Tyrosine hydroxylase and galanin mRNA levels in locus coeruleus neurons are increased following reserpine administration.

The neuropeptide galanin coexists in 80-90% of the norepinephrine-containing neurons in the locus coeruleus. In situ hybridization histochemistry was used to examine the effects of reserpine treatment or swim stress on tyrosine hydroxylase and galanin mRNA concentrations in locus coeruleus neurons. Reserpine administration significantly increased tyrosine hydroxylase and galanin mRNA levels in the locus coeruleus. The reserpine-induced increase in tyrosine hydroxylase mRNA was significantly correlated with the reserpine-induced increase in galanin mRNA. Three consecutive days of swim stress did not significantly alter either tyrosine hydroxylase or galanin mRNA concentrations in the locus coeruleus. These data suggest that both tyrosine hydroxylase and galanin gene expression in locus coeruleus neurons may be regulated by a reserpine-sensitive mechanism.     

Depolarizing stimuli increase tyrosine hydroxylase in the mouse locus coeruleus in culture

The influence of membrane depolarization on the development and regulation of brain noradrenergic neurons was studied in explant cultures of the mouse locus coeruleus (l.c.). Exposure to the depolarizing agents veratridine or elevated K+ significantly increased the catalytic activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. The effects of veratridine were prevented by tetrodotoxin, suggesting that transmembrane Na+ influx was necessary for the rise in TH. Morphometric analysis indicated that the rise in TH activity was not accompanied by altered TH-positive cell number or cell diameter. Rather, TH fluorescence intensity increased in each neuron, suggesting that depolarization increased TH per neuron. Immunoblot and densitometric analysis indicated that depolarization did, indeed, increase TH immunoreactive protein. Moreover, depolarization elevated enzyme activity in cultured neurons expressing the normal developmental increase in TH, as well as those in which plateau levels had already been attained. We conclude that depolarization and/or Na+ influx regulates a critical transmitter macromolecule in brain neurons, as in the periphery, by altering enzyme molecule number.     

Alteration of tyrosine hydroxylase activity in the locus coeruleus after administration of p-chlorophenylalanine

The time course of the variations in tyrosine hydroxylase activity (THA) and serotonin (5-HT) content were measured in the rat locus coeruleus after parachlorophenylalanine (PCPA) administration. Highly significant decreases in the 5-HT content in LC were found 24-48 h after PCPA treatment (300 mg/kg daily). An increase in THA (in the LC) was found to be significant 4 days after 2 successive injections of PCPA and after 4 successive injections of the drug THA gradually increased, reaching a maximum around 4 days after the last injection. This maximum increase in THA was greatly reduced when 5-HTP was simultaneously administered with PCPA. These results join others which suggest that a serotonin-mediated mechanism could be one of the processes controlling noradrenaline metabolism in the locus coeruleus.     

Reduced tyrosine hydroxylase immunoreactivity in locus coeruleus of suicide victims.

Antibodies against tyrosine hydroxylase (TH, the rate-limiting enzyme in norepinephrine synthesis) and dopamine beta-hydroxylase (DBH, the last enzyme in the synthesis) were used for immunohistochemical staining of human brain locus coeruleus sections, obtained postmortem from suicide victims and matched controls. Stain density over individual cells was quantified by a computerized, video-camera-based image analysis system. Mean stain density for TH was significantly lower (by about 30%) in the locus coeruleus of suicide victims. There was no difference between suicides and controls in DBH immunoreactivity or in the number of TH immunoreactive cells. Reduced TH availability, either genetically or environmentally determined, may contribute to the noradrenergic insufficiency postulated to occur in depression and the increased beta-adrenergic receptor concentrations observed in prefrontal cortex of suicide victims.     

Elevated levels of tyrosine hydroxylase in the locus coeruleus in major depression.

BACKGROUND: Levels of tyrosine hydroxylase (TH) are regulated in the noradrenergic locus coeruleus (LC) in response to changes in the activity of LC neurons and in response to changes in brain levels of norepinephrine. To study the potential role of central noradrenergic neurons in the pathobiology of major depression, TH protein was measured in the LC from postmortem brains of 13 subjects with a diagnosis of major depression and 13 age-matched control subjects having no Axis I psychiatric diagnosis. Most of the major depressive subjects died as a result of suicide. METHODS: Protein from sections cut through multiple rostro-caudal levels of LC was transferred to Immobilon-P membrane, immunoblotted for TH, and quantified autoradiographically. RESULTS: The distribution of TH-immunoreactivity (TH-ir) along the rostro-caudal axis of the LC was uneven and was paralleled by a similar uneven distribution of neuromelanin-containing cells in both major depressive and psychiatrically normal control subjects. Amounts of TH-ir in the rostral, middle and caudal levels of the LC from major depressive subjects were significantly higher than that of matched control subjects. There were no significant differences in the number of noradrenergic cells at any particular level of the LC comparing major depressive subjects to control subjects. CONCLUSIONS: Elevated expression of TH in the LC in major depression implies a premortem overactivity of these neurons, or a deficiency of the cognate transmitter, norepinephrine.     

