Tyrosine hydroxylase expression in the Cebus monkey retina

Tyrosine hydroxylase (TH) expression was used as a marker to study the dopaminergic cells in the Cebus monkey retina. Two types of dopaminergic cells were identified by cell body size and location, level of arborization in the inner plexiform layer, and amount of immunolabeling. Type 1 cells displayed intense immunoreactivity and larger somata (12-24 microns) located in the inner nuclear layer or ganglion cell layer, whereas type 2 had smaller cell bodies (8-14 microns) found either in the inner plexiform layer or ganglion cell layer and were more faintly labeled. Interplexiform cells were characterized as type 1 dopaminergic cells. Immunoreactive axon-like processes were seen in the nerve fiber layer, and a net of fibers was visible in the foveal pit and in the extreme periphery of the retina. The population of TH+ cells was most numerous in the temporal superior quadrant and its density peaked at 1-2 mm from the fovea. Type 1 TH+ cells were more numerous than type 2 cells at any eccentricity. Along the horizontal meridian, type 1 cell density was slightly higher in temporal (29 cells/mm2) than in nasal (25 cells/mm2) retina, while type 2 cells had a homogeneous distribution (4.5 cells/mm2). Along the vertical meridian, type 1 cells reached lower peak density (average 17.7 cells/mm2) in the inferior retina (central 4 mm), compared to the superior portion (23.7 cells/mm2). Type 2 cell density varied from 4.5 cells/mm2 in the superior region to 9.4 cells/mm2 in the inferior region. The spatial density of the two cell types varied approximately inversely while the total density of TH+ cells was virtually constant across the retina. No correlation between dopaminergic cells and rod distribution was found. However, we suggest that dopaminergic cells could have a role in mesopic and/or photopic vision in this species, since TH+ fibers are present in cone-dominated regions like the foveola and extreme nasal periphery.     

Increased tyrosine hydroxylase immunoreactivity in the locus coeruleus of cats with interstitial cystitis

PURPOSE: Environmental stressors seem to play a role in exacerbation of symptoms of interstitial cystitis (IC), both in cats and in human beings. These observations suggest a role for the sympathetic nervous system in the pathophysiology of IC. To begin to assess the underlying role in IC of the pontine nucleus locus coeruleus (LC), the most important source of norepinephrine in the central nervous system, we compared the intensity of tyrosine hydroxylase immunoreactivity (THIR) in sections of LC obtained from cats with IC and from healthy cats. Cats with IC were studied during quiescent periods in an attempt to avoid the risk of flare-induced activation of the LC. MATERIALS AND METHODS: Six cats diagnosed with IC and six healthy cats were studied. Cats with IC were monitored to ensure that no behavioral or urinary signs attributable to IC had been observed for at least two weeks prior to the study. Cats were euthanized and perfused with 4% paraformaldehyde, after which brainstem tissues were collected. Coronal sections (10 microns) of LC were prepared and examined for THIR. RESULTS: THIR in total LC, parabrachial nucleus and LC complex was significantly greater (p < 0.05) in samples from cats with IC than from healthy cats. CONCLUSIONS: The increased THIR in the LC of cats with IC provides additional evidence for increased sympathetic nervous system activity in patients with IC, even during periods of absence of clinical signs.     

Estrogen regulates galanin but not tyrosine hydroxylase gene expression in the rat locus ceruleus

The neuropeptide galanin (GAL) is coexpressed by the majority of noradrenergic neurons in the rat locus ceruleus (LC) and may function as an inhibitory modulator of noradrenergic transmission. Because estrogen has been shown to induce GAL expression in other brain regions and modulate noradrenergic transmission, we used in situ hybridization histochemistry to assess the effects of chronic estrogen treatment on GAL and tyrosine hydroxylase (TH) gene expression in the LC of ovariectomized female rats. We found that GAL mRNA levels were significantly elevated in rats implanted with a Silastic capsule containing estradiol compared to sham-implanted controls. Both the average optical density (P < or = 0.05) and the labelling area (P < or = 0.007) differed significantly between the groups. In contrast, TH gene expression measured in alternate brain sections did not differ between the groups. If GAL functions as an inhibitory modulator of noradrenergic transmission as postulated, these findings suggest that chronic estrogen treatment could reduce the noradrenergic tone of the brain in the absence of significant alterations in TH expression by enhancing the level of cosecreted GAL.     

