Morphine Increases Acetylcholine Release in the Trigeminal Nuclear Complex

Study Objectives:

The trigeminal nuclear complex (V) contains cholinergic neurons and includes the principal sensory trigeminal nucleus (PSTN) which receives sensory input from the face and jaw, and the trigeminal motor nucleus (MoV) which innervates the muscles of mastication. Pain associated with pathologies of V is often managed with opioids but no studies have characterized the effect of opioids on acetylcholine (ACh) release in PSTN and MoV. Opioids can increase or decrease ACh release in brainstem nuclei. Therefore, the present experiments tested the 2-tailed hypothesis that microdialysis delivery of opioids to the PSTN and MoV significantly alters ACh release.

Design:

Using a within-subjects design and isoflurane-anesthetized Wistar rats (n = 53), ACh release in PSTN during microdialysis with Ringer''s solution (control) was compared to ACh release during dialysis delivery of the sodium channel blocker tetrodotoxin, muscarinic agonist bethanechol, opioid agonist morphine, mu opioid agonist DAMGO, antagonists for mu (naloxone) and kappa (nor-binaltorphimine; nor-BNI) opioid receptors, and GABA_A antagonist bicuculline.

Measurements and Results:

Tetrodotoxin decreased ACh, confirming action potential-dependent ACh release. Bethanechol and morphine caused a concentration-dependent increase in PSTN ACh release. The morphine-induced increase in ACh release was blocked by nor-BNI but not by naloxone. Bicuculline delivered to the PSTN also increased ACh release. ACh release in the MoV was increased by morphine, and this increase was not blocked by naloxone or nor-BNI.

Conclusions:

These data comprise the first direct measures of ACh release in PSTN and MoV and suggest synaptic disinhibition as one possible mechanism by which morphine increases ACh release in the trigeminal nuclei.

Citation:

Zhu Z; Bowman HR; Baghdoyan HA; Lydic R. Morphine increases acetylcholine release in the trigeminal nuclear complex. SLEEP 2008;31(12):1629–1637.     

Nociception increases during opioid infusion in opioid receptor triple knock-out mice

Opioids are extensively used analgesics yet can paradoxically increase pain sensitivity in humans and rodents. This hyperalgesia is extensively conceptualized to be a consequence of opioid receptor activity, perhaps providing an adaptive response to analgesia, and to utilize N-methyl-D-aspartate (NMDA) receptors. These assumptions were tested here in opioid receptor triple knock-out (KO) mice lacking all three genes encoding opioid receptors (mu, delta, and kappa) by comparing their thermal nociceptive responses to the opioids morphine and oxymorphone with those of B6129F(1) controls. Injecting acute opioid bolus doses in controls caused maximal analgesia that was completely abolished in KO mice, confirming the functional consequence of the KO mouse opioid receptor deficiency. Continuous opioid infusion by osmotic pump in control mice also initially caused several consecutive days of analgesia that was shortly thereafter followed by several consecutive days of hyperalgesia. In contrast, continuously infusing KO mice with opioids caused no detectable analgesic response, but only immediate and steady declines in nociceptive thresholds culminating in several days of unremitting hyperalgesia. Finally, injecting the non-competitive NMDA receptor antagonist MK-801 during opioid infusion markedly reversed hyperalgesia in control but not KO mice. These data demonstrate that sustained morphine and oxymorphone delivery causes hyperalgesia independently of prior or concurrent opioid or NMDA receptor activity or opioid analgesia, indicating the contribution of mechanisms outside of current conceptions, and are inconsistent with proposals of hyperalgesia as a causative factor of opioid analgesic tolerance.     

