Sexual differentiation of monoaminergic neurons--genetic or epigenetic?

It is currently believed that sexual differentiation of the brain is mediated entirely by the epigenetic action of gonadal steroids during a critical period of development. Ingrid Reisert and Christoph Pilgrim review sexual dimorphisms of monoaminergic systems, which also appear to be generated by sex steroids. However, there are a number of observations that are not explainable by the 'androgen theory of sexual differentiation'. Results obtained from cultures of embryonic rat brain tissue appear to indicate that dopaminergic neurons may develop morphological and functional sex differences in the absence of sex steroids. Hormone-independent and -dependent developmental processes may affect diencephalic and mesencephalic dopaminergic neurons in a regionally diverse fashion. Factors other than sex steroids need to be examined. It is possible that some sexual dimorphisms in the nervous system may develop under primary genetic control.

Protein kinase C isoenzyme: selective expression pattern of protein kinase C-θ during mouse development.

Protein kinase C (PKC)-θ, a serine/threonine protein kinase and novel PKC subfamily member, has been recently identified as an essential component of the T cell synapse which activates the NF-kB signaling cascade leading to expression of the IL-2 gene during T cell activation. By RNA in situ hybridization to whole-body embryo sections it is shown that the murine PKCθ is specifically expressed in tissues with hematopoietic and lymphopoietic activity. Expression is also evident in skeletal muscle. A further highly specific expression was observed in the peripheral and central nervous system which is described in detail. Expression in the brain persists up to adult stages.

Volume densities and specific surfaces of neuronal and glial tissue elements in the rat supraoptic nucleus.

The present study was undertaken to provide an explanation for previous autoradiographic results suggesting a several times higher rate of synthesis of glycoconjugates per unit volume of hypothalamic glia than of neurons. Volume densities, specific surfaces (surface-volume ratios), and relative surfaces (contribution of the surface of a tissue element to the total surface of the tissue) were assessed. Neuronal elements occupy about 74% and glial elements about 8% of the total volume. The specific surface, i.e., the amount of plasma membrane per unit volume of structure, is more than 30 times higher in the neuropil than in the neuronal perikaryal fraction. The largest specific surface is found with (unmyelinated) axons and astroglial processes. The specific surface of the average astrocyte is about twice that of the average neuron. If the surface of the entire cell is considered in relation to the perikaryal volume only, this ratio is about seven times as large for glial cells as for neurons. It follows that an astrocyte perikaryon has to renew a several times larger plasma membrane than a neuron, which can account for the above differences in perikaryal synthesis rates of glycoconjugates.

Incorporation of 3H-fucose into nerve and glial cells: assessment by electron microscopic autoradiography.

Incorporation of 3H-fucose into hypothalamic tissue of rats was studied between ten minutes and seven days after intraventricular injection. In EM autoradiographs grain densities over perikarya of neurons, astro-, and oligodendroglia, and neuropil were evaluated. Grain densities were consistently higher over both types of glial cells as compared to neurons. The glia/neuron ratio was especially high after short experimental intervals and declined with increasing time intervals. Concomitantly there was an increase of grain density over the neuropil. The discussion concentrates on the methodical aspects which have to be considered in interpreting the autoradiographic data. It is concluded that both astroglia and oligodendroglia exhibit a considerably higher synthesis rate of glycoproteins per unit volume than nerve cells. The implications of the results for the interpretation of biochemical data on glycoprotein metabolism of brain tissue are discussed.

The glial reaction in the course of axon regeneration: a stereological study of the rat hypoglossal nucleus.

Both hypoglossal nuclei were examined by electron microscope stereology after unilateral axotomy. The principal aim of this study was a quantitative assessment of the accompanying glial reaction. Volume densities (%) of neuronal and glial perikarya, as well as their processes, were evaluated in terms of volume plus surface densities (mm-1). In addition, specific surfaces (surface to volume ratio) of these neuronal and glial processes were assessed. First, a temporary decrease of dendritic volume density was detected on the ipsilateral side only. Further, the astrocytic reaction displayed differences between stem and lamellar processes. One day after axotomy, a bilateral decrease of volume density, as well as surface density of stem processes, was observed, yet their normal dimensions soon were reestablished. However, a more severe lamellar process reaction was evident. During the first 4 days, a significant increase of volume density and surface density was apparent. In the contralateral hypoglossal nucleus, this glial reaction also occurred but disappeared by day 14, whereas the ipsilateral nucleus continued to display a severe reaction of lamellar processes, only returning to normal status at day 77. In addition, a transient, severe reaction of presumptive microglia was established by employing the volume density and surface density quantitation procedure. Nevertheless, in comparison with the volume and surface contribution of astrocytic processes, the presumed microglial component was minimal. This study indicates a two-step involvement of astrocytes in regenerative repair. Namely, the first phase seems to result in an increase of lamellar processes through reshaping of the stem process.(ABSTRACT TRUNCATED AT 250 WORDS)

Sex differences in densities of dopaminergic fibers and GABAergic neurons in the prenatal rat striatum.