Electrocortical desynchronization after microinfusion of kainic acid into the locus coeruleus in rats

CD-1, a genetically-engineered CHO cell line, was cultivated with a Biosilon microcarrier culture system. We successfully cultivated CD-1 cells to a very high density (over 1 x 10(7) cells/ml). Prourokinase was stably secreted at about 180 IU/10(6) cells/24 h. Experiments showed that CD-1 cells growing on Biosilon microcarriers were able to spontaneously release from the microcarriers, then reattach and proliferate on fresh microcarriers. This makes it very easy to scale up production. The microcarriers could be reused several times without affecting adhesion, proliferation and prourokinase secretion. With CM-PECC membrane radial flow chromatography and MPG chromatography, the prourokinase in conditioned medium could be purified to a specific activity of 1 x 10(5) IU/mg of protein. The purification factor was about 600 fold, and approximately 90% of the biological activity was recovered.     

Neonatal handling reduces the number of cells in the locus coeruleus of rats.

Neonatal handling induces long-lasting effects on behaviors and stress responses. The objective of the present study was to analyze the effects of neonatal handling (from the 1st to the 10th day after delivery) on the number of cells and volume of locus coeruleus (LC) nucleus in male and female rats at 4 different ages: 11, 26, 35, and 90 days. Results showed significant reductions in the number of cells and the volume of the LC nucleus in neonatally handled males and females compared with nonhandled rats. Environmental stimulation early in life induced a stable structural change in a central noradrenergic nucleus, which could be one of the causal factors for the behavioral and hormonal alterations observed in adulthood. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Glucocorticoid receptor-immunoreactivity in corticotrophin-releasing factor afferents to the locus coeruleus

Choline acetyltransferase (ChAT) is a specific phenotypic marker of cholinergic neurons. Previous reports showed that different upstream regions of the ChAT gene are necessary for cell type-specific expression of reporter genes in cholinergic cell lines. The identity of the mouse ChAT promoter region controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo is not known. We characterized a promoter region of the mouse ChAT gene in transgenic mice, using beta-galactosidase (LacZ) as a reporter gene. A 3,402-bp segment from the 5'-untranslated region of the mouse ChAT gene (from -3,356 to +46, +1 being the translation initiation site) was sufficient to direct the expression of LacZ to selected neurons of the nervous system; however, it did not provide complete cholinergic specificity. A larger fragment (6,417 bp, from -6,371 to +46) of this region contains the requisite regulatory elements that restrict expression of the LacZ reporter gene only in cholinergic neurons of transgenic mice. This 6.4-kb DNA fragment encompasses 633 bp of the 5'-flanking region of the mouse vesicular acetylcholine transporter (VAChT), the entire open reading frame of the VAChT gene, contained within the first intron of the ChAT gene, and sequences upstream of the start coding sequences of the ChAT gene. This promoter will allow targeting of specific gene products to cholinergic neurons to evaluate the mechanisms of diseases characterized by dysfunction of cholinergic neurons and will be valuable in design strategies to correct those disorders.     

Chronic cold stress alters the basal and evoked electrophysiological activity of rat locus coeruleus neurons

