Fos expression in tyrosine hydroxylase containing hypothalamic neurons in CRH-KO mice: effect of immobilization stress.

OBJECTIVE: Little is known about the response of tyrosine hydroxylase (TH) containing hypothalamic neurons to stress in corticoliberine deficient (CRH-KO) mice. This study was aimed to extend this issue and reveal the data leading to a better understanding of physiological/anatomical plasticity of hypothalamic TH cells in response to acute immobilization stress (IMO) as well as of possible of CRH body deficiency contribution in the regulation of TH cells during stress. We examined the topographic distribution of TH protein immunolabeled perikarya in selected hypothalamic structures including the paraventricular (PVN), supraoptic (SON), periventricular (PeVN), arcuate (ArcN), dorsomedial (DMN), and ventromedial (VMN) nuclei and extrahypothalamic zona incerta (ZI) in CRH-KO and wild type (WT) mice. METHODS: The animals were perfused with fixative 120 min after a single IMO stress. The brains were removed, cryo-sectioned throughout the hypothalamus and Fos-TH co-localizations were processed immunohistochemically. Fos protein was visualized by diaminobenzidine (DAB) intensified with nickel ammonium sulphate, while TH cells were labeled only with DAB chromogen. The evaluation of Fos-TH co-labeled perikarya was performed with the use of computerized Leica light microscope and expressed as the percentage of total amount of TH labeled cells. RESULTS: From the qualitative point of view, the present data indicate similar anatomical distribution of TH immunoreactive perikarya in all brain structures investigated in both WT and CRH-KO mice, while from the quantitative point of view only TH cells in the DMN of CRH-KO mice showed a trend for increased activation by IMO. CONCLUSIONS: In several hypothalamic structures the basic population of TH neurons was not affected by the absence of endogenous CRH. Based on the data of this study it can also be assumed that despite of the presence of direct reciprocal connections between PVN and DMN neurons, PVN CRH neurons possibly are not participating in the regulation of TH neurons in the DMN during IMO stress. KEYWORDS: Hypothalamic nuclei - Fos-immunohistochemistry - Tyrosine hydroxylase - Immobilization stress - CRH knockout mice.

Response of hypothalamic oxytocinergic neurons to immobilization stress is not dependent on the presence of corticotrophin releasing hormone (CRH): a CRH knock-out mouse study.

This study explores the quantitative patterns of immunolabeled Fos protein incidence in the hypothalamic paraventricular (PVN) and supraoptic nuclei (SON) oxytocinergic (OXY) neurons in response to immobilization (IMO) stress in corticotrophin releasing hormone deficient (CRH-KO) mice. Adult male mice, taken directly from cages or 120 min after a single IMO, were sacrificed by intracardial perfusion with fixative. Coronal brain sections of 30 mum thickness were processed for dual Fos/OXY immunohistochemistry. In control wild type (WT) and CRH-KO mice, scattered Fos immunoreactivity was observed in hypothalamus, including the PVN where scanty Fos signal occurred in both parvocellular and magnocellular PVN subdivisions. Dual Fos/OXY immunostainings revealed higher basal Fos expression in the PVN of control CRH-KO mice. IMO evoked a marked rise in Fos expression in OXY neurons of the PVN and SON in both WT and CRH-KO groups of mice. The present data demonstrate that 1/ CRH deficiency upregulates the basal activity of hypothalamic PVN OXY cells in CRH-KO mice and 2/ IMO stress in both WT and CRH-KO mice affects distinctly the activity of OXY cells in both SON and PVN. Our data indicate that CRH deficiency does not alter the responsiveness of PVN and SON OXY cells to IMO stress.

Distribution of neuronal and non-neuronal spliced variants of type 1 IP(3)-receptor in rat hypothalamus and brain stem.

