Different effects of insulin and 2-deoxy- -glucose administration on tyrosine hydroxylase gene expression in the locus coeruleus and the adrenal medulla in rats

The major brain norepinephrinergic nucleus, locus coeruleus, is an important integrating element of extero- and interoceptive stimuli in organisms facing different physiological challenges. We investigated the effects of single and repeated (seven times) exposure to immobilization stress (120 min daily), insulin (5 IU/kg, i.p. daily) or 2-deoxy- -glucose (500 mg/kg, i.p. daily) administration on tyrosine hydroxylase (TH) mRNA levels, the rate-limiting enzyme in catecholamine biosynthesis, by in situ hybridization in locus coeruleus and by Northern blot analysis in the adrenal medulla of rats. Both the single and repeated immobilization caused a significant increase in TH mRNA levels in the locus coeruleus (1.5–2-fold; p < 0.05) and in the adrenal medulla (about 4-fold; p < 0.05) when compared with unstressed controls. Hypoglycemia induced by a single or repeated insulin administration led to about fourfold (p < 0.01) elevation in adrenal medullary TH mRNA levels, whereas TH mRNA in locus coeruleus remained unchanged when compared with saline-treated controls. In contrast to the effect of insulin-induced hypoglycemia, cellular glucoprivation caused by a single or repeated 2-deoxy- -glucose administration significantly elevated TH mRNA levels in both the adrenal medulla (fourfold; p < 0.01) and the locus coeruleus (twofold; p < 0.01). Our data suggest that in contrast to immobilization or cellular glucoprivation caused by 2-deoxy- -glucose administration, insulin-induced hypoglycemia is not a specific or quantitatively sufficient stimulus for induction of TH gene expression in the locus coeruleus, although all these stressors strongly activate the process in the adrenal medulla.     

Effect of novel stressors on gene expression of tyrosine hydroxylase and monoamine transporters in brainstem noradrenergic neurons of long-term repeatedly immobilized rats

Responses of central noradrenergic (NE) neurons to stressors like immobilization (IMO), cold exposure, insulin-induced hypoglycemia, and cellular glucoprivation caused by 2-deoxy-D-glucose (2-DG) were investigated in intact and long-term repeatedly immobilized (LTR, 2 h daily IMO for 41 days) rats. Expression of tyrosine hydroxylase (TH), norepinephrine transporter (NET) and vesicular monoamine transporter (VMAT2) genes were determined by using in situ hybridization histochemistry in brainstem A1, A2, A5 and locus coeruleus (LC) neurons. TH mRNA levels were increased by single IMO or 2-DG administration in all areas studied. Cold was effective only in LC and A2 neurons while insulin had no effect. LTR immobilization elevated TH mRNA levels in all investigated cell groups. These elevations were equally high to those elicited by a single IMO in each noradrenergic group, except the LC where LTR IMO was less effective than the single IMO. The levels of NET and VMAT2 mRNAs were elevated only in the A1 and A2 cell groups of LTR IMO rats. A newly applied IMO in LTR rats did not alter TH, NET, and VMAT2 mRNA levels in any NE cell group investigated. Novel stressors like cold and 2-DG exaggerated the increased TH mRNA levels only in the LC of LTR IMO rats, unlike in the other NE cell groups. The present data indicate that repeated exposure of rats to homotypic stressor induces an adaptation of NE neurons, whereas single exposure of such animals to heterotypic novel stressor produces an exaggerated response of the system at the level of TH (in LC) and NET (in A1, A2) gene expression.     

