RNA editing blood biomarkers for predicting mood alterations in HCV patients.

Treatment-emergent depression is a common complication in patients with chronic hepatitis C virus (HCV) infection undergoing antiviral combination therapy with IFN-α and ribavirin. It has recently been shown that changes in A-to-I RNA editing rates are associated with various pathologies such as inflammatory disorders, depression and suicide. Interestingly, IFN-α induces gene expression of the RNA editing enzyme ADAR1-1 (ADAR1a-p150) and alters overall RNA editing activity. In this study, we took advantage of the high prevalence of pharmacologically induced depression in patients treated with IFN-α and ribavirin to test the interest of RNA editing-related biomarkers in white blood cells of patients. In this 16-week longitudinal study, a small cohort of patients was clinically evaluated using standard assessment methods prior to and during antiviral therapy and blood samples were collected to analyse RNA editing modifications. A-I RNA editing activity on the phosphodiesterase 8A (PDE8A) gene, a previously identified RNA editing hotspot in the context of lupus erythematosus, was quantified by using an ultra-deep next-generation sequencing approach. We also monitored gene expression levels of the ADAR enzymes and the PDE8A gene during treatment by qPCR. As expected, psychiatric evaluation could track treatment-emergent depression, which occurred in 30% of HCV patients. We show that PDE8A RNA editing is increased in all patients following interferon treatment, but differently in 30% of patients. This effect was mimicked in a cellular model using SHSY-5Y neuroblastoma cells. By combining the data of A-I RNA editing and gene expression, we generated an algorithm that allowed discrimination between the group of patients who developed a treatment-emergent depression and those who did not. The current model of drug-induced depression identified A-I RNA editing biomarkers as useful tools for the identification of individuals at risk of developing depression in an objective, quantifiable biological blood test.

Increased Turnover of Cerebral Norepinephrine during Rebound of Paradoxical Sleep in the Rat.

Changes in turnover of cerebral norepinephrine, as measured after intracisternal administration of the H(3) amine, have been studied in rats during selective paradoxical sleep deprivation and its following rebound. Experiments were performed under neurophysiological control. A marked increase of turnover of norepinephrine is associated with the augmentation of paradoxical sleep characteristic of the rebound period.

Region-specific alterations of A-to-I RNA editing of serotonin 2c receptor in the cortex of suicides with major depression.

Brain region-specific abnormalities in serotonergic transmission appear to underlie suicidal behavior. Alterations of RNA editing on the serotonin receptor 2C (HTR2C) pre-mRNA in the brain of suicides produce transcripts that attenuate 5-HT2CR signaling by impairing intracellular G-protein coupling and subsequent intracellular signal transduction. In brain, the distribution of RNA-editing enzymes catalyzing deamination (A-to-I modification) shows regional variation, including within the cerebral cortex. We tested the hypothesis that altered pre-mRNA 5-HT2CR receptor editing in suicide is region-specific. To this end, we investigated the complete 5-HT2CR mRNA-editing profile in two architectonically distinct cortical areas involved in mood regulation and decision-making in a clinically well-characterized cohort of age- and sex-matched non-psychiatric drug-free controls and depressed suicides. By using an original biochemical detection method, that is, capillary electrophoresis single-stranded conformational polymorphism (CE-SSCP), we corroborated the 5-HT2CR mRNA-editing profile previously described in the dorsolateral prefrontal cortex (Brodmann area 9 (BA9)). Editing of 5-HT2CR mRNA displayed clear regional difference when comparing dorsolateral prefrontal cortex (BA9) and anterior cingulate cortex (BA24). Compared with non-psychiatric control individuals, alterations of editing levels of 5-HT2CR mRNA were detected in both cortical areas of depressed suicides. A marked increase in editing on 5-HT2CR was especially observed in the anterior cingulate cortex in suicides, implicating this cortical area in suicide risk. The results suggest that region-specific changes in RNA editing of 5-HT2CR mRNA and deficient receptor function likely contribute to the etiology of major depressive disorder or suicide.

Participation of serotonin in thyrotropin release. II. Evidence for the action of serotonin on the phasic release of thyrotropin.