The absence of endogenous beta-endorphin selectively blocks phosphorylation and desensitization of mu opioid receptors following partial sciatic nerve ligation

Phosphorylation of specific sites in the second intracellular loop and in the C-terminal domain have previously been suggested to cause desensitization and internalization of the mu-opioid receptor (MOP-R). To assess sites of MOP-R phosphorylation in vivo, affinity-purified, phosphoselective antibodies were raised against either phosphothreonine-180 in the second intracellular loop (MOR-P1) or the C-terminal domain of MOP-R containing phosphothreonine-370 and phosphoserine-375 (MOR-P2). We found that MOR-P2-immunoreactivity (IR) was significantly increased within the striatum of wild-type C57BL/6 mice after injection of the agonist fentanyl. Pretreatment with the antagonist naloxone blocked the fentanyl-induced increase. Furthermore, mutant mice lacking MOP-R showed only non-specific nuclear MOR-P2-IR before or after fentanyl treatment, confirming the specificity of the MOR-P2 antibodies. To assess whether MOP-R phosphorylation occurs following endogenous opioid release, we induced chronic neuropathic pain by partial sciatic nerve ligation (pSNL), which caused a significant increase in MOR-P2-IR in the striatum. pSNL also induced signs of mu opioid receptor tolerance demonstrated by a rightward shift in the morphine dose response in the tail withdrawal assay and by a reduction in morphine conditioned place preference (CPP). Mutant mice selectively lacking all forms of the beta-endorphin peptides derived from the proopiomelanocortin (Pomc) gene did not show increased MOR-P2-IR, decreased morphine antinociception, or reduced morphine CPP following pSNL. In contrast gene deletion of either proenkephalin or prodynorphin opioids did not block the effects of pSNL. These results suggest that neuropathic pain caused by pSNL in wild-type mice activates the release of the endogenous opioid beta-endorphin, which subsequently induces MOP-R phosphorylation and opiate tolerance.     

Opioid receptor mechanisms at the hypoglossal motor pool and effects on tongue muscle activity in vivo

Opioids can modulate breathing and predispose to respiratory depression by actions at various central nervous system sites, but the mechanisms operating at respiratory motor nuclei have not been studied. This study tests the hypotheses that (i) local delivery of the μ-opioid receptor agonist fentanyl into the hypoglossal motor nucleus (HMN) will suppress genioglossus activity in vivo , (ii) a component of this suppression is mediated by opioid-induced acetylcholine release acting at muscarinic receptors, and (iii) δ- and κ-opioid receptors also modulate genioglossus activity. Seventy-two isoflurane-anaesthetised, tracheotomised, spontaneously breathing rats were studied during microdialysis perfusion into the HMN of (i) fentanyl and naloxone (μ-opioid receptor antagonist), (ii) fentanyl with and without co-application of muscarinic receptor antagonists, and (iii) δ- and κ-opioid receptor agonists and antagonists. The results showed (i) that fentanyl at the HMN caused a suppression of genioglossus activity ( P < 0.001) that reversed with naloxone ( P < 0.001), (ii) that neither atropine nor scopolamine affected the fentanyl-induced suppression of genioglossus activity, and (iii) that δ-, but not κ-, opioid receptor stimulation also suppressed genioglossus activity ( P = 0.036 and P = 0.402 respectively). We conclude that μ-opioid receptor stimulation suppresses motor output from a central respiratory motoneuronal pool that activates genioglossus muscle, and this suppression does not involve muscarinic receptor-mediated inhibition. This μ-opioid receptor-induced suppression of tongue muscle activity by effects at the hypoglossal motor pool may underlie the clinical concern regarding adverse upper airway function with μ-opioid analgesics. The inhibitory effects of μ- and δ-opioid receptors at the HMN also indicate an influence of endogenous enkephalins and endorphins in respiratory motor control.     

