Arif Siddiqui

NEONATAL ANDROGEN MANIPULATION DIFFERENTIALLY AFFECTS THE DEVELOPMENT OF MONOAMINE SYSTEMS IN RAT CEREBRAL CORTEX, AMYGDALA AND HYPOTHALAMUS

Neural tissue during perinatal life is sufficiently plastic to respond to the presence of testicular androgens. Here we studied the effect of neonatal androgen manipulation (castration of male and androgenization of female rats) on monoamine neurotransmitter systems in the cerebral cortex and sexually dimorphic regions of brain (hypothalamus and amygdala). Norepinephrine (NE) and dopamine (DA) concentrations in cortex, amygdala and hypothalamus of rats were assayed by HPLC at days 25, 60, 120, 180, 240 and 300. Results show that NE levels in all groups of rats at day 25 were higher in the hypothalamus (5-9 ng/mg protein) compared to the amygdala (0.5-3 ng/mg protein) and the cortex (0.5-1 ng/mg protein). Levels of DA at day 25 in the hypothalamus and the amygdala were comparable (up to 3.5 ng/mg) but higher than in the cortex (1.25-1.75 ng/mg protein). NE and DA concentrations in sham-castrated male and androgen-treated females were higher at day 25 compared to castrated male and control females in both amygdala and hypothalamus; however, levels of NE and DA remained unchanged in the cortex. Pattern of NE concentrations started reversing with increase in age, i.e., NE levels in control females and castrated males increased almost 4-fold in amygdala and 2-fold in hypothalamus by day 300, but there was no significant change in the cortex. Compared to that, NE levels decreased in sham-castrated male (2-fold) and androgen-treated females (3-fold) in amygdala as well as in the hypothalamus (2-fold) in both these groups. A similar pattern of reversal of DA levels was found in both amygdala and hypothalamus, however, at day 300 DA levels were comparable in all the four groups. These studies suggest that androgen manipulation (castration or androgen administration) induces age-dependent short- and long-term effects on the development of noradrenergic and dopaminergic systems in the sexually dimorphic regions of brain, amygdala and hypothalamus, without a significant change in the cerebral cortex.

Co-activation of Gi and Gq proteins exerts synergistic effect on human platelet aggregation through activation of phospholipase C and Ca2+ signalling pathways.

Our previous studies have shown that subthreshold concentrations of two platelet agonists exert synergistic effects on platelet aggregation. Here we studied the mechanism of synergistic interaction of 5-hydroxytryptamine (5-HT) and epinephrine mediated platelet aggregation. We show that 5-HT had no or little effect on aggregation but it did potentiate the aggregation response of epinephrine. The synergistic interaction of 5-HT (1-5 µM) and epinephrine (0.5-2 µM) was inhibited by α2-adrenoceptor blocker (yohimbine; IC50= 0.4 µM), calcium channel blockers (verapamil and diltiazem with IC50 of 10 and 48 mM, respectively), PLC inhibitor (U73122; IC50=6 µM) and nitric oxide (NO) donor, SNAP (IC50=1.6 µM)). The data suggest that synergistic effects of platelet agonists are receptor-mediated and occur through multiple signalling pathways including the activation PLC/Ca2+ signalling cascades.

Perinatal Exposure to Morphine Disrupts Brain Norepinephrine, Ovarian Cyclicity, and Sexual Receptivity in Rats

The effect of perinatal exposure to morphine on the development of catecholaminergic and reproductive function in female rats was investigated. Adult rats received morphine intraperitoneally daily for 40 days. The dose of morphine was progressively increased at 10-day intervals from 5, 7.5, 10 to 15 mg/kg body weight until day 40. The rats were mated between days 38 and 45. Administration of morphine at dose rates of 20 and 30 mg/kg continued during pregnancy. The dose was increased to 40 mg/kg for 10 days postpartum. Results showed that morphine disrupted ovarian cyclicity in 52% of the females. Amongst the remaining females, 43% became pregnant when mated. In the female offspring born to such dams, sexual maturation was delayed and body weight was reduced until weaning. At adulthood, lordosis behavior was inhibited when the female offspring were tested against stimulus males. Plasma estradiol and ovarian estradiol and progesterone levels were reduced. Norepinephrine concentration in the hypothalamus was reduced, whereas it remained unchanged in the amygdala. Dopamine concentrations in both hypothalamus and amygdala were not influenced by perinatal morphine exposure. These results suggest that chronic morphine treatment during perinatal life selectively influences the development of noradrenergic mechanisms in the rat brain and this may in turn be responsible for reduced reproductive activity.

