Inhibition of prandial and waterspray-induced rat grooming by 8-OH-DPAT

In addition to its hyperphagic effect in rats, 8-OH-DPAT also reduces grooming, but it is uncertain whether the inhibition of grooming is a specific effect or a consequence of response competition from eating. The present experiments explored the effects of 8-OH-DPAT on periprandial grooming and grooming elicited by spraying rats with water. Momentary time sampling over 30 or 60 min, with behaviour scored in one of 6 or 7 (depending on food availability) mutually exclusive categories (feeding, active, scratching, face grooming, body grooming, genital grooming and resting) at 15s intervals, was used for data collection. Non-deprived rats were tested in the presence and absence of food and baseline grooming levels were manipulated by spraying the dorsal surface of the back with water. Data were submitted to ANOVA. The first experiment confirmed that 8-OH-DPAT increased food intake and that this was associated with a parallel increase in feeding observations; active observations were also increased, but resting and total grooming observations were reduced: scratching was reduced even at 0.003mg/kg, face- and body-grooming were reduced at doses > 0.03mg/kg and genital-grooming was least sensitive, only being reduced at 0.1mg/kg. The second experiment revealed that spraying with water had no effect on food intake, feeding or resting observations, but increased total grooming (largely due to increased body-grooming) and reduced activity observations. In rats sprayed with water, 8-OH-DPAT increased food intake (0.1mg/kg) and observations of feeding (0.003 & 0.1mg/kg), but total grooming was dose-dependently inhibited, with genital-grooming most sensitive(> 0.003mg/kg), followed by face-grooming (> 0.01mg/kg) and body-grooming (>0.03mg/kg), whilst low levels of scratching were unaffected. The final experiment tested water-sprayed rats in the absence of food: 8-OH-DPAT increased resting and reduced total grooming, mostly as a consequence of reductions in face- and body-grooming, but there were also modest reductions in scratching. These results confirm that 8-OH-DPAT has a suppressant effect on all aspects of grooming, except where there are probable floor effects, and that this is independent of response competition from increased eating.

Inhibition of prandial and waterspray-induced rat grooming by 8-OH-DPAT

The effects of 8-OH-DPAT treatment on rat grooming behaviour, elicited either prandially or in response to spraying with water were investigated. Dose (≤0.1 mg/kg s.c.) response studies employed momentary time sampling over 30 or 60 min with behaviour being scored in one of 6 or 7 (depending on food availability) mutually exclusive categories (feeding, active, scratching, face-grooming, body grooming, genital-grooming and resting) at 15 s intervals. In non-deprived rats, tested with wet mash available, feeding and activity frequencies were increased, but resting and total grooming were inhibited by 8-OH-DPAT. Face-, body- and genital-grooming occurred at higher levels than scratching, but all categories were reduced with reductions in scratching occurring at a lower dose (0.01 mg/kg). Misting rats with a fine water spray selectively increased body grooming and decreased activity without altering feeding, while 8-OH-DPAT increased feeding and reduced face-, body- and genital-grooming, without affecting already low levels of scratching. In misted rats, tested without food, 8-OH-DPAT reduced face-, body- and genital-grooming and increased resting. These results confirm i) that the water spray technique is a useful method for increasing grooming and ii) that 8-OH-DPAT has a suppressant effect on grooming independent of response competition from enhanced feeding.

IN SITU AND IN VITRO IMMUNOLOCALIZATION OF OVIDUCTIN BINDING SITES ON HAMSTER UTERINE EPITHELIAL CELLS AND DETECTION OF A HAMSTER OVIDUCTIN HOMOLOGUE IN THE FEMALE RAT REPRODUCTIVE TRACT

Oviductin is an oviduct-specific and high-molecular-weight glycoprotein that has been suggested to play important roles in the early events of reproduction. The present study was undertaken to localize the oviductin binding sites in the uterine epithelial cells of the golden hamster (Mesocricetus auratus) both in situ and in vitro, and to detect a hamster oviductin homologue in the female rat reproductive tract. Immunohistochemical localization of oviductin in the hamster uterus revealed certain uterine epithelial cells reactive to the monoclonal anti-hamster oviductin antibody. In order to study the interaction between hamster oviductin and the endometrium in vitro, a method for culturing primary hamster uterine epithelial cells has been established and optimized. Study with confocal microscopy of the cell culture system showed a labeling pattern similar to what was observed using immunohistochemistry. Pre-embedding immunolabeling of cultured uterine epithelial cells also showed gold particles associated with the plasma membrane and microvilli. These results demonstrated that hamster oviductin can bind to the plasma membrane of certain hamster uterine epithelial cells, suggesting the presence of a putative oviductin receptor on the uterine epithelial cell surface. In the second part of the present study, using the monoclonal anti-hamster oviductin antibody that cross-reacts with the rat tissue, we have been able to detect an oviduct-specific glycoprotein, with a molecular weight of 180~300kDa, in the female rat reproductive tract. Immunohistochemical labeling of the female rat reproductive tract revealed a strong immunolabeling in the non-ciliated oviductal epithelial cells and a faint immunoreaction on the cell surface of some uterine epithelial cells. Ultrastructurally, immunogold labeling was restricted to the secretory granules, Golgi apparatus, and microvilli of the non-ciliated secretory cells of the oviduct. In the uterus, immunogold labeling was observed on the cell surface of some uterine epithelial cells. Furthermore, electron micrographs of ovulated oocytes showed an intense immunolabeling for rat oviductin within the perivitelline space surrounding the ovulated oocytes. The findings of the present study demonstrated that oviductin is present in the rat oviduct and uterus, and it appears that, in the rat, oviductin is secreted by the non-ciliated secretory cells of the oviduct.

