Kappa opioid receptor activation depresses excitatory synaptic input to rat locus coeruleus neurons in vitro

Intracellular recordings were made from neurons of the rat locus coeruleus contained within a brain slice maintained in vitro. When applied to the slice in known concentrations the selective kappa opioid receptor agonist trans-(+)-3,4-dichloro-N-methyl-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide methane sulphonate (U50488) (0.01-1 microM) produced a concentration-dependent depression of the excitatory post-synaptic potential evoked by electrical stimulation of afferent inputs to the locus coeruleus. This effect was antagonized by naloxone with an apparent dissociation equilibrium constant (Kd) of 28 nM. U50488 did not completely abolish the EPSP. Over the same concentration range U50488 had no effect on the resting membrane potential, input resistance or action potential waveform of locus coeruleus neurons, nor did U50488 depress the depolarization produced by local application of L-glutamic acid. The mu opioid receptor agonists [D-Ala2, NMe Phe4, Gly-ol5] enkephalin (0.003-1 microM) and [D-Ala2, NMe Phe4, Met(O)5] enkephalinol (0.003-1 microM) caused a membrane hyperpolarization concomitant with a fall in neuronal input resistance. These effects were concentration-dependent and antagonized by naloxone with an apparent Kd of 1.5 nM. Mu agonists also caused a depression of the tetrodotoxin resistant action potential. An in vitro autoradiographic study of [3H]bremazocine binding within the locus coeruleus revealed that, although the majority of binding appears to be to mu sites, a significant proportion was displaceable by unlabelled U50488 and thus represented kappa binding sites. The possibility that kappa opioid receptors may be located pre-synaptically within the locus coeruleus, and that activation of these receptors depresses excitatory synaptic input, is discussed.     

Changes in noradrenergic terminal excitability induced by amphetamine and their relation to impulse traffic

The effects of amphetamine upon the terminal excitability of noradrenergic neurons of the nucleus locus coeruleus were studied in urethane anesthetized rats. Terminal excitability was measured by determining the stimulus currents necessary to evoke antidromic responses in locus coeruleus neurons from terminals in the frontal cortex. In most cases, terminal excitability was decreased following local infusion of amphetamine into the frontal cortex, while intravenous administration of the drug tended to increase terminal excitability. The decreased terminal excitability induced by local infusion of amphetamine appeared to be due to activation of alpha-adrenergic receptors located on the terminals of locus coeruleus neurons, since this effect mimics that of clonidine, a direct acting alpha-adrenergic agonist, and since the effect was abolished by pretreatment with alpha-methyl-p-tyrosine which disrupts the catecholamine liberating properties of amphetamine. Phentolamine, a direct acting alpha-adrenergic receptor antagonist was also found to block or reverse the effect of amphetamine. The changes in terminal excitability following intravenous injection of amphetamine appeared to be related to changes in the spontaneous activity of locus coeruleus neurons. A large decrease in spontaneous activity following intravenous administration of amphetamine was associated with increased terminal excitability, whereas when smaller changes in spontaneous activity occurred, terminal excitability was found to be decreased. These results are discussed with respect to the pharmacological properties of catecholaminergic neurons and the mechanisms of action of amphetamine.     

Microinjected morphine suppresses the activity of locus coeruleus noradrenergic neurons in freely moving cats

Microinjection of morphine (1.0 microgram/0.1 microliter) produced a significant suppression (approximately 60%) of the single unit activity of locus coeruleus noradrenergic neurons in freely moving cats. This effect was reversible by systemic administration of the opioid receptor antagonist, naloxone (1.0 mg/kg i.v.). The microinjection of naloxone (1.0 microgram/0.1 microliter), however, was without effect on the spontaneous activity of noradrenergic neurons in the locus coeruleus. Non-noradrenergic neurons recorded in the same vicinity showed no consistent response to the microinjection of morphine. These results suggest that the direct effect of opioids in the locus coeruleus is an inhibition of noradrenergic neuronal activity. Furthermore, it appears that opioid influences upon these neurons are not tonically active.     

A highly selective kappa-opioid receptor agonist, CI-977, reduces excitatory synaptic potentials in the rat locus coeruleus in vitro.

