Soon Lee

Alcohol self‐administration acutely stimulates the hypothalamic‐pituitary‐adrenal axis, but alcohol dependence leads to a dampened neuroendocrine state

Clinical studies link disruption of the neuroendocrine stress system with alcoholism, but remaining unknown is whether functional differences in the hypothalamic‐pituitary‐adrenal (HPA) axis precede alcohol abuse and dependence or result from chronic exposure to this drug. Using an operant self‐administration animal model of alcohol dependence and serial blood sampling, we show that long‐term exposure to alcohol causes significant impairment of HPA function in adult male Wistar rats. Acute alcohol (voluntary self‐administration or experimenter‐administered) stimulated the release of corticosterone and its upstream regulator, adrenocorticotropic hormone, but chronic exposure sufficient to produce dependence led to a dampened neuroendocrine state. HPA responses to alcohol were most robust in ‘low‐responding’ non‐dependent animals (averaging < 0.2 mg/kg/session), intermediate in non‐dependent animals (averaging ∼0.4 mg/kg/session), and most blunted in dependent animals (averaging ∼1.0 mg/kg/session) following several weeks of daily 30‐min self‐administration sessions, suggesting that neuroendocrine tolerance can be initiated prior to dependence and relates to the amount of alcohol consumed. Decreased expression of corticotropin‐releasing factor (CRF) mRNA expression in the paraventricular nucleus of the hypothalamus and reduced sensitivity of the pituitary to CRF may contribute to, but do not completely explain, neuroendocrine tolerance. The present results, combined with previous studies, suggest that multiple adaptations to stress regulatory systems may be brought about by excessive drinking, including a compromised hormonal response and a sensitized brain stress response that together contribute to dependence.

Amyloid as a Depot for the Formulation of Long-Acting Drugs

Amyloids are highly organized protein aggregates that are associated with both neurodegenerative diseases such as Alzheimer disease and benign functions like skin pigmentation. Amyloids self-polymerize in a nucleation-dependent manner by recruiting their soluble protein/peptide counterpart and are stable against harsh physical, chemical, and biochemical conditions. These extraordinary properties make amyloids attractive for applications in nanotechnology. Here, we suggest the use of amyloids in the formulation of long-acting drugs. It is our rationale that amyloids have the properties required of a long-acting drug because they are stable depots that guarantee a controlled release of the active peptide drug from the amyloid termini. This concept is tested with a family of short- and long-acting analogs of gonadotropin-releasing hormone (GnRH), and it is shown that amyloids thereof can act as a source for the sustained release of biologically active peptides.

Ontogeny of the Rat Hypothalamic Nitric Oxide Synthase and Colocalization with Neuropeptides

The topographic ontogeny of nitric oxide synthase (NOS) within the paraventricular nucleus (PVN) of the rat hypothalamus was studied by nicotinamide adenine dinucleotide-diaphorase (NADPH-diaphorase) histochemistry. At Day 1 of postnatal life (P1), NOS-positive neurons were already present and achieved their maturity (in terms of perikarya number and dendritic arborization) about the time of weaning (P21). Across all ages studied (P1 to adulthood), intense NADPH-diaphorase staining was primarily confined within magnocellular cells of the PVN largely characterized by medium-sized (12-15 mum in diameter), ovoid bipolar neurons with prominent clear nuclei. To identify the neurosecretory cells of the adult PVN in which NOS was present, double-labeling studies were carried out via fluorescent immunocytochemistry. Magnocellular oxytocin (OT) and arginine vasopressin (AVP), as well as parvocellular corticotropin-releasing factor (CRF), were found to be colocalized with NOS. However, colocalization occurred significantly more frequently in OT-containing neurons, relative to AVP- or CRF-positive cells. Most of the colocalization occurring between NOS and OT was observed in the rostral constituent of the magnocellular subdivision of the PVN, as opposed to a more caudal defined PVN. To provide a distribution comparison of OT, AVP, and CRF to that of NOS in the adult PVN, in situ hybridization was carried out with (35)S-cRNA antisense probes for the aforementioned neuropeptides. The results obtained with this evaluation were correlated with NOS histochemistry in the same brain sections. As expected, specific labeling was observed for all three neuroactive substances over their topographically distinctive nuclei. Among these nuclei, labeling by the OT cRNA probe provided the closest topographical correlation of hybridized signal over NOS perikarya, thus reinforcing the tenet that a relatively small population of OT nerve cells are concurrently colocalized with the enzyme. Taken together, these results indicate that NOS is present in the PVN of the rat at all postnatal ages which we tested. They also indicate that among neurosecretory cells of the PVN, only OT prominently shared with NOS the same common nerve cell type. This suggests that NOS neurons may represent a distinct neuropil group among multiple neuroactive nuclei in the neuroendocrine hypothalamus. Finally, we demonstrate that NADPH-diaphorase histochemistry can be easily combined with immunocytochemical and in situ hybridization procedures to evaluate the colocalization and topographical distribution of NOS with other phenotypic neurons in the mammalian central nervous system.

