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.

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.

Aggressive behavior during social interaction in mice is controlled by the modulation of tyrosine hydroxylase expression in the prefrontal cortex.

The Balb/c strain and the C57BL/6 strain show constitutive differences for tyrosine hydroxylase expression, and noradrenaline (NA) prefrontal transmission. Male mice of these strains also show striking differences in social interaction behaviors, with an increased aggressiveness for the Balb/c strain. To test a potential link between these neurobiological and behavioral parameters, we evaluated the behavioral effects of chronic treatment of mice with BC19, a noreburnamine compound previously known as RU24722, found to modify cell organisation, tyrosine hydoxylase (TH) expression, and its activity into the locus coeruleus (LC). We compared the pharmacological effects between the two strains in social behaviors. Our results show that the emergence of additional TH-expressing (TH+) neurons in the rostral part of the LC of Balb/c mice was associated with an increase in the density of TH+ and noradrenergic (NA+) fibers in the molecular layer in the cingular (Cg1) and prelimbic (PrL) parts of the prefrontal cortex (PFC). BC19 treatment resulted in the near-equalization of the LC number of TH+ neurons and of the density of TH+ and NA+ fibers between both strains. The aggressiveness in Balb/c mice was considerably diminished by BC19 treatment, while the originally non aggressive behavior of C57Bl/6 mice was much less affected by BC19 treatment, despite a moderate increase in some offensive behaviors. In additional control experiments, we checked the effect of BC19 on a separate test for anxiety and assessed the effect of noradrenergic N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4) mediated lesions in C57BL/6 mice on social behaviors. In the present study we show that the BC19 effect in Balb/c mice was independent of anxiety as measured in the light/dark test and that DSP-4 lesions in C57BL/6 mice produced a robust increase in aggressive social interaction. Altogether, these results show that the noradrenergic system, and particularly its projections to the PFC, strongly modulates aggressive behaviors.

Logical analysis of diffuse large B-cell lymphomas.

OBJECTIVE: The goal of this study is to re-examine the oligonucleotide microarray dataset of Shipp et al., which contains the intensity levels of 6817 genes of 58 patients with diffuse large B-cell lymphoma (DLBCL) and 19 with follicular lymphoma (FL), by means of the combinatorics, optimisation, and logic-based methodology of logical analysis of data (LAD). The motivations for this new analysis included the previously demonstrated capabilities of LAD and its expected potential (1) to identify different informative genes than those discovered by conventional statistical methods, (2) to identify combinations of gene expression levels capable of characterizing different types of lymphoma, and (3) to assemble collections of such combinations that if considered jointly are capable of accurately distinguishing different types of lymphoma. METHODS AND MATERIALS: The central concept of LAD is a pattern or combinatorial biomarker, a concept that resembles a rule as used in decision tree methods. LAD is able to exhaustively generate the collection of all those patterns which satisfy certain quality constraints, through a systematic combinatorial process guided by clear optimization criteria. Then, based on a set covering approach, LAD aggregates the collection of patterns into classification models. In addition, LAD is able to use the information provided by large collections of patterns in order to extract subsets of variables, which collectively are able to distinguish between different types of disease. RESULTS: For the differential diagnosis of DLBCL versus FL, a model based on eight significant genes is constructed and shown to have a sensitivity of 94.7% and a specificity of 100% on the test set. For the prognosis of good versus poor outcome among the DLBCL patients, a model is constructed on another set consisting also of eight significant genes, and shown to have a sensitivity of 87.5% and a specificity of 90% on the test set. The genes selected by LAD also work well as a basis for other kinds of statistical analysis, indicating their robustness. CONCLUSION: These two models exhibit accuracies that compare favorably to those in the original study. In addition, the current study also provides a ranking by importance of the genes in the selected significant subsets as well as a library of dozens of combinatorial biomarkers (i.e. pairs or triplets of genes) that can serve as a source of mathematically generated, statistically significant research hypotheses in need of biological explanation.

Lipid peroxidation-mediated oxidative stress and dopamine neuronal apoptosis in the substantia nigra during development.

