Estrogen receptors in the medial basal hypothalamus of the rat following complete hypothalamic deafferentation

This study was designed to investigate the effect of complete hypothalamic deafferentation (CHD) on the estrogen receptor (ER) concentration in the MBH as well as LH and FSH secretion. Adult female rats underwent CHD using a Halasz-Pupp knife. Sham CHD and intact animals served as controls. Five days after CHD all the rats were ovariectomized and 2 days later they were decapitated and trunk blood collected and the plasma analysed for LH and FSH by radioimmunoassay (RIA). The brains were rapidly removed and the MBH and preoptic area (POA) were dissected. Brain tissues were homogenized in 2.0 ml of phosphate buffer, centrifuged and the supernatant (cytosol) withdrawn. The cytosols were then incubated at 0–4°C for 3 h with [³H]E₂ or [³H]E₂ + unlabeled E₂. Bound and free receptor was separated using 5–30% sucrose gradient centrifugation, Sephadex LH-20 column or hydroxylapatite receptor assays. CHD resulted in a significant (P < 0.05) reduction in the concentration of ER in the MBH when compared with controls. ER concentration in the POA of the CHD group was not significantly different from the control group. CHD also resulted in a significant (P < 0.005) reduction in the plasma concentration of LH and FSH when compared with the controls. These data suggest that the estrogen receptors in the MBH are influenced by the connections with extrahypothalamic regions and that the effect of hypothalamic deafferentation on gonadotropin secretion may be in part secondary to this reduction of receptors in the MBH.     

Hippocampal cytosol binding capacity of corticosterone: no depletion with nuclear loading

The recurrence every 24 h of glucocorticoid elevation led us to investigate the temporal relationship between glucocorticoid receptor occupation in brain cell nuclei and the availability of cytosol sites. Adrenalectomized rats were injected i.v. with [³H]corticosterone in doses ranging from 15 to 106 nmol/kg. Peak nuclear binding occured 1–2 h after [³H]corticosterone injection and was preceded by a peak of cytosol receptor labeling at 15–30 min. Yet 1 h after 55 or 105 nmol/kg [³H]-corticosterone, no depletion in the total in vitro binding capacity of the cytosol could be detected even though estimated depletion should have been ≈30% had it occured. Injection of 80 nmol/kg of corticosterone per rat plus 40 nmol/kg dexamethasone also failed to reduce total cytosol binding capacity, even though estimated depletion should have been ≈40%. No change in binding affinity of cytosol sites was observed in the injected animals compared to uninjected controls.The in vivo nuclear binding capacity of hippocampus for [³H]corticosterone (fmol/hippocampus) is about 40% of the cytosol binding capacity measured in vitro. Moreover, no more than 40% of total cytosol sites are occupied in vivo as a result of [³H]corticosterone injections which occupy nuclear sites to 80% of estimated capacity. Yet, even with the larger in vitro cytosol binding capacity, a depletion approaching 40% of cytosol binding sites would have been seen, had it occurred as a result of nuclear translocation. The apparent lack of depletion of cytosol receptors is supported by experiments which showed that two injections of [³H]corticosterone 2 h apart fail to fatigue the nuclear uptake mechanism. The present results suggest (1) that in the hippocampus an excess of extranuclear glucocorticoid binding proteins exists, and (2) that the availability of functional cytosol receptors may be regulated to maintain a relatively constant cellular level.     

Radioimmunoassay of brain tissue and cell nuclear corticosterone

A combined cell fractionation-radioimmunoassay procedure is described to measure tissue and cell nuclear levels of corticosterone (CORT) in rat brain in intact animals in morning, afternoon, and following ether stress as well as in adrenalectomized animals with and without replacement therapy. Tissue and nuclear CORT are, as expected, higher in afternoon than in morning and still higher after stress. Replacement of CORT by a subcutaneous pellet and by single injection of 100 μg CORT give tissue and nuclear levels in the afternoon range. Measurement of cell nuclear CORT in brain regions reveals a pattern in intact rats very similar to that seen in ADX rats given CORT replacement, with highest CORT concentrations in hippocampus. In ADX rats comparison of cell nuclear CORT levels by radioimmunoassay with levels obtained by scintillation counting after [³H]-CORT gives further validation of the radioimmunoassay procedure.     