In vivo extracellular single-unit recording techniques revealed that chronic cold stress significantly alters both the basal and the evoked electrophysiological activity of noradrenergic neurons in the locus coeruleus of the anaesthetized rat. Following 17-21 days of chronic cold exposure (5 degrees C), the single-unit activity of histologically-identified locus coeruleus neurons in chloral hydrate-anaesthetized rats was recorded and analysed in terms of their basal firing rate and pattern of spike activity, as well as their response to footshock stimulation. There was no significant difference in the incidence of spontaneously active cells/electrode track between cold-stressed rats and control rats. However, the basal spike activity of locus coeruleus cells recorded from cold-stressed rats differed significantly from that of control rats along two dimensions: i) they displayed significantly higher basal firing rates (mean = 1.88 Hz vs 1.20 Hz, respectively); and ii) they frequently exhibited spontaneous burst-firing activity that was not observed in control rats (observed in 15/17 cold-stressed rats vs 1/26 control rats). The evoked spike activity of locus coeruleus cells in cold-stressed rats also differed significantly from that of control rats along two dimensions: i) they were more likely to respond to footshock stimulation (mean = 90.3% vs 74.4%, respectively); and ii) these responses were more likely to consist of multispike bursts of action potentials (mean = 8 bursts/50 stimulations vs 1 burst/50 stimulations, respectively). These results indicate that alterations in the electrophysiological activity of noradrenergic locus coeruleus neurons may contribute to the phenomenon of stress-induced sensitization of norepinephrine release that is thought to underlie some of the neuropathological changes that accompany long-term stress.     

Long-term effect of RU24722 on tyrosine hydroxylase in the rat locus coeruleus: differential effects of two enantiomeric forms

RU24722, as a racemic mixture, has been found to act on neuronal activity and the long-term regulation of tyrosine hydroxylase in the locus coeruleus of the rat. In this study, the effects of two enantiomeric derivatives of RU24722 (3 alpha and 16 alpha forms), as compared to the racemic form itself, are studied. The short-term effect was estimated 20 min after treatment by measuring variations in 3,4-dihydroxyphenylacetic acid content in the locus coeruleus. The long-term effect was determined by evaluating tyrosine hydroxylase protein concentration in the locus coeruleus 3 days after a single injection. Comparison of actions of both enantiomers showed that the 16 alpha form was 3-fold more potent in eliciting tyrosine hydroxylase protein elevations at three days, whereas the 3 alpha isomer increased 3,4-dihydroxyphenylacetic acid content 2-fold more in the short-term. These results seem to show that the 16 alpha configuration is crucial for the long term regulation of tyrosine hydroxylase protein elicited by RU24722 within the locus coeruleus.     

Tyrosine hydroxylase expression in primary cultures of olfactory bulb: role of L-type calcium channels

Sensory activity mediates regulation of tyrosine hydroxylase (TH), the first enzyme in the dopamine biosynthetic pathway, in the rodent olfactory bulb. The current studies established for the first time primary cultures of neonatal mouse olfactory bulb expressing TH and tested whether L-type calcium channels mediate the activity-dependent regulation of the dopamine phenotype. After 1 d in vitro (DIV), a small population of TH-immunostained neurons that lacked extensive processes could be demonstrated. After an additional 2 DIV in serum-free medium, the number of TH neurons had doubled, and they exhibited long interdigitating processes. Membrane depolarization for 48 hr with 50 mM KCl produced a further 2.4-fold increase in the number of TH-immunoreactive neurons compared with control cultures. Increased TH neuron number required at least 36 hr of exposure to KCl. Forskolin, which increases intracellular cAMP levels, induced a 1.5- to 1.6-fold increase in the number of TH-immunostained neurons. Combined treatment with KCl and forskolin was not additive. Nifedipine, an L-type calcium channel blocker, completely prevented the depolarization-mediated increase in TH expression but did not block the response to forskolin. Treatment with Bay K8644, an L-type calcium channel agonist, also significantly increased the number of TH-expressing neurons. Depolarization also induced alterations in neuritic outgrowth, resulting in a stellate versus an elongate morphology that, in contrast, was not prevented by nifedipine. These results are the first demonstration that in vitro, as in vivo, depolarization increases TH expression in olfactory bulb and that L-type calcium channels mediate this activity-dependent regulation of the dopamine phenotype.     

Self-stimulation of noradrenergic cell group (A6) in locus coeruleus of rats.