In the nervous system, inositol 1,4,5-trisphosphate (IP(3)) is one of the second messengers produced by PI hydrolysis and triggers IP(3)-receptor (IP(3)R) mediated calcium release from intracellular pools. Throughout the brain, the type 1 IP(3)R is predominantly expressed and its mRNA is widely distributed. Alternative splicing of IP(3)R1 (SI and SII) occurs in two distinct regions. SI splicing in the middle of the ligand binding domain may alter the IP(3) binding activity, while SII splicing probably affects the protein kinase A phosphorylation sites and kinetics. Selective use of IP(3)-receptor subtypes may permit a tissue specific and developmentally specific expression of functionally distinct channels. The present work was focused on detection of the alternatively spliced mRNA of type 1 IP(3)-receptor in individual brain structures and nuclei. Using RT-PCR we detected neuronal (535bp) and non-neuronal (410bp) forms. We identified both spliced variants in the majority of brain structures, except in the cerebellum and medulla. In the cerebellum, the neuronal form of type 1 IP(3)R was found exclusively, while in the medulla, the non-neuronal form was much more abundant. Nevertheless, Western blot analysis and hybridization with specific antibody against IP(3)R revealed no qualitative, but only quantitative differences. Similarly, IP(3) dependent calcium release did not show any differences between the cerebellum and pons. These results demonstrate the distribution of alternatively spliced S2 variants of type 1 IP(3)R in selected brain structures and nuclei. The physiological relevance of these two forms remains to be elucidated by further studies.

Repeated immobilization stress reduces rat vertebral bone growth and osteocalcin.

We previously showed that psychological stressors alter plasma levels of osteocalcin (pOC), a bone-specific mineral binding protein, in ways that differ with the type of stressor. To determine effects of chronic stress, we examined vertebrae, pOC, and corticosterone levels from conscious rats subjected to foot-restraint immobilization (Immo) daily for 1-42 times. After 40-42 Immo, basal pOC was decreased by 25% compared with unstressed rats, and the subsequent rise in pOC during Immo was blunted. Corticosterone was elevated 10-fold during Immo. Immo for seven times did not change vertebral OC concentration, but caused a slight decrease in calcium and phosphorous concentrations in younger rats. Rats Immo for 42 times exhibited reduced body weight, vertebral weight, and vertebral OC concentration but no significant differences in vertebral mineral concentrations. Body fat content was visibly decreased. We do not know the source of or the stimulus for the initial rise in pOC. We conclude that both decreased growth and bone OC concentration are due to repeatedly elevated stress hormones.

Stress increases brain-derived neurotropic factor messenger ribonucleic acid in the hypothalamus and pituitary.

Brain-derived neurotropic factor (BDNF) is a member of the nerve growth factor family that is important for neuronal survival and plasticity. We recently demonstrated that stress decreases BDNF messenger RNA (mRNA) levels in the hippocampus, which raises the possibility that BDNF may play a role in regulation of the hypothalamic-pituitary-adrenal axis. The purpose of this study was to determine whether BDNF expression is present and influenced by stress in other brain areas relevant to control of the hypothalamic-pituitary-adrenal axis. Using in situ hybridization, we found that BDNF mRNA is present in the parvocellular portion of the hypothalamic paraventricular nucleus (PVN), the lateral hypothalamus, and the anterior and neurointermediate lobes of the pituitary in rats. Acute (2-h) or repeated immobilization stress increased BDNF mRNA in all of these areas. This was in distinct contrast to stress-induced decreases in extrahypothalamic areas, including the basolateral amygdala, claustrum, and cingulate cortex as well as the hippocampus. BDNF was expressed in both CRF and TRH neurons in the PVN. Reducing glucocorticoid or thyroid negative feedback increased BDNF mRNA in the PVN and anterior pituitary, but not in the neurointermediate lobe. These results suggest that BDNF is a stress-responsive intercellular messenger that may be an important component of the stress response.

Decreased stress responsivity of central and peripheral catecholaminergic systems in aged 344/N Fischer rats.