During a 12-h light 12-h dark schedule (lights off at 1900 h), male Sprague-Dawley rats show a circadian rhythm of plasma TSH with a zenith near midday. The participation of serotonin (5HT) in the phasic release of TSH was studied using both pharmacological and surgical-stereotaxical approaches. Animals treated with parachlorophenylalanine methyl ester (pCPA), an inhibitor of 5HT synthesis (one or two injections of 250 mg/kg each) showed a reduction or a disappearance of the diurnal peak of TSH, respectively. Additional treatment by 5-hydroxytryptophan, a precursor of 5HT, completely, restored the diurnal TSH peak. Treatment with 5,6-dihydroxytryptamine creatine sulfate, a neurotoxin which selectively destroys 5HT terminals, also induced alterations of the diurnal peak of TSH. There were no major modifications observed in the low nocturnal levels of TSH in rats treated with pCPA, 5-hydroxytryptophan, or 5,6-dihydroxytryptamine. The major serotoninergic innervation of the hypothalamus originates from the raphe dorsalis or centralis; destruction of these two nuclei caused a quasiabolition of the diurnal TSH peak (only a low amplitude TSH circadian rhythm persisted). Hypothalamic 5HT content was measured in the majority of these experiments; the greatest depletions (near 90%) were observed after two injections of pCPA or in rats bearing raphe lesions. We conclude that the diurnal peak of TSH, observed during the physiological circadian rhythm, is serotoninergic dependent.

Quantitative autoradiography of somatostatin receptors in the rat limbic system.

The distribution of somatostatin receptors (SRIF-R) was analyzed in the limbic system of the adult rat by in vitro autoradiography with [125I-Tyr0,DTrp 8]S14 as a radioligand. Precise quantification of the density of binding sites, at 0.2 mm intervals throughout the different areas revealed a marked heterogeneity of labeling in most structures. In particular, SRIF-R were concentrated in the basal (104.4 +/- 3.3 fmol/mg proteins) and basolateral amygdaloid nuclei (94.8 +/- 4.3 fmol/mg proteins), and in the nucleus of the lateral olfactory tract (121.6 +/- 2.4 fmol/mg proteins), whereas moderate densities were detected in the amygdalo-hippocampal nucleus (76.4 +/- 2.8 fmol/mg proteins). The medial (41.3 +/- 1.9 fmol/mg proteins) and the central (24.0 +/- 1.4 fmol/mg proteins) amygdaloid nuclei contained lower SRIF-R concentrations. It appears from these observations, in the light of the anatomical pathways of the amygdala, that intra-amygdalian SRIF-containing neurons project to the amygdalo-hippocampal nucleus, and that SRIF-R in the basolateral complex are the target of afferents from limbic cortical areas. SRIF-R were detected at different levels of the hippocampal formation but their distribution was more restricted than that of SRIF-containing fibers. The maximal density of sites was detected in the ventral and dorsal parts of the subiculum (115.0 +/- 3.4 and 87.0 +/- 2.8 fmol/mg proteins, respectively) and in the parasubiculum (100.1 +/- 5.4 fmol/mg proteins). In Ammon's horn, the stratum oriens and stratum radiatum of the CA1 field were the only sites enriched in SRIF-R (74.1 +/- 2.0 and 74.6 +/- 1.9 fmol/mg proteins, respectively). The apparent lack of receptors in the pyramidal cell layer indicated that, in Ammon's horn, SRIF is involved in intra-hippocampal communication. Low levels of receptors were found in the hippocampal CA2 and CA3 fields. SRIF-R in the dentate gyrus were mainly concentrated in the molecular layer (57.3 +/- 1.2 fmol/mg proteins). A very high density of sites was also observed in the entorhinal cortex (up to 123.1 +/- 1.5 fmol/mg proteins). A clear mismatch between SRIF and SRIF-R was detected in the septum and the habenula. In the profound layers of the cingulum and retrosplenial cortex, a heterogeneous distribution of SRIF-R was observed. High concentrations of sites were detected in the rostral zone of the cingulate cortex (93.4 +/- 2.0 fmol/mg proteins) while the posterior cingulate only exhibited moderate concentrations of sites (66.5 +/- 0.7 fmol/mg proteins).(ABSTRACT TRUNCATED AT 400 WORDS)

Variations in 3,4-dihydroxyphenylacetic acid concentration are correlated to single cell firing changes in the rat locus coeruleus.

