Activation of Extracellular Signal-Regulated Kinases (ERK 1/2) in the Locus Coeruleus Contributes to Pain-Related Anxiety in Arthritic Male Rats.

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.

A HCO(3)(-)-dependent mechanism involving soluble adenylyl cyclase for the activation of Ca²âº currents in locus coeruleus neurons.

Hypercapnic acidosis activates Ca²âº channels and increases intracellular Ca²âº levels in neurons of the locus coeruleus, a known chemosensitive region involved in respiratory control. We have also shown that large conductance Ca²âº-activated K⁺ channels, in conjunction with this pathway, limits the hypercapnic-induced increase in firing rate in locus coeruleus neurons. Here, we present evidence that the Ca²âº current is activated by a HCO(3)(-)-sensitive pathway. The increase in HCO(3)(-) associated with hypercapnia activates HCO(3)(-)-sensitive adenylyl cyclase (soluble adenylyl cyclase). This results in an increase in cyclic adenosine monophosphate levels and activation of Ca²âº channels via cyclic adenosine monophosphate-activated protein kinase A. We also show the presence of soluble adenylyl cyclase in the cytoplasm of locus coeruleus neurons, and that the cyclic adenosine monophosphate analogue db-cyclic adenosine monophosphate increases Ca²âºi. Disrupting this pathway by decreasing HCO(3)(-) levels during acidification or inhibiting either soluble adenylyl cyclase or protein kinase A, but not transmembrane adenylyl cyclase, can increase the magnitude of the firing rate response to hypercapnia in locus coeruleus neurons from older neonates to the same extent as inhibition of K⁺ channels. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease.

Histologic validation of locus coeruleus MRI contrast in post-mortem tissue.

The locus coeruleus (LC) noradrenergic system regulates arousal and modulates attention through its extensive projections across the brain. LC dysfunction has been implicated in a broad range of neurodevelopmental, neurodegenerative and psychiatric disorders, as well as in the cognitive changes observed during normal aging. Magnetic resonance imaging (MRI) has been used to characterize the human LC (elevated contrast relative to surrounding structures), but there is limited understanding of the factors underlying putative LC contrast that are critical to successful biomarker development and confidence in localizing nucleus LC. We used ultra-high-field 7 T magnetic resonance imaging (MRI) to acquire T1-weighted microscopy resolution images (78 µm in-plane resolution) of the LC from post-mortem tissue samples. Histological analyses were performed to characterize the distribution of tyrosine hydroxylase (TH) and neuromelanin in the scanned tissue, which allowed for direct comparison with MR microscopy images. Our results indicate that LC-MRI contrast corresponds to the location of neuromelanin cells in LC; these also correspond to norepinephrine neurons. Thus, neuromelanin appears to serve as a natural contrast agent for nucleus LC that can be used to localize nucleus LC and may have the potential to characterize neurodegenerative disease.

Age-related changes in corticosteroid receptor expression and monoamine neurotransmitter concentrations in various brain regions of postnatal pigs.

Negative early life experience may be associated with altered functioning of stress-related systems and may increase vulnerability to diseases later in life. Corticosteroids are important mediators of homeostasis and stress and exert their effects via two receptors, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR), and through the glucocorticoid-metabolizing enzymes 11ß-hydroxysteroid dehydrogenase (11ß-HSD) types 1 and 2 in a brain-region-specific manner. However, relatively little is known about the postnatal ontogeny of these receptors and enzymes in the central nervous system. Here we describe, for the first time, the postnatal ontogeny of central GR, MR, 11ß-HSD1, and 11ß-HSD2 gene expression and monoamine levels in stress-related brain regions of domestic pigs at 7, 21, and 35 days of age. During the postnatal period, there was an increase in GR, MR, and 11ß-HSD1 mRNA expression in the pituitary and prefrontal cortex and an increase in MR mRNA expression in the hippocampus. We also demonstrated age-dependent changes in levels of noradrenaline and dopamine and their metabolites in the locus coeruleus, with the highest concentrations on day 7 compared with days 21 and 35. In conclusion, the dynamic changes in corticosteroid receptors and monoamines during neural development of postnatal pigs may represent periods of sensitivity to environmental stress that are comparable to some extent with those that are observed in primates and humans. Thus, these findings support the use of the domestic pig as an alternative animal model for humans in stress research.

Distinct glutaminyl cyclase expression in Edinger-Westphal nucleus, locus coeruleus and nucleus basalis Meynert contributes to pGlu-Abeta pathology in Alzheimer's disease.