We investigated the effects of stress on central and peripheral sympatho-adrenal and sympatho-neural functions in healthy, intact young (3-4 mo) and aged (24 mo) male Fischer 344/N rats. Extracellular fluid (ECF) levels of the catecholamines norepinephrine (NE), dihydroxyphenylglycol (DHPG), methoxyhydroxyphenylglycol (MHPG), and dihydroxyphenylacetic acid (DOPAC) were obtained by microdialysis in the paraventricular nucleus (PVN) of the hypothalamus at baseline and during immobilization (IMMO). The baseline levels of these substances were similar in both age groups, and their concentrations increased significantly in response to IMMO. The IMMO-induced increases of NE and MHPG, however, were significantly smaller in old than in young rats. Plasma levels of the catecholamines NE, DHPG, MHPG, DOPAC, dihydroxyphenylalanine (DOPA), epinephrine (EPI), dopamine (DA), and HVA were also determined in young and old rats during IMMO. Basal levels of these substances were significantly higher in old than in young rats. The magnitude of the IMMO-induced increases in the majority of these compounds however, was significantly smaller in old than in young rats. We conclude that, at the basal state, aging in the Fischer rat is associated with normal PVN ECF, but high plasma catecholamine levels; at stress state, however, old rats have substantially lesser activation of their central and peripheral catecholaminergic systems than young rats.

Sex differences in endocrine response to hyperthermia in sauna.

Neuroendocrine response was investigated during and after a single 20 min bath in sauna (80 degrees C) in a group of 8 healthy men and 8 healthy women. In an additional group of 8 young men, the dynamics of plasma ACTH and cortisol levels were studied during a 30 min sauna exposure (90 degrees C). This dynamic study showed a biphasic response of plasma cortisol which decreased during the initial phase of sauna bath (15 min) and increased thereafter, reaching its maximum 15 min after the end of bathing. Maximal increase in plasma ACTH levels occurred 15 min earlier. In the first sauna exposed group the increase in body temperature was the same (about 2 degrees C) in both sexes. Nevertheless, the elevation in plasma ACTH concentration was significantly more pronounced in women than in men. In the plasma collected at the end of sauna bath inside the sauna room, a significant rise in both adrenaline and noradrenaline levels was found. Though the catecholamine responses were similar in both groups, the increase in heart rate during sauna bath was significantly higher in women. Sauna-induced prolactin release was also more pronounced in women compared with men. Thus hyperthermia induced by sauna exposure resulted in a more pronounced neuroendocrine activation in women compared with men. Moreover, it is evident that repeated blood sampling is necessary to reveal the sauna-induced activation of some hormonal systems.

Immobilization stress rapidly decreases hypothalamic corticotropin-releasing hormone secretion in vitro in the male 344/N Fischer rat.

Corticotropin-Releasing-Hormone (CRH) is the principal secretagogue for plasma ACTH and corticosterone secretion and plays an important role in coordinating a variety of physiological and behavioral responses to stress. To explore whether there is a rapid change in the secretory response of the hypothalamic CRH neuron during acute stress, we report here a study of the effects of KCl and norepinephrine (NE) on CRH release in vitro from rat hypothalami explanted after 5, 30, 60, and 120 minutes of immobilization. We also measured the plasma levels of ACTH, beta-endorphin, corticosterone, prolactin, GH, and TSH at these intervals. As the duration of immobilization increased, KCl and NE-induced CRH release in vitro progressively fell. After reaching a maximal rise after 30 minutes of immobilization, plasma ACTH, beta-endorphin, and prolactin progressively fell in plasma, whereas corticosterone remained elevated up to 120 minutes; TSH and GH secretion rapidly declined and remained suppressed. Taken together, these data suggest that during immobilization stress, the responsiveness of the hypothalamic CRH neuron rapidly falls, owing either to CRH depletion and/or desensitization to NE, and this is paralleled by a concomitant decrease in pituitary-adrenal responsiveness.

