Intraventricular injection of 6-hydroxydopamine results in an increased number of tyrosine hydroxylase immune-positive cells in the rat cortex.

Previously we have demonstrated that intraventricular injection of 6-hydroxydopamine (6-OHDA) results in increased proliferation and de-differentiation of rat cortical astrocytes into progenitor-like cells 4 days after lesion (Wachter et al., 2010). To find out if these cells express tyrosine hydroxylase (TH), the rate-limiting enzyme in the catecholamine synthesis pathway, we performed immunohistochemistry in the rat cortex following intraventricular injection of 6-OHDA. Four days after injection we demonstrated a strong emergence of TH-positive (TH(+)) somata in the cortices of 6-OHDA-lesioned animals. The number of TH(+) cells in the cortex of 6-OHDA-lesioned animals was 15 times higher than in sham-operated animals, where virtually no TH(+) somata occurred. Combining TH immunohistochemistry with classical Nissl stain yielded complete congruency, and ∼45% of the TH(+) cells co-expressed calretinin, which indicates an interneuron affiliation. There was no co-staining of TH with other interneuron markers or with glial markers such as glial fibrillary acidic protein (GFAP) or the neural stem/progenitor marker Nestin, nor could we find co-localization with the proliferation marker Ki67. However, we found a co-localization of TH with glial progenitor cell markers (Sox2 and S100ß) and with polysialylated-neural cell adhesion molecule (PSA-NCAM), which has been shown to be expressed in immature, but not recently generated cortical neurons. Taken together, this study seems to confirm our previous findings with respect to a 6-OHDA-induced expression of neuronal precursor markers in cells of the rat cortex, although the TH(+) cells found in this study are not identical with the potentially de-differentiated astrocytes described recently (Wachter et al., 2010). The detection of cortical cells expressing the catecholaminergic key enzyme TH might indicate a possible compensatory role of these cells in a dopamine-(DA)-depleted system. Future studies are needed to determine whether the TH(+) cells are capable of DA synthesis to confirm this hypothesis.

Surgical prosection in a traditional anatomical curriculum-Tübingens' Sectio chirurgica.

Despite the rapid rise of integrated curricula, the teaching of gross anatomy by traditional dissection remains a central element in most medical programs worldwide. However, modern didactic concepts demand the integration of clinical content in preclinical settings. The implementation of interdisciplinary tools often leads to a reduction in teaching of comprehensive anatomy. 'Tübingen's Sectio chirurgica' (TSC) introduces a concept of a teaching activity in which surgical prosection is performed in addition to the traditional dissection course. TSC is designed to integrate clinical and preclinical content in a traditional medical curriculum without affecting the systematic presentation of anatomical content. In the past 2 years, about 10,000 medical students have participated in the use of telemedical transmissions of 'live surgery' in a total of 25 sessions of TSC. Here we describe the organisational plan of TSC and the results of an evaluation which was performed to monitor the influence of TSC on student motivation for surgical disciplines as well as for the learning of anatomical factual content. We demonstrate that additional surgical prosection is a valuable tool in increasing the coherence between preclinical and clinical parts of medical education programs.

Estrogen stimulates brain-derived neurotrophic factor expression in embryonic mouse midbrain neurons through a membrane-mediated and calcium-dependent mechanism.

We have provided evidence that 17beta-estradiol (E) synthesized in the midbrain promotes the differentiation of midbrain dopamine neurons through nonclassical steroid action. Because these developmental effects resemble those reported for brain-derived neurotrophic factor (BDNF), we hypothesized that E influences dopaminergic cell differentiation through a BDNF-dependent mechanism. Competitive RT-PCR and ELISA techniques were employed to study first the developmental pattern of BDNF and trkB expression in the mouse midbrain. BDNF protein/mRNA levels peaked postnatally, whereas trkB did not fluctuate perinatally. To prove the hypothesis that E regulates BDNF expression in vivo, fetuses and newborns were treated with the aromatase antagonist CGS 16949A. CGS 16949A exposure reduced midbrain BDNF mRNA/protein levels. The coapplication of CGS 16949A and E abolished this effect. Midbrain cultures from mouse fetuses were used to investigate intracellular signaling mechanisms involved in transmitting E effects. Estrogen increased expression of BDNF but not of other neurotrophins. As concerns the related signaling mechanism, these effects were antagonized by interrupting intracellular Ca(2+) signaling with BAPTA and thapsigargin but not by the estrogen receptor antagonist ICI 182,780. Insofar as E effects on BDNF mRNA expression were inhibited by cycloheximide, it appears likely that other, not yet characterized intermediate proteins take part in the estrogenic regulation of BDNF expression. We conclude that E exerts its stimulatory effect on the differentiation of dopaminergic neurons by coordinating BDNF expression. This particular E effect appears to be transmitted through Ca(2+)-dependent signaling cascades upon activation of putative membrane estrogen receptors.

Classical and nonclassical estrogen action in the developing midbrain.

