Development of the facial and hypoglossal motor nuclei in the neonatal Brazilian opossum brain.

The development of the facial and hypoglossal motor nuclei were examined in the neonatal Brazilian opossum (Monodelphis domestica), a marsupial in which postnatal central nervous system development has been well characterized. In this study, we utilized postnatal injection of the retrograde tracer cholera toxin subunit B (CtB) to characterize the formation of the facial and hypoglossal motor nuclei in the developing neonatal opossum brainstem. Injections of CtB were made into the cheek/lip region or tongue of opossum pups to retrogradely label the facial or hypoglossal motor nuclei, respectively. Following a 2 h survival time, facial motoneurons in newborn opossum pups (1 PN) exhibited CtB labeling, with their cell bodies localized near the developing cranial abducens nucleus. At 3 and 5 PN, following a 48 h survival time, CtB-labeled facial motoneurons were observed in and migrating to the region of the adult facial motor nucleus in the rostral medulla. Between 7 and 10 PN, almost all facial motoneurons had migrated to their destination within the facial motor nucleus. Hypoglossal motoneurons also exhibited CtB labeling from 1 PN; however, their cell bodies were localized within the hypoglossal motor nucleus at the earliest age examined. Double label studies, to examine guidance of facial motoneurons during migration, demonstrated that CtB-labeled facial motoneurons are in close proximity to vimentin-like immunostained radial glial fibers during migration. These results suggest: (1) migration of facial motoneurons to the facial motor nucleus is a postnatal event, (2) efferent projections from facial and hypoglossal motoneurons project into the peripheral region of their target muscles from the day of birth, and (3) facial motoneurons migrate to their destination in the brainstem thereafter, in close association with radial glial fibers.

Antiporcine relaxin (antipRLX540) treatment decreases relaxin plasma concentration and disrupts delivery in late pregnant pigs.

Antibody against porcine relaxin (antipRLX540; 1:950,000) was produced in sheep and used to determine the effect on relaxin and progesterone secretion, and on parturition in late pregnant pigs. In group 1, Yorkshire gilts with normal estrous cycles were bred on the second observed estrus and fitted with an indwelling jugular cannula and an intraperitoneal cannula on day 100 of pregnancy. Gilts were infused at 6-h intervals with antipRLX540 (n = 10) or PBS (n = 10) beginning on day 103 until parturition. From days 103 to 120, daily blood samples (10 ml) were collected for RIA of relaxin, progesterone, and prolactin. In group 2, bred gilts were randomly assigned to antipRLX540 (n = 11), relaxin (n = 5), and PBS (n = 8) treatment on days 111, 113, and 115. Blood was collected twice daily from day 108 to 120, and every 20 min on days 111, 113, and 115 beginning 60 min before treatment and continuing 180 min. Parturition in gilts given antipRLX540 occurred on day 112.7 compared with day 114.0 in relaxin-treated gilts and day 114.3 in PBS controls (P < 0.05). Duration of delivery from first to last piglet was greatly delayed in antipRLX540 gilts (240 min) compared with PBS controls ([117 min] P < 0.005). Average number of stillborns was greater in antipRLX540- than in PBS-treated controls (2.4 vs. 1.0; P < 0.05). Relaxin concentration in peripheral plasma was lower in antipRLX540-treated gilts from day 105 to 110, but on day 113 the antipRLX540-treated group had a greater relaxin peak release compared with PBS-treated animals (P < 0.05). Plasma progesterone concentrations were similar in antipRLX540- and PBS-treated gilts throughout the period of the study. In group 2, by day 113, progesterone decreased in antipRLX540-treated gilts compared with relaxin- and PBS-treated gilts. Prolactin levels were similar in both antipRLX540- and PBS-treated gilts; however, from 1 to 3 days postpartum the antipRLX540 group had higher prolactin concentration (P < 0.05). The results indicate that antipRLX540 decreased circulating plasma concentrations of unbound or free relaxin during the last 10 days of pregnancy in Yorkshire gilts. AntipRLX540 markedly increased both the duration of delivery of piglets and the average number of stillbirths in this litter-bearing species compared with PBS-treated controls. This study provides strong evidence that increasing circulating concentrations of relaxin during late pregnancy is crucial for unimpaired parturition in the pig.