In a study with 71 male Charles River rats, 20 out of 21 electrodes in the almost-exclusively noradrenergic cell concentrations of the locus coeruleus supported high and stable rates of self-stimulation. Locus coeruleus self-stimulation was facilitated by dextroamphetamine-sulphate and suppressed by chlorpromazine hydrochloride and a-methyl-p-tyrosine, but pimozide at relatively high doses had almost no effect. Results demonstrate that self-stimulation can be localized in a relatively homogeneous noradrenergic site and provide presumptive evidence that at least some noradrenergic neurons specifically mediate rewarding effects. (54 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Injection of corticotropin-releasing hormone into the locus coeruleus or foot shock increases neuronal Fos expression

Previous research suggests that corticotropin-releasing hormone can act in the locus coeruleus to increase the firing of locus coeruleus neurons and elicit physiological responses resembling those associated with stress. The present study used immunocytochemical detection of Fos as a measure of neuronal activation to identify brain areas that were activated by bilateral injections of corticotropin-releasing hormone into the locus coeruleus of rats. Injection of corticotropin-releasing hormone into the locus coeruleus increased the expression of Fos in the locus coeruleus as compared with injection of vehicle into the locus coeruleus or injection of corticotropin-releasing hormone into neighbouring pontine sites. The pattern of Fos expression throughout the brain after injections of corticotropin-releasing hormone into the locus coeruleus was generally consistent with the anatomical organization of efferent projections arising from the locus coeruleus; increased Fos expression was observed in many brain areas including the ventral lateral septum, septohypothalamic nucleus, bed nucleus of the stria terminalis, the central amygdaloid nucleus, the dorsomedial nuclei of the hypothalamus, and the thalamic paraventricular and rhomboid nuclei. Foot shock also increased Fos expression in the locus coeruleus and the other brain regions that expressed Fos after corticotropin-releasing hormone injections into the locus coeruleus. A few brain regions, most notably the hypothalamic paraventricular nucleus, expressed Fos in response to foot shock but not corticotropin-releasing hormone. These results indicate that local injection of corticotropin-releasing hormone into the locus coeruleus stimulates the activity of the locus coeruleus neurons. However, the pattern of Fos expression throughout the brain evoked by injection of corticotropin-releasing hormone into the locus coeruleus does not fully replicate the effects of foot shock.     

Vitamin D increases expression of the tyrosine hydroxylase gene in adrenal medullary cells

We examined expression of the 1,25-dihydroxyvitamin D3 [1,25-(OH)2 D3] receptors in chromaffin cells of the adrenal medulla and the effects of 1,25(OH)2 D3 on expression of the tyrosine hydroxylase (TH) gene. Accumulation of 1,25(OH)2 D3 in the nuclei of adrenal medullary cells, but not in the adrenal cortex, was observed in mice intravenously injected with radioactively labeled hormone. 1,25(OH)2 D3 produced concentration-dependent increases in the TH mRNA levels in cultured bovine adrenal medullary cells (BAMC). The maximal increases (2-3-fold) occurred at 10(-8) M 1,25(OH)2 D3. Combined treatment with 1,25(OH)2 D3 and 20 microM nicotine had no additive effect on TH mRNA levels suggesting that transsynaptic (nicotinic) and vitamin D (hormonal) stimulation of TH gene expression are mediated through converging mechanisms. Induction of TH mRNA by 1,25(OH)2 D3 was not affected by calcium antagonist TMB-8. By increasing expression of the rate limiting enzyme in the catecholamine biosynthetic pathway, 1,25-(OH)2 D3 may participate in the regulation of catecholamine production in adrenal chromaffin cells. This regulation provides mechanisms through which 1,25(OH)2 D3 may control response and adaptation to stress.     