Repeated amphetamine evokes biphasic alterations in the tyrosine hydroxylase mRNA level in the rat adrenal medulla: an in situ hybridization study

Summary In the present study we estimated the effects of single and repeated administration of d-amphetamine (5mg/kg, i.p., twice a day for 14 days) on tyrosine hydroxylase (TH) mRNA levels in the rat adrenal medulla. In situ hybridization experiments, conducted using a [³⁵S]d-ATP-labelled deoxyoligonucleotide probe and a densitometric analysis of autoradiograms, showed that repeated d-amphetamine moderately increased the TH mRNA level (by ca. 24%) in the adrenal medulla at 2 h after the last injection. In contrast, after 48 h the TH mRNA level was decreased (by ca. 21%). No significant changes in the TH mRNA level in the adrenal medulla were found following single administration of d-amphetamine. These results suggest that repeated d-amphetamine administration leads to biphasic changes in the adrenal TH biosynthesis, which may reflect an adaptive response to chronic drug treatment.     

Cellular localization of tyrosine hydroxylase mRNA and its regulation in the rat adrenal medulla and brain by in situ hybridization with an oligodeoxyribonucleotide probe

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamines in neural tissues and adrenal medulla. To study the expression of the TH gene and its regulation in adult and developing neural tissues, we have synthesized an oligodeoxyribonucleotide probe (oligomer) that is specific for TH mRNA. Using Northern blot hybridization of polyadenylated RNAs from adrenal gland, brain stem, liver, and cerebral cortex with the 32P-labeled oligomer, a single TH mRNA of 1.9 kb was detected in adrenal gland and brain stem but not in liver and cerebral cortex. Using this TH-specific oligomer, TH mRNAs were localized to the chromaffin cells in the adrenal medulla and to catecholaminergic neurons in locus coeruleus and substantia nigra by in situ hybridization histochemistry. After reserpine administration, the intensity of hybridization signal was increased to threefold that of normal in sections of adrenal medulla and twofold that of normal in locus ceruleus. No difference in hybridization signal intensity was observed in the substantia nigra of normal and reserpine-treated animals. Use of this specific TH probe in in situ hybridization procedures represents a powerful approach to the study of regulation of TH gene expression at the cellular level.     

Regulatory role of glucocorticoids and glucocorticoid receptor mRNA levels on tyrosine hydroxylase gene expression in the locus coeruleus during repeated immobilization stress

Sustained responsiveness of the hypothalamic-pituitary-adrenal (HPA) axis during chronic or repeated stress is associated with continuous activation of ascending noradrenergic neurons from the brainstem to the hypothalamic paraventricular nucleus (PVN). The fact that glucocorticoid receptor (GR) exists in the brainstem noradrenergic neurons including locus coeruleus (LC) suggests that glucocorticoids play a modulatory role in maintaining the activity of these neurons during chronic stress. To determine whether alterations in the sensitivity of noradrenergic neuronal activity to endogenous CORT occur during chronic or repeated stress, tyrosine hydroxylase (TH) and GR mRNA expressions in the LC were examined in acute (2 h) and repeated (2 h daily, 14 days) immobilization stress, using sham-operated rats and adrenalectomized rats with a moderate dose of CORT replacement (ADX+CORT group). In acute stress, TH mRNA in the LC increased in the ADX+CORT rats, but not in sham operated rats. In repeated stress, however, elevated endogenous CORT failed to inhibit TH mRNA responses in sham rats; LC TH mRNA in sham rats responded to the same extent as in ADX+CORT rats. A reduction of GR mRNA in the LC was observed in the acutely stressed and repeatedly stressed sham group, but not in the ADX+CORT groups. The decrease in LC GR mRNA levels in sham rats tended to be greater after repeated than after acute stress. LC GR mRNA levels decreased in response to systemic CORT treatment (200 mg pellet sc, for 14 days) and increased in response to adrenalectomy; neither CORT treatment nor adrenalectomy influenced TH mRNA levels in the LC. These results suggest that glucocorticoid responses to acute immobilization prevent LC TH mRNA levels from rising significantly, while glucocorticoids appear to decrease their capacity to restrain LC TH mRNA during repeated immobilization. Although the results clearly show glucocorticoid-dependent alterations in LC GR mRNA expression, the association between increased TH mRNA and decreased GR mRNA in the LC remains to be elucidated.     