Ultrastructural data,with special reference to bouton/glial relationships,from the hypoglossal nucleus after a second axotomy of the hypoglossal nerve

Summary The left hypoglossal nerve of adult male albino rats was prevented from regenerating to the tongue after a distal axotomy by implanting the proximal stump into normally innervated left sternomastoid muscle. Eighty-four days after implantation, the hypoglossal nerve was transected again and its regeneration to the tongue unimpeded. From 8 to 70 days after this second axotomy the left hypoglossal nuclei were processed for quantitative ultrastructural analysis. The first aim of this study was to compare regeneration success in the hypoglossal nucleus after second axotomy with that accompanying outgrowth of the hypoglossal nerve into denervated sternomastoid muscle. During quantitative analysis a second aim developed, of elucidating bouton/glial relationships.The second axotomy induced loss and return of subsurface cisterns, dispersal and reassembly of Nissl substance, increase and decrease of microglial numbers, slight further loss and partial return of boutons with clear spherical vesicles and symmetrical synapses, slight increase and decrease of boutons with clear flat vesicles and symmetrical synapses, regrowth of retracted dendrites and restoration of their synapses, and gradual diminution of numbers of electron-dense neurones and dendrites. Astrocytes remained hypertrophied throughout.When compared with events in the hypoglossal nucleus accompanying innervation of denervated sternomastoid muscle by the hypoglossal nerve, the results suggest (1) that regeneration of the hypoglossal nerve to its own tongue muscle instead of to a foreign muscle caused no acceleration of recovery in the hypoglossal nucleus, and (2) that the microglial response is dependent on nerve integrity and not on bouton behaviour.     

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 of parasympathetic postganglionic neurons in the rat hypoglossal nerve trunk

Previous studies have indicated that the geniohyoid (GH) muscle is innervated by efferent axons from both the hypoglossal nerve (CN XII) and ansa cervicalis. To clarify the physiological significance of this dual innervation of the GH muscle, we examined properties of efferent innervations in rat GH muscle using electrophysiological, horseradish peroxidase (HRP) tracing and immunohistochemical techniques. Recordings from the branch of the XII nerve that innervates the GH (GH.Br) revealed that bursts of impulses during fictitious swallowing were conducted via the XII nerve trunk, in which neuronal cell bodies were labeled in the ventrolateral subnucleus of the XII nucleus by HRP tracing. In contrast, in vivo experiments demonstrated that tonic discharges in GH.Br were conducted via the ansa cervicalis. However, HRP-labeled efferent neurons were observed in neither brainstem nor upper spinal cord, but sensory neurons were labeled in the most rostral cervical spinal ganglia via the ansa cervicalis. Tonic activity was abolished in vitro by mecamylamine, an antagonist of nicotinic acetylcholine receptors (nAChR), and by pirenzepine, an antagonist of muscarinic M1 receptors. Incubation of isolated XII nerve segments with antisera to vasoactive intestinal peptide, nAChR, and muscarinic M1 receptor yielded small numbers of labeled neurons with each antiserum. All labeled neurons displayed similar diameters and were located approximately 1.5 mm proximal to the bifurcation of the XII nerve into medial and lateral branches. Our findings indicate that GH muscle in the rat is innervated by both somatic and parasympathetic nervous systems.     

Morphine inhibits resting respiration, but it attenuates reflexive respiratory suppression in anesthetized cat through kappa-receptor

Noxious stimulation of thin-fiber muscular afferents induces a reflexive respiratory suppression that we call "poststimulus respiratory suppression." In anesthetized, vagotomized, paralyzed, and artificially ventilated cats, morphine depressed the level of resting respiration (inhibitory effect on resting respiration) and attenuated the magnitude of the poststimulus respiratory suppression (excitatory effect on the reflexively modified respiration). These two kinds of morphine effects were antagonized by naloxone, suggesting the participation of opioid receptors. To clarify the opioid receptor subtypes responsible for these effects of morphine, three type-selective opioid antagonists-naltrindole (delta antagonist), gamma-funaltrexamine (mu antagonist), and Mr2266 (kappa antagonist)-were tested. The morphine-induced depression in the resting respiration was antagonized by pretreatment with the kappa antagonist, not with the mu or delta antagonist. Furthermore, the morphine-induced attenuation in the magnitude of the poststimulus suppression was also blocked by the kappa antagonist, but not by the mu or delta antagonist. In conclusion, (1) morphine inhibits resting respiration, but it attenuates the magnitude of the poststimulus respiratory suppression; (2) both these morphine effects are mediated by kappa opioid receptors. The possibility that the kappa(3) receptor, one of the kappa receptors subtypes, mediates the two kinds of morphine effects has been discussed.     