Intracellular recordings were made from neurons in a rat locus coeruleus slice preparation in vitro. A postsynaptic potential was evoked by electrical stimulation of afferents to the neurons. CI-977 ([5R-(5a,7a,8b)]-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec -8-yl[-4-benzofuranacetamide monohydrochloride) caused a depression of the evoked postsynaptic potential on locus coeruleus neurons. This action was reversed on washout. Bremazocine had a similar action on less than 50% of locus coeruleus neurons. Concentrations of CI-977 which depressed the postsynaptic potential did not affect either passive membrane conductance or a voltage-sensitive potassium current resembling IA. The depression of the excitatory postsynaptic potential caused by CI-977 remained in the presence of either 30 microM bicuculline and picrotoxin or when potassium acetate-filled recording electrodes were used. Using potassium chloride-filled recording electrodes and in the presence of 30 microM 6-cyano-2,3-dihydro-7-nitroquinoxaline-2,3-dione and either 30 microM DL-2-amino-5-phosphonovaleric acid or 500 microM kynurenic acid, CI-977 had no effect on the postsynaptic potential. The effects of CI-977 were reversed by 30-100 nM naloxone and 1-10 nM norbinaltorphimine but not by 1-10 nM naloxone. The hyperpolarizing response to the mu-opioid receptor-selective agonist D-Ala2,Nme Phe4,Gly-ol5 (DAGOL) was blocked by 1-10 nM naloxone but not by 1-100 nM norbinaltorphimine. The hyperpolarizing response to DAGOL was not affected by high doses of CI-977.(ABSTRACT TRUNCATED AT 250 WORDS)     

Local opiate withdrawal in locus coeruleus neurons in vitro

Noradrenergic neurons of the brain nucleus locus coeruleus (LC) become hyperactive during opiate withdrawal. It has been uncertain to what extent such hyperactivity reflects changes in intrinsic properties of these cells. The effects of withdrawal from chronic morphine on the activity of LC neurons were studied using intracellular recordings in rat brain slices. LC neurons in slices from chronically morphine-treated rats exhibited more than twice the frequency of spontaneous action potentials after naloxone compared with LC neurons from control rats. However, after naloxone treatment, the resting membrane potential (MP) of LC neurons from dependent rats was not significantly different from that in control rats. Neither resting MP nor spontaneous discharge rate (SDR) was altered by naloxone in LC neurons from control rats. Neither kynurenic acid nor a cocktail of glutamate and GABA antagonists (6-cyano-7-nitroquinoxalene-2,3-dione + 2-amino-5-phosphonopentanoic acid + bicuculline) blocked the hyperactivity of LC neurons precipitated by naloxone in slices from morphine-dependent rats. The effects of ouabain on MP and SDR were similar in LC neurons from control and morphine-dependent rats. These results indicate that an adaptive change in glutamatergic or GABAergic synaptic mechanisms or altered Na/K pump activity does not underlie the withdrawal-induced activation of LC neurons in vitro. Specific inhibitors of protein kinase A [Rp-cAMPS or N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide (H-89)] partially suppressed the withdrawal hyperactivity of LC neurons, and activators of cAMP (forskolin) or protein kinase A (Sp-cAMPS) increased the discharge rate of LC neurons from control rats. These results suggest that upregulation of cAMP-dependent protein kinase A during chronic morphine treatment is involved in the withdrawal-induced hyperactivity of LC neurons.     

Efferent projections of nucleus locus coeruleus: morphologic subpopulations have different efferent targets

This study quantitatively addresses the hypothesis that there is a systematic relationship between the morphologic characteristics of locus neurons and the particular target regions they innervate. Following horseradish peroxidase injections into selected terminal fields, locus coeruleus cell bodies are heavily labeled by retrograde transport so that somata size and shape, and in many cases primary dendritic pattern can be observed. This allows the classification of neurons as one of six cell types: large multipolar cells within ventral locus coeruleus, large multipolar cells in the anterior pole of locus coeruleus, fusiform cells in dorsal LC, posterior pole cells, medium-sized multipolar cells (termed core cells in this report), and small round cells. It was found that while core cells contribute to the innervation of all terminal fields examined, other cell types project to more restricted sets of targets. The contributions of each type to selected efferents are presented in detail. In particular, fusiform cells project to hippocampus and cortex, large multipolar cells in ventral locus coeruleus project to spinal cord and cerebellum, and small round cells in central and anterior locus coeruleus, as well as large multipolar cells in anterior locus coeruleus, project to hypothalamus. These results, in conjunction with those described in the preceding report, indicate that locus coeruleus is intrinsically organized with respect to efferent projections with much more specificity than has previously been evident. This high degree of organization is consistent with other recent demonstrations of functional specificity exhibited by locus coeruleus neurons.     

