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Background: There is increasing evidence suggesting that the Locus Coeruleus plays a role in pain-related anxiety. Indeed, we previously found that prolonged arthritis produces anxiety-like behavior in rats, along with enhanced expression of phosphorylated extracellular signal-regulated kinase 1/2 (a marker of plasticity) in the Locus Coeruleus. However, it is unknown how this effect correlates with the electrophysiological activity of Locus Coeruleus neurons or pain-related anxiety., Methods: Using the complete Freund's adjuvant model of monoarthritis in male Sprague-Dawley rats, we studied the behavioral attributes of pain and anxiety as well as Locus Coeruleus electrophysiology in vivo 1 (MA1W) and 4 weeks (MA4W) after disease induction., Results: The manifestation of anxiety in MA4W was accompanied by dampened tonic Locus Coeruleus activity, which was coupled to an exacerbated evoked Locus Coeruleus response to noxious stimulation of the inflamed and healthy paw. When a mitogen-activating extracellular kinase inhibitor was administered to the contralateral Locus Coeruleus of MA4W, the phosphorylated extracellular signal-regulated kinase 1/2 levels in the Locus Coeruleus were restored and the exaggerated evoked response was blocked, reversing the anxiogenic-like behavior while pain hypersensitivity remained unaltered., Conclusion: As phosphorylated extracellular signal-regulated kinase 1/2 blockade in the Locus Coeruleus relieved anxiety and counteracted altered LC function, we propose that phosphorylated extracellular signal-regulated kinase 1/2 activation in the Locus Coeruleus plays a crucial role in pain-related anxiety., (© The Author 2017. Published by Oxford University Press on behalf of CINP.)

Exposure to improvised explosive devices can result in a unique form of traumatic brain injury--blast-induced traumatic brain injury (bTBI). At the mild end of the spectrum (mild bTBI [mbTBI]), there are cognitive and mood disturbances. Similar symptoms have been observed in post-traumatic stress disorder caused by exposure to extreme psychological stress without physical injury. A role of the monoaminergic system in mood regulation and stress is well established but its involvement in mbTBI is not well understood. To address this gap, we used a rodent model of mbTBI and detected a decrease in immobility behavior in the forced swim test at 1 d post-exposure, coupled with an increase in climbing behavior, but not after 14 d or later, possibly indicating a transient increase in anxiety-like behavior. Using in situ hybridization, we found elevated messenger ribonucleic acid levels of both tyrosine hydroxylase and tryptophan hydroxylase 2 in the locus coeruleus and the dorsal raphe nucleus, respectively, as early as 2 h post-exposure. High-performance liquid chromatography analysis 1 d post-exposure primarily showed elevated noradrenaline levels in several forebrain regions. Taken together, we report that exposure to mild blast results in transient changes in both anxiety-like behavior and brain region-specific molecular changes, implicating the monoaminergic system in the pathobiology of mbTBI.

Background and Purpose: Emerging evidence indicates that the balance between pro-inflammatory cytokines (PICs) and anti-inflammatory cytokines (AICs) within the brain is an important determinant in the outcome of hypertension. However, the mechanism by which this dysregulation occurs is not known. We aimed to investigate whether AngII induces imbalance between PIC and AIC by modulating downstream transcription factors, NFκB and cyclic AMP response element-binding protein (CREB), and whether AngII-induced effects are mediated by glycogen synthase kinase-3β (GSK-3β)., Experimental Approach: CATH.a neurons were exposed to AngII (10 nM-1 μM) over a preset time course. In another set of experiments, GSK-3β was knock down by using lentivirus containing short hairpin RNA targeting GSK-3β (L-sh-GSK3β) before AngII exposure. Cell extracts were subjected to RT-PCR, immunoblot and immunoprecipitation., Key Results: AngII caused time-dependent increase in PICs (TNF-α and IL-1β) and reduction in AIC (IL-10). AngII exposure caused reduced phosphorylated CREB(Ser-133) and increased p-NFκB(Ser-276) levels, leading to reduced CREB-CBP and increased NFκB-CBP binding. These results were accompanied by increased activation of GSK-3β, as indicated by increased p-GSK3(Tyr-216) to p-GSK3(Ser-9) ratio. In a subsequent study, pretreatment with L-sh-GSK3β attenuated AngII-induced alterations in PICs and IL-10 by augmenting CREB-CBP and attenuating NFκB-CBP binding., Conclusions and Implications: Collectively, these findings are the first to provide direct evidence that AngII-induced dysregulation in cytokines is mediated by GSK-3β-mediated alterations in downstream transcription factors in neuronal cells. Our data also reveal that AngII-induced effects could be alleviated by GSK-3β inhibition, suggesting GSK-3β as an important therapeutic target for hypertension that is characterized by increased PICs and NFκB activation., (© 2013 The Authors. British Journal of Pharmacology © 2013 The British Pharmacological Society.)

The occurrence of catechol-O-methyltransferase (COMT) in presynaptic neurons remains controversial. This study utilized dopaminergic and noradrenergic toxins to assess the presence of COMT in the presynaptic neurons originating from the substantia nigra, ventral tegmental area or locus coeruleus. Destruction of dopaminergic and noradrenergic neurons was assessed by measuring the dopamine and noradrenaline content in the projection areas of these neurons. Additionally, COMT protein expression and activity were examined in several projection areas to determine whether there are any changes in COMT values. Colocalization studies were done to identify COMT-containing postsynaptic neurons. Despite successful lesioning of dopaminergic and noradrenergic neurons, no changes in COMT protein expression or activity could be noted. These results strongly suggest that COMT is not present in presynaptic dopaminergic and noradrenergic neurons. There was a high colocalization of COMT with the GABAergic marker of short neurons both in the striatum and cortex but only a weak, if any, with the cholinergic marker in the cortex.