The purpose of this study is to see whether variations in the catechol oxidation peak in the locus coeruleus would be closely related with variations in single cell firing, and would be independent of the mechanism triggering the variation of neuronal activity. Single unit and electrochemical (differential pulse voltammetry) recordings were performed, respectively on the left and right locus coeruleus of anaesthetized rats. The variations in single unit activity were induced using well known stimulatory mechanisms (yohimbine, hypovolemia, indirect activation using a 5-hydroxytryptamine central agonist RU 24969, RU 24722 known to activate noradrenergic metabolism), or inhibitory (clonidine, morphine) models. This was done in addition to successive activation or inhibition (reversal of oxotremorine with scopolamine, antagonism of clonidine by piperoxane). In all the experimental conditions, variations in the catechol oxidation current followed variations in single cell activity. Furthermore, the catechol oxidation current variations correlated significantly to changes in the firing rate. Such a close correlation between a catechol oxidation peak, and the electrical activity of the locus coeruleus cell bodies supports the use of catechol oxidation current variations as a good indirect index of functional activity in noradrenergic locus coeruleus perikarya. This suggests a close relationship between dopamine metabolism and electrical activity in locus coeruleus cell bodies.

Changes in steady-state levels of tryptophan hydroxylase protein in adult rat brain after neonatal 6-hydroxydopamine lesion.

A recently developed technique of immunoautoradiography on nitrocellulose transfers of serial frozen sections was used to determine tryptophan hydroxylase concentration in selected areas of the adult rat brain following neonatal 6-hydroxydopamine destruction of nigrostriatal dopamine neurons. Particular attention was paid to the neostriatum, known to be serotonin-hyperinnervated under these conditions, and to the nucleus raphe dorsalis, containing the cell bodies of origin for these nerve terminals. The hippocampus was also investigated as a territory of structurally intact serotonin innervation arising primarily from the nucleus raphe medianus. Tryptophan hydroxylase protein was measured at successive transverse levels across the entire caudorostral extent of all these regions. Similar measurements of tyrosine hydroxylase protein across the substantia nigra and the neostriatum verified the disappearance of the nigrostriatal dopamine neurons. The average tryptophan hydroxylase tissue concentration in the dorsal third of the serotonin-hyperinnervated neostriatum was up by 36% above control, i.e. significantly less than the number of its serotonin axon terminals or varicosities. This was therefore indicative of a lowering of the tryptophan hydroxylase protein content per serotonin ending. Interestingly, a tight correlation between the respective level-by-level concentrations of tryptophan hydroxylase and tyrosine hydroxylase protein in the control neostriatum allowed the prediction the tryptophan hydroxylase concentration after dopamine denervation with a serotonin hyperinnervation. Tryptophan hydroxylase concentration was also significantly reduced in both the nucleus raphe dorsalis and nucleus raphe medianus, notably at those raphe dorsalis levels known to give rise to the serotonin hyperinnervation of neostriatum. It is hypothesized that the lower steady-state level of tryptophan hydroxylase inside the terminals and cell bodies of hyperinnervating serotonin neurons was the result of a feedback inhibition of the synthesis of the enzyme by its end-product, presumably because of the increased amount of serotonin in these terminals.

Phenotypic characteristics of expressed tyrosine hydroxylase protein in the adult rat nucleus tractus solitarius: plasticity revealed by RU24722 treatment.

The phenotypic characteristics of expressed tyrosine hydroxylase protein have been precisely analysed in the rat nucleus tractus solitarius, which contains the majority of A2 noradrenergic and C2 adrenergic neurons of the medulla oblongata. This study was based upon quantitative analysis of immunohistochemical and immunoradioautographic staining of tyrosine hydroxylase protein in serial coronal sections. In control rats, there were few tyrosine hydroxylase-expressing cell bodies which express less than 2% of the immunoradiolabeled tyrosine hydroxylase protein measured in the structure. These cell bodies were scattered throughout an extensive immunopositive neuropile, which precisely delimited the topological space of the nucleus tractus solitarius quantiatively reconstructed using a polar coordinate system. The quantification of tyrosine hydroxylase tissue concentration from immunoradioautograms allowed us to subdivide the structure into two distinct regions. The posterior region of the nucleus tractus solitarius, which mainly corresponds to the A2 cell group, contains a relatively high tissue concentration of tyrosine hydroxylase protein (18.56 +/- 0.154 units per mg of tissue). The anterior region, which mainly corresponds to the C2 cell group, exhibits a relatively low concentration (12.09 +/- 0.81) of this protein. Three days after an intraperitoneal injection of RU24722, there was a strong increase (90 +/- 17%) in tyrosine hydroxylase protein content only in the anterior region of the nucleus tractus solitarius. This increase was associated with a dramatic elevation (142 +/- 20%) in the number of tyrosine hydroxylase-expressing cell bodies. The additional cell bodies were mainly located inside the initial perikarya-containing area.(ABSTRACT TRUNCATED AT 250 WORDS)

Origin and influence of the serotoninergic innervation of the subcommissural organ in the rat.

