Ovarian steroid regulation of serotonin-1A autoreceptor messenger RNA expression in the dorsal raphe of rhesus macaques.

It is widely hypothesized that ovarian steroids act on serotonin neurons to modulate mood and alter neuroendocrine function in women. However, information is needed on the molecular consequences of estrogen and progesterone action in serotonin neurons. This study examined the effect of estrogen, with and without progesterone, on the expression of messenger RNA for the serotonin-1A autoreceptor in monkeys using in situ hybridization and a 432-bp serotonin-1A probe generated with polymerase chain reaction. Monkeys were spayed/ovariectomized (control; n=4), estrogen treated (28 days, n=4) and estrogen+progesterone treated (14 days estrogen+14 days estrogen+progesterone, n=4). Perfusion-fixed midbrain sections containing the dorsal raphe (10 microm) were hybridized at 60 degrees C with 35S antisense complementary RNA. After a final wash in 0.1 x standard saline citrate at 70 degrees C, sections were apposed to betamax film for four days and then emulsion fixed. Adjacent sections were immunostained for serotonin to confirm the location of the dorsal raphe. Densitometric analysis of autoradiographs with gray level thresholding was performed at five levels of the dorsal raphe. The number of pixels exceeding background in defined areas was obtained (pixel number), as well as the mean optical density. In the estrogen- and the estrogen+progesterone-treated groups compared to the control group, there was a 38% and 43% decrease in serotonin-1A messenger RNA signal, respectively, represented by pixel number (P<0.05). Mean optical density for serotonin-1A was significantly decreased by estrogen treatment (21%; P<0.05) and then further decreased with the addition of progesterone treatment (45%; P<0.01). Also, the number of positive cells and the grains/cell were counted. There were significantly fewer serotonin-1A messenger RNA-positive cells in the serotonergic neurons of the dorsal raphe in estrogen- and estrogen+progesterone-treated groups (P<0.001) than controls. There were significantly lower single-cell levels of serotonin-1A messenger RNA in serotonergic neurons of the dorsal raphe only in the estrogen+progesterone-treated group (P<0.05). These results suggest that estrogen reduces serotonin-1A gene expression and that the addition of progesterone further reduces serotonin-1A gene expression in non-human primates. If the changes in gene expression are manifested by alterations in protein expression, then, together, these actions of estrogen and progesterone could increase serotonin neurotransmission, thereby elevating mood and/or altering neuroendocrine functions.

Ovarian steroid effects on serotonin 1A, 2A and 2C receptor mRNA in macaque hypothalamus.

This study mapped the location of serotonin (5HT) 1A, 2A and 2C receptor mRNA expression in the female macaque hypothalamus and determined whether the expression was regulated by estrogen plus or minus progesterone treatment using in situ hybridization (ISH) and densitometric analysis of autoradiographic films. The experimental groups of pigtail macaques (Macaca nemestrina) were spayed controls (n=4), estrogen treated (28 days, n=4) and estrogen+progesterone-treated animals (14 days estrogen+14 days estrogen and progesterone, n=4). Monkey specific 5HT1A (432 bp), 2A (411 bp) and 2C (294 bp) receptor probes were generated with PCR. Moderate 5HT1A receptor hybridization signal was detected in the preoptic area and the ventromedial nuclei. Less intense 5HT1A receptor signal was detected in a contiguous area from the dorsomedial nuclei through the posterior hypothalamus and in the supramammillary area. There was no change in 5HT1A receptor hybridization signal in any area with ovarian steroid treatment. Dense 5HT2A receptor hybridization signal was morphologically confined to the paraventricular, supraoptic, and mammillary nuclei and the external capsule of the thalamus. Light 5HT2A mRNA signal was inconsistently observed in the ventromedial nuclei. There was no change in the 5HT2A receptor hybridization signal in any area with ovarian steroid treatment. The 5HT2C receptor mRNA was widely distributed in the macaque hypothalamus. The preoptic area and anterior hypothalamus were largely positive for 5HT2C mRNA with a more concentrated signal in a narrow periventricular area. Dense 5HT2C receptor signal was detected lateral to the ventromedial nuclei (capsule), in the tuberomammillary nuclei, arcuate nucleus, dorsomedial nuclei, infundibular area and choroid plexus. Moderate 5HT2C receptor signal was detected in the ventromedial nuclei, lateral hypothalamus and dorsal to posterior hypothalamus. There was a significant decrease in total 5HT2C mRNA hybridization signal with ovarian steroid treatment in the ventromedial nuclei, dorsal and posterior hypothalamus. In summary, macaque 5HT1A, 2A and 2C receptor mRNAs are located in distinct hypothalamic loci which play a role in a number of autonomic functions and behavior. Ovarian steroids decreased the expression of 5HT2C receptor mRNA in the ventromedial nuclei, dorsal and posterior hypothalamus. The expression of 5HT1A and 5HT2A receptor mRNA was not altered by treatment with ovarian steroids.

