Neurons Expressing Tyrosine Hydroxylase or Aromatic L-Amino Acid Decarboxylase in the Mediobasal Hypothalamus of Wistar Rats in Ontogeny: Topographic Interrelations and Axonal Projections to the Medial Eminence

Topographic interrelations of the arcuate nucleus (AN) neurons expressing the dopamine-synthesized enzymes, tyrosine hydroxylase (TH) and/or aromatic L-amino acid decarboxylase (AAD), as well as projections of axons of these neurons to the medial eminence were studied in male rats at the 21st embryonal and 9th postnatal days as well as in adult animals. The method of double immunocytochemical labeling and its modification were used to reveal these enzymes. For identification of immunoreactive neurons, a confocal microscope was used. At all ontogenetic stages, three populations of neurons were found, which differed by composition of the dopamine-synthesizing enzymes as well as by the character of topographic interrelations of the TH-expressing monoenzyme neurons with the AAD-expressing neurons. In ontogeny, the topographic tight junctions are formed between the monoenzyme TH- and AAD expressing neurons at the level of both the cell body and the distal axons, which seems to increase effectiveness of the L-dihydroxyphenylalanine (L-DOPA) transfer from the TH- to the AAD-expressing neurons. The TH- and AAD expressing monoenzyme neurons project their axons to the medial prominence to provide entrance of the products of the specific syntheses into the pituitary portal circulating system. Thus, the morphological data obtained confirm indirectly our hypothesis about a cooperative participation of the TH- and AAD-expressing monoenzyme neurons of the AN in the dopamine synthesis.     

Optimization of dopaminergic neuron counting in substantia nigra of parkinsonian mice

Parkinson’s disease (PD) results from the degeneration of dopaminergic (DA-ergic) neurons of substantia nigra pars compacta (SNc). The disease is modeled in mice by the administration of a neurotoxin precursor 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Neurotoxin efficiency is estimated by reduced number of DA-ergic neurons in SNc. Cell counting on serial sections is very laborious and expensive and, therefore, is not widely used in spite of its high precision. The well-known Konigsmark’s formula (KF) allows one to perform counting on sections chosen with a certain interval rather than to utilize serial sections. However, its precision decreases with increasing interval and other parameters. In this paper, we described the mathematical method of approximation (MA) by improving KF. MA maintains counting precision and allows one to reduce the time and expenses for material processing and analysis.     

Involvement of Accessory Neurosecretory Nuclei of Hypothalamus in the Formation of Hypothalamohypohysial System during Prenatal and Postnatal Development in Rats

We studied the development of direct axonal connections of the accessory neurosecretory hypothalamic nuclei with the posterior pituitary lobe on the fixed rat brain from day 15 of embryogenesis until day 10 of postnatal development using the retrograde diffusion method of the lipophilic fluorescent carbocyanine dye 1,1"-dioctadecyl-3,3,3",3"-tetramethylindocarbocyanine perchlorate along the neuronal membranes. The marker was applied onto the posterior pituitary lobe and, after incubation in a fixative, fluorescing bodies of nerve cells were visualized in the hypothalamus. Neuronal axons of the retrochiasmatic nucleus were the first of the accessory nuclei to ingrow in the posterior pituitary lobe (on days 16–17 of embryogenesis). Neurons of the circular and dorsolateral nuclei and the nuclei of the median bundle of the forebrain sent their axons to the posterior pituitary lobe starting from the first days of postnatal development. No direct connections of the anterior commissure and perifornical accessory nuclei with the posterior pituitary lobe were found in perinatal development. These facts are discussed in the light of concepts about the different functional role of accessory peptidergic hypothalamic nuclei in rats.     

Interaction of human lymphocytes and viruses in vitro.

Interaction between phytohaemagglutinin (PHA) stimulated human lymphocytes and two DNA viruses (adenovirus type 5 and herpes simplex virus type 1) considered to be closely connected with lymphoid tissues has been studied. The fate of the same viruses was investigated also in non-stimulated separated lymphocytes for comparative purposes. To elicit this interaction infectivity titrations, immunofluorescent technique and electron microscopy were used. The production of viral antigens was investigated by complement fixation. It has been shown that in PHA-stimulated lymphocytes from peripheral blood of healthy donors adenovirus type 5 is capable to replicate in its infectious form. Prolongation of the interval between stimulation and infection of cells significantly influenced the dynamics of replication. Non-stimulated lymphocytes produced antigens but no infectious particles.     