Corticotrophin and corticosterone secretory patterns following acute neurogenic stress, in intact and in variously hypothalamic deafferented male rats

Adult male rats, intact (N) or bearing complete (CHD), anterior (AHD), or posterior (PHD) hypothalamic deafferentations, were acutely exposed to either visual or audiogenic stimulation. At 2, 4, 10 or 30 min following the onset of stress exposure the animals were decapitated and trunk blood was collected for ACTH (RIA) and corticosterone (CS, CBG) determinations. Basal serum concentrations of both hormones were elevated in CHD and AHD, but not in PHD animals as compared to N animals. In N rats, exposure to both stresses resulted in elevated serum ACTH and CS concentrations, with the ACTH response to audiogenic but not visual stimulation being biphasic. In CHD animals, serum ACTH concentrations decreased, and those of CS were unchanged following stress exposure. While audiogenic stimulation caused elevation in serum levels of both hormones in AHD rats, the normal ACTH and CS response to visual stimulation were completely abolished by anterior hypothalamic deafferentation. In PHD animals, no ACTH response to either of the stress exposure was apparent; in spite of this, partial CS responses were elicited. These data thus describe the temporal aspects of the ACTH and CS secretory responses to different neurogenic stresses, and provide insight into the neural pathways mediating these responses.     

Posterior hypothalamic deafferentation abolishes the amnestic effect of electroconvulsive shock in rats

The amnestic effect of immediate post-training transcorneal electroconvulsive shock ECS (15.0 mA, 60 Hz, 2 sec) on step-down inhibitory avoidance learning (0.5 mA, 60 Hz training footshock) was studied in intact rats and in rats submitted to bilateral surgical transection of the dorsal fornix, to anterior or posterior hypothalamic deafferentation, and in sham-operated animals. Animals were tested for retention 24 hr after training. The amnestic effect of ECS was observed in all groups except in the one with the posterior hypothalamic lesion. Fornix-lesioned animals showed a moderate retention deficit which was considerably worsened by the ECS treatment. The results indicate that the amnestic effect of ECS requires integrity of posterior hypothalamic pathways. One possibility is that the amnestic effect of ECS may be mediated by posterior afferent fibers to the hypothalamus acting on hypothalamic opioid systems such as have been previously proposed to play a role in ECS-induced amnesia.     

Single-cell responsiveness to sensory stimuli in the mediobasal hypothalamus of rats with partial hypothalamic deafferentations and medial forebrain bundle lesions

Neuroendocrine studies have demonstrated that photic, acoustic, and sciatic nerve stimulation produce adrenocortical discharges and these can be blocked by partial hypothalamic deafferentations and medial forebrain bundle (MFB) lesions. The effects of these sensory stimuli on single-cell activity of mediobasal hypothalamic (MBH) neurons were studied in intact male rats and in animals with anterior, anterolateral, and posterolateral hypothalamic deafferentations and bilateral MFB lesions. The data were analyzed as to changes in responsiveness, facilitation: inhibition ratio, patterns of firing, magnitude of response, frequency distribution, and sensory convergence. Photic projections enter the MBH both anteriorly and posteriorly and via the MFB, acoustic afferent fibers utilize the MFB, and the sciatic impulses arrive posteriorly. The MFB has also an inhibitory modulation on the photic, acoustic, and sciatic responses and AHD has the same effect on the latter. MFB lesions reduced most markedly the sensory convergence of the three modalities. The relation of these electrophysiologic findings to neuroendocrine studies on the neural pathways mediating adrenocortical responses are discussed.     

Effect of Hypothalamic Deafferentation on the Pulsatile Secretion of Luteinizing Hormone in Ovariectomized Lactating Rats*

The pulsatile luteinizing hormone (LH) secretion in ovariectomized lactating rats bearing complete (CD), anterior (AD), anterolateral (ALD), posterior (PD), or roof (RD) deafferentation of the hypothalamus was determined. All lactating rats were ovariectomized on Day 2 of lactation (Day 0, day of parturition). The deafferentation of nerve fibres to the mediobasal hypothalamus was performed on Day 6 or 7 of lactation. Twenty-four h after the surgery, blood samples were taken through the indwelling atrial catheter every 6 min for 3 h. Plasma concentrations of LH and prolactin (PRL) were measured by radioimmunoassay. The loss of LH pulses associated with lactation was still apparent following AD, PD and sham-deafferentation (SD); pulsatile LH secretion was, however, present in rats with CD, ALD and RD despite continued suckling. The only significant difference in plasma PRL concentrations among the various groups was a reduction in the PRL level in rats with RD in comparison to those with SD. We conclude that the neural signal responsible for the inhibition of pulsatile LH release by suckling is conveyed through the dorsal part of the hypothalamus and PRL does not mediate the suppression of LH pulses in mid-lactation.     

