Unique behavioral effects of beta endorphin and their relationship to thermoregulation and hypothalamic function.

Intraventricular (ivt) injections of sub-cataleptic doses of β endorphin in rats were observed to result in wet-dog shakes. Subsequent to the wet-dog shakes, copious salivation accompanied by a clonic, seizure-like state was occasionally observed to occur. This sialogogic effect of β endorphin was blocked by naloxone and diminished by injections of thyrotropin releasing hormone. None of these behaviors were observed following ivt injection of morphine in equi-antinociceptive doses. Furthermore, hypophysectomy was observed to attenuate or block these behaviors. Both the wet-dog shakes and “sialogogic seizures” were demonstrated to be dependent upon dose as well as ambient temperature. It appears possible that the occurrence of initial wet-dog shakes may result in the elevated body temperature which then precipitates excess salivation. Since both β endorphin induced wet-dog shakes and salivation were shown to be correlated with alterations in temperature, it is possible that these effects of β endorphin indicate a physiological role for that peptide in thermoregulation.     

Brain Indoleamines in Alloxan- and Streptozotocin-Induced Diabetic Rats

Previous work by other authors has shown that alloxan-induced diabetes increases whereas streptozotocin-induced diabetes does not alter nonesterified fatty acid (NEFA) plasma levels. The present study replicates these results and demonstrates that fasted, streptozotocin-induced diabetic animals also have increased NEFA levels. In addition, brain levels of 5-hydroxytryptamine (5-HT) and of its immediate precursor and metabolite were measured. Alloxan- and fasted, streptozotocin-induced diabetic rats showed significant increases in brain indoleamine concentrations, whereas fed, streptozotocin-induced diabetic rats had unchanged levels of the same compounds. Levels of brain indoleamines exhibited a strong positive correlation with wet-dog shakes (an index of 5-HT activity) elicited by hippocampal stimulation. Blockade of wet-dog shakes by 5-HT receptor antagonists strengthens the proposal that this behavior is a good index of central 5-HT activity. The increased content of brain indoleamines in alloxan- and fasted, streptozotocin-induced diabetic rats may be related to the increased NEFA plasma levels seen in the same animals. This hypothesis is supported by the positive correlation demonstrated between NEFA and 5-HT levels. In conclusion, it is suggested that alloxan-induced diabetes may represent a useful model for studying the various behavioral changes known to occur in diabetics.     

Responsiveness of genetically epilepsy-prone rats to intracerebroventricular morphine-induced convulsions

The sensitivity to intracerebroventricular morphine-induced convulsions was determined in members of the severe seizure (GEPR-9) and moderate seizure (GEPR-3) colonies of genetically epilepsy-prone rats as well as in non-epileptic control rats. GEPR-9s were more sensitive to morphine-induced wet-dog shakes, rearing with bilateral forelimb clonus and generalized clonus than controls of GEPR-3s. GEPR-3s were less sensitive to morphine-induced wet-dog shakes and rearing with bilateral forelimb clonus than controls. Both high and extremely low doses of morphine in GEPR-9s elicited tonic extensor convulsions resembling the characteristic sound-induced convulsion of GEPR-9s. The results suggest that opiotergic systems may contribute to the pathophysiology of the seizure-prone condition in GEPR-9s. Further, differences in responsiveness of opiotergic systems in GEPR-3s and GEPR-9s may partially account for differences in seizure severity in the characteristic sound-induced seizures of these two types of GEPRs.     

Icilin evokes a dose- and time-dependent increase in glutamate within the dorsal striatum of rats

Summary. Icilin, the peripheral cold channel agonist, activates TRPM8 and TRPA1, localized on dorsal root ganglia and trigeminal neurons in rats. Icilin precipitates immediate wet-dog shakes in this species, which are antagonized by centrally acting mu and kappa opioid agonists, implicating the central nervous system in the behavioral response. We studied the effect icilin has on glutamate levels in the dorsal striatum, a brain region involved in movement. Icilin (0.25, 0.5 and 0.75 mg/kg, i.p.) elicited a dose- and time-dependent increase in glutamate within the striatum, indicative of icilin’s neurochemical effect in rats.     