Differential regulation of neuropeptide Y mRNA expression in the arcuate nucleus and locus coeruleus by stress and antidepressants

In rats, circulating corticosterone and insulin are involved in regulation of the hypothalamic neuropeptide Y (NPY) system, which in turn, is involved in regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Since the HPA axis and stress responsivity is altered in diseases such as depression, we investigated interactions between the effects of stress and antidepressant drug treatment on arcuate nucleus and locus coeruleus NPY mRNA expressions using in-situ hybridization histochemistry. After acute (2 h) and repeated immobilization (2 h daily, for 14 days), plasma concentrations of corticosterone increased, and those of insulin decreased. The expression of NPY mRNA was significantly increased in the arcuate nucleus, but was unchanged in the locus coeruleus following acute and repeated immobilization. Adrenalectomized rats with systemic corticosterone replacement (ADX+CORT), whose corticosterone concentration was maintained at approximately 50-100 ng/ml during repeated stress, showed a decrease in plasma insulin and an increase in arcuate nucleus NPY mRNA similar to that observed in sham rats, suggesting that changes in NPY mRNA levels are more closely tied to circulating insulin than to circulating corticosterone. In contrast, locus coeruleus NPY mRNA expressions in ADX+CORT rats were significantly higher than those in sham rats after repeated stress. Desmethylimipramine (DMI) treatment for 24 days did not affect basal plasma concentrations of corticosterone or insulin, or arcuate nucleus NPY mRNA expressions, but significantly decreased basal levels of locus coeruleus NPY mRNA compared to saline-treated rats. After repeated immobilization (2 h daily, for 4 days), DMI significantly reduced the stress-induced rise in locus coeruleus NPY mRNA levels, but potentiated the stress-induced rise in arcuate nucleus NPY mRNA expression. These results demonstrate that: (1) the increase in arcuate nucleus NPY mRNA expressions in stressed rats closely follows the decrease in plasma concentrations of insulin; (2) increases in NPY mRNA expressions occur in the absence of changes in plasma corticosterone; and (3) desipramine treatment potentiated the effect of stress on arcuate nucleus NPY mRNA expressions, but blocked the repeated stress-induced increase in locus coeruleus NPY mRNA expressions. Thus, NPY mRNA expression in the arcuate nucleus and the locus coeruleus is sensitive to the effects of stress and to the antidepressant drug desipramine, but the arcuate nucleus NPY system is regulated by different mechanisms than the locus coeruleus NPY system. The results provide further evidence for the importance of circulating insulin in the regulation of the arcuate nucleus NPY system.     

Adrenal medullary responses to insulin-induced hypoglycaemia in the young rat. Influence of thyroid hormones

The adrenal medulla of normal, hypothyroid and hyperthyroid young rats was stimulated by insulin-induced hypoglycaemia. In normal rats, insulin-induced adrenal epinephrine secretion increases during the first 10 days of post-natal life. Hypothyroidism retards the development of adrenal response; hyperthyroidism facilitates the development of this response. At 14 days, when insulin-induced adrenal epinephrine depletion is the same for all groups, the recovery of adrenal catecholamines stores after depletion is linear and takes less than 48 h. Recovery rate is slightly slower for hyperthyroid rats than for either hypothyroid or control rats at 14 days. Following epinephrine depletion, adrenal tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) activities are increased for a few days in the control rats, corresponding to a transsynaptic induction. Hypothyroidism impairs TH induction and completely suppresses DBH induction; hyperthyroidism impairs TH induction, but has no effect on DBH induction. These data show that the various processes related to CA synthesis, in the adrenal medulla of the developing rat, are controlled in different ways by the thyroid hormones.     