The origin of the serotoninergic innervation of the rat subcommissural organ was studied using radioautography of tritiated serotonin and biochemical determination of endogenous serotonin content after electrolytic lesions of raphe nuclei. The results suggest that this innervation is mainly derived from nuclei raphe centralis superior and raphe dorsalis, each nucleus contributing about one-third of the input. A possible contribution from nucleus raphe pontis is also suggested. Given the different patterns of innervation revealed by silver staining of nerve fibers and the different patterns of secretory activity observed with histochemical methods after the electrolytic lesions, the following working hypothesis is formulated. Nucleus raphe dorsalis would inhibit the synthesis of secretory material in the rat subcommissural organ via medium-sized serotoninergic fibers restricted to the hypendymal region, whereas nucleus raphe centralis superior might inhibit the release of secretory material via rather thin serotoninergic fibers reaching the nuclear level of the ependyma. This hypothesis is in line with the inhibitory effect postulated for the serotoninergic innervation in the rat subcommissural organ in early investigations using serotonin neurotoxins.

Light- and electron-microscopic immunocytochemistry of glutamic acid decarboxylase (GAD) in the basal hypothalamus: morphological evidence for neuroendocrine gamma-aminobutyrate (GABA).

GABAergic cells and axon terminals were localized in the basal hypothalamus of differnet species (rat, mouse and cat), by means of an immunocytochemical approach using a specific and well-characterized antiserum to the GABA biosynthetic enzyme, glutamate decarboxylase. Light-microscopic visualization was performed with an indirect immunofluorescence method and electron-microscopic observations were made on material with pre-embedding staining and use of the peroxidase-antiperoxidase procedure. At the light-microscopic level, a dense immunofluorescent plexus was observed over both the medial and lateral parts of the external layer of the median eminence. The labelling extended from the rostal part of the median eminence up to the pituitary stalk. Over the subependymal and internal layers only a few immunoreactive dots were visible, except around the blood vessels where they appeared more concentrated. Immunoreactive varicosities could be found following the outlines of the capillary loops and lining tanycyte processes, especially in the median eminance midportion. At the electron-microscopic level, the immunolabelling was exclusively found over neuronal profiles in the median eminence. The latter represented a small fraction of the total number of varicosities visible on the same section. Labelled profiles typically contained numerous small clear synaptic vesicles and only a few or no dense-core vesicles. In the subependymal and internal layers, rare labelled endings were found close to ependymal cells or among transversally cut fibers, respectively. In the palisadic zone, elongated positive boutons were visible intermingled with bundles of unlabelled axons and glial or ependymal processes. In the neurohemal contact zone, immunoreactive endings were observed among unlabelled neurosecretory endings in close vicinity to fenestrated capillary perivascular space. Small moderately intense immunofluorescent varicosities were observed all over the hypothalamus. The density of the glutamate decarboxylase-positive network was higher than in most diencephalic regions. Intraventricular or topical injection of colchicine allowed the visualization of small lightly immunoreactive cells in the diffusion area of colchicine. In the arcuate nucleus labelled axonal endings containing small pleomorphic synaptic vesicles and sometimes a few dense-core vesicles were observed at the electron-microscopic level. Typical synaptic junctions were commonly found between positive endings and unlabelled perikarya, or more frequently, unlabelled dendrites. These findings show that glutamate decarboxylase-containing endings are localized ed in several strategic sites for potential GABAergic neuroendocrine regulations. The GABAergic endings found among neurosecretory endings in the neurohemal contact zone may provide the morphological support for the release of gamma-aminobutyrate into the portal blood flow as an hypothalamic hypophysiotropic hormone.

Effect of haloperidol and sulpiride on dopamine metabolism in nucleus accumbens and olfactory tubercle: a study by in vivo voltammetry.