Acute renal failure (ARF) is mainly characterized by acute tubular necrosis. No significant change was found for mortality rates over the past few decades despite significant advances in supportive care. In recent years, great effort has been focused on traditional and herbal medicine, which is much less toxic than those agents conventionally used and which is nowadays considered as a novel therapeutic agent for ARF. However, the effect of ginsenosides (GS) administered orally on ARF has not been reported yet and little is known about its cellular and molecular mechanism. The purpose of the study is to investigate the protective effect of ginsenoside in rats with ARF on the changes of tyrosine hydroxylase immunoreactivity (TH-IR) as well as on the involvement of mitogen-activated protein kinases (MAPK) in the locus coeruleus. In our assay, glycerol-induced acute renal failure in rats was employed to study the protective effects of ginsenoside. Our results indicated that the treatment of ARF rats with ginsenosides for 48 h significantly reduced the serum blood urea nitrogen, creatinine level, and lipid peroxidation, restored the GSH level and the normal renal morphology. Immunohistochemistry showed that an obvious increase of TH-IR was further enhanced in ARF+GS group. The same effect was also observed in the changes of p-ERK1/2-IR in the locus coeruleus. Our results suggest that ginsenoside administered orally may have a strong renal protective effect against glycerol-induced ARF, and ginsenoside can also activate the brain catecholaminergic neurons in the locus coeruleus. Our future attention will be focused to the question whether there is a correlation between the renal protective effect of ginsenosides against acute renal failure and the activation of tyrosine hydroxylase in the locus coeruleus.

The objective of the present study was to determine with precision the localization of neurons and fibers immunoreactive (ir) for aromatic L-amino acid decarboxylase (AADC), the second-step enzyme responsible for conversion of L-dihydroxyphenylalanine (L-DOPA) to dopamine (DA) and 5-hydroxytryptophan (5-HTP) to serotonin (5-hydroxytryptamine: 5-HT) in the midbrain, pons, and medulla oblongata of the adult human brain. Intense AADC immunoreactivity was observed in a large number of presumptive 5-HT neuronal cell bodies distributed in all of the raphe nuclei, as well as in regions outside the raphe nuclei such as the ventral portions of the pons and medulla. Moderate to strong immunoreaction was observable in presumptive DA cells in the mesencephalic reticular formation, substantia nigra, and ventral tegmental area of Tsai, as well as in presumptive noradrenergic (NA) cells, which were aggregated in the locus coeruleus and dispersed in the subcoeruleus nuclei. In the medulla oblongata, immunoreaction of moderate intensity was distributed in the mid and ventrolateral portions of the intermediate reticular nucleus, which constitutes the oblique plate of A1/C1 presumptive adrenergic and/or NA neurons. The dorsal vagal AADC-ir neurons were fewer in number and stained more weakly than cells immunoreactive for tyrosine hydroxylase (TH). AADC immunoreactivity was not identified in an aggregate of TH-ir neurons lying in the gelatinous subnucleus of the solitary nucleus, a restricted region just rostroventral to the area postrema. Nonaminergic AADC-positive neurons (D neurons), which are abundant in the rat and cat midbrain, pons, and medulla, were hardly detectable in homologous regions in the human brain, although they were clearly distinguishable in the forebrain.

We investigated chronic psycho-social stress effects on stress-related parameters and on pathohistological changes in the murine colon. Moreover, we aimed to reveal the involvement of adrenal glucocorticoids in chronic stress effects. Chronic subordinate colony housing (CSC, 19 d) resulted in reduced body weight gain, thymus atrophy, adrenal hypertrophy, increased plasma norepinephrine, and increased anxiety. With respect to the time course of CSC effects, CRH mRNA in the hypothalamic paraventricular nucleus, light phase corticosterone and tyrosine hydroxylase expression in colonic tissue were found to be increased, whereas tyrosine hydroxylase expression in the locus coeruleus was found to be decreased on d 2 of CSC; these parameters returned to control levels thereafter. Nevertheless, after 19 d of CSC exposure, the adrenal corticosterone responses in vivo and in vitro, and glucocorticoid sensitivity of isolated splenic cells were found to be decreased. Importantly, in CSC mice a significant histological damage of the colon was found beginning on d 14 of CSC exposure. Additionally, pro- and antiinflammatory cytokine secretion by mesenteric lymph node cells was increased after CSC exposure. Adrenalectomy before CSC at least partially prevented these chronic stress effects as reflected by less increase in proinflammatory cytokine secretion and an equal histological damage score in adrenalectomized compared with sham-operated CSC mice. In conclusion, chronic exposure to CSC alters relevant neuronal, neuroendocrine and immune functions that could be directly or indirectly involved in the damage of the histological integrity of the colon comparable with that seen during the development of colitis.

Central administration of losartan effectively blocked the increase of blood pressure and drinking response induced by angiotensin II (Ang II) or carbachol. However, the relationship between angiotensin AT(1) receptors and the natriuresis induced by brain cholinergic stimuli is still not clear. The purpose of the study is to reveal the role of brain angiotensin AT(1) receptor in the carbachol-induced natriuresis and expression of neuronal nitric oxide synthase (nNOS) in the locus coeruleus (LC) and proximal convoluted tubule (PCT). Our results indicated that 40 min after intracerebroventricular (ICV) injection of carbachol (0.5 microg), urinary sodium excretion was significantly increased to 0.548+/-0.049 micromol x min(-1) x 100 g(-1). Immunohistochemistry showed that carbachol induced an increase of neuronal nitric oxide synthase immunoreactivity (nNOS-IR) in the LC and renal proximal tubular cells. After pretreatment with losartan (20 microg), carbachol-induced urinary sodium excretion was reduced to 0.249+/-0.067 micromol x min(-1) x 100 g(-1). The same was true for carbachol-induced increase of nNOS-IR in the LC and PCT. The present data suggest that ICV cholinergic stimulation could induce a natriuresis and upregulate the activity of nNOS in the LC and PCT. The blockade of AT(1) receptors might downregulate the effects induced by carbachol in the LC and PCT. Consequently, we provide a new evidence that brain angiotensinergic pathway and NO-dependent neural pathway contribute to the natriuresis following brain cholinergic stimulation and thus play an important role in the regulation of fluid homeostasis. Furthermore, the final effect of nitric oxide on proximal tubular sodium reabsorption participated in the natriuresis induced by brain cholinergic stimulation.

