Metabotropic glutamate receptor in GHRH and beta-endorphin neurones of the hypothalamic arcuate nucleus

Growth hormone-releasing hormone (GHRH) and beta-endorphin are mainly synthesized in neurones of the hypothalamic arcuate nucleus. Arcuate neurones also contain both ionotropic and metabotropic glutamate receptors. The aim of present study was to investigate whether glutamate receptors are present in GHRH and beta-endorphin containing nerve cells of this hypothalamic area. Using double-label immunocytochemistry as well as the mirror technique, we found that almost all GHRH and beta-endorphin immunoreactive arcuate neurones contain the metabotropic glutamate receptor la. The observations provide morphological evidence for the view that glutamate, which appears to be a major excitatory neurotransmitter in the hypothalamus, may directly stimulate GHRH and beta-endorphin neurones of the medial hypothalamus.     

Developmental changes in synaptic formation in the hypothalamic arcuate nucleus of female rats

The hypothalamic arcuate nucleus (ARCN) of female rats at 5, 20, 45 and 90 days of age was examined ultrastructurally. Axodendritic and axosomatic synapses were counted in 18,000 mum2 area of the ARCN in each brain. Axodendritic and axosomatic synapses in the ARCN of day 5 rats were very small in number. Axon terminals contained small spherical vesicles (SSVs, 40-60 nm in diameter). Occasionally large granular vesicles (LGVs, 75-130 nm in diameter) were found to coexist with SSVs in the endings. Pre- and postsynaptic membranes were thin. The ARCN at this age exhibited a large extracellular space which decreased with advancing age. In day 20 rats, axodendritic and axosomatic synapses increased in number up to about one-half of those of day 45 or day 90 animals. Synaptic vesicles increased in number and mitochondria were frequently encountered in the axon terminals. Pre- and postsynaptic membranes became thicker than those of day 5 rats. Further increase in the number of axodendritic and axosomatic synapses in the ARCN of day 45 rats was observed, and there were no significant difference in the morphology and incidence of synapses between day 45 and day 90 rats. Synaptic vesicles were numerous and pre- and postsynaptic membranes were thick. In tissue incubated with 5-hydroxydopamine (5-OH-DA) before fixation, small granular vesicles (SGVs, about 50 nm in diameter) which were labeled with 5-OH-DA were detected in a certain number of endings in all material taken from each age group, but the incidence of synapses containing SGVs was usually low. From these results, it can be proposed that an increase in the number of synapses in the ARCN is correlated wihh functional maturation of the ARC neurons.     

Lectinhistochemistry and ultrastructure of microglial response to monosodium glutamate-mediated neurotoxicity in the arcuate nucleus

The reproducibility of serotonin (5-HT) and (+)8-OH-DPAT-mediated inhibition of adenylyl cyclase activity was assessed in membranes, stimulated by forskolin, of rat frontal cortex postmortem as well as of human fronto-cortical, hippocampal and dorsal raphe tissues obtained from autopsy brains. The results revealed that differences between basal and forskolin-stimulated enzyme activities were still significant after 48 h postmortem in rat cortex and in all human brain regions up to 46 h after death. However, a decrease of about 17 and 26% in forskolin-stimulated adenylyl cyclase activity was observed at 24 and 48 h, respectively, in rat cortex. 5-HT and the 5-HT1A receptor agonist, (+)8-hydroxy-2(di-N-propylamino)tetraline (8-OH-DPAT), were able to inhibit forskolin-stimulated adenylyl cyclase activity in a dose-dependent manner for 48 h after death in rat and human brain. In rat cortex, both 5-HT and (+)8-OH-DPAT potencies (EC50, nM) and efficacies (percent of maximum inhibition capacity, %) varied significantly with postmortem delay. Conversely, in human tissues, postmortem delay and subject age did not modify agonist potencies and efficacies. Furthermore, a regionality of 5-HT potency and efficacy was revealed in the human brain. 5-HT was equally potent in cortex and raphe nuclei, while being more potent but less effective in hippocampus. (+)8-OH-DPAT was more active in hippocampus and raphe nuclei than in cortex. (+)8-OH-DPAT behaved as an agonist in all areas, as its efficacy was similar or greater than those obtained with 5-HT. The (+)8-OH-DPAT dose-response curve was completely reversed by 5-HT1A receptor antagonists in rat cortex and all human brain areas. In conclusion, we suggest here that differences between rat and human brain might exist at the level of postmortem degradation of 5-HT-sensitive adenylyl cyclase activity. In human brain, 5-HT1A receptor-mediated inhibition of adenylyl cyclase seems to be reproducible, suggesting that reliable experiments can be carried out on postmortem specimens from patients with neuropsychiatric disorders.     

