The endocannabinoid (eCB) system is widely expressed throughout the central nervous system (CNS) and the functionality of type-1 cannabinoid receptors in neurons is well documented. In contrast, there is little knowledge about type-2 cannabinoid receptors (CB 2 Rs) in the CNS. Here, we show that CB 2 Rs are located intracellularly in layer II/III pyramidal cells of the rodent medial prefrontal cortex (mPFC) and that their activation results in IP 3 Rdependent opening of Ca 2+ -activated Cl − channels. To investigate the functional role of CB 2 R activation, we induced neuronal firing and observed a CB 2 R-mediated reduction in firing frequency. The description of this unique CB 2 R-mediated signaling pathway, controlling neuronal excitability, broadens our knowledge of the influence of the eCB system on brain function.calcium-activated chloride current | firing rate | whole-cell current | voltage clamp | intracellular calcium stores T he endocannabinoid (eCB) system is involved in many functions of the CNS, including executive functions associated with the prefrontal cortex, such as decision-making and working memory (1). The eCB system consists of at least two G proteincoupled receptors (GPCRs), type-1 cannabinoid receptor (CB 1 R) and type-2 cannabinoid receptor (CB 2 R), lipid endogenous ligands (e.g., anandamide and 2-arachidonoylglycerol), and various enzymes responsible for the synthesis and degradation of the endogenous ligands (2-6). CB 1 Rs are among the most abundantly expressed GPCRs in the rat brain and their role, predominantly as presynaptic receptors, in modulating neurotransmission is clearly established (5,7,8). In contrast with CB 1 R, the presence and function of CB 2 R in the brain has long been a matter of debate (9). CB 2 Rs are found primarily in the immune system and were initially regarded as the "peripheral" cannabinoid receptor (10,11). This generally accepted idea is challenged by the description of CNS CB 2 R gene expression in rats and wild-type mice (12-14) and the identification of functional CB 2 Rs on glial cells and neurons (15)(16)(17)(18). In addition to the current view that supports the expression of functional CB 2 Rs in neurons upon brain stress or damage (19), it has been reported that CB 2 Rs could play a role in general CNS physiology (20)(21)(22). These developments emphasize the importance of understanding how CB 2 R activation affects neuronal functioning. To demonstrate the presence of functional CB 2 Rs in the rodent medial prefrontal cortex (mPFC) and to elucidate their functional role, we used Western blotting, a radioactive binding assay, and electrophysiological techniques (whole-cell current and voltage clamp) on layer II/III pyramidal neurons. ResultsFunctional CB 2 Rs in the mPFC. The presence of CB 2 Rs in the rat mPFC was demonstrated by a Western blot performed on homogenated mPFC samples (Fig. 1A). A band of the expected molecular weight for CB 2 R was detected, which was absent when the primary antibody was incubated with immunizing peptide (Fig. 1A)....

Activation of the endocannabinoid (eCB) system by exogenous cannabinoids (drug abuse) can alter the physiology of the brain circuits involved in higher-order cognitive functions such as the medial prefrontal cortex (mPFC). A proper balance between excitation and inhibition (E/I balance) is critical for neuronal network oscillations underlying cognitive functions. Since type-1 cannabinoid receptors (CB1Rs), expressed in many brain areas including the mPFC, can modulate excitatory and inhibitory neurotransmission, we aimed to determine whether CB1R activation results in modifications of the E/I balance. We first confirm the presence of functional presynaptic CB1Rs that can modulate both excitatory and inhibitory inputs to layer II/III pyramidal neurons of the prelimbic (PL) area of the mPFC. By decomposing the synaptic response evoked by layer I stimulation into its excitatory and inhibitory components, we show that in vitro CB1R activation with the cannabinoid receptor agonists WIN55,212-2 (WIN) and CP-55940 (CP) modulates the balance between excitation and inhibition (E/I balance) of layer II/III pyramidal neurons. This treatment caused a significant shift of the E/I balance towards excitation, from 18/82 % to 25/75 % (WIN) and from 17/83 to 30/70 % (CP). Finally, when animals were injected with a cannabinoid receptor agonist, we observed a shift of the E/I balance (measured in vitro) towards excitation 1 h after WIN (24/76 %) or after CP injection (30/70 %) when compared to vehicle-injected animals (18/82 %). This modulation of the E/I balance by CB1Rs may thus be fundamental in the regulation of local PL cortical network excitability and could be the mechanism through which excessive CB1R activation (cannabis abuse) affects cognitive functions.