Wet Dog Shakes Research Articles

5-HT 1A receptor agonists modify epileptic seizures in three experimental models in rats

The effects of two serotonergic (5-HT 1A) receptor agonists (8-OH-DPAT; 0.01, 0.1, 0.3, 1 mg/kg, s.c., and Indorenate; 1, 3, 10 mg/kg, i.p.) were evaluated in three type of seizures in male Wistar rats: clonic–tonic convulsions induced by pentylenetetrazol (PTZ, 60 mg/kg, i.p.), status epilepticus (SE) of limbic seizures produced by kainic acid (KA, 10 mg/kg, i.p.) and tonic–clonic seizures by amygdala kindling. 8-OH-DPAT decreased the incidence of tonic seizures and the mortality rate induced by PTZ. Indorenate increased the latency to the PTZ-induced seizures and decreased the percentage of rats showing tonic extension and death. Concerning KA, 8-OH-DPAT augmented the latency and reduced the frequency of wet-dog shake (WDS) and generalized seizure (GS). At high doses it diminished the occurrence and delayed the establishment of SE. Indorenate augmented the latency to WDS, GS and SE, and diminished the number of GS. 8-OH-DPAT and Indorenate did not alter the expression of kindled seizures. However, Indorenate enhanced the refractoriness to subsequent seizures during the postictal depression. Some effects induced by 8-OH-DPAT and Indorenate on seizures evaluated and postictal depression were fully or partially blocked by WAY100635. These results suggest that 5-HT 1A receptor agonists modify epileptic activity depending on the type of seizure.

Differential GABAA Subunit Expression Following Status Epilepticus in Seizure‐prone and Seizure‐resistant Rats: A Putative Mechanism for Refractory Drug Response

Two rat strains were selectively bred to be prone (Fast) or resistant (Slow) to amygdala kindling. The first objective of this experiment was to determine whether that selection was specific to kindling or was sensitive more broadly to another seizure induction agent, kainic acid (KA). Second, we investigated whether these strains exhibit distinct molecular responses to KA with respect to GABA(A) receptor subunit expression. Development of status epilepticus (SE) was profiled in Fast and Slow rats injected with 20 mg/kg KA (i.p.). Two hours post-SE onset, rats received a sedative dose of sodium pentobarbital. Behavioral profiles included latency to SE, number of wet dog shakes (WDS), and number and duration of stage 3-5 generalized seizures. Rats were killed 24 h post-SE, and alpha(1) and alpha(4) mRNA levels were compared in the hippocampus and amygdala using QPCR. Slow rats exhibited a much greater latency to SE onset (p < 0.01) and many more WDS (p < 0.01) than Fast rats. During SE, Fast rats spent more time in and exhibited more repeated bouts of generalized stage 3-5 seizures (p < 0.01) than Slow rats. Constitutive levels of alpha1 and alpha4 were not different between the strains in either structure and equivalent reductions in alpha4 were evident 24 h post-SE. However, while Fast rats showed KA-induced reductions in alpha1 in both structures, Slow rats showed significant elevations. Genetic selection for temporal lobe excitability, manifested as differential amygdala kindling rates, is paralleled by vulnerability to KA-induced SE. Further, these strains exhibited at least one opposing molecular response to SE, namely alpha1 expression. This finding may offer a putative mechanism through which seemingly similar epilepsies can be intractable in some patients but treatable in others.

Altered responsiveness of serotonin receptor subtypes following long-term cannabinoid treatment

This study examined the effects of long-term cannabinoid administration on the responsivity of 5-HT1A and 5-HT2A receptors, which have been implicated in depression. Animals received 12 d administration of the potent cannabinoid receptor agonist HU-210 (100 microg/kg), following which they were monitored on their behavioural, physiological and hormonal responses to a single challenge of a 5-HT1A and 5-HT2A receptor agonist, 8-OH-DPAT (0.3 mg/kg) and DOI (1 mg/kg) respectively. Chronic HU-210 treatment lead to a significant enhancement of DOI-induced wet-dog shakes, but a reduction of DOI-induced back muscle contractions. DOI-induced corticosterone release was unaffected by HU-210 treatment. The hyperthermic response to DOI appeared to be potentiated by long-term HU-210 treatment, as 50% of these subjects died from an apparent serotonin syndrome with core temperatures exceeding 43 degrees C. The 8-OH-DPAT-induced hypothermic response and elevation of corticosterone were both significantly attenuated by long- term HU-210 treatment. These data imply that chronic cannabinoid treatment may up-regulate 5-HT2A receptor activity while concurrently down-regulating 5-HT1A receptor activity, a finding similar to that sometimes observed in depression. This may partially explain the association between excessive cannabis consumption and the induction of affective disease.