Vasopressin and 1-deamino-8-D-arginine-vasopressin (DDAVP) reduce elevated plasma catecholamine levels in rats with hypothalamic deafferentation.

1. Anterolateral cut (ALC) of the medial basal hypothalamus (MBH) in rats produces an elevation of plasma catecholamine levels, especially of norepinephrine (NE), in unstressed animals and a more pronounced rise of plasma NE levels in response to immobilization (IMO). Animals with ALC have a destroyed corresponding vasopressin (AVP) and other peptides containing innervation of the median eminence and the posterior pituitary, resulting in the prevention of increased AVP secretion during the early intervals of IMO. 2. The administration of AVP (Pitressin, 7 days, 1 IU per rat i.m.) or of 1-deamino-8-D-arginine-vasopressin (DDAVP), an AVP analogue without pressoric activity, taken in drinking water (about 100 micrograms per day) was almost equally potent in decreasing the elevated water consumption and plasma NE levels in unstressed rats with ALC. However, the stress-induced potentiation of plasma NE levels in rats with ALC was not influenced by AVP substitution and only partly reduced by DDAVP in the late IMO intervals. 3. The lack of circulating vasopressin is the main factor in the mechanism of increased activity of the sympathoadrenal system induced by ALC in unstressed rats. 4. The regulation of sympathoadrenal activity by vasopressin and DDAVP in rats with ALC seems to be mediated predominantly by V2-subtype receptors. 5. In stressed rats with ALC the potentiation of plasma NE levels was not reduced after AVP or DDAVP administration, suggesting that some addition regulatory mechanisms were involved.

Acetylcholine and its enzymes in some brain areas of the rat under stress.

A period of 1 or 24 h of cold stress (5 degrees C) resulted in a significant decrease of acetylcholine (ACh) concentration in the hypothalamus and hippocampus in rats. In the hippocampus the activity of the choline acetyltransferase (ChAT) was significantly increased after 24 h and that of acetylcholinesterase (AChE) after 1 and 24 h exposure to cold, whereas in the hypothalamus, AChE activity was found to be decreased, albeit only after 24 h exposure. Separate investigation of the dorsal and ventral hippocampus under 24 h of cold revealed that the ACh decreased in the dorsal hippocampus only, where no change in ChAT activity was observed. On the other hand, ACh showed no change in the ventral hippocampus where an increase of ChAT activity was found. Forced swimming for 20 min also induced a significant decrease of ACh in the hippocampus and cerebral cortex, along with a significant increase of choline concentration in the given regions. We conclude that under certain stress situations the cholinergic system in rat brain areas, mainly in the hippocampus and hypothalamus, is activated.

Acetylcholinesterase activity in several hypothalamic and brain stem nuclei after acute and chronic immobilization stress in rats.

Acute and repeated immobilization stress increased acetylcholinesterase (AChE) specific activity in the nucleus supraopticus and median eminence of the rat and this increase was still present 24 hours after the 7th or 45th day of immobilization. The increase of AChE activity in those nuclei was probably related to the neuronal activation of the hypothalamic-pituitary axis during stress conditions. Acute and repeated immobilization was not accompanied by the change of AChE activity in A1 area and locus coeruleus. 46 days of immobilization periods produced a significant increase of AChE specific activity in the raphae dorsalis nucleus only which returned to normal values 24 hours after the last day of immobilization.

Neurotransmitter levels in the hypothalamus during postnatal development of rats.

