The release of beta-endorphin and the neuropeptide-receptor mismatch in the brain.

Microprobes bearing immobilized antibodies to the carboxy-terminus of beta-endorphin were used to study the release of beta-endorphin in the urethane anaesthetized rat following electrical stimulation of the ipsilateral arcuate nucleus. The microprobes were inserted through the cerebral hemisphere, the superior colliculus and the midbrain periaqueductal grey. Since such microprobes detect extracellular molecules along their entire length they give information on the persistence and spread of compounds following release. Little immunoreactive-beta-endorphin was detected in the areas of brain sampled during electrical stimulation of arcuate nucleus but a remarkable spread throughout the midbrain and cerebral cortex occurred within 30 min of the cessation of stimulation. The results suggest that although beta-endorphin-containing fibres are absent in many parts of the brain, this neuropeptide can access receptors in these sites and it is not necessary for release to be directly adjacent to opiate receptors. As such it is important evidence supporting the hypothesis of volume transmission as a means of neuronal communication. The results also suggest that an important mechanism of the transport of beta-endorphin is the cerebrospinal fluid.

The release of immunoreactive neuropeptide Y in the spinal cord of the anaesthetized rat and cat.

The release of immunoreactive (ir-) neuropeptide Y (NYP) was studied in the anaesthetized rat and cat by means of microprobes bearing immobilized antibodies to the C terminus of NPY. An extensive basal release of ir-NYP was detected throughout the dorsal and upper ventral horn of the rat. This spontaneous release was not significantly altered by sectioning the spinal cord at the thoraco-lumbar junction nor by electrical stimulation of peripheral nerves. Since NPY is virtually absent in primary afferents it is probable that spontaneous release within the spinal cord comes from active NPY-containing intrinsic spinal neurones. In the spinal cat spontaneous release of ir-NPY was detected in the mid-dorsal horn and this was unaltered by peripheral noxious thermal or noxious mechanical stimuli. As in the rat, release from intrinsic spinal neurones is most probable. The extensive spontaneous release of ir-NPY in both species suggests a widespread role in spinal cord function.

Afferent volley patterns and the spinal release of immunoreactive substance P in the dorsal horn of the anaesthetized spinal cat.

Microprobes bearing immobilized antibodies to the C-terminus of substance P were used to measure release of this neuropeptide in the spinal cord of the anaesthetized spinal cat in response to peripheral nerve stimulation. Release of substance P was just detectable in laminae I, II with 150 stimuli (0.5 Hz, 5 min) and was near maximal with 300 stimuli. Using two periods of stimulation of 10 min separated by 15 min, greater levels of substance P were detected during the second period. Fifteen to 25 min after two periods of peripheral nerve stimulation levels of substance P detected by microprobes were still elevated above those present prior to stimulation. Stimulation with bursts of three impulses when delivering a fixed number of stimuli resulted in detection of increased levels of substance P at sites adjacent to the areas of maximal release. The results suggest that maximal release of substance P from the central terminals of primary afferent fibres occurs with relatively few impulses and at low frequencies in agreement with what is known of release from the peripheral terminals of these fibres.

Effects of the kappa-opioid agonist U50,488 on parturition in rats.

1. The effects of the kappa-opioid agonist U50,488 on parturition were studied in the rat. 2. Given directly after the birth of the second pup U50,488 (5 mg or 10 mg kg-1, i.p.) delayed the birth of the subsequent 4 pups by ca. 100 min, acting like morphine (10 mg kg-1, i.p.). In controls given the vehicle i.p., the birth of the 4 pups after treatment took 45.4 +/- 4.6 min. The effects of U50,488 could be prevented by simultaneous naloxone injection (10 mg kg-1). Injection of either U50,488 or morphine at 1 mg kg-1, i.v. also significantly delayed parturition. The effects of U50,488 but not of morphine were fully prevented by preinjection with nor-binaltorphimine (0.5 mg kg-1, i.v.) showing selective kappa-opioid receptor-mediated inhibition by U50,488 of established parturition. 3. In rats with an indwelling jugular venous cannula, i.v. injection of U50,488 (5 mg kg-1) after the birth of the second pup slowed parturition in a similar way to i.p. injection and significantly reduced blood plasma oxytocin concentration measured by radioimmunoassay compared with vehicle-injected controls. 4. Bolus i.v. injections of oxytocin (4 mu once per 5 min) significantly reduced the delay in parturition caused by i.v. U50,488, but continuous i.v. infusion of oxytocin (4 mu 5 min-1) was less effective. 5. Since i.v. oxytocin did not immediately reverse the effects of U50,488 on parturition, direct effects of U50,488 on isometric uterine contractions in vitro were sought. U50,488 inhibited spontaneous or oxytocin-stimulated contractions of uteri from rats within 24 h after parturition in a dose-related manner; the inhibitory effect was not naloxone-reversible.6. Thus U50,488 inhibited established parturition in the rat in a Kappa-opioid selective manner by reducing oxytocin secretion. The inhibitory effect may well have been potentiated by a direct non-opioid depressant action on contractile activity of the uterus.

Intracellular recordings from neurons of the arcuate nucleus in superfused explants of rat hypothalamus.

The membrane properties of arcuate nucleus (ARN) neurons and their connections with extranuclear regions were examined in vitro, using intracellular recordings obtained in superfused explants of rat hypothalamus. Current-clamp analysis revealed that the electroresponsive properties of a large proportion of ARN neurons are influenced by the expression of various forms of low threshold rectification. When the initial membrane potential of a neuron was maintained negative to -70 mV, depolarizing current injection could precipitate an immediate bursting response, due to the activation of a low-threshold spike, or delay firing as a result of transient outward rectification. These properties represent a means by which changes in membrane potential may modulate the responsiveness of ARN neurons to synaptic input. Indeed, electrical stimulation revealed that in addition to efferent projections to the supraoptic nucleus, medial preoptic area, or neurointermediate pituitary, subgroups of ARN neurons may receive a reciprocal, or collateral innervation from each of these areas. This functional diversity of intrinsic and synaptic mechanisms provides ARN neurons with a high capacity for the complex local processing of afferent information.

Opioid peptides have differential actions on sub-populations of arcuate neurones.

Extracellular recordings were made from spontaneously active arcuate neurones in hypothalamic slices in vitro. The majority of these neurones (82%) were inhibited in a dose-related manner by opioid peptides added to the perfusion medium at concentrations of 0.1-100 microM. The inhibitory responses were reversed by equimolar concentrations of naloxone. In terms of their latency to 50% inhibition and duration of action the order of potency of the opioid peptides on a molar basis was beta-Endorphin greater than DAGO greater than DADLE greater than Dynorphin. Several sub-populations of arcuate neurones can be identified on the basis of their neuronal activity. Opioid peptides selectively abolished the bursts in those neurones (putative peptidergic neurones) which discharged with a combination of bursts and single irregular action potentials suggesting a selective membrane action rather than an overall hyperpolarizing influence. A population of neurones which displayed episodic or regular activity, and which had action potentials characteristic of dopamine neurones, were profoundly inhibited by the opioid peptides; the activity of a second population of regularly firing neurones with conventional action potential profiles was totally unaffected. These electrophysiological studies thus provide evidence that the arcuate nucleus is one possible site of action at which endogenous opioid peptides could exert their modulatory role on neuroendocrine function; and the predominate influence is likely to be mediated through mu receptors.