Mechanisms of Hypothalamic-Pituitary-Adrenal Axis Stimulation by Immune Signals in the Adult Rata

Abstract: Immune stimulation increases the activity of the HPA axis, a phenomenon directly or indirectly mediated through cytokines. We have used two models, the peripheral administration of endotoxin (LPS) or turpentine‐induced tissue injury to show that corticotropin‐releasing factor (CRF) and vasopressin (VP), hypothalamic peptides released by cytokines, play a dominant role in the increased ACTH measured in these two paradigms. In turn, CRF and VP synthesis and/or release is modulated by catecholamines, prostaglandins (PGs), and nitric oxide (NO). These secretagogues are produced in the periphery and/or the central nervous system (CNS) in response to increased cytokine levels and act on CRF/VP neurons and nerve terminals. Finally, endotoxemia and local tissue inflammation may upregulate brain levels of tumor necrosis factor α, interleukin‐1β, and/or interleukin‐6, providing yet another mechanism through which the occurrence of systemic inflammation is conveyed to the brain. The relative importance of brain or peripheral intermediates appears to depend on the site at which cytokine levels are increased. We have shown, for example, that peripheral, but not brain, PGs are important in mediating the neuroendocrine influence of blood‐borne cytokines, while PGs in the CNS play a role in situations characterized by elevated brain immune proteins. NO, on the other hand, restrains the response of the HPA axis to circulating, but not brain cytokines. These results illustrate the complexity of the mechanisms involved in the stimulation of the HPA axis and suggest that their specific involvement depends on the type, intensity, and duration of immune stimulation.

Systemic endotoxin increases steady-state gene expression of hypothalamic nitric oxide synthase: comparison with corticotropin-releasing factor and vasopressin gene transcripts

The enzyme responsible for nitric oxide (NO) formation, NO synthase (NOS), is found in hypothalamic neurons that control ACTH secretion. This led to the hypothesis that brain NO may modulate the response of the hypothalamic-pituitary (HP) axis to various stimuli. We tested this hypothesis by measuring changes in constitutive (c) NOS mRNA levels in the hypothalamus of rats systemically injected with endotoxin, a lipopolysaccharide (LPS) that releases endogenous cytokines, and analyzed these results in the context of the appearance of ACTH-releasing secretagogues such as corticotropin-releasing factor (CRF) and vasopressin (VP), as well as CRF receptors type A (CRF-RA). We purposefully chose doses of LPS thought to only minimally disrupt the blood-brain barrier and not be accompanied by an endotoxin shock, so that the results we obtained did not primarily stem from abnormal passage of compounds into the brain, or non-specific stress. Three to four hours following LPS injection (100 micrograms/kg, i.v.), cNOS mRNA levels increased in the paraventricular nucleus (PVN) of the hypothalamus. LPS treatment also upregulated PVN CRF gene transcription (measured by CRF heteronuclear RNA) and increased steady-state gene expression of the immediate early genes (IEG) c-fos and NGFI-B, with the first changes noted 1-2 h after treatment. Transcripts of CRF receptors type A were present in the hypothalamus 6 h after endotoxin treatment. On the other hand, no alterations in cytoplasmic VP mRNA levels were noted in rats injected with LPS. Because the dose of LPS we used stimulates ACTH secretion within 30 min, our results suggest that systemic LPS acts first within the median eminence, where it stimulates peptidic nerve terminals. Neuronal activation of hypothalamic cell bodies takes place later, and whether this phenomenon is due to the production of brain neurotransmitters and/or cytokines, or whether it primarily results from increased demand on the synthetic machinery, remains to be established. These studies extend prior work showing that systemic LPS increases the neuronal activity of hypothalamic regions known for their involvement in the responses of the HP axis, and bring forth two important additional points. First, increases in CRF primary nuclear transcripts are delayed with regard to the temporal release of ACTH. This suggests, though it does not demonstrate, that under the experimental conditions we used, the first site of action of LPS is the median eminence. Second, the observation of increased cNOS gene expression following LPS treatment, and the presence of this enzyme in neurons that regulate ACTH secretion, bring support to the hypothesis that this gas plays an important function in mediating the HP axis response to an immune challenge.