The content of norepinephrine (NE), epinephrine (E), dopamine (DA) and serotonin (5-HT) in the rat hypothalamus was determined at four hours time interval during 24 hours period at the age of 12, 23, 27 and 75 days. It was found that the content of NE in the hypothalamus is low in 12 day-old rats, and during the postnatal period a gradual increase of NE was noted. The content of E in the hypothalamus is low in 12- to 27-day-old rats and an important increase was found in adult animals. The high values of DA and 5-HT concentration were found in the hypothalamus of 12-day-old rats and a transient decrease of 5-HT and DA at the age of 23 days was noted, further an important increase of the content of both neurotransmitters in the hypothalamus was demonstrated later in life. The presence of circadian rhythm of hypothalamic NE, E, DA and 5-HT content was found in 12-, 23-, 27-day-old and in adult rats overfed or underfed during the suckling period. However, no significant correlation was observed between the NE and E levels in the hypothalamus and the diurnal fluctuations of plasma corticosterone concentrations. In adult animals an inverse correlation between the plasma corticosterone level and hypothalamic 5-HT content was noted. In young 12- and 23-day-old rats the correlation between 5-HT in the hypothalamus and corticosterone in the plasma was positive. These results indicate that the shift of peak in plasma corticosterone levels during postnatal ontogenesis of rats observed in previous experiments is not dependent on the changes in diurnal fluctuation of hypothalamic neurotransmitter concentrations.

Increased glucagon secretion during hyperthermia in a sauna.

Plasma glucagon, adrenaline, noradrenaline, insulin and glucose concentrations were measured in 7 healthy young males during hyperthermia in a sauna bath: plasma glucagon levels increased from baseline values of 127.0 +/- 12.9 (SEM) pg X ml-1 to a maximum of 173.6 +/- 16.1 (SEM) pg X ml-1 at the 20th min of exposure. No change in plasma insulin and a slight increase in plasma glucose concentration were seen. Since a concomitant moderate increase in plasma catecholamine levels was also present, the adrenergic stimulus is believed to trigger glucagon release during hyperthermia. Diminished visceral blood flow, known to occur in sauna baths, may cause a decrease in the degradation of plasma glucagon and thus contribute to the elevated plasma glucagon levels.

Effect of repeated immobilization stress on central and peripheral adrenoceptors in rats.

Repeated forced immobilization stress (40 times for 2.5 h daily) reduced the number of beta-receptors in the heart, hypothalamus and brain stem, but not in the spleen of rats. Repeated immobilization stress has also been found to decrease the number of alpha1-receptors in the heart and increase the number of alpha2-receptors in the spleen and brain stem, as compared to control unstressed rats. The number of heart alpha1-, spleen and brain stem alpha2-receptors was still decreased or increased, respectively, 24 h after the 39th immobilization stress. However, at the same time the number of beta-receptors in the heart and brain stem returned to the control levels. We concluded, that the changes in the number of rat adrenoceptors in the heart, hypothalamus and brain stem correlate with peripheral catecholamines released during repeated immobilization stress.

Serotonin and tryptophan hydroxylase in isolated hypothalamic and brain stem nuclei of rats exposed to acute and repeated immobilization stress.

The effect of acute and repeated immobilization stress on serotonin concentration and tryptophan hydroxylase activity in some isolated hypothalamic and brain stem nuclei was measured using a special microdissection technique and sensitive isotopic-enzymatic microassays. In acutely immobilized rats serotonin concentration was increased in the median eminence, ventromedial and dorsomedial nuclei. In repeatedly immobilized rats increased serotonin concentration was recorded in the dorsomedial nucleus immediately after seven, and in the median eminence after forty consecutive daily exposures to immobilization. Decreased tryptophan hydroxylase activity was found in the suprachiasmatic nucleus after seven exposures to immobilization stress. Acute and repeated immobilization stress failed to produce any changes of serotonin concentration in the isolated brain stem nuclei studied, and of tryptophan hydroxylase activity in the dorsal raphe nucleus and n. centralis superior. The increased tryptophan hydroxylase activity observed without any changes in serotonin concentration in the locus coeruleus after the 7th immobilization may suggest an increased synthesis and release rate of serotonin in serotonergic nerve terminals in this area. The changes of serotonin concentration in some hypothalamic nuclei under the first exposure of rats to stress indicate the involvement of serotonin in the activation of the pituitary-adrenocortical system as well as in other neuroendocrine reactions initiated in the hypothalamus during acute stress. On the basis of the results presented, the presumed role of the serotonergic system in the regulation of pituitary-adrenocortical stress response in repeatedly stressed rats has not been established. The reported response of brain tryptophan hydroxylase to the release of endogenous corticosterone could not be confirmed in our experiments.