Studies of wet-dog shake behavior induced by septohippocampal stimulation in the rat

Stimulation of the septum and the hippocampus were found to elicit a great number of "wet-dog" shakes (WDS). Their occurrence is strongly related to the evocation and to the time course of the afterdischarges elicited by the stimulation. Morphine, apomorphine, diazepam, and antiserotoninergic drugs greatly reduce the incidence of these WDS but do not alter the afterdischarge duration. Based on electroencephalographic and pharmacological data we propose that WDS induced by stimulation of the septohippocampal system may share some common mechanisms with many other models inducing WDS and offer a useful method to study further the neuroanatomical substrate of this behavior.     

β-endorphin: Dose-dependent suppression of fixed-ratio operant behavior

Centrally administered β-endorphin or morphine suppressed fixed-ratio 15, food-reinforced responding by rats in a dose- dependent manner. β-Endorphin was 21 times more potent than morphine on a molar basis. Scratching and wet-dog shakes were observed within 30 minutes of β-endorphin administration but were not seen after morphine and did not appear to be responsible for the suppression of the conditioned behavior.     

In vivo and in vitro studies with agents that cause quasi-morphine withdrawal syndromes

Compounds that elicit a "quasi-morphine withdrawal syndrome" (QMWS) after acute administration to rats constitute a new class of behavior-modifying agent. In studying the pharmacological bases of quasi-opiate withdrawal syndromes, we found that the dihydrocodeinone, RX 336-M, a standard QMWS-inducing agent, caused a naloxone-insensitive increase in twitch tension on the field-stimulated rat was deferens preparation. In contrast, IBMX (3-isobutyl-1-methylxanthine), the historical inducer of quasi-abstinence in rats, gave a naloxone-insensitive decrease in twitch tension. Tolerance developed to the "wet-dog" shakes elicited by twice-daily s.c. injections of RX 336-M or IBMX in rats for 5 or 15 days, respectively. The parallel development of tolerance to the in vitro effects of these compounds could not be demonstrated.     

Kainic acid-induced seizures: changes in brain extracellular ions as assessed by intracranial microdialysis

The effect of kainic acid on extracellular [K+], [Ca2+], and [Na+] in the rat piriform cortex and hippocampus was studied by means of intracranial microdialysis. Either a dialysis fiber loop or horizontal Vita fiber were stereotaxically implanted within the piriform cortex or hippocampus, respectively. About 24 h later, fibers were perfused (1 ml/min) with Krebs-Ringer bicarbonate solution. Effluent samples were collected before (four at 30 min intervals), and after (six at 30 min intervals) administration of kainic acid (16 mg/kg, i.p.) or kainic acid vehicle. Kainic acid induced sequential signs of lethargy, staring, "wet-dog shakes," forepaw clonus, and tonic-clonic convulsions. In these awake free-moving rats, kainic acid induced a rapid and prolonged increase in extracellular [K+] and an apparent, but not statistically significant, decrease in extracellular [Ca2+] within the hippocampus. In the piriform cortex, kainic acid induced increases in extracellular [K+] and [Na+], which were associated with early pre-convulsive signs. In contrast to the pronounced ion changes commonly seen when the brain is activated by factors such as local application of excitatory substances or when the brain is made ischemic or hypoxic, extracellular ion concentrations are relatively well maintained during parenteral kainic acid-induced seizures.     

A quantitative analysis of the shaking behavior induced in rats by β-endorphin and [D-Ala2, Met5] enkephalinamide

