Neonatal Rat Hypothalamus Research Articles

Evidence for sexual dimorphism of estrogen receptors in hypothalamus and thymus of neonatal and immature Wistar rats

It is hypothesized that the rat thymus is sexually differentiated. To begin testing this, we used the 5-day-old rat, whose hypothalamus is sexually differentiated during this period. Cytosolic estrogen receptors (ER) were measured in cytosols prepared from the brain, thymus and uterus of Wistar rats at 5, 18 and 30 days post-partum. ER concentrations were significantly higher in hypothalamus in cytosols of female 5-day-old rats, and this difference had disappeared by day 18. The pattern in thymus was identical to that observed in hypothalamus, suggesting the presence in the thymus of the aromatase system that converts androgen to estrogen, and that estrogen-mediated sexual differentiation of thymus might be proceeding at 5 days. Unlike the case for hypothalamus, no experimental model exists at present for testing functional sexual differentiation in thymus. Therefore we tested the effects of aromatase inhibitors on estrogen receptor activity in thymus well after the five-day period, and before atrophy of the thymus has commenced. Male and female rats were implanted at 15 days of age with SILASTIC implants containing 5 mg of estradiol or with 25 mg of the aromatase inhibitor 4-hydroxyandrostenedione (4-OHA) and cytosolic ER prepared at 30 days and activity measured. Administration of estradiol resulted in failure to detect available receptor, suggesting that the binding components measured were ER. After 4-OHA administration, ER concentrations were significantly increased in cytosols from male but not female hypothalamus and thymus. There is therefore a basis for exploring further the hypothesis that rat thymus is sexually differentiated.

Leptin stimulates prostaglandin E 2 and F 2α, but not nitric oxide production in neonatal rat hypothalamus

Leptin, an adipocyte-derived 16 kDa polypeptide hormone, has been found to regulate food intake and thermogenesis by modulating stimulatory and inhibitory pathways in the feeding circuitry of the hypothalamus, among which corticotropin releasing hormone (CRH). Nitric oxide (NO) and prostaglandins have been shown to be involved in both CRH neurosecretion and feeding regulation. We have investigated the role of NO, prostaglandin E 2 and prostaglandin F 2α as mediators of the hypothalamic effects of leptin and their possible involvement in leptin-stimulated CRH secretion. Using primary cultures of neonatal (5- to 6-day-old) rat hypothalamic cells, we confirmed that leptin (0.1–10 nM) stimulates CRH secretion. This effect was not blocked by l- N G-nitro-methyl-arginine ( l-NAME, 100 μM), a NO-synthase competitive inhibitor; and leptin did not stimulate NO production. Cyclooxygenase inhibition by indomethacin (10 μM) did not modify leptin-induced CRH secretion, while leptin stimulated prostaglandin E 2, and prostaglandin F 2α secretion. In conclusion, leptin-induced hypothalamic CRH secretion is not modulated by NO-synthase- or cyclooxygenase-mediated mechanisms; leptin does not stimulate NO production, but it stimulates prostaglandin E 2 and F 2α production, which could add to the growing list of mediators of leptin signaling in the hypothalamus.

Identification of a sex steroid-inducible gene in the neonatal rat hypothalamus

By the cDNA subtraction between cDNA preparations from androgenized and intact neonate rats hypothalami, granulin/epithelin (grn) gene was identified as a gene enriched by androgenization. Strong grn mRNA signals were detected by in situ hybridization in the ventromedial nucleus (VMH) and the arcuate nucleus (ARC) of the hypothalamus of a 5-day-old male. The grn gene expression level in the hypothalamus was similar between males and females at birth. At 10 days of age, this level was maintained in males, but decreased to 1/4 in females. Thus, grn can be the gene of which expression in the VMH and ARC is sustained by endogenous androgen in male neonates.

A-type K+ current in neurons cultured from neonatal rat hypothalamus and brain stem: modulation by angiotensin II.