Glutaminyl cyclase (QC) was discovered recently as the enzyme catalyzing the pyroglutamate (pGlu or pE) modification of N-terminally truncated Alzheimer's disease (AD) Abeta peptides in vivo. This modification confers resistance to proteolysis, rapid aggregation and neurotoxicity and can be prevented by QC inhibitors in vitro and in vivo, as shown in transgenic animal models. However, in mouse brain QC is only expressed by a relatively low proportion of neurons in most neocortical and hippocampal subregions. Here, we demonstrate that QC is highly abundant in subcortical brain nuclei severely affected in AD. In particular, QC is expressed by virtually all urocortin-1-positive, but not by cholinergic neurons of the Edinger-Westphal nucleus, by noradrenergic locus coeruleus and by cholinergic nucleus basalis magnocellularis neurons in mouse brain. In human brain, QC is expressed by both, urocortin-1 and cholinergic Edinger-Westphal neurons and by locus coeruleus and nucleus basalis Meynert neurons. In brains from AD patients, these neuronal populations displayed intraneuronal pE-Abeta immunoreactivity and morphological signs of degeneration as well as extracellular pE-Abeta deposits. Adjacent AD brain structures lacking QC expression and brains from control subjects were devoid of such aggregates. This is the first demonstration of QC expression and pE-Abeta formation in subcortical brain regions affected in AD. Our results may explain the high vulnerability of defined subcortical neuronal populations and their central target areas in AD as a consequence of QC expression and pE-Abeta formation.

Differential central NOS-NO signaling underlies clonidine exacerbation of ethanol-evoked behavioral impairment.

BACKGROUND: The molecular mechanisms that underlie clonidine exacerbation of behavioral impairment caused by ethanol are not fully known. We tested the hypothesis that nitric oxide synthase (NOS)-derived nitric oxide (NO) signaling in the locus coeruleus (LC) is implicated in this phenomenon. METHODS: Male Sprague-Dawley rats with intracisternal (i.c.) and jugular vein cannulae implanted 6 days earlier were tested for drug-induced behavioral impairment. The latter was assessed as the duration of loss of righting reflex (LORR) and rotorod performance every 15 minutes until the rat recovered to the baseline walk criterion (180 seconds). In a separate cohort, we measured p-neuronal NOS (nNOS), p-endothelial NOS (eNOS), and p-ERK1/2 in the LC following drug treatment, vehicle, or NOS inhibitor. RESULTS: Rats that received clonidine [60 Ig/kg, i.v. (intravenous)] followed by ethanol (1 or 1.5 g/kg, i.v.) exhibited synergistic impairment of rotorod performance. Intracisternal pretreatment with nonselective NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME, 0.5 mg) or selective nNOS inhibitor N-propyl-L-arginine (1 microg) exacerbated the impairment of rotorod performance caused by clonidine-ethanol combination. Exacerbation of behavioral impairment was caused by L-NAME enhancement of the effect of ethanol, not clonidine. L-NAME did not influence blood ethanol levels; thus, the interaction was pharmacodynamic. LORR caused by clonidine (60 microg/kg, i.v.)-ethanol (1 g/kg, i.v.) combination was abolished by selective inhibition of central eNOS (L-NIO, 10 microg i.c.) but not by nNOS inhibition under the same conditions. Western blot analyses complemented the pharmacological evidence by demonstrating that clonidine-ethanol combination inhibits phosphorylation (activation) of nNOS (p-nNOS) and increases the level of phosphorylated eNOS (p-eNOS) in the LC; the change in p-nNOS was paralleled by similar change in LC p-ERK1/2. NOS inhibitors alone did not affect the level of nitrate/nitrite, p-nNOS, p-eNOS, or p-ERK1/2 in the LC. CONCLUSIONS: Alterations in NOS-derived NO in the LC underlie clonidine-ethanol induced behavioral impairment. A decrease in nNOS activity, due at least partly to a reduction in nNOS phosphorylation, mediates rotorod impairment, while enhanced eNOS activity contributes to LORR, elicited by clonidine-ethanol combination.

Protective effect of ginsenoside against acute renal failure and expression of tyrosine hydroxylase in the locus coeruleus.

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.

Electroconvulsive shock increases tyrosine hydroxylase activity in the brain and adrenal gland of the rat.

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.

Compensatory increase in tyrosine hydroxylase activity in rat brain after intraventricular injections of 6-hydroxydopamine.

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.