Amygdaloid corticotropin-releasing factor targets locus coeruleus dendrites: substrate for the co-ordination of emotional and cognitive limbs of the stress response

Corticotropin-releasing factor (CRF), the neurohormone that initiates the endocrine limb of the stress response via its actions on the anterior pituitary, also acts as a neurotransmitter in the noradrenergic locus coeruleus (LC) to activate this system during stress. Because the central nucleus of the amygdala contains numerous CRF-immunoreactive neurones, the present study examined whether CRF projections from the central nucleus of the amygdala target LC dendrites, thereby providing a mechanism for limbic-CRF modulation of brain noradrenergic activity. Retrograde tracers injected into the rostrolateral pericoerulear region, where CRF-immunoreactive fibres are dense, labelled numerous CRF-immunoreactive neurones in the central nucleus of the amygdala. Consistent with this, ultrastructural analysis of the rostrolateral pericoerulear region in sections that were dually labelled for an anterograde tracer (biotinylated dextran amine, BDA) injected into the central nucleus of the amygdala and CRF immunoreactivity revealed that a substantial percentage (35%) of amygdaloid axon terminals were CRF-immunoreactive. These terminals formed synaptic specializations with unlabelled dendrites that were more often of the asymmetric (excitatory) type. Additionally, ultrastructural analysis of sections that were dually labelled to visualize CRF-and tyrosine hydroxlase-immunoreactivity demonstrated synaptic specializations between CRF-immunoreactive terminals and LC dendrites in the rostrolateral peri-LC, which were also frequently asymmetric. Taken together with previous ultrastructural findings that LC dendrites in the rostrolateral pericoerulear region are targeted by anterogradely labelled terminals from the central nucleus of the amygdala, the present results implicate this nucleus as a source of CRF that can impact on LC activity via effects on dendrites in the rostrolateral pericoerulear region. This cellular substrate for amygdaloid-CRF modulation of brain noradrenergic activity may serve as a mechanism for the integration of emotional and cognitive responses to stress.     

Changes in the central and peripheral serotonergic system in rats exposed to water-immersion restrained stress and nicotine administration

The effects of water-immersion restraint stress (WS) on chronically nicotine-administered rats were studied in the blood and various regions of the brain. Serotonin (5-HT) levels increased in the hypothalamus, hippocampus, cortex and cerebellum following the administration of nicotine. 5-HT levels increased in all the brain regions following stress. Nicotine decreased stress-induced increased levels of 5-HT in the hippocampus and cerebellum. Nicotine administration alone increased 5-hydroxyindole acetic acid (5-HIAA) levels in the hippocampus and cerebellum. Stress alone also increased 5-HIAA levels in all the brain regions. In the cortex, 5-HT and 5-HIAA levels further increased following the administration of a combination of stress and nicotine compared to rats given stress alone. In the blood as well as in all the brain regions, except the cerebellum, stress or nicotine administration did not affect tryptophan levels. Stress given to nicotine-administered rats resulted in a decrease in tryptophan levels in the blood and plasma. Although 5-HT and 5-HIAA levels were not influenced by stress and/or nicotine administration, the 5-HIAA/5-HT ratio increased in the blood and plasma of rats administered with nicotine and exposed to stress. The effects of nicotine on the serotonergic system depend upon the kind of stress given together with the organs and brain regions involved.     

Induction of tyrosine hydroxylase gene expression in human foetal cerebral cortex

INTRODUCTION: The poisoning severity score is a four-point severity-classification scale, developed by the International Programme on Chemical Safety, the Commission of the European Union, and the European Association of Poison Centres and Clinical Toxicologists (IPCS/EC/EAPCCT), for the retrospective assessment of cases of poisoning reported to poisons information centers. OBJECTIVES: The aims of this study were to test the validity of using the poisoning severity score obtained at initial referral to assess clinical severity and the likelihood of subsequent deterioration, to select cases for follow-up, and also to determine the need for referral to a clinical toxicologist. METHODS: The poisoning severity score was determined at the time of initial inquiry. Follow-up was then undertaken until either the patient was discharged from medical attention or died. A second poisoning severity score was then calculated taking note of the most severe features present after the initial inquiry. RESULTS: Of 718 consecutive telephone inquiries, 397 were given an initial poisoning severity score of 0 (no signs and symptoms), 225 a score of 1 (mild symptoms), 71 a score of 2 (moderate symptoms), and 25 a score of 3 (severe symptoms). Follow-up data are available only in 638 cases because the patient or referring doctor could not be traced in 80 instances. Of the 638 cases, 41 deteriorated; 31 of these were graded initially as poisoning severity score 0, four as 1, and six as 2. Five patients died (two with an initial score of 2 and three with an initial score of 3). CONCLUSIONS: This study demonstrates that it is useful to score telephone inquiries to a poisons information service at initial referral with the poisoning severity score. First, the poisoning severity score is helpful in assessing accurately the clinical severity and the likelihood of further deterioration. Second, the poisoning severity score is useful in determining the need for referral of the inquiry to a clinical toxicologist, thus ensuring that more serious and complicated cases of poisoning receive expert medical advice on management. Third, the poisoning severity score is helpful in selecting those cases which warrant follow-up for medical and epidemiological reasons.     