天麻的抗癲癇作用與Interleukin-1β、Tumor Necrosis Factor-α及一氧化氮關係

Purpose Our previous studies have shown that Gastrodia elata (GE) reduces kainic acid (KA)-induced epileptic seizures and inhibits activation of microglia in rates. The present study investigated the relationship between the anticonvulsion effects of GE and interleukin-1β (IL-1β), tumor necrosis factor-α (TNE-α), as well as nitric oxide (NO). Methods Rats were administered KA (12mg/kg) to induce epileptic seizure. After oral administration of GE 1.0g/kg, 0.5g/kg and valproic acid (VA) 250mg/kg for 4 days prior to K injection of the number of KA-induced we dog shakes was calculated, and the changes in IL-1β, TNF-α and NO levels in the peripheral blood, and IL-1β and TNF-α levels in the frontal cortex and hippocampus regions of the rat brain were measured. Results Both pretreatment of GE 1.0g/kg and VA 250mg/kg reduced the number of wet dog shakes. GE also reduced the levels of IL-1β and NO in the peripheral blood, and the levels of IL-1β and TNF-α in the frontal cortex, and TNF-α in the hippocampus. Conclusions GE inhibits the generation of cytokine IL-1β, TNF-α, and NO in rats with KA-induced seizures.

Pituitary adenylate cyclase-activating polypeptide (PACAP) mimics neuroendocrine and behavioral manifestations of stress: Evidence for PKA-mediated expression of the corticotropin-releasing hormone (CRH) gene

The physiologic response to stress is highly dependent on the activation of corticotropin-releasing hormone (CRH) neurons by various neurotransmitters. A particularly rich innervation of hypophysiotropic CRH neurons has been detected by nerve fibers containing the neuropeptide PACAP, a potent activator of the cAMP-protein kinase A (PKA) system. Intracerebroventricular (icv) injections of PACAP also elevate steady-state CRH mRNA levels in the paraventricular nucleus (PVN), but it is not known whether PACAP effects can be associated with acute stress responses. Likewise, in cell culture studies, pharmacologic activation of the PKA system has stimulated CRH gene promoter activity through an identified cAMP response element (CRE); however, a direct link between PACAP and CRH promoter activity has not been established. In our present study, icv injection of 150 or 300 pmol PACAP resulted in robust phosphorylation of the transcription factor CREB in the majority of PVN CRH neurons at 15 to 30 min post-injection and induced nuclear Fos labeling at 90 min. Simultaneously, plasma corticosterone concentrations were elevated in PACAP-injected animals, and significant increases were observed in face washing, body grooming, rearing and wet-dog shakes behaviors. We investigated the effect of PACAP on human CRH promoter activity in αT3-1 cells, a PACAP-receptor expressing cell line. Cells were transiently transfected with a chloramphenicol acetyltransferase (CAT) reporter vector containing region − 663/+124 of the human CRH gene promoter then treated for with PACAP (100 nM) or with the adenylate cyclase activating agent, forskolin (2.5 μM). Both PACAP and forskolin significantly increased wild-type hCRH promoter activity relative to vehicle controls. The PACAP response was abolished in the CRE-mutant construct. Pretreatment of transfected cells with the PKA blocker, H-89, completely prevented both PACAP- and forskolin-induced increases in CRH promoter activity. Furthermore, CREB overexpression strongly enhanced PACAP-mediated stimulation of hCRH promoter activity, an effect which was also lost with mutation of the CRE. Thus, we demonstrate that icv PACAP administration to rats under non-stressed handling conditions leads to cellular, hormonal and behavioral responses recapitulating manifestations of the acute stress response. Both in vivo and in vitro data point to the importance of PACAP-mediated activation of the cAMP/PKA signaling pathway for stimulation of CRH gene transcription, likely via the CRE.

