Effects of the opiates on the paraventricular nucleus in genetically polydipsic mice

The inbred mice, STR/N, are known to possess extreme polydipsia with no known abnormality in vasopressin system and the kidney function. Our previous studies indicate that the opiate antagonists given intracerebroventricularly strongly attenuated spontaneous drinking. To determine the site(s) of action, the present study was undertaken. Microinjections of naltrexone methobromide and a selective kappa-receptor antagonist, nor-binaltorphimine (nor-BNI), into the paraventricular nucleus of the hypothalamus (PVN) greatly attenuated drinking of the STR/N for 0.5 to 16 h after injections, while in the two control groups, non-polydipsic STR/1N and Swiss/Webster strains, drinking was not affected by the injections. Food intake was not much altered in all groups. Studies of PVN neurons in vitro (n = > 160 for each group) showed that basal firing rates and patterns were similar in the STR/N and the control groups. Morphine added to the medium inhibited some but excited none in all strains tested. The threshold for the inhibitory action was higher in the polydipsic STR/N mice (10(-8) M), compared to that in the control, S/W mice (10(-9) M). Further, a proportion of neurons inhibited by morphine in the PVN was significantly smaller (P < 0.01) in the STR/N (41.7%), compared to that in the control (64.9%). Dynorphin had very similar effect to that of morphine, but the proportion of cells inhibited was 25.4% in the STR/N, and 70.4% in the S/W. Prior applications of naloxone to the medium prevented the action of both morphine and dynorphin. Under the synaptic blockade (in a low Ca2+ and high Mg2+ medium) the inhibitory effect of the opiates persisted. We concluded that the PVN is at least one of the possible sites where the opiates are acting to cause the polydipsia in the STR/N mice.     

Coexistence of putative neuroactive substances in lateral olivocochlear neurons of rat and guinea pig

We have used the retrograde axonal transport of Fast Blue, injected intra-cochlearly, to identify in the rat lateral superior olive (LSO) neurons which belong to the lateral olivocochlear system (LOCS). Using immunohistofluorescence technique, we have localized within Fast Blue-labeled neurons immunostainings for enkephalins (Met-enkephalin, Met-enkephalin-Arg⁶-Gly⁷-Leu⁸), dynorphins ( -neo-endorphin, dynorphin 1-17) or choline acetyltransferase (ChAT). Many Fast Blue-labeled neurons did not show any immunostaining, but all the immunostained neurons found in the LSO were Fast Blue-labeled. In immunohistofluorescence colocalization experiments of two antigens, we could colocalize within the same neurons of the rat LSO immunostainings for ChAT and enkephalins and for ChAT and dynorphins. In each case, neurons only immunostained for ChAT, enkephalins or dynorphins could also be observed. A colocalization of the immunostainings for Met-enkephalin and dynorphins within neurons of the guinea pig and rat LSO was also found. However, in this case, neurons which did not show colocalization were only Met-enkephalin-immunoreactive, thus suggesting that all the dynorphins immunoreactive LSO neurons also contain enkephalins. These findings support the idea that the neurons of the LSO which contain ChAT-, enkephalin- or dynorphin-immunostainings project to the cochlea and belong to the LOCS. It can also be concluded that acetylcholine, enkephalins and dynorphins coexist within a same population of neurons of the LOCS, although other patterns of co-containment of neuroactive substances within LOCS neurons may also exist.     

In vivo microdialysis study on changes in septal dynorphin and β-endorphin activities in active and hibernating Columbian ground squirrels

State-dependent changes in extracellular concentration of endogenous opioids in the septum of Columbian ground squirrels were examined in the hibernating and euthermic states using in vivo microdialysis. The order of estimated extracellular concentration was found to be: hibernating > interbout euthermia > non-hibernating euthermia for dynorphin A and interbout euthermia > hibernating > non-hibernating euthermia for β-endorphin. The apparent turnover rates of dynorphin A during hibernation was 15 times greater than that during euthermic non-hibernation phase and that of β-endorphin was 8-fold greater. These results demonstrate that subfamilies of endogenous opioids may vary differentially in their activities at different stages of an annual hibernation cycle and may reflect their different roles in the regulation of hibernation.     

