1. CB1R activation enhances hippocampal excitatory neurotransmission in female adolescent rats Christian G. Reich and Joseph Boscarino Psychology Program, Ramapo College of New Jersey, Mahwah, NJ 07430 References Reich CG, Taylor, ME, McCarthy, MM (2009) Differential effects of chronic unpredictable stress on hippocampal CB1 receptors in male and female rats. Behav. Brain Res. 203(2): 264-69. Rubino T, and Parolaro, D (2011) Sexually dimorphic effects of cannabinoid compounds on emotion and cognition. FrontBehavNeurosci 28(5):64. Acknowledgements This research was supported by R15 MH085280-01 and Ramapo College Foundation Grant to CGR. Background Hippocampal CB1 receptor (CB1R) levels are lower in female adolescent animals compared to males. Following 21 day exposure to chronic mild stress, CB1 increased in females while decreased in males, suggesting that hippocampal CB1 responds differentially to stress depending on sex (Reich et al., 2009). Hippocampal CB1 receptor (CB1R) levels are lower in female adolescent animals compared to males. Following 21 day exposure to chronic mild stress, CB1 increased in females while decreased in males, suggesting that hippocampal CB1 responds differentially to stress depending on sex (Reich et al., 2009). Converging evidence clearly indicates a functional sex difference in the endocannabinoid system and behavioral reactions to exogenous cannabinoids in both humans and animals (Rubino and Paralaro, 2011). However, it is unknown how these changes in CB1 receptor levels affect CB1-regulated synaptic physiology in the hippocampus. We therefore investigated the effects of exogenous CB1 activation on glutamatergic neurotransmission at the CA3- Schaffer Collateral-CA1 synapse in female adolescent rats. Methods Methods Subjects Adolescent (48-56 day old) Female Sprague-Dawley rats (Charles River, Boston, MA) were group-caged (3) in plastic cages upon arrival in the Ramapo College animal facility. Animals were allowed to acclimate to the vivarium for ≥ 5 days prior to any experimental manipulations. arrival in the Ramapo College animal facility. Animals were allowed to acclimate to the vivarium for ≥ 5 days prior to any experimental manipulations. Electrophysiology Animals were deeply anesthetized with halothane and decapitated in accordance with the guidelines set forth by the Institutional Animal Care and Use Committee at Ramapo College of New Jersey. The brain was rapidly removed and hippocampi dissected. Transverse hippocampal slices, 400 µM thick, were cut on a Vibrotome (Leica). Slices were kept in a holding chamber at room temperature at the interface of a physiological medium and a humidified 95/5% O2/CO2 atmosphere for > 1 hr. The slices were then transferred to a submerged recording chamber and perfused with warm saline (30° C). The extracellular saline contained (mM): NaCl, 120; KCl, 3; MgSO4, 2; NaH2PO4, 1; NaHCO3, 25; CaCl2, 2.5; and glucose, 10 and saturated with 95% O2-5% CO2 (pH 7.4). Field potentials (fESPs) were recorded from stratum radiatum of CA1 with glass microelectrodes (tip diameter ~ 4 -7 μm) filled with extracellular saline. Stimuli were delivered via a bipolar stimulating electrode located between CA3 and CA1. Signals were recorded with an amplifier (Model 773, WPI Inc.), digitized at 10 kHz with an AD interface (Digidata 1440A, Axon Instruments) and analyzed with pClamp 10.0 (Axon, Instruments). Statistical comparisons were performed using one-way ANOVAs or repeated measures MANOVA (SPSS). Pharmacological Treatment: WIN 55,212-5 (1 μM –final concentration) was dissolved in dimethyl sulfoxide (DMSO) and prepared stock solutions. Final DMSO concentrations in extracellular saline did not exceed 0.1 %. Summary and Conclusions Hippocampal dendritic CB1R function at GABAergic synapses is enhanced in females. CB1R activation in females enhances fEPSP magnitudes while it decreases magnitudes in males. - This effect in reversed by blocking GABA neurotransmission. - This effect is prevented by a CB1 antagonist. LTP induction to a weak TBS stimulation is equal males and females. Sex difference in CB1 function may serve as parallel mechanism to achieve similar physiological endpoint. CB1R activation increases CA3-CA1 glutamatergic neurotransmission in females n = 6 n = 7 n = 6 Increased CB1R sensitivity in females does not result in enhanced LTP induction Left : In Females, WIN,212-5 increases CA3-CA1 fEPSPs, whereas it decreases fEPSP magnitudes in Males. Right : WIN effect in females is blocked by the CB1 antagonist AM251 (3  M) applied continuously throughout the experiments. Asterisks indicate differences between groups; independent-sample t-test, p < 0.05 Blocking GABA(A) receptors results in a decrease of glutamatergic neurotransmission by CB1 receptor activation in females. Left : Continuous application of the GABA(A) antagonist, Picrotoxin (100 mM, PTX) results in a WIN-induced reduction of fEPSP magnitudes in females. Right: Summary graph of WIN experiments. Shown is the average last six fEPSPs (30 min) compared to the average last six baseline fEPSPs. Asterisks indicate differences from the female group, p < 0.05. Female Male Sex Differences of Hippocampal CB1R under basal and chronic stress conditions Reich et al., 2009 LTP induction via a weak Theta-Burst- Stimulation (TBS;10 bursts of 5 stimuli, 100 Hz within burst, 200 ms interburst interval)in both females and males yields similar levels of LTP.