Functional role of the endocannabinoid system and AMPA/kainate receptors in 5-HT 2A receptor-mediated wet dog shakes

These experiments sought to determine the influence of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors and the endocannabinoid system in the functional expression of the serotonin (5-HT) type 2A receptor-mediated wet dog shake response. Male Long–Evans rats were pretreated with either 1 mg/kg i.p. of the 5-HT 2A/2C receptor antagonist ketanserin; 1, 10 or 30 mg/kg i.p. of the AMPA/kainate antagonist 6,7-dinitroquinnoxaline-2,3-dione (DNQX); 1, 5 or 10 mg/kg i.p. of the endocannabinoid uptake inhibitor AM404; or 1, 5 or 10 mg/kg i.p. of the cannabinoid CB 1 receptor antagonist AM 251 prior to injection of the 5-HT 2A/2C receptor agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI, 1 mg/kg i.p.). Results demonstrated that 10 mg/kg of AM404 significantly reduced the expression of DOI-induced wet dog shakes, but lower doses were ineffective. Administration of AM251 did not induce wet dog shakes behavior when administered alone, but significantly potentiated DOI-induced wet dog shaking behavior at a dose of 10 mg/kg. Pretreatment with DNQX significantly reduced the expression of DOI-induced wet dog shakes at all doses tested. These data suggest that AMPA/kainate receptors play a role in the mediation of 5-HT 2A receptor activity, whereas the endocannabinoid system may act as a regulatory buffer system during periods of elevated activity, but not under basal conditions.

Effects of monoamine reuptake inhibitors on wet-dog shakes mediated by 5-HT 2A receptors in ACTH-treated rats

We examined the influence of imipramine, a serotonin (5-HT) and noradrenaline (NA) reuptake inhibitor, desipramine, a NA reuptake inhibitor, bupropion, a dopamine reuptake inhibitor, fluvoxamine, a selective 5-HT reuptake inhibitor, and mazindol, a catecholamine reuptake inhibitor, on a 5-HT 2A receptor-mediated behavior, (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-induced wet-dog shakes, in naive and adrenocorticotropic hormone (ACTH)-treated rats. Chronic administration of imipramine, desipramine and mazindol suppressed the number of wet-dog shakes in naive rats. Chronic ACTH (100 μg/rat, s.c.) treatment increased the number. Chronic administration of imipramine did not decrease the number of wet-dog shakes in ACTH-treated rats. On the other hand, desipramine and mazindol inhibited the increase in wet-dog shakes in ACTH-treated rats. Fluvoxamine and bupropion did not have any effect on the (±)-DOI-induced response in naive and ACTH-treated rats. NA reuptake inhibitors may improve the hyperfunction of 5-HT 2A receptors induced by chronic ACTH treatment.