Repeated administration of methamphetamine blocked cholecystokinin-octapeptide injection-induced c-fos mRNA expression without change in capsaicin-induced junD mRNA expression in rat cerebellum

In the cerebellum, there are numerous cholecystokinin (CCK-8)-containing fibers. Since systemic CCK-8 injection-induced anxiety (psychological stress) activates the locus coeruleus cells that send mossy fiber inputs to the cerebellum, we examined whether systemic CCK-8 injections activate the rat and mouse cerebellum. First, injections of CCK-8 were found to induce c-fos mRNA expression in a vague patchy pattern that is different from single methamphetamine-induced Zebrin band-like c-fos mRNA expression, suggesting that the CCK-8 activating mossy fibers induce gene expression differently from the dopamine-containing mossy fibers in the ventral tegmental area. Second, since CCK-8 facilitates neural activity of dopamine in the midbrain, we examined whether repeated methamphetamine administration that induced behavioral sensitization had similar effects on the cerebellar CCK system. Repeated administration of methamphetamine suppressed the CCK-8-induced c-fos mRNA expression in the rat cerebellum. Third, capsaicin injections (physical stress) into a hind limb of the rat increased junD mRNA expression with no effect on c-fos mRNA expression, and repeated methamphetamine injections had no effect on the capsaicin-induced expression of junD mRNA. Fourth, either single injection of methamphetamine or CCK-8 to mice increased c-fos mRNA expression in the locus coeruleus, and so noradrenalin, but not dopamine, might interact with CCK-8-activating system. However, we considered the possibility unlikely. Thus, we conclude that repeated methamphetamine administration though dopamine selectively inhibits the c-fos mRNA expression after CCK-8 injection in the cerebellum.     

Neuropeptide Y and tyrosine hydroxylase mRNA levels in the locus coeruleus show similar increases after reserpine treatment.

In situ hybridisation histochemistry was used to study the effect of intra-peritoneal reserpine administration on messenger RNA (mRNA) levels in the locus coeruleus (LC) of the rat. 48h after injection, levels of mRNA encoding tyrosine hydroxylase (TH) and neuropeptide Y (NPY) in the LC increased to approximately 250% of control values (p less than 0.05). The level of beta-tubulin mRNA was unaffected by reserpine administration. The similar and selective increase in TH and NPY mRNA in the LC following reserpine administration suggests that NPY may play a role in the neurotransmitter function of this catecholaminergic nucleus.     

Effects of acute restraint stress on tyrosine hydroxylase mRNA expression in locus coeruleus of Wistar and Wistar-Kyoto rats

Norepinephrine (NE) is thought to play a role in the stress response, and may be involved in stress-related psychopathological conditions such as depression or anxiety. Heterogeneity in individual responses to the same stressor suggest that a genetic susceptibility to the effects of stress may contribute to such pathology. To address possible mechanisms underlying this genetic aspect of the stress response, we examined acute stress-induced changes in mRNA expression for several components of the NE system in the locus coeruleus (LC) and adrenal medullae of stress-susceptible Wistar-Kyoto (WKY) rats and their parent Wistar (W) strain. Expression of tyrosine hydroxylase (TH), NE transporter (NET) and alpha(2A) receptor mRNA were measured in the LC by in situ hybridization 30 min and 2 h after the onset of 30 min restraint stress. Adrenal TH mRNA was measured by slot blots. No basal differences were observed for any measure, but in the LC, expression of TH mRNA increased by 40% in W rats at 30 min (n=8, p<0.05) and returned toward baseline by 2 h, while WKY rats showed only a non-significant 29% increase at 2 h. In contrast, adrenal TH mRNA expression increased in WKY rats at 2 h (n=3, p<0.05), with no significant change in W rats. NET and alpha(2A) mRNA were unaltered by restraint stress in both strains. Differences in the stress-reactivity of TH gene expression in the central and peripheral noradrenergic systems may be related to differences in behavioral coping strategies and autonomic responsivity to stress in these strains, and suggest that differences in noradrenergic reactivity may contribute to genetic susceptibility to stress-related pathology.     

Effects of reserpine on tyrosine hydroxylase mRNA levels in locus coeruleus and medullary A1 and A2 neurons analyzed by in situ hybridization histochemistry and quantitative image analysis methods.

