Abstracts

Rationale: A growing interest in developing cannabinoid based medication along with marijuana s recreational use makes it important to investigate molecular adaptations the endocannabinoid system undergoes following prolonged use and withdrawal. Repeated administration and prolong exposure to cannabinoids results in development of tolerance to its physiological effects and produces clinically relevant withdrawal symptoms that include irritability, restlessness and occasionally seizures. Laboratory studies have also indicated enhanced CNS excitability following cannabinoid withdrawal. Methods: To identify mechanisms responsible for this increased neuronal excitability we employed electrophysiological and immunocytochemical techniques to investigate effects of prolonged cannabinoid type-1 receptor (CB1) agonist exposure on cultured hippocampal neurons. Neuronal cultures were exposed to the cannabinamimetic ( WIN or -WIN) at 1 uM concentration in maintenance medium for 24-h. At the end of this period, /- WIN was removed and cultures were immediately utilized for experimentation. Miniature inhibitory postsynaptic currents (mIPSCs) were recorded using whole-cell voltage-clamp technique. Hippocampal neuronal cultures were also evaluated immunocytochemically for membrane CB1, GABAA receptor and markers for inhibitory and excitatory synapses. Results: CB1 co-localized extensively at GABAergic synapses in our neuronal preparations. Prolonged exposure and subsequent withdrawal of the CB1 agonist WIN 55212-2 ( WIN 1uM, 24-h) produced neuronal hyperexcitability. Prolonged WIN exposure caused profound CB1 downregulation (87%) that was accompanied by an increased GABA release as indicated by increased mIPSC frequency (2-fold), a diminished GABAergic inhibition as indicated by a reduction in mIPSC amplitude (40%) and a reduction in GABAA channel number (45%). Immunocytochemical analysis demonstrated a decrease in surface GABAA ?2/3 receptor subunit expression (25%) but no change in vesicular GABA transporter following WIN (1uM, 24-h) suggesting that even though GABAA receptors are downregulated, the GABAergic terminals remained intact. Conclusions: This study demonstrates that prolonged cannabinoid exposure is associated with profound downregulation of CB1. Subsequent withdrawal following prolonged CB1 agonist exposure results in massive neuronal hyperexcitability. This hyperexcitability observed following agonist-induced downregulation of the CB1 may result from alterations in both presynaptic GABA release mechanisms and postsynaptic GABAA receptor function, thus demonstrating a novel form of cannabinoid mediated neural plasticity.