1. The presence of ionotropic P2X receptors, targets of ATP in fast synaptic transmission, as well as metabotropic P2Y receptors, known to activate K(+) currents in cultured neostriatal neurones, was investigated in medium-spiny neurones and cholinergic interneurones contained in neostriatal brain slices from 5-26-day-old rats. 2. In these cells, adenosine-5'-triphosphate (ATP) (100-1000 microm), 2-methylthioadenosine-5'-triphosphate (2MeSATP), alpha,beta-methyleneadenosine-5'-triphosphate (alpha,betameATP, 30-300 microm, each) and adenosine-5'-O-(3-thiotriphosphate (ATPgammaS) (100 microm) failed to evoke P2X receptor currents even when 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 0.1 microm), apyrase (10 U ml(-1)) or intracellular Cs(+) was used to prevent occluding effects of the ATP breakdown product adenosine, desensitisation of P2X receptors by endogenous ATP and an interference with the activation of K(+) channels, respectively. P2X receptor agonists were also ineffective in outside-out patches withdrawn from the brain slice tissue. Muscimol (10 microm) evoked GABA(A) receptor-mediated currents under all these conditions. 3. When used as a control, locus coeruleus neurones responded with P2X receptor-mediated currents to ATP (300 microm), 2MeSATP and alpha,betameATP (100 microm, each). 4. ATP and adenosine-5'-diphosphate (ADP) (100 microm, each) did not activate K(+) currents in the neostriatal neurones. 5. Despite the observed lack of function, P2X(2) and P2Y(1) immunofluorescence was found in roughly 50% of the medium-spiny neurones and cholinergic interneurones. 6. A role of ATP in synaptic transmission to striatal medium-spiny neurones and cholinergic interneurones appears unlikely, however, the otherwise silent P2X and P2Y receptors may gain functionality under certain yet unknown conditions.

Repeated treatment of rats with antidepressant drugs down-regulates tyrosine hydroxylase (TH) in the locus coeruleus (LC). Using this effect as a model system, this study evaluated the antidepressant potential of the atypical antipsychotic drug, olanzapine. In an initial study, rats were treated i.p. with saline, olanzapine (3 mg/kg), or imipramine (15 mg/kg) once daily for 18 d. Three additional groups of rats received the same treatments that were overlapped with reserpine administration (0.5 mg/kg.d for 21 d). In a second study, groups of rats were treated twice daily with saline, olanzapine (5 mg/kg.d), fluoxetine (15 mg/kg.d), or fluoxetine (15 mg/kg.d) plus olanzapine (5 mg/kg.d) for 1, 6, 12 and 18 d. In the initial study, imipramine produced a 45% reduction in LC TH levels, while olanzapine had no effect. In reserpinized rats, olanzapine exhibited an action that was opposite to that of imipramine, although this effect did not reach statistical significance. In the second study, olanzapine treatment alone or in combination with fluoxetine up-regulated TH in the LC, while fluoxetine alone had no effect. When fluoxetine was co-administered, olanzapine-induced increases in LC TH were more robust and occurred earlier in the time-course of treatment. Based on this preclinical model alone, olanzapine did not exhibit typical antidepressant properties. The unique effect of olanzapine to elevate LC TH may result from olanzapine-induced increases in LC activity. Such an action may contribute to novel behavioural effects of this atypical antipsychotic drug, including enhanced attention.

Aim: To investigate the effects of different muscarinic receptor (M) subtypes in the spinal cord on the scores of naloxone-precipitated morphine-withdrawal symptoms and the changes of nNOS expression in locus coeruleus (LC)., Methods: nNOS immunohistochemistry, intrathecal injection (it), and antisense oligonucleotides (AS-ONs) techniques were used., Results: Intrathecal injection of M2-antisense oligonucleotides (M2-AS) decreased the scores of morphine withdrawal symptoms. M1-AS attenuated morphine-withdrawal symptoms, but the effect was less than that of M2-AS. The expression of nNOS positive neurons in the LC increased in morphine-dependent rats and increased to a greater extent during morphine withdrawal. Intrathecal injection of M2-AS inhibited the increase of nNOS expression in LC during morphine-withdrawal, but there was no effect in case of M1-AS., Conclusion: M2 muscarinic receptor of spinal cord mediated the increase of nNOS expression in LC during morphine withdrawal.