Inhibition of noradrenergic locus coeruleus neurons by C1 adrenergic cells in the rostral ventral medulla.

Recent anatomical and physiological experiments indicate that the nucleus locus coeruleus receives a predominant excitatory amino acid input, as well as a substantial inhibitory input, from the nucleus paragigantocellularis in the ventrolateral medulla. To determine whether C1 adrenergic neurons are involved in the inhibitory projection, the effects of the alpha-2 adrenoceptor antagonist, idazoxan, on inhibitory responses of locus coeruleus neurons to paragigantocellularis stimulation were characterized in the rat. Intravenous administration of idazoxan (0.2-1 mg/kg) attenuated paragigantocellularis-evoked inhibition, and often revealed an underlying weak excitation. Intraventricular administration of kynurenate, an excitatory amino acid antagonist, eliminated excitation from paragigantocellularis and disclosed an underlying inhibitory response in many locus coeruleus neurons that were previously excited by paragigantocellularis stimulation. These results revealed that about 90% of locus coeruleus neurons receive inhibition from the paragigantocellularis. Intravenous idazoxan significantly reduced such paragigantocellularis-evoked inhibition, completely blocking this response in three of eight locus coeruleus cells tested. Idazoxan was much more potent when locally infused into the locus coeruleus. Local infusion of idazoxan (0.1-2.5 ng) into locus coeruleus produced a dose-dependent decrease of paragigantocellularis-evoked inhibition and completely blocked the inhibition in 10/33 locus coeruleus neurons, indicating that the site of idazoxan action was in the locus coeruleus. These results extend our previous anatomical studies of adrenergic input to locus coeruleus, and indicate that C1 adrenergic neurons in the paragigantocellularis provide a direct inhibitory input to the great majority of locus coeruleus noradrenergic neurons. In addition, this is the first report of a neuronal response to activation of C1 adrenergic cells indicating that these neurons are strongly inhibitory when acting at alpha-2 receptors in vivo.     

Age-dependent changes in axonal branching of single locus coeruleus neurons projecting to two different terminal fields

Age-dependent changes in the axonal branching patterns of single locus coeruleus neurons, which innervate both the frontal cortex and hippocampus dentate gyrus, have been studied in male F344 rats. We used an electrophysiological approach involving antidromic activation to differentiate single from multi-threshold locus coeruleus neurons in each terminal field with age (7-27 mo of age). Most of these neurons have a single threshold in the young rats, whereas in the older brains, the neurons have multi-threshold responses. This implies an increased amount of axonal branching in the older brains. The time course of the increase differs in the two terminal fields, suggesting that the degree of plasticity or age-dependent increase in branching can differ across terminal fields.     

Effects of morphine and D-Ala, D-Leu enkephalin on the electrical activity of supraoptic neurosecretory cells in vitro

Experiments were performed to investigate the effects of morphine and [D-Ala2, D-Leu5]enkephalin on supraoptic cells in hypothalamic slices in vitro. To ensure the presence of a steady background activity, the cells were recorded with glutamate-filled glass microelectrodes and the level of activity was controlled by selecting a suitable retaining current (0.1-9.8 nA). Under these conditions, supraoptic cells showed either the non-phasic (65%) or phasic (35%) firing pattern previously associated with oxytocin or vasopressin cells, respectively. During perifusion of the slice with morphine (10 microM), 10 out of 17 non-phasic supraoptic cells were profoundly inhibited, five cells showed no response and the remaining 2 cells were excited. Similarly with [D-Ala2, D-Leu5]enkephalin (10 microM), 11 out of 15 non-phasic cells were inhibited, 3 cells showed no response and 1 cell was excited. The inhibition produced by morphine or [D-Ala2, D-Leu5]enkephalin could be reversed by concomitant application of naloxone (10 microM). In contrast to the profound effects seen in the non-phasic cells, only 1 out of 13 phasic cells tested with either morphine or [D-Ala2, D-Leu5]enkephalin was inhibited. The remaining 12 phasic cells showed no change in either their overall firing rate or pattern of activity during opiate perifusion. These results provide further evidence that, in addition to their inhibitory effects within the posterior pituitary, opiates can directly suppress the electrical activity of magnocellular neurosecretory cells at the level of the hypothalamus. However, the absence of an opiate effect on the phasic cells might suggest that the action of opioid peptides within the hypothalamus would be exerted predominantly on the oxytocin, rather than the vasopressin cells.     