Developmental nicotine exposure alters neurotransmission and excitability in hypoglossal motoneurons

Hypoglossal motoneurons (XII MNs) control muscles of the mammalian tongue and are rhythmically active during breathing. Acetylcholine (ACh) modulates XII MN activity by promoting the release of glutamate from neurons that express nicotinic ACh receptors (nAChRs). Chronic nicotine exposure alters nAChRs on neurons throughout the brain, including brain stem respiratory neurons. Here we test the hypothesis that developmental nicotine exposure (DNE) reduces excitatory synaptic input to XII MNs. Voltage-clamp experiments in rhythmically active medullary slices showed that the frequency of excitatory postsynaptic currents (EPSCs) onto XII MNs from DNE animals is reduced by 61% (DNE = 1.7 ± 0.4 events/s; control = 4.4 ± 0.6 events/s; P < 0.002). We also examine the intrinsic excitability of XII MNs to test whether cells from DNE animals have altered membrane properties. Current-clamp experiments showed XII MNs from DNE animals had higher intrinsic excitability, as evaluated by measuring their response to injected current. DNE cells had high-input resistances (DNE = 131.9 ± 13.7 MΩ, control = 78.6 ± 9.7 MΩ, P < 0.008), began firing at lower current levels (DNE = 144 ± 22 pA, control = 351 ± 45 pA, P < 0.003), and exhibited higher frequency-current gain values (DNE = 0.087 ± 0.012 Hz/pA, control = 0.050 ± 0.004 Hz/pA, P < 0.02). Taken together, our data show previously unreported effects of DNE on XII MN function and may also help to explain the association between DNE and the incidence of central and obstructive apneas.     

G protein activation in rat ponto-mesencephalic nuclei is enhanced by combined treatment with a mu opioid and an adenosine A1 receptor agonist

STUDY OBJECTIVES: Opioids delivered to the pons inhibit REM sleep, whereas pontine administration of adenosine enhances REM sleep. In other brain areas opioids and adenosine interact to produce antinociception. Adenosine A1 receptors and mu opioid receptors each activate Gi/Go proteins. This study tested the hypothesis that combined treatment with the adenosine A1 receptor agonist SPA and the mu opioid agonist DAMGO would enhance G protein activation to a greater level than produced by either agonist alone. G protein activation was quantified in seven brainstem regions regulating sleep and nociception. This study also tested the hypothesis that G protein activation caused by SPA would be concentration dependent and blocked by the adenosine A1 receptor antagonist DPCPX. DESIGN: Activation of G proteins was assessed autoradiographically by agonist stimulation of [35S]GTPgammaS binding in slide-mounted sections of rat brainstem. G protein activation was quantified in nCi/g tissue for pontine reticular formation, dorsal raphe, ventrolateral and dorsomedial periaqueductal gray, and laterodorsal and pedunculopontine tegmental nuclei. SETTING: N/A PATIENTS OR PARTICIPANTS: N/A MEASUREMENTS AND RESULTS: Combined treatment with SPA and DAMGO caused a partially additive increase in G protein activation that was significantly (p<0.01) greater than G protein activation caused by either agonist alone. Treatment with SPA alone caused a concentration dependent (p<0.001) increase in [35S]GTPgammaS binding that was blocked by DPCPX. CONCLUSION: Agonist activation of adenosine A1 receptors stimulates G proteins in brainstem nuclei regulating sleep and nociception. In these same nuclei, G protein activation by combined treatment with DAMGO and SPA was partially additive, suggesting that mu opioid and adenosine A1 receptors activate some common G protein pools.     

