Biomedical subjects
Publications and source records attributed to J Thibault.
In the postero-dorso-lateral neostriatum (PDLNS) of the pigeon we found a high density of tyrosine hydroxylase and perineuronal nests of fibres labelled with anti-dopamine antibody. These data confirm our earlier indirect evidence that PDLNS receives a dense dopaminergic innervation and therefore can be compared with the mammalian prefrontal cortex.
The present study has attempted to determine the birthdates of the tyrosine hydroxylase (TH)-immunopositive neurons in the arcuate nucleus (AN) of rats, thus evaluating the time of the last mitotic divisions of their cell precursors. For this aim, 'long-survival' [3H]thymidine autoradiography was used in combination with immunocytochemistry of TH, the first enzyme of catecholamine synthesis. According to our data, some TH-immunopositive neurons in the AN were born as early as the 13th fetal day. At day 14, the number of double-labeled neurons almost tripled while reaching a maximum at day 15 of the intrauterine development. The fraction of the double-labeled neurons dropped on the following day, and practically disappeared by fetal day 17, showing cessation of the production of the TH-immunopositive neurons. The birth of the TH-immunopositive neurons is considered as the onset of the development of the tuberoinfundibular dopaminergic system.
Aromatic L-amino acid decarboxylase (AADC) decarboxylates L-DOPA and 5-hydroxytryptophan into dopamine and serotonin, respectively. Starting from a rat AADC cDNA clone isolated in our laboratory, we produced a beta-galactosidase-AADC fusion protein in E. coli. It was purified from inclusion bodies and injected into a rabbit. The antiserum identified AADC on a Western blot of extracts from rat organs as a unique 50 kDa band; it also strongly reacted by immunohistochemistry with dopaminergic and serotonergic neurons. This new beta-galactosidase-AADC fusion protein then constitutes a useful tool for producing AADC as an antigen free of contamination by mammalian proteins.
The preoptic area contains most of the luteinizing hormone releasing hormone immunoreactive neurons and numerous monoaminergic afferents whose cell origins are unknown in sheep. Using tract tracing methods with a specific retrograde fluorescent tracer, fluorogold, we examined the cells of origin of afferents to the medial preoptic area in sheep. Among the retrogradely labeled neurons, immunohistochemistry for tyrosine hydroxylase, dopamine-beta-hydroxylase, phenylethanolamine N-methyltransferase, and serotonin was used to characterize catecholamine and serotonin fluorogold labeled neurons. Most of the afferents came from the ipsilateral side to the injection site. It was observed that the medial preoptic area received major inputs from the diagonal band of Broca, the lateral septum, the thalamic paraventricular nucleus, the lateral hypothalamus, the area dorsolateral to the third ventricle, the perimamillary area, the amygdala, and the ventral part of the hippocampus. Other numerous, scattered, retrogradely labeled neurons were observed in the ventral part of the preoptic area, the vascular organ of the lamina terminalis, the ventromedial part of the hypothalamus, the periventricular area, the area lateral to the interpeduncular nucleus, and the dorsal vagal complex. Noradrenergic afferents came from the complex of the locus coeruleus (A6/A7 groups) and from the ventro-lateral medulla (group A1). However, dopaminergic and adrenergic neuronal groups retrogradely labeled with fluorogold were not observed. Serotoninergic fluorogold labeled neurons belonged to the medial raphe nucleus (B8, B5) and to the serotoninergic group situated lateral to the interpeduncular nucleus (S4). In the light of these anatomical data we hypothesize that these afferents have a role in the regulation of several functions of the preoptic area, particularly those related to reproduction. Accordingly these afferents could be involved in the control of luteinizing hormone releasing hormone (LHRH) pulsatility or of preovulatory LHRH surge.
By use of rat cDNA probes and a panel of cell hybrids segregating rat chromosomes, the genes encoding three pyridoxal 5'-phosphate (PLP)-dependent decarboxylases--namely, DOPA-decarboxylase (Ddc), glutamic acid decarboxylase 1 and 2 (Gad1 and Gad2)--were assigned to rat Chromosomes (Chrs) 14, 3, and 17, respectively. If one takes into account chromosome localizations in the human and the mouse, the present results (i) show that a synteny group is retained on rat Chr 14, human Chr 7, and mouse Chr 11 (Ddc); (ii) strengthen the homology relation known between rat Chr 3 and human and mouse Chrs 2 (Gad1); (iii) suggest that rat Chr 17 has no extensive homology to any human chromosome; and (iv) suggest the order (Prl, Fdp)--Tpl2--Gad2 on the rat Chr 17.
