Differential effects of acute morphine, and chronic morphine-withdrawal on obsessive-compulsive behavior: inhibitory influence of CRF receptor antagonists on chronic morphine-withdrawal.

Recent studies have provided convincing evidences for co-morbidity between opioid addiction and obsessive-compulsive disorder (OCD), and the involvement of the corticotrophin-releasing factor (CRF) in the effects of morphine-withdrawal. Some scanty evidences also point towards the role of CRF in OCD and related disorders. But, no evidence indicated the role of CRF in morphine withdrawal associated obsessive-compulsive behavior (OCB). Therefore, the present study investigated the role of CRF in morphine-withdrawal induced OCB in mice. Marble-burying behavior in mice was used to assess OCB as this model has good predictive and face validity. The results revealed that acute morphine dose dependently attenuated the marble burying behavior, whereas withdrawal of chronic morphine was associated with significant rise in marble burying behavior. This indicates the differential effect of acute morphine and chronic morphine-withdrawal on OCB. Further, acute treatment with CRF receptor antagonists like antalarmin (2 and 4 µg/mouse, i.c.v.) or astressin-2B (3 and 10 nmol/mouse, i.c.v.) dose dependently attenuated the peak morphine-withdrawal induced increase in marble burying behavior. Moreover, concomitant treatment with antalarmin (4 µg/mouse, i.c.v.) or astressin-2B (10 nmol/mouse, i.c.v.) along with morphine blocked the morphine-withdrawal associated exacerbation of OCB. These results indicate that OCB associated with morphine withdrawal state is partly mediated by the activation of central CRF receptors.

Role of nitric oxide in obsessive-compulsive behavior and its involvement in the anti-compulsive effect of paroxetine in mice.

In view of the reports that nitric oxide modulates the neurotransmitters implicated in obsessive-compulsive disorder, patients with obsessive-compulsive disorder exhibit higher plasma nitrate levels, and drugs useful in obsessive-compulsive disorder influence nitric oxide, we hypothesized that nitric oxide may have some role in obsessive-compulsive behavior. We used marble-burying behavior of mice as the animal model of obsessive-compulsive disorder, and nitric oxide levels in brain homogenate were measured using amperometric nitric oxide-selective sensor method. Intraperitoneal administration of nitric oxide enhancers viz. nitric oxide precursor-l-arginine (800 mg/kg), nitric oxide donor-sodium nitroprusside (3 mg/kg) or phosphodiesterase type 5 inhibitor-sildenafil (3 mg/kg) significantly increased marble-burying behavior as well as brain nitrites levels, whereas treatment with 7-nitroindazole-neuronal nitric oxide synthase inhibitor (20-40 mg/kg, i.p.) or paroxetine-selective serotonin reuptake inhibitor (5-10 mg/kg, i.p.) dose dependently attenuated marble-burying behavior and nitrites levels in brain. Further, co-administration of sub-effective doses of 7-nitroindazole (10 mg/kg) and paroxetine (2.5 mg/kg) significantly attenuated marble-burying behavior. Moreover, pretreatment with l-arginine (400 mg/kg, i.p.), sodium nitroprusside (2.0 mg/kg, i.p.) or sildenafil (2.0 mg/kg, i.p.) significantly attenuated the inhibitory influence of 7-nitroindazole (40 mg/kg) or paroxetine (10 mg/kg) on marble-burying behavior as well as on brain nitrites levels. None of the above treatment had any significant influence on locomotor activity. In conclusion, obsessive compulsive behavior in mice appears related to nitric oxide in brain, and anti-compulsive effect of paroxetine appears to be related to decrease central levels of nitric oxide.

A possible participation of gonadotropin-releasing hormone in the neuroleptic and cataleptic effect of haloperidol.

Haloperidol, an antipsychotic agent, stimulates the release of gonadotropin-releasing hormone (GnRH), and this hormone is known to mimic some of the behavioral effects of haloperidol. Hence, the present study was carried out to find out the contribution of GnRH in the behavioral effects of haloperidol. The studies revealed that haloperidol (0.15, 0.25 and 0.5 mg/kg, i.p.) and leuprolide (GnRH agonist; 50, 100, 200 and 400 microg/kg, s.c.) dose-dependently inhibited conditioned avoidance response (CAR) in male Sprague-Dawley rats. In higher doses, haloperidol (0.5, 1 mg/kg, i.p.) and leuprolide (200, 400 microg/kg, s.c.) produced catalepsy in rats. Co-administration of sub-effective dose of leuprolide (50 or 100 microg/kg, s.c.) and haloperidol (0.15 or 0.5 mg/kg, i.p.) similarly inhibited CAR and induced catalepsy. Pre-treatment of rats with antide (GnRH antagonist; 10 microg/rat, s.c.), attenuated the inhibitory effect of both the agents on CAR; blocked leuprolide-induced catalepsy; and attenuated the intensity and reduced the duration of haloperidol-induced catalepsy. In conclusion, the studies suggest a possible role of GnRH in the neuroleptic and cataleptic effect of haloperidol.