There is widespread acceptance that estrogen is involved in various steps of cellular differentiation during brain development. In the past years, we have demonstrated such a developmental role for estrogen in the rodent midbrain. Precisely, estrogen affects midbrain dopamine neurons with respect to functional and morphological maturation. On the cellular level, estrogen may act classically by binding and activating its respective nuclear receptors, thereby controlling the transcription of target genes. On the other hand, many estrogen effects in the CNS are transmitted nonclassically by interactions with putative membrane receptors and by stimulating distinct intracellular signaling cascades. In the midbrain, classical and nonclassical estrogen signaling routes operate side by side to ensure the proper development of dopaminergic cells. In the present report, we detail some of the cellular and molecular events which are activated by estrogen and are thought to take part in the estrogen-mediated stimulation of dopamine neuron differentiation.

Dopamine regulates brain-derived neurotrophic factor (BDNF) expression in cultured embryonic mouse striatal cells.

The differentiation of striatal GABAergic neurons coincides with the perinatal establishment of nigrostriatal dopaminergic synaptic connections. We have shown previously that dopamine stimulates the maturation of striatal GABAergic neurons. Since BDNF also regulates the development of GABAergic cells, we hypothesized that dopamine might affect striatal BDNF expression. The influence of dopamine on BDNF protein/mRNA and trkB mRNA levels was studied in neuronal and astroglia cultures of the mouse striatum. Stimulation with dopamine and a dopamine D1 receptor agonist increased BDNF mRNA and protein but not trkB mRNA in neuronal cultures. Our data indicate a potential role for dopamine in the developmental regulation of striatal BDNF expression and suggest that dopamine effects on GABAergic cells may be intertwined with BDNF action.

Developmental regulation of glutamic acid decarboxylase mRNA expression and splicing in the rat striatum by dopamine.

Dopamine (DA) promotes the morphological differentiation of striatal GABAergic neurons through D(1) receptor activation and cAMP/PKA signaling. In this study, we investigated the developmental role of DA on the expression of the two GAD(65/67) genes and the alternative splicing of GAD(67) transcripts in the rat striatum. In vivo, embryonic and adult GAD(67) splice variants and GAD(65) transcripts increased until E17 and E19, respectively. Thereafter, the embryonic GAD(67) isoform disappeared, whereas GAD(65) mRNA levels remained unchanged postnatally. The hypothesis that the prenatal ingrowth and functional maturation of nigrostriatal afferents may be responsible for these developmental events through DA-dependent signaling pathways was tested in E17 rat striatal cultures. Treatment with DA and D(1) but not D(2) agonists decreased the ratio of embryonic to adult GAD(67) mRNAs and increased GAD(65) mRNA levels as well as GABA synthesis rates. Our findings demonstrate a distinct developmental switch in the regulation of GAD(65) expression and GAD(67) splicing in the rat striatum which clearly depends upon D(1) receptor but not D(2) signaling. The dopaminergic input thus appears to control the functional differentiation of GABAergic neurons not only by upregulation of expression of the two GAD genes but also by regulating GAD(67) splicing.

Estrogen: a multifunctional messenger to nigrostriatal dopaminergic neurons.

Gonadal steroids affect a wide variety of functions in the mammalian brain ranging from the regulation of neuroendocrine systems and the modulation of behavior to the stimulation of differentiation and plasticity of distinct neuronal populations and circuits. The last decades have also demonstrated that estrogen serves as a neuroprotective factor for distinct neurodegenerative disorders. Such neuroprotective effects of estrogen are most obvious for Parkinson's and Alzheimer's disease. Despite this knowledge, little is known about the mechanisms and cellular targets by that estrogen might elicit its protective influence. In the past, we have intensively studied the effects of estrogen on midbrain dopaminergic neurons which represent the most affected cell population during Parkinson's disease. These studies were mainly performed on developing dopaminergic cells and revealed that estrogen is an important regulator of plasticity and function of this neuronal phenotype. Precisely, we found that dopaminergic neurons are direct targets for estrogen and that estrogen stimulates neurite extension/branching and the expression of tyrosine hydroxylase, the key enzyme in dopamine synthesis. Together with other in vivo studies, we might draw the conclusion that estrogen is required for the plasticity and activity of the developing and adult nigrostriatal system. The presence of the estrogen-synthesizing enzyme aromatase within the nigrostriatal system further supports this idea. Surprisingly, estrogen effects on nigrostriatal cell function are not only transmitted by classical nuclear estrogen receptors but also depend on nonclassical estrogen actions mediated through putative membrane receptors coupled to diverse intracellular signaling cascades. In the future, it has to be elucidated whether nonclassical mechanisms besides genomic actions also contribute to estrogen-mediated neuroprotection in the adult CNS.

Expression of estrogen receptor-alpha and beta mRNA in the developing and adult mouse striatum.