Aromatic L-amino acid decarboxylase (AADC) is involved in the biosynthesis of catecholamines and indolamines. AADC is present in the nervous system, in the chromaffin cells, and in non-neuronal tissues. We tested the capacity of a new polyclonal antibody, obtained by immunization of rabbits with a recombinant protein beta-galactosidase-AADC, to detect monoaminergic neurons in the brainstem as well as monoaminergic paraneurons in the adrenal medulla from goldfish, frog, skink, quail, and mouse. In the adrenal gland we found an immunoreactivity that was consistent with the distributions of the chromaffin cells previously reported. In the brainstem, groups of immunoreactive neurons and several labelled fibers were observed in the five species studied. The raphe region showed cell bodies and processes similar to those previously identified as monoaminergic by other authors. In addition, in medulla oblongata and isthmic tegmentum we found, in goldfish, skink, and quail, neuronal groups similar to mammalian D groups which contain AADC but are devoided of serotonin and catecholamines.
As an indirect way to establish whether catecholamines are actually elaborated in the islets of Langerhans of adult albino and pigmented mice and guinea pigs, or albino rats, the presence of tyrosine-hydroxylase was immunohistochemically investigated using a highly specific polyclonal serum against the enzyme. The antiserum was applied to paraffin sections of pancreases fixed either in acrolein or Bouin's fluid. Such sections were then treated with the avidin-biotin method. Tyrosine-hydroxylase was found exclusively in the beta cells of rats and in those of the two strains of mice. Results in the guinea pig varied according to whether the animals were pigmented or albino. In pigmented specimens, the enzyme was detected in beta and non-beta cells, whereas in albino animals it was undetectable in any of the islet cells. All these observations were performed in material fixed in acrolein. Bouin's fluid resulted a rather poor fixative for the detection of the enzyme in the islets. The results are compared with those of other authors who investigated the presence of monoamines in the islets of Langerhans. Since tyrosine-hydroxylase is a specific marker for catecholamine-synthesizing cells, it is concluded that insular cells have the ability to elaborate these substances.
This work describes the partial purification of a heat-stable peptide which has the same properties as the scorpion toxin, scyllatoxin, a specific blocker of one class of Ca(2+)-activated K+ channels: (i) it competes with [125I]apamin for binding to the same site, (ii) like apamin and scyllatoxin, it blocks the after-potential hyperpolarization in skeletal muscle cells in culture, (iii) like apamin and scyllatoxin, it contracts guinea-pig taenia coli relaxed by epinephrine, (iv) it cross-reacts with antibodies raised against scyllatoxin but not with antibodies raised against apamin.
In sheep, the arcuate nucleus contains numerous tyrosine hydroxylase (TH) and estradiol receptor (rE2) immunoreactive (IR) perikarya and it has been shown previously in this species that catecholaminergic neurons can mediate the gonadal steroid action on the reproductive function. In the present study, double immunohistochemical labelling with antibodies against TH and rE2 have been used to demonstrate the presence of rE2 in TH-IR neurons in the arcuate nucleus where the distribution of TH-IR and rE2-IR neurons overlap each other. Only less than 10% of all the rE2-IR perikarya presented TH immunoreactivity. It was therefore hypothesized that either such a low number of double labelled neurons can support the effects of estradiol in this area or that the effect of this steroid was indirect. In the latter case it might be first mediated by beta-endorphin neurons which have been previously described in this nucleus.
A human DOPA decarboxylase (DDC) cDNA probe of 747 base pairs has been used to map the DDC gene by in situ hybridization on mouse metaphase chromosomes. This result indicates that the gene is located on band 11A, near the erythroblastosis oncogene B (erb b) locus. This provides evidence for a synteny group on mouse chromosome 11 and human chromosome 7.
Tyrosine hydroxylase (TH) was purified from tumours of rat phaeochromocytoma (PC12) cells by a three-step purification procedure giving 30 mg of pure enzyme in 3 days. The enzyme sedimented with an S(eo),w value of 9.2 S and revealed an apparent subunit molecular mass of 62 kDa with a minor 60 kDa component. Two-dimensional gel isoelectric focusing/electrophoresis and tryptic digestion revealed that the heterogeneity could be accounted for by limited proteolysis of the 62 kDa component and the presence of covalently bound phosphate. The enzyme had a strong blue-green colour (epsilon 700 = 3.1 +/- 0.2 mM-iron-1.cm-1). The resonance Raman spectrum obtained with lambda excitation = 605 nm revealed the presence of an Fe(III)-catecholamine complex in the isolate enzyme, similar to that observed in the bovine adrenal enzyme [Andersson, Cox, Que, Flatmark & Haavik (1988) J. Biol. Chem. 263, 18621-18626]. In the rat PC12 enzyme, all of the iron present (0.53 +/- 0.03 atom per subunit) seems to be chelated by the feedback inhibitors (0.49 +/- 0.05 mol of dopamine and 0.10 +/- 0.03 mol of noradrenaline per mol of subunit). The e.p.r. spectra at 3.6 K show g-values at 7.0, 5.2 and 1.9 as observed for other catecholate-complexed enzymes. After phosphorylation of serine-40 and addition of L-tyrosine a new rhombic (magnitude of E/D = 0.33) e.p.r. species could be observed. Phosphorylation of serine-40 by cyclic AMP-dependent protein kinase increased the catalytic activity; depending on assay conditions, up to 80-110-fold activation could be observed when measured at high TH (i.e. high endogenous catecholamine) concentration.
