Chronic morphine administration induces adaptations in neurons resulting in opioid tolerance and dependence. Functional studies have implicated a role for the periaqueductal gray area (PAG) in the expression of many signs of opioid withdrawal, but the cellular mechanisms are not fully understood. This study describes an increased efficacy, rather than tolerance, of opioid agonists at mu-receptors on GABAergic (but not glutamatergic) nerve terminals in PAG after chronic morphine treatment. Opioid withdrawal enhanced the amplitudes of electrically evoked inhibitory synaptic currents mediated by GABAA receptors and increased the frequency of spontaneous miniature GABAergic synaptic currents. These effects were not blocked by 4-aminopyridine or dendrotoxin, although both Kv channel blockers abolish acute opioid presynaptic inhibition of GABA release in PAG. Instead, the withdrawal-induced increases were blocked by protein kinase A inhibitors and occluded by metabolically stable cAMP analogs, which do not prevent acute opioid actions. These findings indicate that opioid dependence induces efficacious coupling of mu-receptors to presynaptic inhibition in GABAergic nerve terminals via adenylyl cyclase- and protein kinase A-dependent processes in PAG. The potential role of these adaptations in expression of withdrawal behavior was supported by inhibition of enhanced GABAergic synaptic transmission by the alpha2 adrenoceptor agonist clonidine. These findings provide a cellular mechanism that is consistent with other studies demonstrating attenuated opioid withdrawal behavior after injections of protein kinase A inhibitors into PAG and suggest a general mechanism whereby opioid withdrawal may enhance synaptic neurotransmission.

1 In this study we have examined the effects of nociceptin, an endogenous ligand for the opioid-like receptor ORLI, on the membrane properties of rat locus coeruleus (LC) neurones in vitro, using intracellular and whole cell patch clamp recording. 2 When locus coeruleus neurones were voltage clamped to -60 mV, application of nociceptin caused an outward current in all cells examined (n = 49), with an EC50 of 90 nm. Neither the potency nor the maximal effect of nociceptin was altered in the presence of the peptidase inhibitors, bestatin (20 gM) or thiorphan (2 pM). 3 The outward currents caused by nociceptin in 2.5 mM extracellular K+ reversed polarity at -123 mV, more negative than the predicted K+ reversal potential of -105 mV. Increasing extracellular K+ to 6.5 mM resulted in a shift of the reversal potential of + 25 mV, a shift consistent with a K+ conductance. The conductance activated by nociceptin showed mild inward rectification.4 Application of a high concentration of nociceptin (3 gM) occluded the current produced by simultaneous application of high concentrations of Met-enkephalin (10 gM), (3 uM) somatostatin and UK 14304 (3 gM), indicating that nociceptin activated the same conductance as p-opioid and somatostatin receptors and a2-adrenoceptors. 6 Dynorphin A (3 gM), another putative endogenous ligand for ORL,, caused a robust outward current in locus coeruleus neurones that was, however, completely antagonized by moderate concentrations of naloxone (300 nM-1 gM). 7 Continuous application of nociceptin (3 gM) resulted in a decrease of the outward current to a steady level of 70% of the maximum response with a t1/2 of 120s. Desensitization was largely homologous because simultaneous application of Met-enkephalin (30 gM) during the desensitized period of the nociceptin response resulted in an outward current that was 92% of control responses to Met-enkephalin in the same cells. Conversely, continuous application of Met-enkephalin (30 gM) resulted in a decrease of Met-enkephalin current to a steady level that was 54% of the initial current. During this desensitized period application of nociceptin (3 gM) resulted in a current that was 78% of the control responses to nociceptin in the same cells. 8 Thus nociceptin potently activates an inwardly rectifying K+ conductance in locus coeruleus neurones, with a pharmacological profile consistent with activation of the ORL, receptor. Dynorphin A does not appear to be a ligand for ORLI in rat locus coeruleus neurones.

The human neuroblastoma cell line SH-SY5Y expresses the 'orphan' opioid receptor (ORLI). We have demonstrated that nociceptin, the putative endogenous ligand for ORLI, produces a concentrationdependent inhibition of the N-type calcium channel current in these cells (IC,0 42 nM). In addition, in the presence of carbachol, nociceptin increased the intracellular concentration of Ca2" (ECQ0 60 nM). Both effects of nociceptin were blocked by pertussis toxin pretreatment but not by the opioid antagonists CTAP (1 pM), naltrindole (1 gM) and naloxone (10 gM).

We have isolated a novel family of insect-selective neurotoxins that appear to be the most potent blockers of insect voltage-gated calcium channels reported to date. These toxins display exceptional phylogenetic specificity, with at least a 10,000-fold preference for insect versus vertebrate calcium channels. The structure of one of the toxins reveals a highly structured, disulfide-rich core and a structurally disordered C-terminal extension that is essential for channel blocking activity. Weak structural/functional homology with -agatoxin-IVA/B, the prototypic inhibitor of vertebrate P-type calcium channels, suggests that these two toxin families might share a similar mechanism of action despite their vastly different phylogenetic specificities.

1 In this study we have investigated 6 and y opioid receptor-mediated elevation of intracellular Ca2" concentration ([Ca2+]) in the human neuroblastoma cell line, SH-SY5Y. 2 The Ca2 +-sensitive dye, fura-2, was used to measure [Ca2+], in confluent monolayers of SH-SY5Y cells. Neither the 6-opioid agonist, DPDPE