The cellular pathways underlying naturally occurring neuronal apoptosis in the rat substantia nigra (SN) during the perinatal period remain largely unknown. Determining the mediators of this process in development may shed light on causes of premature neuronal death in adult neurodegenerative disorders, including the loss of dopamine neurons in Parkinson's disease. In the present study, we investigated whether lipid peroxidation-mediated oxidative stress mediates developmental death of nigral neurons by (1) establishing the profile of lipid peroxidation and other oxidative stress markers throughout the postnatal period both in the SN and striatum, and (2) examining whether the inhibitor of lipid peroxidation, alpha-tocopherol, protects these neurons from death. In addition to monitoring, the level of lipid peroxidation throughout development, we also measured the activities of three antioxidant enzymes, namely superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx). We have shown that lipid peroxidation and SOD activity progressively increased from postnatal day (PND) 3 to PND 42 in both SN and striatum. During this period, GPx activity remained stable, while catalase activity transiently increased at PND 8 only in the SN. Furthermore, alpha-tocopherol treatment from embryonic day 18 to PND 2 did not reduce the number of apoptotic neurons at PND 3. These results do not support the hypothesis that lipid peroxidation-mediated oxidative stress is the major mediator of nigral dopamine neuronal apoptosis during the perinatal period.

Recurrences in nodal T-cell lymphoma. Changes in histologic appearance and immunophenotype over the course of disease.

We examined the patterns of relapse or persistence in 37 cases of nodal peripheral T-cell lymphoma (PTCL) to address the morphologic and immunophenotypic findings. Relapses were documented in lymph node (25 cases) and/or a variety of extranodal sites at a mean of 21 months after presentation; several cases recurred as late as 13 years. Persistent bone marrow involvement was a feature of angioimmunoblastic lymphoma (AIL) and histiocyte-rich and small-cell tumors. Relapses in anaplastic tumors often involved unusual extranodal sites. The majority of relapsed PTCLs retained a similar histologic appearance, pattern of nodal involvement, and immunophenotype. Histologic progression, as assessed by increased numbers of large cells, was seen in 3 cases of AIL, in 1 case with an initial small cell morphologic appearance, and in 2 cases of PTCL with an initial mixed small and large cell appearance. Immunostains for T-cell activation markers showed increased immunoreactive cells in 5 of the 6 cases, whereas increased numbers of p53-positive tumor cells were noted in 3 of the 6 cases. The discrete large cell transformation occasionally seen in B-cell lymphoma and extranodal T-cell lymphoma was not observed in these cases.

[(18)F]p-MPPF: aA radiolabeled antagonist for the study of 5-HT(1A) receptors with PET.

This paper summarizes the present status of the researches conducted with [(18)F]4-(2'-methoxyphenyl)-1-[2'-[N-(2"-pyridinyl)-p-fluorobenzamido ]ethyl]-piperazine known as [(18)F]p-MPPF, a new 5-HT(1A) antagonist for the study of the serotonergic neurotransmission with positron emission tomography (PET). This includes chemistry, radiochemistry, animal data (rats, cats, and monkeys) with autoradiography and PET, human data with PET, toxicity, and metabolism.

Tissue distribution, autoradiography, and metabolism of 4-(2'-methoxyphenyl)-1-[2' -[N-2"-pyridinyl)-p-[(18)F]fluorobenzamido]ethyl]piperazine (p-[(18)F]MPPF), a new serotonin 5-HT(1A) antagonist for positron emission tomography: An In vivo study in rats.

The in vivo behavior of 4-(2'-methoxyphenyl)-1-[2'-[N-(2"-pyridinyl)-p-[(18)F]fluorobenzamido ]ethyl]-piperazine (p-[(18)F]MPPF), a new serotonin 5-HT(1A) antagonist, was studied in awake, freely moving rats. Biodistribution studies showed that the carbon-fluorine bond was stable in vivo, that this compound was able to cross the blood-brain barrier, and that a general diffusion equilibrium could account for the availability of the tracer. The great quantity of highly polar metabolites found in plasma did not contribute to the small amounts of metabolites found in hippocampus, frontal cortex, and cerebellum. Exvivo p-[(18)F]MPPF and in vitro 8-hydroxy-2-(di-n-[(3)H]propylamino)tetralin autoradiography were compared both qualitatively and quantitatively. Qualitative evaluation proved that the same brain regions were labeled and that the p-[(18)F]MPPF labeling is (a) in total agreement with the known distribution of 5-HT(1A) receptors in rats and (b) characterized by very low nonspecific binding. Quantitative comparison demonstrated that the in vivo labeling pattern obtained with p-[(18)F]MPPF cannot be explained by differences in regional blood flow, capillary density, or permeability. The 5-HT(1A) specificity of p-[(18)F]MPPF and binding reversibility were confirmed in vivo with displacement experiments. Thus, this compound can be used to evaluate parameters characterizing 5-HT(1A) binding sites in the brain.

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.

Evidence of deprenyl-insensitive apoptosis of nigral dopamine neurons during development.