Galanin receptor-mediated inhibition of glutamate release in the arcuate nucleus of the hypothalamus

It is thought that galanin, a 29 amino acid neuropeptide, is involved in various neuronal functions, including the regulation of food intake and hormone release. Consistent with this idea, galanin receptors have been demonstrated throughout the brain, with high levels being observed in the hypothalamus. However, little is known about the mechanisms by which galanin elicits its actions in the brain. Therefore, we studied the effects of galanin and its analogs on synaptic transmission using an in vitro slice preparation of rat hypothalamus. In arcuate nucleus neurons, application of galanin resulted in an inhibition of evoked glutamatergic EPSCs and a decrease in paired-pulse depression, indicating a presynaptic action. The fragments galanin 1-16 and 1-15 produced a robust depression of synaptic transmission, whereas the fragment 3-29 produced a lesser degree of depression. The chimeric peptides C7, M15, M32, and M40, which have been reported to antagonize some actions of galanin, all produced varying degrees of depression of evoked EPSCs. In a minority of cases, C7, M15, and M40 antagonized the actions of galanin. Analysis of mEPSCs in the presence of TTX and Cd2+, or after application of alpha-latrotoxin, indicated a site of action for galanin downstream of Ca2+ entry. Thus, our data suggest that galanin acts via several subtypes of presynaptic receptors to depress synaptic transmission in the rat arcuate nucleus.     

Nitric oxide synthase-, N-methyl-D-aspartate receptor-, glutamate- and aspartate-immunoreactive neurons in the mouse arcuate nucleus: effects of neonatal treatment with monosodium glutamate

Glutamate-, aspartate-, N-methyl-D-aspartate receptor (NMDAR1 and 2 subunits)-, and nitric oxide synthase (NOS)-immunoreactive neurons were studied in the arcuate nucleus (AN) of mice treated neonatally with monosodium glutamate (MSG) which is known to cause extensive neuronal loss in this hypothalamic nucleus. It was found that intensely stained glutamate- and aspartate-immunoreactive neurons present in the AN of control mice were completely absent in the MSG-lesioned AN as well as the ventromedial nucleus lateral to the AN. Similarly, NMDAR1-immunoreactive neurons were mostly absent in the MSG-lesioned AN but remained intact in the ventromedial nucleus. There was also a substantial loss of NMDAR2 immunoreactivity within the AN. In contrast, NOS-immunoreactive neurons in the AN survived the neonatal glutamate treatment, although they appeared to be less intensely stained.     

Effect of 6-hydroxydopamine injection into the arcuate hypothalamic nucleus on the osmotic release of vasopressin in conscious rats