Respiratory activation of the genioglossus muscle involves both non-NMDA and NMDA glutamate receptors at the hypoglossal motor nucleus in vivo

Brainstem respiratory neurons innervate the hypoglossal motor nucleus which in turn transmits this respiratory drive signal to the genioglossus muscle of the tongue. The mechanism of this transmission is important to help maintain an open airspace for effective breathing, and is thought to rely almost exclusively on non-N-methyl-d-aspartate (non-NMDA) glutamate receptor activation during respiration. However those studies were performed in slices of medulla from neonatal animals in vitro which may have led to an underestimation of the contribution of NMDA glutamate receptors that may normally operate in intact preparations. The current study tests the hypothesis that both NMDA and non-NMDA receptors contribute to respiratory drive transmission at the hypoglossal motor nucleus in vivo. Experiments were performed in urethane-anesthetized and tracheotomized adult Wistar rats in which vagus nerves were either intact or sectioned. In the presence of augmented genioglossus activity produced by vagotomy, microdialysis perfusion of either an NMDA receptor antagonist (D-2-amino-5-phosphonovaleric acid, 0.001-10 mM) or a non-NMDA receptor antagonist (6-cyano-7-nitroquinoxaline-2, 3-dione disodium salt, 0.001-1 mM) to the hypoglossal motor nucleus reduced respiratory-related genioglossus activity in a dose-dependent manner (P < 0.001) indicating that both NMDA and non-NMDA glutamate receptors are necessary for transmission of the respiratory drive signal to genioglossus muscle in vivo. Similar effects were observed in the vagus nerve intact rats. Further experiments demonstrated that each delivered antagonist had effects that were specific to its respective receptor. Regression analysis also revealed that the activity of both NMDA and non-NMDA receptors at the hypoglossal motor nucleus is related to levels of the prevailing respiratory drive. These results show that both NMDA and non-NMDA glutamate receptors at the hypoglossal motor nucleus are involved in transmission of the respiratory drive signal to genioglossus muscle in vivo.     

N-methyl-D-aspartate triggers neonatal rat hypoglossal motoneurons in vitro to express rhythmic bursting with unusual Mg2+ sensitivity

The brainstem nucleus hypoglossus innervates the tongue which must contract rhythmically during respiration, chewing and swallowing. Such rhythmic discharges are due to network bursting mediated by AMPA receptor-dependent glutamatergic transmission. The contribution by hypoglossal motoneurons themselves to rhythmicity remains, however, unclear as they might simply express cyclic patterns produced by premotoneurons or, in analogy to spinal motoneurons, might participate to bursting due to activation of their N-methyl-D-aspartate (NMDA) receptors. Using patch clamp recording from hypoglossal motoneurons in slice preparations of neonatal rat brainstem, we observed that NMDA directly depolarized motoneurons to generate various discharge patterns. Most motoneurons produced transient bursts which were consistently restored by repolarizing membrane potential to rest. Fewer motoneurons generated either sustained bursting or random firing. Rhythmic bursts were recorded from XII nerve rootlets even when single motoneuron bursting required hyperpolarization. NMDA evoked bursts were blocked by the Ca2+ antagonist Cd2+, the gap junction blocker carbenoxolone, or Mg2+ free solution, and partially inhibited by tetrodotoxin or nifedipine. Under voltage clamp, NMDA-induced bursting persisted at negative or positive potentials and was resistant to high extracellular Mg2+ in accordance with the observation of widespread motoneuron expression of NMDA 2D receptor subunits that confer poor Mg2+ sensitivity. It is proposed that NMDA depolarized motoneurons with the contribution of Mg2+ insensitive channels, and triggered bursting via cyclic activation/deactivation of voltage-dependent Na+, Ca2+ and K+ currents spread through gap junctions. The NMDA-evoked bursting pattern was similar to the rhythmic discharges previously recorded from the XII nerve during milk sucking by neonatal rats.     

