M. V. Ugrumov

Non-dopaminergic neurons partly expressing dopaminergic phenotype: distribution in the brain, development and functional significance.

Besides the dopaminergic (DA-ergic) neurons possessing the whole set of enzymes of DA synthesis from l-tyrosine and the DA membrane transporter (DAT), the neurons partly expressing the DA-ergic phenotype have been first discovered two decades ago. Most of the neurons express individual enzymes of DA synthesis, tyrosine hydroxylase (TH) or aromatic l-amino acid decarboxylase (AADC) and lack the DAT. A list of the neurons partly expressing the DA-ergic phenotype is not restricted to so-called monoenzymatic neurons, e.g. it includes some neurons co-expressing both enzymes of DA synthesis but lacking the DAT. In contrast to true DA-ergic neurons, monoenzymatic neurons and bienzymatic non-dopaminergic neurons lack the vesicular monoamine transporter 2 (VMAT2) that raises a question about the mechanisms of storing and release of their final synthetic products. Monoenzymatic neurons are widely distributed all through the brain in adulthood being in some brain regions even more numerous than DA-ergic neurons. Individual enzymes of DA synthesis are expressed in these neurons continuously or transiently in norm or under certain physiological conditions. Monoenzymatic neurons, particularly those expressing TH, appear to be even more numerous and more widely distributed in the brain during ontogenesis than in adulthood. Most populations of monoenzymatic TH neurons decrease in number or even disappear by puberty. Functional significance of monoenzymatic neurons remained uncertain for a long time after their discovery. Nevertheless, it has been shown that most monoenzymatic TH neurons and AADC neurons are capable to produce l-3,4-dihydroxyphenylalanine (L-DOPA) from l-tyrosine and DA from L-DOPA, respectively. L-DOPA produced in monoenzymatic TH neurons is assumed to play a role of a neurotransmitter or neuromodulator acting on target neurons via catecholamine receptors. Moreover, according to our hypothesis L-DOPA released from monoenzymatic TH neurons is captured by monoenzymatic AADC neurons for DA synthesis. Such cooperative synthesis of DA is considered as a compensatory reaction under a failure of DA-ergic neurons, e.g. in neurodegenerative diseases like hyperprolactinemia and Parkinsons disease.Thus, a substantial number of the brain neurons express partly the DA-ergic phenotype, mostly individual complementary enzymes of DA synthesis, serving to produce DA in cooperation that is supposed to be a compensatory reaction under the failure of DA-ergic neurons.

Modeling of presymptomatic and symptomatic stages of parkinsonism in mice

A degradation of the nigrostriatal dopaminergic (DA-ergic) system is the key component of pathogenesis of Parkinsons disease (PD). Initial clinical symptoms appear 20-30 years after the onset of neurodegeneration, at a 70% DA depletion in the striatum and a 50% loss of nigral DA-ergic neurons. Low efficacy of the therapy might be improved if preclinical diagnostics and preventive therapy are developed. The development of appropriate experimental models should precede clinical trials. This multidisciplinary study first managed to model in mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) all together the following stages of parkinsonism: (a) the early presymptomatic stage manifested by a subthreshold degeneration of axons and DA depletion in the striatum without loss of nigral cell bodies; (b) the advanced presymptomatic stage manifested by a subthreshold degeneration of striatal axons and DA depletion and by a subthreshold loss of nigral cell bodies; (c) the advanced presymptomatic stage characterized by threshold depletion of striatal DA and a loss of DA-ergic axons and nigral cell bodies resulting in motor dysfunction. The degeneration of axons proceeds and prevails that of cell bodies suggesting higher sensitivity to MPTP of the former. Compensatory processes were developed in parallel to neurodegeneration that was manifested by the increase of the DA content in individual nigral cell bodies and DA turnover in the striatum. The developed models might be exploited for: (a) an examination of pathogenetic mechanisms not only in the nigrostriatal system but also in other brain regions and in the periphery; (b) a study of the compensatory mechanisms under DA deficiency; (c) a search of precursors of motor disorders and peripheral biomarkers in presymptomatic parkinsonism; (d) the development of preventive therapy aiming to slow down the neurodegeneration and strengthen compensatory processes. Thus, the models of the early and advanced presymptomaic stages and of the early symptomatic stage of parkinsonism were developed in mice with MPTP.

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.

Ontogenesis of tyrosine hydroxylase-immunopositive structures in the rat hypothalamus. An atlas of neuronal cell bodies

