Maternal regulation of the hypothalamic-pituitary-adrenal axis in the 20-day-old rat: consequences of laboratory weaning.

There is a large body of evidence that the development of the hypothalamic-pituitary-adrenal (HPA) system in the rat is under maternal regulation. One method used to study the influence of the dam-pup interaction in neonates and weanlings is the separation of mother and litter for 24 h. Previous studies showed that, even at the time of weaning, maternal deprivation results in a dysregulation of the HPA axis at multiple levels. However, the maternal deprivation paradigm usually includes deprivation of food and water, and it was not clear to which extent the observed effects are due to either maternal cues or dehydration and fasting. The primary purpose of the present study was to determine the role of fasting and/or maternal separation on the HPA axis at the time of weaning. Pups at 20 days after parturition are capable of self-feeding and no longer require tactile stimulation to induce eliminative functions. The results indicated that 24 h of fasting led to increased basal levels and further increases in stress induced corticosterone secretion. Fasting also appeared to contribute to the down regulation of basal glucocorticoid receptor mRNA in the hippocampus. In contrast, abrupt weaning irrespective of fasting or dehydration resulted in a suppressed adrenocorticotropin hormone response to an injection of isotonic saline. Although there was an effect of maternal separation on corticotropin-releasing factor mRNA in the paraventricular nucleus, this effect was further exacerbated by the absence of food. Finally, all rats that were separated from their dams showed more efficient negative-feedback. Thus, different aspects of the HPA system appear to respond differentially to either the absence of food or the absence of the mother or both.

Gene expression profiling in the amygdala: an approach to examine the molecular substrates of mammalian behavior.

The molecular substrates of behavior have been difficult to assess because of the large number of messenger RNAs (mRNAs) expressed in a given brain region, the heterogeneous composition of the CNS, and the complexity of mammalian behavior. To gain insight into the molecular components of behavior requires an understanding of the anatomy associated with a specific behavior and the ability to examine multiple gene expression in discrete brain regions. Neuroanatomical and behavioral studies have demonstrated that the amygdaloid complex is an essential component of the neural pathways mediating behaviors, such as fear, anxiety, learning, and memory. The amygdala is composed of several interconnected subnuclei and it is the modulation of information, as it flows through these subnuclei, that underlies amygdala function. To examine the molecular components of the amygdala, we have combined the antisense RNA (aRNA) amplification procedure with microarray technology. This experimental approach permits the simultaneous detection and quantification of numerous mRNAs in fixed tissue sections. Our initial experiment examines region-specific gene expression in naïve mice in order to map the molecular relationship between the subregions of the amygdala. This report provides a general overview of the techniques used to examine regional gene expression, suggests future experiments, and describes a theoretical framework for examining the molecular analysis of behavior.

The acetylcholine release enhancer linopirdine induces Fos in neocortex of aged rats.

Centrally acting cholinergic agents induce the immediate early gene c-fos in the rat brain resulting in transient increases of Fos protein, most notably in the cerebral cortex. In this study we have monitored by Fos immunohistochemistry the effect of the acetylcholine release enhancer linopirdine (DUP996) on the immediate early gene c-fos in brains of 3 months and 30 months old rats. In young rats linopirdine had only a marginal effect on Fos expression. In contrast, in aged rats linopirdine caused widespread expression of Fos throughout neocortex. In somatosensory cortex, the induction of the c-fos gene by linopirdine was nearly completely blocked by atropine and scopolamine and strongly attenuated by the NMDA receptor blockers CPP and MK-801. The results suggest that the age-related decline in acetylcholine release in rodents can be partially compensated for by administration of linopirdine.

Stress-induced alterations in locus coeruleus gene expression during ontogeny.

Brainstem noradrenergic neurons, particularly the locus-coeruleus (LC), play a pivotal role in modulating the central stress response and have been implicated in regulating the hypothalamic-pituitary-adrenal (HPA) axis. In adult rats, acute stress causes an increase in LC firing and tyrosine hydroxylase (TH) gene expression. While the role of the LC-norepinephrine (LC-NE) system in the adult stress response has been well characterized, there is limited evidence for its participation during development. Previous studies described the neonatal HPA axis as hyporeactive because of stimulus-selective pituitary activation. However, maternal deprivation does reinstate stress-induced endocrine activity and can amplify the neural stress response. Considering that LC neurons can modulate neuroendocrine activity, we hypothesized that the LC-NE system would be stress-responsive during development. Because maternal deprivation (DEP) can alter the central stress response, we examined the LC-NE stress response in both DEP and non-deprived (NDEP) pups. Following an isotonic saline injection (stressor) the time course of TH, c-fos and glucocorticoid receptor (GR) mRNA was examined. Stress-induced TH mRNA was increased in DEP pups at postnatal day (pnd) 12 and in both NDEP and DEP pups at pnd 18. At 15, 30 and 240 min c-fos mRNA was markedly increased in all groups examined. GR mRNA was not altered at pnd 12; however, at pnd 18 NDEP pups showed reduced GR mRNA expression. These data indicate that during ontogeny the LC-NE system is stress-responsive to an acute mild challenge. Activation of LC-NE neurons suggests that this system may participate in modulating the neuroendocrine stress response during development.