The neurotoxic effects of inorganic lead are known to include peripheral neuropathy in adults and encephalopathy in children. The purpose of this study was to determine the effect of inorganic lead (PbCl2) administration on norepinephrinergic neurons of the locus ceruleus in neonatal rats by immunocytochemical and electron microscopic analyses. Lead chloride solutions, 0.05%, 0.1% and 0.2% in concentrations, were prepared in distilled water and administered orally via drinking water. After 4, 8, or 12 weeks of continuous administration, the rats were sacrificed and brains were immunostained with the tyrosine hydroxylase antibody. The number of immunoreactive cell bodies in the locus ceruleus was estimated. Densitometric analysis of immunoreactive profiles visualized by electron microscopy was performed using an image analyzer. The numbers of immunoreactive neurons in the locus ceruleus were increased statistically by lead administration. The intensity of the immunoreaction, both under the light and electron microscopes was also increased. Degenerative changes, including intra-axonal vacuole formation and widening of the extracellular spaces, were found by electron microscopy in and around the tyrosine hydroxylase immunoreactive axons. Increased tyrosine hydroxylase immunoreactivity may correlate with the hyper-reactivity of lead intoxicated children. Degenerative changes may account for the reported deficits in intellectual attainment and achievement in lead intoxicated children.

Somatic symptoms and aversion of opiate withdrawal, regulated by noradrenergic signaling, were attenuated in mice with a CNS-wide conditional ablation of neurotrophin-3. This occurred in conjunction with altered cAMP-mediated excitation and reduced upregulation of tyrosine hydroxylase in A6 (locus coeruleus) without loss of neurons. Transgene-derived NT-3 expressed by noradrenergic neurons of conditional mutants restored opiate withdrawal symptoms. Endogenous NT-3 expression, strikingly absent in noradrenergic neurons of postnatal and adult brain, is present in afferent sources of the dorsal medulla and is upregulated after chronic morphine exposure in noradrenergic projection areas of the ventral forebrain. NT-3 expressed by non-catecholaminergic neurons may modulate opiate withdrawal and noradrenergic signalling.

The telencephalic nucleus HVc (sometimes referred to as the high vocal center) plays a key role in the production and perception of birdsong. Although many afferent and efferent connections to this nucleus have been described, it has been clear for many years, based on chemical neuroanatomical criteria, that there are projections to this nucleus that remain undescribed. A variety of methods including high performance liquid chromatography, immunohistochemistry and receptor autoradiography have identified high levels of catecholamine transmitters, the presence of enzymes involved in the synthesis of catecholamines such as tyrosine hydroxylase and a variety of catecholamine receptor sub-types in the HVc of several songbird species. However, no definitive projections to HVc have been described from cells groups known to synthesize catecholamines. These projections were analyzed in the present study by retrograde tract tracing combined with immunocytochemistry for tyrosine hydroxylase. The origin of the catecholaminergic inputs to HVc were determined based exclusively on birds in which injections of the retrograde tracer (latex fluospheres) were confined within the cytoarchitectonic boundaries of the nucleus. Retrogradely transported latex fluospheres were found mainly in cells of two dopaminergic nuclei, the mesencephalic central gray (A11) and, to a lesser extend, the area ventralis of Tsai (A10; homologous to the ventral tegmental area of mammals). A few retrogradely-labelled cells were also found in the noradrenergic nucleus subceruleus (A6). Most of these retrogradely-labelled cells were also tyrosine hydroxylase-positive. Other catecholaminergic nuclei were devoid of retrograde label. These data converge with others studies to indicate that HVc receives discrete dopaminergic and noradrenergic inputs. These inputs may influence the steroid regulation of HVc, attentional processes related to song and modulate sensory inputs to the song system.

Valproate has proven effective in treating bipolar disorder. Though some biochemical effects of valproate are rapid, mood-stabilizing effects can take weeks, suggesting that regulatory changes in gene expression in brain neurotransmitter systems may be involved. Given a presumed role for norepinephrine (NE) in bipolar disorder, as well as the actions of mood-stabilizing drugs, we examined changes in mRNA expression for tyrosine hydroxylase (TH), the NE transporter (NET) and alpha 2A autoreceptor in the rat locus coeruleus after valproate treatment. TH mRNA increased slightly (16%) following acute treatment, and more so after chronic valproate treatment (26%), while neither NET nor alpha 2A mRNA expression changed. Further, chronic valproate treatment attenuated the elevation in TH mRNA expression induced in the LC in response to acute restraint stress. Both acute and chronic valproate treatment attenuated restraint stress-induced elevations in plasma ACTH secretion. These observations suggest that the therapeutic effects of valproate may involve regulatory alterations in TH message expression in the brain, and attenuation of stress-reactivity of the central noradrenergic system and the hypothalamic-pituitary-adrenal axis.

Unlabelled: There is some evidence of a relationship between nitric oxide and pain control pathways. However, it is still controversial whether nitric oxide synthase (NOS) inhibitors affect minimum alveolar anesthetic concentration (MAC). We examined the effects of 7-nitro indazole (7-NI), a selective neuronal NOS (nNOS) inhibitor, on halothane MAC. With nicotinamide adenine dinucleotide phosphate diaphorase histochemistry, we also investigated the nNOS activity of the dorsal horn and the locus ceruleus in 26 Sprague-Dawley rats. 7-NI (100, 500, 1000 mg/kg intraperitoneally) reduced halothane MAC to 0.34% +/- 0.12%, 0.1% +/- 0.03%, and 0.05% +/- 0.12%, dose dependently (P < 0.01). 7-NI also reduced the number of nicotinamide adenine dinucleotide phosphate diaphorase-positive cells by 20% to 65% (P < 0.05 or 0.01) and the staining intensity of the axons in the locus ceruleus and lumbar and thoracic spinal cord as compared with the control group. 7-NI reduced the MAC observed with halothane anesthesia, which was accompanied by nNOS activity suppression in the spinal cord and the locus ceruleus. Our results support the hypothesis that the nitric oxide signaling pathway is related to MAC., Implications: We examined the effects of a selective neuronal nitric oxide synthase inhibitor, 7-nitro indazole, on halothane minimum alveolar anesthetic concentration and measured the nitric oxide synthase activity in the spinal cord and the locus ceruleus of Sprague-Dawley rats using nicotinamide adenine dinucleotide phosphate diaphorase staining method. 7-Nitro indazole decreased both the minimum alveolar anesthetic concentration and neuronal nitric oxide synthase activity.