The aim of the present study was to evaluate a role in vasopressin secretion of the catecholaminergic neurons, including the tuberohypophysial dopaminergic neurons situated in the arcuate hypothalamic nucleus. A neurotoxin, 6-hydroxydopamine (6 g/l), was injected locally into the arcuate nucleus and its effects on catecholamine levels of the hypothalamic tissue and the neurointermediate lobe, and on the plasma vasopressin concentrations before and during i.v. infusion (0.1 ml kg-1 min-1) of isotonic (0.15 mol/l) or hypertonic saline (2.5 mol/l), were examined in conscious rats. The infusion of hypertonic saline produced increases of plasma vasopressin 15 and 30 min later, accompanied by elevations of plasma osmolality, sodium, chloride and arterial pressure. The vasopressin response was potentiated markedly by the 6-hydroxydopamine injection performed 8 days before, which hardly affected the responses of the other variables. Histological examination indicated that the injection sites of 6-hydroxydopamine in those rats had been located in the area ranging from rostral to medial arcuate nucleus. The i.v. infusion of isotonic saline did not change plasma vasopressin, osmolality, sodium, chloride or arterial pressure, regardless of the presence or absence of pretreatment with 6-hydroxydopamine. It was confirmed that when 6-hydroxydopamine was injected into the arcuate nucleus region 8 days before, noradrenaline and adrenaline concentrations of the hypothalamic tissue containing the injection site were decreased remarkably, although we could not detect any significant alteration in the dopamine concentration of the hypothalamic tissue or the neurointermediate lobe. On the basis of these results, we concluded that catecholaminergic neurons in the arcuate nucleus may act to inhibit osmotic vasopressin secretion.     

Effect of neonatal treatment with MSG (monosodium glutamate) on thyroid of the adult male rats

Percutaneous transluminal angioplasty (PTA) has been well described in the treatment of mesenteric artery stenoses but has met with limited success in ostial lesions. The authors describe a case of a 79-year-old woman diagnosed with chronic mesenteric ischemia associated with a 22-pound weight loss and postprandial pain. The celiac axis and inferior mesenteric artery were occluded. A high-grade, calcified stenosis was present in the proximal superior mesenteric artery. This was treated with primary stent placement using a Palmaz stent deployed from an axillary approach. A brief discussion of mesenteric ischemic and visceral artery PTA is included.     

The arcuate nucleus is the major source for neuropeptide Y-innervation of thyrotropin-releasing hormone neurons in the hypothalamic paraventricular nucleus

Neuropeptide Y (NPY) immunoreactive (-ir) nerve fibers densely innervate hypophysiotropic TRH perikarya and dendrites in the hypothalamic paraventricular nucleus (PVN). To evaluate the contribution of the arcuate nucleus (Arc) to this innervation, the effect of Arc ablation by neonatal monosodium glutamate (MSG) treatment on the density of NPY-fibers contacting TRH neurons in the PVN was investigated. After the lesioned animals and vehicle-treated controls reached adulthood, the number of contacts between NPY-ir boutons and TRH-ir perikarya in the PVN was determined in double-immunostained sections. In controls, numerous contacts between NPY-ir terminals and TRH perikarya and dendrites were observed, confirming earlier findings. MSG treatment resulted in a marked reduction of the size of the Arc and also the number of NPY-perikarya with a concomitant reduction of 82.4 +/-2.1% in the relative number of NPY terminals contacting TRH perikarya and first order dendrites in the medial parvocellular and periventricular subdivisions of the PVN. In contrast, lesioning of the ascending adrenergic bundle in the brain stem caused no statistically significant change in the number of NPY-terminals in close apposition to hypophysiotropic TRH neurons in the PVN. These data confirm earlier findings that NPY-containing axon terminals innervate TRH neurons in the PVN and further demonstrate a potentially important anatomical relationship between NPY-producing neurons in the Arc and hypophysiotropic TRH neurons.     

Exogenous glutamate enhances glutamate receptor subunit expression during selective neuronal injury in the ventral arcuate nucleus of postnatal mice

Much attention has been paid to proteinases derived from not only neurons but also microglia in relation to neuronal death. There is accumulating evidence that intra- and extracellular proteinases in these cells are part of the basic machinery of neuronal death pathways. Some members of the ced-3/interleukin-1 beta converting enzyme (ICE) (caspase) family of cysteine proteinases have been thought to play a major role in apoptosis of not only non-neuronal cells but also neurons. Calpain has also been demonstrated to be a mediator of the neurodegenerative response. Recent studies have shown that excitotoxic and ischemic neuronal injury could be attenuated by inhibitors of caspases and calpain. Several recent studies have suggested the involvement of endosomal/lysosomal proteinases, including cathepsins B, D and E, in neuronal death induced by excitotoxins and ischemia. Furthermore, it has been reported that the extracellular tissue-type plasminogen activator/plasmin proteolytic cascade is involved in excitotoxic injury of the hippocampal neurons. In addition to such neuronal proteinases, microglial proteinases are believed to be important for the modification of neuronal functions positively or negatively. Cathepsins E and S derived from microglia have been suggested to contribute to neuronal survival through degradation and removal of beta-amyloid, damaged neurons and cellular debris. On the other hand, 6-hydroxydopamine-induced microglial cell death was inhibited by inhibitors of aspartic proteinases and caspases, suggesting the involvement of cathepsins E and D and caspases in microglial cell death. Therefore, identification of which proteinases play a causative role in neuronal death execution and clarification of the regulators and substrates for such proteinases is very important for understanding the molecular basis of the neuronal death pathways and to develop novel neuroprotective agents.     