Stress-induced alterations in corticotropin-releasing hormone and vasopressin gene expression in the paraventricular nucleus during ontogeny.

From postnatal day (PND) 4 to 14, neonates display a minimal pituitary-adrenal response to mild stress, the so-called 'stress hyporesponsive period' (SHRP). During the SHRP, maternal deprivation (MD) alters the pituitary-adrenal system, enabling neonates to become endocrine responsive to specific stimuli. We have previously reported that during the SHRP, mild stress enhances corticotropin-releasing hormone (CRH) messenger RNA (mRNA) expression in the paraventricular nucleus (PVN). Insofar as elevated CRH mRNA was observed both in the presence and absence of adrenocorticotropin (ACTH) release, we hypothesized that other ACTH secretagogues may participate in the pituitary stress response. During the SHRP, does arginine vasopressin (AVP) complement the actions of CRH which might be reflected centrally by the enhanced biosynthesis of both neuropeptides? To test this hypothesis we examined the time course of stress-induced CRH and AVP mRNA in the PVN at PND 6, 12, and 18. As an index of neural activity, c-fos mRNA in the PVN was also examined. Restraint was used as the stressor and MD was employed to enable an endocrine response during the SHRP. Despite the absence of stress-induced ACTH, in nondeprived pups during the SHRP, CRH mRNA was rapidly enhanced. In their maternally deprived (DEP) counterparts, ACTH levels were increased, and a significant induction of CRH mRNA was only observed at day 12. AVP mRNA levels were elevated in DEP 12-day-old pups at 15, 30 and 60 min. In rats beyond the SHRP, plasma ACTH levels, CRH and AVP mRNA were all enhanced following restraint. At PND 18, elevated CRH mRNA was not observed until 4 h after stimulus. Following restraint, c-fos mRNA was increased at all three ages, although the magnitude of c-fos response was less during the SHRP. These results demonstrate that when restraint elicits prototypical ACTH release, the neonatal central response is to enhance the biosynthesis of both AVP and CRH. If nucleic acid changes correlate with release, the increased synthesis of both neuropeptides may indicate the potential for AVP to synergize with CRH during the neonatal stress response.

Rapid induction of corticotropin-releasing hormone gene transcription in the paraventricular nucleus of the developing rat.

Neonates from postnatal days (pnd) 4-14 display a minimal pituitary-adrenal response to mild stress, the so-called stress hyporesponsive period (SHRP). However, during the SHRP, maternal deprivation (deprived) alters the pituitary-adrenal system, enabling neonates to become endocrine responsive to specific stimuli. Although neonates do display stress-induced ACTH, there is limited evidence for enhanced CRH gene expression early in development. The present experiment examined whether a mild stimulus (isotonic saline injection) administered to deprived and nondeprived neonates would enhance CRH biosynthesis in the paraventricular nucleus. Using in situ hybridization we measured the time course of CRH heteronuclear RNA (hnRNA) and messenger RNA at 15, 30, and 240 min poststimulus. Pnd 6, 12, and 18 were included to examine the CRH gene response during and outside of the SHRP. Despite the minimal endocrine response of nondeprived pups during the SHRP, CRH hnRNA and messenger RNA were elevated at 15 min (all ages). Both transcripts were enhanced at 15-30 min in deprived (pnd 12 and 18) pups; however, the magnitude of the response was less than that in nondeprived pups. These data indicate that during ontogeny there is a rapid stimulus-induced CRH biosynthesis. Thus, during development, the central components of the hypothalamic-pituitary-adrenal axis may be stress hyperresponsive rather than hyporesponsive.

The ontogeny of the neuroendocrine response to endotoxin.

During development, the hypothalamic-pituitary-adrenal (HPA) axis is normally hyporesponsive between postnatal days (pnd) 4 and 14. This interval has been designated as the stress-hyporesponsive period (SHRP). Recent evidence indicates that the neonate can respond to selective stimuli, i.e., exposure to immune signals. The purpose of this study was to investigate the neural correlates of the neonatal stress axis in response to a stimulus that activates the pituitary-adrenal hormones. Thus, lipopolysaccharide (LPS) was administered to neonates at three ages (pnd 6, 12, and 18) during or after the SHRP. In an effort to understand the neonatal hypothalamic paraventricular nucleus (PVN) response to an endotoxin, we measured c-fos immunoreactivity and corticotrophin-releasing hormone (CRH) gene expression. At all ages tested, there was an increase in ACTH and corticosterone (CORT) following LPS compared to controls. During the SHRP, LPS treatment resulted in a marked increase in Fos-positive cells in the PVN, whereas a saline injection had no effect. However, at pnd 18, both LPS and a saline injection elicited equivalent PVN Fos expression. In contrast to the effect on Fos, LPS and a saline injection decreased CRH mRNA at pnd 6 and 12. Outside the SHRP, LPS resulted in an increase in CRH gene expression relative to saline-injected controls. Thus, while the LPS-induced activation of Fos protein and plasma hormones were concordant, CRH mRNA did not positively correlate with the peripheral response. This suggests that the SHRP is not absolute, and the brain is responsive to some stimuli during this period.