Differential pulse voltammetry used with electrochemically pretreated carbon fibre microelectrodes enables separation between the two peaks corresponding to the ascorbic acid and catechol oxidation currents. The effects of haloperidol and sulpiride on the 3,4-dihydroxyphenylacetic acid peak recorded in the nucleus accumbens and olfactory tubercle of rats were studied. Chloral hydrate anaesthetized preparations and chronic preparations were used. A microdevice was designed to implant electrodes in freely moving rats. Voltammograms were recorded every minute in each structure in acute preparations and every 2 min in chronic preparations. In acute preparations haloperidol induced a similar dose-dependent increase in the catechol oxidation peak in both structures. Sulpiride at all doses only induced an increase in the olfactory tubercle. In chronic preparations haloperidol and sulpiride had even larger effects on the 3,4-dihydroxyphenylacetic acid peak in both regions. In these preparations sulpiride induced a significant increase in nucleus accumbens. The effects induced by haloperidol in the two regions were greater than those induced by sulpiride. The main conclusions of this study are that the results of voltammetry agree with biochemical results on the effects of haloperidol and sulpiride on dopamine metabolism. An interaction of chloral hydrate with the effects of the two neuroleptics was also observed.

Pharmacological and functional evidence for extracellular 3,4-dihydroxyphenylacetic acid as an index of metabolic activity of the adrenergic neurons: an in vivo voltammetry study in the rat rostral ventrolateral medulla.

Catecholamine metabolism was studied in vivo in the C1 adrenergic area of the rostral ventrolateral medulla oblongata in rats, using differential normal pulse voltammetry coupled with an activated carbon fiber microelectrode. Pharmacological evidence indicates that 3,4-dihydroxyphenylacetic acid, the major dopamine metabolite, is responsible for the electrochemical signal appearance in the C1 group, and that it reflects the catecholamine synthesis rate, as previously reported in the locus coeruleus. Indeed, 3,4-dihydroxyphenylacetic acid was estimated to be formed from 77% of the intracellular dopamine, since its synthesis was increased by only 23%, after blockade of the dopamine-beta-hydroxylase activity. Neuronal activation by retrograde electrical stimulation increased the electrochemical signal, as well as hemorrhage and hypotension, suggesting that the level of extracellular 3,4-dihydroxyphenylacetic acid is a good biochemical index of the C1 adrenergic cellular activity in baseline conditions and during cellular activation.

Quantitative radioautographic study of somatostatin receptors heterogeneity in the rat extrahypothalamic brain.

The possible heterogeneity of extrahypothalamic somatostatin receptors was studied in rat brain by quantitative radioautography. The respective distribution and relative proportion of two somatostatin receptor sub-types (SS1 and SS2) were assessed by using two radioligands, the non-selective probe [125I]Tyr3-D-Trp8-somatostatin14 and the SS1 selective analogue [125I]Tyr3-SMS 201-995. For both ligands, adjacent brain sections were processed in the presence of micromolar concentrations of either a non-discriminative competitor (somatostatin14) or SS1-selective analogue (SMS 201-995). The comparative analysis of the specific binding remaining in the presence of each non-radioactive competitor permitted a semi-quantitative analysis of the proportion of SS1 and SS2 receptor sub-types in each brain region examined. Data obtained correlate well with homogenate binding results reported previously [Reubi J. C. (1984) Neurosci. Lett. 49, 259-263]. Although the distribution patterns obtained with both radioligands were similar, [125I]Tyr3-SMS 201-995 labelled only a fraction of [125I]Tyr0-D-Trp8-somatostatin14-labelled sites in certain brain regions. For example, both superficial and deep cortical laminae, as well as the basolateral amygdaloid nucleus and CA1 hippocampal area exhibited different binding densities with [125I]Tyr0-D-Trp8-somatostatin14 depending on the competitor used in the assay (somatostatin14 or SMS 201-995). On the other hand, [125I]Tyr3-SMS 201-995 binding was eliminated in an identical fashion by either competitor in these very same brain areas. This suggests the existence of SS1 and SS2 somatostatin receptor sub-types in these regions. In all other brain areas examined, somatostatin receptor sites are apparently of the SS1 sub-type. The heterogeneity of somatostatin receptors observed in certain regions may have relevance for the various biological effects induced by somatostatin in the central nervous system.