Down-regulation of neural corticosterone receptors by corticosterone and dexamethasone

Stress or elevated corticosterone titers can reduce the concentration of corticosterone receptors in the brain. We demonstrate that corticosterone and the related glucocorticoid, dexamethasone, induce different anatomical patterns of such 'down-regulation'. Corticosterone administration reduces receptor number in the hippocampus, particularly the CA1 and CA2 pyramidal cell fields, but nowhere else in the brain or pituitary. In contrast, equivalent dosages of dexamethasone down-regulate pituitary, amygdaloid and hypothalamic corticosterone receptor numbers. These different anatomical profiles of sensitivity to down-regulation appear due to differential access of the two steroids to the receptor pools.     

Effects of Various Types of Hypothalamic Deafferentation on Luteinizing Hormone Pulses in Ovariectomized Rats

The pulsatile luteinizing hormone (LH) secretion in chronically ovariectomized rats bearing various types of hypothalamic deafferentation was examined. Ovariectomized rats were subjected to complete, anterolateral or anterior hypothalamic deafferentation and bled every 6 min for 3 h through an indwelling atrial cannula 5 days after the brain surgery. Another group of ovariectomized animals was subjected to posterior-anterior hypothalamic deafferentation (PAD), which cut off the anterior part of the arcuate nucleus from the mediobasal hypothalamus, and bled 1, 3 or 5 weeks after the deafferentation. Coronal sections of the brain were immunostained with anti-LH-releasing hormone (LHRH) serum. The pulsatile LH secretion was observed in rats bearing anterior, anterolateral or complete hypothalamic deafferentation and these types of deafferentation did not affect the frequency of LH pulses. The mean LH level during the 3-h sampling period and the amplitude of LH pulses decreased as the incision extended postero-laterally. Rats bearing PAD showed an irregular fluctuating pattern in plasma LH concentration 1 week after PAD. Parameters of LH pulses were restored with time after PAD. This suggests that the system generating LHRH pulses severed by PAD had been reorganized. LHRH-immunopositive neuronal fibres were found in the external layer of the median eminence in the rats bearing any type of deafferentation. The present results suggest that the frequency of LH pulses could be controlled by the LHRH pulse generator, which consists of non-LHRH neurons and is located in the mediobasal hypothalamus.     

Mediobasal hypothalamic neurons are excited by the iontophoretic application of sodium

In the course of studies on the responsiveness of mediobasal hypothalamic neurons to the iontophoretic application of cortisol, it was found that positive currents applied to a sodium chloride (1 M) barrel alone, but not to a choline chloride (1 M) barrel, frequently increased the firing of these neurons. Subsequently, systematic examination demonstrated that out of 102 MBH neurons 52 (51%) increased their firing by at least 30% with application of NaCl, using currents no greater than 10 nA. No such effect was obtained in response to Na application from a dilute solution (0.05 or 0.1 M). When glutamate was absent from the electrodes, the incidence of Na+ sensitivity fell to 17%, despite the routine use of backing currents to the glutamate barrel. K+ ions were more active than Na+ ions in producing excitation. When Na+ sensitivity was found, however, Na+ effects were produced by currents greater than K+ currents producing equivalent excitation. Like glutamate, K+ ions were capable of greatly enhancing responses to Na+. Comparison was made between cortisol and Na+ sensitivity in 70 MBH neurons; 28 cells responded to both, and 24 of them were inhibited by cortisol. Thus Na+ sensitivity is a frequent characteristic of MBH neurons inhibited by cortisol, and was present in 83% of cortisol-sensitive cells in this region. Iontophoresis of Na+ is commonly used as a control in pharmacological studies of the nervous system. Even more common is the case of concentrated NaCl solutions for recording. These procedures may not be as inert as previously thought, particularly in the hypothalamus.     