Levels of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, are known to be upregulated in specific brain regions by chronic administration of drugs of abuse. Chronic morphine administration increases TH levels in the locus coeruleus and ventral tegmental area, whereas chronic cocaine administration increases TH levels in the ventral tegmental area only. While such upregulation of TH has been related to behavioral effects of the drugs, the mechanism underlying these adaptations has remained controversial. To study the possibility that upregulation of TH occurs at the transcriptional level, we investigated the effect of chronic morphine or cocaine treatment on the activity of the TH gene promoter (9.0 kb), coupled to the LacZ reporter gene, in transgenic mice. These TH9.0-LacZ mice have been shown to exhibit correct tissue-specific expression and regulation of the reporter gene. We show here that chronic (but not acute) exposure of the TH9.0-LacZ mice to morphine increases the expression of beta-galactosidase (which is encoded by the LacZ gene) in the locus coeruleus by twofold compared with sham-treated mice. In contrast, beta-galactosidase expression in the ventral tegmental area was decreased 20-25% by chronic morphine and unaffected by chronic cocaine administration. Similar results were obtained after analysis of TH mRNA levels in these brain regions by in situ hybridization. These results suggest that chronic morphine upregulates TH expression via transcriptional mechanisms in the locus coeruleus but by post-transcriptional mechanisms in the ventral tegmental area.

Adaptation of the skin colour to the background light condition in the amphibian Xenopus laevis is achieved by migration of pigment granules in the skin melanophores, a process regulated by alpha-MSH secretion from melanotrope cells in the pituitary pars intermedia (PI). alpha-MSH secretion in turn, is regulated by various stimulatory and inhibitory messengers synthesized in brain nuclei, especially the hypothalamic suprachiasmatic and magnocellular nuclei and the locus coeruleus in the hindbrain. In the present study, the roles in background adaptation of nitric oxide (NO) and NO synthase (NOS) enzyme activity were evaluated. In situ, using both immunohistochemistry with anti-human brain NOS (bNOS) serum in paraffin-embedded material and using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry in cryo-sections, we showed NOS in neurons in the optic tectum and in the locus coeruleus. NADPH-d reactivity was also found in neurons in the lateral amygdala, the ventral hypothalamic nucleus and in fibers in the median eminence. Using a Western blot stained with an anti-human bNOS serum, we demonstrated a 150 kDa band in Xenopus hindbrain lysates, which is similar to the NOS protein present in the rat anterior pituitary, but which was not detectable in the lysates from both the neurointermediate and distal lobes in Xenopus. No differences in histochemical staining pattern or on Western blotting were observed between animals adapted to a black or a white background. Paraffin sections of the endocrine PI and pars distalis did not reveal bNOS-like immunoreactivity. NADPH-d reactivity was observed in the endothelia of this gland. However, using a new procedure of thin cryo-sections of pituitary neurointermediate lobes, we observed bNOS-immunoreactive fibers as well as cyclic 3',5' guanosine monophosphate (cGMP)-accumulating fibers in the PI. The PI may be regulated by NOergic neurons from higher brain centers. The possibility that NOergic neurons in the locus coeruleus are involved in the innervation of the PI needs further investigation. The latter neurons are probably not noradrenergic because double labeling studies show no co-localization of NADPH-d reactivity and tyrosine hydroxylase immunoreactivity in locus coeruleus neurons.

Previous studies have shown the presence of a large number of tyrosine hydroxylase immunoreactive neurons and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase positive elements within the mesencephalic and pontine regions of the Japanese quail. In the present study histochemical and immunohistochemical procedures reveal that cells expressing at least one of these two neurochemical markers coexist throughout a large part of the substantia nigra and of the area ventralis of Tsai. Also about 40% of the neurons in these two regions that contain immunoreactive tyrosine hydroxylase also exhibit NADPH-diaphorase activity. This is not a general property of the quail catecholaminergic system: in the locus coeruleus (the main noradrenergic group) there is a complete separation between these two neuronal populations. The number of neurons expressing either neurochemical marker is not different between males and females in any of the regions that have been investigated. NADPH-diaphorase is known to be an indicator of the enzyme nitric oxide synthase; these results therefore suggest that nitric oxide may play an important role in the regulation of the activity of a significant part of the avian mesencephalic dopaminergic system.

To date only global dosages of tyrosine hydroxylase (TH) protein have been realized in the locus coeruleus (LC) without discriminating the enzyme contained in the cell body area from the one in the surrounding neuropil. The preceding immunohistochemical study (Bezin et al., 1994) revealed a dramatic plasticity of the cellular expression of TH in the LC during the postnatal development of the rat. It was therefore necessary to develop a quantitative biochemical approach, strengthened by a great anatomical resolution, to follow the developmental evolution of TH levels exactly in the space containing the coerulean TH-immunoreactive perikarya. In the present work two biochemical parameters necessary for precisely defining the phenotypic characterization of TH expression within the rat LC have been established during the postnatal development at six different stages: postnatal day 4 (PND4), PND10, PND14, PND21, PND30, and PND42. TH tissue concentration and content were precisely determined along the caudorostral extent of the LC within the previously (Bezin et al., 1994) defined spaces delimited by the TH-containing perikarya. TH tissue concentration remained quite stable during the postnatal development. TH quantity exhibited few age-related variations with a transient peak at PND10 and followed the same evolution as the volume containing the TH-expressing perikarya. The mean cell contribution to the total quantity of TH measured in the whole LC showed important age-related fluctuations with a dramatic peak at PND10 followed by a drastic decrease until PND21.(ABSTRACT TRUNCATED AT 250 WORDS)