Quantitative immunocytochemistry of tyrosine hydroxylase in rat brain. II. Variations in the amount of tyrosine hydroxylase among individual neurons of the locus coeruleus in relationship to neuronal morphology and topography

We sought to determine by a computer assisted quantitative immunocytochemical method using the peroxidase-antiperoxidase (PAP) technique if individual neurons of the nucleus locus coeruleus (LC) varied with respect to the amount of tyrosine hydroxylase (TH) within each cell and, if so, whether the amount of TH is related to morphometric characteristics and/or location of the neuron within the LC. Appropriate immunohistochemical reaction conditions were used so that the intensity of DAB reaction product formed in the PAP reaction was directly and linearly related to the amount of TH enzyme protein present in the tissue. The distribution of the average optical density of 1425 individual LC neurons obtained from 5 animals and standardized to an overall mean density varies unimodally over a 3-fold range. The relationship between staining intensity and morphological and topographic characteristics of individual neurons was examined. There was no correlation between staining intensity and morphological features of individual neurons, including cytoplasmic area. However, differences in the amount of TH in individual neurons correlated significantly (P less than 0.01) with the location of the cell in LC: the lightest staining neurons were located in the central core of the LC. The density of staining did not differ significantly in the other 4 traditionally defined regions of LC (the anterior, posterior, and ventral poles of the LC and the nucleus subcoeruleus) which form a rim around the central core. Similarly, cells which are closest to the center of mass of the LC showed a lighter staining intensity than cells located progressively further from the center. These lighter staining cells in the center were also packed more closely together (P less than 0.01). Therefore dense packing of neurons correlates with light staining intensity (P less than 0.01). We conclude that neurons of the LC vary with respect to the amount of TH, which in turn relates to the location of the cell in the LC and possibly its packing density, but not its morphometric characteristics. Since in noradrenergic neurons, the amount of TH is directly influenced by firing rate, the results suggest that the biochemical heterogeneity of LC neurons reflects differences in their functional activities. In turn, this may relate to heterogeneity of LC with respect to projection fields.     

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

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

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.     

Isolation stress increases tyrosine hydroxylase mRNA in the locus coeruleus and midbrain and decreases proenkephalin mRNA in the striatum and nucleus accumbens.

Isolation of adult animals represents a form of psychological stress from which the animals cannot escape. In order to assess the effect of this stressor on neurochemical substrates in the brain, we assessed behavior and measured tyrosine hydroxylase and proenkephalin mRNA levels in selected brain areas by in situ hybridization histochemistry. Tyrosine hydroxylase (TH) mRNA levels in the locus coeruleus (LC) were significantly and progressively increased by 18, 42 and 68% after 7, 14 or 28 days of isolation, respectively. TH mRNA in the midbrain was transiently increased by isolation. Levels were significantly elevated by 34 and 48% above group-housed controls in the ventral tegmentum and the substantia nigra, respectively, after 14 days of isolation. In the forebrain, proenkephalin (PE) mRNA levels were found to be transiently decreased by 29% in the anterior and medial aspects of the caudate-putamen and the nucleus accumbens after 7 or 14 days of isolation stress, but the levels returned toward control levels after 28 days of isolation. Behavioral tests indicate that isolated animals progressively became more aggressive with duration of stress and showed a small but significant decrease in locomotor activity. The results demonstrate that a physically noninvasive stressor such as isolation of adult male rats can produce significant alterations in brain neurochemistry. The neurochemical responses observed may represent a brain mechanism designed to help the organism adapt to or protect from the deleterious effects of chronic psychological stress.     

Anomalous rectification in rat locus coeruleus neurons

Rat locus coeruleus neurons recorded under current clamp conditions show anomalous rectification (AR) as indicated by a progressive decrease in slope resistance measured with hyperpolarizing current pulses of increasing amplitude. AR was most prominent at potentials more negative than the K+ equilibrium potential. AR was strongly dependent on external K+ concentration and was blocked by external Ba2+ and Cs+, and intracellular injection of acetate.     

A cholinergic antagonist blocks cold stress-induced alterations in rat adrenal tyrosine hydroxylase mRNA

The role of nicotinic cholinergic transmission in cold stress-induced alterations in rat adrenomedullary tyrosine hydroxylase (TH) mRNA was investigated by RNA dot-blot hybridization, using a cloned TH cDNA probe. Chlorisondamine, a ganglionic blocking agent, greatly attenuated the induction of TH mRNA levels caused by cold exposure, whereas carbachol and nicotine, cholinergic agonists, increased TH mRNA in control animals. These results suggest that cholinergic nicotinic receptors play a key role in the transsynaptic induction of adrenal TH gene expression.     

