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Spinally administered muscarinic receptor agonists or acetylcholinesterase inhibitors can produce antinociception. However, the mechanisms of the action of cholinergic agents in the spinal cord are not fully understood. Activation of spinal muscarinic receptors evokes γ-aminobutyric acid (GABA) release, which reduces the glutamatergic synaptic input to dorsal horn neurons through GABAB receptors. In this study, we determined the functional role of spinal GABAB receptors in the antinociceptive action of intrathecal cholinergic agents in normal rats and in a rat model of diabetic neuropathic pain. Diabetes was induced by intraperitoneal streptozotocin in rats. The intrathecal catheter was inserted with its tip positioned at the lumbar spinal level. Nociceptive threshold was measured by the paw withdrawal latency in response to a radiant heat stimulus in normal rats. Mechanical allodynia in diabetic rats was determined by von Frey filaments applied to the hindpaw. The effect of intrathecal muscarine or neostigmine was examined through pretreatment with the specific GABAB receptor antagonist, CGP55845, or its vehicle. Intrathecal injection of muscarine or neostigmine significantly increased the withdrawal latency in response to a heat stimulus in normal rats and the withdrawal threshold in response to application of von Frey filaments in diabetic rats. Intrathecal pretreatment with CGP55845 significantly attenuated the effect of both muscarine or neostigmine in normal rats. Furthermore, the antiallodynic effect of intrathecal neostigmine and muscarine was largely eliminated by CGP55845 in diabetic rats. These data suggest that the GABAB receptors in the spinal cord mediate both the antinociceptive and antiallodynic actions of intrathecal muscarine or neostigmine in normal rats and in a rat model of diabetic neuropathic pain. This study provides new functional evidence that activation of spinal GABAB receptors is one of the important mechanisms underlying the antinociceptive action of intrathecal cholinergic agents. [Copyright &y& Elsevier]

Systemically administered mu opioids may produce analgesia through inhibition of the ascending nociceptive transmission and activation of descending pathways. However, the relative importance of the spinal and supraspinal sites in the analgesic action of systemic opioids remains uncertain. It has been shown that systemic morphine can inhibit dorsal horn neurons independent of the descending system. In this study, we determined the extent to which spinal mu opioid receptors mediate the analgesic effect of systemic mu opioids. Rats were instrumented with an intrathecal catheter with the tip placed in the lumbar spinal cord. Nociception was measured by testing the paw withdrawal threshold in response to a noxious radiant heat or pressure stimulus. Surprisingly, intrathecal pretreatment with naloxone or H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP, a specific mu opioid receptor antagonist) completely blocked the inhibitory effect of intravenous morphine on mechanical nociception. Intrathecal naloxone or CTAP also abolished the effect of intravenous morphine on the withdrawal latency of the hindpaw, but not the forepaw, measured with a radiant heat stimulus. Furthermore, the inhibitory effect of subcutaneous fentanyl on mechanical nociception was eliminated by CTAP injected intrathecally. Intrathecal CTAP similarly abolished the effect of subcutaneous fentanyl on thermal nociception of the hindpaw but not the forepaw. Therefore, this study provides new information that when spinal mu opioid receptors are blocked, subsequent systemic administration of mu opioids fails to produce an analgesic effect. This finding highlights the important role of mu opioid receptors in the spinal cord in the antinociceptive action of opioids.

The nucleus locus coeruleus (LC) plays an important role in analgesia produced by opioids and by modulation of the descending noradrenergic pathway. The functional role of micro-opioid receptors (muOR) in regulation of the excitability of spinally projecting LC neurons has not been investigated. In the present study, we tested the hypothesis that activation of presynaptic mu-opioid receptors excites a population of spinally projecting LC neurons through attenuation of gamma-aminobutyric acid (GABA)-ergic synaptic inputs. Spinally projecting LC neurons were retrogradely labeled by a fluorescent dye injected into the spinal dorsal horn of rats. Whole-cell current- and voltage-clamp recordings were performed on labeled LC neurons in brain slices. All labeled LC noradrenergic neurons were demonstrated by dopamine-beta-hydroxylase (DbetaH) immunofluorescence. In 37 labeled LC neurons, (D-Ala(2),N-Me-Phe(4),Gly-ol(5))-enkephalin (DAMGO) significantly increased the discharge activity of 17 (45.9%) neurons, but significantly inhibited the firing activity of another 15 (40.5%) cells. The excitatory effect of DAMGO on seven labeled LC neurons was diminished in the presence of bicuculline. DAMGO significantly decreased the frequency of GABA-mediated miniature inhibitory postsynaptic currents (mIPSCs) in all nine labeled LC neurons. However, DAMGO had no effect on glutamate-mediated miniature excitatory postsynaptic currents (mEPSCs) in 12 of 15 neurons. Furthermore, DAMGO significantly inhibited the peak amplitude of evoked inhibitory postsynaptic currents (eIPSCs) in all 11 labeled neurons, but had no significant effect on the evoked excitatory postsynaptic currents (eEPSCs) in 10 of these 11 neurons. Thus, data from this study suggest that activation of micro-opioid receptors excites a population of spinally projecting LC neurons by preferential inhibition of GABAergic synaptic inputs. These findings provide important new information about the descending noradrenergic modulation and analgesic mechanisms of opioids.