Regulation of serotonin re-uptake transporter mRNA expression by ovarian steroids in rhesus macaques.

It has been widely hypothesized that the ovarian steroids, estrogen (E) and progesterone (P), act on serotonin neurons to modulate mood and increase prolactin secretion in women. However, information is needed on the molecular consequences of ovarian hormone action in serotonin neurons. This study examined the effect of E and P on the expression of mRNA for the serotonin re-uptake transporter (SERT) in monkeys using in situ hybridization and a 253 bp human SERT cRNA probe. Monkeys (n=5 animals/group) were ovariectomized and hysterectomized (spayed) and then untreated (control), or treated, with E for 28 days (E treated) or treated with E for 28 days and supplemented with P for the last 14 days of the E regimen (E+P treated). Densitometric analysis of autoradiographs with gray-level thresholding was performed at five levels of the dorsal and median raphe. The number of pixels exceeding background in defined areas was obtained (pixel number). The average pixel number for spayed, E- and E+P-treated groups was 22 280+/-3517, 15 227+/-1714, and 14 827+/-2042, respectively, in the combined dorsal and median raphe. In the E- and E+P-treated groups compared to the control group, there was a 32% and 33% decrease in SERT mRNA signal represented by pixel number (ANOVA, P<0.05). Hence, E- and E+P-treated groups were significantly less than the control group, but they were not different from one another. Also, there were significantly fewer SERT mRNA-positive cells in the dorsal raphe of E- and E+P-treated groups (ANOVA, P<0.001). Therefore E, with or without P, reduces SERT mRNA expression. These results suggest that the ability of P to increase prolactin secretion in E-primed monkeys does not involve an action at the level of SERT gene transcription. Hence, the mechanism by which the CNS transduces the action of P on prolactin secretion remains to be elucidated. However, these data suggest that one action of E replacement therapy in postmenopausal women may be to decrease expression of the SERT gene.

Ovarian steroids and serotonin neural function.

The serotonin neural system originates from ten nuclei in the mid- and hindbrain regions. The cells of the rostral nuclei project to almost every area of the forebrain, including the hypothalamus, limbic regions, basal ganglia, thalamic nuclei, and cortex. The caudal nuclei project to the spinal cord and interact with numerous autonomic and sensory systems. This article reviews much of the available literature from basic research and relevant clinical research that indicates that ovarian steroid hormones, estrogens and progestins, affect the function of the serotonin neural system. Experimental results in nonhuman primates from this laboratory are contrasted with studies in rodents and humans. The sites of action of ovarian hormones on the serotonin neural system include effects within serotonin neurons as well as effects on serotonin afferent neurons and serotonin target neurons. Therefore, information on estrogen and progestin receptor-containing neurons was synthesized with information on serotonin afferent and efferent circuits. The ability of estrogens and progestins to alter the function of the serotonin neural system at various levels provides a cellular mechanism whereby ovarian hormones can impact mood, cognition, pain, and numerous other autonomic functions.

RU 486 blocks and fluoxetine augments progesterone-induced prolactin secretion in monkeys.

Progesterone (P) stimulates prolactin secretion through an unknown neural mechanism in estrogen (E)-primed female monkeys. Serotonin also stimulates prolactin secretion and this laboratory demonstrated that E induces nuclear progestin receptors (PR) in serotonin neurons. Thus, PR in serotonin neurons could transduce the action of P on prolactin secretion. Studies were performed to determine (1) whether blocking nuclear PR would block P-induced prolactin secretion and conversely; (2) whether increasing serotonin concentrations in the synapse would augment P-induced prolactin secretion. In both studies, female monkeys were spayed, adapted to a vest and tether remote sampling system and catheterized prior to experiments. Monkeys received 2 E-filled silastic implants (3.0 cm) 1-3 weeks prior to study. P (20 mg) in corn oil was injected (s.c.) to transiently increase prolactin secretion. In both studies, each monkey served as its own control. To block nuclear PR and not membrane PR, RU 486 (2 mg/kg, i.m.) or ethanol (control) was administered with the P injection. Relative to the P injection, blood samples were taken twice daily from -30 to +24 h, then every 4 h from +36 to +48 h and once at +65 h. To increase serotonin in the synapse, the serotonin reuptake inhibitor, fluoxetine (5 mg/day, i.v.), was infused for 4 weeks. P was injected during the week of vehicle infusion and during the last week of fluoxetine infusion. Blood samples were obtained twice daily prior to and following P treatment. Prolactin, E, P and RU 486 concentrations were determined by RIA. RU 486 completely blocked the P-induced prolactin surge (n = 3). In addition, fluoxetine significantly increased prolactin secretion during the P-induced prolactin peak compared to equal time points during saline infusion (n = 5). These data indicate that P induces prolactin via a genomic mechanism and not through a membrane action. The data also support a pivotal role for serotonin in the neural regulation of P-induced prolactin secretion.

