Modification of 5-HT2 receptor mediated behaviour in the rat by oleamide and the role of cannabinoid receptors.

Oleamide (cis-9,10-octadecenoamide) is an endogenous brain lipid which has been suggested to induce sleep in experimental animals. The mechanism of action is unclear but shares many of the characteristics of endogenous cannabinoids such as anandamide and has been shown to enhance in vitro responses to 5-HT and GABA. In the present study we investigated the effects of oleamide on two motor behaviours, back muscle contractions (BMC) and wet-dog shakes (WDS) induced in rats by treatment with the 5-HT2 receptor agonist DOI ((+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride). We then examined the potential involvement of CB1 cannabinoid receptors in the responses to oleamide and the mechanism of interaction between CB1 and 5-HT2 receptors. Oleamide and the cannabinoid receptor agonist HU210 (6aR)-trans-3-(1,1-dimethylheptyl)6a,7,10,10a-tetrahydro-1-h ydroxy-6,6-dimethyl-6H-dibenzo[b,d]pyran-9-methanol) produced a hypolocomotion which was prevented by the CB1 antagonist SR141716A (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide hydrochloride). Despite having no effect alone, oleamide and HU210 potentiated BMC induced by treatment with DOI. SR141716A alone did not affect the response to DOI but it blocked the potentiations caused by oleamide or HU210. WDS were unaffected by oleamide and slightly reduced by HU210. In vitro, oleamide and HU210 enhanced the high affinity binding of 5-HT to 5-HT2 receptors on rat cerebral cortex membranes labelled with 3H-ketanserin. Neither agent, however, altered 5-HT-stimulated phosphoinositide hydrolysis in rat cerebral cortex slices. Oleamide occupied CB1 cannabinoid receptors on rat brain membranes labelled with 3H-CP55940 with an IC50 of 10 microM. The data presented are consistent with oleamide acting via a cannabinoid recognition site to enhance 5-HT2 receptor function in vivo. The mechanism of the modulation is still unclear but it does not appear to involve a potentiation of 5-HT2 receptor-stimulated phosphoinositide hydrolysis.

Influence of cannabinoids on electrically evoked dopamine release and cyclic AMP generation in the rat striatum.

Using the endogenous cannabinoid receptor agonist anandamide, the synthetic agonist CP 55940 [[1alpha,2beta(R)5alpha]-(-)-5-(1,1-dimethylheptyl+ ++)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]phenol], and the specific antagonist SR 141716 [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide hydrochloride], second messenger activation of the central cannabinoid receptor (CB1) was examined in rat striatal and cortical slices. The effects of these cannabinoid ligands on electrically evoked dopamine (DA) release from [3H] dopamine-prelabelled striatal slices were also investigated. CP 55940 (1 microM) and anandamide (10 microM) caused significant reductions in forskolin-stimulated cyclic AMP accumulation in rat striatal slices, which were reversed in the presence of SR 141716 (1 microM). CP 55940 (1 microM) had no effect on either KCl- or neurotransmitter-stimulated 3H-inositol phosphate accumulation in rat cortical slices. CP 55940 and anandamide caused significant reductions in the release of dopamine after electrical stimulation of [3H]dopamine-prelabelied striatal slices, which were antagonised by SR 141716. SR 141716 alone had no effect on electrically evoked dopamine release from rat striatal slices. These data indicate that the CB1 receptors in rat striatum are negatively linked to adenylyl cyclase and dopamine release. That the CB1 receptor may influence dopamine release in the striatum suggests that cannabinoids play a modulatory role in dopaminergic neuronal pathways.

Analysis of protein-bound metabolites of furazolidone and furaltadone in pig liver by high-performance liquid chromatography and liquid chromatography-mass spectrometry.

Studies undertaken using radiolabelled furazolidone have demonstrated the covalent binding of residues of the drug to cellular protein in vivo. A portion of these bound residues and those formed by furaltadone, a related nitrofuran drug, possess intact side-chains, 3-amino-2-oxazolidinone (AOZ) and 5-morpholino-methyl-3-amino-2-oxazolidinone (AMOZ), respectively. These side-chains have molecular characteristics in common with the parent compounds and may be released from liver tissue under mild acidic conditions. Derivatization with 2-nitrobenzaldehyde (NBA) serves to isolate the released side-chains and the derivatives NPAOZ and NPAMOZ are chromophoric, thereby permitting UV detection. This paper reports the introduction of an extract clean-up step to the existing procedure which eliminates or decreases interference from NBA in the HPLC-UV determination of NPAOZ. The modified procedure was also applied to the determination of AMOZ. The development of an LC-MS method for the quantitative and confirmatory determination of AOZ and AMOZ extracted and derivatized according to the same procedure as that for HPLC-UV is described. The methods were validated for AOZ and AMOZ in fortified (intra- and inter-assay studies) and incurred (inter-assay studies) pig liver samples. The limit of determination for fortified control liver samples was 5 ng AOZ g-1 and 10 ng AMOZ g-1 by HPLC-UV and 10 ng AOZ or AMOZ g-1 by LC-MS. In addition, a study to determine the ratio of released AOZ to the total bound residues present in incurred liver samples from pigs treated with furazolidone is described.

