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OREXIN A (HYPOCRETIN-1) APPLICATION AT THE MEDIAL PREOPTIC AREA POTENTIATES MALE SEXUAL BEHAVIOR IN RATS Neuroscience 116 (2003) 921–923 K. K. GULIA, H. N. MALLICK* AND V. M. KUMAR Abstract—The medial preoptic area plays an important role in the regulation of male sexual behavior in rats, and this area receives orexinergic inputs. The role of orexinergic inputs in the medial preoptic area in sexual behavior has not been studied, though they have been shown to play a role in some other physiological functions. In this study, the changes in male sexual behavior in rats were studied after local injection of orexin A (Hypocretin-1) at the medial preoptic area. The results of the study showed that orexin A application at the medial preoptic area increased sexual arousal as well as the copulatory performance. Sexual arousal is one of the physiological stimuli, which influences wakefulness. It is possible that the earlier reports showing increased wakefulness (Espana et al., 2001; Thakkar et al., 2001), on application of orexin A at the medial preoptic area/basal forebrain, has a contribution from sexual arousal. Orexins, also known as hypocretins, are the recently discovered neuropeptides, which are synthesized exclusively by some neurons located in the lateral hypothalamic area (de Lecea et al., 1998; Sakurai et al., 1998). Therefore, it was initially proposed that orexins participate in the control of feeding behavior (Sakurai et al., 1998). However, a widespread distribution of the axons of orexinergic neurons throughout the brain suggests that they may have a role in autonomic functions, energy homeostasis, sleepwakefulness, neuroendocrine release and reproduction. EXPERIMENTAL PROCEDURES The study was conducted on ten adult male Wistar rats, weighing 180– 250 g. The animals were kept in controlled temperature 25_2 °C) under 14:10h light-dark schedule (light on at 6:00 A.M.). Food and water were provided ad libitum. The rats were screened for sexual behavior using standard criteria (Mittimohan et al., 1989; Mallick et al., 1996) and those having a sex drive score above four were chosen for the study. Under sodium pentobarbital anesthesia (40 mg/kg body wt, i.p.), 26 gauge bilateral stainless steel guide cannulae with indwelling styli were implanted in the brain, 1.5 mm above the mPOA, at the co-ordinates 7.8 mm anterior, 0.75 mm lateral and 1.5 mm below the interaural zero, as per DeGroot atlas (DeGroot, 1959). The whole assembly was firmly fixed to the skull with four implanted anchoring screws and dental cement. Seven days after the implantation of the cannulae, the rats were tested for their copulatory activity on three occasions, at an interval of three days between the tests. Rats showing consistency in sexual behavior as shown by analysis of variance were only used for further study. Mean of these 3 days recordings were taken as control readings for comparison with post-treatment values. OX-A (O-6012, Sigma Chemical Co., St. Louis, MO, USA) at the dose of 0.3 nmol in 200 nl of 0.9% NaCl was bilaterally injected at the mPOA by a slow injector at the rate of 100 nl/min. The injector cannula was left in place for 1 min following injection. The cannula was then replaced with the stylus. The injection was given only once in any one brain site in each of the animals. The scoring was taken after 10 min of administration of the drug. Sex behavior scoring was performed under dim illumination in a wooden box (45_30_30 cm) with a sliding glass front, during the dark phase of the light/dark cycle (20.0–23.0 h). Bilaterally ovariectomized females of the same strain, primed with 25 _g of estradiol benzoate and one mg of progesterone, were used as receptive partners. The male rat was introduced into the test arena, 5 min prior to the introduction of the female. Recording was initiated at the entry of the female into the box. A computer program was used to record the latencies of pursuit, mount, intromission and ejaculation, frequencies of pursuit, mount and intromission, mean inter-intromission and post-ejaculatory intervals, and sex drive score. The latencies and frequencies of the events (pursuit, mount, intromission and ejaculation) were registered by manually pressing the assigned keys. The computer did timing operation, using its internal clock. At the end of the experiment, the brain sites and spread of injection were verified histologically by injecting 200 nl of 2% ferric chloride through the implanted guide cannulae and then perfusing the brain with 10% formal-saline containing 3% potassium ferrocyanide. Injection sites were bilaterally confirmed to be at the mPOA in seven rats. RESULTS AND DISCUSSION Seven rats in which OX-A was bilaterally injected at the mPOA were grouped together for statistical analysis. The bilateral microinjection of OX-A (0.3 nmol) in the mPOA (n_7) produced significant decrease in latencies to mount, intromission and ejaculation (Table 1). There was signifi- cant decrease in postejaculatory interval and mean interintromission interval (P_0.05) and increase in pursuit, mount, intromission frequencies (P_0.05) and sex drive scores (P_0.05). The OX-A treated rats were moving around in the chamber during the post-ejaculatory interval. During this period, they were not indulging in active pursuit of the female. Rats generally go into a phase of inactivity, lying prostrate for some time after ejaculation (Sachs and Meisel, 1988). No change in the sex behavior was observed in one rat in which OX-A was injected above anterior commissure. There was an increase in sex drive score from 7.88–8.28 in one, 14.5–24.27 in the other, where injections were given unilaterally. They also showed a decrease in ejaculation latency, post-ejaculatory interval and mean interintromission interval.