Gastric pentadecapeptide BPC 157 effective against serotonin syndrome in rats

Serotonin syndrome commonly follows irreversible monoamine oxidase (MAO)-inhibition and subsequent serotonin (5-HT) substrate (in rats with fore paw treading, hind limbs abduction, wet dog shake, hypothermia followed by hyperthermia). A stable gastric pentadecapeptide BPC 157 with very safe profile (inflammatory bowel disease clinical phase II, PL-10, PLD-116, PL-14736, Pliva) reduced the duration of immobility to a greater extent than imipramine, and, given peripherally, has region specific influence on brain 5-HT synthesis (α-[ 14C]methyl- l-tryptophan autoradiographic measurements) in rats, different from any other serotonergic drug. Thereby, we investigate this peptide (10 μg, 10 ng, 10 pg/kg i.p.) in (i) full serotonin syndrome in rat combining pargyline (irreversible MAO-inhibition; 75 mg/kg i.p.) and subsequent l-tryptophan (5-HT precursor; 100 mg/kg i.p.; BPC 157 as a co-treatment), or (ii, iii) using pargyline or l-tryptophan given separately, as a serotonin-substrate with (ii) pargyline (BPC 157 as a 15-min posttreatment) or as a potential serotonin syndrome inductor with (iii) l-tryptophan (BPC 157 as a 15 min-pretreatment). In all experiments, gastric pentadecapeptide BPC 157 contrasts with serotonin-syndrome either (i) presentation (i.e., particularly counteracted) or (ii) initiation (i.e., neither a serotonin substrate (counteraction of pargyline), nor an inductor for serotonin syndrome (no influence on l-tryptophan challenge)). Indicatively, severe serotonin syndrome in pargyline + l-tryptophan rats is considerably inhibited even by lower pentadecapeptide BPC 157 doses regimens (particularly disturbances such as hyperthermia and wet dog shake thought to be related to stimulation of 5-HT 2A receptors), while the highest pentadecapeptide dose counteracts mild disturbances present in pargyline rats (mild hypothermia, feeble hind limbs abduction). Thereby, in severe serotonin syndrome, gastric pentadecapeptide BPC 157 (alone, no behavioral or temperature effect) has a beneficial activity, which is likely, particular, and mostly related to a rather specific counteraction of 5-HT 2A receptors phenomena.

Correlation between shaking behaviors and seizure severity in five animal models of convulsive seizures

Wet dog shakes (WDS) and head shakes (HS) are associated with experimentally induced convulsive seizures. We sought to determine whether these behaviors are correlated or not with major (status epilepticus (SE) or fully kindled animals) or minor (non-SE or partially kindled animals) seizure severity. WDS are directly correlated with SE induced by intracerebral star fruit extract ( Averrhoa carambola) injection and with kindled animals in the amygdala fast kindling model. On the other hand, WDS are inversely correlated with SE induced by intracerebral bicuculline and pilocarpine injections. Systemic pilocarpine in animals pretreated with methyl-scopolamine barely induced WDS or HS. The role of shaking behaviors may vary from ictal to anticonvulsant depending on the experimental seizure model, circuitries involved, and stimulus intensity. The physical presence of acrylic helmets may per se inhibit the HS response. Also, methyl-scopolamine, a drug incapable of crossing the blood–brain barrier, can induce HS in animals without acrylic helmets.

Selected Papers: Laboratory Studies

Abstract *†Soichi Obara, *Shin-ichi Imamura, ‡Koichi Akaike, §Shigeya Tanaka, †Ikuro Maruyama, and *Jun-ichi Kuratsu Departments of *Neurosurgery , †Laboratory and Molecular Medicine and ‡Neuropsychiatry, University of Kagoshima, Faculty of Medicine , and §Tanaka Neurosurgical Clinic, Kagoshima, Japan . Purpose: Prior studies indicated that 2-arachidonoglycerol (2-AG), an endogenous cannabinoid, might have neurophysiologic effects on the hippocampus, possibly by activating cannabinoid receptor-1 (CB1). This study was designed to clarify the effect of 2-AG on hippocampal neuronal damages caused by kainic acid (KA)-induced limbic seizures. Methods: Twenty-four rats underwent stereotactic implantation of electrodes in the left amygdala (LA), left hippocampus (LH), and left sensorimotor cortex (LCx). A stainless steel cannula also was introduced into the LA and LH. The animals then were divided into four groups according to the pretreatment agent infused into LH: sham control [dimethyl sulfoxide (DMSO); 1 μl, n = 6], KA control (DMSO; 1 μl, n = 6), group A (2-AG; 100 μM, n = 6), and group B [2-AG (100 μM) + SR-141716A (a CB-1 antagonist; 10 μM), n = 6]. After 10 min, all rats except the sham-treated group received 1 μg of kainic acid (KA) via the cannula. Sham-treated rats received phosphate-buffered saline (PBS) instead. After electroclinical observation for 7 days, histologic examination and statistical analysis were performed. Results: In KA controls, immediate multiple spike discharges in LA propagated concurrently to the LH. The propagation involved the LCx, with development of status epileptics 1–2 h after KA injection. Seizures were characterized by mastication, salivation, facial twitching, forelimb clonus, and sometimes rearing and falling, and lasted 1–2 days. Microscopic examination revealed severe neuronal cell damage in the LA and LH. In group A, seizure discharges were eliminated overall and neuronal cell damage in LH was reduced compared with the KA controls. Rats in group A only showed behavioral changes such as wet-dog shake. In group B, seizure discharges and neuronal cell damage in LH were virtually the same as in controls. Sham-treated rats showed no electroclinical and histologic changes. Conclusions: These results indicate that the 2-AG exhibits a neuroprotective effect against limbic seizures by activating CB1.