On the basis of observations on dopaminergic neurons developing in gender-specific cultures of embryonic rat mesencephalon, we have hypothesized that as yet unknown sexual dimorphisms might be found in projection areas of dopaminergic neurons. Therefore we searched for possible sex differences in the striatum during the period when massive ingrowth of mesencephalic afferents occurs and the striatal gamma-aminobutyric acid (GABA)ergic neurons differentiate. Male and female rats of embryonic days (E) 16, 18, 20, and 21 were fixed by perfusion through the heart. Vibratome sections were cut from the striatal anlage and sequentially immunostained for GABA by the immunogold-silver technique and tyrosine hydroxylase (TH) by the avidin-biotin-peroxidase method. Ultrathin sections were scanned for numbers of GABA- and TH-immunoreactive (IR) elements. Densities of TH-IR axons as well as of GABA-IR cell body profiles progressed with time. Contacts between TH-IR axons and GABA-IR and immunonegative cells were observed as early as E-16, increasing in numbers toward later stages. Throughout prenatal development, female striata displayed higher densities of both TH-IR axon and GABA-IR cell body profiles than male ones. This is the first report of a distinct anatomical sex difference regarding two major components of a key center of motor control. Prenatal sexual differentiation of the striatum may lead to a sexually dimorphic extrapyramidal circuitry, the existence of which, in the adult, is suggested by experimental and clinical data.

Different developmental schedules of dopaminergic and noradrenergic neurons in dissociation culture of fetal rat midbrain and hindbrain.

The development of dopaminergic and noradrenergic neurons in dissociation cultures of mesencephalon and rhombencephalon obtained from 18-day-old rat fetuses was characterized by their capacity to take up and release catecholamines. In both types of cultures, uptake of [3H]dopamine and [3H]noradrenaline was obtained which could be inhibited by reserpine. Autoradiographic studies demonstrated an almost exclusive neuronal localization of the labeled catecholamines. The transmitters could be released by depolarization with K+ in a Ca2+-dependent manner during the entire cultivation period. In contrast, catecholamine uptake by cultures of neocortex was minimal, could not be inhibited by reserpine, and the accumulated radioactivity could not be released upon depolarization. These points provide evidence for an active accumulation of the exogenous transmitters and for the presence of stimulus-secretion coupling in a distinct population of neurons of both brain stem cultures. Striking differences between the two brain stem cultures concerned their sensitivity to desmethylimipramine and benztropine as well as the time course of the development of the uptake capacity. Desmethylimipramine inhibited the uptake of both catecholamines in rhombencephalic, but not in mesencephalic cultures. The reverse was true for benztropine. It is concluded that cultures of rhombencephalon contain predominantly noradrenergic, and those of mesencephalon dopaminergic cells. Comparison of the uptake behaviour suggested that noradrenergic neurons mature considerably later than dopaminergic neurons. The results show that dissociation cultures of mid- and hindbrain, inspite of their heterogeneous composition, can serve as valuable models for the study of development and function of dopaminergic and noradrenergic neurons, respectively.

The development of immunoreactivity for neuron-specific enolase of preoptic and septal neurons in dissociated cultures.

The immunocytochemical visualization of neuron-specific enolase, which is a marker protein for differentiated neurons, was applied to follow the differentiation of preoptic and septal neurons in dissociated cultures. From 4 to 24 days in vitro, the relative numbers of stained neurons were counted and the staining intensity of individual neurons determined by absorbency measurements using a television-based densitometer. Whereas few stained cells could be observed at 4 DIV, 80% of the neurons were neuron-specific enolase-positive at 13 days in vitro. This value remained constant up to 24 days in vitro. The density of the immunoreaction product increased dramatically from 13 to 17 days in vitro and was still higher at 24 days in vitro. The glial and ependymal cells of the carpet, as well as neuroblasts, remained unstained. Comparison with morphological observations and immunocytochemical demonstration of neuronal peptides made earlier shows that expression of neuron-specific enolase closely parallels neuronal differentiation. These observations indicate that cultures derived from preoptic and septal neurons represent a viable model system for the study of neuronal maturation in vitro.

Rapid maturation of synaptic functions of prenatal serotoninergic neurons in short-term cultures: absence of sex differences and hormone effects.