Localization of substance P- and enkephalin-like immunoreactivity in relation to catecholamine-containing cell bodies in the cat dorsolateral pontine tegmentum: an immunofluorescence study.

The distribution of tyrosine hydroxylase-, substance P- and enkephalin-immunoreactive neurons in the cat dorsolateral pons was studied using the indirect immunofluorescence method of Coons. To allow for the visualization of substance P- and enkephalin-immunoreactive cell bodies, colchicine was injected either in the ventricular space or in the cerebral tissue. The distribution of the tyrosine hydroxylase-immunoreactive cell bodies corresponded with the well-known distribution of catecholamine cells in this area of the brain. The observation of adjacent sections treated separately with tyrosine hydroxylase- and enkephalin-antiserum revealed that most catecholaminergic cells contain enkephalin-immunoreactivity. In addition to this catecholamine-enkephalin cell population, a moderate number of substance P-immunoreactive cell bodies was found in dorsolateral pons. The peribrachial nuclei were found to be densely supplied with substance P- and enkephalin-immunoreactive fibers, whereas the medial subdivisions, which contain the majority of the catecholamine cells in the dorsolateral pons, display a moderate number of immunoreactive fibers. These results are suggestive of interactions between peptide-containing and catecholaminergic neurons and also between-peptide-containing and non-catecholamine-containing neurons in the cat dorsolateral pons.

Immunohistochemistry of tryptophan hydroxylase in the rat brain.

An antiserum raised against tryptophan tetrahydropterine oxygen oxidoreductase was used to examine in rat brain the immunohistochemical localization of this rate-limiting enzyme catalysing the biosynthesis of serotonin. Tryptophan tetrahydropterine oxygen oxidoreductase was detected in numerous nerve cell bodies, proximal dendrites and axon varicosities or terminals corresponding to those of serotonin neurons as judged by their anatomical distribution and concomitant immunoreactivity to an antiserum against serotonin. In hypothalamus, a serotonin-containing nerve cell group previously visualized in the pars ventralis of the nucleus dorsomedialis by radioautography after serotonin uptake, and by serotonin immunohistochemistry after tryptamine loading, remained tryptophan tetrahydropterine oxygen oxidoreductase-unreactive even in rats treated with colchicine. On the other hand, a small group of tryptophan tetrahydropterine oxygen oxidoreductase-positive cells was identified in the rostrolateral portion of nucleus dorsomedialis, which could play a part in the intrinsic serotonin innervation of hypothalamus. There was no overlap between tryptophan tetrahydropterine oxygen oxidoreductase immunostaining and the cellular distribution of N-acetyl serotonin as reported in earlier studies. It is therefore likely that the synthesis of N-acetyl serotonin from tryptophan does not take place in N-acetyl serotonin-containing neurons.

Importance of fixation in immunohistochemistry: use of formaldehyde solutions at variable pH for the localization of tyrosine hydroxylase.

Adequate fixative in immunohistochemistry requires not only a rapid and total immobilization of the antigen, but also a sufficient preservation of its immunoreactivity and maintenance of its accessibility to the immunochemical reagents for localization. Thus, the optimal fixation condition for a specific antigen necessitates a compromise between these opposing variables and can be determined by the preparation of a series of tissues with a progressively increasing degree of fixation. Unless the results of localization using such a series is available, one must be satisfied with adequate but less than optimal results. In the present study, this principle is demonstrated using the localization of tyrosine hydroxylase in the dopaminergic system with formaldehyde as the fixative. The rate and degree of fixation with formaldehyde was shown to be highly pH dependent. By perfusing the tissue with formaldehyde at pH 6.5 (where the rate of fixation is extremely slow) it is possible to rapidly distribute the fixative homogeneously into the tissue. By suddenly changing to a formaldehyde perfusate of higher pH, the cross-linking reaction is rapidly increased. This two-step fixation procedure provides a means of obtaining a rapid and uniform immobilization of the antigen, so that its translocation can be avoided. The final degree of fixation is controlled by the duration and pH of the second fixative solution. The results obtained by increasing the pH of the second solution demonstrated that complete fixation of tyrosine hydroxylase in the dopaminergic system with formaldehyde maybe obtained using a very basic formaldehyde solution (pH 11) while still retaining immunoreactivity of the enzyme. The localization that was achieved at lower pH appeared adequate until it was compared to the results obtained by perfusion at pH 11 in the second step.

