Monique Krieger

Tyrosine hydroxylase‐expressing and/or aromatic L‐amino acid decarboxylase‐expressing neurons in the mediobasal hypothalamus of perinatal rats: Differentiation and sexual dimorphism

In this quantitative and semiquantitative immunocytochemical study, the authors evaluated the differentiation of neurons expressing tyrosine hydroxylase (TH) and/or aromatic L‐amino acid decarboxylase (AADC) in the mediobasal hypothalamus (MBH) of male and female rats on embryonic day 18 ( E18 ), E20, and postnatal day 9 ( P9 ). Four neuronal populations were distinguished according to either enzyme expression or neuron location. The earliest and most prominent first population was represented by TH‐immunoreactive (IR)/AADC‐immunonegative (IN) neurons that were detected initially at E18 and always were located in the ventrolateral region of the MBH. The second population of TH‐IN/AADC‐IR neurons was observed first at E20 and, after that time, was distributed dorsomedially. The third minor population of TH‐IR/AADC‐IR neurons initially was detected at E20 and was located dorsomedially. The fourth population was represented by TH‐IR/AADC‐IN neurons that were distributed in the dorsomedial region at any studied age. The numbers of TH‐IR and AADC‐IR neurons increased from their initial detection at E18 and E20 until P9. The area of TH‐IR and AADC‐IR neurons also increased from E18 to E20 and from E20 to P9, respectively. Both TH‐IR and AADC‐IR neurons showed sex differences in the neuron number, size, and optic density (OD). The numbers of TH‐IR neurons in males exceeded those of females at E20 and at P9, although, at P9, sexual dimorphism was a characteristic only of the ventrolateral population. The area and OD of TH‐IR neurons from females exceeded those from males in the entire mediobasal hypothalamus (MBH) at E18 and E20 but only in its dorsomedial region at P9. Sexual dimorphism also was an attribute of AADC‐IR neurons at E20 and P9. Their number, size, and OD were significantly higher in females than in males. Thus, the MBH of perinatal rats contained two major populations of TH‐IR/AADC‐IN or TH‐IN‐AADC‐IR neurons and a minor population of TH‐IR/AADC‐IR neurons. The differentiating neurons expressing either enzyme showed sexual dimorphism. J. Comp. Neurol. 425:167–176, 2000.

Differentiation of tyrosine hydroxylase-synthesizing and/or aromatic L-amino acid decarboxylase-synthesizing neurons in the rat mediobasal hypothalamus: Quantitative double-immunofluorescence study

In this double‐immunofluorescence study, we first quantified the neurons of the arcuate nucleus as immunoreactive (+) for tyrosine hydroxylase (TH) and/or aromatic L‐amino acid decarboxylase (AADC) in rats at embryonic day 21 (E21), at postnatal day 9 (P9), and in adulthood by using conventional fluorescent or confocal microscopy. On E21, monoenzymatic (TH+AADC immunonegative (−) and TH−AADC+) neurons and bienzymatic (TH+AADC+) neurons accounted for 99% and 1%, respectively, of the whole neuron population expressing enzymes of dopamine synthesis. Further development was characterized by the dramatic increase in TH+AADC− dorsomedial and TH+AADC+ dorsomedial populations from E21 to P9 as well as by the increase in the TH+AADC+ dorsomedial population (in females) and a drop in the TH+AADC− ventrolateral and TH+AADC− dorsomedial (in males) populations from P9 to adulthood. In contrast to TH+AADC− (in males) and TH+AADC+ neurons, the TH−AADC+ neurons did not change in number from E21 to adulthood. Thus, in rat fetuses, the neurons synthesizing TH and/or AADC were mainly monoenzymatic, whereas during postnatal life the fraction of bienzymatic neurons increased by up to 60%. J. Comp. Neurol. 446:114–122, 2002.

Degeneration of dopaminergic neurons triggers an expression of individual enzymes of dopamine synthesis in non-dopaminergic neurons of the arcuate nucleus in adult rats.