Morphine induces reproductive changes in female rats and their male offspring

The effect of intrauterine morphine exposure on the development of reproductive functions has been investigated in the rat. Female rats were treated daily ip with morphine sulfate, doses increasing at 10-d intervals from 5, 7.5, 10, to 15 mg/kg. These rats were mated between day 38 and 45, and morphine treatment continued at 20 and 30 mg/kg over pregnancy and at up to 40 mg/kg for 10 d postpartum. The treatment mainly disrupted ovarian cyclicity; only 48% exhibited normal cyclicity. Of these, 43% became pregnant when mature male rats were placed with them. Litter size was normal but with significantly more stillbirths in each litter and live pups had decreased body weights. Male offspring had reduced body weight at the time of weaning that persisted until 60 d of age. At 120 d, animals showed complete abolition of spermatogenesis and drastically reduced testicular steroidogenesis. Plasma LH levels were low, and hypothalamic noradrenaline was high.

Neonatal 5HT activity antagonizes the masculinizing effect of testosterone on the luteinizing hormone release response to gonadal steroids and on brain structures in rats

Hypothalamic 5HT concentrations are transiently lower in male compared to female Wistar rats in the second week post partum (pp) and our previous findings have shown that pharmacologically potentiating 5HT activity over this period feminizes certain aspects of sexually differentiated behaviours in adult males and androgenized females. In order to investigate whether neonatal testosterone and 5HT interact to influence physiological and morphological brain sexual differences, females, androgenized females and males were treated with the 5HT2 agonist (−) [2,5 dimethoxy‐4‐iodophenyl]‐2‐amino propane HCl [(−) DOI], over days 8–16 pp. In androgenized females (250 µg testosterone proprionate, day 2 pp) (−) DOI prevented the delay in vaginal opening, but did not prevent the androgen‐induced constant oestrus in females treated with 100 µg TP, day 2 pp. (−) DOI overcame the neonatal androgen effect in suppressing the positive feedback of ovarian steroids in a few males and androgenized females. (−) DOI had a feminizing effect on the volume of the anteroventral periventricular nucleus (normally smaller in males), by significantly increasing its volume in male and androgenized females. It also had a significant antagonistic effect on the testosterone‐induced increase in the volume of the sexually dimorphic nucleus of the preoptic area in males and androgenized females. These findings support the view that raised 5HT activity in the second week of life antagonizes the masculinizing effect of neonatal testosterone.

The 5HT7 receptor subtype is involved in the regulation of female sexual behaviour in the rat

5-Hydroxytryptamine (5-HT) regulates sexual behaviour in the female rat via a number of its receptors. The role of the 5HT(7) receptor was investigated in ovariectomised rats primed with 10 mug oestradiol benzoate (OB) followed at 48 h by 0.5 mg progesterone, which induced receptivity in approximately half of the animals. These animals were treated with three agonists all effective at 5HT(1A) and 5HT(7) receptors; 5-hydroxytryptophan, 8-hydroxy-2-(di-n-propylamino)tetralin 1-Br (8-OH DPAT) and 5-carboxy-aminotryptamine (5-CT) in the presence or absence of selective 5HT(1A) and 5HT(7) antagonists: WAY 100135 and SB 269970-A. The three agonists inhibited lordosis in the receptive group, and this was prevented by both the selective 5HT(1A) and 5HT(7) antagonists. When given alone, both WAY 100135 and SB 269970-A increased the lordosis in the non-receptive rats indicating that endogenous 5-HT acting on 5HT(1A) and 5HT(7) receptors may have a tonic inhibitory effect on receptivity. A comparison of OB priming doses on the effect of serotoninergic agents showed that the higher OB doses attenuated the inhibitory effect of 8-OH DPAT and enhanced the stimulatory effect of WAY 100135, but did not affect the actions of 5-CT or SB 269970-A. The interaction between oestradiol and 5-HT activity on sexual behaviour may therefore be selective to the 5HT(1A) pathway.

Neonatal organizational effects of the 5-HT2 and 5-HT1A subsystems on adult behavior in the rat.

Males, females, neonatally androgenized females, and neonatally castrated males were treated over the second week of life with 0.25 mg/kg of either the 5-HT2 agonist 1-(2,5-dimethoxy-3-iodophenyl)-2-aminopropane HCl (DOI), the 5-HT2 antagonist ritanserin (Rit), the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), or the 5-HT1A antagonist WAY100135 (WAY). Exploration, anxiety, sociosexual preferences, and sexual behavior were measured in adulthood. Agents acting on 5-HT1A receptors do not appear to affect organization of any of the behavioral systems studied. DOI increased exploratory activity but in females only, which suggests that testosterone antagonizes the stimulatory effect of 5-HT2 activity on exploration. Neonatal ritanserin selectively reduced anxiety in females, and DOI had a similar effect in androgenized females. This indicates that neonatal 5-HT2 activity is anxiogenic in normal females, anxiolytic in androgenized females, and has no effect on anxiety in males. Males and androgenized females both showed a preference for the female teaser that was abolished by the 5-HT2 agonist, DOI. These results point out that 5-HT2 activity selectively suppresses heterosexual preference induced in the presence of neonatal testosterone. DOI also reduced both male sexual behavior in males and female sexual behavior in androgenized females. Thus, the 5-HT2 system antagonizes the action of testosterone in stimulating heterosexual orientation and sexual activity, and this is independent of genetic sex.