Serotonin is believed to modulate neuronal differentiation during early stages of brain development. In order to assess basic functional requirements for such a role, it was investigated how early serotoninergic neurons mature with respect to transmitter storage and stimulus-secretion coupling. Dissociated cell cultures were raised from embryonic rat rhombencephalon obtained at gestational day 14 and cultured for 3-8 days, which may roughly correspond to the prenatal period in vivo. Because of a possible involvement of serotonin in processes leading to sexual differentiation of the brain, gender-specific cultures were raised in addition and treated with sex steroids. Sensitivity of [3H]serotonin uptake to fluoxetine could already be observed at 3 days in vitro. Vesicular storage as probed with reserpine and nigericin, and the capability of releasing preaccumulated serotonin in a Ca2+-dependent manner were also present as early as 3 days in vitro. Seven per cent of the pre-accumulated transmitter could be released per minute upon stimulation with 54 mM K+. Immunocytochemical and autoradiographic preparations demonstrated that, after the same short culture period, the neurons had formed large fiber networks. No differences could be detected regarding any of the above parameters between female and male serotonin neurons and between cultures treated with and without estradiol, testosterone and dihydrotestosterone. It is concluded that, in contrast to other neuronal phenotypes, serotoninergic neurons are functionally mature when or shortly after they are taken into culture, i.e. around gestational day 14. The functional competence of prenatal serotonin systems should be a prerequisite for their suspected role in modulating neural development at pre and postsynaptic sites. The present results provide no evidence for the occurrence of a sexual dimorphism of serotonin neurons at this early developmental stage.

Activation of dopaminergic D1 receptors promotes morphogenesis of developing striatal neurons.

The early dopaminergic input from the midbrain may play an important role in the development of the basal ganglia. We therefore investigated whether and how dopamine affects the morphogenesis of striatal target neurons. Dissociated cell cultures of embryonic day 17 rat striatum were raised for seven days. Cells were then incubated with dopamine or various receptor-specific ligands for 1 h. At various times after termination of the treatment, cells were immunostained for growth-associated protein-43. Morphological parameters including numbers of growth cones, length of neurites, number of bifurcations, and neuronal soma size were assessed by means of a computer-based morphometric device. Treatment with dopamine in low concentrations as well as with the D1-like receptor agonist SKF 38393 increased the numbers of growth cones and neurite length and arborization. The morphogenetic effect took several hours to evolve and remained stable for at least 24 h. It could be blocked by the D1-like receptor antagonist SCH 23390 or by cycloheximide but not by pretreatment of the cultures with tetrodotoxin. The D2-like receptor agonist quinpirole had no effect on the morphological parameters and did not contribute to that of SKF 38393. Dopamine and SKF 38393 but not quinpirole also induced an increase in the number of neurons immunoreactive for Fos-like proteins. However, this effect was restricted to growth-associated protein-43-negative neurons. This is the first observation of a positive regulatory effect of D1-like receptors on neuronal morphogenesis. We conclude that the changes reflect true differentiation rather than short-term modulation of cellular properties and that c-fos induction is not an obligatory step in the transduction pathway coupling D1-like receptors to neurite outgrowth. Our results suggest that the differentiation of embryonic striatal neurons is promoted by the dopaminergic nigrostriatal projection through D1-like receptors.

Effects of sex and estrogen on tyrosine hydroxylase mRNA in cultured embryonic rat mesencephalon.

In order to elucidate cellular events responsible for sex differentiation of the nigro-striatal system, we studied the influence of estrogen on the expression of tyrosine hydroxylase (TH) in sex-specific dissociated cell cultures of embryonic day 14 rat mesencephalon. Cultures were raised in the absence or presence of 17 beta-estradiol (10(-12) M) and hybridized with a [35S]oligonucleotide specific to TH. Cultured cells and tissues were probed for estrogen receptor (ER) transcripts by hemi-nested PCR. More TH mRNA containing cells were present in control cultures from female than from male donors. Estrogen treatment resulted in an up-regulation of TH expression in male cells only and induced a reversal of the sex difference in TH mRNA levels present in early control cultures. ER message was detectable in hypothalamic and uterine tissues but not in mesencephalic tissue or cultured cells. Estrogen exposure failed to induce ER expression in cultured mesencephalic cells. It is concluded that there are sex differences in TH mRNA expression of developing midbrain dopaminergic neurons which are independent of the steroid environment. Estrogen can up-regulate TH mRNA in a sex-specific fashion by modulating signal transduction mechanisms other than the classical nuclear receptor pathway.

Transcription of the Y chromosomal gene, Sry, in adult mouse brain.