The cellular phenotypic characteristics of tyrosine hydroxylase (TH) expression have been studied within the rat locus coeruleus (LC) during postnatal development at six different stages: postnatal day 4 (PND4), PND10, PND14, PND21, PND30, and PND42. Coronal brain sections were selected at intervals of 80 microns along the caudorostral extent of the LC and processed for TH immunohistochemistry. At each anatomical level we (1) reconstructed the mean space of the LC delineated by the TH positive cell bodies, (2) enumerated the mean number of these cell bodies, and (3) determined the mean volume circumscribed by these cell bodies and their density. The topological study revealed a steady remodeling of the structure until the third week, with a progressive reducing of a ventral cellular group in the anterior LC, which was no longer observable at PND21, concomitant to the stretch of the structure toward its caudal limit. We have noted invariant and variant cellular phenotypic characteristics of TH expression. At any stage, the LC could be separated into a posterior and an anterior subregion and its total volume remained quite stable during the studied period. At PND14 and PND21, we observed a transient 33% increase in the total number of TH positive perikarya as compared to PND42. Conjoint analysis of the topological reconstruction and the density of TH positive cells suggested there were three distinct and precisely localized subsets of "quiescent" neurons. TH gene expression in these cells would have lowered between PND14 and PND21 inside two subsets and between PND21 and PND30 inside the last one. So topologically defined populations of cells could be involved in specific functions. If they have not definitively lost their TH expression capacity, they could contribute to increasing TH levels in LC occurring in response to physiological perturbations or pharmacological treatments.

1. Intracellular recordings were obtained from a pontine slice preparation of the rat brain containing the locus coeruleus (LC). Two openers of ATP-sensitive potassium (K(ATP)) channels, RO 31-6930 (10 microM) and cromakalim (100 microM) decreased the spontaneous discharge of action potentials without altering their amplitude or duration. Neither compound changed the resting membrane potential. 2. Of two K(ATP) channel blockers, tolbutamide (300 microM) increased the firing rate, while glibenclamide (3 microM) only tended to do so. In addition, both compounds antagonized the effect of RO 31-6930 (10 microM). Neither glibenclamide (3 microM) nor tolbutamide (300 microM) altered the resting membrane potential. 3. Tetrodotoxin (0.5 microM) depressed the firing, but did not influence the inhibitory action of RO 31-6930 (10 microM). The excitatory amino acid antagonist, kynurenic acid (500 microM), did not change the spontaneous discharge of action potentials. 4. Small shifts (2-4 mV) of the membrane potential by hyper- or depolarizing current injections markedly decreased and increased the firing rate, respectively. 5. Noradrenaline (100 microM) hyperpolarized the cells and decreased their input resistance. This effect was not antagonized by glibenclamide (3 microM) or tolbutamide (300 microM). Ba2+ (2 mM), a blocker of both ATP-sensitive and inwardly rectifying potassium channels, abolished the effects of RO 31-6930 (10 microM) and noradrenaline (100 microM). 6. These data suggest that K(ATP) channels are present on the noradrenergic LC neurones, but are not coupled to alpha 2-adrenoceptors.

Previous studies have revealed the presence of pontospinal neurons with either methionine-enkephalin- or tyrosine hydroxylase-like immunoreactivity in the dorsolateral pontine tegmentum of the cat. Using a combined fast blue retrograde transport technique and simultaneous immunofluorescence histochemistry, the present study was designed to reveal the coexistence of enkephalin and tyrosine hydroxylase in cat coerulospinal neurons and to determine if and to what extent the coerulospinal pathway is heterogeneous. Fast blue-labelled neurons with tyrosine hydroxylase- and enkephalin-like immunoreactivities were found in the nucleus locus coeruleus, nucleus subcoeruleus, Kölliker-Fuse nucleus, and the medial and lateral parabrachial nuclei. Approximately 87% of tyrosine hydroxylase-like immunoreactive neurons had enkephalin-like immunoreactivity, whereas about 76% of the enkephalin-like immunoreactive neurons had tyrosine hydroxylase-like immunoreactivity. About 71% of all coerulospinal neurons exhibited both tyrosine hydroxylase- and enkephalin-like immunoreactivities. These findings indicate that coerulospinal activity may lead to spinal cord effects reflecting both norepinephrine and enkephalin activity in most cases but do not rule out each transmitter's isolated functions.

Regulation of tyrosine hydroxylase expression by antidepressant treatments was investigated in the locus coeruleus (LC), the major noradrenergic nucleus in brain. Rats were treated chronically with various antidepressants, and tyrosine hydroxylase levels were measured in the LC by immunoblot analysis. Representatives of all major classes of antidepressant medication-including imipramine, nortriptyline, tranylcypromine, fluvoxamine, fluoxetine, bupropion, iprindole, and electroconvulsive seizures-were found to decrease levels of tyrosine hydroxylase immunoreactivity by 40-70% in the LC. Decreased levels of enzyme immunoreactivity were shown to be associated with equivalent decreases in enzyme mRNA levels. Antidepressant regulation of LC tyrosine hydroxylase appeared specific to these compounds, inasmuch as chronic treatment of rats with representatives of other classes of psychotropic drugs, including haloperidol, diazepam, clonidine, cocaine, and morphine, failed to decrease levels of this protein. The results demonstrate that chronic antidepressants dramatically downregulate the expression of tyrosine hydroxylase in the LC and raise the possibility that such regulation of the enzyme represents an adaptive response of LC neurons to antidepressants that mediates some of their therapeutic actions in depression and/or other psychiatric disturbances.