Apoptosis of dopamine neurons occurs naturally in the substantia nigra during development, culminating in approximately 30% loss of these cells during the perinatal period. Deprenyl, independent of its monoamine oxidase (MAO)-B inhibitory properties, can prevent dopamine neuronal apoptosis in models of neurodegeneration. Our current study demonstrate that apoptotic death of dopamine neurons during development is insensitive to daily treatment of pregnant mothers and then newborns with deprenyl (0.1, 1, or 10 mg/kg). This result is not due to poor crossing of the placental and blood-brain barriers, since deprenyl caused a dose-dependent inhibition of brain MAO-B activity in pups at birth. Determining the pathway(s) leading to deprenyl-insensitive apoptosis of nigral dopamine neurons in development may shed light on mechanisms underlying the premature death of dopamine neurons in neurodegenerative disorders.

In vivo characterization of p-[(18)F]MPPF, a fluoro analog of WAY-100635 for visualization of 5-HT(1a) receptors.

The in vivo and ex vivo distributions and the pharmacological profile of the fluorinated phenylpiperazine derivative p-[(18)F]MPPF (4-(2'-methoxyphenyl)-1-[2'-(N-2"-pyridinyl)-p-fluorobenzamido]-et hyl piperazine) were evaluated in the cat brain as a potential selective antagonist for 5-HT(1A) receptors using PET. After intravenous injection of p-[(18)F]MPPF in cats, there was a rapid accumulation of radioactivity in the brain, with 4% of the total radioactivity injected present in the brain at 4 minutes postinjection. The highest uptakes of radioactivity were observed in the hippocampus and cingulate cortex, regions known to be rich in 5-HT(1A) receptors, whereas lower levels of radioactivity were observed in the cerebellum. The mean ratio of radioactivity in the hippocampus to the cerebellum was 4.29 (SD = 0.21; n = 5) from 40 to 90 minutes postinjection of p-[(18)F]MPPF. The corresponding ratio for the cingulate cortex was 3.01 (SD = 0.16; n = 5). Specific binding in the hippocampus and the cingulate cortex was markedly reduced following injection of unlabeled WAY-100635 and pindolol but was unaffected by treatment with alpha1, 5-HT(2), or reuptake inhibitor agents indicating reversibility and selectivity of p-[(18)F]MPPF binding to 5-HT(1A) receptors. Ex vivo autoradiographic study with p-[(18)F]MPPF in cat brain sections showed labeling of areas rich in 5-HT(1A) receptors with a regional brain distribution that closely matched that observed using PET. These results indicate that p-[(18)F]MPPF may be a useful candidate for noninvasive PET imaging of 5-HT(1A) receptors in the living human brain.

In situ examination of tyrosine hydroxylase activity in the rat locus coeruleus using (3',5')-[(3)H(2)]-alpha-fluoromethyl-tyrosine as substrate of the enzyme.

Tyrosine hydroxylase (TH) activity can be modified by changes in the specific activity of the enzyme (SA(TH)) or in the levels of active enzyme. We developed a methodology making it possible to measure with excellent anatomical resolution TH enzymatic activity and TH protein quantity by quantitative autoradiography and immunoautoradiography, respectively, from adjacent sections taken at serial intervals along the longitudinal extent of a same brain. SA(TH) was estimated by the slope of linear regressions established between TH activity and TH quantity measured at each anatomical plane. To evaluate TH activity, we used (3',5')-[(3)H(2)]-(D, L)-alpha-fluoromethyl-tyrosine [(3)H(2)]-MFMT, which is transformed by TH to [(3)H]-MFM-dopa, a potent and irreversible substrate for aromatic amino acid decarboxylase. We found that the SA(TH) in the cell body area of the LC (PKA) was 48% lower than that evaluated in the surrounding pericoerulean neuropil (PCN). In the PCN, 22% only of TH level exhibited a level of enzymatic activity above threshold. We also examined how SA(TH) was distributed in the LC 15 min and 3 days after RU 24722 treatment, a potent phasic and tonic activator of TH enzyme in noradrenergic neurons. Two distinct mechanisms have been observed: the short-term effect was due to an increase in the SA(TH) in the PKA only, while the long-term effect was mainly caused by an increase in the number of active TH proteins in the PCN. These results suggest that the fine regulation of TH activity which occurs in the different compartments of LC neurons may be critical in the functions involving the LC.

Biochemical and autoradiographic measurements of brain serotonin synthesis rate in the freely moving rat: a reexamination of the alpha-methyl-L-tryptophan method.

