Christoph Pilgrim

Prenatal development of mesencephalic and diencephalic dopaminergic systems in the male and female rat.

Previous results had suggested mesencephalic dopaminergic neurons in cultures of gestational day (GD) 14 rat embryonic brains to be characterized by an early maturation and acquisition of sex-related differences in transmitter uptake. Therefore development of dopaminergic systems was reexamined in the rat in vivo with special emphasis on the prenatal period, mesencephalo-hypothalamic relationships, and possible sex differences. Perfusion-fixed brains of GD 14, 17, 20, 21 and newborn rats were sectioned or processed as whole-mounts and immunostained for tyrosine hydroxylase (TH). Total numbers of mesencephalic TH-immunoreactive cell bodies as assessed by a stereological method rose between GD 14 and 17 and fell again between GD 17 and 21. As early as GD 14, a prominent mesencephalo-hypothalamic projection was observed coming off the medial forebrain bundle and terminating in the retrochiasmatic region. Two additional TH-immunoreactive fiber bundles leaving the medial forebrain bundle, one rostral and one caudal to the former, and terminating in the paraventricular and premammillary region, respectively, were noticed on GD 17. Careful examination of developing TH-immunoreactive neurons in the lower brainstem ascertained that there was no interference from ascending catecholaminergic fibers other than dopaminergic of mesencephalic origin during this early prenatal period. All 3 mesencephalo-hypothalamic projections had largely disappeared by GD 20 and were no longer detectable as distinct fiber bundles thereafter. There were subtle sex differences in numbers and distribution of both mesencephalic and diencephalic TH-immunoreactive neurons present at GD 17, which thus occurred prior to manifestation of other well-known sexual dimorphisms of the rat brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Sex steroids promote neurite growth in mesencephalic tyrosine hydroxylase immunoreactive neurons in vitro

The influence of steroid hormones on the differentiation of catecholaminergic and serotonergic (5‐HT) neurons was studied in dissociated cell cultures from embryonic day 14 (E14) rat diencephalon, mesencephalon and metencephalon treated for 6 days with 17β‐estradiol (E), testosterone (T), 5α‐dihydrotestosterone (DHT), progesterone (P), dexamethasone (DEX), or E + T. The effects of these hormones on morphologic differentation were determined by morphometric measurements of total length of neurites of immunocytochemically identified neurons in culture, which were stained with antisera against tyrosine hydroxylase (TH) or 5‐HT. A significant increase in neurite length was observed in cultures of TH‐immunoreactive (TH‐IR) neurons from the mesencephalon treated with E, T, E + T, but not with P, DHT or DEX. Based on labeling with [3H]dopamine (DA) uptake and competition with specific inhibitors, these mesencephalic TH‐IR cells appear to represent DA neurons of the A8–A10 groups (which includes the substantia nigra). No statistically significant effects of these steroids were observed on TH‐IR neurons from the diencephalon (assumed to be precursors of the tuberoinfundibular and incertohypothalamic dopaminergic groups). The 5‐HT neurons of the raphe nuclei (metencephalon) showed no statistically significant response to steroids. We conclude that during the early fetal period, sex steroids can affect the morphologic differentiation of mesencephalic DA neurons in vitro, indicating that these hormones are capable of selectively influencing the development of a specific population of monoamine neurons during this critical period.

Sexual differentiation of mesencephalic neurons in vitro: Effects of sex and gonadal hormones

In order to study the influence of gender on the development of transmitter uptake by dopaminergic neurons, dissociated cell cultures were raised separately from male and female midbrains of gestational day 14 rats. It was ascertained by use of specific inhibitors and by autoradiography that the uptake of [3H]dopamine was restricted to neurons immunoreactive for tyrosine hydroxylase and that these neurons have dopaminergic properties. The uptake capacity was higher in male than in female dopaminergic neurons by a factor of 1.5. This sexual dimorphism in dopamine uptake was present in cultures of tissue removed before the perinatal rise of testosterone occurs in males and was present even in the absence of hormonal additives to the culture medium. It therefore appears to be independent of the presence of gonadal steroids. It occurred likewise in cultures raised with serum‐supplemented and serum‐free medium, which may indicate that glia are not decisive in generating these differences. In addition, sexual differences were found regarding hormone responsiveness. Whereas testosterone and dihydrotestosterone were ineffective, estradiol was seen to promote dopamine uptake in female but not in male neurons. The results would suggest that mesostriatal and/or mesolimbic dopaminergic systems assume an early role in the development of some sexual dimorphisms of the brain.