β-Endorphin (5–80 μg) or [D-Ala², Met⁵] enkephalinamide (DALA) (5–40 μg) was administered intracerebroventricularly to rats. With both opioid peptides, there was no direct relationship between log dose and mean number of wet-dog shakes (WDS) that occured during the following 15 min. When the results were analyzed quantally, the dose of DALA that caused 50% of the rats to shake at least twice was 8.6 μg (4.9–15 μg). β-Endorphin had such poor efficacy that an ED 50 could not be obtained. Morphine (1 and 5 mg/kg, s.c.) antagonized shaking caused by the optimal dose of DALA (20 μg). Naloxone (0.1–10 mg/kg, s.c.) attenuated both DALA- and β-endorphin-induced WDS in a dose-related manner. This latter result differentiates shaking associated with opioid peptides from that caused by thyrotropin releasing hormone (TRH), another endogenous stimulant of WDS in rats. There was no cross-tolerance between RX 336-M (7,8-dihydro-5′,6′-dimethylylohex-5′-eno-1′,2′,8′,14 codeinone), a novel shake inducing agent, and β-endorphin. This finding again differentiates β-endorphin-induced shaking from that caused by TRH and also from that associated with several exogenous stimulants of WDS.     

Studies of kainate-induced wet-dog shakes in the rat

Four experiments were undertaken to investigate the specificity of kainate-induced wet-dog shakes (WDS) to glutamate and morphine sensitive brain systems. It was shown that intraventricular (icv) administration was much more effective than intracisternal (ic) administration of kainate in producing WDS. This difference was shown not to be due to etherization of the animals during intracisternal injections. Also, like endorphin-induced WDS, kainate-induced WDS were blocked by naloxone. Kainate-induced WDS were substantially reduced by the glutamate receptor blocking agent GDEE. In a study of the relationship between morphine and kainate, behaviors induced by a low dose of morphine (such as grooming and darting movements) were blocked by subsequent administration of kainate. However, kainate-induced WDS were not affected by the morphine treatment.     

Amygdaloid kindling in alloxan-diabetic rats

Wistar rats, made diabetic by intravenous administration of alloxan, 40 mg/kg, were submitted to amygdala kindling. The EEG and behavioral responses elicited by stimulating the amygdala nuclei in these animals were compared with those observed in control rats. Alloxan-treated rats required more stimulation to kindle, had increased duration of afterdischarges (AD), presented intense interictal spiking, and exhibited greater number of wet-dog shakes than controls. Although the AD threshold was not different between control and experimental rats, the above results seem to indicate an increase in the local epileptic susceptibility represented by longer ADs. On the other hand, this increased local discharge seems to be unable to access the generalization mechanism, which can be verified by the increased kindling rate. Hyperosmolarity, pH alterations, or other generalized metabolic changes frequently associated with diabetes could be implicated in these results.     

Pharmacological profile of a model for central serotonin receptor activation

The head shake response in rats after systemic administration of the serotonin (5HT) precursor 5-hydroxytryptophan (5HTP) was pharmacologically characterized and shown to be a useful animal model to quantify brain 5HT receptor activation. The behavior occurred in a dose-dependent manner after injection of 5HTP and the 5HT agonist quipazine. Head shakes were also observed after injection of L-tryptophan, 5-methoxydimethyltryptamine and fenfluramine. The 5HT antagonists cyproheptadine and metergoline were potent blockers of the response. Xylamidine, a peripheral 5HT antagonist, had no effect on head shaking. Inhibition of 5HT uptake with fluoxetine potentiated the head shake response after 5HTP. Manipulation of central cholinergic or GABAergic mechanisms did not alter 5HTP-induced shakes. Alpha-noradrenergic receptor blockade had no significant effect on head shakes. However, desmethylimipramine was equipotent with methysergide as an antagonist of the behavior. Beta-noradrenergic receptor blockade had no specific effect on 5HTP head shakes. Concomitant dopamine receptor activation with SK&;F 38393 did not affect head shakes but the neuroleptics chlorpromazine and pimozide reduced the number of head shakes after 5HTP. The H₁ receptor antagonist pyrilamine had no effect on head shakes. It is concluded that 5HTP-induced head shakes in rats is a quantitative model of brain 5HT receptor activation which is particularly sensitive to 5HT antagonists.     