Atropine fails to block the overconsumption of sugar solutions by hypothalamic hyperphagic rats.

Adult female Sprague-Dawley rats given bilateral parasagittal knife cuts in the medial hypothalamus (VMH group) were hyperphagic and became obese on a chow diet, compared with sham-operated controls. VMH Ss also overconsumed, relative to controls, sucrose and glucose solutions during 30-min/day tests. Pretreating VMH and control Ss with atropine methyl nitrate (1.0, 5.0, or 10.0 mg/kg) reduced their intake of the sugar solutions in 3 of 5 experiments, and in all experiments it suppressed their 24-hr chow intake. However, VMH Ss continued to drink more of the sugar solutions than the controls after all atropine treatments, and in 3 of 4 experiments their hyperphagia on the chow diet was not blocked by the atropine. Results do not support the hypothesis that vagally stimulated insulin release or other cholinergically mediated cephalic responses of digestion are essential for the expression of hypothalamic hyperphagia and finickiness. (44 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Seasonal neuroendocrine rhythms in the male Siberian hamster persist after monosodium glutamate-induced lesions of the arcuate nucleus in the neonatal period

The aim of these experiments was to examine the role of the arcuate nucleus in the control of seasonal cycles of body weight, feed intake, moulting and reproduction in the Siberian hamster. The arcuate nucleus has previously been implicated as a central site where systemic feedback signals (e.g. leptin) might act to regulate feed intake and body weight, so it was predicted that hamsters with lesions of this structure would be unable to display the inhibitory effects of short days on these parameters. In the first series of studies, lesions that destroyed approximately 80% of the cells in the arcuate nucleus were produced by treating hamsters neonatally with monosodium glutamate (MSG; 4 mg/g body weight sc), and vehicle- and MSG-treated males were raised from birth in long days (LD) or short days (SD). In hamsters raised in LD, the initial gain in body weight and testicular growth were significantly reduced by MSG treatment, however, growth rate and testis weight were still significantly greater than in vehicle- or MSG-treated hamsters raised in SD. In the second study, hamsters treated neonatally with vehicle or MSG were raised in LD for 8 weeks and, subsequently, approximately half in each group were transferred to SD for 18 weeks. As expected, vehicle-treated hamsters showed a characteristic decline in body weight when exposed to SD, while those remaining in LD continued to increase body weight. Feed intake decreased in parallel with the decline in body weight in SD, a complete moult to the white winter pelage occurred by 16 weeks in SD, and testicular regression occurred. Responses to SD also occurred in the MSG-treated hamsters: body weight decreased in SD but increased in their lesioned litter mates remaining in LD, and feed intake paralleled body weight changes in these groups. The moult to winter pelage was significantly retarded in MSG-treated hamsters transferred to SD. The testes were completely regressed in sham- and MSG-treated hamsters exposed to SD, whereas testes weights in MSG-treated hamsters maintained in LD were intermediate between those in vehicle-treated hamsters in SD and LD. Thus, despite initial effects on growth, the MSG-treated hamsters bearing substantial lesions of the arcuate nucleus were able to show appropriate responses to photoperiod, although not always of the same magnitude as the unlesioned controls. We conclude that feedback mechanisms operating via the arcuate nucleus are not the major regulators of seasonal cycles of body weight, feed intake, pelage and reproduction.     