Different developmental potentials of diencephalic and mesencephalic dopaminergic neurons in vitro

Morphological and functional differentiation of dopamine (DA) neurons was compared in dissociated cultures from gestational day 14 rat mesencephalon and diencephalon. Numbers of tyrosine hydroxylase-immunoreactive (TH-IR) neurons relative to all neurons were 4 and 1.7 times higher in mesencephalic than in diencephalic cultures at 6 and 13 days in vitro (DIV), respectively. Morphological maturation of diencephalic DA neurons was retarded in comparison to mesencephalic DA neurons. There were gross differences in soma size and length of processes between the two types of DA neurons, the appearance of which was strongly reminiscent of DA cell types described in vivo. Functional maturation of DA neurons was quantified by measuring uptake and Ca2+-dependent K+-stimulated release of [3H]DA per TH-IR neuron. As early as 6 DIV, DA uptake by mesencephalic DA neurons was saturable, was sensitive to benztropine and reserpine, and could be displaced by unlabeled DA. Twenty to 30% of the radioactivity accumulated could be released upon depolarization within a period of 5 min. At 6 DIV, influx of [3H]DA into diencephalic DA neurons was almost insensitive to benztropine, reserpine and unlabeled DA. Even after 13 DIV, diencephalic DA uptake was characterized by a markedly lower initial velocity, a longer time needed to reach saturation, a lower uptake capacity, and a lower sensitivity to benztropine than mesencephalic DA uptake. The releasable pool was very small and did not increase between DIV 6 and 13. The results demonstrate that mesencephalic DA neurons in vitro differentiate considerably faster than diencephalic DA neurons and gain functional competence very early in brain development. Comparison with data on adult nigrostriatal and hypothalamic DA systems suggests that the in vitro differences reflect a fundamental regional diversity of DA neurons.

The influence of genetic sex on sexual differentiation of diencephalic dopaminergic neurons in vitro and in vivo

Soma sizes of embryonic male and female tyrosine hydroxylase-immunoreactive neurons were measured in 3 diencephalic regions in situ and in diencephalic dissociated cell cultures. Male neurons were about 30% larger than female neurons both in vitro and in situ. Treatment of cultures with sex steroids did not affect the sex differences. It is concluded that sexual differentiation of dopaminergic neurons may be under primary genetic control.

The significance of the subcompartments of the marginal zone for directing lymphocyte traffic within the splenic pulp of the rat.

SummaryThese studies were designed to gain more detailed information on the sites of lymphocyte migration into the marginal zone, on lymphocyte segregation within this area and on subsequent migration of the cells in individual compartments of the rat spleen. A lymphocyte population enriched in T-lymphocytes was obtained from rat lymph nodes and was labeled with 5-(3H)uridine in vitro. Observations on localization of labeled lymphocytes at short time intervals following their intravenous transfusion into syngeneic recipients, indicate that the sites of emigration from the blood are the internal and external layers of the marginal zone. From here, the cells migrate towards the periarteriolar lymphoid sheath and the red pulp.

Macrophages and interdigitating cells; their relationship to migrating lymphocytes in the white pulp of rat spleen.