We have compared the time course of the behavioral manifestations of opiate withdrawal to the in vivo activity of locus coeruleus (LC) neurons and to increases in the levels of G-proteins, adenylate cyclase, and cAMP-dependent protein kinase known to occur in the LC in opiate-dependent animals. Rats were given morphine by daily subcutaneous implantation of morphine pellets for 5 d. On the sixth day, morphine withdrawal was induced by subcutaneous administration of naltrexone, an opiate receptor antagonist, with additional doses given 6 and 24 hr later, conditions that resulted in sustained, maximal levels of withdrawal over the duration of the experiment. We found a striking parallel between the time courses of the behavioral signs and the increased activity of LC neurons during withdrawal, both of which appeared to follow 2 phases. There was an early, rapid phase, during which withdrawal signs and increased LC activity became most pronounced within 15-30 min after naltrexone administration, and then recovered rapidly by over 50% within 4 hr of withdrawal. Subsequently, there was a slower phase, during which the persisting withdrawal signs and elevated LC activity remained roughly constant from 4 to 24 hr and did not recover completely until after 72 hr of continuous withdrawal. Adenylate cyclase and cAMP-dependent protein kinase activities in isolated LC subcellular fractions, both elevated in dependent animals, recovered to control levels after 6 hr of withdrawal, in parallel with the rapid phase of withdrawal. Levels of G1 and Go, also elevated in dependent animals, remained only slightly elevated at 6 hr and returned to normal by 24 hr. Taken together, these data suggest that increased neuronal activity in the LC is associated temporally with the behavioral morphine withdrawal syndrome and that increased levels of G-proteins and an up-regulated cAMP system may contribute to the early withdrawal activation of these neurons.

Dopamine-beta-hydroxylase (DBH) activity in serum, DBH and tyrosine hydroxylase (TH) activities in mesenteric vessels, and DBH and TH activities in locus coeruleus and hypothalamus of brain did not differ significantly between spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKR) at 16 weeks of age when hypertension of SHR was fixed. In contrast, DBH and TH activities in vas deferens and adrenal glands were significantly higher in SHR than in WKR. These changes in SHR at 16 weeks of age after establishment of hypertension are directly opposite those reported previously in SHR at 3 weeks of age before the onset of hypertension.

In vivo biosynthesis of the 2 subunit forms of dopamine beta-hydroxylase (DBH) was examined in the rat brain. 35S-methionine was injected into the noradrenergic neurons of the locus coeruleus (LC) using a stereotactic device. Several hours later, newly synthesized 35S-Met-labeled DBH was immunoprecipitated and quantitated. Both Mr = 77,000 (77K) and 73,000 (73K) subunit forms were present in near-equal proportions after 4 hr of labeling, and these were indistinguishable from those isolated from rat PC12 pheochromocytoma cells by electrophoretic mobility. Both forms sedimented with the vesicular subcellular fraction of LC homogenates, and a portion of the 73K form could be released by hypotonic lysis of these vesicles. The 77K form predominated in the first 30 min labeling period, while the 73K form appeared more slowly over the next several hours. By 16 hr, the 73K form comprised about 2/3 of the total 35S-Met-labeled DBH present. Inhibition of protein synthesis with puromycin 30 min after 35S-Met injection into the LC did not prevent the subsequent appearance of the 73K form, suggesting that this subunit form was the product of posttranslational modification of the 77K subunit form in a fashion similar to that seen in PC12 cells. Also, newly synthesized 35S-Met-labeled DBH that underwent axonal transport from the LC to the anterior hypothalamus was predominantly the 73K subunit form. A single injection of the catecholamine-depleting drug reserpine (10 mg/kg, i.p.) produced a 17-fold increase in the relative synthesis of DBH 2 d later without affecting the proportion of its 2 subunit forms.

The visualization of protein antigens has been performed on semithin sections embedded in Araldite. After partial removal of the resin and a light proteolytic treatment of the tissue we were able to localize several biosynthetic enzymes: tyrosine hydroxylase (TH), phenylethanolamine N-methyltransferase (PNMT), and glutamic acid decarboxylase (GAD), which are, respectively, markers of catecholaminergic, adrenergic, gamma-aminobutyric acid (GABA)ergic systems. This technique afforded a high resolution of light microscopy details and immunostaining of TH, GAD, and PNMT on serial sections enabled us to compare with great precision GABAergic and adrenergic innervations in the rat locus coeruleus. In addition, it allows us to study the possible relationship between these terminals and the noradrenergic neurons. We also compared the general pattern of distribution of TH- and GAD-positive endings in the hypothalamic median eminence. The preliminary results obtained with this technique revealed some interesting facts previously unseen when preparations with lower histological resolution were used.

A single application of electroconvulsive shock produced a rapid but short-lasting increase in tyrosine hydroxylase activity above control values in the rat adrenal medulla and striatum. After repeated electroconvulsive shock treatment (once per day for 7 days), tyrosine hydroxylase activity increased significantly in the locus ceruleus, nucleus of the tractus solitarius, hippocampus, cerebellum, and frontal cortex and remained elevated for 4 to 8 days. Adrenal tyrosine hydroxylase activity increased 1 day after the termination of repeated electroconvulsive shock treatments and remained elevated for at least 24 days, possibly reflecting the establishment of a new and higher steady-state level of catecholamine biosynthesis in the adrenal. These findings suggest that the persistent changes in tyrosine hydroxylase activity produced by repeated electroconvulsive shock may be a factor contributing to the long-lasting antidepressant effects of this treatment.