Age-dependent loss of corticosterone modulation of central serotonin 5-HT1A receptor binding sites

A loss of endocrine and neurotransmitter system interactions, including corticosterone regulation of 5-HT_1Areceptors, may underlie the age-related deficits in the hypothalamic-pituitary-adrenal (HPA) axis including adapting to stress. In this study, female Fischer 344 rats, (ages 3, 13, and 18 months), were bilaterally adrenalectomized and supplemented for 3 weeks with placebo or corticosterone (200 mg or 600 mg) containing 21 day sustained-release pellets implanted subcutaneously (LC, MC, or HC, respectively). Scatchard analysis using the 5-HT_1A receptor agonist [³H]8-hydroxy-2-(di-N-propylamino) tetralin (8-OH-DPAT) demonstrated a significant decrease in hippocampal receptor density in 3 month and 13 month MC groups (-35.2 and -32.1%, respectively) as compared to age-matched LC groups; a significant decline in 5-HT_1A receptor density in 3 month and 13 month HC groups was found compared to age-matched MC groups (−16.7 and −22.0%, respectively). However, these hormone treatments (LC or HC) failed to alter hippocampal 5-HT_1A binding site density in the 18 month groups. Cortical 5-HT_1A receptor densities were altered in a similar age-dependent manner. In contrast, the density of hypothalamic 5-HT_1A receptors in the 18 month LC group was significantly increased above that in the 3 month LC group. An additional indicator of the hippocampal response to corticosterone, the distribution of glial fibrillary acidic protein (GFAP), revealed an age-related decline in responsiveness to hormone treatment in the oldest group. The present study has identified an age-associated deficit in the regulation of hippocampal 5-HT_1A receptors by corticosterone which may underlie the diminished capacity of the aging HPA axis to cope with stress. J. Neurosci. Res. 53:86–98, 1998. © 1998 Wiley-Liss, Inc.     

Expression of immunoregulatory cytokines in neurons of the lateral hypothalamic area and amygdaloid nuclear complex of rats immunized against human IgG

Present results showed that interleukin-1 (IL-1), IL-6 and transforming growth factor-beta (TGF-beta) were constitutively expressed in the supraoptic and paraventricular nuclei of the rat hypothalamus. Immunoreactive cells were also detected, but to a lesser extent, in other parts of hypothalamus as well as in the cerebral cortex. In rats immunized with IgG, there was moderate increase in immunoreactivities of the cytokines. A notable feature, however, was the induction of the cytokine expression in the lateral hypothalamic area and the amygdaloid nuclear complex, suggesting that the neurons in these two areas are involved in possible immune regulation.     

A substance P-containing hypothalamic neuronal system projects to the median eminence

Immunoreactive substance P (SPi) was measured in the various hypothalamic structures and amygdala one week after placing a cut around the medial basal hypothalamus (MBH). SPi content decreased in the stalk-median (SME), anterior and posterior parts of the arcuate nucleus (inside the isolated region) as well as in the paraventricular nucleus (outside the cut), while there was no change in the posteromedial amygdaloid nucleus. We suggest that there is a tuberoinfundibular neuronal system containing SPi with cell bodies in the MBH and terminals in the SME. In addition, nerve fibers containing SPi from outside the MBH may reach both the SME and the arcuate nucleus and might influence neuroendocrine regulation.     

Activation of Opioid Receptors in the Mediobasal Hypothalamus Stimulates Prolactin Secretion in the Conscious Rat

In an attempt to localize the opioid receptor(s) (mu, delta and kappa) involved in opioid-stimulated prolactin release in the conscious male rat, opioid agonists were microinjected into the mediobasal hypothalamus and prolactin levels measured before and after injection. The specific mu agonist, DAGO ((D-Ala², NMe-Phe⁴, Gly-ol⁵)-enkephalin) was the most effective in eliciting prolactin release, the smallest effective dose being 0.01 nmoles. The specific delta agonist, DPDPE ((D-Pen², D-Pen⁵)-enkephalin) had no significant effect even at the highest dose of 10 nmoles. The specific kappa agonist, U50,488H ((trans-3,4-dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzene acetamide) was effective at the doses 1.0 and 10 nmoles. We conclude that mu and kappa opioid receptors in the mediobasal hypothalamus are involved in opioid-stimulated prolactin release and that delta receptors are not.     