Behavioral effects of local microinfusion of pituitary adenylate cyclase activating polypeptide (PACAP) into the paraventricular nucleus of the hypothalamus (PVN)

Pituitary adenylate cyclase activating polypeptide (PACAP) has been implicated in the regulation of several autonomic and neuroendocrine functions. In the hypothalamic paraventricular nucleus (PVN), for example, PACAP-immunoreactive fibers densely innervate corticotropin-releasing hormone (CRH)-containing neurons in the medial parvocellular region, suggesting that PACAP acts to mediate stress responses. Therefore, we examined the behavioral effects of an intra-PVN PACAP injection (25 pmol) in combination with a mild stressor. PACAP or artificial cerebrospinal fluid (aCSF) was microinjected into the PVN (0.25l) and then animals were restrained or placed in their home cage for 5 min. Exploratory activity (total distance traveled) and scored behaviors (face washing, body grooming, wet dog shakes, and rearing) were observed in a familiar open field for 10 min. In animals receiving aCSF, there were no behavioral differences between restrained and unrestrained groups. For the entire 10-min observation period, animals receiving PACAP, whether restrained or not, displayed elevated face washing and body grooming with decreased locomotor activity and rearing. Among PACAP-injected animals, restrained animals displayed increased body grooming compared to unrestrained animals during the first 2 min in the open field suggesting a summation of the effects of peptide injection and stressor. The observed elevation in grooming is consistent with previous studies reporting similar increases following electrical-, NMDA-, CRH-, or stressor-induced activation of the PVN. Thus, at the level of the PVN, PACAP may act as an excitatory neuropeptide and augment behavioral responses to stressors.

Hippocampal Afterdischarges after GABAB‐Receptor Blockade in the Freely Moving Rat

To determine whether hippocampal afterdischarges (ADs) and excitability changes were induced by gamma-aminobutyric acid (GABA)(B)-receptor blockade in adult, freely moving rats. A specific GABA(B)-receptor antagonist CGP35348, CGP55845A, or CGP55699A was injected intracerebroventricularly (i.c.v.), and EEGs and behaviors of rats were analyzed. CGP35348 (56-110 microg, i.c.v.) induced afterdischarges (ADs) approximately 60% of the time, starting at the hippocampus or neocortex. Neocortical-onset ADs began with sporadic discharges and were <3 mV. Hippocampal-onset ADs were bilateral, >5 mV, and spread to the entorhinal cortex and amygdala, often ending in a rebound AD and accompanied with stereotypic jumping, forelimb clonus, and wet-dog shakes. The CGP35348-induced hippocampal AD had an onset frequency (5-9 Hz) that was higher than the electrically evoked AD (2-4 Hz). CGP35348 i.c.v. also increased the mean starting frequency of an electrically evoked hippocampal AD from 3.6 Hz to 5.3 Hz. Hippocampal gamma activity (25-80 Hz) increased up to twofold for 30 min after a hippocampal but not a neocortical AD. A single dose of CGP35348 induced repeated ADs of increasing duration. Paired-pulse inhibition of the evoked potentials in CA1, at interpulse interval of <100 ms, was decreased after but not before a hippocampal AD. CGP56999A (i.c.v.) gave results similar to those with CGP35348, whereas CGP55845A (i.c.v.) rarely induced ADs. GABA(B)-receptor blockade increases seizure susceptibility by reducing AD threshold and increasing the frequency and spread of a hippocampal AD. Hippocampal excitability (based on paired-pulse test) and gamma activity increased after but not before a hippocampal AD.

