Natalia O. Tsivkovskaia

Urocortin 1 distribution in mouse brain is strain-dependent

Urocortin 1 has been implicated in a number of specific behaviors, which include energy balance, stress reactivity and ethanol consumption. To elucidate genetically influenced differences in the mouse urocortin 1 system, we performed immunohistochemical characterization of urocortin 1 distribution in C57BL/6J and DBA/2J mouse brain. Urocortin 1 analysis reveals strain-dependent differences in distribution of urocortin 1 immunoreactive neurons and neuronal fibers. In both strains, the highest number of urocortin 1-positive neurons was observed in the Edinger-Westphal nucleus and lateral superior olive. Urocortin 1-positive neurons were detected in the dorsal nucleus of the lateral lemniscus of DBA/2J mice, but were absent in the C57BL/6J strain. Differences in urocortin 1 fibers were detected in many areas throughout the brain, and were most apparent in the septal areas, thalamic areas, several midbrain regions, and medulla. Strain-dependent distribution of urocortin 1-containing cells and fibers suggests that differences in this neuropeptide system may underlie differences in behavior and physiological responses between these strains. Further, we found that in both mouse strains, urocortin 1 in the Edinger-Westphal nucleus and choline acetyltransferase are not coexpressed. We show that the urocortin 1-positive neurons of this brain area form a separate population of cells that we propose to be called the non-preganglionic Edinger-Westphal nucleus.

Urocortin 1-containing neurons in the human Edinger-Westphal nucleus

The topographical location of neurons containing urocortin 1, a peptide related to corticotropin-releasing factor was investigated in human postmortem brain by immunohistochemistry, and compared with the location of neurons containing choline acetyltransferase, a marker for cholinergic cells. A three-dimensional computer reconstruction of the urocortin 1 and choline acetyltransferase-positive population of neurons within the oculomotor area was made. It was shown that the urocortin 1-positive neurons are located within the area identified as the Edinger-Westphal nucleus according to the human brain stem atlas, and that the neurons identified as Edinger-Westphal nucleus in the atlas are not choline acetyltransferase-positive. This finding agrees with recent animal studies showing that urocortin 1-positive neurons are not identical with the parasympathetic cholinergic neurons projecting to the ciliary ganglion. They indicate that the neurons identified as Edinger-Westphal nucleus in the human brain stem atlas belong to the non-preganglionic Edinger-Westphal nucleus, whereas the location of preganglionic Edinger-Westphal nucleus remains unidentified.

Strain differences in urocortin expression in the Edinger–Westphal nucleus and its relation to alcohol-induced hypothermia

The Edinger-Westphal nucleus is the primary source of urocortin in rodent brain. Mapping of inducible transcription factors has shown that the Edinger-Westphal nucleus is preferentially sensitive to ethanol self-administration. In the present study we have immunohistochemically compared expression of urocortin and c-Fos in naive and ethanol-treated C57BL/6J and DBA/2J mouse inbred strains. We found that C57BL/6J mice possess significantly higher numbers of urocortin-expressing cells in the Edinger-Westphal compared to DBA/2J mice. Subsequent histological analysis confirmed a lower number of large neurons in the DBA/2J Edinger-Westphal nucleus. Surprisingly, despite the differences in structure, no strain differences were observed in the number of c-Fos-containing cells after acute (0.6-4.8 g/kg, i.p.) and repeated (2.4 g/kg, 14 days, one injection/day) administration of ethanol. Double-label immunohistochemistry showed that ethanol-induced c-Fos expression is present in different sets of Edinger-Westphal cells between the strains. Specifically, expression of c-Fos in C57BL/6J mice is preferentially induced in urocortin cells, while c-Fos in DBA/2J mice occurs in a mixed population of cells. Behavioral analysis of the B6D2 F2 intercross, a heterogeneous mouse strain, showed that the number of urocortin cells is positively correlated with basal temperatures and ethanol-induced hypothermia. Involvement of the Edinger-Westphal in alcohol-induced hypothermia is further confirmed by analysis of urocortin cells in the HOT/COLD selected lines. These results provide evidence that C57BL/6J and DBA/2J mice have structural differences in the Edinger-Westphal that can result in activation of different populations of neurons upon alcohol intoxication contributing to differential thermoregulation between these inbred strains.

Identification of temperature-sensitive neural circuits in mice using c-Fos expression mapping

Expression of the inducible transcription factor c-Fos was mapped in mouse brain to identify neural circuits selectively involved in response to cold and hot external temperatures. Male C57BL/6J mice were exposed acutely or repeatedly (seven sessions) to 10 or 34 degrees C in sound-attenuated chambers. Control mice were acclimated to exposure to the experimental room at 20 degrees C. All animals were sacrificed at 90 min for immunohistochemical analysis. A statistically significant induction of c-Fos was observed in the shell of nucleus accumbens and posterior medial cortical amygdala only following the acute thermal exposure, showing a significant habituation of the response to repeated treatments, a finding arguing against specificity of responses in these nuclei to thermal exposures. In contrast, expression of c-Fos was significantly increased following both acute and repeated thermal exposures in subregions of hypothalamus (the median and medial preoptic nuclei, the paraventricular nucleus of hypothalamus and the posterior hypothalamic area), septum (the ventral and dorsal portions of the lateral septum), midbrain (the periaqueductal gray and the intermediate layers of superior colliculus), as well as in the dentate gyrus and the paraventricular nucleus of thalamus, suggesting specificity of their responses to external temperatures. Expression of c-Fos was also significantly increased in the Edinger-Westphal nucleus following acute thermal exposures versus control mice, but not versus mice repeatedly exposed to cold and hot temperatures, providing modest support for thermal specificity of c-Fos response in this nucleus. While thermal sensitivity of hypothalamic structures has been previously confirmed by many authors, the present study identifies a number of structures previously not found to be responsive to changes in external temperature, and lays ground for future work important for identification of neural circuits involved in thermoregulation.

Distribution of Corticotropin-Releasing Factor and Urocortin 1 in the Vole Brain

Brain receptor patterns for the corticotropin-releasing factor (CRF) receptors, CRF1 and CRF2, are dramatically different between monogamous and promiscuous vole species, and CRF physiologically regulates pair bonding behavior in the monogamous prairie vole. However, it is uncertain whether species differences also exist in the neuroanatomical distribution of the endogenous ligands for the CRF1 and CRF2 receptors, such as CRF and urocortin-1 (Ucn1). We compared the expression of CRF and Ucn1 in four vole species, monogamous prairie and pine voles, and promiscuous meadow and montane voles, using in situ hybridization of CRF and Ucn1 mRNA. Our results reveal that CRF mRNA expression patterns in all four vole species appear highly conserved throughout the brain, including the olfactory bulb, nucleus accumbens, bed nucleus of the stria terminalis, medial preoptic area, central amygdala, hippocampus, posterior thalamus, and cerebellum. Similarly, Ucn1 mRNA primarily localized to the Edinger-Westphal nucleus in all four vole species. Immunocytochemistry in prairie and meadow voles confirmed localization of CRF and Ucn1 protein to these previously identified brain regions. These data demonstrate a striking dichotomy between the extraordinary species diversity of brain receptor patterns when compared to the highly conserved brain distributions of their respective ligands. Our findings generate novel hypotheses regarding the evolutionary mechanisms underlying the neural circuitry of species-typical social behaviors.