Non-dopaminergic neurons expressing individual complementary enzymes dopamine (DA) synthesis were shown to produce DA in cooperation [Ugrumov, M., Melnikova, V., Ershov, P., Balan, I., Calas A., 2002. Tyrosine hydroxylase- and/or aromatic L-amino acid decarboxylase-expressing neurons in the rat arcuate nucleus: ontogenesis and functional significance. Psychoneuroendocrinology 27, 533-548; Ugrumov, M.V., Melnikova, V.I., Lavrentyeva, A.V., Kudrin, V.S., Rayevsky, K.S., 2004. Dopamine synthesis by non-dopaminergic neurons expressing individual complementary enzymes of the dopamine synthetic pathway in the arcuate nucleus of fetal rats. Neuroscience 124, 629-635]. This study was aimed at testing our hypothesis that the cooperative synthesis of DA in non-dopaminergic neurons is an adaptive reaction under functional insufficiency of the dopaminergic system. Functional insufficiency of the tuberoinfundibular dopaminergic system was provoked by 6-OHDA-induced degeneration of dopaminergic neurons in the arcuate nucleus in adult rats. Bienzymatic (dopaminergic) neurons and monoenzymatic neurons expressing tyrosine hydroxylase (TH) or aromatic L-amino acid decarboxylase (AADC) were detected with a double-immunofluorescent technique on cryostat sections. The 6-OHDA-induced degeneration of dopaminergic neurons was accompanied by a significant increase of the number of monoenzymatic TH neurons and AADC neurons that appears to support our hypothesis. The reaction of bienzymatic and monoenzymatic neuron populations to the 6-OHDA administration occurred to be region-specific. The former disappeared in the dorsomedial region of the arcuate nucleus while the latter increased in the ventrolateral region. Thus, degeneration of dopaminergic neurons in the arcuate nucleus of adult rats is accompanied by the expression of individual enzymes of DA synthesis in non-dopaminergic neurons that may be an adaptive reaction.

Neurons possessing enzymes of dopamine synthesis in the mediobasal hypothalamus of rats: Topographic relations and axonal projections to the median eminence in ontogenesis

We evaluated the topographic relations between tyrosine hydroxylase (TH)- and/or aromatic L-amino acid decarboxylase (AADC)-immunoreactive neurons in the arcuate nucleus (AN), as well as between TH- and/or AADC-immunoreactive axons in the median eminence (ME) in rats at the 21st embryonic day, 9th postnatal day, and in adulthood. The double-immunofluorescent technique in combination with confocal microscopy was used. Occasional bienzymatic neurons but numerous monoenzymatic TH- or AADC-immunoreactive neurons were observed in fetuses. There was almost no overlap in the distribution of monoenzymatic neurons, and therefore few appositions were observed in between. In postnatal animals, numerous bienzymatic neurons appeared in addition to monoenzymatic neurons. They were distributed throughout the AN resulting in the increased frequency of appositions. Furthermore, specialized-like contacts between monoenzymatic TH- and AADC-immunoreactive neurons appeared. The quantification of the fibers in the ME showed that there were large specific areas of the monoenzymatic TH-immunoreactive fibers and bienzymatic fibers in fetuses, followed by the gradual reduction of the former and the increase of the latter to adulthood. The specific area of the monoenzymatic AADC-immunoreactive fibers in fetuses was rather low, and thereafter increased progressively to adulthood. The fibers of all the types were in apposition in the ME at each studied age. Close topographic relations between the neurons containing individual complementary enzymes of dopamine synthesis at the level of cell bodies and axons suggest functional interaction in between.

Preparation of an antiserum using a fusion protein produced by a cDNA for rat aromaticl-amino acid decar☐ylase

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.

A confocal approach to the morphofunctional characterization of the transient tyrosine hydroxylase system in the rat suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) of the neonatal rat is transiently innervated by tyrosine hydroxylase (TH) fibers of unknown origin and whose catecholaminergic nature is rather doubtful. In order to characterize this system morphofunctionally, immunocytochemical double labelling and confocal laser scanning microscopy analysis were employed on cryostat brain sections of 10-day-old rats. Simultaneous stainings for neuropeptide Y (NPY) and tyrosine hydroxylase (TH) immunoreactivity showed that they are not colocalized, neither in the SCN fibers nor in the intergeniculate leaflet (IGL) neurons, site of origin of the NPY projection to the SCN. Therefore, the possibility that SCN transient TH fiber system originates from the IGL could be excluded. Double labelling for TH and aromatic L-aminoacid decarboxylase (AADC) demonstrated that transient SCN TH immunoreactive (IR) fibers are AADC negative, thus supporting the hypothesis of their non-catecholaminergic nature. Moreover two new group of cells which are TH positive and AADC negative were found: one in the SCN and the other in the periventricular hypothalamic nucleus (PHN). The presence of somatostatin (SRIF) and TH in PHN neurons and SCN fibers suggested their possible colocalization, but double immunolabellings gave negative results. Simultaneous immunocytochemical staining for vasoactive intestinal polypeptide (VIP) and TH showed that TH fibers may interact with ventrolateral SCN VIP neurons. This result suggests a possible involvement of TH fibers in regulating VIP cells activity in the entrainment of circadian rhythms.