SummaryThe pathway of lymphocyte migration through the white pulp of rat spleen and the relationship of migrating cells to the accessory cells (marginal zone macrophages and interdigitating cells, IDCs) of the white pulp compartments were analysed. Donor lymphocytes were obtained from lymph nodes, enriched for T lymphocytes and labeled in vitro with 5-(3H)uridine. They were injected intravenously into syngeneic recipients from which samples of spleen were taken at short intervals from 3 to 300 min after injection. Autoradiographs of semithin and ultrathin sections showed that, in the internal layer of the marginal zone (MZ), lymphocytes tended to accumulate within some regions in close proximity to marginal-zone macrophages before migrating into the periarteriolar lymphoid sheath (PALS). The lymphocytes enter PALS between protrusions of the accessory cells located in the peripheral area of the sheath. During migration towards the central area of PALS, a close contact between both cell types was noted. In the central area of PALS, preferential accumulation of lymphocytes around IDCs was observed. Labeled lymphocyte distribution within PALS and the rate of cell migration through the white pulp seem to depend on lymphocyte-IDC contact. A common feature of accessory cells which may affect the migration of lymphocytes in both MZ and PALS is the presence of Birbeck granules.

Pathways of lymphocyte migration within the periarterial lymphoid sheath of rat spleen

SummaryMale Wistar rats were injected intravenously with 5-(3H)uridine-labeled lymphocytes isolated from lymph nodes of syngeneic donors and enriched in T cells. After short periods of time (3 to 120 min after injection), labeled lymphocytes were localized in spleen compartments using autoradiography to identify routes of lymphocyte movement from blood into splenic parenchyma and to follow migration pathways of recirculating lymphocytes within the periarterial lymphoid sheath (PALS). Topographical analysis of labeled lymphocytes was performed in specific planes of PALS characterized by the diameter of the arterial vessel and termed PALS large, PALS medium, and PALS small (PALS L, PALS M, PALS S, respectively). Attention was also paid to accumulations of labeled lymphocytes close to the arterial vessel wall. Initially, labeled lymphocytes were localized in PALS S and PALS M near the terminal branching of arterial vessels and in the marginal zone (MZ). We conclude that lymphocytes emigrate from blood into splenic parenchyma within two white pulp compartments: in MZ, and directly within PALS through the wall of capillary vessels. The sequential accumulation of labeled cells near arterial vessels of increasing diameter suggests that the recirculating pool of lymphocytes migrates into the central part of PALS L by two routes: from MZ, and along arterial vessels from PALS S and PALS M.

Angiotensin II promotes development of neurophysin neurons in dissociated culture

Dissociated cultures of retrochiasmatic hypothalamus of 18-day-old rat embryos were continuously treated with 2 X 10(-9) M angiotensin II (ang II) from the third day in vitro on. Cultivation was terminated at days 9 and 16 in vitro, respectively. Neurons immunoreactive for neurophysin, arg-vasopressin and oxytocin were visualized by immunocytochemistry, using the unlabeled antibody technique, and counted. Large, well-differentiated magnocellular neurons and small, probably immature cells could be distinguished. A certain number of large neurons were, in addition, immunoreactive for either vasopressin or oxytocin whereas the small cells were devoid of such staining. Ang II treatment brought about a modest increase in neurophysin-immunoreactive (NEU-IR) cell numbers at day 9 and a drastic augmentation of both large and small NEU-IR cells at day 16 in vitro, without appreciably affecting the total counts of neurons per culture.

GABAergic neurons in dissociated cultures of rat hypothalamus, septum, and midbrain

SummaryCultures of dissociated tissue blocks from 18 day-old rat embryos containing either septum plus anterior hypothalamus, retrochiasmatic hypothalamus, or midbrain, were maintained for 8–29 days in vitro. GABAergic neurons were visualized by incubation with (3H)γ-aminobutyric acid followed by autoradiography and by immunocytochemistry using an antiserum against glutamic acid decarboxylase. Positive neurons were of variable shapes and sizes. Labeled neurons in the three cultured regions represented 3.4, 1.1 and 1.7%, respectively, of all neurons. Apart from a few exceptions, autoradiographic labeling and immunocytochemical staining coincided. Glial cells did not stain and showed very low grain densities. Radioactivity could be released from preloaded cultures upon depolarization by 50 mM potassium and 100 μM Veratridine. The demonstration of GABAergic neurons in cultures that are known to contain a variety of peptidergic neurons opens up the possibility of studying the interaction between the two classes of neurons in vitro.