Biochemical approaches were used in freely moving rats to determine, under steady-state conditions, the brain/arterial plasma partition coefficients of L-tryptophan and alpha-[3H]methyl-L-tryptophan, from which the lumped constant for the alpha-methyl-L-tryptophan method of estimating the rate of brain serotonin synthesis is calculated. The lumped constants were significantly different in the various structures examined: 0.149 +/- 0.003 in the raphe dorsalis, 0.103 +/- 0.002 in the raphe centralis, 0.087 +/- 0.003 in the reticular formation, and 0.62 +/- 0.08 in the pineal gland. From these data we proposed a two-compartment model to calculate the rate of serotonin synthesis by quantitative autoradiography using a three-time point experiment. Rates of synthesis for the raphe dorsalis and the reticular formation (620 +/- 57 and 80 +/- 35 pmol/g of tissue/min, respectively) were similar to those measured simultaneously by biochemical means, but rates were 50% higher for the raphe centralis (568 +/- 90 vs. 381 +/- 31 pmol/g of tissue/min). The lack of dynamic equilibrium of the tracer between plasma and tissue pools may explain the discrepancy between the two methods. Our findings did not confirm previous data, indicating that the application of the autoradiographic method to measure the rate of brain serotonin synthesis using alpha-methyl-L-tryptophan as tracer has limitations.

High-yield radiosynthesis and preliminary in vivo evaluation of p-[18F]MPPF, a fluoro analog of WAY-100635.

No-carrier-added 4-[18F]fluoro-N-[2-[1-(2-methoxyphenyl)-1 piperazinyl]ethyl-N-2-pyridinyl-benzamide (p-[18F]MPPF) was synthesized by nucleophilic substitution of the corresponding nitro compound in the presence of Kryptofix 222 and K2CO3 by microwave heating (3 min, 500 W) using a remotely controlled radiosynthesis. Baseline separation of p-[18F]MPPF from the nitro derivative was performed on a semipreparative HPLC C18 column. After Sep-Pak formulation, the radiopharmaceutical was obtained with a radiochemical yield of 25% (EOS) in about 70 min. Specific radioactivity averaged between 1-5 Ci/micromol EOS. Labelling of the ortho and meta derivatives was also attempted. Brain uptake of p-[18F]MPPF was studied with PET on fluothane-anesthetized cats. Following intravenous injection of p-[18F]MPPF, high accumulation of radioactivity was observed in the hippocampus and cerebral cortex. Low levels of radioactivity were observed in cerebellum. At 30 min, the mean hippocampus/cerebellum and cortex/cerebellum ratios were 5 and 3.8, respectively. The accumulation of the tracer was blocked by prior administration of reference WAY-100635, demonstrating the specificity of the ligand.

Plasticity of tyrosine hydroxylase gene expression within BALB/C and C57Black/6 mouse locus coeruleus.

The plasticity of tyrosine hydroxylase (TH) phenotype in the locus coeruleus (LC) of two pure inbred strains of mice, Balb/C (C) and C57Black/6 (B6), was investigated at the molecular level by radioactive in situ hybridization. The results demonstrated that in basal conditions, C mouse LC contains less TH-mRNA-expressing cells than B6. After RU 24722-treatment, which induces long lasting TH gene expression in the LC, we previously reported an increase in TH-expressing cell number in C mouse LC only, equalizing TH phenotype between the two strains. Here, we demonstrate that strain specific plasticity of TH phenotype detected in spatially organized cells is associated with the regulation of TH-mRNA expression above a detectable level. These results suggest that interstrain differences and pharmacologically-induced phenotypic plasticity in TH phenotype may occur at the transcriptional level.

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.

p-Chlorophenylalanine-induced alteration of somatodendritic levels of tryptophan hydroxylase within the rat mesencephalic raphe nuclei.

Tryptophan hydroxylase distribution was examined across the nuclei raphe dorsalis, medianus, and pontis of the adult rat, under basal conditions and 2 days after a single injection of p-chlorophenylalanine, an irreversible tryptophan hydroxylase inhibitor. Tryptophan hydroxylase-expressing cells were numbered in transverse sections processed for immunohistochemistry, and the area of tryptophan hydroxylase distribution was delineated in adjacent sections transferred onto nitrocellulose and processed for immunoautoradiography. Two distinct areas were visualized: an inner zone, corresponding to the area displaying tryptophan hydroxylase-immunoreactive cells (so-called somatic area), and an outer zone, here called perisomatic, devoid of perikarya yet rich in tryptophan hydroxylase-positive neuropil in the histological sections. After treatment with p-chlorophenylalanine, a significant decrease in the number of tryptophan hydroxylase-immunoreactive cells could be observed only in the rostral raphe dorsalis, particularly within its ventromedian and dorsomedian subdivisions. In all raphe nuclei, the topological reconstruction of the somatic area was not modified. Based on the densitometric measurements in the immunoautoradiographs, however, a dramatic decrease in the content, concentration, and volume of expression of tryptophan hydroxylase could be documented in the three raphe nuclei. Detailed analysis of these results led to the conclusion that (a) tryptophan hydroxylase expression is differentially regulated in different serotoninergic cell body subpopulations of the raphe, some of which are more sensitive to p-chlorophenylalanine, and (b) distribution of tryptophan hydroxylase protein is modified also in the somatodendritic area in all raphe nuclei.