Progesterone receptor immunoreactivity in aromatic L‐amino acid decarboxylase‐containing neurons of the guinea pig hypothalamus and preoptic area

A double‐labeling immunofluorescence procedure was used to determine whether progesterone receptor (PR)‐immunoreactive (IR) neurons in the preoptic area and hypothalamus of female guinea pigs also contained aromatic L‐amino acid decarboxylase (AADC), an enzyme involved in the synthesis of both catecholamines and serotonin. Immunostaining was performed on cryostat sections prepared from ovariectomized guinea pigs primed by estradiol to induce PR. The nuclear presence of PR was visualized by a red fluorescence while the AADC‐containing perikarya showed a yellow‐green fluorescence. The topographic distribution of AADC‐IR neurons was investigated by using a specific antiserum obtained by immunization of rabbits with a recombinant protein β‐galactosidase‐AADC in the two regions known to contain the densest populations of estradiol‐induced PR‐IR cells: the preoptic area and the mediobasal hypothalamus. The localization of PR‐IR and AADC‐IR cell populations showed considerable overlap in these areas, mainly in the medial and periventricular preoptic nuclei and in the arcuate nucleus. A quantitative analysis of double‐labeled cells estimated that about 15% to 23% of AADC‐IR cells in the preoptic area and about 11% to 21% of AADC‐IR cells in the arcuate nucleus possessed PR. This colocalization persisted throughout the rostrocaudal extent of these areas and represented 3% to 9% of the population of PR‐IR cells.

Aromatic amino acid decarboxylase (AADC) immunohistochemistry in vertebrate brainstem with an antiserum raised against AADC made in E. coli.

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.

Structure modelling and site-directed mutagenesis of the rat aromatic L-amino acid pyridoxal 5´-phosphate-dependent decarboxylase: A functional study

The pyridoxal‐5´‐phosphate‐dependent enzymes (B6 enzymes) are grouped into three main families named α, β, and γ. Proteins in the α and γ families share the same fold and might be distantly related, while those in the β family exhibit specific structural features. The rat aromatic L‐amino acid decarboxylase (AADC; EC(4.1.1.28)) catalyzes the synthesis of two important neurotransmitters: dopamine and serotonin. It binds the cofactor pyridoxal‐5`‐phosphate and belongs to the α family. Despite the low level of sequence identity (approximately 10%) shared by the rat AADC and the sequences of the enzymes belonging to the B6 enzymes family, including the known three‐dimensional structures, a multiple sequence alignment was deduced. A model was built using segments belonging to seven of the eleven known structures. By homology, and based on knowledge of the biochemistry of the aspartate aminotransferase, structurally and functionally important residues were identified in the rat AADC. Site‐directed mutagenesis of the conserved residues D271, T246, and C311 was carried out in order to confirm our predictions and highlight their functional role. Mutation of D271A and D271N resulted in complete loss of enzyme activity, while the D271E mutant exhibited 2% of the wild‐type activity. Substitution of T246A resulted in 5% of the wild‐type activity while the C311A mutant conserved 42% of the wild‐type activity. A functional model of the AADC is discussed in view of the structural model and the complementary mutagenesis and labelling studies. Proteins 1999;37:191–203. ©1999 Wiley‐Liss, Inc.

Aromatic l-amino acid decar☐ylase immunohistochemistry in the suprachiasmatic nucleus of the sheep Comparison with tyrosine hydroxylase immunohistochemistry

Using antisera against tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC), we have demonstrated the presence of numerous AADC immunoreactive neurons and a few TH immunoreactive neurons, homogeneously distributed throughout the suprachiasmatic nucleus. Similar results have been described in other species. These observations show that this nucleus is able to synthesize trace amines (such as phenylethylamine or tyramine) in addition to dopamine. It is hypothesized that these trace amines are possibly involved in the integration of day length variation in sheep, a species whose reproduction is closely related to photoperiod.