Serotonin Inhibits Luteinizing Hormone Release via 5-HT1A Receptors in the Zona incerta of Ovariectomised, Anaesthetised Rats Primed with Steroids

The zona incerta (ZI), an area in the dorsal hypothalamus, contains neuronal systems that appear to control gonadotropin release. Previous findings show that there is an inverse relationship between serotonin (5-HT) activity in the ZI and plasma luteinizing hormone (LH) levels, indicating that the 5-HT system in this area has an inhibitory effect on LH release. Employing anaesthetised, ovariectomised rats primed with 5 µg oestradiol benzoate followed at 48 h by 0.5 mg progesterone, we have shown that 2 µg/side 5-HT in the ZI inhibits the LH surge that normally occurs 4 h after the progesterone treatment. This effect was mimicked by 2 µg/side 8-OH-DPAT, a 5-HT1A agonist, but not by DOI, a 5-HT2 agonist, BMY7378, a presynaptic 5-HT1A agonist or MCPP, a 2B & 2C agonist. The inhibitory effect of 5-HT and 8-OH-DPAT was prevented by pretreatment, 1 h before, with either 2 mg/kg i.p. WAY100135, a 5-HT1A antagonist or 0.25 mg/kg i.p. ritanserin, a 5-HT2 antagonist. These results indicate that 5-HT in the ZI exerts its inhibitory effect on LH release via 5-HT1A receptors but that another 5-HT subtype may also be involved.

Involvement of the 5-HT1A subtype receptor in the neonatal organization of agonistic behaviour in the rat

5-hydroxytryptamine (5-HT) interacts with testosterone (T) in the development of a number of neuronal systems controlling sexually dimorphic adult behaviours. In this report, we investigated this interaction on the organization of agonistic behaviour in males, females, androgenized females (250 micrograms/pup of T proprionate on the day of birth), and males castrated on the day of birth. We have shown previously that manipulating 5-HT2 activity over the 2nd week of life modulates adult agonistic behaviour, depending on genetic sex and the presence of T. In this report, we investigated the effects seen in adulthood of a 5-HT1A agonist [8-OH-DPAT, 0.25 mg/kg, intraperitoneally (i.p.)] and antagonist (WAY100135, 0.25 mg/kg, i.p.) given over days 8-16 postpartum. The test for agonistic behaviour was carried out in a neutral territory against a matched conspecific, and introductory, offensive and defensive activities were note. Results show that neonatal administration of the 5-HT1A antagonist WAY100135 increases introductory activity and defense in the presence of neonatal T, independent of genetic sex, because these effects were seen in sham-castrated males and androgenized females. Offence followed a similar pattern, in that it was increased by WAY100135, but only in males. In the case of defence, the effects of the antagonist were reinforced by the action of the agonist (8-OH-DPAT) in both males and females, indicating an inhibitory role of 5-HT1A perinatal activity on defence in the presence of malelike levels of circulating T and a facilitatory role when levels of T are low or negligible. These findings indicate that 5-HT1A activity is involved in the development of agonistic behaviour and the effects are influenced by T. The results also show that the offensive and defensive facets of agonistic activity are controlled differently.

Effects of perinatal diazepam exposure on the liver of rats.

Diazepam is routinely used in obstetrics in the treatment of maternal eclampsia or as anesthetic induction during deliveries or cesarean sections. We investigated here the effects of perinatal diazepam treatment on the liver of Sprague Dawley rats. From 15th day of gestation to birth pregnant rats were treated daily in the first experimental group (DZ1) with 2.5 mg DZ/kg b.w., s.c., the second (DZ2) with 5 and the third (DZ3) with 10 mg DZ/kg b.w. After birth respective treatment was continued to the pups till 5th day postnatal. One half of pups from DZ2 and control was sacrificed at 12th day, the other were raised to 120th day and then sacrificed. After fixation in formaldehyde serial paraffin sections were processed with proliferating cell nuclear antigen (PCNA), autofluorescence of lipofuscin and Elastica v. Gieson staining. Livers from 12 day old rats showed increased levels of PCNA marked hepatocytes and lipofuscin containing cells. Livers from 120 day old animals exhibited increased levels of PCNA cells especially with weakly stained nuclei, the number of lipofuscin containing cells were decreased in DZ1 and DZ2 but reached in DZ3 the similar number as control. Statistical analysis showed that quantification of Kupffer cells lacked significance. To conclude livers of 12 day old animals demonstrated a stimulated DNA synthesis and an accumulation of nondegradable residues. Livers from 120 day old rats demonstrated an increase in number of potentially proliferating hepatocytes by increased levels of weakly stained PCNA nuclei. At lower dosage DZ seems to inhibit lipofuscinogenesis, at highest dosage DZ accumulates non-degradable residues. Elastica v. Gieson staining was not suitable to quantify Kupffer cells with certainty.