These studies examined the effects of reserpine on concentrations of norepinephrine (NE), dopamine (DA) and epinephrine (EPI) and on levels of tyrosine hydroxylase (TH) mRNA in locus coeruleus (LC) and medullary A1 and A2 neurons. Noradrenergic neurons in these regions first were identified by immunocytochemistry and, thereafter, by in situ hybridization histochemistry. Levels of TH mRNA were measured by quantitative image analysis methods. Changes in catecholamine concentrations in micropunches of these brain regions were analyzed by HPLC. Epinephrine was not detected in any of the nuclei examined. Twenty-four hours after reserpine treatment, NE concentrations declined in A1, A2 and LC neurons by 46, 69 and 34% respectively while DA declined only in the region of A2 neurons. This reserpine-induced depletion of NE was accompanied by a 2- to 3-fold increase in TH mRNA levels in LC and A1 neurons but no change in message levels occurred in A2 cells 24 h after reserpine. Forty eight hours later, message levels in A1 and LC neurons did not differ significantly from the elevated 24 h values but TH mRNA levels in A2 neurons now were significantly elevated compared to 24 h values. TH mRNA levels 72 h after reserpine did not differ from 48 h values in A1, A2 and LC neurons. Thus, TH gene expression in A1 neurons increases after reserpine treatment in a manner equivalent to that observed in LC, adrenal medulla and superior cervical ganglia. The reason why it required 48 h for TH mRNA to increase in A2 neurons remains unclear.     

Repeated immobilization stress alters tyrosine hydroxylase, corticotropin-releasing hormone and corticosteroid receptor messenger ribonucleic Acid levels in rat brain

In situ hybridization histochemistry was used to localize and quantify the effects of acute and repeated immobilization stress on mRNA levels of tyrosine hydroxylase (TH) in catecholaminergic neurons in the locus ceruleus and substantia nigra and on mRNA levels of relevant markers of the hypothalamic-pituitary-adrenal axis, namely corticotropin-releasing hormone (CRH) in the hypothalamic paraventricular nucleus (PVN), proopiomelanocortin in the pituitary, and mineralocorticoid receptors (MR, type I) and glucocorticoid receptors (GR, type II) in the hippocampus, PVN and pituitary. Control, acutely stressed (1 × lMO, sacrificed immediately after 2 h of immobilization), and repeatedly stressed (6 × IMO plus delay, sacrificed 24 h after 6 daily 2-h immobilizations and 6 × lMO plus challenge, sacrificed immediately after the seventh daily 2-h immobilization) male Sprague-Dawley rats were examined. TH mRNA expression was increased in the locus ceruleus in the acutely stressed and repeatedly stressed animals. The increase in TH mRNA levels was greatest in the repeatedly stressed (6 × IMO plus challenge) group. TH mRNA levels were not altered in the substantia nigra. CRH mRNA levels in the PVN were significantly increased in the three stressed groups and the increase was greatest in the 6 × IMO plus challenge group. CRH mRNA levels were increased in the central nucleus of the amygdala only after acute stress. Proopiomelanocortin mRNA levels were elevated in the anterior pituitary during acute and repeated stress, but the magnitude of the effect was largest after acute stress. The changes in the hypothalamic-pituitary-adrenal axis were accompanied by an acute stress-induced increase in MR mRNA levels in the hippocampus, MR and GR mRNA levels in the PVN and GR mRNA levels in the pituitary. MR mRNA levels continued to be elevated in the PVN in the 6 × IMO plus challenge animals. Plasma corticosterone levels were elevated in the acute and repeated stress conditions. The results show that repeated immobilization stress produces a rapid and persistent increase in mRNA expression of TH in the locus ceruleus, CRH in the PVN, and proopiomelanocortin in the anterior pituitary. The TH-containing neurons in the locus ceruleus and the CRH-containing neurons in the PVN appear to preserve the capability to respond to repeated stimulation (6 × IMO plus challenge) indicating altered feedback mechanisms under repeated stress conditions. GR and MR mRNA levels are differentially regulated in the hippocampus, PVN and pituitary by acute and repeated stress. It is of interest that the central nervous system systems which are activated during repeated stress, namely the locus ceruleus-norepinephrine system and hypothalamic-pituitary-adrenal axis, are dysregulated in melancholic depression. Further studies of the central nervous system effects of prolonged exposure to stress may help elucidate the mechanisms underlying dysregulation of the locus ceruleus-norepinephrine system and hypothalamic-pituitary-adrenal axis in depression and other stress-related psychiatric diseases.     

