Within the cerebellum, a major component of presynaptic inhibition is mediated by G-protein coupled cannabinoid CB₁ receptors. In particular, CB₁ is prominently expressed in inhibitory synapses between interneurones and Purkinje cells, the latter representing the sole output of the cerebellum. Here, we combine single and multi-electrode electrophysiological recording in acute brain slices with GTPγS binding assays in isolated membranes to investigate the effects of synthetic agents and the phytocannabinoid Δ⁹-tetrahydrocannabinvarin (Δ⁹-THCV) on CB₁ receptors in the mouse (male TO strain, 3-5 weeks old) cerebellum. Using whole-cell patch clamp recordings from Purkinje cells in mouse cerebellar brain slices, we have recently shown that the CB₁ agonist WIN55,212-2 caused a reduction in mean miniature inhibitory post-synaptic current (mIPSC) frequency and that this reduction was increased to levels above control by subsequent application of AM251 or Δ⁹-THCV (Ma et al. BPS Oxford Meeting 2006 abstract). Moreover, when applied alone, AM251, Δ⁹-THCV or SR141617A all acted to increase mIPSC frequency. We have now made extracellular multi-electrode array (8×8) recordings of spontaneous excitatory activity in the same preparation. Recording electrodes were positioned proximal to the Purkinje cell layer and the effects of AM251 (2μM) or Δ⁹-THCV (5μM) examined (bath-applied for a minimum of 15 min, n=6 slices with a minimum 6 electrodes’ data each slice, significance by Mann-Whitney U test). Both agents reduced spontaneous excitatory spike firing frequency (Δ⁹-THCV: 25±12% of control; AM251: 71±9% of control; both P<0.05). In parallel studies, we measured the effects of synthetic CB₁-selective agents and Δ⁹-THCV on [³⁵S]-GTPγS binding to mouse cerebellar membranes. Log concentration-response curves (10pM-10μM) for agonists WIN55,212-2 and CP-55,940 showed a dose-dependent increase in [³⁵S]-GTPγS binding, whilst AM251 or Δ⁹-THCV alone had little effect. In contrast, log concentration-response curves for WIN55,212-2 were shifted rightward with no clear decrease in E_max by AM251 (100pM-10nM) and Δ⁹-THCV (100nM-5μM), consistent with a CB₁ antagonist action. Preliminary Schild analysis gave apparent K_B values of 63 pM (AM251) and 68 nM (Δ⁹-THCV). Overall, we provide functional evidence that AM251 and Δ⁹-THCV act to increase GABA release onto Purkinje cells and that this action correlates with a reduction in spontaneous excitatory activity in the cerebellum. AM251 and Δ⁹-THCV act as CB₁ receptor antagonists suggesting that the increased GABA release is due to removal of an endocannabinoid tone. Moreover, the potent effect of Δ⁹-THCV on spontaneous activity may suggest a differential effect on CB₁ receptors at inhibitory vs excitatory synapses.