Quantitation of tyrosine hydroxylase protein in the locus coeruleus from postmortem human brain

In this study, we developed an immuno-autoradiographic method to obtain quantitative estimates of tyrosine hydroxylase (TH) protein in tissue sections from post-mortem human brain. Protein from tissue sections containing the locus coeruleus (LC) was directly transferred to a polyvinylidene fluoride (PVDF) membrane. Immunoreactive TH on PVDF membranes was identified with optimized concentrations of TH antibody followed by application of [125I]labeled secondary antibody. Quantities of TH on autoradiograms were estimated by comparing optical densities of transferred immunoblots to a calibrated standard curve produced with purified recombinant TH dotted onto the same PVDF membranes. Amounts of TH-immunoreactivity in the LC were proportional to the thickness of tissue sections up to 15 micrometer. However, the amounts of total protein, as measured by Ponceau S staining, were linearly related to section thicknesses up to 30 micrometer. Comparisons of quantities of immunoreactive TH in the LC using this method to amounts determined using traditional Western blotting, in which LC tissue was punched from adjacent sections from the same subject, showed a positive correlation (r(2)=0.99, P<0.01). Using the transfer immunoblot method, an uneven distribution of TH protein was observed along the rostrocaudal axis of the human LC (P<0.01). This method may provide a sensitive and useful tool for the study of the role of human TH expression in the pathophysiology of psychiatric disease.     

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

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

Somatotopic organization of tyrosine hydroxylase expression in the rat locus coeruleus: long term effect of RU24722

Tyrosine hydroxylase (TH) tissue concentration was determined by immunostaining of tissue sections directly transferred onto nitrocellulose membranes in the restricted region of the noradrenergic perikarya of the locus coeruleus (LC) along its postero-anterior axis. TH containing cells were systematically counted on adjacent post fixed sections stained by immunohistochemistry. The absolute quantity of TH was estimated in each section and was found to be linearly related to the number of TH immuno-positive cells found in the adjacent section. The ratio between these two parameters was thus used as an index of the cellular concentration of TH in noradrenergic cells. In the LC of control rats, the TH cellular concentration was lower (-39%) in the anterior than in the posterior half of the structure. Three days after an injection of 20 mg/kg of RU24722, an eburnamine derivative known to increase the quantity of TH in the LC, increases in quantities of TH were found in both portions of the LC. Moreover in the posterior LC the increase in the amount of TH resulted from a significant increase in the number of TH-immunopositive cells. In the anterior part, however, it was primarily the result of a significant increase in TH cellular concentration. Throughout the LC there was an increase in the cellular concentration of TH which was inversely proportional to the concentrations found in control animals. TH mRNA content was measured by a quantitative in situ hybridization in sections of both the posterior and anterior LC one day after a single injection of RU24722 at the same dose. The quantity of TH mRNA was significantly increased in both parts. The number of TH mRNA-expressing neurons also increased, especially in the anterior LC. Thus the effects at the level of TH protein and TH mRNA were strikingly parallel though increase in TH protein occurred later than the increase in the TH mRNA. These results suggest that in the rat LC: (1) there is a significant population of 'sleeping cells' in which TH expression is either inactivated or, at a low level of activation; (2) TH cellular concentration could exert a retrocontrol on its own expression in cells of the LC that contained TH and (3) TH expression appears to be regulated by different selective mechanisms in these two different subpopulations of noradrenergic cells within the LC.     

Periodic bursting activities of locus coeruleus neurons in the rat

Unit activity of locus coeruleus (LC) neurons in rats was investigated. After the animal recovered from anesthesia, the spontaneous activity exhibited periodic bursting discharges at about 15–30 s intervals. The oscillation was observed to last for a long time (1–3 h). It is suggested that many LC neurons exhibited the oscillation synchronously during stress in the awake animal.     

Transient homologous mu-opioid receptor desensitization in rat locus coeruleus neurons.

Opioid agonists hyperpolarize neurons of the locus coeruleus (LC) in the slice preparation. When opioids were applied at concentrations that caused a maximum hyperpolarization, the membrane potential hyperpolarized to a peak (about 30 mV) in the first minute and then declined over a period of 5 min. In addition, following the washout, the amplitude of the hyperpolarization induced by a lower concentration of opioid was significantly reduced as compared to control. The original response to both the low and the high concentrations of opioid recovered after removal of opioids for about 20 min. The decline in response, termed "acute desensitization," was observed only with concentrations of opioids that caused a maximum hyperpolarization and was dependent on the concentration of opioid applied (EC50 for [Met5]-enkephalin (ME), between 3 and 5 microM). The response to ME (300 nM) was reduced to 6% of control following washout of a 5-min application of ME (30 microM), whereas the response to noradrenaline (300 nM) was reduced to 75% of control. The acute desensitization therefore was selective for the opioid receptor with marginal cross-desensitization to the alpha 2-adrenoceptor-mediated hyperpolarization. The desensitization still occurred following treatment with beta-chlornaltrexamine (beta-CNA), to decrease receptor reserve, as well as in cells taken from animals treated chronically with morphine. The mechanism for the acute desensitization was investigated using agents thought to alter kinase activity. This acute desensitization may represent an initial stage in the development of tolerance produced by chronic administration of opioids.