Quantification of urinary excretion of ethosuximide enantiomers and their major metabolites in the rat.

A chiral gas chromatographic assay has been developed for quantitative analysis of ethosuximide and its major metabolites in rat urine. The extraction procedure was found to be precise and reproducible. Recovery was in the range of 94-98%, intraday CV(%) was 0.92% for (S)-ethosuximide (50 mug/ml) and 0.51% for (R)-ethosuximide (50 mug/ml). Interday CV(%) was 1.12% for (S)-ethosuximide and 0.72% for (R)-ethosuximide. The limit of detection was determined to be around 0.01 mug/ml for each enantiomer. Following administration of rac-ethosuximide by i.v., i.p. and oral routes, unchanged ethosuximide was detected in urine up to 72h after drug administration. The appearance of all detected metabolites occurred Within 24h of drug administration. Significantly more (S)-ethosuximide was excreted unchanged than (R)-ethosuximide with all three routes studied. A substantial amount of the drug was eliminated as the 2-(1-hydroxyethyl)-2-methylsuccinimide (2 pairs of diastereoisomers). Much less drug was eliminated as the 2-ethyl-3-hydroxy-2-methylsuccinimide with only one diastereoisomer observed. Examination of the one pair of diastereoisomers of 2-(1-hydroxyethyl)-2-methylsuccinimide that was resolved showed preferential excretion of one isomer. Comparison of both pairs of diastereoisomers showed that one pair was formed in preference to the other with a ratio of approximately 0.8:1. It is concluded that stereoselective metabolism of ethosuximide occurs. Copyright (C) 2001 John Wiley & Sons, Ltd. Author Keywords: chiral pharmacokinetics; ethosuximide enantiomers; metabolism; rat; urinary excretion; gas chromatography

Changes in fos expression in the rat brain after unilateral lesions of the anterior thalamic nuclei

Activity of the immediate early gene c-fos was compared across hemispheres in rats with unilateral anterior thalamic lesions. Fos protein was quantified after rats performed a spatial working memory test in the radial-arm maze, a task that is sensitive to bilateral lesions of the anterior thalamic nuclei. Unilateral anterior thalamic lesions produced evidence of a widespread hippocampal hypoactivity, as there were significant reductions in Fos counts in a range of regions within the ipsilateral hippocampal formation (rostral CA1, rostral dentate gyrus, 'dorsal' hippocampus, presubiculum and postsubiculum). A decrease in Fos levels was also found in the rostral and caudal retrosplenial cortex but not in the parahippocampal cortices or anterior cingulate cortices. The Fos changes seem most closely linked to sites that are also required for successful task performance, supporting the notion that the anterior thalamus, retrosplenial cortex and hippocampus form key components of an interdependent neuronal network involved in spatial mnemonic processing.

Carbachol triggers RyR-dependent Ca(2+) release via activation of IP(3) receptors in isolated rat gastric myocytes.

Possible interactions between different intracellular Ca(2+) release channels were studied in isolated rat gastric myocytes using agonist-evoked Ca(2+) signals. Spontaneous, local Ca(2+) transients were observed in fluo-4-loaded cells with linescan confocal imaging. These were blocked by ryanodine (100 microM) but not by the inositol 1,4,5-trisphosphate receptor (IP(3)R) blocker, 2-aminoethoxydiphenyl borate (100 microM), identifying them as Ca(2+) sparks. Caffeine (10 mM) and carbachol (10 microM) initiated Ca(2+) release at sites which co-localized with each other and with any Ca(2+) spark sites. In fura-2-loaded cells extracellular 2-aminoethoxydiphenyl borate and intracellular heparin (5 mg ml(-1)) both inhibited the global cytoplasmic [Ca(2+)] transient evoked by carbachol, confirming that it was IP(3)R-dependent. 2-Aminoethoxydiphenyl borate and heparin also increased the response to caffeine. This probably reflected an increased Ca(2+) store content since 2-aminoethoxydiphenyl borate more than doubled the amplitude of transients evoked by ionomycin. Ryanodine completely abolished carbachol and caffeine responses but only reduced ionomycin transients by 30 %, suggesting that blockade of carbachol transients by ryanodine was not simply due to store depletion. Double labelling of IP(3)Rs and RyRs demonstrated extensive overlap in their distribution. These results suggest that carbachol stimulates Ca(2+) release through co-operation between IP(3)Rs and RyRs, and implicate IP(3)Rs in the regulation of Ca(2+) store content.