Carol D. Jacobson

Distribution of Fos-like immunoreactivity in the rat brain following intravenous lipopolysaccharide administration.

The central nervous system, particularly the hypothalamus, is intimately involved in the coordination of various aspects of the inflammatory response, including the generation of fever. We used intravenous injections of bacterial cell wall lipopolysaccharide (LPS; 5 or 125 μg/kg) to stimulate the acute phase response and mapped the resultant distribution of Fos‐like immunoreactivity in the rat brain. In addition, we compared the patterns of Fos distribution with the thermoregulatory responses elicited by the LPS. Administration of LPS resulted in a dose‐ and time‐dependent pattern of Fos‐like immunoreactivity throughout the rat brain consistent with a coordinated autonomic, endocrine, and behavioral response to the LPS challenge that was most pronounced 2 hours following injection. Specifically, Fos‐like immunoreactivity was observed in key autonomic regulatory nuclear groups, including the insular and prelimbic cortices, paraventricular hypothalamic nucleus, parabrachial nucleus, nucleus of the solitary tract, and the rostral and caudal levels of the ventrolateral medulla. In addition, a significant sustained elevation of Fos‐like immunoreactivity was observed in a cell group adjacent to the organum vasculosum of the lamina terminalis, which we termed the ventromedial preoptic area. This sustained elevation of Fos‐like immunoreactivity coupled with the alterations in body temperature elicited by LPS leads us to hypothesize that the ventromedial preoptic area may be a key site for the initiation of fever during endotoxemia.

Effects of small medial preoptic area lesions on maternal behavior: Retreiving and nest building in the rat

Previous studies in rats have demonstrated that large lesions in the medial preoptic area (MPOA) disrupt all aspects of maternal behavior. In the present study, small bilateral electrolyte lesions in the MPOA of lactating females abolished nest building and retrieving components of maternal behavior while crouching and nursing were unaffected. Animals which failed to show retrieval and nest building behaviors tended to have a greater area of lesion within the more dorsal part of the MPOA. Although the dorsal MPOA may play a role in the maintenance of the active components of maternal behavior, i.e. nest building and retrieving, another critical factor in determining which components of maternal behavior are disrupted may be the size of the lesion. As one increases the area of damaged tissue there is also an accompanying increase in the components of maternal behavior which are disrupted. No correlation was found between damage to the Sexually Dimorphic Nucleus of the preoptic area and retrieving and nest building.

Masculinization of the female rat spinal cord following a single neonatal injection of testosterone propionate but not estradiol benzoate

We previously reported that a motor nucleus of the rat spinal cord, the spinal nucleus of the bulbocavernosus (SNB), has more and larger neurons in males than females. The SNB is significantly masculinized in adult female rats receiving 1 mg of testosterone propionate (TP) on the second day of life, compared to that of their oil treated sisters. This masculinization consists of more neurons in the region of the SNB. These results are consistent with the hypothesis that the action of androgens during the early perinatal period is responsible for the sexually dimorphic development of the SNB. Neonatal TP caused no detected masculinization, i.e. enlargement of the size of individual neurons in this region in females. Male rats given TP on day 2 had significantly smaller testes and smaller SNB somas in adulthood than oil treated males. In female rats given 100 micrograms of estradiol benzoate (EB) on day 2, the SNB was not masculinized in either number of size when observed in adulthood. The lack of a masculinizing effect of EB suggests that the aromatization of testosterone to estradiol is not sufficient for this dimorphic development.

Formation of the sexually dimorphic nucleus of the preoptic area: Neuronal growth, migration and changes in cell number

The sexually dimorphic nucleus of the preoptic area (SDN-POA) appears to be a morphological marker of the process of sexual differentiation of the rat brain. A portion of the presumptive SDN-POA neurons can be specifically identified utilizing autoradiography following the administration of [3H]thymidine on day 18 of gestation. In the present study we have utilized this fact in order to describe the general pattern of formation of the SDN-POA during the perinatal period. Following the administration of [3H]thymidine, fetal pups were sacrificed and perfused with neutral formalin either two hours after the injection or on day 20 or 22 postfertilization. Neonatal pups were either sacrificed and perfused or assigned to one of the treatment groups. Male pups were either gonadectomized or sham gonadectomized; females were all sham gonadectomized. These pups were then sacrificed and perfused on either day 26, 28 or 32 postfertilization. All brains were processed for autoradiography. The size, number and location of labeled cells within the medial preoptic area (MPOA) was determined. In general it appears that the labeled cells grow in size during the early postnatal period. These cells also migrate from the more ventral aspects of the MPOA to aggregate and form the SDN-POA. Furthermore, there is a significant decrease in the number of labeled cells by day 32 postfertilization (day 10 of postnatal life) which appears to contribute to the specific labeling of the SDN-POA of the adult animal. However, results obtained in this study from quantitative analyses do not indicate that sex or the postnatal steroid hormone environment influence the processes of growth, migration and decrease in MPOA cell number of those presumptive SDN-POA cells born specifically on day 18 of gestation and analyzed in this study.

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.

