032; p = 0.691). Next, to determine whether activity-dependent production of NO in DMH neurons relies on an increase in intracellular Ca2+, which is often secondary to the activation of NMDARs (Bains and Ferguson,

1997, Nugent et al., 2007 and Szabadits et al., 2011), we conducted two independent experiments. First, we delivered HFS in the presence of AM251 and the NMDAR antagonist APV (50 μM). Under these conditions, HFS failed to elicit LTPGABA (94% ± 10.7% of baseline, n = 7, p = 0.436; Figure 3B). In the second experiment, the postsynaptic www.selleckchem.com/erk.html cell was loaded with the Ca2+ chelator BAPTA (10 mM), and HFS was delivered. This manipulation also completely abolished LTPGABA in the presence of AM251 (99% ± 14.8% of baseline, n = 5, p = 0.944; Figure 3C), indicating that a rise in postsynaptic Ca2+ is necessary for NO-mediated potentiation of GABA synapses. The effects of NO on GABA release VX809 require the activation of presynaptic soluble guanylate

cyclase (sGC), with a subsequent rise in cyclic GMP (cGMP) (Nugent et al., 2007). Consistent with these observations, we failed to elicit LTPGABA (87% ± 6.7% of baseline, n = 6, p = 0.053; Figure 3C) in the presence of both AM251 and the sGC inhibitor, ODQ (10 μM). When taken together, these findings are consistent with the hypothesis that GABA synapses are potentiated by NO recruited in a heterosynaptic fashion by the activation of NMDARs. We next examined whether these synapses were sensitive to pharmacological manipulations using exogenous ligands that either activate CB1Rs or liberate NO directly. Bath application of the CB1R agonist WIN 55,212-2 (5 μM) elicited

a robust depression in evoked IPSC amplitude (51% ± 7.0% of baseline, n = 10, p = 0.0001; Figure 4A). This was accompanied by an increase in the PPR (baseline: 0.887 ± Bay 11-7085 0.110; post-drug: 1.087 ± 0.112; p = 0.020; Figure 4A) and CV (baseline: 0.394 ± 0.058; post-drug: 0.707 ± 0.174; p = 0.001; Figure 4A), suggesting that these effects are localized at the presynaptic terminal. This is consistent with its action elsewhere in the hypothalamus (Hirasawa et al., 2004, Huang et al., 2007, Oliet et al., 2007 and Wamsteeker et al., 2010) and throughout the brain (Kano et al., 2009). To determine whether the NO donor SNAP (200 μM) modulates GABA release in the DMH, we assessed its effects on evoked IPSCs. SNAP caused a rapid increase in IPSC amplitude (145% ± 14.7% of baseline, n = 13, p = 0.0003, Figure 4B) and a decrease in PPR (baseline: 0.757 ± 0.074; post-drug: 0.655 ± 0.056; p = 0.042; Figure 4B) and CV (baseline: 0.343 ± 0.037; post-drug: 0.284 ± 0.031; p = 0.039; Figure 4B). This is also consistent with previous reports that NO increases GABA release in the CNS (Bains and Ferguson, 1997, Di et al., 2009, Horn et al., 1994, Nugent et al., 2007 and Stern and Ludwig, 2001). Together, our findings confirm that GABA synapses in the DMH are sensitive to manipulations that directly activate CB1Rs or deliver NO to the tissue.