Catecholamines and their enzymes in discrete brain areas of rats after space flight on biosatellites Cosmos.

The activity of the catecholaminergic system was measured in the hypothalamus of rats which had experienced an 18.5-19.5-day-long stay in the state of weightlessness during space flights on board Soviet biosatellites of the type Cosmos. In the first two experiments, Cosmos 782 and 936, the concentration of norepinephrine and the activities of synthesizing enzymes tyrosine hydroxylase and dopamine-beta-hydroxylase and of the degrading enzyme monoamine oxidase were measured in the total hypothalamus. None of the given parameters was changed after space flight. In the light of the changes of these parameters recorded after exposure to acute stress on Earth, this finding indicates that long-term state of weightlessness does not represent an intensive stressogenic stimulus for the system studied. In the space experiment Cosmos 1129, the concentration of norepinephrine, epinephrine, and dopamine was studied in isolated nuclei of the hypothalamus of rats within 6-10 hr following return from space. Norepinephrine was found to be significantly reduced in the arcuate nucleus, median eminence and periventricular nucleus, epinephrine in the median eminence, periventricular and suprachiasmatic nuclei, whereas dopamine was not significantly changed after space flight. The decreased catecholamine levels found in some hypothalamic nuclei of rats which had undergone space flight indicate that no chronic intensive stressor could have acted during the flight, otherwise the catecholamine concentration would have been increased in the nuclei. The decreased levels must have been induced by the effect of a stressogenic factor acting for a short time only, and that either during the landing maneuver or immediately after landing. Thus long-term exposure of the organism to the state of weightlessness does not represent a stressogenic stimulus for the catecholaminergic system in the hypothalamus, which is one of the regulators of the activation of neuroendocrine reactions under stress.

Distribution of catecholamines and serotonin in 17 portions of rat ventromedial nucleus and effect of acute immobilization stress.

The distribution of catecholamines (i.e. adrenalin -- A, noradrenalin -- NA, dopamine -- DA) and serotonin (5-HT) in 17 portions of hypothalamic ventromedial nucleus (VMN) and the effect of acute immobilization stress on their levels were studied in rats using of a special dissection technique and a sensitive radioenzymatic microassay. In unstressed rats distinct differences of biogenic amine concentrations between individual portions within the hypothalamic ventromedial nucleus subdivisions were found. The concentration of all catecholamines investigated, i.e. NA, A and DA was lower in the lateral half of VMN, while in the distribution of 5-HT we did not find such apparent regional differences. After acute immobilization stress for 30 min significant changes in all measured neurotransmitters, in NA and A decreases and in DA and 5-HT increases, were detected in several subdivisions of the nucleus. On the basis of the present data we suppose that not all subdivisions of the VMN are equally engaged in neuroendocrine processes activated during acute stress.

[Noradrenaline and the enzymes of its synthesis and breakdown in the rat hypothalamus after a flight on the Kosmos-936 biosatellite]. / Noradrenalin i fermenty ego sinteza i raspada v gipotalamuse krys posle poleta na biosputnike "Kosmos-936".

In the hypothalamus of the weightless and centrifuged rats flown for 18.5 days on board the biosatellite Cosmos-936 the noradrenaline concentration and activity of the enzymes involved in the catecholamine synthesis and degradation were measured. It was found that under the space flight influence the noradrenaline concentration and tyrosine hydroxylase, dopamine-beta-hydroxylase and monoamine oxidase activities remained unaltered. These findings indicate that a prolonged exposure to weightlessness was not a stressogenic agent that could activate the adrenergic system in the rat hypothalamus.