Transient, high levels of SNAP-25 expression in cholinergic amacrine cells during postnatal development of the mammalian retina.

In the present study, we have examined the development of cholinergic amacrine cells in the retina of the Brazilian opossum, Monodelphis domestica. An antibody directed against choline acetyltransferase (ChAT) revealed that ChAT-like immunoreactivity (ChAT-IR) was first observed at 15 days postnatal (15PN). By 25PN, ChAT-IR identified two matching populations of amacrine cells in the inner nuclear and ganglion cell layer. Bromodeoxyuridine birth-dating analysis coupled with immunolabeling with the anti-ChAT antibody revealed that the cholinergic amacrine cells are born postnatally, between 2PN and 15PN. In addition, we have examined the differentiation of the cholinergic amacrine cells by using an antibody directed against a presynaptic terminal-associated protein, synaptosomal-associated protein of 25 kDa (SNAP-25). Double-labeling analysis revealed that relatively high levels of SNAP-25-IR were selectively present in cholinergic amacrine cells prior to eye opening. However, in the mature retina, high levels of SNAP-25-IR were no longer observed in the ChAT-IR amacrine cells. These results reveal a distinct period in development, prior to eye opening, when high levels of SNAP-25-IR are selectively expressed in cholinergic amacrine cells. The specificity and time course of the high levels of SNAP-25 in cholinergic amacrine cells may be critical in mediating the transient properties of these cells during visual system development.

Prostaglandin F2alpha-induced luteolysis of aging corpora lutea in hysterectomized pigs.

Prostaglandins primarily of uterine origin play an important role in parturition. Hysterectomy of nongravid pigs early in the luteal phase maintains luteal function until about Day 150, whereas the duration of normal pregnancy is about 114 days. A precisely timed peak release of relaxin and coincident decrease in progesterone secretion in unmated hysterectomized gilts are similar to hormonal changes that occur a few hours before parturition. It is hypothesized that prostaglandin F2alpha (PGF2alpha) in hysterectomized pigs mimics abrupt changes in ovarian and pituitary hormone secretion seen before normal parturition and in early lactation. Unmated Yorkshire gilts were hysterectomized on Days 6-8 of a normal estrous cycle, and at 1200 h on Day 113, they were given an i.m. injection of 30 mg PGF2alpha-trihydroxymethylaminomethane (THAM) salt or PBS. None of these gilts expressed behavioral estrus immediately after PGF2alpha or vehicle treatment. On Day 113, PGF2alpha increased peak relaxin (60 ng/ml) compared with that of controls (34 ng/ml; p < 0.01), whereas progesterone decreased abruptly (4 vs. 16 ng/ml in PGF2alpha and PBS; p < 0.01). Prolactin remained at < 5 ng/ml from Day 98 to 120 in controls but peaked at 33 ng/ml immediately after PGF2alpha treatment on Day 113, and then decreased to levels similar to those of controls on Day 120. Sequential bleeding revealed an acute growth hormone release (4.5 ng/ml) immediately after PGF2alpha injection and return to basal levels (< 0.6 ng/ml) on Days 114-120. PGF2alpha induced abrupt shifts in progesterone, relaxin, prolactin, and growth hormone secretion in hysterectomized gilts that mimicked hormone changes seen in late pregnancy, parturition, and early lactation. These findings provide new insight into the role of PGF2alpha in abruptly changing hormone secretions by aging corpora lutea and the pituitary gland even in the absence of conceptuses or the uterus in the pig.

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.