The development of the catecholaminergic system in the hypothalamus and in the septal region was studied in rats from the 12th fetal day until the 9th postnatal day. Catecholaminergic structures were visualized with pre-embedding immunocytochemistry using antiserum to tyrosine hydroxylase. An intensification of diaminobenzidine product with silver and gold was additionally applied to make the immunocytochemical technique more sensitive. In this paper only the data on the appearance and distribution of the tyrosine hydroxylase-immunopositive neurons (cell bodies) are presented, whereas the catecholaminergic innervation of the hypothalamus with the tyrosine hydroxylase-immunopositive fibers is the topic of an accompanying paper. Sparse tyrosine hydroxylase-immunopositive neurons were first observed in the anlage of the hypothalamus and septal region on the 13th fetal day. Their number increased progressively with age and by the 15th fetal day they already gave rise to a large dorsal accumulation. From the 18th fetal day on, tyrosine hydroxylase immunopositive neurons began to occupy their definitive positions, mainly concentrating within the hypothalamus: in the zona incerta, periventricular and arcuate nuclei. To a lesser extent, they were concentrated in the medial preoptic area, suprachiasmatic, supraoptic, paraventricular, dorsomedial, and anterior hypothalamic nuclei. The data on the distribution of the tyrosine hydroxylase-immunopositive neurons both in the hypothalamus and in the septal region during ontogenesis are summarized in the precise atlas. Primarily small bi- and unipolar catecholaminergic neurons first observed in the youngest fetuses undergo cytodifferentiation during ontogenesis, giving rise to at least two different populations localized ventrally, mainly in the arcuate nucleus, and dorsally, in the zona incerta. The neurons of the former population remain similar to those of the youngest fetuses, whereas the neurons of the latter increase significantly in size, forming several long, highly ramified processes.

Distribution of serotonin 5-hydroxytriptamine 1B (5-HT1B) receptors in the normal rat hypothalamus

This is the study first attempting to evaluate distribution of neurons expressing serotonin 5-hydroxytriptamine 1B (5-HT(1B)) receptors in hypothalamus by using immunocytochemistry. The 5-HT(1B)-immunoreactive neurons were widely distributed in hypothalamus. Accumulations of 5-HT(1B) neurons occurred in magnocellular nuclei, supraoptic nucleus, paraventricular nucleus (dorsolateral part) and accessory perifornical, circular and retrochiasmatic nuclei. Magnocellular neurons manifested an intense immunostaining suggesting a high level of 5-HT(1B) receptors. Large and middle-sized neurons with different 5-HT(1B) staining patterns were scattered throughout lateral hypothalamus, periventricular nucleus and lateral preoptic area. Immunofluorescent double-labeling revealed a great overlapping of the distribution 5-HT(1B) neurons and dense network of neuropeptide Y-immunoreactive fibers in paraventricular, supraoptic and arcuate nuclei. The potential functional significance of 5-HT(1B) receptors in the 5-HT control of endocrine functions and feeding are discussed.

Ontogenesis of the hypothalamic catecholaminergic system in rats: synthesis, uptake and release of catecholamines.

The development of the hypothalamic catecholaminergic system during ontogenesis in rats has been studied with glyoxylic acid histofluorescent method in vivo and with isotopic biochemical technique in vitro. It has been demonstrated that at the 15th fetal day the catecholaminergic system was functionally inactive at least in its ability for the uptake and K(+)-stimulated release of catecholamines. Since the 16th fetal day, hypothalamic neuronal elements gained an ability for synthesis of catecholamines, their specific uptake and K(+)-evoked release. Over the subsequent two days, the intensity of the fluorescent intraneuronal product rose considerably showing the increase of either synthesis or accumulation of catecholamines. Simultaneously, the values of the uptake and K(+)-stimulated release of the exogenous radioactively-labelled dopamine increased significantly. The intensity of the fluorescence of the hypothalamic neuronal elements dropped from 20th fetal until the ninth postnatal day, whereas the specific uptake doubled over the same period reaching its adult level. By the 21st postnatal day the reaccumulation of the fluorescent product occurred.

Dopamine synthesis by non-dopaminergic neurons expressing individual complementary enzymes of the dopamine synthetic pathway in the arcuate nucleus of fetal rats

This study was aimed to test our hypothesis about dopamine (DA) synthesis by non-DAergic neurons expressing individual complementary enzymes of the DA synthetic pathway in cooperation, i.e. L-dihydroxyphenylalanine (L-DOPA) synthesized in tyrosine hydroxylase (TH)-expressing neurons is transported to aromatic L-amino acid decarboxylase (AADC)-expressing neurons for conversion to DA. The mediobasal hypothalamus of rats at the 21st embryonic day was used as an experimental model because it contains mainly monoenzymatic TH neurons and AADC neurons (>99%) whereas the fraction of bienzymatic (DAergic) neurons does not exceed 1%. The fetal substantia nigra containing DAergic neurons served as a control. DA and L-DOPA were measured by high performance liquid chromatography in: (1) cell extracts of the cell suspension prepared ex tempora; (2) cell extracts and incubation medium after the static incubation of the cell suspension with, or without exogenous L-tyrosine; (3) effluents of the incubation medium during perifusion of the cell suspension in the presence, or the absence of L-tyrosine. Total amounts of DA and L-DOPA in the incubation medium and cell extracts after the static incubation were considered as the indexes of the rates of their syntheses. L-Tyrosine administration caused the increased L-DOPA synthesis in the mediobasal hypothalamus and substantia nigra. Moreover, L-tyrosine provoked an increase of DA synthesis in the substantia nigra and its decrease in the mediobasal hypothalamus. This contradiction is most probably explained by the L-tyrosine-induced competitive inhibition of the L-DOPA transport to the monoenzymatic AADC-neurons after its release from the monoenzymatic TH neurons. Thus, this study provides convincing evidence of cooperative DA synthesis by non-DAergic neurons expressing TH or AADC in fetal rats at the end of the intrauterine development.