Zinc metabolism and homeostasis in rats fed a wide range of high dietary zinc levels

Zinc metabolism and homeostasis were studied in young growing rats fed a 38 ppm zinc diet with added zinc levels ranging from 0 to 8400 ppm for 21 days. High dietary zinc did not cause toxicity symptoms. Stable zinc in feces increased linearly with dietary zinc intake but fecal 65Zn, from a single oral dose, did not increase above the 1200 ppm dietary level. Stable zinc in liver, kidney, and tibia increased two to three times with 2400 ppm added zinc, but was not further elevated until 8400 ppm was fed. Stable zinc in muscle and heart was not affected appreciably by dietary zinc level. In all tissues, 65Zn retention was drastically reduced with 1200 ppm added dietary zinc. Additional dietary zinc reduced 65Zn in muscle and heart but had little effect on liver and kidney 65Zn. The data indicate that rats have fairly effective homeostatic control mechanisms for tissue zinc below about 7200 ppm dietary zinc. Whereas, with dietary zinc up to about 1200 ppm, decreasing absorption is the main route of homeostatic control, above this level, more rapid zinc turnover rates and increasing endogenous zinc excretion appear to have major importance.     

Estradiol implants in the arcuate nucleus induce lactogenesis in virgin rats. Role of progesterone

Peroxisomal proliferators induce in rodents hepatic hyperplasia and hypertrophy; the significant increase in the peroxisomal population is accompanied by specific and reversible induction of some peroxisomal enzymes. In suckling rats born from clofibrate-treated mothers, a massive removal of proliferated organelles occurs within 3 days of recovery. In the present paper we examined the early stages of the recovery period in liver of male rats treated with clofibrate for 5 days. The lysosomal involvement in the removal of drug-induced peroxisomes was investigated under physiological conditions, i.e. in the absence of inhibitors of the autophagic process. Biochemical results indicate that peroxisomal beta-oxidation, but not catalase activity, returns to the control values within the examined period. Total acid phosphatase activity is not affected by clofibrate treatment, but following fractionation on a linear density gradient the lysosomal marker enzyme activity is shifted towards lower density values, particularly at day 1 and 2 of recovery. This class of organelles possibly represents lysosomes involved in active autophagic processes. Acid phosphatase cytochemistry shows an increase of lysosome number at day 1 of recovery. Combination of acid phosphatase cytochemistry either with catalase cytochemistry or with catalase immunogold labelling allows to reveal organelles containing both marker enzymes. These results strongly support the involvement of autophagic processes in the removal of proliferated peroxisomes.     

Modulation of multiple potassium currents by metabotropic glutamate receptors in neurons of the hypothalamic supraoptic nucleus

We studied the effects of activation of the metabotropic glutamate receptors on intrinsic currents of magnocellular n urons of the supraoptic nucleus (SON) with whole cell patch-clamp and conventional intracellular recordings in coronal slices (400 micron) of the rat hypothalamus. Trans-(+/-)-1-amino-1,3-cyclopentane dicarboxylic acid (trans-ACPD, 10-100 microM), a broad-spectrum metabotropic glutamate receptor agonist, evoked an inward current (18.7 +/- 3.45 pA) or a slow depolarization (7.35 +/- 4.73 mV) and a 10-30% decrease in whole cell conductance in approximately 50% of the magnocellular neurons recorded at resting membrane potential. The decrease in conductance and the inward current were caused largely by the attenuation of a resting potassium conductance because they were reduced by the replacement of intracellular potassium with an equimolar concentration of cesium or by the addition of potassium channel blockers to the extracellular medium. In some cells, trans-ACPD still elicited a small inward current after blockade of potassium currents, which was abolished by the calcium channel blocker, CdCl2. Trans-ACPD also reduced voltage-gated and Ca2+-activated K+ currents in these cells. Trans-ACPD reduced the transient outward current (IA) by 20-70% and/or the IA-mediated delay to spike generation in approximately 60% of magnocellular neurons tested. The cells that showed a reduction of IA generally also showed a 20-60% reduction in a voltage-gated, sustained outward current. Finally, trans-ACPD attenuated the Ca2+-dependent outward current responsible for the afterhyperpolarization (IAHP) in approximately 60% of cells tested. This often revealed an underlying inward current thought to be responsible for the depolarizing afterpotential seen in some magnocellular neurons. (RS)-3,5-dihydroxyphenylglycine, a group I receptor-selective agonist, mimicked the effects of trans-ACPD on the resting and voltage-gated K+ currents. (RS)-alpha-methyl-4-carboxyphenylglycine, a group I/II metabotropic glutamate receptor antagonist, blocked these effects. A group II receptor agonist, 2S,1'S,2'S-2carboxycyclopropylglycine and a group III receptor agonist, (+)-2-amino-4-phosphonobutyric acid, had no effect on the resting or voltage-gated K+ currents, indicating that the reduction of K+ currents was mediated by group I receptors. About 80% of the SON cells that were labeled immunohistochemically for vasopressin responded to metabotropic glutamate receptor activation, whereas only 33% of labeled oxytocin cells responded, suggesting that metabotropic receptors are expressed preferentially in vasopressinergic neurons. These data indicate that activation of the group I metabotropic glutamate receptors leads to an increase in the postsynaptic excitability of magnocellular neurons by blocking resting K+ currents as well as by reducing voltage-gated and Ca2+-activated K+ currents.     