6β-N-heterocyclic substituted naltrexamine derivative NAP as a potential lead to develop peripheral mu opioid receptor selective antagonists

A 6β-N-heterocyclic substituted naltrexamine derivative, NAP, was proposed as a peripheral mu opioid receptor (MOR) selective antagonist based on the in vitro and in vivo pharmacological and pharmacokinetic studies. To further validate this notion, several functional assays were carried out to fully characterize this compound. In the charcoal gavage and intestinal motility assay in morphine-pelleted mice, when administered 0.3 mg/kg or higher doses up to 3 mg/kg subcutaneously, NAP significantly increased the intestinal motility compared to the saline treatment. The comparative opioid withdrawal precipitation study and the lower locomotor assay demonstrated that NAP showed only marginal intrinsic effect in the central nervous system either given subcutaneously or intravenously: no jumps were witnessed for the tested animals even given up to a dose of 50 mg/kg, while similar noticeable wet-dog shakes only occurred at the dose 50 times of those for naloxone or naltrexone, and significant reduction of the hyper-locomotion only happened at the dose as high as 32 mg/kg. Collectively, these results suggested that NAP may serve as a novel lead to develop peripheral MOR selective antagonist which might possess therapeutic potential for opioid-induced bowel dysfunction (OBD), such as opioid-induced constipation (OIC).     

Identification of a novel 2-pyridyl-benzensulfonamide derivative,RQ-00203078, as a selective and orally active TRPM8 antagonist

A novel series of 2-pyridyl-benzensulfonamide derivatives have been identified as selective and orally active TRPM8 antagonists via high throughput screening (HTS). Exploration of the structure–activity relationships of compound 1 has led to the identification of RQ-00203078 (compound 36) as a highly selective, potent and orally available TRPM8 antagonist.RQ-00203078 demonstrated excellent in vivo activity in a dose dependent manner with an ED₅₀ value of 0.65 mg/kg in the icilin-induced wet-dog shakes model in rats after oral administration and may become an important pharmacological tool for fully assessing the potential therapeutic use of the targets activated by cold stimulation.     

Hippocampal afterdischarges in rats. I. Effects of antiepileptics

Hippocampal afterdischarges (ADs) are considered to be a model of complex partial seizures. To study the pharmacology of these ADs, stimulation electrodes were implanted into the dorsal hippocampus of 33 male Wistar rats. Stimulation (15-s series of monophasic rectangular pulses with a duration of 1 ms and frequency of 8 Hz) was applied four times with interstimulation intervals of 15 min. Drugs (carbamazepine 50 and 100 mg/kg; clonazepam 0.2 and 0.5 mg/kg; ethosuximide 125 and 250 mg/kg; phenobarbital 40 and 80 mg/kg) as well as solvent and isotonic saline were injected intraperitoneally 2 min after the cessation of the first AD. Duration of AD, of the latent period between AD and recurrent AD and duration of recurrent AD and the number of wet dog shakes were measured. ADs were markedly shortened by both doses of clonazepam and phenobarbital and by the higher dose of carbamazepine. The action of ethosuximide was negligible. Wet dog shakes were influenced in the same way as AD duration. Recurrent ADs were more sensitive to antiepileptics than ADs and wet dog shakes.     

Naloxone-precipitated morphine withdrawal increases pontine glutamate levels in the rat

Extracellular fluid (ECF) levels of glutamate (Glu) and aspartate (Asp) were measured in the locus coeruleus (LC) during morphine withdrawal by using microdialysis in conscious morphine-dependent Sprague-Dawley rats. Guide cannulae were implanted chronically and rats were given intracerebroventricular (i.c.v.) infussions of morphine (26 nmol/1 μl/ht) of saline (1 μl/hr) for 3 days. Microdialysis probes (2 mm tip) were inserted into the LC 24 hr before precipitation of withdrawal by i.c.v. injection of naloxone (12 or 48 nmol/5 μl). Behavioral evidence of withdrawal (teeth-chattering, wet-dog shakes, etc.) was detected following naloxone challenge in morphine, but not in saline-infused rats. Increases (P<0.01) in ECF levels of Glu (and Asp, to a lesser degree) were noted after naloxone-precipitated withdrawal only in the morphine group. The ECF Glu levels in the LC increased from 9.6 ± 2.7 to 15.5 ± 5.0 μM following 12 nmol/5 μl naloxone, and from 9.5 ± 1.9 to 20.5 ± 3.3 μM following 48 nmol/5 μl naloxone, before and in the first 15 min sample after the precipitation of withdrawal in the morphine-dependent rats, respectively. These results provide direct evidence to support the role of excitatory amino acids within the LC in morphine withdrawal.     