The effects of iontophoretic application of putative neurotransmitters on the electrical activity of rat mediobasal hypothalamic neurons in relation to their steroid sensitivity

In order to characterize the neurons responsible for ACTH release and the suppression produced by adrenocortical steroids, we have studied the pharmacological sensitivity of neurons in the region containing the highest concentration of corticotropin releasing factor (CRF) in the brain and probably responsible for most of these activities. The effects of 8 putative neurotransmitters applied iontophoretically to more than 400 mediobasal hypothalamic (MBH) neurons in the rat were examined, and compared with cortisol sensitivity of the same neurons. Glutamate was the only agent that produced excitation exclusively, while GABA, serotonin, glycine and dopamine were inhibitory in action. Mixed excitation and inhibition were produced by histamine, acetylcholine (ACh) and norepinephrine (NE). All 8 cells excited by ACh were inhibited by cortisol and constitute only about 10% of neurons sampled; these neurons are considered to be strong candidates for CRF-releasing cells. On 7 of these neurons histamine was strongly excitatory and its action greatly outlasted the iontophoretic application. Histamine, however, excited many other neurons, including those neither excited by ACh nor steroids.     

Effects of naloxone on basal and stress-induced ACTH and corticosterone secretion in the male rat - site and mechanism of action

The acute effects of naloxone upon basal and stress-induced secretion of ACTH and corticosterone (CS) in the adult male rat were investigated. Forty-five minutes subsequent to naloxone injection (5 mg/kg body wt, i.p.), basal serum levels of ACTH (by radioimmunoassay) and of CS (by corticosterone-binding globulin) were more than doubled, as compared to vehicle-treated animals. Upon exposure to either photic or audiogenic stress, the ACTH and CS secretory responses were greater in the naloxone-injected groups. In animals with complete hypothalamic deafferentation basal serum ACTH concentrations were significantly greater than in intact controls (2-fold), and naloxone elicited a further doubling of this parameter. In dexamethasone-pretreated rats (50 μg/animal,4 h prior to naloxone), naloxone had no effect upon ACTH and CS secretion. This study demonstrates: (1) that acute naloxone administration leads to hypersecretion of ACTH, as well as of CS, in the adult male rat; and (2) that its effect is due to an action within the hypothalamo-hypophyseal unit. The data also suggest that these naloxone effects are not mediated by glucocorticoid hormones.     

Multiple hypothalamic sites control the frequency of hippocampal theta rhythm

Stimulation of a neural pathway originating in the brainstem reticular formation, with synapses in the medial hypothalamus, activates the hippocampal theta rhythm. The frequency of reticular-elicited theta is determined in the medial supramammillary nucleus (mSuM) completely in anaesthetised rats, but only partially when the animal is awake. We tested other medial hypothalamic sites for their capacity to control theta frequency in awake rats. Blockade of sodium channels (1 microl fast infusion of the local anaesthetic procaine, experiment 1) or increased inhibition by GABA (Chlordiazepoxide [CDP], experiment 2) was found to reduce or increase the frequency of reticular-elicited theta, depending on the precise site of injection, in the region of the dorsomedial hypothalamic nucleus (DMH) and the posterior hypothalamic nucleus (PH). A band of null sites for CDP was located in the region of the ventral border of PH and dorsal border of mSuM. Using 0.5 and 1 microl CDP, and slow infusions (experiment 3), it was found that effective PH sites were also separate from mSuM in the rostrocaudal direction. In experiment 4, the DMH/PH region was mapped with unilateral and bilateral slow infusions of 0.5 microl CDP. CDP significantly reduced frequency in medial (periventricular) and dorsal PH, but not DMH. Bilateral injections appeared to generally sum the usual effects of unilateral injection, producing effects of intermediate size. However, the absolute frequency change in any given site, or with any pair of sites, did not exceed 1 Hz, which is similar to what is seen with single injections in mSuM. Overall, it appears that at, any one time, theta frequency may be determined by a complex interplay between distinct but interacting modulatory regions in the medial hypothalamus.