Morphine-induced changes of adenylate and guanylate cyclase in locus ceruleus, periaqueductal gray, and substantia nigra in rats.

OBJECTIVES: To observe the changes of adenylate cyclase (AC) and guanylate cyclase (GC) in the cerebral regions including the locus ceruleus, periaqueductal gray, and substantia nigra in rats that were physiologically dependent on morphine. We also investigated the relationship of enzymatic changes in these cerebral regions to the mechanism of morphine dependence. METHODS: A morphine-dependent rat model was established and withdrawal symptoms evaluated. Enzyme histochemistry was used to detect the variations of AC and GC in cerebral regions. RESULTS: Compared to controls, AC and GC significantly increased in morphine-dependent groups. Comparisons of four different morphine-dependent groups also showed AC and GC significantly differed at weeks 1, 2, 4, and 8. CONCLUSIONS: Results found that the content of AC and GC increased in these cerebral regions in rats that demonstrated morphine dependence and appeared to be closely linked to increases in AC and GC activity.

Angiotensin II AT(1) receptor blockade selectively enhances brain AT(2) receptor expression, and abolishes the cold-restraint stress-induced increase in tyrosine hydroxylase mRNA in the locus coeruleus of spontaneously hypertensive rats.

Spontaneously hypertensive rats, a stress-sensitive strain, were pretreated orally for 14 days with the AT(1) receptor antagonist candesartan before submission to 2 h of cold-restraint stress. In non-treated rats, stress decreased AT(1) receptor binding in the median eminence and basolateral amygdala, increased AT(2) receptor binding in the medial subnucleus of the inferior olive, decreased AT(2) binding in the ventrolateral thalamic nucleus and increased tyrosine hydroxylase mRNA level in the locus coeruleus. In non-stressed rats, AT(1) receptor blockade reduced AT(1) receptor binding in all areas studied and enhanced AT(2) receptor binding in the medial subnucleus of the inferior olive. Candesartan pretreatment produced a similar decrease in brain AT(1) binding after stress, and prevented the stress-induced AT(2) receptor binding decrease in the ventrolateral thalamic nucleus. In the locus coeruleus and adrenal medulla, AT(1) blockade abolished the stress-induced increase in tyrosine hydroxylase mRNA level. Our results demonstrate that oral administration of candesartan effectively blocked brain AT(1) receptors, selectively increased central AT(2) receptor expression and prevented the stress-induced central stimulation of tyrosine hydroxylase transcription. The present results support a role of brain AT(1) and AT(2) receptors in the regulation of the stress response, and the hypothesis that AT(1) receptor antagonists may be considered as potential therapeutic compounds in stress related disorders in addition to their anti-hypertensive properties.

Glutamate signaling proteins and tyrosine hydroxylase in the locus coeruleus of alcoholics.

It has been postulated that alcoholism is associated with abnormalities in glutamatergic neurotransmission. This study examined the density of glutamate NMDA receptor subunits and its associated proteins in the noradrenergic locus coeruleus (LC) in deceased alcoholic subjects. Our previous research indicated that the NMDA receptor in the human LC is composed of obligatory NR1 and regulatory NR2C subunits. At synapses, NMDA receptors are stabilized through interactions with postsynaptic density protein (PSD-95). PSD-95 provides structural and functional coupling of the NMDA receptor with neuronal nitric oxide synthase (nNOS), an intracellular mediator of NMDA receptor activation. LC tissue was obtained from 10 alcohol-dependent subjects and eight psychiatrically healthy controls. Concentrations of NR1 and NR2C subunits, as well as PSD-95 and nNOS, were measured using Western blotting. In addition, we have examined tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of norepinephrine. The amount of NR1 was lower in the rostral (-30%) and middle (-41%) portions of the LC of alcoholics as compared to control subjects. No differences in the amounts of NR2C, PSD-95, nNOS and TH were detected comparing alcoholic to control subjects. Lower levels of NR1 subunit of the NMDA receptor in the LC implicates altered glutamate-norepinephrine interactions in alcoholism.

Bee venom injection produces a peripheral anti-inflammatory effect by activation of a nitric oxide-dependent spinocoeruleus pathway.