Citalopram elicits a discriminative stimulus in rats at a dose selectively increasing extracellular levels of serotonin vs. dopamine and noradrenaline

Citalopram (2.5 mg/kg, i.p.) increased (+145-+180%) extracellular levels of serotonin (5-hydroxytryptamine, 5-HT) in the frontal cortex, nucleus accumbens and striatum of freely-moving rats, whereas dopamine and noradrenaline were unaffected. At this dose, employing a two-lever, food-reinforced, drug discrimination procedure, citalopram generated reliable recognition and fully (> 80%) generalized to itself with an Effective Dose50 (ED50) of 0.1 mg/kg, s.c. Two further selective 5-HT reuptake inhibitors, sertraline and paroxetine, fully generalized with ED50s of 0.01 and 0.04 mg/kg, s.c., respectively. In contrast, the anxiolytic, diazepam (0.63), and the antipsychotic, clozapine (2.5), did not (< or = 20%) generalize. In conclusion, the selective 5-HT reuptake inhibitor, citalopram, elicits a pharmacologically-specific discriminative stimulus in rats at a dose selectively elevating extracellular concentrations of 5-HT.     

An improved method for the detection of changes in brain extracellular glutamate levels

We developed a method for in vivo real-time monitoring of the concentration of extracellular glutamate ([Glu]e) in the brain under anoxic conditions. A dialysis electrode (Sycopel Int., UK) was employed as a sensing device to measure the concentration of glutamate by enzyme amperometry, and an electron mediator, ferrocene, was introduced into the electrode together with glutamate oxidase. The ferrocene was covalently conjugated with a high molecular weight molecule, bovine serum albumin, to avoid outward diffusion through the dialysis membrane. With this set-up, the amperometric response was independent of the pO2 around the electrode in vitro up to 400 microM glutamate. Using this method, we investigated the dynamics of [Glu]e in the rat striatum during anoxia. [Glu]e increased rapidly at 102+/-5.4s (n = 6) after the start of nitrogen inhalation. The increase continued for about 30 s, and then [Glu]e decreased. The peak value of delta[Glu]e was 141+/-37 micro M. [Glu]e subsequently underwent another gradual increase, reaching 213+/-69 microM at 15 min after the start of nitrogen inhalation. This distinct biphasic profile was reproducible. We conclude that this method is very useful for monitoring [Glu]e in the brain under low pO2 conditions.     

