Lynne R. Ross

Galanin-like immunoreactivity in the adult and developing Brazilian opossum brain

The distribution of galanin-like immunoreactivity has been characterized in the brain of the adult and developing Brazilian opossum (Monodelphis domestica). Two commercially available antisera were used to examine the distribution of galanin-like immunoreactive (GAL-IR) cells and fibers. Nuclear groups containing GAL-IR cell bodies and fibers were seen throughout the adult opossum brain. The distribution of GAL-IR elements seen is similar to that reported for other mammals. Based on these findings, we believe that galanin may have similar physiological functions in the adult Brazilian opossum as has been reported for other mammals. In the developing brain, GAL-IR structures were seen as early as 1 day postnatal (PN) in the developing hypothalamus and brainstem. By days 5 and 10 PN, there was a robust expression of galanin-like immunoreactivity in specific regions of the brain. Since neurogenesis and brain morphogenesis are actively occuring postnatally in the opossum, galanin may be playing a role in the differentation of specific regions of the brain.

Localization of cells containing estrogen receptor-like immunoreactivity in the Brazilian opossum brain

The Brazilian opossum (Monodelphis domestica) is a small, pouchless marsupial whose young are born in an immature, sexually undifferentiated state. Etgen and Fadem, and Handa and coworkers have biochemically detected and characterized estrogen receptors in the forebrain of the Brazilian opossum. In this study, we have examined the distribution of estrogen receptor-like immunoreactive (ER-LI) cells in the brains of gonadectomized male and female Brazilian opossums using Abbott H222 rat monoclonal estrogen receptor antibody (H222 is a gift of Abbott Labs). An indirect immunohistochemical procedure employing the Vectastain Elite system and a nickel-enhanced DAB chromogen was used. A large number of ER-LI cell nuclei were observed in the medial preoptic area, ventral septal nucleus, medial division of the bed nucleus of the stria terminalis, lateral part of the ventromedial hypothalamus, premammillary nucleus, arcuate nucleus, posterior amygdaloid nucleus, and the midbrain central grey. Lower numbers of ER-LI cell nuclei were observed in the intermediate subdivision of the lateral septal nucleus, and in the anterior, medial, and posterior cortical amygdaloid nuclei. The anatomical distribution of ER-LI in the Brazilian opossum brain is similar to that which has been reported for estrogen binding sites following biochemical analysis. Based on these findings, we believe specific regions of the Brazilian opossum brain may serve as substrata for the action of estrogen in the adult. In addition, these results are supportive of the use of this animal model to investigate the organizational effects of estrogen on the developing central nervous system.

Ontogeny of cells containing estrogen receptor-like immunoreactivity in the Brazilian opossum brain

In this study, we have used the Brazilian short-tailed opossum (Monodelphis domestica) as a model to study the ontogeny of estrogen receptors in the mammalian brain. Monodelphis is a small, pouchless marsupial which breeds well under laboratory conditions and whose young are born in an immature sexually undifferentiated state. The Abbott H222 monoclonal rat estrogen receptor antibody (gift of Abbott Laboratories) was utilized in an indirect immunohistochemical procedure to detect estrogen receptors in developing opossum brains. Estrogen receptors were first expressed in the dorsomedial and ventromedial hypothalamus of the opossum 10 days after birth (10PN). Most regions that contained estrogen receptor-like immunoreactivity (ER LI) in the adult opossum contained ER LI at 15 PN. These areas include the lateral septum, medial preoptic area, bed nucleus of the stria terminalis, periventricular preoptic area and hypothalamus, amygdala, dorsomedial and ventromedial hypothalamic nuclei, arcuate nucleus, ventral premammillary nucleus, and the midbrain central grey. The number of cells that contain ER LI increased through 60PN in all regions that will contain ER LI in the adult opossum. These results indicate that estrogen receptors are present in early development of the Monodelphis brain and may mark the beginning of a critical period for sexual differentiation of the opossum brain.

Relaxin-induced expression of Fos in the forebrain of the late pregnant rat.

Relaxin, administered parenterally, has been shown to increase the release of oxytocin (OT) into the circulation and increase the firing rate of OTergic neurons. The objective of the present study was to determine if relaxin administration can result in the expression of a transcription factor, suggesting that it alters transcriptional activity within OTergic neurons at the level of the hypothalamus. Primigravid rats were ovariectomized and a jugular cannula was inserted on day 11 of gestation (g11). Pregnancy was maintained by implanting 17 beta-estradiol and progesterone caplets subcutaneously at the time of ovariectomy. At gl9, rats were challenged with intravenous relaxin or isotonic saline and the brains were removed for study. Immunohistochemistry was performed on coronal brain sections, utilizing Fos as a marker of cellular activation. In the group receiving relaxin, Fos-like immunoreactivity (Fos-IR) was abundant only in the supraoptic (SON) and paraventricular nuclei (PVN) of the hypothalamus as well as in the subfornical organ (SFO). In contrast, Fos-IR in the group given isotonic saline was lacking in these three brain regions. A double label study using antibodies against Fos and OT demonstrated that a majority of the Fos-labeled cells in the hypothalamus were OTergic. Because Fos can act as a transcription factor, we interpret these data to indicate that transcription within OTergic cells is altered following relaxin administration, with abundant Fos-IR being limited to the SON and PVN of the hypothalamus and the SFO during late pregnancy in the rat.