Effects of progesterone on blood pressure, plasma volume, and responses to hypotension.

Gonadal steroids have been implicated in the control of blood pressure and fluid homeostasis. These experiments test the effect of progesterone in ovariectomized ewes on blood pressure, volume, and hormone responses to hypotension. Eight ewes were each studied in four conditions: ovariectomized, progesterone and estrogen replaced, progesterone replaced, or sham treated. During each treatment mean arterial pressure (MAP), plasma volume (PV), baroreflex responsiveness, and adrenocorticotropic hormone (ACTH), arginine vasopressin (AVP), and renin responses to hypotension were determined. Progesterone treatment significantly reduced resting MAP and increased PV compared with ovariectomy or sham treatments. Heart period at 85 mmHg was reduced with progesterone treatment. There was no effect of progesterone treatment on ACTH, AVP, or renin or heart rate responses to hypotension. Basal AVP levels were increased, and angiotensin II concentrations were decreased, by estrogen and progesterone and by sham treatments; plasma Na+ also tended to be increased during these treatments. These results suggest that a small increase in progesterone can reset resting MAP and PV without altering reflex heart rate or endocrine responses to hypotension.

Ovarian steroid regulation of tryptophan hydroxylase mRNA expression in rhesus macaques.

Progesterone (P) stimulates prolactin secretion through an unknown neural mechanism in estrogen (E)-primed female monkeys. Serotonin is a stimulatory neurotransmitter in prolactin regulation, and this laboratory has shown previously that E induces progestin receptors (PR) in serotonin neurons. Therefore, we questioned whether E and/or E+P increased serotonin neural function. The expression of mRNA for tryptophan hydroxylase (TPH) was examined in ovariectomized (spayed) control, E-treated (28 d), and E+P-treated monkeys (14 d E and 14 d E+P) using in situ hybridization and a 249 bp TPH cRNA probe generated with RT-PCR (n = 5 animals/group). Densitometric analysis of film autoradiographs revealed a ninefold increase in TPH mRNA in E-treated macaques compared to spayed animals (p < 0.05). With supplemental P treatment, TPH mRNA signal was increased fivefold over spayed animals (p < 0.05), but was not significantly different compared to E-treated animals. These results were verified by grain counts from photographic emulsion-coated slides. There were significantly higher single-cell levels of TPH mRNA in serotonergic neurons of the dorsal raphe in E- and E+P-treated groups (p < 0.05). These data indicate that E induces TPH gene expression in nonhuman primates and that the addition of P has little additive effect on TPH gene expression. Thus, the action of P on prolactin secretion is probably not mediated at the level of TPH gene transcription. However, because P increases raphe serotonin content in E-primed rodents, the possibility remains that P may have other actions on post-translational processing or enzyme activity.

Beta-endorphin, but not oxytocin, substance P or vasoactive-intestinal polypeptide, contributes to progesterone-induced prolactin secretion in monkeys.

Progesterone (P) stimulates prolactin secretion through a neural mechanism in estrogen (E)-primed female monkeys. Several peptides, including beta-endorphin (BE), oxytocin (OT), substance P (SP) and vasoactive intestinal polypeptide (VIP) are potential prolactin stimulatory factors and could mediate the effect of P. We hypothesized that the antagonism of a pivotal peptidergic neural system would block P-induced prolactin secretion and that the function of a pivotal peptidergic system would be altered by changes in gonadal steroid concentrations. Therefore it was of interest (1) to examine the effect of infusion of antagonists to these peptides on P-induced prolactin secretion, and (2) to determine BE, OT, SP and VIP levels in the hypothalamus of monkeys of various reproductive states. For the antagonist studies, female monkeys (n = 8) were spayed, adapted to a vest and tether remote sampling system and catheterized prior to antagonist challenges. E-primed monkeys received P injections 48 h prior to antagonist administration. Prolactin increased within 36-48 h of P injection. All antagonist challenges were administered in varying doses during the P-induced prolactin elevation and blood samples were collected every 10 min for prolactin determinations. The opiate antagonist, naloxone (n = 5), reduced serum prolactin in a dose-related manner with a mean IC50 of 1.5 +/- 0.6 micrograms/kg/min. The OT (n = 4), SP (n = 4) or VIP (n = 4) antagonists did not reduce serum prolactin in a dose-related manner. We previously reported that the hypothalamic content of OT is increased by ovarian hormones. To determine whether the hypothalamic content of BE, SP or VIP was related to gonadal status, the peptide levels in 4 hypothalamic regions of monkeys in various physiological states were measured. BE (ng/mg protein) in the medial basal hypothalamus (MBH) was significantly greater in adult females (17.7 +/- 6.9; n = 6) as compared to spayed females (0.6 +/- 0.2; n = 3) and juvenile females (1.8 +/- 1.1; n = 3). Hypothalamic content of SP in the preoptic area and mammillary bodies, but not the MBH, was significantly greater in gonadal intact females than spayed females. VIP content (pg/mg protein) was not significantly different between adult, spayed and juvenile females nor between adult and juvenile males in any hypothalamic area. Taken together these results support a pivotal role for BE in the neural regulation of P-induced prolactin secretion. The involvement of OT, SP, and VIP in a specific manner at the pituitary level is not indicated.