Kappa-Opioid and NMDA glutamate receptors are differentially targeted within rat medial prefrontal cortex

Activation of kappa-opioid receptors (KOR) in the medial prefrontal cortex (mPFC) modulates excitatory transmission, which may involve interactions with N-methyl-D-aspartate (NMDA) glutamate receptors. We investigated possible anatomical correlates of this modulation by using dual labeling electron microscopy to examine the cellular distributions of antibodies raised against KOR and the R1 subunit of the NMDA receptor (NR1). KOR immunoreactivity primarily was localized to plasma and vesicular membranes of axons and axon terminals that were morphologically heterogeneous. A small proportion of KOR immunoreactivity was associated with cytosolic compartments of dendrites and membranes of glial processes. NR1 labeling was mainly postsynaptic, associated most often with membranes of cytoplasmic organelles in cell bodies and large dendrites and plasmalemmal surfaces of distal dendrites. The remaining NR1-labeled profiles were axonal profiles and glial processes. Of all cellular associations between labeled profiles, the majority were KOR-labeled axons that contacted NR1-immunoreactive dendrites or cell bodies. Occasionally the two antigens were colocalized in axon terminals that formed either asymmetric synapses or displayed varicose morphology. KOR and NR1 also were colocalized within dendrites, and rarely were observed in the same cell bodies. Occasionally glial processes coursing adjacent to axo-spinous appositions expressed both KOR and NR1 immunoreactivity. These results indicate that ligand activation of KOR or NMDA receptors differentially modulates excitatory transmission in the mPFC through pre- and postsynaptic mechanisms, respectively. The data also suggest more minor roles for colocalized KOR and NMDA receptors in shared regulation of presynaptic transmitter release, postsynaptic responsivity, and glial function.     

Characterization of Opioid Peptides and Opioid Receptors in the Brain of Jerboa (Jaculus orientalis), a Hibernating Rodent

The present study was undertaken to investigate the biochemical characteristics of the opioid receptors and opioid peptides in the jerboa (Jaculus orientalis) brain, a subdesert rodent of Morocco. We have demonstrated the presence of δ, μ, and κ sites in the jerboa brain. The endogenous opioid peptides methionine-enkephalin, β-endorphin, and dynorphin were evaluated in different physiological states of the animal (active and hibernating). The circulating methionine-enkephalin in different states of the animal (active, hibernating, exposure to cold conditions, and fasting) was evaluated in the plasma. Our results indicate that in the hibernating state the opioid receptors level decreased, whereas the concentration of opioid peptides increased. These findings suggest that both opioid receptors and opioid peptides could be involved in the adaptation of the jerboa to survive under thermal stress.     

Intrastriatal injection of a muscarinic receptor agonist and antagonist regulates striatal neuropeptide mRNA expression in normal and amphetamine-treated rats

Systemic administration of the muscarinic receptor antagonist, scopolamine, augments, whereas the muscarinic receptor agonist, oxotremorine, attenuates behaviors (locomotion and stereotypies) and preprodynorphin (PPD) and substance P (SP) gene expression in striatonigral neurons induced by the indirect dopamine receptor agonist, amphetamine (AMPH). In contrast, systemic scopolamine blocks, whereas oxotremorine augments, AMPH-stimulated preproenkephalin (PPE) gene expression in striatopallidal neurons. This study investigated the site of action of these effects by administering scopolamine and oxotremorine directly into the striatum and assessing the expression of neuropeptide mRNAs with quantitative in situ hybridization. Unilateral injection of scopolamine into the dorsal striatum augmented, and oxotremorine attenuated, AMPH (2.5 mg/kg, i.p.)-stimulated behaviors. Intrastriatal scopolamine at a concentration of 62 mM, but not 6.2 mM, increased basal levels of PPD and SP mRNAs in the dorsal striatum. In addition, both 6.2 and 62 mM scopolamine significantly augmented AMPH-stimulated PPD and SP mRNA levels. Intrastriatal infusion of 1.6 or 8.1 mM oxotremorine did not alter basal levels of striatal PPD and SP mRNAs. However, both concentrations of oxotremorine completely blocked AMPH-stimulated SP mRNA and oxotremorine at 8.1 mM blocked AMPH-stimulated PPD mRNA. In contrast, PPE induction by AMPH was blocked by 62, but not 6.2, mM scopolamine. Both concentrations of oxotremorine tended to augment basal and AMPH-stimulated PPE mRNA in the dorsal striatum but the trend was not significant. These data demonstrate an inhibition of striatonigral, and facilitation of striatopallidal, gene expression through activation of local striatal muscarinic receptors, which is consistent with the changes seen after systemic administration of muscarinic agents. Therefore, muscarinic cholinergic regulation of basal and stimulated expression of neuropeptide mRNA is processed within the striatum.     