The regulation of A-type K+ current (I(A)) and the single channel underlying I(A) in neonatal rat hypothalamus/brain stem cultured neurons were studied with the use of the patch-clamp technique. I(A) had a threshold of activation between -30 and -25 mV (n = 14). Steady-state inactivation of I(A) occurred between -80 and -70 mV and had a membrane voltage at which I(A) was half-maximum of -52.2 mV (n = 14). The mean values for the activation and inactivation (decay) time constants during a voltage step to +20 mV were 2.1 +/- 0.3 (SE) ms (n = 8) and 13.6 +/- 1.9 ms (n = 8), respectively. Single-channel recordings from outside-out patches revealed A-type K+ channels with voltage-dependent activation, 4-aminopyridine (4-AP) sensitivity, and inactivation kinetics similar to those of I(A). The single-channel conductance obtained from cell-attached patches was 15.8 +/- 1.3 pS (n = 4) in a physiological K+ gradient and 41.2 +/- 3.7 pS (n = 5) in symmetrical 140 mM K+. Angiotensin II (Ang II, 100 nM) reduced peak I(A) by approximately 20% during a voltage step to +20 mV (n = 8). Similarly, Ang II (100 nM) markedly reduced single A-type K+ channel activity by decreasing open probability (n = 4). The actions of Ang II on I(A) and single A-type K+ channels were reversible either by addition of the selective angiotensin type 1 (AT1) receptor antagonist losartan (1 microM) or on washout of the peptide. Thus the activation of AT1 receptors inhibits a tetraethylammonium-chloride-resistant, 4-AP-sensitive I(A) and single A-type K+ channels, and this may underlie some of the actions of Ang II on electrical activity of the brain.

Pharmacological model of catecholamine depletion in the hypothalamus of fetal and neonatal rats and its application.

1. The present study aimed to develop a pharmacological model of catecholamine (CA) depletion in the hypothalamus during the period of its morphofunctional development, i.e. in fetal and neonatal rats of both sexes. 2. In the first series of experiments, pregnant females and, hence, fetuses were systemically treated daily from the embryonic day (E) 13 to E20 with the inhibitor of the CA synthesis alpha-methyl-m-tyrosine. The CA concentrations were subsequently measured in the fetal hypothalamus at E21 by high performance liquid chromatography with electrochemical detection (HPLC-ED). In the second series of experiments, neonatal rats were injected with neurotoxin, 6-hydroxydopamine and/or alpha-methyl-m-tyrosine daily from the 2nd postnatal day (P2) to P10. 3. The HPLC-ED assay of hypothalamic catecholamines (CA's) at E21 and P11 showed that both in fetuses and neonates, alpha-methyl-m-tyrosine caused more than 50% depletion of hypothalamic noradrenaline and adrenaline, while the dopamine (DA) level remained unchanged. The combined treatment of neonatal rats with alpha-methyl-m-tyrosine and 6-hydroxydopamine resulted additionally in a 25% decreased level of DA. 4. The influence of CA deficiency on the developing hypothalamic CA system was further evaluated by measuring [3H]DA uptake by nervous tissue in vitro. 5. The CA deficiency caused a 50% drop of [3H]DA uptake by the hypothalamic tissue in treated fetuses suggesting a stimulating effect of CA's on the early development of the CA system. In pharmacologically treated neonatal rats [3H]DA uptake remained at the control level showing no influence of the CA deficiency on the developing CA system after birth. 6. The usefulness of the proposed pharmacological model for studying of CA influence on differentiating hypothalamic target neurons is discussed.

Mitogen-activated Protein Kinases in Rat Brain Neuronal Cultures Are Activated by Angiotensin II Type 1 Receptors and Inhibited by Angiotensin II Type 2 Receptors

Neurons cultured from neonatal rat hypothalamus and brainstem contain many angiotensin II (Ang II) type 2 (AT2) receptors, and we previously determined that activation of these sites elicited a stimulation of serine/threonine phosphatase 2A (PP2A). Here, we have investigated the effects of Ang II on neuronal mitogen-activated protein (MAP) kinases, potential targets for PP2A. Using in-gel kinase assays and immunoprecipitation analyses we have shown that Ang II (10 nM-1 microM) elicits significant increases in p44(MAPK) (Erk1) and p42(MAPK) (Erk2) activities in cultured neurons, mediated via Ang II type 1 (AT1) receptors. This stimulatory effect of Ang II on Erk1 and Erk2 activities was potentiated by blockade of AT2 receptors with (S)-1-[4-(dimethylamino)-3-methylphenyl]methyl-5-(diphenylacetyl)- 4, 5,6,7-tetrahydro-1H-imidazo[4,5-C]pyridine-6-carboxylic acid (PD 123319, 1 microM). Furthermore, the AT2 receptor agonist N-alpha-nicotinoyl-Tyr-Lys-(N-alphaCBZ-Arg)-His-Pro-Ile-OH (CGP42112A) (10-50 nM) caused significant decreases in neuronal Erk1 and Erk2 activities, which were abolished by PD 123319 (1 microM) and by the PP2A inhibitor okadaic acid (3 nM). This indicates that AT1 and AT2 receptors have opposite actions on Erk1 and Erk2 activities in neonatal neurons. Since MAP kinases are involved in the regulation of growth/differentiation and apoptosis, our data may provide an intracellular basis for modulatory effects of Ang II receptors on these processes.