Social interaction increases 5-HT release and cAMP efflux in the rat ventral hippocampus in vivo.

The aim of the present study was to combine in vivo microdialysis in the rat with behaviour in the social interaction test in order to investigate changes in both 5-HT release and cyclic AMP (cAMP) efflux in the ventral hippocampus with simultaneous measurement of behaviour. Exposure of the rat to a 10min period of social interaction with an unfamiliar partner in a brightly lit, unfamiliar arena resulted in an increase in extracellular 5-HT and extracellular cAMP in the ventral hippocampus. Pretreatment with diazepam (1mg/kg i.p.) 30min prior to the social interaction test significantly inhibited the increases in both extracellular 5-HT and cAMP while significantly increasing the amount of time the pair of rats spent in active social contact over the 10min period. During the 30min prior to the social interaction test diazepam reduced basal levels of 5-HT, but had no effect on the basal efflux of cAMP. Pretreatment with a selective 5-HT(1A) antagonist, WAY 100135 (5mg/kg s.c.), 30min the social interaction test, significantly potentiated the increase in extracellular 5-HT observed in saline-treated rats during the social interaction test. In contrast, WAY 100135 pretreatment significantly attenuated the increase in extracellular cAMP observed in saline-treated rats during the social interaction test but had no effect on the time spent in active social contact between pairs of rats. The results suggest that social interaction results in activation of a post-synaptic 5-HT receptor (5-HT(1A) or 5-HT(6)/5-HT(7)) coupled to adenylate cyclase but that this receptor is not responsible for the aversion-induced behaviour. Furthermore antagonism of the 5-HT(1A) somatodendritic autoreceptor under conditions of 5-HT neuronal activation, but not under basal conditions, potentiates 5-HT release.

Serotonin 5-HT1A receptor activation increases cyclic AMP formation in the rat hippocampus in vivo.

In vivo microdialysis was used to examine the efflux of cyclic AMP (cAMP) into the extracellular fluid of the ventral hippocampus in the freely moving rat. The changes in extracellular cAMP concentration were monitored in response to forskolin and the serotonin 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The basal level of hippocampal extracellular cAMP was 2.3 +/- 0.2 pmol/ml (n = 6), after a 3-h postsurgery stabilisation period. Perfusion of forskolin (100 microM) through the probe for 30 min significantly increased the efflux of cAMP, which returned to baseline levels within 90 min. 8-OH-DPAT (0.3 mg/kg s.c.) also significantly increased cAMP efflux, whereas a similar volume of saline had no effect. Desensitisation of the 8-OH-DPAT-induced increase in cAMP efflux was observed following a second administration of 8-OH-DPAT after a 4-h interval. Administration of 8-OH-DPAT did not alter the efflux of cAMP when forskolin was perfused through the probe. Pretreatment with WAY 100135 [N-tert-butyl 3-4-(2-methoxyphenyl)piperazine-1-yl-2-phenylpropanamide dihydrochloride] (5 mg/kg s.c.), a specific 5-HT1A receptor antagonist, prevented the 8-OH-DPAT-induced increase in cAMP efflux. The data indicate that the 8-OH-DPAT-induced increase in cAMP efflux in vivo is mediated by a 5-HT1A receptor.

Evidence that chronic administration of paroxetine or fluoxetine enhances 5-HT2 receptor function in the brain of the guinea pig.

The effects of chronic treatment with antidepressants on 5-HT-stimulated hydrolysis of phosphoinositide (PI) in the cerebral cortex of the guinea pig were examined. The pharmacological profile of the response was consistent with it being mediated by a 5-HT2 receptor. Chronic (but not acute) treatment with the selective 5-HT uptake inhibitors, paroxetine and fluoxetine (10 mg/kg, p.o., for 21 days), resulted in a significant increase in the maximum response to 5-HT without altering the EC50. There were no significant alterations in the density or affinity of 5-HT2 receptors, labelled with [3H]ketanserin. In contrast, treatment with the tricyclic antidepressant, amitriptyline (10 mg/kg, p.o., for 21 days), did not produce a significant change in 5-HT-stimulated hydrolysis of PI or in the number and affinity of 5-HT2 receptors. The findings are discussed in relation to the possible neurochemical targets for antidepressant drugs and species differences in the responses to these agents.