Effects of the antidepressant trazodone, a 5-HT2A/2C receptor antagonist, on dopamine-dependent behaviors in rats

5-Hydroxytryptamine, via stimulation of 5-HT 2C receptors, exerts a tonic inhibitory influence on dopaminergic neurotransmission, whereas activation of 5-HT 2A receptors enhances stimulated DAergic neurotransmission. The antidepressant trazodone is a 5-HT 2A/2C receptor antagonist. To evaluate the effect of trazodone treatment on behaviors dependent on the functional status of the nigrostriatal DAergic system. The effect of pretreatment with trazodone on dexamphetamine- and apomorphine-induced oral stereotypies, on catalepsy induced by haloperidol and apomorphine (0.05 mg/kg, i.p.), on ergometrine-induced wet dog shake (WDS) behavior and fluoxetine-induced penile erections was studied in rats. We also investigated whether trazodone induces catalepsy in rats. Trazodone at 2.5-20 mg/kg i.p. did not induce catalepsy, and did not antagonize apomorphine (1.5 and 3 mg/kg) stereotypy and apomorphine (0.05 mg/kg)-induced catalepsy. However, pretreatment with 5, 10 and 20 mg/kg i.p. trazodone enhanced dexamphetamine stereotypy, and antagonized haloperidol catalepsy, ergometrine-induced WDS behavior and fluoxetine-induced penile erections. Trazodone at 30, 40 and 50 mg/kg i.p. induced catalepsy and antagonized apomorphine and dexamphetamine stereotypies. Our results indicate that trazodone at 2.5-20 mg/kg does not block pre- and postsynaptic striatal D2 DA receptors, while at 30, 40 and 50 mg/kg it blocks postsynaptic striatal D2 DA receptors. Furthermore, at 5, 10 and 20 mg/kg, trazodone blocks 5-HT 2A and 5-HT 2C receptors. We suggest that trazodone (5, 10 and 20 mg/kg), by blocking the 5-HT 2C receptors, releases the nigrostriatal DAergic neurons from tonic inhibition caused by 5-HT, and thereby potentiates dexamphetamine stereotypy and antagonizes haloperidol catalepsy.

Primary structure, behavioral and electroencephalographic effects of an epileptogenic peptide from the sea anemone Bunodosoma cangicum

The primary structure of cangitoxin (CGX), a 4958 Da peptide from the sea anemone Bunodosoma cangicum, was determined: GVACRCDSDGPTVRGNSLSGTLWLTGGCPSGWHNCRGSGPFIGYCCKK. CGX contains all the 11 residues that are conserved and the 5 that are conservatively substituted within or between the type 1 and type 2 sequences of sea anemone peptides with specific action on voltage-sensitive sodium channels. Furthermore, it also has 6 identities (Asp9, Arg14, Asn16, Leu18, Trp33 and Lys48) and 1 homology (Arg36) in the 8 residues of the pharmacophore of the sea anemone ApB which are essential for interaction with mammalian sodium channels. The intrahippocampal injection of CGX induces several sequential behavioral alterations—episodes of akinesia alternating with facial automatisms and head tremor, salivation, rearing, jumping, barrel-rolling, wet dog shakes and forelimb clonic movements–and the electroencephalography analysis shows that they were followed by important seizure periods that gradually evolved to status epilepticus that lasted 8–12 h, similar to that observed in the acute phase of the pilocarpine model of epilepsy. These results suggest that CGX may be an important tool to develop a new experimental model of status epilepticus which may contribute to understanding the etiology of epilepsy and to test the effects of new antiepileptic drugs.