Repeated restraint and nerve growth factor administration in male and female mice: effect on sympathetic and cardiovascular mediators of the stress response

Chronic stress and increased sympathetic nerve activity have been associated with cardiovascular disorders such as hypertension, myocardial infarction and stroke. The aim of this study was to investigate the role of nerve growth factor (NGF) on the expression of tyrosine hydroxylase (TH), vascular-endothelial growth factor (VEGF) and leptin receptor (OB-R) in brain, adrenal and cardiovascular tissues of adult male and female mice following a chronic stress procedure. It was found that daily restraint for 10 consecutive days alters TH levels in hypothalamic and brainstem areas related to sympathetic activation, in both male and female mice. Chronic stress procedure also modifies heart and aorta VEGF levels in male mice, and adrenal glands TH in female mice. The NGF administration in stressed mice reverted the stress-induced up-regulation of TH levels in male and female mice hypothalamic nuclei and in male locus coeruleus. Administration of NGF in stressed animals also down-regulated OB-R levels in the hypothalamus of both male and female mice and in the female aorta. Our findings indicate that repeated restraint in mice has an effect on TH and VEGF protein content at different brain and peripheral sites involved in the sympathetic and cardio-vascular response to stressful stimuli. NGF administration is able to counteract some of these stress-induced changes. Since NGF is known to be up-regulated during stress, a possible functional significance of our observations is that the circulating NGF released during and following stress may serve to prevent possible deficits and/or damage linked to stress-induced sympathetic and cardiovascular activation.     

Repeated administration of desmethylimipramine blocks the reserpine-induced increase in tyrosine hydroxylase mRNA in locus coeruleus neurons of the rat.

The levels of tyrosine hydroxylase and galanin mRNA were measured by in situ hybridization histochemistry in the rat locus coeruleus after repeated (21 days) administration of desmethylimipramine (10 mg/kg/day), of reserpine (0.25 mg/kg/day), of coadministered desmethylimipramine and reserpine, or of vehicle. Reserpine administration resulted in increased levels of both tyrosine hydroxylase and galanin mRNAs in locus coeruleus neurons as compared to vehicle-treated controls. Administration of desmethylimipramine alone failed to alter either the tyrosine hydroxylase or galanin mRNA. However, coadministration of desmethylimipramine with reserpine blocked the elevation in tyrosine hydroxylase mRNA induced by reserpine alone.     

Tyrosine hydroxylase mRNA levels in locus ceruleus of rats during adaptation to long-term immobilization stress exposure

The major central norepinephrinergic nucleus, locus ceruleus (LC), is thought to participate in modulation of such brain areas as cerebral cortex, septum, hippocampus, thalamus, hypothalamus, and cerebellum in animals facing various physiological challenges, including stress. Exposure of experimental animals to different stressors causes an increase in LC activity and gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. The aim of this work was to investigate the effect of a single and repeated (7 time) or long-term repeated (42 times) daily immobilization stress (IMMO) on TH mRNA levels in LC of laboratory rats byin situ hybridization method. A single IMMO caused significant elevation of LC TH mRNA levels in comparison to unstressed controls. This was found immediately and at 3 and 6 h after IMMO, and progressively increased up to 24 h after the first IMMO terminated. Further exposure to IMMO did not cause additional increases in LC TH mRNA levels, which stayed significantly elevated in comparison to unstressed rats. In animals that underwent IMMO for 42 times, the LC TH gene expression, 24 h after the last stress exposure, was significantly lower when compared to that of singly or seven times stressed rats. Thus, our results indicate a possible adaptation of catecholamine-synthesizing system at the level of TH gene expression in LC of rats exposed to long-term repeated IMMO.     