The Y chromosomal gene Sry encodes a putative transcription factor which appears to serve as a master switch initiating testicular development. Here we show that this gene is transcribed in hypothalamus, midbrain, and testis of adult male but not adult female mice. In contrast to its circular transcripts in adult testis, those in brain are linear and may be translated. We propose that Sry exerts a role in the regulation of sex differentiation of the mammalian nervous system.

Ontogeny of aromatase messenger ribonucleic acid and aromatase activity in the rat midbrain.

Estrogen formation catalyzed by neural aromatase is crucial for the sexual differentiation of the brain. Ontogenic expression of aromatase mRNA and aromatase activity were studied in male and female rat midbrains. Aromatase mRNA was transiently expressed in both sexes showing maximum levels on postnatal day (P)2 and being absent on P20 and in adults. Developmental expression of aromatase mRNA preceded that of aromatase activity. These data demonstrate that the capacity for estrogen formation is present during a distinct phase of midbrain development. Our findings suggest an active role for estrogens in the differentiation of midbrain neurons.

Incorporation of [3H]leucine into hypothalamic nerve and glial cells. A comparison by EM autoradiography.

Incorporation of L-[3H]leucine into the arcuate and supraoptic nuclei of rat and mice hypothalamus was studied by EM autoradiography at 30 min after intracerebral and intravenous application, respectively. The analysis applied provides information about (1) the distribution and (2) the concentration of radioactivity in the various tissue compartments, in particular nerve and glial cells. In the supraoptic nucleus, the proportion of radioactivity present in neuronal perikarya markedly exceeds that in the neuropil. Similar amounts of radioactivity in neuronal perikarya and neuropil were found for the arcuate nucleus. In both hypothalamic nuclei, only a small percentage of radioactivity is localized in glial cell bodies. However, the concentration of radioactivity in glial cells is nearly as high as in nerve cells and is lowest in the neuropil. Furthermore analysis of the subcompartments of the neuropil reveals that a considerable proportion of its radioactivity can be attributed to astrocytic processes. Conditions are discussed under which grain densities can be taken as measurements of relative synthesis rates of proteins. The results which are at variance with earlier autoradiographic studies are largely consistent with those of more recent biochemical studies on brain tissue separated into neuronal and glial fractions.

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.

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.

Sex differences of hypothalamic prolactin cells develop independently of the presence of sex steroids.

There is evidence for a hypothalamic prolactin (PRL) system that expresses sexually dimorphic traits. The aim of this in vitro study is to gain an insight into the process of sexual differentiation of hypothalamic PRL cells. In particular, we wanted to determine whether sexual differentiation of these cells can occur independently of the surge of gonadal testosterone which, in the male rat embryo, takes place at embryonic day (E) 18 and is commonly believed to start the critical period of sexual differentiation of the brain. Gender-specific cell cultures were prepared from E 14 or E 17 rat diencephalon and raised in the absence of gonadal steroids. After 10 days in vitro, numbers of PRL-immunoreactive (IR) cells and PRL levels were quantified by immunocytochemistry and Western blotting, respectively. Numbers of PRL-IR cells and PRL levels were 2-3 times higher in cultures prepared from female than from male embryos of either age. It is concluded that sexual differentiation of hypothalamic PRL cells starts well before the generally acknowledged onset of the critical period and may proceed independently of the action of gonadal testosterone. Besides gonadal steroids, other mechanisms, such as cell-intrinsic realization of a sex-specific genetic program, may be responsible for initiating the development of sexually dimorphic neuronal phenotypes.

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.

Sex-specific schedule in steroid response of rhombencephalic catecholaminergic neurons in vitro.

Morphological differentiation of tyrosine hydroxylase-immunoreactive neurons was investigated in dissociated cell cultures of rhombencephalon of male and female day 14 rat embryos grown in the presence or absence of sex steroids. Numbers of cells were counted and morphometrical measurements carried out of soma size and length of tyrosine hydroxylase-immunoreactive neurites (processes). Subtle sex differences in length of stained neurites, which were not yet present after 3 days in vitro, were observed after 6 days in cultures grown in the absence of sex steroids. Female tyrosine hydroxylase-immunoreactive neurites could be traced over longer distances than male ones. Daily treatment of cultures with testosterone or 17 beta-estradiol resulted in an increase of lengths of stained neurites of female neurons after 3 days and of male neurons after 6 days in vitro. Regarding cell numbers or soma size, there were no differences between genders or between controls and hormone-treated cultures. It is concluded that sex steroids promote the outgrowth of neurites from noradrenergic neurons within a gender-specific time frame. It appears that the critical period for developmental effects of sex steroids differs between males and females.