Estrogen not only modulates nigrostriatal function but also developmental processes in the striatum. Recently, we have demonstrated the presence of the estrogen-synthesizing enzyme aromatase in the developing mouse striatum. This study is concerned with the expression of estrogen receptor-alpha/beta (ER) mRNA in the developing and adult mouse striatum by semiquantitative reverse transcription-polymerase chain reaction. Expression of both ER subtypes occurred already prenatally and further increased until birth. Early postnatally, ER-alpha/beta levels remained high but decreased to lower levels in adults. No sex difference in ER expression was observed. These data together with our previous findings demonstrate the simultaneous expression of both ER subtypes and aromatase in the mouse striatum. It is concluded that estrogen signalling through both nuclear receptors plays a potential role for striatal differentiation.

Expression of aromatase in the embryonic and postnatal mouse striatum.

Estrogen influences striatal activity and the development of the nigrostriatal system. This study is concerned with the ontogenetic and postnatal expression of aromatase in the mouse striatum. Aromatase activity and mRNA expression were detectable in the embryonic striatum and increased postnatally with no differences between sexes. Aromatase-positive cells were uniformly distributed within the striatum. These data demonstrate that estrogen formation is an intrinsic property of striatal cells and suggest that estrogen may be important for striatal development and function.

Developmental expression and regulation of aromatase- and 5alpha-reductase type I mRNA in the male and female mouse hypothalamus.

Androgen metabolites synthesized by neural aromatase and 5alpha-reductase are implicated in many aspects of mammalian brain development and, in particular, in the masculinization of distinct central nervous system structures and brain functions. The present study was designed to determine (1) the developmental profile of aromatase- and 5alpha-reductase type I mRNA expression in the mouse hypothalamus and (2) to relate ontogenetic sex differences in aromatase activity which have been described in the past to sex-specific aromatase gene expression. In addition, we analysed the effect of androgens on the perinatal regulation of hypothalamic aromatase and 5alpha-reductase type I mRNA expression. By applying semiquantitative reverse transcription-polymerase chain reaction analysis, we found hypothalamic aromatase mRNA expression to be developmentally regulated and to display sex differences at birth and on postnatal day 15 with higher mRNA levels in males. Newborn males and females, which were treated in utero with the androgen receptor antagonist cyproterone actetate, exhibited significantly reduced aromatase mRNA levels compared with untreated controls. In contrast to aromatase, expression levels of hypothalamic 5alpha-reductase mRNA did not reveal a clear-cut developmental profile or sex differences, and no regulatory role for androgens in controlling 5alpha-reductase mRNA expression was found. In conclusion, these results demonstrate perinatal sex differences in hypothalamic aromatase- but not 5alpha-reductase gene expression and suggest that sex differences in perinatal aromatase activity are reflected by corresponding differences in mRNA levels. Androgens are found to control brain estrogen formation pretranslationally at the level of aromatase gene expression. Our findings imply that sex differences in androgen availability and responsiveness are important regulatory factors for aromatase expression in the developing male hypothalamus.

Genotype-dependent sex differentiation of dopaminergic neurons in primary cultures of embryonic mouse brain.

In order to investigate genetic factors that interfere with hormone-mediated sex differentiation of dopaminergic neurons, we raised sex-specific primary cultures from embryonic day 13 diencephalon (D) or mesencephalon (M) of three different strains of mice, NMRI, CBA/J, and BALBc/J. Part of the cultures were maintained for 6 or 13 days in vitro (DIV) in medium containing 17 beta-estradiol or testosterone. The cultures were analyzed for sex differences in numbers of tyrosine hydroxylase-immunoreactive neurons, endogenous dopamine (DA) levels, and specific uptake of [3H]DA. Previous results obtained with cultures of embryonic Sprague-Dawley rats had shown that these parameters develop sex-specific characteristics in the absence of sex differences in hormone environment. Similar steroid-independent sex differences as they occur in the rat were found in M cultures of NMRI but not in CBA and BALBc mice. Long-term sex steroid treatment did not affect any of the above parameters in any strain. It is concluded that cell-autonomous realization of the genetic sex of dopaminergic neurons depends on the genetic background.

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.

[Three basic kinds of behaviour, a scheme of their neurobiological correlates (author's transl)]. / Drei Grundarten unseres Verhaltens und ein Schema ihrer neurobiologischen Korrelate

The psychic sphere is to be regarded as the source of meaningful behaviour, which is carried out in the service of the cell community that makes up our body. Three fundamental types of behaviour can be distinguished: the purely practical, the theoretical-practical, and the purely theoretical. These three types of behaviour have three different reasons: the first a determining reason, the second a motivating reason, and the third a supporting reason. The threefold nature of the reasons is related to the threefold needs of the cell community (in this context man is considered as an example of an animal). Possible neurobiological bases of human behaviour are depicted in a schematic figure showing the relation of cerebral centres and sensorimotor functions of the human face including eye movements. The psychic centre may be located in the thalamus, the areas of the central sulcus are regarded as an objectivation zone. This indicates that the motor zones of the cortex, including the frontal adversive fields, are intention zones, and the sensory zones reproduction, expectation, and recollection zones. A system so composed would render possible what we see in every animal being: decision-making, foresight, and learning.