Release of nociceptin-like substances from the dorsal horn of rat spinal cords in situ was measured by the immobilized-antibody microprobe technique. Spinal cords removed from anesthetized 4-6 week-old rats were superfused with oxygenated Krebs solution at room temperature (21 ± 1°C). Glass microelectrodes, coated with antibodies to nociceptin, were inserted into the dorsal horn of the lumbar spinal cord (1.9 mm lateral to the midline to a depth 2.5 mm below the surface of the cord) for 15 rain periods before, during and after electrical stimulation applied to the dorsal root entry zone of the same segment. There was a basal release of immunoreactive nociceptin- like substance (irNC) from the dorsal horns during the pre-stimulation period. A significant increase in irNC release was detected during the period of electrical stimulation and this increase was maintained for at least 15 min following the cessation of electrical stimulation. These results provide the first evidence on the release of irNC, albeit non-quantitative, from the in situ rat spinal cord dorsal horn and an enhanced release upon electrical stimulation.

In vitro and in situ experiments were conducted to evaluate the hypothesis that the nonclassical opioid peptide nociceptin acting on sympathetic preganglionic neurons (SPNs) inhibits spinal sympathetic outflow. First, whole cell patch recordings were made from antidromically identified SPNs from immature (12-16 day old) rat spinal cord slices. Nociceptin (0.1, 0.3, and 1 μM) concentration dependently suppressed the excitatory postsynaptic potentials (EPSPs) evoked by focal stimulation and hyperpolarized a population of SPNs; these effects were naloxone insensitive. L-Glutamate-induced depolarizations were not significantly changed by nociceptin. Results from this series of experiments indicate that nociceptin inhibits the activity of SPNs by either a presynaptic or postsynaptic site of action, whereby the peptide reduces, respectively, the amplitude of EPSPs or the excitability of SPNs. Second, intrathecal injection of nociceptin (3, 10, and 30 nmol) to urethan-anesthetized rats dose dependently reduced the mean arterial pressure and heart rate; these effects were not prevented by prior intravenous administration of naloxone (1 mg/kg). Physiological saline given intrathecally was without appreciable effects. These results, together with earlier observations of the detection of nociceptin-immunoreactive nerve fibers and nociceptin receptor immunoreactivity in the rat intermediolateral cell column, raise the possibility that the opioid peptide, which may be released endogenously, reduces spinal sympathetic outflow by depressing the activity of SPNs.

Nociceptin, also referred to as orphanin FQ, is believed to be the endogenous ligand for the ORL1. Nociceptin, when injected intracerebroventricularly to mice, produced hyperalgesia in behavioral tests. Recent studies have demonstrated the presence of ORL1 transcript in the spinal cord, and ORL1-like immunoreactivity has been localized to nerve fibers and somata throughout the spinal cord. Here, we report the localization of nociceptin-like immunoreactivity to fiber-like elements of the superficial layers of the rat dorsal horn by immunohistochemical techniques. Whole-cell recordings from substantia gelatinosa neurons in transverse lumbar spinal cord slices of 22-26-day-old rats showed that exogenous nociceptin at low concentrations (100-300 nM) depressed excitatory postsynaptic potentials evoked by stimulation of dorsal rootlets without causing an appreciable change of resting membrane potentials and glutamate- evoked depolarizations. At a concentration of 1 μM, nociceptin hyperpolarized substantia gelatinosa neurons and suppressed spike discharges. The hyperpolarizing and synaptic depressant action of nociceptin was not reversed by the known opioid receptor antagonist naloxone (1 μM). Our result provides evidence that nociceptin-like peptide is concentrated in nerve fibers of the rat dorsal horn and that it may serve as an inhibitory transmitter within the substantia gelatinosa.

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1. Whole-cell patch recordings were made from antidromically identified sympathetic preganglionic neurons (SPN) of immature rat spinal cord slices. Bath application of nociceptin (0.1-1 μmol/L) suppressed excitatory postsynaptic potentials (EPSP) and hyperpolarized a population of SPN; these effects were naloxone (1 μmol/L) insensitive. 2. Nociceptin suppressed the amplitude of EPSP without causing a concomitant change in glutamate-induced depolarizations, suggesting a presynaptic inhibitory action. 3. Analysis of current-voltage relationships showed that nociceptin hyperpolarized SPN by increasing an inwardly rectifying K+ current. 4. Intrathecal injection of nociceptin (3, 10 and 30 nmol) to urethane-anaesthetized rats dose-dependently reduced the mean arterial pressure and heart rate; these effects were not prevented by prior intravenous injection of naloxone (1 mg/kg). 5. Results from our in vitro and in vivo experiments suggest that nociceptin suppresses spinal sympathetic outflow either by attenuating excitatory synaptic responses or hyperpolarizing SPN.

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Genetic and environmental factors interact to determine the individual vulnerability to develop ethanol dependence. The neurobiological mechanisms underlying these processes are not fully understood. Endogenous opioid peptides have been suggested to contribute. Brain opioids mediate ethanol reward and reinforcement via actions on the mesocorticolimbic dopamine system. This thesis focuses on environmental factors and investigates the impact of the early-life environment on adult voluntary ethanol consumption. The possible involvement of opioid peptides in environmental influences on adult ethanol consumption was examined using an experimental animal model. Maternal separation with short 15 min separations (MS15) was used to simulate a safe environment whereas prolonged 360 min separations (MS360) simulated an unsafe environment. Control rats were subjected to normal animal facility rearing (AFR). The separations were performed daily from postnatal day 1 to 21. Long-term ethanol consumption was registered using a two-bottle or a four-bottle free-choice paradigm in adult male and female ethanol-preferring AA (Alko, Alcohol), ethanol-avoiding ANA (Alko, Non-Alcohol) and non-preferring Wistar rats. In addition, analyses of immunoreactive Met-enkephalin-Arg6Phe7 (MEAP), dynorphin B (DYNB) and nociceptin/orphanin FQ (N/OFQ) peptide levels were performed after maternal separation as well as after voluntary ethanol drinking. In male rats, MS15 was related to lower ethanol consumption and these rats preferred lower concentrations, whereas MS360 was associated with an increased risk for higher consumption and/or preference for higher ethanol concentrations. Differences in basal opioid levels were observed in MS15 and MS360 rats. Furthermore, the ethanol-induced effects on opioid peptides in adults were dependent on the early environment. Female rats, on the other hand, were less affected or unaffected by maternal separation both in terms of ethanol consumption and neurobiological effects. Taken together, voluntary ethanol drinking, preference for low or high ethanol concentrations and opioid peptides in brain areas related to reward and reinforcement, motivation and stress were influenced by postnatal maternal separation in a sex dependent manner. The early environment thus had profound impact on the adult brain and the individual propensity for high ethanol drinking. A deranged endogenous opioid system contributed to these effects and may act as a mediator for long-term environmental influence on voluntary ethanol consumption.