Ultrasonographic pregnancy detection and inhalation anesthesia in the gray short-tailed opossum (Monodelphis domestica).

Female gray short-tailed opossums (Monodelphis domestica) with known breeding dates were anesthetized with isoflurane for ultrasonographic pregnancy detection. Beginning on gestational day 9, gravid females could be identified using a 9-MHz mechanical sector-scanning transducer equipped with a standoff pad. Fluid-filled vesicles 3 to 4 mm in diameter were seen within the thick-walled uteri on gestational days 9 and 10. Visualization revealed loss of individual vesicles, with replacement by thick, irregular uterine walls and some free luminal fluid by gestational days 12 and 13. On the basis of subsequent birth of pups, sonographic diagnosis of pregnancy was accurate in 27 of 28 oppossums examined.

Ontogeny of androgen receptor-like immunoreactivity in the reproductive tract of male Monodelphis domestica.

The distribution of nuclear androgen receptor-like immunoreactivity was studied in the reproductive tract of the developing and adult gray short-tailed Brazilian opossum (Monodelphis domestica), using the well-characterized rabbit polyclonal androgen receptor antibody, PG21. Androgen receptor-like immunoreactivity was first detected on the fifth day of postnatal age, in the mesenchymal tissues of the ductus deferens, gubernaculum testis, inguinal, and scrotal areas; the urogenital sinus; and the genital tubercle. Androgen receptor-like immunoreactivity was first seen in the interstitial cells of the epididymis at 45 days of age; the testes developed androgen receptor-like immunoreactivity at 60 days of age. The epithelium of prostatic glands contained androgen receptor-like immunoreactivity only in the adult. The presence of androgen receptor-like immunoreactivity during development correlated well with the known androgen dependence of the differentiation of most reproductive organs.

Autoradiographic localization of arginine vasopressin binding sites in the brain of adult and developing Brazilian opossums.

We are utilizing the Brazilian short-tailed opossum, Monodelphis domestica, to study the development of the vasopressinergic system. Earlier studies demonstrated that arginine vasopressin-like immunoreactivity was present very early in the Brazilian opossum brain, suggesting a role for vasopressin in the developing central nervous system of mammals. In this study, we have utilized [3H]arginine vasopressin autoradiography to describe the distribution of arginine vasopressin binding sites in adult and developing Brazilian opossum brains. In general, arginine vasopressin binding patterns in adult opossum brains resembled those of other species. However, we found very few labelled areas in neonatal Brazilian opossum brains. At birth, only the ventral tegmental area and the nucleus of the solitary tract were labelled. Binding was not evident in the forebrain until 25 days of postnatal age. The anterior pituitary was heavily labelled from birth onward, but binding in the brain itself remained at low levels until 35 days postnatal. Heavy binding was observed in only a few areas of the brain in adults, including the dorsal part of the lateral septal nucleus, the suprachiasmatic nucleus, the dorsal and median raphe, the nucleus of the solitary tract, and the caudal part of the spinal trigeminal nucleus. Surprisingly, arginine vasopressin binding sites in the Brazilian opossum appeared much later than arginine vasopressin immunoreactivity and, in many cases, after neurogenesis was complete. These findings suggest that the arginine vasopressin binding sites are not playing a developmental role in opossums, although the peptide is present at an early age.

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.

Replication and pathogenicity after intranasal and intracranial inoculation of swine with a recombinant pseudorabies virus containing a deletion at the UL/IR junction.