Our recent data, obtained using a zymosan-induced inflammatory air pouch model in mice, have demonstrated that subcutaneous bee venom (BV) injection into the hind limb selectively activates the contralateral brain stem locus coeruleus (LC) and then via a descending noradrenergic pathway and subsequent adrenal medullary catecholamine release induces a potent anti-inflammatory effect. While the efferent limb of this BV-induced neuroimmune anti-inflammatory pathway is well documented, the afferent limb of this pathway is poorly understood. In particular the spinal mechanisms involved with BV activation of the LC are currently unknown. Spinal nitric oxide (NO) and its synthase (NOS) have been shown to play an important role in the transmission and amplification of neuronal information from the spinal cord to the brain stem. In the present study we evaluated whether spinal NO plays a role in BV-induced LC activation, since we have previously shown that LC activation underlies this 'BV-induced anti-inflammatory effect' (BVAI) using the mouse air pouch model. Intrathecal (i.t.) pretreatment with l-nitro arginine methyl ester (l-NAME, non-selective NOS inhibitor), hemoglobin (NO scavenger) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, soluble guanylate cyclase inhibitor) abolished BVAI on zymosan-induced leukocyte migration into the air pouch. Moreover, i.t. injection of l-N-iminoethyl-lysine (l-NIL, inducible NOS inhibitor), but not 7-nitroindazole (7-NI, neuronal NOS inhibitor), also inhibited BVAI. BV injection significantly increased both the number of Fos immunoreactive neurons and tyrosine hydroxylase-Fos double labeling neurons in the contralateral LC in zymosan-induced inflamed mice. Importantly this increase in Fos expression in the LC was also completely inhibited by i.t. injection of l-NIL, but not by i.t. injection of 7-NI. Collectively these results indicate that spinal NO generated from inducible NOS is involved in the BV-induced LC activation that underlies BVAI.

Role of locus coeruleus heme oxygenase-carbon monoxide-cGMP pathway during hypothermic response to restraint.

Central heme oxigenase-carbon monoxide (HO-CO) pathway has been shown to play a pyretic role in the thermoregulatory response to restraint. However, the specific site in the central nervous system where CO may act modulating this response remains unclear. LC is rich not only in sGC but also in heme oxygenase (HO; the enzyme that catalyses the metabolism of heme to CO, along with biliverdin and free iron). Therefore, the possible role of the HO-CO-cGMP pathway in the restraint-induced-hypothermia by LC neurons was investigated. Body temperature dropped about 0.7 degrees C during restraint. ZnDPBG (a HO inhibitor; 5 nmol, intra-LC) prevented the hypothermic response during restraint. Conversely, induction of the HO pathway in the LC with heme-lysinate (7.6 nmol, intra-LC) intensified the hypothermic response to restraint, and this effect was prevented by pretreatment with ODQ (a sGC inhibitor; given intracerebroventricularly, 1.3 nmol). Taken together, these data suggest that CO in the LC produced by the HO pathway and acting via cGMP is implicated in thermal responses to restraint.

Tyrosine hydroxylase immunoreactivity in the locus coeruleus is elevated in violent suicidal depressive patients.

Our postmortem study aimed to determine the impact of suicide on the number of noradrenergic neurons of the locus coeruleus (LC) in suicidal depressive patients. Noradrenergic neurons were shown by immunostaining tyrosine hydroxylase in the LC of 22 non-elderly patients with mood disorders compared to 21 age- and sex-matched normal controls. Eleven patients were suicide victims and the other eleven died of natural causes. Seven violent suicide victims revealed an increased number of tyrosine hydroxylase immunoreactive (TH-ir) neurons compared with non-violent suicide victims and controls. No difference was found between the number of TH-ir neurons in all suicidal patients and controls and between non-suicidal patients and controls. The differences of TH-immunoreactivity could neither be attributed to medication nor to the polarity of depressive disorder (unipolar/bipolar). The numbers of TH-ir neurons in suicidal patients correlated negatively with the mean doses of antidepressants. The study suggested a presynaptic noradrenergic dysregulation in the LC related to the level of self-aggression. Traditional antidepressants may, therefore, regulate noradrenergic activity of the LC in suicide patients, however, without demonstrating the suicide-preventing effect.

Enhanced expression of heme oxygenase-1 in the locus coeruleus can be associated with anxiolytic-like effects.