Role of Sex Steroids on the Survival, Neuritic Outgrowth of Neurons, and Dopamine Neurons in Cultured Preoptic Area and Hypothalamus

Morphological sex differences in some discrete brain regions are thought to be developed by the influence of circulating androgen during the perinatal period. In order to know whether the effect of androgen is a direct one, cells derived from neonatal rat preoptic area (POA) and/or hypothalamus were cultured in a serum-free medium, and the effects on survival, process growth of neurons, and dopaminergic function were examined. When the POA cells were exposed to testosterone (T), neuronal survival was greater than the controls, and the frequency distribution of total process length and the number of process branchings were significantly deviated from the controls. Estradiol-17β (E2) and 5α-dihydrotestosterone had less significant effects on these parameters than T. Moreover, the addition of T increased the dopamine (DA) contents of cells and medium DA in the hypothalamic culture. E2 also greatly increased DA content of the medium. These steroids failed to alter the DA levels in the POA cell cultures. These results generally conform to the notion of previous investigators that T has direct effects on the expansion of dendritic elements of the POA and the development of sex difference in the hypothalamic DA neurons, although the effect of T after conversion to E2 cannot be excluded.

Methods for implanting steroid-containing cannulae into the paraventricular nucleus of neonatal rats

Implantation of cannulae into brains of neonatal rats presents methodological difficulties. We discuss such issues as avoiding tissue injury, and describe successful techniques. Cannulae size, methods of preparation, insertion, and securing are evaluated. We present a modified cannula holder applicable to the soft neonatal brain. Application of these methods to the study of glucocorticoid receptors in the neonatal rat hypothalamus is illustrated.

Induction by nerve growth factor (NGF) of NGF receptor-like immunoreactivity in the preoptic area and hypothalamus of neonatal rats

Nerve growth factor (NGF) was injected in the brain of neonatal rats. In eleven of thirty-one rats that received 1 μg of mouse NGF (2.5S form) on postnatal day 3, NGF receptor-like immunoreactivity was found in the preoptic area (POA) and hypothalamus until 12 days after birth. However, no immunoreactivity was seen in the region of rats in the same group examined on day 20 or 45 and of uninjected control rats on days 1–45. These results indicate that NGF induces the transient appearance of the receptor for NGF in the POA and hypothalamus of neonatal rats.

The influence of angiotensin II on catecholamine synthesis in neuronal cultures from rat brain

Incubation of primary neuronal cultures prepared from the hypothalamus and brainstem of neonatal rats with angiotensin II (Ang-II) resulted in a concentration-dependent effect on the incorporation of [ 3H]-tyrosine ([ 3H]-Tyr) into [ 3H]-catecholamines ([ 3H-CA). At concentrations of 1 nM–1μM, Ang-II (60 min. incubation) caused significant decreases (31–52%) in neuronal [ 3H]-CA content compared with controls. Conversely, higher concentrations of Ang-II (10–100 μM; 60 min.) caused significant increases (20–60%) in neuronal [ 3H]-CA content compared with controls. Both of these effects were blocked by co-incubation with the Ang-II receptor antagonist Sar 1IIe 8-Ang-II. These observations demonstrate that neuronal cells in primary culture have the ability to synthesize [ 3H]-CA from [ 3H]-Tyr, and that Ang-II has a receptor-mediated biphasic influence on newly synthesized [ 3H]-CA (norepinephrine and dopamine).

Tryptophan hydroxylase activity in hypothalamus and brainstem of neonatal and adult rats treated with hydrocortisone or parachlorophenylalanine

The present study has been undertaken to determine whether glucocorticoid, and parachlorophenylalanine (PCPA, a tryptophan hydroxylase inhibitor) affects tryptophan hydroxylase (TPH) levels in brainstem and hypothalamus of neonatal and adult rats. Our results show that: (1) administration of hydrocortisone causes small but significant increases in TPH activity of neonatal brainstem: (2) treatment with PCPA plus glucocorticoid results in a marked decrease of TPH activity in brainstem and hypothalamus of both neonatal and adult rats.