Intrastriatal and intraventricular injections of oligodeoxynucleotides in the rat brain: tissue penetration, intracellular distribution and c-fos antisense effects.

We have determined the time course, the spatial spread in brain tissue, and the intracellular distribution of biotin- and fluorescein-labeled phosphorothioate oligodeoxynucleotides (ODNs) following single injections into the rat striatum or the lateral ventricle. These time and space parameters were correlated with the ability of c-fos phosphorothioate antisense ODNs to suppress the induction of Fos protein by cocaine. A rapid and dose-dependent tissue penetration of labeled ODNs was observed following either intrastriatal or intraventricular injections of a constant sample volume. Inspection of tissue sections by confocal microscopy uncovered a distinct change in the intracellular disposition of labeled ODNs during the 24 h post-injection period. At 1, 6 and 12 h, the vast majority of the fluorescent signal was confined to the interstitial spaces throughout the zone penetrated by ODNs. Neuronal nuclei displayed faint labeling along the outer portion of the nucleus at 1 and 6 h post-injection. At these time-points, ODNs were not detected in the cytoplasm. By 16 h, ODNs were barely detectable in the extracellular space and absent from neuronal nuclei. Instead, ODNs were seen in large cytoplasmic granules of neurons throughout the tissue zone penetrated by the ODNs. Experiments with intrastriatal injections of antisense ODNs to c-fos mRNA revealed Fos suppression between 3 and 12 h, but not at 16 and 24 h. This combined analysis has revealed that (1) restricted tissue penetration by ODNs limits their antisense effects on protein expression, and (2) depletion of extracellular ODNs and sequestration of c-fos antisense ODNs into large intracellular granules coincides with the loss of their biological activity.

Corticotropin-releasing factor (CRF) receptors in infant rhesus monkey brain and pituitary gland: biochemical characterization and autoradiographic localization.

A large body of data suggests that the corticotropin-releasing factor (CRF) system serves to coordinate the autonomic, endocrine, immune and behavioral aspects of the stress response. In rats, the distribution of CRF receptors in brain and pituitary has been well characterized, however, little information is available in primates. In this study, CRF receptors were characterized by radioligand binding and localized using autoradiography with [125I]-oCRF in the pituitary gland and in discrete brain regions of 2-week-old, 12-week-old and adult rhesus monkeys. Autoradiographic localization studies in slide-mounted tissue sections in the 2- and 12-week-old monkeys demonstrated high CRF receptor densities in both anterior and intermediate lobes of the pituitary as well as in discrete regions of the brain. The distribution of CRF receptors in the anterior pituitary demonstrated a 'cluster-like' appearance reminiscent of corticotrope distribution. In contrast, receptors in the intermediate lobe were more uniformly distributed. No significant differences were evident in the pattern of localization or the number of CRF receptors in the pituitaries of 2- compared to 12-week-old animals. However, marked differences were observed in the dentate gyrus of the hippocampus. Receptors in this region were absent in 1- to 2-week old animals but quite dense by 11-12 weeks of age. Conversely, in the lateral and medial geniculate nuclei, high levels of CRF receptors were identified early in life that virtually disappeared by 11-12 weeks of age. Thus, there is considerable correspondence in the development of the CRF system between the rat and rhesus monkey and this presents further evidence for the functional role of this peptide in brain development.

Effects of the memory enhancer linopirdine (Dup 996) on cerebral glucose metabolism in naive and hypoxia-exposed rats.

Linopirdine [DuP 996; 3,3-bis(4-pyrindinylmethyl)-1-phenylindolin-2-one] represents a novel class of compounds which enhance depolarization-activated (but not basal) release of acetylcholine, dopamine and serotonin in brain slices and improve learning and memory in rodents. The effects of linopiridine on local cerebral glucose metabolism were studied by the quantitative autoradiographic 2-deoxy-D-[1-14C]glucose method. Linopirdine administration in naive rats (0.01, 0.1, or 1.0 mg/kg, s.c.) did not significantly alter cerebral glucose metabolism in any of the regions analyzed. Since linopirdine protects against hypoxia-induced passive avoidance deficits in rats, we also examined the effects of linopirdine on cerebral metabolism after the rats were exposed to 30 min of hypoxia. Glucose metabolism was not significantly altered after hypoxic exposure, except for a small increase in some brain regions. Linopirdine administered after hypoxia decreased glucose metabolism in the hippocampus, limbic cortex, ventral hippocampal commissure, medial septum, striatum, subthalamic nucleus, zona incerta, lateral habenula, cerebral cortex, cerebellar vermis and a few thalamic nuclei. Statistically significant effects of linopirdine on glucose metabolism were observed in 22 of 56 brain regions sampled. In hypoxia-exposed rats, linopirdine altered glucose metabolism in brain regions that are implicated in learning and memory and are affected in Alzheimer's disease. Several of the affected regions are associated with the cholinergic system and may play a role in the cognitive enhancing properties of linopirdine.