Prolonged absence of ovarian hormones in the ewe reduces the adrenocorticotropin response to hypotension, but not to hypoglycemia or corticotropin-releasing factors.

The ACTH responses to hypotension, hypoglycemia, CRF, arginine vasopressin (AVP), and the combination of CRF and AVP were compared to determine whether there was a general decrease in ACTH responses to these stimuli in ovariectomized ewes compared to intact animals. The ovariectomized ewes were studied either 2-4 weeks post-ovariectomy (acute) or 4-7 months post-ovariectomy (chronic). Each ewe was subjected to saline control infusion, nitroprusside-induced hypotension (100 micrograms/min for 10 min), insulin-induced hypoglycemia (25 U porcine insulin), CRF (1 microgram/min for 60 min), AVP (0.2 microgram/min), and a combination of CRF plus AVP. In each experiment, plasma ACTH concentrations were measured at 10-min intervals for 1 h. The peak ACTH concentrations were significantly lower in response to hypotension in the chronic ewes compared to those in either the intact or acute group. The ACTH response to hypoglycemia was not significantly reduced in either ovariectomized group. The ACTH responses to CRF, AVP, and the combination of both were not significantly reduced in either ovariectomized group. The results suggest that the effect of ovariectomy on the ACTH response to stress occurs at a site within the brain and does not involve altered pituitary responsiveness to CRF or AVP.

Behavioral and neurochemical changes caused by repeated ethanol and cocaine administration.

Combined cocaine and ethanol abuse has become increasingly popular, yet research on the behavioral and neurochemical interactions of these two substances is limited. Four groups of male rats received either daily cocaine (10 mg/kg, IP) or saline injections with either water (groups C and S) or only 15% ethanol to drink (groups CE and E). Initially, locomotor activity was increased equally by ethanol or cocaine and to the greatest extent by both. After 2 weeks of drug treatment, group C exhibited behavioral sensitization to cocaine, group E exhibited ethanol tolerance and group CE exhibited greater cocaine sensitization with no indication of ethanol tolerance. In support of enhanced sensitization to cocaine, amphetamine-stimulated 3H-dopamine (DA) release in striatum and D2 DA receptor binding in the nucleus accumbens (NAC) were increased in group CE compared to group C. In support of a loss of ethanol tolerance, increases in striatal D2 DA and 35S-TBPS binding seen in group E (which exhibited ethanol tolerance) were absent in group CE (which did not exhibit tolerance). Thus, the synergistic effect of ethanol and cocaine on behavior may be due to complex interactions of these two drugs both on DA and GABA transmission in mesolimbic and nigrostriatal areas.

Selective changes in GABAergic transmission in substantia nigra and superior colliculus caused by ethanol and ethanol withdrawal.

One of ethanol's actions after acute exposure is anticonvulsant activity whereas withdrawal from chronic ethanol exposure increases convulsant activity. An increase in neuronal transmission in the GABAergic pathways from striatum to the substantia nigra (SN) and a decrease in GABAergic transmission from SN to superior colliculus (SC) both appear to play a major role in inhibiting seizure propagation. If this is the case, then the changes in seizure sensitivity caused by ethanol may be expected to affect GABAergic transmission in opposite ways in SN and SC. We measured the effects of in vitro ethanol on pre- and postsynaptic indices of GABA transmission using SN and SC tissue from both ethanol-naive rats and rats given ethanol in their drinking water for 24 days and then withdrawn for 24 hr, a treatment that decreases seizure latency. While ethanol inhibited 3H-GABA release from slices of SC at low concentrations (20-100 nM), much higher concentrations were required to inhibit release from SN (100-500 mM). In fact, release from SN was increased by low concentrations of ethanol. Ethanol in vitro (20-1000 mM) also inhibited specific binding of 35S-TBPS to the GABAA receptor but this effect was similar in both potency and efficacy in SC and SN. Next, the in vitro effects of ethanol were measured in rats that had consumed an average of 9.8 g ethanol/kg body weight/day and were then withdrawn for 24 hr. Ethanol inhibition of 3H-GABA release from SC was significantly less in ethanol-treated rats compared to controls whereas the inhibitory effect of ethanol was increased in SN from ethanol-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)