Ontogeny of cholecystokinin-like immunoreactivity in the Brazilian opossum brain

We have studied the anatomical distribution of cholecystokinin-like immunoreactive (CCK-IR) somata and fibers in the brain of the adult and developing Brazilian short-tailed opossum, Monodelphis domestica. Animals ranged in age from the day of birth (1PN) to young adulthood (180PN). A nickel enhanced, avidin-biotin, indirect immunohistochemical technique was used to identify CCK-IR structures. Somata containing CCK immunoreactivity were observed in the cerebral cortex, hippocampus, hypothalamus, thalamus, midbrain, and brainstem in the adult. Cholecystokinin immunoreactive fibers had a wide distribution in the adult Monodelphis brain. The only major region of the brain that did not contain CCK-IR fibers was the cerebellum. The earliest expression of CCK immunoreactivity was found in fibers in the dorsal brainstem of 5-day-old opossum pups. It is possible that the CCK-IR fibers in the brainstem at 5PN are of vagal origin. Cholecystokinin immunoreactive somata were observed in the brainstem on 10PN. The CCK-IR cell bodies observed in the brainstem at 10PN may mark the first expression of CCK-IR elements intrinsic to the brain. A broad spectrum of patterns of onset of CCK expression was observed in the opossum brain. The early occurrence and varied ontogenesis of CCK-IR structures indicates CCK may be involved in the function of a variety of circuits from the brainstem to the cerebral cortex. The early expression of CCK-IR structures in the dorsal brainstem suggests that CCK may modulate feeding behavior in the Monodelphis neonate. Cholecystokinin immunoreactivity in forebrain structures such as the suprachiasmatic nucleus, medial preoptic area, thalamus and cortical structures indicates that CCK may also be involved in circadian rhythmicity, reproductive functions, as well as the state of arousal of the Brazilian opossum. The ontogenic timing of CCK immunoreactivity in specific circuitry also indicates that CCK expression does not occur simultaneously throughout the brain. This pattern of CCK onset may relate to the temporal need for CCK in specific circuits of the central nervous system (CNS) during development.

Postnatal neurogenesis of the hypothalamic paraventricular and supraoptic nuclei in the brazilian opossum brain

We have used bromodeoxyuridine (BrdU) single and BrdU-arginine vasopressin-oxytocin (BrdU-AVP-OT) double and triple label immunohistochemistry to characterize postnatal neurogenesis of the supraoptic and paraventricular nuclei in the Brazilian opossum. Developing pups received a single injection of BrdU between days 1 and 11 postnatally. All brains were collected on day 60 of postnatal life (60 PN). Single label BrdU immunohistochemistry revealed that an injection at 1 PN resulted in heavy labelling in the hypothalamus including the area of the paraventricular nucleus, whereas only approximately one third of the cells in the supraoptic nucleus were labelled. Analysis of data indicated that neurogenesis of the supraoptic and paraventricular nuclei is completed by days 5 and 7 PN, respectively. Double and triple label immunohistochemistry demonstrated that following BrdU injection on day 1 or 2 PN, few of the AVP and OT secreting cells in the supraoptic nucleus were double labelled with either peptide and BrdU, and no double labelled cells were seen following BrdU injection on day 5 PN. Similarly, in the paraventricular nucleus most of the AVP and OT secreting magnocellular cells were not double labelled with either peptide and BrdU. Whereas several double labelled cells were observed in the parvicellular part following BrdU injection on day 1 or 2 PN. No double labelled cells were present in any component of the paraventricular nucleus following injection on day 7 PN or later. These results indicate that the majority of the AVP and OT secreting magnocellular neurons are born prenatally and the OT and AVP parvicellular group of neurons are born during postnatal life. Our results also demonstrate that in contrast to that of eutherian rodents such as the rat and mouse, neurogenesis in the opossum hypothalamus continues into the postnatal period and provides a unique opportunity to study the neuroanatomical development of diverse regions such as the paraventricular nucleus.

Differential down-regulation of estrogen receptor-like immunoreactivity by estradiol in the female Brazilian opossum brain

The effects of estrus and prolonged estradiol benzoate (EB) treatment on estrogen receptor-like immunoreactivity (ER-LI) were studied in the female Brazilian opossum (monodelphis domestica) brain. In general, both estrus animals and EB treated animals had reduced ER-LI in specific brain regions when compared to ovariectomized controls. These results support a down-regulatory role for estrogen in relation to its receptor in the brain, and indicate that ER-LI is differentially regulated in the Monodelphis forebrain.