Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse.

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable. Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Early forebrain wiring: genetic dissection using conditional Celsr3 mutant mice.

Development of axonal tracts requires interactions between growth cones and the environment. Tracts such as the anterior commissure and internal capsule are defective in mice with null mutation of Celsr3. We generated a conditional Celsr3 allele, allowing regional inactivation. Inactivation in telencephalon, ventral forebrain, or cortex demonstrated essential roles for Celsr3 in neurons that project axons to the anterior commissure and subcerebral targets, as well as in cells that guide axons through the internal capsule. When Celsr3 was inactivated in cortex, subcerebral projections failed to grow, yet corticothalamic axons developed normally, indicating that besides guidepost cells, additional Celsr3-independent cues can assist their progression. These observations provide in vivo evidence that Celsr3-mediated interactions between axons and guidepost cells govern axonal tract formation in mammals.

Early embryonic administration of xenoestrogens alters vasotocin system and male sexual behavior of the Japanese quail.

The copulatory behavior and the parvocellular vasotocin (VT) system of the nucleus of the stria terminalis (BST) are sexually dimorphic in the Japanese quail. Embryonic administration of estradiol benzoate (EB) induces an organizational effect determining the disappearance of such a dimorphism (male shows behavior and cerebral phenotype of the female). The VT parvocellular system can therefore be considered an accurate marker of the sexual differentiation of brain circuits and a very sensitive indicator of the activity of estrogen-like substances on neural circuits. To test this hypothesis we administered diethylstilbestrol (DES), a powerful synthetic xenoestrogen, genistein (GEN), a phytoestrogen produced by soy, and bisphenol A (BPA). After 3 days of incubation, quail eggs were injected with vehicle, EB, DES, GEN or BPA. Administration of BPA caused an early blockage of development and no further analyses were done on the BPA groups. At puberty, the copulatory behavior of EB- or DES-treated male quail was totally abolished, whereas only the highest doses of GEN determined a significant decrease of the behavior. After the tests, the animals were sacrificed and perfused. The fractional area (FA) covered by VT immunoreactivity was analyzed in BST, medial preoptic nucleus, and lateral septum by computerized image analysis. The FA was significantly reduced after treatment with EB, DES and GEN at high doses. These results confirm that the sexually dimorphic VT system of the Japanese quail is a sensible indicator of the effects of xenoestrogens at the level of the central nervous system.

Target-dependent sexual differentiation of a limbic-hypothalamic neural pathway.

Neural pathways between sexually dimorphic forebrain regions develop under the influence of sex steroid hormones during the perinatal period, but how these hormones specify precise sex-specific patterns of connectivity is unknown. A heterochronic coculture system was used to demonstrate that sex steroid hormones direct development of a sexually dimorphic limbic-hypothalamic neural pathway through a target-dependent mechanism. Explants of the principal nucleus of the bed nuclei of the stria terminalis (BSTp) extend neurites toward explants of the anteroventral periventricular nucleus (AVPV) derived from male but not female rats. Coculture of BSTp explants from male rats with AVPV explants derived from females treated in vivo with testosterone for 9 d resulted in a high density of neurites extending from the BSTp to the AVPV explant, as was the case when the BSTp explants were derived from females and the AVPV explants were derived from males or androgen-treated females. These in vitro findings suggest that during the postnatal period testosterone induces a target-derived, diffusible chemotropic activity that results in a sexually dimorphic pattern of connectivity.