Gastrointestinal dysfunction in diabetic rats relates with a decline in tissue L-arginine content and consequent low levels of nitric oxide.

Diabetic subjects exhibit low levels of nitric oxide (NO), its precursor L-arginine, and nitric oxide synthase (NOS) in tissues like endothelium and kidney. In view of this, we speculated that gastrointestinal (GI) dysfunction in diabetes could be related to similar changes in NO turnover in GI tissues. Hence, the studies were carried out in rats after eight weeks of streptozotocin-induced hyperglycemia, wherein the GI functions were assessed in terms of gastric emptying and intestinal transit using barium sulfate semisolid test meal, and the levels of L-arginine and NO in pylorus and ileum were estimated, respectively, by HPLC and amperometry. The results revealed that diabetic group exhibited significant delay in gastric emptying and intestinal transit, and the pylorus and ileum tissues had significantly low levels of NO and L-arginine. Daily treatment of non-diabetic rats with NOS inhibitor [Nomega-nitro-L-arginine methyl ester (10mg/kg/day, p.o.)] for eight weeks produced similar delay in gastric emptying and intestinal transit with associated low levels of NO in GI tissues. Daily supplementation of L-arginine (100mg/kg, p.o.) for eight weeks to diabetic and NOS inhibitor treated non-diabetic group was found to restore the gastric emptying and intestinal transit and improved the levels of NO in GI tissues. The findings indicate that diabetes-induced L-arginine deficiency and consequent low levels of NO in GI tissues could be possible cause for the GI dysfunction, and L-arginine supplementation can prevent the same. However, extensive clinical investigations are necessary to recommend the use of L-arginine for the treatment of GI dysfunctions in diabetes.

Gonadotropin-releasing hormone agonist blocks anxiogenic-like and depressant-like effect of corticotrophin-releasing hormone in mice.

Corticotrophin-releasing factor (CRF) is reported to inhibit the release of gonadotropin-releasing hormone (GnRH). In addition to the endocrine effects, GnRH is reported to influence the behavior via its neuronal interactions. We therefore, hypothesized that anxiety and depression produced by CRF could be also subsequent to the decrease in GnRH. To support such possibility, we investigated the influence of GnRH agonists on CRF or CRF antagonist induced changes in social interaction time in social interaction test, and immobility time in forced swim test in mice, as the indices for anxiety and depression, respectively. Results indicated that GnRH agonists [leuprolide (20-80 ng/mouse, i.c.v.), or d-Trp-6-LHRH (40-160 ng/mouse, i.c.v.)] dose dependently increased social interaction time and decreased immobility time indicating anxiolytic- and antidepressant-like effect, respectively. Such effects of GnRH agonists were even evident in castrated mice, which suggest that these effects were unrelated to their endocrine influence. Administration of CRF (0.1 and 0.3 nmol/mouse, i.c.v.) produced just opposite effects as that of GnRH agonist on these parameters. Further, it was seen that pretreatment with leuprolide (10 or 20 ng/mouse, i.c.v.) or d-Trp-6-LHRH (20 or 40 ng/mouse, i.c.v.) dose dependently antagonized the effects of CRF (0.3 nmol/mouse, i.c.v.) in social interaction and forced swim test. CRF antagonist [alpha-Helical CRF (9-41), (1 or 10 nmol/mouse, i.c.v.)] was found to exhibit anxiolytic- and antidepressant-like effect, and its sub-effective dose (0.1 nmol/mouse, i.c.v.) when administered along with sub-threshold dose of leuprolide (10 ng/mouse, i.c.v.), or d-Trp-6-LHRH (20 ng/mouse, i.c.v.) also produced significant anxiolytic- and antidepressant-like effect. These observations suggest reciprocating role of GnRH in modulating the CRF induced anxiogenic- and depressant-like effects.

Leuprolide: a luteinizing hormone releasing hormone agonist attenuates ethanol withdrawal syndrome and ethanol-induced locomotor sensitization in mice.