Oestrogen upregulates noradrenaline release in the mediobasal hypothalamus and tyrosine hydroxylase gene expression in the brainstem of ovariectomized rhesus macaques

Noradrenaline plays a key role in the initiation of ovulation in nonprimate species. A similar noradrenaline role in the primate has not been established experimentally. We utilized the ovariectomized-oestrogen-supplemented (OVX + E) rhesus macaque to examine the effects of intravenous (i.v.) infusion of oestradiol-17beta (E2) on the activity of the brain noradrenaline system. Experiment 1 established the induction of a preovulatory surge-like release of luteinizing hormone in OVX + E monkeys by i.v. infusion of E2 (OVX + E + E2). In experiment 2, a marked increase in hypothalamic microdialysate noradrenaline concentrations occurred after identical E2 infusion into the OVX + E monkeys that were used in experiment 1. In experiment 3, tyrosine hydroxylase (TH) mRNA expression in the locus coeruleus of the brainstem increased at various times after E2 infusion as determined by semiquantitative in situ hybridization. The amount of TH mRNA in OVX + E + E2 animals was higher (P < 0.05) than that in either the OVX + E or OVX monkeys; no difference was found in the latter two groups. Moreover, selected locus coeruleus sections from E2-infused monkeys were examined for the localization of oestrogen receptors (ER) by in situ hybridization. Both ER-alpha and ER-beta mRNAs were expressed in the locus coeruleus, although the expression was greater for ER-alpha than for ER-beta. We conclude that i.v. infusion of E2, which induces a preovulatory surge-like release of LH, stimulates brain noradrenaline activity; this enhanced activity likely involves an ER-mediated process and is reflected by hypothalamic noradrenaline release and locus coeruleus TH mRNA expression. The results support the concept that noradrenaline can influence the E2-stimulated ovulation in nonhuman primates and that the brainstem is one of the components in this neuroendocrine process.     

Expression of tyrosine hydroxylase in cerebellar Purkinje neurons of the mutant tottering and leaner mouse.

In situ hybridization histochemistry, Northern blot analysis and immunohistochemistry were used to examine tyrosine hydroxylase (TH) mRNA concentrations and immunoreactivity in the locus coeruleus and cerebellum of the tottering (tg/tg), leaner (tgla/tgla), compound heterozygous (tg/tgla) and wild type control (+/+) mice, bred on a C57BL/6J background. Cerebellar Purkinje neurons, long considered to be GABAergic, showed high levels of TH mRNA in the caudal vermis and the lateral hemispheres of the cerebellum of tg/tg, tg/tgla, and tgla/tgla mice. Analysis of grain density over individual Purkinje cells showed significantly greater concentrations of TH mRNA in tg/tg, tg/tgla, and tgla/tgla mice as compared to +/+ wild type control mice. Comparison of adult (greater than or equal to 2 months) and young, pre-seizure (less than or equal to 3 weeks) mutant mice showed Purkinje cells densely labelled for TH mRNA at both ages, suggesting that TH gene expression in Purkinje cells is independent of the onset of seizures. Northern blot analysis confirmed the findings from the in situ hybridization studies, demonstrating a single band identical to TH mRNA. Immunohistochemistry confirmed the presence of TH protein in Purkinje cells of the caudal vermis and the lateral hemispheres of the cerebellum in both control and mutant mice. Quantitation of mRNA for TH and the coexisting neuropeptide, galanin, in the locus coeruleus detected no significant differences between adult tg/tg, tg/tgla and +/+ control mice. The present findings demonstrate that the classically GABAergic Purkinje cells in the cerebellum express low levels of TH, and that the mutant tottering and leaner strains of mice express extremely high levels of mRNA and protein for TH.