “a” showing spike pattern in the fast and slow spike region, respectively. Spike frequencies ranged from 3 to 5 Hz in slow area (c) to up to 10–15 Hz in fast area (b). Individual epileptiform bursts; each consisting of depolarization shifts overlaid with multiple spike activity can be seen. This data was representative of six experiments.

Fig. 2. Antagonism of CB1 receptors by AM251 causes SE-like activity in “epileptic” neurons in cultures. (A) representative recording from a control neuron showing occasional spontaneous action potentials. (B) Whole cell current clamp recording from a neuron displaying SREDs 1-day following a 3-h exposure to low Mg²⁺ solution. (C) Application of AM251 (1 μM) to an epileptic neuron caused continuous epileptiform discharges, characteristic of SE. (a) is an expansion of a 3-min segment from C showing the transition to SE-like activity. (b and c) are further expansions of segments from “a” showing spike pattern and frequency in the fast and slow spike areas, respectively. This data was representative of six experiments.

Fig. 3. Restoration of endocannabinoid tone prevents SE-like activity in epileptic neurons. (A) Representative control neuron displaying occasional action potentials when treated with AM251 (1 μM) alone. (B) Removal of AM251 stops the continuous epileptiform discharges and restores the neuron to SREDs/ seizure phenotype. After the first seizure event AM251 (1 μM) is applied to neuron. This causes disruption of endocannabinoid tone resulting in the progression of the neuron to SE-like activity. Upon washout of AM251, SE-like activity stops and the neuron revert to the original electrophysiological pattern manifesting SREDs. Thus, the production of SE-like activity by AM251 was reversible. (C) WIN55,212-2 (5 μM) overcomes the AM251 mediated block of CB1 receptors and completely suppresses the spontaneous epileptiform discharges. Using WIN55,212-2 (5 μM) not only blocked SE-like activity, but also suppressed the SREDs. This data was representative of five experiments.