Pseudorabies virus (PRV) is a neurotropic herpesvirus of swine. Previously, we described construction of a recombinant strain of PRV (LLT beta delta 2) which contains a 3.0-kb deletion spanning the junction of the unique long and internal repeat sequences. Compared to the parental strain, Indiana-Funkhauser, and a virus rescued for the deleted sequences (LLT beta res), LLT beta delta 2 replicated efficiently at the site of inoculation, yet exhibited significantly reduced virulence when inoculated intranasally in pigs. In this report, we investigated the effect of the deletion on PRV replication and virulence after intracranial inoculation of swine, in comparison to replication and virulence after intranasal inoculation, in order to more precisely locate the defect in LLT beta delta 2. Four-day-old pigs were infected intranasally with LLT beta delta 2 or LLT beta res and necropsied at various times postinfection. Compared to LLT beta res-infected pigs, tissue distribution of virus, PRV antigen, and lesions of LLT beta delta 2-infected pigs were comparable in all peripheral tissues examined, including trigeminal ganglia, but were reduced in tissues from the central nervous system (CNS). LLT beta delta 2 was able to replicate in the CNS after intracranial inoculation into the cerebral cortex of 2-day-old piglets and to spread from CNS to peripheral tissues. Neurovirulence of LLT beta delta 2 was somewhat reduced, as demonstrated by delayed onset of neurological signs and death in intracranially inoculated pigs. These results indicate that decreased neurovirulence after intranasal inoculation is not due to inability of LLT beta delta 2 to replicate in CNS tissues. The difference in the amount of antigen detected in CNS tissues after intracranial inoculation compared to intranasal inoculation suggests that one defect in LLT beta delta 2 is reduced ability to spread from peripheral neurons to the CNS after intranasal inoculation.

The prepubertal ontogeny of luteinizing hormone releasing hormone-like immunoreactivity in the diencephalon and telencephalon of the male Meishan pig brain.

Luteinizing hormone releasing hormone (LHRH) is a decapeptide that regulates reproductive function and behaviors in mammalian species. Because of the importance of proper reproductive function and efficiency in agricultural species, we have investigated the prepubertal ontogeny of LHRH-like immunoreactivity (IR) in the male Meishan pig. The Meishan is a Chinese breed known for reproductive traits including increased litter size and precocious puberty, but slow growth and obesity. Brains of animals from gestational day (g) 30, 50, 70, 90 and 110 and postnatal day (pn) 1, 10, 20 and 50 (duration of pregnancy averaged 114 days) were processed using a standard immunohistochemical technique utilizing a commercially available rabbit anti-LHRH antibody. LHRH-IR in cell bodies and fibers was detected at g30 entering the brain via the terminal nerve and in the septal region of the basal telencephalon. The number of immunoreactive cells increased at g50 and cells were localized primarily to the septum, organum vasculosum of the lamina terminalis, preoptic area and lateral hypothalamus, whereas immunoreactive fibers were present throughout the septum and hypothalamus and had reached the median eminence. The density and distribution of immunoreactive fibers increased by g70 and g90, but did not change dramatically from g90 to pn50. These results indicate that LHRH may be present in the Meishan brain earlier during development and fibers containing LHRH-IR appear to reach the median eminence earlier than previously reported for the domestic pig. These results suggest a breed difference in the ontogeny of reproductive control systems in the pig. Future studies utilizing direct comparisons between domestic and Chinese breeds will be required to investigate the apparent differences in the ontogeny of LHRH-containing systems in the pig.

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 micrograms/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.

The prepubertal ontogeny of galanin-like immunoreactivity in the male Meishan pig brain.

Galanin (GAL) is a neuropeptide found in the mammalian brain and is involved in numerous functions including the control of feeding, growth and reproduction, and therefore may be an important peptide to study in agricultural species. We describe the immunohistochemical localization of GAL throughout prepubertal development in the Meishan pig, a Chinese breed known for its superior reproductive characteristics, but slow growth rate and abundant adipose tissue. Brains of animals from gestational day (g) 30, 50, 70, 90 and 110 and postnatal day (pn) 1, 10, 20 and 50 (duration of pregnancy averaged 114 days) were processed using a standard immunohistochemical technique utilizing a commercially available rabbit anti-porcine GAL antibody. Galanin-like immunoreactivity (GAL-IR) in cell bodies and fibers was evident in the brain at g30, primarily in the hypothalamus. Throughout prenatal development, cell bodies containing GAL-IR generally increased in number and distribution in the brain. During postnatal development, the number of cell bodies displaying GAL-IR decreased, particularly in hypothalamic areas. The distribution of GAL-IR in fibers became more widespread throughout gestational development, showing a pattern by pn1 that continued during later postnatal ages. The intensity of GAL-IR in fibers also increased throughout gestation. Some additional increases in immunoreactivity occurred postnatally, especially in the periventricular hypothalamus. The results of this study indicate that the distribution of GAL-IR in cell bodies and fibers in the Meishan pig brain was similar to that seen in other species, including the rat. These results support the hypothesis that GAL participates in the control of feeding, growth and reproduction in the pig.