Internalization of mu-opioid receptors produced by etorphine in the rat locus coeruleus.

Chronic administration of mu-opioid receptor agonists is known to produce adaptive changes within noradrenergic neurons of the locus coeruleus. Although mu-opioid receptors are densely expressed by locus coeruleus neurons, the effects of acute and chronic administration of agonists on the subcellular distribution of mu-opioid receptors remain poorly understood. Therefore, we examined the ultrastructural distribution of mu-opioid receptor immunoreactivity in the locus coeruleus of rats subjected to either acute morphine, or etorphine, or chronic morphine treatment. In the locus coeruleus of control rats receiving acute saline injections or placebo pellet implants, immunogold-silver labeling for mu-opioid receptors was localized to parasynaptic and extrasynaptic portions of the plasma membranes of perikarya and dendrites. Only 8% of the gold-silver particles analyzed were distributed within the cytoplasm of dendrites and perikarya in vehicle-treated rats. Immunolabeling for mu-opioid receptors was distributed along portions of the plasma membrane that were often apposed by astroglial sheaths. After acute injections of etorphine, there was a dramatic internalization of mu-opioid receptors to intracellular compartments. Quantitative analysis of gold-silver particles indicative of mu-opioid receptors showed that a substantial number of gold particles shifted from the plasma membrane to early endosomes in dendrites from etorphine-treated rats. In dendrites sampled from etorphine-treated rats, 85% of the gold-silver grains indicative of mu-opioid receptor labeling were located in intracellular compartments as compared to 15% that were distributed along the plasma membrane. In animals that received either acute morphine injections or chronic morphine via pellet implantation, no change in the subcellular distribution of immunogold particles indicative of mu-opioid receptors was detected when compared to matched control animals.These results provide the first ultrastructural evidence that mu-opioid receptors are internalized by agonists such as etorphine, but not the partial agonist morphine, in the locus coeruleus.     

Excitatory amino acid release in the locus coeruleus during naloxone-precipitated morphine withdrawal in adjuvant arthritic rats

OBJECTIVE AND DESIGN: Excitatory amino acid levels in the locus coeruleus (LC) and the behavioral signs during naloxone-precipitated withdrawal in arthritic rats treated with chronic morphine were investigated by in vivo microdialysis. METHODS: Increases in glutamate (Glu) and aspartate (Asp) were noted after naloxone (48 nmol/5 microl, LC)-precipitated withdrawal from normal and adjuvant arthritic rats which had been intracerebroventricularly infused for 3 days with morphine (26 nmol/l microl/h). RESULTS: The increases in Glu and Asp levels on morphine withdrawal in normal rats were attenuated following naloxone challenge in the morphine-dependent arthritic rats. Moreover, behavioral signs during morphine withdrawal were detected following the naloxone challenge in both the morphine-dependent normal and adjuvant arthritic rats, but not in the saline-infused controls. CONCLUSIONS: These results show that the attenuation of Glu and Asp release from the LC in the adjuvant arthritic rats might explain the anti-inflammatory and analgesic effects of mu-opioids in adjuvant arthritic rats.     

Effects of morphine administration on catecholamine levels in rat brain; specific reduction of epinephrine concentration in hypothalamus

The effects of morphine administration on concentrations of epinephrine, norepinephrine and dopamine were examined in the rat brain. Morphine injection reduced the epinephrine level only in the hypothalamus, while the norepinephrine level was reduced in the hypothalamus, medulla, and locus coeruleus. The dopamine concentration was elevated in all regions examined. These changes were blocked by administration of naloxone. Repeated injection of morphine for 14 days did not affect any catecholamine level. In naloxone-induced withdrawal, epinephrine was most markedly depleted in hypothalamus. These observations suggest that the epinephrine level in hypothalamus is affected by morphine acting on opioid receptors.     