The formation of histamine from its precursor histidine is catalyzed by histidine decarboxylase (HDC), a pyridoxal phosphate (PLP)-dependent decarboxylase. The knowledge of sequence similarities between various rodent HDCs permitted us to prepare a rat brain HDC cDNA probe. After reverse transcription of rat brain polyA + mRNA, the HDC cDNA obtained was amplified by the polymerase chain reaction using two specific primers. The resulting 1019-bp DNA was cloned in the p-MAL vector. Its sequence corresponds to the published data on rat fetal liver HDC. This 1019-bp rat probe detected two BamHI sequences in man; these were assigned to chromosome 15 by somatic hybrid cell analysis. According to the well-known homology between human chromosome 15 and mouse chromosome 2, the result obtained is in agreement with the published localization of HDC on mouse chromosome 2. The mapping of the human HDC gene on chromosome 15 is an original contribution to the chromosomal assignment of related PLP-dependent decarboxylases.
Mapping of monoaminergic systems in the brain of the newt Triturus alpestris was achieved with antisera against (1) thyrosine hydroxylase (TH), (2) formaldehyde-conjugated dopamine (DA), and (3) formaldehyde-conjugated serotonin (5-HT). In the telencephalon, the striatum was densely innervated by a large number of 5-HT-, DA- and TH-immunoreactive (IR) fibers; IR fibers were more scattered in the amygdala, the medial and lateral forebrain bundles, and the anterior commissure. In the anterior and medial diencephalon, TH-IR perikarya contacting the cerebrospinal fluid (CSF-C perikarya) were located in the preoptic recess organ (PRO), the organum vasculosum laminae terminalis and the suprachiasmatic nucleus. Numerous TH-IR perikarya, not contacting the CSF, were present in the posterior preoptic nucleus and the ventral thalamus. At this level, DA-IR CSF-C neurons were only located in the PRO. In the posterior diencephalon, large populations of 5-HT-IR and DA-IR CSF-C perikarya were found in the paraventricular organ (PVO) and the nucleus infundibularis dorsalis (NID); the dorsal part of the NID additionally presented TH-IR CSF-C perikarya. Most regions of the diencephalon showed an intense monoaminergic innervation. In addition, numerous TH-IR, DA-IR and 5-HT-IR fibers, originating from the anterior and posterior hypothalamic nuclei, extended ventrally and reached the median eminence and the pars intermedia of the pituitary gland. In the midbrain, TH-IR perikarya were located dorsally in the pretectal area. Ventrally, a large group of TH-IR cell bodies and some weakly stained DA-IR and 5-HT-IR neurons were observed in the posterior tuberculum. No dopaminergic system equivalent to the substantia nigra was revealed. The possible significance of the differences in the distribution of TH-IR and DA-IR neurons is discussed, with special reference to the CSF-C neurons.