Effect of serotonin on the formation of neurons producing gonadotropin-releasing hormone in Wistar rats

The effect of serotonin on the formation of neurons producing gonadotropin-releasing hormone (GnRH) during embryogenesis of Wistar rats was studied. The neurons producing GnRH were detected immunocytochemically on days 18 and 21 of embryonic development and on day 15 of postnatal development of rats with normal serotonin metabolism and rats in which the synthesis of serotonin was inhibited by p-chlorophenylalanine. The total number of GnRH neurons in serotonin deficiency was larger than in the case of its normal metabolism at all developmental stages studied. This is an indirect evidence for the inhibitory effect of serotonin on the formation of GnRH neurons. To confirm the morphogenetic effect of serotonin, we studied the rate of formation of GnRH neurons by injecting bromodeoxyuridine in the formation period of these neurons. It was found that serotonin deficiency had no effect on the time of formation of GnRH neurons: over 97% of neurons formed on days 11 to 15 of embryonic development both in the experimental and control groups. Note that, in serotonin deficiency, the maximum number of GnRH neurons formed one day later than in the normal state. Thus, serotonin inhibits the proliferation of GnRH neuron progenitor cells and thereby has a morphogenetic effect on the development of these neurons.     

The brain is one of the sources of L-dihydroxyphenylalanine in systemic circulation in fetuses and neonatal rats

The performed study was aimed at checking our hypothesis that the developing brain is a source of L-dihydroxyphenylalanine (L-DOPA), a precursor of dopamine in the total circulation system. At the initial stage, the L-DOPA concentration in peripheral blood was analyzed at the 18th and 21st embryonal days (E18 and E21), at the 3rd postnatal day (P3), and at the prepubertal period (P30). The highest L-DOPA concentration was revealed at the perinatal period, while decreased 4–12 times for the first month of life. The subsequent analysis of dynamics of the total blood L-DOPA content showed that maintenance of the constant L-DOPA concentration at the perinatal period on the background of a gradual increase of the blood serum volume is due to a rise of its secretion. At the postnatal period (P3–P30), the blood L-DOPA content increased twice in males, whereas it decreased to the same extent in females. Analysis of the L-DOPA concentration in two most important brain centers, hypothalamus and mesencephalon-rhombencephalon, showed its twofold decrease in hypothalamus during E18–E21 of development; then it slightly increased from E21 to P3 and fell 4–5 times by P30. In mesencephalon-rhombencephalon, the L-DOPA concentration was slightly reduced from E18 to E21 (only in females), while on P3 it returned to the E18 level and decreased 7–9 times by P30. The direct proof for the L-DOPA release from the developing brain into the systemic circulation follows from comparison of the blood L-DOPA concentration in shamoperated and encephalectomized rat fetuses after mechanical destruction of neurons of the two abovementioned most important dopaminergic centers. Thus, encephalectomy led to a twofold reduction of the blood L-DOPA concentration (statistically significant differences were observed only in females). Thus, the work presents evidence that the developing brain is one of L-DOPA sources in the total circulation system in rats during prenatal and early postnatal periods of ontogenesis.     

Development of central and peripheral serotonin-producing systems in rats in ontogenesis

The work deals with study of development of central and peripheral serotonin-producing systems in rat ontogenesis before and after formation of the blood-brain barrier. By the method of highly efficient liquid chromatography it has been shown that the serotonin level in peripheral blood before formation of the blood-brain barrier (in fetuses and neonatal rats) is sufficiently high for realization of physiological effect on target cells and organs. At the period of formation of the blood-brain barrier the serotonin level in brain sharply rises, whereas the serotonin concentration and amount in blood plasma and duodenum increase insignificantly. Completion of formation of the blood-brain barrier is accompanied by a significant increase of the serotonin content in duodenum, probably for maintenance of the high serotonin level in blood. To evaluate secretory activity, the mean rate of daily serotonin increment in the studied tissues was calculated. In brain, this parameter was maximal at the period of formation of the blood-brain barrier-from the 4th to the 16th postnatal days. This allows thinking hat brain before formation of the blood-brain barrier is the most important source of serotonin in peripheral blood.