On the distribution and morpho-functional characteristics of 5-HT-immunoreactive cells in the hypothalamus of fetuses and neonatal rats

This study attempted to visualize serotonin (5-HT)-immunoreactive (IR) neurons in the hypothalamus of intact fetuses (E18) and neonatal rats (P9) as well as after their pretreatment with some drugs interfering with the 5-HT metabolism and uptake in the serotoninergic neurons ( l-tryptophan, pargyline, 5-hydroxytryptophan, fluoxetine). The 5-HT-IR cells were not observed in the hypothalamus of normal, untreated fetuses and neonatal rats. However, two large accumulations of 5-HT-IR neurons appeared in the anterolateral hypothalamus and in the dorsomedial nucleus after the subsequent injections of the monoamine oxidase inhibitor, pargyline, and the amino acid precursor of the 5-HT synthesis, l-tryptophan. A significantly less intensive reaction was observed after injections either of the second precursor of the 5-HT synthesis, 5-hydroxytryptophan instead of l-tryptophan, or pargyline only. Immunostaining, provoked by the pargyline and l-tryptophan pretreatment, was completely blocked by the injection of the specific 5-HT uptake inhibitor, fluoxetine. It means that the 5-HT immunostaining of the hypothalamic neurons may be accounted for by their capacity to take up specifically 5-HT from the environment rather than by its intraneuronal synthesis from l-tryptophan. Nevertheless, the 5-HT synthesis from 5-hydroxytryptophan in these cells cannot be excluded. The uptake of extracellular 5-HT into catecholaminergic neurons can be excluded as nomifensine, the specific inhibitor of the uptake to these neurons, did not modify the immunostaining.

Whole cell voltage clamp recordings from cultured neurons of the supraoptic area of neonatal rat hypothalamus

Whole-cell voltage and current clamp recordings were made from cultured neurons from the hypothalamic supraoptic area of neonatal rats. These neurons fired spontaneous Na +-action potentials, appearing as mixed Na +/K +-currents in voltage clamp. Isolated Na +-currents (< 3nA) were rapidly activated and inactivated during positive potential pulses from -80 mV. Two voltage-activated Ba 2+-currents (< 1nA) were also recorded. These techniaues offer a promising new approach for studying the striking electrical behavior of cultured hypothalamic neurons.

Effect of progesterone on the testosterone and estradiol levels in the hypothalamus of neonatal rats.

Pregnant female rats were injected with progesterone or oil from 18.5 up to 21.5 days of gestation. Rat pups were delivered by caesarean section, and pups delivered from progesterone-treated mothers were injected with 100 micrograms of progesterone and pups from oil-treated mothers were injected with oil. Two hours after delivery, pups were killed and hypothalamic testosterone and estradiol were determined by RIA. Control males had substantially higher concentrations of testosterone and estradiol in hypothalamus than control females. Treatment with progesterone did not affect the accumulation of testosterone in the hypothalamic tissue of neonatal males, but completely prevented the formation of estradiol in hypothalamic tissue. This result suggests that progesterone can inhibit hypothalamic aromatase activity in the neonate and may explain why progesterone can protect against some of the neural defeminizing effects of neonatally administered androgens.

Cells in neonatal rat hypothalamus primary culture - ab immunofluorescence study

Hypothalami from 1 day neonatal rats were dissociated and cultured for 4–16 days. Using immunofluorescence and antisera against neurofilament (NF) peptides, glial fibrillary acidic protein (GFAP), galactocerebroside and fibronectin we have distinguished neurons, astricytes, oligodendrocytes and fibroblast-like cells in culture. Astrocytes initially grew as islets of 15–30 cells which dispered as the culture aged. These cells, together with fibronectin-reactive flat cells, formed a monolayer upon which ovoid and process-bearing cells grew after 4 days in culture. Neurofilament-positive neurons constituted 5–10% of the total cell population. In maturing cultures the number of neurons decreased and fibroblasts increased. Oligodendrocytes represented less than 1% of total cell population. These studies emphasize the necessity of using the complementary techniques of morphology and immunocytochemistry for the characterization of hypothalamic neural cells in vitro.

Influence of intrahypothalamic implants of antioestrogen or aromatase inhibitor on development of sterility following neonatal androgenization in female rats

The validity of the hypothesis that aromatization of androgens in the hypothalamus is a prerequisite for induction of sterilization in female rats androgenized neonatally was tested by implanting paraffin micropellets containing 16β-ethylestradiol-17β (EED) or 1,4,6-androstatriene-3,17-dione (ATD), directly into the hypothalamus of neonatal female rats 6 h prior to a single subcutaneous injection of testosterone propionate (TP). The antagonists did not impair the sterilizing action of TP but enhanced the induction of sterility. By contrast, intrahypothalamic implantation of paraffin micropellets containing 1% TP together with 50% MER-25 an antiocstrogen, brought about a significant suppression of induction of sterility. The reasons why intrahypothalamic implants of antioestrogen or aromatase inhibitor failed to suppress the sterilizing effect of TP subcutaneously injected were discussed.