Effects of short-and long-term administration of tricyclic antidepressants and lithium on norepinephrine turnover in brain

The findings summarized in this paper show that norepinephrine turnover in brain is decreased after acute administration of imipramine or desmethylimipramine but tends to increase during chronic administration of these tricyclic antidepressants. Similarly, it appears that there also may be important differences between the effects of acute and chronic administration of lithium salts on norepinephrine turnover in the central nervous system. Such changes in norepinephrine turnover that develop gradually over the course of long-term drug administration may help to explain the need for chronic administration of tricyclic antidepressants or lithium salts in the treatment of patients with affective disorders.     

Non-selective enhancement of locus coeruleus and substantia nigra self-stimulation after termination of chronic dopaminergic receptor blockade with pimozide in rats

The selectivity of hexane isomers towards cottonseed pigments and colour fixed pigments was illustrated by using spectrophotometric analysis of crude, refined and bleached oils, and by refinability and bleachability criteria. Crude cottonseed oil contains besides the alkali-refinable gossypol and gossypurpurin, several colour fixed pigments. Anhydrogossypol, gossyfulvin, anthocyanins and carotenoids seem to be responsible for the colour-fixation of the oil. Only carotenoids are eliminated by bleaching. Selectivities of hexane isomers towards colour fixed pigments are in the following order: isohexane less than n-hexane less than cyclohexane less than benzene less than methylcyclopentane.     

Response of locus coeruleus neurons to footshock stimulation is mediated by neurons in the rostral ventral medulla

While it is well documented that locus coeruleus neurons are potently activated by foot-pinch or sciatic nerve stimulation, little is known about the circuit producing this sensory response. Previous work in our laboratory has identified the medullary nucleus paragigantocellularis as a major excitatory afferent to the locus coeruleus. Here, we use local microinjections into the paragigantocellularis to test whether this nucleus is a link in the pathway mediating the activation of locus coeruleus neurons by subcutaneous footpad stimulation, or footshock, in anesthetized rats. Lidocaine HCl microinjected into the paragigantocellularis reversibly attenuated footshock-evoked activation of 50 out of 56 locus coeruleus cells, with responses in 20 cells completely blocked. Microinjections of GABA into the paragigantocellularis reduced the footshock-evoked responses of 17 out of 27 locus coeruleus cells (seven complete blocks); microinjections of the GABAB agonist baclofen had no effect (0 out of 11 cells blocked). Microinjections of a synaptic decoupling cocktail of manganese and cadmium also attenuated locus coeruleus activation in eight out of nine cells with two complete blocks. With each agent, the most effective injection placement for complete blockade of responses was the ventromedial paragigantocellularis; injections bordering this region attenuated responses, while those outside of the paragigantocellularis (dorsal medullary reticular formation, nucleus tractus solitarius, or facial nucleus), or vehicle injections, were ineffective. These results are consistent with previous findings that pharmacologic blockade of paragigantocellularis-evoked locus coeruleus activity also blocks footshock-evoked responses of locus coeruleus neurons [Ennis and Aston-Jones (1988) J. Neurosci. 8, 3644-3657], and support the view that this somatosensory response, and perhaps other sensory-evoked responses of locus coeruleus neurons, involve the nucleus paragigantocellularis.