L-α-Aminoadipate Inhibits Kynurenate Synthesis in Rat Brain Hippocampus and Tissue Culture

Intracerebral administration of L--aminoadipic acid (L-AAA) at 500 mg/kg body weight to rats caused a complex behavioral change with sporadic wet-dog shakes. Animals developed severe limbic seizures between 1 and 6 h after L-AAA injection, characterized by generalized convulsions. Twenty days after L-AAA injection kynurenine aminotransferase (KAT) activity measured in hippocampal brain tissue slices prepared with a McIlwain chopper at 30 m showed a significant 43% decrease. Subcutaneous injection of kynurenine at 500 mg/kg showed a 63% increase in KAT activity twenty days later. This increase was offset by a concomitant administration of 500 mg/kg L-AAA stereotaxically on day one. In astrocyte culture kynurenic acid synthesis is inhibited by L-AAA and L-pipecolic acid. The possible involvement of kynurenic acid in the modulation of neuronal degeneration is discussed.     

Involvement of histamine H1 and H2 receptors in hypothermia induced by ionizing radiation in guinea pigs

Radiation-induced hypothermia was examined in guinea pigs. Exposure to the head alone or whole-body irradiation induced hypothermia, whereas exposure of the body alone produced a small insignificant response. Systemic injection of disodium cromoglycate (a mast cell stabilizer) and cimetidine (H2-receptor antagonist) had no effect on radiation-induced hypothermia, whereas systemic and central administration of mepyramine (H1-receptor antagonist) or central administration of disodium cromoglycate or cimetidine attenuated it, indicating the involvement of central histamine through both H1 and H2 receptors in this response. Serotonin is not involved, since the serotonin antagonist methysergide had no effect on radiation-induced hypothermia. These results indicate that central histaminergic systems may be involved in radiation-induced hypothermia.     

Binding of [3H]forskolin to human platelet membranes. Regulation by guanyl-5'-yl imidodiphosphate, NaF, and prostaglandins E1 and D2

[3H]Forskolin binds to human platelet membranes in the presence of 5 mM MgCl2 with a Bmax of 125 fmol/mg of protein and a Kd of 20 nM. The Bmax for [3H]forskolin binding is increased to 455 and 425 fmol/mg of protein in the presence of 100 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p) and 10 mM NaF, respectively. The increase in the Bmax for [3H]forskolin in the presence of Gpp(NH)p or NaF is not observed in the absence of MgCl2. The EC50 values for the increase in the number of binding sites for [3H]forskolin by Gpp(NH)p and NaF are 600 nM and 4 mM, respectively. The EC50 value for Gpp(NH)p to increase the number of [3H]forskolin binding sites is reduced to 35 mM and 150 nM in the presence of 50 microM PGE1 or PGD2, respectively. The increase in the number of [3H]forskolin binding sites observed in the presence of NaF is unaffected by prostaglandins. The binding of [3H]forskolin to membranes that are preincubated with Gpp(NH)p for 120 min or assayed in the presence of PGE1 reaches equilibrium within 15 min. In contrast, a slow linear increase in [3H]forskolin binding is observed over a period of 60 min when Gpp(NH)p and [3H]forskolin are added simultaneously to membranes. A slow linear increase in adenylate cyclase activity is also observed as a result of preincubating membranes with Gpp(NH)p. In human platelet membranes, agents that activate adenylate cyclase via the guanine nucleotide stimulatory protein (Ns) increase the number of binding sites for [3H]forskolin in a magnesium-dependent manner. This is consistent with the high affinity binding sites for [3H]forskolin being associated with the formation of an activated complex of the Ns protein and adenylate cyclase. This state of the adenylate cyclase may be representative of that formed by a synergistic combination of hormones and forskolin.