Intraphypothalamic implants of testosterone or dihydrotestosterone in neonatal female rats with reference to induction of sterility.

Implants of paraffin micropellets containing about 5 microgram 5 alpha-dihydrotestosterone (DHT) into the hypothalamus of 5-day-old female rats were without effect on the sexual differentiation of the brain. By contrast, approximately the same amount of testosterone propionate (TP) given as subcutaneous or intrahypothalamic micropellets masculinized the female brain. In the light of these results as well as the author's previous findings that an antiestrogen implanted into the hypothalamus of neonatal female rats failed to block masculinization by subcutaneous injection of TP, the possibility cannot be excluded that testosterone is capable of masculinizing the brain of neonatal females without being converted into estrogens.

Cytosol and nuclear receptors for 5α-dihydrotestosterone and testosterone in the hypothalamus and hypophysis, and testosterone receptors isolated from neonatal female rat hypothalamus

Characterization of cytosol receptor macromolecules for 5α-dihydrotestosterone, (DHT) and testoster one (T) was compared in the hypothalamus of 27-day-old male rats. The binding of [ 3H]DHT increased rapidly after starting incubation, and approached a maximum at 1 h. In contrast, the [ 3H]T binding increased slowly with a maximum at 4h. On analysis of the saturation kinetics of the binding of androgens by the Lineweaver-Burk plot the dissociation constant (K D) and the number of binding sites (NBS) for DHT binding were determined to be 0.3 nM and 3.8 ( n = 2) fmol/mg protein, respectively, and the values for T binding 1.5 ± 0.28 (S.E.) nM and 2.2 ± 0.23 ( n = 3) fmol/mg protein, respectively; indicating higher affinity of the hypothalamic cytosol for DHT than for T. Maximal binding capacities for DHT and T were 3.9 ± 0.36 [4] and 2.1 ± 0.15 [4], respectively. Relative affinity of competition of the [ 3H]T binding by DHT was 2.8 times than by T. Hypothalamic and hypophyseal [ 3H]DHT binding was inhibited in a competitive way by the addition of 17β-estradiol, but not by estrone, estriol, diethylstilbestrol or clomiphene citrate. The injections of estradiol even in large amount did not affect the binding of [ 3H]DHT. From these results it seems that the hypothalamic cytosol can bind DHT and T in a different way, indicating different characterization of cytosol receptors for DHT and T in the hypothalamus of 27-day-old male rats, and further suggesting the possibility of individual receptors for DHT and T Receptors macromolecules of 5–6 S for DHT were isolated from purified nuclei of the hypothalamus and the anterior hypophysis. From these results, together with nuclear T receptors, it is tempting to speculate that androgens can directly act on the brain through the interaction with the receptors for DHT and T in the hypothalamus and hypophysis. The presence or absence of receptors for T was investigated in the hypothalamus of neonatal female rats at 3 and 7 days of age. On sucrose density gradients there was a small but definite peak of radioactive androgen in the 8 S region. The 8 S binding components were found to be saturable and steroid-specific. These suggest the presence of ‘receptors’ for T in the hypothalamus on neonatal female rats. The function of neonatal hypothalamic T ‘receptor’ was discussed in relation with the mechanism of induction of androgenization.

Growth in hypothalamic neurons as reflected by nuclear size and labelling with 3H-uridine

(1) Differential patterns of nuclear growth were observed in the neonatal rat hypothalamus. Significant growth of the neuronal nucleus occurred in the supraoptic and paraventricular regions during the first week of life, while marginal growth occurred in the ventrolateral portion of the ventromedial region. No growth was observed in the suprachiamatic and arcuate regions. (2) After injection of 3H-uridine, the degree of labelling of nuclear RNA was different in the various regions of the hypothalamus. The pattern of distribution of radioactivity changed with age. (3) Little or no change in the level of labelling of RNA took place in regions demostrating nuclear growth (supraoptic nucleus (SO), paraventricular nucleus (PV), ventromedial hypothalamic region (VM)). In the absence of nuclear growth, the degree of labelling of RNA declined (suprachiasmatic nucleus (SC), arcuate nucleus (ARC)). (4) Nuclei in female neurons were larger than in males in the five regions of the neonatal rat hypothalamus that were studied.

Late effects of thyroxine infected into the hypothalamus of the neonatal rat.

Thyroxine (T4) in a systemically ineffective dose was implanted or injected into the hypothalamus of neonatal rats. Such treatment caused late and persistent alterations in pituitary-thyroidal and gon