The mGlu5 receptor antagonists MPEP and MTEP attenuate behavioral signs of morphine withdrawal and morphine-withdrawal-induced activation of locus coeruleus neurons in rats

N-Methyl- d-aspartate (NMDA) antagonists have been demonstrated to suppress the signs of opiate withdrawal; however, side effects limit their clinical use. Since the metabotropic glutamate (mGlu) 5 receptor has been shown to affect glutamate release and modulate NMDA receptor function, we examined the effects of two selective mGlu5 receptor antagonists, 2-methyl-6-(phenyl-ethynyl)-pyridine (MPEP) and 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP), on morphine withdrawal. Pretreatment with MPEP or MTEP (1, 3, and 10 mg/kg, i.p.) significantly attenuated behavioral signs of morphine withdrawal. Specifically, both MPEP and MTEP attenuated the occurrence/severity of chews, digging, salivation, and weight loss, and increased the occurrence of erections. Neither compound changed the occurrence of wet-dog shakes, ptosis, irritability, or lacrimation. Both MPEP and MTEP produced a modest, but significant, attenuation of morphine-withdrawal-induced activation of locus coeruleus neurons in anesthetized rats. These results indicate a role for mGlu5 receptors in morphine withdrawal and suggest the potential for mGlu5 antagonists in the treatment of withdrawal from opiates and other drugs of abuse.

P.5.034 Czech university students and drugs of abuse:Preliminary results of urinalysis

In 1997, endomorphin-1 (EM-1) and -2 (EM-2) were identified as the most specific endogenous μ-opioid ligands. These two peptides have shown analgesic effects and many other opioid functions. In the present study, we attempt to investigate the possible ability of endomorphins to induce naloxone-precipitated withdrawal in comparison with that induced by morphine. Using the previously established scoring system in rats, 12 withdrawal signs (chewing, sniffing, grooming, wet-dog shakes, stretching, yawning, rearing, jumping, teeth grinding, ptosis, diarrhea, and penile erection) were observed and scored following naloxone (4 mg/kg, i.p.) challenge. Compared with the sham control, EM-1 and EM-2 (20 μg, i.c.v., b.i.d. for 5 days) both produced significant naloxone-induced withdrawal syndromes with similar severity to that induced by the same dose of morphine. There was no significant difference between EM-1, EM-2, and morphine-treated group for naloxone-induced withdrawal signs, except for grooming. EM-1 and EM-2 induced more grooming than that caused by morphine. Although EM-1 and EM-2 both led to the withdrawal, they displayed different potency for certain signs and suggest their distinct regulations. The present results indicate EM-1 and EM-2 could initiate certain mechanism involved opiate dependence.

Effects of venlafaxine on ethanol withdrawal syndrome in rats

The present study was designed to investigate the effects of venlafaxine, a serotonin and noradrenaline reuptake inhibitor (SNRI), on ethanol withdrawal syndrome in rats. Adult male Wistar rats (187-319 g) were used for the study. Ethanol (7.2%, v/v) was given to rats by a liquid diet for 21 days. Control rats were pair-fed an isocaloric liquid diet containing sucrose as a caloric substitute to ethanol. Venlafaxine (5, 10, 20 and 40 mg/kg) and saline were injected to rats intraperitoneally just before ethanol withdrawal. After the 2nd, 4th and 6th hour of ethanol withdrawal, rats were observed for 5 min, and withdrawal signs that included locomotor hyperactivity, agitation, stereotyped behaviour and wet dog shakes were recorded or rated. A second series of injections was given at the 6th hour after the first one, and rats were then tested for audiogenic seizures. Venlafaxine produced some inhibitory effects on locomotor hyperactivity, stereotypic behaviours and wet dog shakes. However, a two-way anova of the data did not indicate any significant effect. It reduced the incidence of the audiogenic seizures at the 6th hour of ethanol withdrawal. Venlafaxine (20 mg/kg) also prolonged the latency of the seizures significantly. Our results suggest that acute venlafaxine treatment has limited beneficial effects on ethanol withdrawal syndrome in rats.