Characterization and ontogeny of synapse-associated proteins in the developing facial and hypoglossal motor nuclei of the Brazilian opossum.

The characterization and ontogeny of synapse-associated proteins in the developing facial and hypoglossal motor nuclei were examined in the Brazilian opossum (Monodelphis domestica). Immunohistochemical markers utilized in this study were the synaptic vesicle-associated proteins synaptophysin and synaptotagmin; a synaptic membrane protein, plasma membrane-associated protein of 25 kDa (SNAP-25); a growth cone protein, growth-associated phosphoprotein-43 (GAP-43); and the microtubule-associated proteins axonal marker tau and dendritic marker microtubule-associated protein-2 (MAP-2). In this study, we have found that, during the first 10 postnatal days (1-10 PN), the facial motor nucleus lacked immunoreactivity for synaptophysin, synaptotagmin, GAP-43, tau, and SNAP-25. After 10 PN, immunoreactivity increased in the facial motor nucleus for synaptophysin, synaptotagmin, GAP-43, and tau, whereas immunoreactivity for SNAP-25 was not evident until between 15 and 25 PN. Conversely, immunoreactivity for MAP-2, was present in the facial motor nucleus from the day of birth. In contrast, the hypoglossal motor nucleus displayed immunoreactivity from 1 PN for synaptophysin, synaptotagmin, SNAP-25, GAP-43, tau, and MAP-2. These results suggest that the facial motor nucleus of the opossum may not receive afferent innervation as defined by classical synaptic markers until 15 PN and, further, that characteristic mature synapses are not present until between 15 and 25 PN. These results indicate that there may be a delay in synaptogenesis in the facial motor nucleus compared to synaptogenetic events in the hypoglossal motor nucleus. Because the facial motor nucleus is active prior to completion of synaptogenesis, we suggest that the facial motoneurons are regulated in a novel or distinct manner during this time period.

The prepubertal ontogeny of neuropeptide Y-like immunoreactivity in the male Meishan pig brain.

Neuropeptide Y (NPY) is widely distributed in the mammalian brain and is involved in numerous functions including the control of feeding, growth and reproduction. Therefore, NPY may be an important peptide to study in agricultural species. This study describes the immunohistochemical localization of NPY throughout prepubertal development in the Meishan pig, a Chinese breed known for its superior reproductive characteristics. Brains of animals from gestational day (g) 30 through postnatal day (pn) 50 (duration of pregnancy averaged 114 days) were processed using a standard immunohistochemical technique utilizing a commercially available rabbit anti-porcine NPY antibody. Neuropeptide Y-like immunoreactivity (NPY-IR) in cell bodies and fibers is evident in many areas of the brain at g30, including the basal telencephalon, hypothalamus, mesencephalon, pons, and medulla. Throughout prenatal development, cell bodies containing NPY-IR generally increase in number and distribution in the brain. During postnatal development the number of cell bodies displaying NPY-IR decreases. The arcuate nucleus of the hypothalamus, shows a dramatic reduction in the number of immunoreactive cell bodies between pn1 (day of birth) and pn20, just before weaning. The distribution of NPY-IR in fibers becomes more widespread throughout gestational development, showing a pattern by g110 that was characteristic of postnatal ages. The intensity of NPY-IR in fibers also increases throughout gestation. Some additional increases in immunoreactivity occur postnatally, especially in the periventricular hypothalamus and the hippocampus. Other brain areas like the caudate nucleus and putamen show decreases in immunoreactivity postnatally. The distribution of NPY-IR in cell bodies and fibers is similar to that seen in other species, including the rat, and supports the hypothesis that NPY participates in controlling feeding, growth and reproduction in the pig.