Ethanol inhibits the synthesis, content and release of hypothalamic luteinizing hormone releasing hormone (LHRH), and LHRH modulates the activity of several neurotransmitters that experience adaptive changes on chronic exposure to ethanol, and also implicate in ethanol dependence. Hence, it was contemplated that LHRH agonist such as leuprolide may influence the behavioral consequences of withdrawing ethanol in dependent state. In the present study, ethanol dependence was produced in mice by providing ethanol liquid diet for 10 days; and its withdrawal on day 11 led to physical signs of hyperexcitability with its peak at 6th h. Acute treatment with leuprolide (20 ng/mouse, i.c.v.), 10 min prior to peak, significantly attenuated hyperexcitability. Such effect of leuprolide was evident even in castrated mice, and castration significantly increased the hyperexcitability in ethanol withdrawal state. Chronic treatment with leuprolide (10 ng/mouse, twice daily, i.c.v.) till day 10 significantly reduced the signs of hyperexcitability in ethanol withdrawal state. In another set of experiment, ethanol (2.4 g/kg, i.p.) was administered on day 1, 4, 7, 10 and 15, which caused gradual increase in locomotor activity indicating ethanol-induced sensitization. Leuprolide (20 ng/mouse, i.c.v.), 10 min prior to the challenge dose of ethanol (2.4 g/kg, i.p.) on day 15 significantly attenuated the expression of sensitization to hyperlocomotor effect of ethanol. Similarly, administration of leuprolide (20 ng/mouse, i.c.v.), 10 min prior to ethanol on day 1, 4, 7 and 10 not only reduced the gradual increase in locomotor activity but also attenuated the sensitized locomotor response on day 15, indicated attenuation of development of sensitization. Leuprolide per se did not affect physical signs and locomotor activity in control group. In conclusion, the present study demonstrated that leuprolide treatment attenuates expression and development of ethanol dependence and sensitization in mice.

Leuprolide--a GnRH agonist--prevents restraint stress-induced immunosuppression via sex steroid-independent peripheral mechanism in mice.

Gonadotropin-releasing hormone (GnRH) and sex steroids are known to modulate the immune system. To find out whether GnRH analogue can be useful to prevent the stress-induced immunosuppression, mice were treated with leuprolide (50 microg/mouse, s.c.) 30 min prior to restraint stress and immunological parameters were studied. Leuprolide prevented stress-induced decrease in relative weights of thymus, total leukocyte count and sheep red blood cells challenged humoral and cell-mediated immune reaction. Prior administration of GnRH antagonist (10 ng/mouse) through i.c.v. route did not alter the immunological effects of leuprolide. Further, the observed effect of leuprolide remained unaffected in castrated and ovariectomized mice. Thus, the investigations revealed that leuprolide prevented stress-induced immunosuppression through a peripheral mechanism, which is independent of systemic sex steroids.

Rapid and Simple RPHPLC Method for the Estimation of Metformin in Rat Plasma.

A simple reverse phase high-performance liquid chromatographic method has been developed for determining the concentration of metformin in rat plasma. The method employs C(18) column (300 mm x 2.4 mm i.d.), ammonium acetate (0.15 M) and acetonitrile (90:10; pH-5.5; 1.0 ml/min) as mobile phase and ultraviolet detection at 236 nm. Acetonitrile was used to simultaneously deproteinize rat plasma and extract metformin. The assay was linear in the concentration range of 0.33 mug-16.6 mug/ml with co-efficient of correlation 0.994. The retention time was 4.7 min. The method was found to be precise (% CV < 15%), accurate and suitable for pharmacokinetic study of orally administered metformin in rats.

A synergistic decline in humoral and cellular immunity of diabetic mice on exposure to polluted air.

It is clinically known that diabetic patients are more prone to infectious diseases, due to low immune status. Since, some of the common air pollutants are reported to suppress immune system, how exposure to artificially polluted air influences the immune responses in experimental diabetic mice was studied. A diabetic state was induced by alloxan and mice were exposed to artificially polluted air for 30 days. During the period of exposure, the humoral (antibody titer) and cellular (foot and swelling) immune responses to antigenic challenges with sheep RBC were investigated. The exposure to polluted air produced a significant decline in the immune responses in non-diabetic mice whereas a synergistic decline was observed in diabetic group. Since, daily oral treatment with vitamin E (150 mg/kg) significantly prevented the pollution induced immunosuppression, the involvement of free radicals is suggested.