Blockade of ionotropic glutamatergic transmission in the ventral tegmental area attenuates the physical signs of morphine withdrawal in rats

The present study sought to assess whether the blockade of ionotropic glutamate receptors in the ventral tegmental area (VTA) could modulate the morphine withdrawal in male Sprague–Dawley rats. The effects of dizocilpine (MK-801) or 6,7-dinitroquinnoxaline-2,3-dione (DNQX), ionotropic glutamate receptor antagonists, microinjected unilaterally into the VTA 30 min before naloxone [2 mg/kg, intraperitoneally (i.p.)] administration on the morphine withdrawal were assessed. Morphine dependence was developed with increasing morphine injection (i.p.), and morphine withdrawal was induced by injection of naloxone (2 mg/kg, i.p.). Jumping, wet-dog shakes, writhing posture, wall clamber, weight loss and Gellert–Holtzman scale were used as the indices to evaluate the intensity of morphine withdrawal. The results showed that unilateral microinjection of MK-801 or DNQX into the VTA significantly increased the incidence of wall clamber, had no effect on weight loss, and reduced all other symptoms of morphine withdrawal. These data suggest that the ionotropic glutamate receptors in the VTA are involved in mediating naloxone-precipitated opiate withdrawal.

Buprenorphine blocks withdrawal in morphine-dependent rat pups.

Infants placed on extracorporeal membrane oxygenation (ECMO) or mechanical ventilation often need continuous morphine infusions for pain relief and sedation. The resulting physical dependence requires an additional 2-3-week hospital stay to taper the morphine to avoid withdrawal. Buprenorphine effectively blocks abstinence in dependent adults, and in infants it could accelerate or eliminate the tapering schedule, thereby enabling earlier hospital dismissals. Morphine-dependent infant rats were used in this study to determine the effectiveness of buprenorphine in blocking abstinence. Postnatal day-14 (P14) rats were implanted with osmotic minipumps that delivered saline (1 microl x h(-1)) or morphine (2 mg x kg(-1) h(-1)) for 72 h. The minipumps were then removed to allow the rats to undergo spontaneous morphine withdrawal. The withdrawal period lasted approximately 72 h out of a 96-h observation period. The following signs were significant during these hours: wet-dog shakes, 1-72 h; abdominal stretches, 1-72 h; forepaw tremors, 1-24 h; splayed hind-limbs, 1-72 h; ptosis, 4-72 h; and evoked vocalization, 4 and 8 h. A single 1 mg x kg(-1) buprenorphine dose significantly decreased wet-dog shakes from 1 to 72 h, abdominal stretches from 1 to 48 h, forepaw tremors and splayed hind-limbs 1-8 h, and ptosis and evoked vocalization at 4 and 8 h. Repeated administration of 1 mg x kg(-1) buprenorphine before pump removal and at 24, 48 and 72 h resulted in a greater magnitude of blockade of abstinence throughout the 96-h observation period. Buprenorphine may prove to be a suitable drug for treating opioid withdrawal in human infants.

Influence of nociceptin(1-17) fragments and its tyrosine-substituted derivative on morphine-withdrawal signs in rats.

Our previous studies demonstrated that endogenous ligand of nociceptin (NOP) receptor, nociceptin(1-17) (also known as orphanin FQ), inhibits morphine-withdrawal syndrome measured as wet dog shakes in rats [Life Sci. 66 (2000) PL119]. This peptide is metabolized in the spinal cord, both in vitro and in vivo, to shorter fragments, including nociceptin(1-11) and nociceptin(1-6). These fragments, formed after cleavage by endogenous peptidase, are behaviorally active and modulate nociception in a bi-phasic process [Peptides 20 (1999) 239]. As these peptides induced transient naloxone-reversible analgesia in behavioral tests [Peptides 20 (1999) 239], in the present study we tested the influence of nociceptin(1-11) (10 and 20 microg) and nociceptin(1-6) (10, 20 and 40 microg) on the morphine-withdrawal syndrome in rats. Furthermore, the modified fragment of nociceptin(1-6) with an opioid-message domain achieved by replacement of Phe1 with Tyr was tested. Morphine-withdrawal syndrome was precipitated by the i.p. injection of naloxone hydrochloride (2 mg/kg), 72 h after implantation of morphine pellets. The wet-dog shakes were chosen for statistical analyses of the abstinence signs. The results show that nociceptin(1-11) and (1-6) attenuate this morphine-withdrawal symptom. The replacement of Phe1 with Tyr in nociceptin(1-6) fragment did not potentiate the influence of nociceptin(1-6) on wet dog shakes precipitated by naloxone in morphine-dependent rats.