Descriptive and functional neuroanatomy of locus coeruleus-noradrenaline-containing neurons involvement in bradykinin-induced antinociception on principal sensory trigeminal nucleus

The present study was carried out in Wistar rats, using the jaw-opening reflex and dental pulp stimulation, to investigate noradrenaline- and serotonin-mediated antinociceptive circuits. The effects of microinjections of bradykinin into the principal sensory trigeminal nucleus (PSTN) before and after neurochemical lesions of the locus coeruleus noradrenergic neurons were studied. Neuroanatomical experiments showed evidence for reciprocal neuronal pathways connecting the locus coeruleus (LC) to trigeminal sensory nuclei and linking monoaminergic nuclei of the pain inhibitory system to spinal trigeminal nucleus (STN). Fast blue (FB) injections in the locus coeruleus/subcoeruleus region retrogradely labeled neurons in the contralateral PSTN and LC. Microinjections of FB into the STN showed neurons labeled in both ipsilateral and contralateral LC, as well as in the ipsilateral Barrington's nucleus and subcoeruleus area. Retrograde tract-tracing with FB also showed that the mesencephalic trigeminal nucleus sends neural pathways towards the ipsilateral PSTN, with outputs from cranial and caudal aspects of the brainstem. In addition, neurons from the lateral and dorsolateral columns of periaqueductal gray matter also send outputs to the ipsilateral PSTN. Microinjections of FB in the interpolar and caudal divisions of the STN labeled neurons in the caudal subdivision of STN. Microinjections in the STN interpolar and caudal divisions also retrogradely labeled serotonin- and noradrenaline-containing nucleus of the brainstem pain inhibitory system. Finally, the gigantocellularis complex (nucleus reticularis gigantocellularis/paragigantocellularis), nucleus raphe magnus and nucleus raphe pallidus also projected to the caudal divisions of the STN. Microinjections of bradykinin in the PSTN caused a statistically significant long-lasting antinociception, antagonized by the damage of locus coeruleus-noradrenergic neuronal fibres with (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine) (DSP4), a neurotoxin that specifically depleted noradrenaline from locus coeruleus terminal fields. These data suggest that serotonin- and noradrenaline-containing nuclei of the endogenous pain inhibitory system exert a key-role in the antinociceptive mechanisms of bradykinin and the locus coeruleus is crucially involved in this effect.     

Changes in the brain kappa-opioid receptor levels of rats in withdrawal from physical dependence upon butorphanol

Changes in kappa-opioid receptor levels have been implicated in the development of physical dependence upon and withdrawal from the mixed agonist-antagonist opioid, butorphanol. Immunoblotting analysis was performed to determine the levels of kappa- and mu-opioid receptors in brain regions of rats in withdrawal from dependence upon butorphanol or morphine. Physical dependence was induced by a 72 h i.c.v. infusion with either butorphanol or morphine (26 nmol/microl/h). Withdrawal was subsequently precipitated by i.c.v. challenge with naloxone (48 nmol/5 microl/rat), administered 2 h following cessation of butorphanol or morphine infusion. Immunoblotting analysis of kappa-opioid receptors from butorphanol-withdrawal rats showed significant increases in 11 of 21 brain regions examined, including the nucleus accumbens, amygdala, dorsomedial hypothalamus, hypothalamus, paraventricular thalamus, thalamus, presubiculum, and locus coeruleus, when compared with saline treated, non-dependent controls. In addition, significant reductions were found in the hippocampus and in cortical brain regions, including the parietal cortex and temporal cortex from butorphanol-withdrawal rats. These findings contrasted with those from morphine-withdrawal rats, in which the only changes noted were increases in the thalamus and paraventricular thalamus. Changes in the levels of the mu-opioid receptor protein were observed in 11 of 21 brain regions examined in morphine-withdrawal rats, but only in three of 21 in butorphanol-withdrawal rats. These results implicate a substantive and largely unique role for kappa-opioid receptors in mediation of the development of physical dependence upon, and the expression of withdrawal from, butorphanol, as opposed to the prototypical opioid analgesic, morphine.     

Concomitant depression of locus coeruleus neurons and of flexor reflexes by an alpha 2-adrenergic agonist in rats: a possible mechanism for an alpha 2-mediated muscle relaxation