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.

Ipsilateral alterations in tryptophan hydroxylase activity in rat brain after hypothalamic 5,7-di-hydroxytryptamine lesion.

The in vivo relationship between the amounts of tryptophan hydroxylase (TPH) protein and its intrinsic synthetic activity, measured by quantifying the amounts of alpha-[3H]methyl-5-hydroxytryptamine (alpha-[3H]M5-HT), is reported in cell body and terminal areas of intact and disturbed serotonergic neurons following a unilateral 5,7-dihydroxytryptamine (5,7-DHT) lesion of the dorsolateral hypothalamus. Five days after the lesion, the relationships between TPH and its synthetic product 5-HT were evaluated on adjacent brain sections in serotonergic cells bodies of the dorsal raphe nucleus (DRN) and nerve fibres of the medial forebrain bundle (MFB). On the side contralateral to the lesion, TPH and alpha-[3H]M5-HT levels in the intact hemi-DRN exhibited a caudo-rostral distribution and were positively and significantly correlated (p < or = 0.001); the calculated TPH-specific activity was 0.76 nCi of alpha-[3H]M5-HT formed per U TPH. In the MFB, quantitative measurements of TPH and alpha-[3H]M5-HT showed no correlation between enzyme and product and no specific activity for TPH could be determined. On the side ipsilateral to the lesion, the density of TPH-immunoreactive fibers was drastically decreased in the dorsolateral hypothalamus where a significant reduction in TPH content (45.5% of control side, P < 0.001) was found. In the overall ipsilateral hemi-DRN, TPH and alpha-[3H]M5-HT levels, their correlation as well as TPH-specific activity were unaltered by the lesion but a significant increase in alpha-[3H]M5-HT and TPH contents was observed in the lateral wings of the DRN. The lesion also induced a significant increase in alpha-[3H]M5-HT and TPH levels (136% and 93.8%, P < 0.001, respectively) in the ipsilateral MFB, which resulted in a positive and significant correlation between these two markers and yielded a TPH-specific activity of 1.0 nCi of alpha-[3H]M5-HT formed per U TPH. TPH topological area was also significantly increased in the lateral aspect of the ipsilateral MFB 5 days post lesion. These results show that 5-HT synthesis in the intact DRN is proportional to and dependent on TPH activity while in the MFB, 5-HT accumulation appears unrelated to TPH content which is most likely in an inactive enzymatic form. Moreover, the data show that a local disruption of serotonergic terminals in the dorsolateral hypothalamus does not affect 5-HT synthesis in the overall ipsilateral DRN neurons but results in local activation of TPH within the serotonergic projection neurons and the ipsilateral MFB, as evidenced by active de novo synthesis of 5-HT. Altogether the results point to circumscribed activation of compensatory mechanisms in 5-HT synthesis after selective destruction of serotonergic terminals.

Tyrosine hydroxylase expression within Balb/C and C57black/6 mouse locus coeruleus. I. Topological organization and phenotypic plasticity of the enzyme-containing cell population.

Tyrosine hydroxylase phenotype expression was investigated in the catecholaminergic population of the locus coeruleus neurons of two pure inbred mouse strains, Balb/C and C57Black/6. Therefore, we have characterized the precise organization of tyrosine hydroxylase-expressing perikarya population, in control animals and following RU24722 treatment, which is known to induce tyrosine hydroxylase expression. Serial coronal sections were selected along the caudo-rostral extent of the structure and were processed for tyrosine hydroxylase immunocytochemistry. Three days after the treatment, an increase in the number of cells which expressed tyrosine hydroxylase was observed all along the locus coeruleus in the Balb/C strain only. This increase equalized the catecholaminergic neuron populations of the two strains. In the caudal subdivision of the structure, these newly detected perikarya were intermingled with the perikarya which expressed tyrosine hydroxylase in control conditions. In the rostral half, the additional immunoreactive perikarya enlarged the mean coerulean space, defined as the area delimited by the tyrosine hydroxylase-containing perikarya. These results demonstrate a plasticity of the tyrosine hydroxylase phenotype expression, topologically organized and specific to the Balb/C strain.