Anionic responses of electrochemically synthesized polypyrrole films.

Conducting polypyrrole films with thickness of about 8 mum were prepared on platinum by continuous scanning in 0.1M pyrrole and 0.1M electrolyte (LiCl, LiBF(4) or NaBF(4)). Scan-rates of 10, 20 and 50 mV/sec with scan-times of 30, 45 and 60 min were used. The potentiometric response of the films was tested over a range of anions. Films were found to be anion-sensitive but very non-selective. The influence of the doping cation was also investigated.

Oxygen consumption and survival prediction in neonatal rats exposed to prenatal hypervitaminosis A.

Fetal exposure to excess vitamin A results in a highly variable degree of lung pathology and high neonatal mortality in the Long-Evans rat. The present study evaluated O2 consumption in newborn of vitamin A-treated, vehicle-treated, and untreated pregnancies on five consecutive postnatal days beginning with the day of delivery (D0). Pregnant female rats were treated by gavage with 160,000 USP units of retinyl acetate dissolved in 0.5 ml corn oil on days 15 through 19 of gestation. Vehicle and undisturbed controls were run concurrently. All animals delivered spontaneously, and the pups were tattooed and individually tested in a closed system consisting of three chambers submerged within a thermostatically controlled water bath at 33 degrees C. Vitamin A-exposed pups, as a group, have significantly lower QO2 (ml O2 consumed/min/kg body weight) values than controls through postnatal day 2 (p less than 0.05). By days 3 and 4 of age, the mean QO2 values of surviving vitamin A-treated pups were similar to those of controls. A QO2 of 30 or greater on day 0 appears to be critical for early neonatal survival of vitamin A-exposed pups, as 87% of the pups with initial QO2 less than 30 died prior to day 4. Oxygen consumption rates in teratogen-exposed pups exhibiting low QO2 on day 0 rarely reached normal levels. In contrast, the occasional control pup with such low initial levels were well within normal limits (means +/- 1 SD) by the following day.(ABSTRACT TRUNCATED AT 250 WORDS)

Regeneration by dissociated adult Hydra cells: a histologic study.

Adult Hydra attenuata with vitally stained gastrodermal cells were dissociated into their component cells which were then randomly reaggregated into pellets by low-speed centrifugation. Representative examples of these preparations, which develop into normal adult hydra if left undisturbed, were examined fresh at low magnification and at higher magnification in fixed, stained, and sectioned specimens. The actual pellet stage lasts less than 1 hour because the adult ectodermal and gastrodermal cells rapidly sort themselves into an inner and outer layer and seem to secrete a new mesoglea immediately thereafter. The "embryo" becomes trilaminar and attains a central cavity by extruding a large amount of cellular debris at the end of the first day. At about this same time, new tentacles begin to differentiate from rapidly dividing and undifferentiated interstitial cells. Regulation of tentacle number and position occurs at the end of two days, and the body form is essentially reestablished within 60 hours by further differentiation of the hypostomes and body wall. Complete separation of the preparation into individual polyps does not occur until about 190 hours of development.

Intralitter variation of neonatal rat lung morphology following prenatal maternal hypervitaminosis A.

Prenatal exposure to excess vitamin A (160,000 USP units/day) from days 15 through 19 of gestation results in altered lung morphology, characterized by thickened septal walls and/or large areas of atelectasis and an associated high neonatal mortality. Marked variability in both morphological and physiological expression from this prenatal insult is commonly seen between litters and littermates, making analyses difficult. The present study documents morphological intralitter variation observed in vitamin A-exposed 2-day-old rats as compared to controls. Representative midhilar coronal histological sections of each lung were examined by two methods and the results compared. The first method, although less sensitive, demonstrated that five out of eight experimental rat lungs had a significantly greater percentage of tissue to airway space as compared with undisturbed controls (greater than 43%). The second method, using several morphological criteria and a grid system to score parenchyma into classifications based on the degree of morphological variation and projected functional capability, clearly found significantly increased percentages of poor or nonfunctional lung tissue (P greater than 0.01) in seven out of eight pups exposed to excess vitamin A. This method of ranking the severity of adverse effects on tissue morphology allowed identification of drug-affected newborn and provided a means to quantify the alterations. Such a means for identifying affected individuals from littermates is essential to research methodologies for detecting substances which, when administered during fetal life, produce decrements of postnatal function but no gross structural abnormalities.