The administration of 6-hydroxydopamine into the cerebroventricles of the rat produced a rapid and permanent decrease of norepinephrine in hippocampus due to an apparent degeneration of central catecholaminergic nerve terminals. The decrease in norepinephrine levels was accompanied by a decrease in the activity of the rate-limiting biosynthetic enzyme, tyrosine hydroxylase. However, the decrease in enzyme activity was less pronounced than the decrease in norepinephrine levels, resulting in an increase in the ratio of tyrosine hydroxylase activity to norepinephrine content. This relative increase in enzyme activity was shown to result from two processes. Within 36 hr after the lesion, the apparent Vmax had decreased in parallel to the norepinephrine loss. However, there was an apparent activation of the remaining enzyme molecules. This activation was only detectable in the presence of subsaturating cofactor concentrations and at a pH above the pH optimum. The activation resembled that produced in control samples by in vitro adenosine 3':5'-monophosphate-dependent protein-phosphorylating conditions, and incubation under these conditions had no further effect on enzyme activity. The activation was followed by a gradual increase in the apparent Vmax of tyrosine hydroxylase toward control values. This increase was preceded by a 2-fold rise in the amount of enzyme present in the region of the locus coeruleus, an area rich in noradrenergic cell bodies. The time course of the increased Vmax in terminal fields appeared to be related to their proximity to the locus coeruleus, since it was more rapid for cerebellum (peak activity, 7 days) than for hippocampus (21 days) and probably represented a 3- to 4-fold increase in the amount of tyrosine hydroxylase per residual terminal. The increase in the Vmax was accompanied by a return to a basal activation state of the enzyme molecules and a restoration of the ability of in vitro protein-phosphorylating conditions to increase enzyme activity. These short and long term alterations in tyrosine hydroxylase activity after 6-hydroxydopamine treatment may represent adaptive responses to the lesion.

The neurotoxin 6-hydroxydopamine produced a permanent loss of endogenous norepinephrine and of 3H-labeled norepinephrine uptake sites in the hippocampus within 5 days. These losses were initially accompanied by parallel decreases in tyrosine hydroxylase activity and synaptosomal norepinephrine synthesis. Within 21 days, however, hippocampal tyrosine hydroxylase activity and norepinephrine synthesis rate increased three- to fivefold. These data suggest a novel form of plasticity in brain-damaged animals characterized by an increase in the capacity for transmitter biosynthesis in residual neurons.

It has previously been demonstrated that monkeys exhibit certain event-related potential (ERP) components showing latency, polarity, and contingency similarities to those observed in humans. In the present study, monkey P300-like components were studied in order to evaluate the hypothesis that the noradrenergic locus coeruleus (LC) system participates in their generation or modulation. ERPs were recorded from untrained squirrel monkeys (Saimiri sciureus) twice a week for 4 weeks before and after bilateral LC lesions and interruption of dorsal bundle (DB) fibers. Stimuli consisted of 2 and 6 kHz tone pips (40 msec duration, 60 dB above nHL) presented once a second in random order. In most sessions, one tone constituted 90% of the stimuli and the other tone 10%, while in some sessions tones were made equiprobable to test the effects of manipulating stimulus probability. LC and DB lesions were made by first localizing the nucleus and creating an electrolytic lesion. Then, the electrode was placed at the anterior pole of the nucleus and a knife cut effected. The extent of damage to LC perikarya and ascending axons was assessed by reconstructing lesions from Nissl-stained sagittal sections through the brain stems. The effect of lesions on cortical noradrenergic axons was immunohistochemically verified utilizing antisera directed against dopamine-B-hydroxylase and tyrosine hydroxylase to label noradrenergic and dopaminergic axons, respectively. The prelesion ERP results replicated previous findings of P300-like components recorded in response to low-probability tones. The postlesion ERP data indicated that following damage to LC cell bodies, combined with interruption of histochemically detectable ascending noradrenergic axons, monkey P300-like potentials exhibited decreased areas, altered brain-surface distribution, and reduced sensitivity to stimulus probability. The correlation between the extent of cell body lesions and percentage reduction in the magnitude of P300-like responses was significant. However, interruption of DB fibers alone did not have similar effects. Neither type of lesion had any effect on amplitudes, latencies, or brain-surface distributions of P52, P172, or N250-900. There was, however, a significant effect on N106. Stimulus probability effects on the frontally distributed P52 and N106 were not altered by the lesions. These data support the hypothesis that the integrity of the LC nucleus and its ascending fibers is important in the generation and modulation of surface-recorded P300-like activity.

Immunocytochemical localization of tyrosine hydroxylase (TH) was performed on paraffin sections pretreated with various proteolytic enzymes. It was found that pretreatment with trypsin (1.2 mg/ml) for 5 min resulted in a dramatic increase in the number of TH-positive terminals throughout the brain, especially in the cerebellum, which contains fine preterminal and terminal axons that are difficult to stain. This pretreatment also led to a significant reduction in background staining and allowed for the use of the TH antiserum at high working dilutions. Several other proteolytic enzymes were tested and only chymotrypsin was nearly as effective as trypsin with respect to TH staining.

A rat tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2] cDNA probe was used for in situ hybridization studies on histological sections through the locus coeruleus, substantia nigra, and the ventral tegmental area of the rat brain. Experimental conditions were established that yielded no background and no signal when pBR322 was used as a control probe. Using the tyrosine hydroxylase probe, we ascertained the specificity of the labeling over catecholaminergic cells by denervation experiments and comparison of the hybridization pattern with that of immunoreactivity. The use of 35S-labeled probe enabled the hybridization signal to be resolved at the cellular level. A single injection of reserpine into the rat led to an increase of the intensity of the autoradiographic signal over the locus coeruleus area, confirming an RNA gel blot analysis. The potential of in situ hybridization to analyze patterns of modulation of gene activity as a result of nervous activity is discussed.