Singular subsets of locus coeruleus neurons may recover tyrosine hydroxylase phenotype transiently expressed during development.

The number of tyrosine hydroxylase (TH)-expressing neurons appears to be precisely determined in basal conditions within the noradrenergic pontine nucleus locus coeruleus (LC). However, additional neurons exhibiting TH phenotype have been observed in the adult rat LC following a single administration of RU 24722, a potent inducer of TH expression specific to the LC. The neurons acquiring TH phenotype following treatment had a topographical localization similar to that of the neurons, which transiently expressed TH during postnatal development and lost TH phenotype during the third postnatal week. The idea that the fluctuation of TH phenotype in singular subsets of LC neurons during development may be selectively restored in adults is of particular interest. The present study attempted to determine whether the cells in which TH expression was repressed during the third postnatal week could correspond to those which exhibited TH phenotype in response to RU 24722 treatment in adults. We first verified that no massive cell death occurred in the LC during the period ranging from days 13 to 30. Then, we observed that both cell populations exhibited the same altered steady-state concentration of TH-mRNA as compared to cells that permanently expressed TH. Finally, we demonstrated the presence of TH-negative neurons expressing the homeodomain transcription factor Phox2a, specific for the determination of noradrenergic phenotype, providing further evidence that "resting-noradrenergic" neurons exist in the adult rat LC under basal conditions. These neurons provide interesting prospective for gain of noradrenergic function when classical noradrenergic LC neurons are impaired.

Controlled targeting of tyrosine hydroxylase protein toward processes of locus coeruleus neurons during postnatal development.

Dendrites of locus coeruleus (LC) neurons laying within the pericoerulean neuropil (PCA) organize the major site where tyrosine hydroxylase (TH) is present throughout postnatal development. Those dendrites constitute the neuronal compartment in which TH levels increase beyond postnatal day (P) 21 or after RU24722-induced TH expression. Distal LC dendrites are present in the PCA by at least P20 but are devoid of TH and can rapidly accumulate TH protein when gene induction is triggered. Contrasting with the increase in TH levels within LC perikarya and dendrites, TH-mRNA concentration remains constant in LC perikarya from P4 to P42. Thus, supposing TH synthesis and degradation are also constant, any change in TH levels targeted toward axons might be balanced by a shift in the TH deposition within LC dendrites. This mechanism may be crucial in functions that the different processes of LC neurons have at critical steps of postnatal ontogeny.

Pharmacological modulations of adrenergic phenotype in medullary C2 and C3 cell groups of adult rat.

The adrenergic phenotype was analysed in the rat's rostral dorsomedial medulla under normal conditions and 3 days after a single intraperitoneal injection of an eburnamine derivative, RU 24722, which increases tyrosine hydroxylase protein expression in the rostral portion of the nucleus tractus solitarius. This approach was investigated by a double immunofluorescence labelling of tyrosine hydroxylase and phenylethanolamine N-methyltransferase proteins. Under normal conditions, most adrenergic cell bodies are anatomically distributed in the dorsal and rostral medulla oblongata between the rostral part of the dorsal motor nucleus of the vagus nerve and the medial longitudinal fasciculus. Adrenergic neurons detected in this medullar region were distributed between both cell groups. Three days after the pharmacological RU 24722 treatment, an upregulation in tyrosine hydroxylase and phenylethanolamine N-methyltransferase protein expression was detected in both cell groups characterized by a highly increased number of tyrosine hydroxylase- and phenylethanolamine N-methyltransferase-containing cell bodies. The number of TH-mRNA containing neurons was also increased, indicating the transcriptional level of this regulation. These results demonstrated a particular neuronal plasticity of adrenergic phenotype in the medullary cell groups of adult rat.

Evaluation of the relative amount of specific gaba-benzodiazepine receptor mrna after injection of total poly(A+)-rna into xenopus oocytes.

GABA-benzodiazepine receptor-chloride channel complexes have been detected by electrophysiological recording in Xenopus oocytes previously injected with messenger RNA extracted either from optic lobes of chick embryos or from adult rat hippocampi. The ability of the oocyte to correctly translate exogenous messengers was used to develop a routine method which could allow a quantitative evaluation of specific mRNA coding for GABA-benzodiazepine receptor proteins following an injection of a fixed amount of total poly(A+)-RNA. The conditions of the validation of this method have been determined.