Androgen receptor-like immunoreactivity in the Brazilian opossum brain and pituitary: distribution and effects of castration and testosterone replacement in the adult male

Androgens are involved in a variety of centrally mediated functions after binding to their intracellular receptors. In the present report, we have employed the androgen receptor antibody, PG-21, and indirect immunohistochemistry to examine the distribution of cells containing androgen receptor-like immunoreactivity (AR-IR) in the intact adult male Brazilian opossum brain and pituitary. Additional adult males were castrated to examine the effects of withdrawal of circulating androgens and testosterone replacement on AR-IR. Immunoblots and immunohistochemical controls demonstrated that the androgen receptor in the opossum brain and peripheral tissues are of a similar molecular mass as to has been reported for the rat. Cells containing AR-IR were widely distributed throughout the brain of intact adult males. The highest number of immunoreactive cells were present in the dorsal and ventral nuclei of the lateral septum, medial division of the bed nucleus of the stria terminalis, medial preoptic area, median preoptic nucleus, nucleus of the lateral olfactory tubercle, central amygdaloid nucleus, anterior cortical amygdaloid nucleus, posterior amygdaloid nucleus, subiculum, ventromedial hypothalamic nucleus, arcuate-median eminence region, and ventral premammillary nucleus. The anterior pituitary gland also contained a high number of cells containing AR-IR. The general distribution of AR-IR both in the brain and anterior pituitary gland resembled that reported for other mammalian species. Castration of the adult males four days prior to perfusion eliminated androgen receptor immunostaining throughout the brain except for a few lightly immunostained cells in the ventral nucleus of the lateral septum and stria terminalis. Androgen receptor immunostaining was decreased in the anterior pituitary gland following castration and became cytoplasmic. Testosterone administration 2 h before perfusion restored AR-IR both in the brain and anterior pituitary gland. These data suggested that immunohistochemical detection of bound (nuclear) androgen receptors as seen with PG-21 antibody in the brain and anterior pituitary gland of the opossum is dependent upon circulating androgens. Further, the wide distribution and similarity in localization of androgen receptors in the opossum brain and anterior pituitary gland to that of other species suggests that androgen receptors might be involved in similar functions in the opossum as has been reported for other species.

Postnatal development and the differential expression of presynaptic terminal-associated proteins in the developing retina of the Brazilian opossum, Monodelphis domestica

In the present study we have characterized the postnatal (PN) development of the retina in the Brazilian opossum, Monodelphis domestica. Monodelphis, a small, pouchless marsupial, undergoes a protracted period of postnatal development. Using bromodeoxyuridine immunohistochemistry, we have investigated postnatal neurogenesis of the retina. In addition, we have examined the differentiation of the retina by using antibodies directed against the presynaptic terminal-associated proteins synaptotagmin, Rab3A, synaptophysin and synaptosomal-associated protein-25 (SNAP-25), and have characterized their spatial and temporal distribution during postnatal development. This study is the first systematic comparison of the developmental expression of multiple presynaptic terminal-associated proteins in relation to retinal neurogenesis and differentiation. At birth (1PN), the Monodelphis retina was relatively undifferentiated morphologically and birthdating analysis revealed mitotically active cells throughout the retina. The 8PN retina was organized into two cellular layers: an outer region of mitotically active neuroepithelial cells and an inner region of postmitotic cells. The inner plexiform layer formed between 5PN and 10PN, and exhibited unique patterns of immunoreactivity with the antibodies used in this analysis. By 25PN the retina was well laminated, and synaptotagmin-, Rab3A-, synaptophysin- and SNAP-25-like immunoreactivities exhibited distinct and specific patterns within the plexiform layers, although they had not yet achieved their mature, adult patterns. These results indicate that each of these proteins exhibits developmentally regulated changes in its cellular localization, and therefore may play important roles during morphogenesis and synaptogenesis of the vertebrate retina.

Steroid autoradiography of the sexually dimorphic nucleus of the preoptic area

Autoradiography was performed to determine if the neurons of the sexually dimorphic nucleus of the preoptic area (SDN-POA) in the adult rat accumulate estradiol (E2), testosterone (T), and/or dihydrotestosterone (DHT). Three days prior to steroid administration, adult male and female Sprague-Dawley rats were gonadectomized and adrenalectomized. Animals were then given either [3H]T, [3H]E2, or [3H]DHT through an indwelling jugular cannula. One hour later, animals were decapitated and brain sections processed for thaw mount autoradiography. The autoradiograms which contained the SDN-POA and an adjacent area of the medial preoptic area (MPOA) were quantitatively analyzed using the 3 times background, 5 times background, and Poisson criteria for labeled cells. In general, cells in the SDN-POA and the MPOA accumulate T, E2, or DHT. For both sexes, there is a greater percentage of labeled cells in the SDN-POA than in the MPOA, and a greater percentage of labeled cells following E2 exposure than following T or DHT exposure. In addition, there is a sex difference (male greater than female) in the percentage of labeled cells following T exposure. In summary, these data indicate that adult SDN-POA neurons do accumulate gonadal steroids.

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.