Opiate agonists activate feeding in Limax: comparison of in vivo and in vitro effects.

The neural control system for feeding in the terrestrial mollusc Limax maximus is modulated by at least two major families of peptides. Sequence homology between one of the peptides known to modulate Limax feeding and some members of the opioid peptide family suggested that opioid peptides might also modulate Limax feeding. Experiments with the mu agonist morphine and the kappa agonist U50,488H showed that the probability of feeding, but not meal size, was increased by morphine injection into intact animals, whereas the length of feeding motor program responses elicited from the isolated lip-brain preparation of Limax was augmented by U50,488H. The behavioral effect of morphine was blocked by naltrexone injection, whereas the physiological effect of U50,488H was blocked by naloxone. Factors that influence the behavioral and electrophysiological effects of opioids on mollusc feeding are discussed.     

Bioactivity of new mu and delta opioid peptides

Endogenous opioids have been studied extensively since their discovery, in the hope of findings a perfect analgesic, devoid of the secondary effects of alkaloid opioids. However, the design of selective opioid agonists and or antagonists has proved very difficult. First, structural studies of peptides in general are hampered by their intrinsic flexibility. Second, the relationship between constitution and the so called "bioactive conformations" is far from obvious. Ideally, a direct structural study of the complex between a peptide and its receptor should answer both questions, but such a study is not possible, because opioids receptors are large membrane proteins, difficult to study by standard structural techniques. Thus, conformational studies of opioid peptides are still important for drug design and also for indirect receptor mapping. This review deals the pharmacological activity of : a) a new mu and deltaagonist: The single amino acid replacement of 2',6'-dimethyl-L-tyrosine in deltorphin B (H-Dmt-D-Ala-Phe-Glu-Val-Val-Gly-NH2) yielded high affinity for mu- and delta-binding sites. [Dmt1]Deltorphin B lacks activity at kappa-opioid binding sites. Bioactivity in vitro with guinea-pig ileum confirmed that [Dmt1]deltorphin B interacted with mu-opioid receptors by reducing electrically induced contractions in a naloxone-reversible manner and was 150-fold more potent than morphine and comparable to [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAGO). The inhibition of spontaneous contractions of rabbit jejunum provided evidence for delta-opioid receptor interaction. Analgesia (hot plate and tail flick tests) revealed that [Dmt1]deltorphin B was 180- to 200-fold more potent than morphine. Pretreatment with naloxone, naltrindole or H-Dmt-Tic-Ala-OH (a highly selective delta-opioid receptor antagonist) prevented [Dmt1]deltorphin B antinociception. Thus, [Dmt1]deltorphin B exhibited remarkably high dual affinity and bioactivity toward delta- and mu-opioid receptors. b) two new delta opioid peptide receptor antagonists (Dmt-Tic-OH (DTOH) and Dmt-Tic-Ala-OH (DTAOH): Dmt-Tic-OH (DTOH) and Dmt-Tic-Ala-OH (DTAOH), effective antagonists in vitro, represent a new potent opioid dipeptides for the delta-opioid receptor (Ki delta of 0.022 nM and a selectivity, Ki mu/Ki delta, of 150,000 for DTOH; Ki delta of 0.285 nM and a selectivity Ki mu/Ki delta, of 20,4 for DTAOH). In the present study we considered the pharmacological activity of these two new delta opioid peptide receptor antagonists in vivo. Therefore, we have evaluated their possible antagonistic activity against the antinociception induced by the highly selective delta opioid receptor agonist, [D-Ala2]deltorphin II (DEL). Furthermore, these two delta opioid peptide receptor antagonists were injected centrally or peripherally in order to assess their ability to act also after systemic administration. Concurrent i.c.v. injection of DTOH or DTAOH (0.5-1.0-2.0 nM) with DEL (5 nmol) induced a significant reduction of DEL antinociception. By contrast, while DTOH (10-20-40 mg/kg) administered peripherally (i.p., s.c. or i.v.) was also able to reduce DEL antinociception, DTAOH failed. The present results indicate that DTOH is the first opioid dipeptide with delta antagonist activity after systemic administration and it could be important in the clinical and therapeutic applications. c) a new mu selective opioid dipeptide antagonists: the potent delta selective opioid antagonist dipeptides were designed on the basis of a simple conformational analysis. Following a similar procedure we found a mu selective dipeptide antagonist, 2,6-dimethyl-Tyr-D-Phe-NH2. Although its selectivity is not as high as those of the quoted delta selective dipeptides it has good in vitro activity and looks very promising for further development since the 2,6-dimethyl-Tyr-D-Phe message, like the delta selective 2,6-dimethyl-Tyr-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid counterpart, seems able to impart antagonism to longer peptides.     