Previous studies, aimed at identifying which diffusible signals may influence the differentiation of embryonic neurons towards the monoaminergic phenotypes during brain development, have shown that serotonin itself could promote the 'serotoninergic-like properties' of hypothalamic cells from mouse embryos. We presently reinvestigated such 'autocrine/paracrine' regulatory mechanisms by exposing dissociated cell cultures from embryonic rat hypothalamus and brain stem to dopamine--or related agonists--in an attempt to influence their differentiation towards the catecholaminergic phenotype. Chronic treatment of cells by dopamine or apomorphine (a mixed D1/D2 agonist), but not selective D1 and D2 agonists, significantly increased the number of cells that expressed tyrosine hydroxylase (TH: as assessed with a specific anti-TH antiserum) and the activity of aromatic L-amino acid decarboxylase (AADC) in the cultures. Furthermore, apomorphine treatment also decreased the levels of cholecystokinin-like material in primary cultures from the brainstem (but not the hypothalamus) where both dopamine and cholecystokinin are--partly--colocalized in mesencephalic dopaminergic neurons. The maximal effects of both dopamine and apomorphine on TH expression and AADC activity occurred earlier in the brainstem (on cells from 14- to 15-day-old embryos) than in the hypothalamus (on cells from 15- to 16-day-old embryos), in line with the well established caudo-rostral maturation of the rat brain. Furthermore both the expression and the dopamine-induced modulation of AADC activity and TH immunoreactivity appeared to occur independently of each other. Present and previous data are in agreement with a possible autocrine/paracrine action of dopamine and serotonin during brain development.(ABSTRACT TRUNCATED AT 250 WORDS)
A cDNA clone for dopa decarboxylase (EC 4.1.1.28) has been isolated from a rat pheochromocytoma cDNA library and the cDNA sequence has been determined. It corresponds to an mRNA of 2094 nucleotides. The length of the mRNA was measured by primer-extension of rat pheochromocytoma RNA and the 5' end of the sequence of the mRNA was confirmed by the PCR. A probe spanning the translation initiation site of the mRNA was used to hybridize with mRNAs from various organs of the rat. S1 nuclease digestion of the mRNAs annealed with this probe revealed two classes of mRNAs. The comparison of the cDNA sequence and published sequences for rat liver, human pheochromocytoma, and Drosophila dopa decarboxylase supported the conclusion that two mRNAs are produced: one is specific for tissue of neuronal origin and the other is specific for tissues of nonneuronal (mesodermal or endodermal) origin. The neuronal mRNA contains a 5' untranslated sequence that is highly conserved between human and rat pheochromocytoma including a GA stretch. The coding sequence and the 3' untranslated sequence of mRNAs from rat liver and pheochromocytoma are identical. The rat mRNA differs only in the 5' untranslated region. Thus a unique gene codes for dopa decarboxylase and this gene gives rise to at least two transcripts presumably in response to different signals during development.
Major technical progress in the development of computer-based image analysis has made possible the entry of autoradiography and immunohistochemistry into a new era where quantification by densitometry has become easily accessible. Autoradiography could become quantitative and displayed adequate reproducibility with the help of emulsion-coated films and the use of scales of standards of known radioactivity exposed and analyzed in parallel to the tissue sections. Immunohistochemistry after revelation by a color-based enzymatic technique can also become quantitative, providing that standardization of the crucial steps of the procedure and calibration through a parallel treatment of a scale of antigen standards can be ensured. Such an approach is described here in the rat with reference to tyrosine hydroxylase (TH), the main synthesizing enzyme for catecholamines, and with dopamine (DA) itself, a catecholaminergic neurotransmitter. The different parts of the procedure, which can influence the results, such as the fixation of the animals by perfusion and the evaluation of the fluctuations via the calibration curve, are discussed in detail. Biological validation of the proposed procedure is described by reference to experiments already well documented biochemically, such as the induction effect of reserpine on TH in the rat locus coeruleus and the depleting effect of alpha-methyltyrosine (AMPT), a well-known blocker of TH activity, on rat striatal DA content. Finally the importance of restricting the measurements to the (pseudo)linear portion of the calibration curve is illustrated by the autoradiographic identification of the differential intrastriatal repartition of the dopaminergic D1 and D2 receptor sites, particularly the dual patch-matrix compartments.
Levels of tyrosine hydroxylase (TH) were quantified in discrete areas of unfixed rat brain tissue sections using a rapid and sensitive radioimmunohistochemical method. The immunological reaction with the TH monoclonal antibody was revealed by a 35S-labelled secondary antibody and thus permitted autoradiographic detection of the enzyme. Autoradiograms were generated by apposition of tissue sections to high-sensitivity films or by dipping into autoradiographic emulsion. A detailed analysis of antibody concentration, incubation time, tissue section thickness, and exposure time of the film was undertaken to determine optimal conditions to produce a linear radiolabelling intensity with respect to the amount of antigen. Quantification of the antigen at regional levels was assessed by computer-assisted image analysis. Autoradiographic optical density of radiolabelling in brain areas was converted to enzyme concentrations by interpolation with a constructed TH calibration curve processed in parallel with tissue sections. The specificity of the labelling and the validity and reproducibility of the quantification were investigated. The distribution of TH radiolabelling was comparable to that described using immunofluorescence histochemistry or measuring TH enzymatic activity on homogenates. Using a 35S-labelled antibody, the detection of TH could be performed at the cellular level.
The L-DOPA decarboxylase mRNA levels were determined by a sensitive S1 nuclease method in four organs and one tumor of adult rat. S1 mapping analysis, with probes corresponding to the mRNA coding region, showed that this region is conserved in all L-DOPA decarboxylase mRNA of neuronal and non-neuronal tissues. The mRNA was not very abundant; its representation varies approximately from 0.00035% of total RNA in the mid brain to 0.013% of total mRNA in the pheochromocytoma. A strong correlation between mRNA level and enzyme amount was observed (correlation coefficient = 0.99). The results indicate that the level of mRNA is a primary factor determining the L-DOPA decarboxylase level.