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.     

Single low doses of MPTP decrease tyrosine hydroxylase expression in the absence of overt neuron loss

Parkinson's disease (PD) is the second most common age-related neurodegenerative disease. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a prototypical neurotoxicant used in mice to mimic primary features of PD pathology including striatal dopamine depletion and dopamine neuron loss in the substantia nigra pars compacta (SNc). In the literature, there are several experimental paradigms involving multiple doses of MPTP that are used to elicit dopamine neuron loss. However, a recent study reported that a single low dose caused significant loss of dopamine neurons. Here, we determined the effect of a single intraperitoneal injection of one of three doses of MPTP (0.1, 2 and 20 mg/kg) on dopamine neurons, labeled by tyrosine hydroxylase (TH⁺), and total neuron number (Nissl⁺) in the SNc using unbiased stereological counting. Data reveal a significant loss of neurons in the SNc (TH⁺ and Nissl⁺) only in the group treated with 20 mg/kg MPTP. Groups treated with lower dose of MPTP (0.1 and 2 mg/kg) only showed significant loss of TH⁺ neurons rather than TH⁺ and Nissl⁺ neurons. Striatal dopamine levels were decreased in the groups treated with 2 and 20 mg/kg MPTP and striatal terminal markers including, TH and the dopamine transporter (DAT), were only decreased in the groups treated with 20 mg/kg MPTP. These data demonstrate that lower doses of MPTP likely result in loss of TH expression rather than actual dopamine neuron loss in the SN. This finding reinforces the need to measure both total neuron number along with TH⁺ cells in determining dopamine neuron loss.     

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.     

Tyrosine hydroxylase mRNA is increased in old age and norepinephrine uptake transporter mRNA is decreased in middle age in locus coeruleus of Brown-Norway rats

In normal aging, cell loss occurs in the locus coeruleus (LC), the major noradrenergic nucleus in the brain. This study examined changes in the LC of aged rats by measuring mRNA expression for tyrosine hydroxylase (TH) and the norepinephrine uptake transporter (NET). TH and NET mRNA expression were measured by in situ hybridization in young, middle-aged and aged rats. It appears that in middle age, the transporter system responds initially to LC cell loss by decreasing NET mRNA expression. Then, with further aging and cell loss, TH mRNA expression increases which may potentially increase NE synthesis in the remaining neurons. These findings suggest that multiple regulatory components are used to maintain stable noradrenergic synaptic levels despite neuronal loss. Published by Elsevier Science B.V.     

Effect of repeated seizure experiences on tyrosine hydroxylase immunoreactivities in the brain of genetically epilepsy-prone rats

The genetically epilepsy-prone rat (GEPR) is a model of generalized tonic/clonic epilepsy, and has functional noradrenergic deficiencies that act as partial determinants for the seizure predisposition and expression. The present study investigated the effect of repeated seizure experiences by acoustic stimulation (110 dB, 10 times) on the immunoreactivities of tyrosine hydroxylase (TH), a rate-determining enzyme in the synthesis of norepinephrine, in brain regions of GEPRs. TH immunoreactivity in locus coeruleus, the major noradrenergic nucleus in brain, was lower in GEPRs than control Sprague-Dawley rats. It was also decreased in several regions including inferior colliculus of GEPRs. Repeated experiences of audiogenic seizures further decreased TH immunoreactivities in locus coeruleus and inferior colliculus of GEPRs. The results from the present study suggest that the lower immunoreactivities of TH in locus coeruleus and inferior colliculus contribute, at least in part, to the noradrenergic deficits in GEPRs, and repeated seizure experiences further intensified these noradrenergic deficits, which may be related to the altered seizure expression by repetitive audiogenic seizure in GEPRs.     