Modulation of humoral immune responses by endogenous opioids

The effects of opioid agonists and antagonists were investigated on humoral immune mechanisms in mice and rats. Opioid agonists like morphine, Leu-enkephalin, and Met-enkephalin, enhanced antigen-induced histamine release from mixed peritoneal cells of rats in vitro; this enhancement was effectively antagonized by naloxone, an opioid antagonist. Naloxone, per se, decreased anaphylactic mortality in doses of 10 mg/kg, while it increased mortality in a dose of 1 mg/kg. Reduced IgE antibody titer, measured by passive cutaneous anaphylaxis, decreased hemagglutination titer to sheep red blood cells, blocked histamine release from mixed peritoneal cells of rats in vitro induced by antigen, but had no significant effect when histamine release was induced by compound 48/80. Thus, it appears that endogenous opioids are involved in humoral immune responses.     

Endogenous opioid regulation of norepinephrine release in guinea pig hippocampus.

Release of endogenous norepinephrine was detected in guinea pig hippocampal slices using a radioligand displacement assay. Focal electrical stimulation released endogenous norepinephrine and caused a calcium-dependent reduction in specific [3H]propranolol binding at beta-adrenergic receptors in the brain slice. The mu-opioid agonist PL017 decreased norepinephrine release, and the inhibition by PL017 could be blocked by the opioid antagonist naloxone. Endogenous opioid peptides concomitantly released by tissue stimulation also decreased norepinephrine release in a naloxone-sensitive manner. These results support the hypothesis that endogenous opioids can regulate excitability in the hippocampus by presynaptic modulation of norepinephrine release.     

Mu opioid receptors in the ventrolateral periaqueductal gray mediate stress-induced analgesia but not immobility in rat pups

Rat pups become immobile and analgesic when exposed to an adult male rat. The aim of this study was to determine whether these reactions are under the control of endogenous opioids and to determine the role of the midbrain periaqueductal gray (PAG), which mediates stress-induced immobility and analgesia in adult animals. In Experiment 1, 14-day-old rats were injected systemically with the general opioid receptor antagonist naltrexone (1 mg/kg), which blocked male-induced analgesia to thermal stimulation but did not affect immobility. In Experiment 2, the selective mu opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP; 50 or 100 ng/200 nl) was microinjected into the ventrolateral and lateral PAG. CTOP suppressed male-induced analgesia when injected into the ventrolateral PAG. Male-induced immobility was not affected by CTOP. Male proximity therefore seems to induce analgesia in rat pups by releasing endogenous opioids that bind to mu opioid receptors in the ventrolateral PAG.     

Serotonergic excitatory drive to hypoglossal motoneurons in the decerebrate cat.

In decerebrate, paralyzed, vagotomized and artificially ventilated cats, serotonin (5-HT) and its analogues, microinjected into the hypoglossal (XII) motor nucleus, altered the activity of the genioglossal branch of XII nerve. 5-HT, carboxamidotryptamine maleate (5-CT) and DOI (1-5 mM) increased the activity by over 200%. Methysergide reversed this increase. Methysergide, mianserin, or ketanserin (100-250 nl, 1 mM) reduced the spontaneous hypoglossal activity by 20-50%. Buspirone, 8-OH-DPAT and (-)-propranolol were without effect. Thus, 5-HT provides a substantial tonic excitatory drive to XII motoneurons. The 5-HT receptors involved are likely to be type 1C or 2, but uncertainty regarding the affinity profiles of the drugs used in in vivo conditions in the cat precludes a definite identification.     