The alpha 2-agonist tizanidine, clinically used as an antispastic drug, also strongly reduces polysynaptic flexor reflexes. The hypothesis was tested that the noradrenergic coerulespinal system exerts a tonic facilitation on spinal reflexes and that the depressant effects of tizanidine may be explained by an alpha 2-mediated autoinhibition of the tonic activity of locus coeruleus neurons, resulting in a disfacilitation of the spinal reflexes. The following results support this working hypothesis: (1) systemic injections of tizanidine markedly decreased the spontaneous activity of locus coeruleus neurons, but not of non-locus coeruleus neurons. The alpha 2-antagonist yohimbine reversed this effect. (2) The time course of diminished locus coeruleus activity paralleled that of depressed flexor reflexes. (3) Flexor reflexes were also markedly depressed by the alpha 1-adrenergic antagonist prazosin, administered alone, which is in line with the proposition that the noradrenergic system exerts a tonic facilitation on spinal neurons by way of alpha 1-adrenergic receptor activation. (4) Flexor reflexes were facilitated by conditioning microstimulation of locus coeruleus neurons, and this effect was reversed by prazosin. (5) Flexor reflexes significantly diminished in size following placement of an irreversible lesion in the ipsilateral locus coeruleus. Although these results strongly support the above hypothesis regarding a descending modulatory function of the descending locus coeruleus system on spinal reflexes, possible additional mechanisms, perhaps also involving the ascending projection of the locus coeruleus to supraspinal motor structures, remain to be elucidated.     

The role of alpha 1-adrenoceptor-mediated collateral excitation in the regulation of the electrical activity of locus coeruleus neurons

The physiological role of two types of autoreceptors, alpha 1- and alpha 2-adrenoceptors, located on the somadendritic membranes of locus coeruleus neurons, was studied in the developing and adult rat brain. Animals from birth to adulthood were anesthetized with urethan, and single-unit activity was recorded extracellularly in the locus coeruleus. The spontaneous firing of most locus coeruleus neurons was inhibited by iontophoretic application of noradrenaline at a high concentration, while noradrenaline at a low concentration frequently caused excitation of the neurons, predominantly in the developing brain. A similar excitation was also produced by iontophoretic application of the alpha 1-agonist phenylephrine. These excitations were antagonized by the alpha 1-antagonist, 2-beta [4-hydroxyphenylethylaminomethyl]-tetralone, while this antagonist had little effect on glutamate-induced excitation. The noradrenaline- and phenylephrine-induced excitation occurred more frequently in the neurons having little or no spontaneous activity. Electrical stimulation of the dorsal noradrenergic bundle arising in the locus coeruleus produced both inhibition and excitation. The excitatory responses were manifest primarily in early developmental stages, and occurred predominantly when the neurons had little or no spontaneous activity. When the neurons began firing at relatively high rates, the effects of dorsal noradrenergic bundle stimulation became principally inhibitory. Since the excitation evoked by dorsal noradrenergic bundle of stimulation was blocked by the alpha 1-antagonist, the excitation was thought to result from activation of alpha 1-adrenoceptors by noradrenaline released from the terminals of recurrent axon collaterals of locus coeruleus neurons themselves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the mesolimbic loop of antinociception--II. A serotonin-enkephalin interaction in the nucleus accumbens

In a previous report we have shown that the antinociceptive effect elicited by microinjection of morphine into the periaqueductal gray is due, at least in part, to the activation of an ascending serotonergic pathway which releases 5-hydroxytryptamine in the nucleus accumbens. We now report that antinociception induced by intra-periaqueductal gray injection of morphine can be attenuated also by the narcotic antagonist naloxone or the enkephalin antibodies administered into the nucleus accumbens, and potentiated by D-phenylalanine, a putative inhibitor of the degradation of enkephalins. Moreover, the antinociceptive effect induced by 5-hydroxytryptamine administered into nucleus accumbens could be blocked by naloxone injected into the same site, whereas the antinociception elicited by intra-accumbens injection of [D-Ala2,D-Leu5]enkephalin was not affected by cinanserin, a 5-hydroxytryptamine blocking agent. It is concluded that morphine administered to the periaqueductal gray is capable of activating an ascending serotonergic pathway to release 5-hydroxytryptamine in the nucleus accumbens, which in turn activates an enkephalinergic mechanism within the same nucleus, resulting in an antinociceptive effect.     

Activation of adenylate cyclase attenuates the hyperpolarization following single action potentials in brain noradrenergic neurons independently of protein kinase a