Different effects of insulin and 2-deoxy-D-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-d-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-d-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-d-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 starvation on hypothalamic tyrosine hydroxylase activity in adult male rats

The activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in norepinephrine synthesis, was determined in the mediobasal hypothalamus of adult male rats during acute and semistarvation. 3,4-Dihydroxyphenylalanine (DOPA) formed by TH was measured using high-performance liquid chromatography (HPLC). Acute starvation, as well as 3 weeks of semistarvation on a high-protein low-carbohydrate diet (CST PR) reduced TH activity significantly. Three weeks of a low-protein high-carbohydrate diet (CST KH) did not affect TH activity. While maximal velocity (Vmax) is significantly diminished in acute starvation and in semistarvation with a high-protein low-carbohydrate diet, Kd-values for tyrosine were not changed. These results suggest that TH activity in the brain contributes to decreased norepinephrine (NE) turnover in starvation.     

Role of nitric oxide in the regulation of gonadotropin-releasing hormone and tyrosine hydroxylase gene expression in the male rat brain

It has been recently demonstrated that nitric oxide (NO), a free radical gas which may act as neurotransmitter in the brain, can stimulate the in vivo release of luteinizing hormone as well as the in vitro hypothalamic release of gonadotropin-releasing hormone (GnRH). In order to study the influence of NO on GnRH mRNA expression, two inhibitors of NO synthase (NOS) N^G-monomethyl-

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-arginine (NMMA) and HP-228, were microinjected into the left lateral ventricle of sham-operated and castrated male rats 4 h before sacrifice. Since the dopaminergic system can positively influence GnRH gene expression, we have also measured in the same animals tyrosine hydroxylase (TH) mRNA in tuberoinfundibular dopamine (TIDA) neurons. GnRH and TH mRNA levels were measured at the cellular level by quantitative in situ hybridization. The injection of HP-228 or NMMA induced a similar decrease (−19.5%) in GnRH mRNA. In castrated animals, the hybridization signal was 88% higher than that observed in sham-operated animals. Both HP-228 and NMMA produced in castrated animals a 39% decrease in GnRH mRNA. In contrast the injection of NOS inhibitors resulted in an increase in the amount of TH mRNA in TIDA neurons. The stimulating effect was more striking in HP-228-treated (+60%) than in NMMA-treated (+32%) animals. Castration did not induce any changes in the number of silver grains overlying TIDA neurons, while the administration of either HP-228 or NMMA induced a 43% increase in castrated animals. These results together with previous ones on GnRH release in vitro suggest that NO exerts a positive influence not only on the secretion but also on the biosynthesis of GnRH. Since NO appears to play a role in the negative regulation of dopamine, it is likely that the increase in GnRH mRNA expression is not mediated by the TIDA system.     

Effects of stress and adrenalectomy on tyrosine hydroxylase mRNA levels in the locus ceruleus by in situ hybridization

The locus ceruleus-norepinephrine system is one of the principal effectors of the stress response. Acute stress induces norepinephrine synthesis and release, and noradrenergic cells compensate by increasing the activity of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis. Here we use in situ hybridization histochemistry to show the effects of acute and chronic intermittent stress on the expression of tyrosine hydroxylase mRNA in the rat locus ceruleus. Restraint stress increased tyrosine hydroxylase mRNA in the locus ceruleus but not in dopaminergic nuclei such as the substantia nigra or ventral tegmental area. One hour of footshock or restraint caused a rapid increase in locus ceruleus tyrosine hydroxylase mRNA which returned to basal levels within 24 h. Chronic intermittent stress (1 hour of restraint or footshock per day for 14 days) produced no change in tyrosine hydroxylase mRNA. Neither adrenalectomy nor dexamethasone replacement significantly affected mRNA expression. These findings indicate that acute stress can increase the expression of tyrosine hydroxylase mRNA in the locus ceruleus but that adaptation occurs to repeated stress, and that the expression of tyrosine hydroxylase mRNA in the locus ceruleus is independent of direct glucocorticoid modulation.     

Effect of Deep Brain Stimulation of the ventromedial prefrontal cortex on the noradrenergic system in rats

Background

Deep Brain Stimulation (DBS) of the subgenual cingulate cortex (SCC) is a promising therapeutic alternative to treat resistant major depressive disorder. In preclinical studies, DBS of the ventromedial prefrontal cortex (vmPFC, the rodent SCC correlate) provokes an antidepressant-like effect, along with changes in noradrenaline levels at the site of stimulation. Hence, DBS appears to activate the noradrenergic-locus coeruleus (LC) system.