Gi proteins and calcium in dynorphin-induced hypothermia and behaviour

The intracerebroventricular (i.c.v.) administration of pertussis toxin (0.5 μg) to rats significantly reduced the hypothermic and behavioural effects (episodic bizarre postures characterized by limb rigidity and followed by barrel rolling) induced by i.c.v. dynorphin A (10 μg). These central effects of dynorphin A thus appear to be initiated at a receptor site that interacts with G proteins substrates sensitive to pertussis toxin. Dynorphin A-induced hypothermia was also significantly reduced by i.c.v. pretreatment with the Ca²⁺ antagonist, verapamil (10 μg), although verapamil per se did not modify the behavioural effects elicited by the peptide.     

Effects of dynorphin A(1–13) on opiate withdrawal in humans

The objectives of the current study were to determine 1) the effects of various doses of dynorphin A (1–13) on opiate withdrawal in humans and 2) the safety of dynorphin at these doses. Opiate dependent subjects who had been stabilized on morphine received a single IV dose of placebo, 150, 500 or 1000μg/kg dynorphin after exhibiting spontaneous withdrawal using a randomized, double-blinded, between-subjects study design. Observer Withdrawal Scores were lower in the 150 and 1000μg/kg groups as compared to placebo (P<0.05) but no significant differences were observed on the observer-rated Wang or Sickness Scales. Significant decreases were also found for self-reported symptoms of nervousness, runny nose, sneezing, and painful joints in the 500μg/kg group. Significant increases in serum prolactin levels were seen after all dynorphin doses; however, these were not dose-related. Dynorphin A (1–13) was well tolerated and safe, with no changes in physiologic parameters. We conclude that dynorphin A (1–13) has a modest effect in reducing mild opiate withdrawal in humans and is well tolerated at doses up to 1000μg/kg. Received: 24 March 1997/Final version: 22 November 1997     

Endogenous opioid system down-regulation during hibernation in amphibians

In late summer, Northern grass frogs, Rana pipiens, intraspinally administered dynorphin produces a potent, dose-dependent antinociceptive action as measured by the acetic acid test used to evoke a hindlimb wiping response. Surprisingly, in fall, frogs which have entered hibernation, intraspinal dynorphin produces no antinociception action. Intraspinal morphine shows a decreased effect in fall frogs while systemic morphine is equi-effective in summer and fall frogs. Immobilization stress, previously shown to be mediated by endogenous opioid systems in this amphibian, produces a robust increase in nociceptive thresholds in summer frogs while the nociceptive thresholds of fall frogs are unaffected by this procedure. Summer frogs adapted to cold room (4 degrees C) show a significant decrease in nociceptive thresholds compared to cohorts kept at room temperature, and cold-adapted frogs returned to room temperature show a naloxone-attenuated increase in nociceptive threshold. Collectively, these data suggest that endogenous opioid systems in these northern frogs are down-regulated during fall hibernation.     

Studies of peptidergic input to the lateral spinal nucleus

Lesions were made to interrupt potential sources of peptidergic input to the lateral spinal nucleus (LSn) in rats. Rhizotomies and spinal transections, as well as lesions of the lateral funiculus, failed to reduce immunohistochemical staining for substance P, dynorphin₁₋₈, Met-enkephalin, somatostatin and FMRF-amide in the LSn at lumbar levels. Thus, all examined peptidergic afferent input to the LSn appears to originate locally within the spinal cord.