Influence of serotonin on the development and migration of gonadotropin-releasing hormone neurones in rat foetuses.

This study used a pharmacological approach to evaluate the consequences of the metabolic perturbations of neurotransmitters on brain development. Pregnant rats received p‐chlorophenylalanine (pCPA), an inhibitor of serotonin (5‐hydroxytryptamine, 5‐HT) synthesis, or saline (control) from the 11th day of gestation once or daily up to the 15th, 17th and 20th day, followed by processing of the forebrain and/or nasal cranium of foetal males and females for high‐performance liquid chromatography of monoamines, radioimmunoassay of gonadotropin‐releasing hormone (GnRH) and quantitative and semiquantitative immunocytochemistry for GnRH. The pCPA treatment resulted in a 50–70% depletion of 5‐HT in the nasal crania and forebrains at any studied age. Radioimmunoassay showed no change in GnRH content in 5‐HT deficient foetuses at E16 compared to controls, being higher in both cases in the rostral forebrain than in the hypothalamus. In controls at E21, the GnRH content in the hypothalamus exceeded that in the rostral forebrain, whereas in the 5‐HT deficient group the opposite was found. These data suggest that 5‐HT provided a stimulating effect on GnRH neurone migration, and this was confirmed by quantification of GnRH‐immunoreactive neurones in the forebrain along the trajectory of their migration. At E18 and E21, the fractions of GnRH neurones in the rostral part of the trajectory in pCPA‐treated foetuses were greater than those in control foetuses but the opposite was true for the caudal part of the trajectory. Moreover, 5‐HT appeared to control the proliferation of the precursor cells of GnRH neurones and their differentiation, as derived from the observations of the increased number of GnRH neurones in the forebrain of foetuses of both sexes, as well as the region‐specific decreased neuronal size and content of GnRH in 5‐HT‐deficient females. Thus, 5‐HT appears to contribute to the regulation of the origin, differentiation and migration of GnRH neurones.

Decreased NPY innervation of the hypothalamic nuclei in rats with cancer anorexia

Whether the decrease in food intake that occurs at the onset of anorexia in tumor bearing (TB) rats is related to a change in the hypothalamic neuropeptide Y (NPY) system was tested by comparing NPY expression in sham operated Fischer Control and anorectic TB rats. Coronal cryocut sections of their fixed brain were processed by the peroxidase-antiperoxidase method with NPY polyclonal antibodies. NPY-immunoreactive fibers were widely distributed throughout the forebrain, but were most prominent in the hypothalamic paraventricular, suprachiasmatic, arcuate and periventricular nuclei. NPY-immunoreactive neurons were visualized in Control and anorectic TB rats in the preoptic region, the arcuate nucleus, and occasionally in the lateral hypothalamus. Semiquantitative image analysis showed a significant decrease in the NPY immunostaining in some hypothalamic nuclei of the anorectic TB rats, most prominently in the supraoptic nucleus, the parvocellular portion of the paraventricular nucleus, and, to a lesser extent, the suprachiasmatic and arcuate nuclei. No changes in NPY innervation were seen in the ventromedial nucleus and the lateral hypothalamus. The data support the hypothesis of an altered hypothalamic NPY system at the onset of anorexia in TB rats and also reveal the hypothalamic nuclei through which NPY influences food intake.

Ontogenesis of tyrosine hydroxylase-immunopositive structures in the rat hypothalamus. Fiber pathways and terminal fields.

The innervation of the hypothalamus and septal region by catecholaminergic fibers was studied in rats from the 12th fetal day until the 9th postnatal day. Catecholaminergic fibers were visualized with preembedding immunocytochemistry using antibodies to tyrosine hydroxylase. An intensification of diaminobenzidine product with silver and gold was additionally applied to increase the sensitivity and resolution power of the routine immunocytochemical technique. It has been demonstrated that, from the 13th fetal day, the hypothalamus and the septal region receive catecholaminergic fibers either belonging to the hypothalamic neurons or coming with the medial forebrain bundle from the outside of the hypothalamus. As the development of the hypothalamus proceeds, these fibers form the extensive networks within some neurosecretory centers either containing (the zona incerta, periventricular nucleus, etc.) or almost lacking (suprachiasmatic and paraventricular nuclei) the catecholaminergic neurons. In the former case, they terminate on the processes or perikarya of catecholaminergic neurons, while in the latter case their varicosities surround the immunonegative presumptive neurons in a basket-like manner. Moreover, from the 18th fetal day catecholaminergic fibers penetrate between the ependymal cells towards the 3rd ventricle and the primary capillary plexus of the hypophysial portal circulation, apparently providing the release of catecholamines to the cerebrospinal fluid and portal blood, respectively. The data obtained in this study are considered as the morphological basis for the involvement of the hypothalamic catecholamines in neuroendocrine regulations during ontogenesis.