In the paraventricular nucleus (PVN) of the hypothalamus, nitric oxide (NO) inhibits sympathetic outflow through increased GABA release. However, the signal transduction pathways involved in its action remain unclear. In the present study, we determined the role of cGMP, soluble guanylyl cyclase, and protein kinase G in the potentiating effect of NO on synaptic GABA release to spinally projecting PVN neurones. The PVN neurones were retrogradely labelled by a fluorescent tracer injected into the thoracic spinal cord of rats. Wholecell voltage-clamp recordings were performed on labelled PVN neurones in the hypothalamic slice. Bath application of the NO donor, S-nitroso-N -acetyl-penicillamine (SNAP), reproducibly increased the frequency of miniature GABAergic inhibitory postsynaptic currents (mIPSCs) without changing the amplitude and the decay time constant. Neither replacement of Ca 2+ with Co 2+ nor application of Cd 2+ to block the Ca 2+ channel altered the effect of SNAP on mIPSCs. Also, the effect of SNAP on mIPSCs was not significantly affected by thapsigargin, a Ca 2+ -ATPase inhibitor that depletes intracellular Ca 2+ stores. Application of a membrane-permeant cGMP analogue, pCPT-cGMP, mimicked the effect of SNAP on mIPSCs in the presence of a phosphodiesterase inhibitor, IBMX. Furthermore, both the soluble guanylyl cyclase inhibitor, ODQ, and the specific protein kinase G inhibitor, Rp pCPT cGMP, abolished the effect of SNAP on mIPSCs. Thus, these data provide substantial new information that NO potentiates GABAergic synaptic inputs to spinally projecting PVN neurones through a cGMP-protein kinase G pathway.

Opioids are potent analgesics, but the sites of their action and cellular mechanisms are not fully understood. The central nucleus of the amygdala (CeA) is important for opioid analgesia through the projection to the periaquaductal gray (PAG). In this study, we examined the effects of opioid receptor stimulation on inhibitory and excitatory synaptic inputs to PAG-projecting CeA neurons retrogradely labeled with a fluorescent tracer injected into the ventrolateral PAG of rats. Whole-cell voltageclamp recordings were performed on labeled CeA neurons in brain slices. The specific opioid receptor agonist, [D-Ala 2 ,NMe-Phe 4 ,Gly 5 -ol]-enkephalin (DAMGO, 1 M), significantly reduced the frequency of miniature inhibitory postsynaptic currents (mIPSCs) without altering the amplitude and decay constant of mIPSCs in 47.6% (10 of 21) of cells tested. DAMGO also significantly decreased the peak amplitude of evoked IPSCs in 69% (9 of 13) of cells examined. However, DAMGO did not significantly alter the frequency of miniature excitatory postsynaptic currents (EPSCs) and the amplitude of evoked EPSCs in 69% (9 of 13) and 83% (10 of 12) of labeled cells, respectively. The IPSCs were blocked by the GABA A receptor antagonist bicuculline, whereas the EPSCs were largely abolished by the non-N-methyl-D-aspartate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. The immunoreactivity of opioid receptors was colocalized with synaptophysin, a presynaptic marker, in close appositions to labeled CeA neurons. These results suggest that activation of opioid receptors on presynaptic terminals primarily attenuates GABAergic synaptic inputs to PAG-projecting neurons in the CeA.

The rostral ventromedial medulla (RVM) is a major locus for the descending control of nociception and opioid analgesia. However, it is not clear how opioids affect synaptic inputs to RVM neurons. In this study, we determined the effect of -opioid receptor activation on excitatory and inhibitory synaptic transmission in spinally projecting RVM neurons. RVM neurons were retrogradely labeled with a fluorescent tracer injected into the dorsal horn of the spinal cord in rats. Whole-cell voltage-clamp recordings were performed on labeled RVM neurons in brain slices in vitro. The -receptor agonist [D-Ala 2 ,N-Me-Phe 4 ,Gly 5 -ol]-enkephalin (DAMGO, 1 ⌴) significantly decreased the amplitude of evoked excitatory postsynaptic currents (EPSCs) in 52% (9 of 17) of labeled cells. DAMGO also significantly reduced the amplitude of evoked inhibitory postsynaptic currents (IPSCs) in 69% (11 of 16) of cells examined. Furthermore, DAMGO significantly decreased the frequency of miniature EPSCs in 55% (15 of 27) of cells and significantly decreased the frequency of miniature IPSCs in all 12 cells studied. Although most EPSCs and IPSCs were mediated by glutamate and GABA, the nicotinic and glycine receptor antagonists attenuated EPSCs and IPSCs, respectively, in some labeled RVM neurons. Immunocytochemical labeling revealed that only 35% of recorded RVM neurons were tryptophan hydroxylase-positive, and 15% cells had GABA immunoreactivity. Thus, this study provides important functional evidence that activation of -opioid receptors decreases the release of both excitatory and inhibitory neurotransmitters onto most spinally projecting RVM neurons.The rostral ventromedial medulla (RVM) plays an important role in the descending modulation of nociceptive information and opioid analgesia (Fields et al., 1983a;Jones and Gebhart, 1988;Urban and Smith, 1994). The RVM receives synaptic inputs from the periaqueductal gray (PAG) and relays information to the dorsal horn of the spinal cord through the dorsolateral funiculus. The analgesia produced by microinjection of morphine into the PAG is dependent upon the connection to the RVM (Urban and Smith, 1994;Pan and Fields, 1996). Direct stimulation of the RVM also can produce potent analgesia. For example, medullary electrical stimulation or microinjection of glutamate and morphine into the RVM results in either an increase in the tail-flick latency or a decrease in spinal dorsal horn neuronal activity (Llewelyn et al., 1986;Jones and Gebhart, 1988;McGowan and Hammond, 1993;Urban and Smith, 1994). However, the cellular mechanisms of the action of opioids in the RVM remain unclear.Three classes of RVM neurons have been identified, including ON-, OFF-, and NEUTRAL-cells, that show, respectively, increases, decreases, and no changes in the firing activity in response to noxious stimuli (Fields et al., 1983b(Fields et al., , 1995. Furthermore, activation of -opioid receptors inhibits ON-cells, but stimulates OFF-cells during the application of noxious stimulation (Fields et al., 1983b). Althoug...