Stimulation of endogenous opioid release displaces mu receptor binding in rat hippocampus

Physiological release of endogenous opioids in the rat hippocampus was detected by an in vitro radioligand displacement assay using [3H][D-Ala2,N-methyl-Phe4,glyol5]enkephalin ([3H]DAGO), a mu selective opioid agonist. In this assay, radioligand binding to opioid receptors in the in vitro hippocampal slice was reduced by competition with endogenous opioids released following tissue depolarization. Veratridine-induced opioid release caused displacement of [3H]DAGO that could be blocked by either tetrodotoxin addition or calcium removal from the incubation buffer. Maximal displacement of [3H]DAGO also required the presence of peptidase inhibitors in the incubation buffer. None of the buffer composition changes directly affected [3H]DAGO binding to rat brain membranes. Calcium-dependent displacement of [3H]DAGO binding from mu receptor sites elicited by focal electrical stimulation depended on the intensity and frequency of stimulation and positioning of the electrode in the slice. Maximal displacement of [3H]DAGO binding was observed following high intensity (150-300 microA), high frequency (10-50 Hz) stimulation of the perforant path, a major afferent fiber system to the hippocampus previously shown to contain proenkephalin-derived opioids. Low frequency stimulation (0.1-1 Hz) was ineffective. Stimulation of the mossy fibers (containing both dynorphins and enkephalins) also significantly reduced mu receptor binding, but to a lesser extent. Electrical stimulation of the hippocampal slice at sites not containing opioid peptides did not cause mu receptor displacement. These results demonstrate that under physiological conditions, the release of endogenous opioids from the major opioid containing pathways can be detected in a single hippocampal slice following high frequency stimulation.     

Endogenous opioids: do they modulate the rat pup's response to social isolation?

Previous studies with several different species have suggested that opioids and their receptors are involved in the mediation of the infant's vocal response to social isolation. In the case of the rat pup, 2 models have been hypothesized to relate opioids and the ultrasonic call emitted during social isolation. One model views the comforting effects of social contact as opioid mediated and the apparent distress of social isolation as analogous to opiate withdrawal. The 2nd model considers social separation as a stressor that recruits endogenous opioids. This article describes 3 experiments that tested both of these models in 7-10-day-old rat pups. In Experiment 1, morphine (0.04-0.40 mg/kg) decreased the rate of isolation calls in a dose-dependent, naloxone-reversible fashion. However, the decrease in calling rate was observed only at doses that decreased locomotor activity. Administration of the reversible opiate antagonist naloxone (0.05-5.0 mg/kg) did not alter the rate of calls during either 2- or 6-min isolation tests at either 24 or 32 degrees C. In Experiment 2, the irreversible mu opioid receptor antagonist beta-funaltrexamine (beta-FNA) was administered into the lateral ventricle of 6-day-old pups. Again, no change in the rate of isolation calls was found, although sensitivity to morphine was markedly decreased, and mu (but not delta or kappa) receptors were decreased in selected brain regions by about 40%. In Experiment 3, in vivo receptor binding was used to directly investigate the availability of mu opioid receptors during social contact and social isolation. Pups injected with 3H-diprenorphine showed relatively high levels of specific in vivo binding that followed the regional pattern of in vitro binding, but no effects of social isolation were apparent in the 5 brain regions assayed. Taken together, the consistent negative results with opiate receptor antagonists, as well as the inability to detect an alteration of in vivo binding, suggest that the mu opioid receptor is not an essential part of the rat pup's vocal response to social separation.