Ontogeny of cholecystokinin binding sites in the hindbrain of the laboratory rat.

Studies in our laboratory have revealed a robust, transient expression of cholecystokinin binding sites in the facial motor nucleus during development in the Brazilian opossum, Monodelphis domestica. To investigate the ubiquity of this phenomenon, we have performed receptor autoradiography on the hindbrains of embryonic and neonatal rat pups. Cholecystokinin binding sites are present at very low levels in the embryonic day-16 rat hindbrain, but binding sites are abundant prior to birth. The greatest increase in labelled nuclei occurs prior to 5 days of postnatal age. Binding levels are heavy in the nucleus of the solitary tract, medial vestibular nucleus, posterior dorsal tegmental nucleus, area postrema, and caudal spinal trigeminal nucleus by 30 days postnatal. Both A-type and B-type receptors are present in the neonatal brainstem, although most labelled areas appear to be B-type. A-type binding sites are present in the ventral cochlear nucleus, the nucleus of the solitary tract, the dorsal motor nucleus of the vagus, the area postrema, the spinal nucleus of the trigeminal, and the cuneate and gracile nuclei by 5 days postnatal. As reported for the Brazilian opossum, cholecystokinin binding sites are expressed in the facial motor nucleus of neonatal rats and are transient. In this study of the brainstem in laboratory rats, a transient expression is also observed in the rubrospinal tract, parvocellular reticular nucleus, raphe obscurus, cuneate and gracile nuclei, and the ventral median fissure of the spinal cord. As vasopressin binding sites and estrogen receptors have also been shown to be expressed transiently in the laboratory rat facial motor nucleus, the physiological and developmental significance of transient binding site expression remains to be elucidated.

Ontogeny of oxytocin-like immunoreactivity in the Brazilian opossum brain.

The neuropeptide oxytocin (OT) has been shown to function as a neurotransmitter and/or neuromodulator in addition to its hormonal function in the periphery in the adult central nervous system (CNS). Previously, we have studied the postnatal neurogenesis of the paraventricular and supraoptic nuclei and ontogeny of arginine vasopressin-like immunoreactivity in the Brazilian opossum brain, Monodelphis domestica. In this study, we have described the ontogeny of oxytocin-like immunoreactivity (OT-IR) in the opossum brain. As a marsupial, opossum pups are in an extremely immature state, with neurogenesis and morphogenesis continuing into the second week of postnatal life. Thus, opossum pups are a good model for developmental studies. In the adult opossum brain, OT-IR was localized in regions as reported for the adult rat and other species, except for a few differences. These findings suggest similar functional roles for OT in the adult opossum brain as in other mammals. Unlike the prenatal expression of arginine vasopressin, OT-IR was first detected in the forming median eminence on day 1 of postnatal life (1 PN). Between 3 and 5 PN, OT-IR was present in the hypothalamic supraoptic and paraventricular nuclei and posterior pituitary. At this time, neurogenesis of these nuclei is not completed. By 10 to 15 PN, OT-IR was seen in several brain areas, and begins to resemble that of the adult between 45 and 60 PN. These results indicate that the time course of appearance of the OTnergic system does not directly parallel the early expression of the vasopressinergic system. However, the expression of OT-IR in the opossum brain before neurogenesis and morphogenesis is completed suggests a potential role for OT in developmental events. Similar to arginine vasopressin, oxytocin may also be involved in the regulation of autonomic functions that are essential for the opossum's adaptation to an ex utero environment. Future studies utilizing experimental manipulations of the OTnergic system will help determine the significance of this peptide in the neonatal opossum.