Some researchers have shown that carbon monoxide (CO) plays a role in emotional behavior modulation through intracellular 3'-5'-guanosine monophosphate mechanisms in the locus coeruleus (LC). In fact, the LC region has a high expression of the heme-oxygenase (HO) enzymes, which are responsible for the production of CO. However, the physiological mechanism by which the HO-CO pathway participates in the modulation of emotional responses in the LC still needs clarification. This study evaluates whether a systemic intraperitoneal treatment is able to alter behavioral responses (in the elevated plus-maze and the light-dark box test) and the expression of the HO-1 and HO-2 enzymes in the LC. The tested treatments are acute (3h before) or chronic (twice daily for 10days) and with a carbon monoxide releaser (tricarbonyldichlororuthenium [II] dimer, or CORM-2) or with a HO-1 inducer compound (cobalt protoporphyrin IX, CoPP). The results for the elevated plus-maze show that CO-for both acute or chronic administration of either drug-ncreased the number of entries into the open arms and the percentage of time spent in the open arms. Regarding the light-dark box test, chronic treatment with either drug increased the time spent in the light compartment. Additionally, treatment with CORM-2 or CoPP, either acutely or chronically, increased HO-1 enzyme expression in the LC cells. This study shows that systemic CO treatment can promote an anxiolytic-like effect and the expression of HO-1 enzymes in LC cells.

Adrenal insufficiency and colonic inflammation after a novel chronic psycho-social stress paradigm in mice: implications and mechanisms.

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.

Role of brain angiotensin AT1 receptor in the carbachol-induced natriuresis and expression of nNOS in the locus coeruleus and proximal convoluted tubule.

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.

Characterization of neurochemically specific projections from the locus coeruleus with respect to somatosensory-related barrels.

Tactile information from the rodent mystacial vibrissae is relayed through the ascending trigeminal somatosensory system. At each level of this pathway, the whiskers are represented by a unique pattern of dense cell aggregates, which in layer IV of cortex are known as "barrels." Afferent inputs from the dorsal thalamus have been demonstrated repeatedly to correspond rather precisely with this modular organization. However, axonal innervation patterns from other brain regions such as the noradrenergic locus coeruleus are less clear. A previous report has suggested that norepinephrine-containing fibers are concentrated in the center/hollow of the barrel, while other studies have emphasized a more random distribution of monoaminergic projections. To address this issue more directly, individual tissue sections were histochemically processed for cytochrome oxidase in combination with dopamine-beta-hydroxylase, the synthesizing enzyme for norepinephrine, or the neuropeptide galanin. These two neuroactive agents were of particular interest because they colocalize in a majority of locus coeruleus neurons and terminals. Our data indicate that discrete concentrations or local arrays of dopamine-beta-hydroxylase- or galanin-immunoreactive fibers are not apparent within the cores of individual barrels. As such, the data suggest that cortical inputs from the locus coeruleus are not patterned according to cytoarchitectural landmarks or the neurochemical identity of coeruleocortical efferents. While transmitter-specific actions of norepinephrine and/or galanin may not be derived from the laminar/spatial connections of locus coeruleus axons, the possibility remains that the release of these substances may mediate distinctive events through the localization of different receptor subclasses, or the contact of their terminals onto cells with certain morphological characteristics or ultrastructural components.

Effects of long-term cigarette smoking on the human locus coeruleus.

BACKGROUND: It has been hypothesized that cigarette smoking among subjects with major depression is a form of self-medication. To explore a possible biological basis for this hypothesis, noradrenergic proteins in the locus coeruleus (LC) were measured in long-term cigarette smokers and in nonsmokers. The LC was studied because elevated amounts of alpha2-adrenoceptors and tyrosine hydroxylase have been observed postmortem in the LCs of subjects with major depression or who commit suicide, and because long-term administration of antidepressant drugs to rats down-regulates these proteins in the LC. METHODS: Postmortem LCs were obtained from long-term cigarette smokers (n=7) and from nonsmokers (n = 9), all of whom lacked diagnoses of major depression. Amounts of tyrosine hydroxylase immunoreactivity and radioligand binding to the norepinephrine transporter, monoamine oxidase A, and alpha2-adrenoceptors were measured. RESULTS: Amounts of tyrosine hydroxylase immunoreactivity and radioligand binding to alpha2-adrenoceptors were significantly lower (approximately 60% and 40%, respectively) along the axis of the LCs of long-term smokers compared with nonsmokers. Smoking had no statistically significant effects on binding to monoamine oxidase A or to the norepinephrine transporter. CONCLUSION: This is the first demonstration that cigarette smoking affects noradrenergic proteins in the LC. The direction of these changes is opposite to that observed when comparing subjects who have major depression with normal controls and the same as that produced by long-term antidepressant treatment in animals. If the present observations reflect long-term effects of smoking on premortem noradrenergic biochemistry, smoking-induced changes in LC biochemistry may strengthen the smoking habit among subjects with major depression.