Afterhyperpolarizations that follow action potentials are a prominent mechanism for the control of neuronal excitability. Such afterhyperpolarizations in many neurons are modulated by a variety of second messenger systems. Here, we examined the regulation of afterhyperpolarizations in noradrenergic locus coeruleus neurons by the adenylate cyclase system. Although superfusion of the adenylate cyclase activator, forskolin, had no effect on hyperpolarizations following trains of action potentials, both forskolin and a membrane permeable analog of cyclic AMP, 8-bromo-cyclic AMP, attenuated the amplitude of afterhyperpolarizations which followed single action potentials of locus coeruleus neurons recorded intracellularly in brain slices. In contrast, superfusion of 1,9-dideoxyforskolin, the forskolin analog that does not activate adenylate cyclase, had no effect on these single action potential afterhyperpolarizations. Co-application of a protein kinase inhibitor (H8, KT5720, staurosporin or Rp-cAMPS) with either forskolin or 8-bromo-cyclic AMP failed to block the reduction of afterhyperpolarization amplitude, but blocked the cyclic AMP-dependent enhancement of opiate responses in the same locus coeruleus neurons. Furthermore, application of a membrane permeable analog of 5'-AMP, 8-bromo-5'-AMP, the cyclic AMP metabolite that does not activate a protein kinase, potently reduced the amplitudes of single action potential afterhyperpolarizations. The afterhyperpolarization amplitude was also reduced in locus coeruleus neurons taken from chronically morphine-treated rats, a treatment known to increase adenylate cyclase activity.These results indicate that elevation of intracellular cyclic AMP or 5'-AMP reduces the single action potential afterhyperpolarization in locus coeruleus neurons. This action may be mediated through a mechanism independent of protein kinase activation.     

Role of agonist-dependent receptor internalization in the regulation of mu opioid receptors

Organotypic cultures and ileal neuromuscular preparations were used to determine (i) whether endogenous release of opioids by electrical stimulation induces mu receptor endocytosis, and (ii) whether and under which conditions ligand-induced mu receptor endocytosis influences the responsiveness of neurons expressing native mu receptors. In longitudinal muscle-myenteric plexus preparations, electrical stimulation at 20 Hz induced a prominent endocytosis of mu receptors in enteric neurons, indicating endogenous release of opioids. A similar massive endocytosis was triggered by exogenous application of the mu receptor agonist, [D-Ala(2),MePhe(4), Gly-ol(5)] enkephalin, whereas exogenous application of morphine was ineffective. [D-Ala(2),MePhe(4),Gly-ol(5)] enkephalin and morphine induced a concentration-dependent inhibition of neurogenic cholinergic twitch contractions to electrical stimulation at 0.1 Hz. beta-Chlornaltrexamine shifted to the right the inhibitory curve of both agonists with a concentration-dependent reduction of the maximum agonist response, which is consistent with the existence of spare mu opioid receptors. Under these conditions, the induction of mu receptor endocytosis by exogenously applied [D-Ala(2), MePhe(4),Gly-ol(5)] enkephalin diminished the inhibitory effect of this agonist on twitch contractions and tritiated acetylcholine release. In contrast, there was no reduction of the inhibitory effect of morphine, which failed to induce mu receptor endocytosis, on neurogenic cholinergic response.These results provide the first evidence for the occurrence of mu receptor endocytosis in neurons by endogenously released opioids and show that agonist-dependent mu receptor endocytosis could serve as a mechanism to regulate mu opioid receptor responsiveness to ligand stimulation when the opioid receptor reserve is reduced.     

Evidence for cholinergic regulation of basal norepinephrine release in the rat olfactory bulb.

The effects of locally infused cholinergic agonists on extracellular levels of norepinephrine in the olfactory bulb of anesthetized rats were determined using in vivo microdialysis coupled with high-performance liquid chromatography and electrochemical detection. Using chronically implanted microdialysis probes, the basal norepinephrine level in the olfactory bulb was 0.55 pg/10 microl dialysate. Local infusion of K+ (30 mM) or the norepinephrine re-uptake inhibitor desipramine (1 microM) through the dialysis probe significantly increased basal norepinephrine levels. Focal activation of noradrenergic locus coeruleus neurons, the sole source of norepinephrine innervation of the olfactory bulb, increased norepinephrine levels by 247% of control. Local infusion of the acetylcholinesterase inhibitor soman (0.4 mM) into the olfactory bulb increased basal norepinephrine levels by 134% of control, suggesting that endogenously released acetylcholine modulates norepinephrine release. Intrabulbar infusion of acetylcholine (40 mM) or nicotine (40 mM) increased norepinephrine levels (317% and 178% of control, respectively), while infusion of the muscarinic receptor agonist pilocarpine (40 mM) reduced norepinephrine levels (54% of control). These results demonstrate that basal norepinephrine release in the olfactory bulb is potently modulated by stimulation of local cholinergic receptors. Nicotinic receptors stimulate, and muscarinic receptors inhibit, norepinephrine release from locus coeruleus terminals.