Ventromedial hypothalamic lesions produced by gold thioglucose do not impair induction of NPY mRNA in the arcuate nucleus by fasting

The motorneuron degeneration (mnd) mutation causes one of the few late-onset progressive neurodegenerations in mice; therefore, the mnd mouse is a valuable paradigm for studying neurodegenerative biology. The mnd mutation may also model human neuronal ceroid lipofuscinosis (NCL) or Batten disease. mnd maps to the centromeric region of mouse chromosome 8, which likely corresponds to portions of human chromosomes 13,8, or 19; we note that the chromosome 13 portion maps close to a region thought to contain the human Type V NCL locus. We have identified candidate genes for the mnd locus from human chromosomes 13,8, and 19, and we are mapping these genes in the mouse to determine their proximity to the mutated locus and to refine the comparative human-mouse map in this area. A candidate gene from human chromosome 13 is LAMP1, which encodes lysosomal membrane protein 1. We found that LAMP1 in the mouse lies within the region of the mnd mutation. Therefore, we sequenced LAMP1 cDNAs from homozygous mnd mice and unrelated wildtype C57BL/6 mice. We find no differences between the two cDNA species in the regions examined, and expression analysis shows a similar LAMP1 protein distribution in wildtype and mutant mice, suggesting that an abnormal accumulation of material within normal lysosome structures is unlikely to be the pathogenetic mechanism in the mnd mouse.     

Time course of induction of oxytocin messenger ribonucleic acid levels in the hypothalamic paraventricular nucleus of ovariectomized rats following gonadal steroid administration

The nonapeptide oxytocin (OT) is important for uterine contractility at parturition, milk ejection during lactation, and the induction of maternal behavior. OT messenger ribonucleic acid (mRNA) levels increase in the paraventricular and supraoptic nuclei (PVN and SON) of late pregnant and lactating rats and are modulated by the steroid milieu that accompanies these states. Specifically, sequential exposure to estradiol (E2) and progesterone (P) followed by P withdrawal 48 hrs prior to sacrifice increases PVN, and to a lesser but significant degree, SON OT mRNA. To better define the time course of induction of OT mRNA levels following P withdrawal, ovariectomized Sprague-Dawley rats were treated with empty or steroid-filled capsules. On day 1, animals received an E2-filled or empty capsule, followed by P-filled or empty capsules on day 3. On day 14, P-filled or empty capsules were removed and animals were sacrificed 24, 36, or 48 hrs later. The hypothalamic PVN were analyzed for OT mRNA by in situ hybridization histochemistry. Significant differences in PVN OT mRNA were found among the groups (P<0.0001, Kruskal-Wallis). Animals in the 48 hr (P=0.007) and 36 hr (P=0.005), but not the 24 hr, steroid-treated groups had significantly increased OT mRNA relative to their respective sham-treated cohorts (Mann-Whitney U test). The relative abundance of PVN OT mRNA differed among the steroid-treated groups (Kruskal-Wallis, P<0.0003), with highest levels at 48 hr. We conclude that increases in PVN OT mRNA occur by 36 hrs, and are highest at 48 hrs, after P withdrawal in the E2-primed rat. Future studies will determine if OT-mediated changes in behavior or physiology that surround parturition are related to these changes in OT mRNA.     

Serotonin stimulates corticotropin-releasing factor gene expression in the hypothalamic paraventricular nucleus of conscious rats

To examine the direct effects of serotonin (5-HT) on the release and synthesis of corticotropin-releasing factor (CRF) in the hypothalamic paraventricular nucleus (PVN), 5-HT was microinjected just onto the bilateral PVN of conscious rats. Plasma adrenocorticotropic hormone (ACTH) levels peaked at 30 min and returned to the basal levels in 90 min. Northern blot analysis revealed that the CRF messenger RNA (mRNA) level in the PVN as well as the proopiomelanocortin mRNA level in the anterior pituitary significantly increased 120 min after the 5-HT injections (50-250 nmol/side). Pretreatment with intracerebroventricular (i.c.v.) injection of pindobind 5-HT1A (5 nmol) or LY-278584 (500 nmol) completely abolished the 5-HT-induced ACTH response, whereas LY-53857 (100 nmol) was without effect. These results suggest that 5-HT stimulates CRF release, which has interactions with 5-HT1A and 5-HT3 receptors on CRF neurons in the PVN, and activates CRF synthesis in conscious rats.