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.

Reduced physeal area and chondrocyte proliferation in Pasteurella multocida toxin-treated rats.

Pasteurella multocida toxin depresses weight gain in rats and pigs. It also affects tissues with rapidly dividing cells. In the present study, we investigated the role of this protein toxin on chondrocyte growth in vivo. Rats were divided into a single- or multiple-dose group and were given, respectively, either a single injection (0.15 or 0.6 micrograms/kg toxin subcutaneously) or multiple injections (0.01-0.2 micrograms/kg subcutaneously) of toxin. Bone (humerus) and other selected tissues were stained for bromodeoxyuridine immunoreactivity (BrDU-IR) in order to gauge cell proliferation. Physeal area was measured in rats from the multiple-dose group. Serum from single- and multiple-dose groups were tested for tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) activity using a bioassay system. Decreased weight gain, feed intake, and feed efficiency were observed in single- and multiple-dose groups of rats. Decreased BrDU-IR indices were present in the resting and proliferative zone chondrocytes of the humeral physis in rats from the multiple-dose group, as was decreased physeal area. Increased serum IL-6 bioactivity was present in rats after 24 hours, and no changes in TNF-alpha bioactivity were seen in any group. No alterations in BrDU-IR were seen in rats fed restricted (80% of control) diets. These studies show that sublethal doses of toxin decrease weight gain and affect growth of long bones through suppression of chondrocyte proliferation. These effects may be mediated by direct binding of the toxin to target cells or IL-6 but are not associated with altered feed intake or TNF-induced cachexia.

Ontogeny of arginine vasopressin-like immunoreactivity in the Brazilian opossum brain.

The neuropeptide arginine vasopressin is involved in many centrally mediated functions and brain development. In this study, we have examined the ontogeny of arginine vasopressin-like immunoreactivity (AVP-IR) in the Brazilian opossum (Monodelphis domestica) brain to further understand the involvement of AVP in the forming central nervous system. Monodelphis is a small pouchless marsupial and its pups are born in an extremely immature state before neurogenesis is completed. In the adult brain, cell bodies containing AVP-IR were found in several nuclear groups and areas, and immunoreactive fibers were found to be widely distributed throughout the brain. The distribution of AVP-IR in the adult opossum brain generally resembled that reported for other species including the rat, however, some differences in localization of immunoreactive cells were observed. In the developing opossum brain, AVP-IR was first seen in the mesencephalon and diencephalon between embryonic days 12 and 13. Subsequently, a distinct group of AVP immunoreactive cells was present in the forming supraoptic nucleus on day 1 of postnatal life (1 PN) and at 3 PN in the paraventricular nucleus. Between 1 and 3 PN, a few cells transiently expressed AVP-IR in the forming thalamus and tegmental area. At these ages a few immunoreactive fibers were also detected in the forming cerebellum. These fibers were not seen at later ages in these areas. By 5 PN, an increased expression of AVP-IR was seen in the forming supraoptic and paraventricular hypothalamic nuclei, median eminence, and posterior pituitary. At 7 PN, immunoreactive cells and fibers were seen in several forebrain areas. The distribution pattern of AVP-IR became adult-like by 60 PN. A sex difference in the amount of AVP-IR in the lateral septum was also observed in the opossum brain at 60 PN. This difference persisted in the adult brain. Due to the early presence of AVP-IR in the Monodelphis brain before neurogenesis and morphogenesis is completed, we suggest that AVP may be involved in morphogenesis of the central nervous system. In addition, AVP may have a significant physiological function in regard to homeostasis before the forebrain contributes to these control mechanisms. Further studies, including physiological and developmental manipulations, will define the significance of the early presence of AVP during the differentiation and maturation of the central nervous system in Monodelphis.