Increased Activity of the Hypothalamic-Pituitary-Adrenal Axis of Rats Exposed to Alcohol in Utero: Role of Altered Pituitary and Hypothalamic Function

Prenatal exposure to ethanol (E) enhances the offsprings ACTH and corticosterone responses to stressors. Here, we determined the role of increased pituitary responsiveness and/or PVN neuronal activity in this phenomenon. Pregnant rats were exposed to E vapors during days 7-18 of gestation, and we compared the responses of their 55- to 60-day-old offspring (E rats) to those of control (C) dams. PVN mRNA levels of the immediate early genes (IEGs) c-fos and NGFI-B, which were low under basal conditions in all groups, showed a peak response 15 min after shocks and 45 min after LPS treatment. These responses were significantly enhanced in E, compared to C offspring of both genders. CRF, but not VP hnRNA levels were also significantly higher in the PVN of shocked E offspring. Resting median eminence content of CRF and VP, and pituitary responsiveness to CRF, were unchanged, while responsiveness to VP was marginally increased in females. These results indicate that prenatal alcohol selectively augments the neuronal activity of hypothalamic CRF perikarya.

Gender differences in the effect of prenatal alcohol exposure on the hypothalamic-pituitary-adrenal axis response to immune signals

Immature (3 week old) rat offspring of alcohol (E)-fed dams show a blunted ACTH response to immune signals such as interleukin-1 beta (IL-1 beta) and endotoxin (LPS). In contrast, mature offspring respond to physical stresses with an exaggerated activation of their hypothalamic-pituitary-adrenal (HPA) axis. The present work was aimed at determining if there was a differential influence of prenatal E exposure on the HPA axis responses to various stressors or if, alternatively, sexual maturation modified these responses. When administered IL-1 beta at 5 weeks age, E-treated intact male offspring released less ACTH, compared to control (C) or pair-fed (PF) animals. However, they showed an augmented response to LPS and a local inflammatory process induced by turpentine injection. At this same age, intact E females secreted significantly more ACTH in response to IL-1 beta, LPS and turpentine, than C or PF offspring. By 9 weeks of age, both E males and E females exhibited larger (p < .05) ACTH responses to all three immune stimuli. In order to determine whether sex steroids modulate the influence of E in females, ovariectomy was done prior to puberty. This treatment decreased the difference in the ACTH released by E and C rats in response to IL-1 beta, LPS and turpentine. These results show that while immature rats exposed to E prenatally released less ACTH in response to cytokines than C or PF animals did, this response was qualitatively reversed after puberty. At that time, the larger amounts of ACTH secreted by E offspring, compared to the other groups, were reminiscent of the hyperactive response of the HPA axis when these offspring were exposed to physical stress. Interestingly, removal of circulating ovarian steroids prevented the influence of E from being exerted. This suggests the presence of a functional relationship between the pathways influenced by prenatal E and those influenced by female sex steroids, that are important in regulating the activity of the HPA axis.

Isolation and Characterization of Novel Presenilin Binding Protein

Approximately 50% of familial Alzheimers disease (AD) cases are linked to the presenilin (PS) gene. This suggests that an altered function of mutated PSs accounts for a fundamental process leading to AD. Here we identify a new PS binding protein, PBP, which is highly expressed in cerebral cortex and hippocampus. immunohistochemical studies and cell fractionation analysis show that PBP redistributes from cytoplasm to membranes in the presence of PS. In addition, PBP is deficient in the soluble fraction of sporadic AD brains.

Stimulatory effect of cocaine on ACTH secretion: role of the hypothalamus.

The administration of cocaine to rats causes the release of adrenocorticotropin hormone (ACTH). The observation that immunoneutralization of endogenous corticotropin-releasing factor (CRF) significantly blunts this response has suggested the importance of CRF-dependent pathways in this response. As the paraventricular nucleus (PVN) of the hypothalamus represents a major source of the CRF delivered to the pituitary, the present studies investigated the role of this hypothalamic nucleus in mediating cocaine-induced ACTH secretion. We first observed that one single iv injection of 5 mg cocaine/kg, or two sequential sc injections of 40 mg cocaine/kg, caused a measurable increase in steady-state levels of CRF mRNA. Bilateral lesions of the PVN significantly (P < 0.01) decreased the ability of cocaine to release ACTH measured 15 min later. Though we failed to measure any changes in the levels of the mRNA encoding for vasopressin (VP) following either regimen of drug administration, we noted that rats in which the magnocellular portion of the PVN (which contains VP perikarya) was spared, showed a normal ACTH response to cocaine. We therefore studied the possibility that VP might regulate the activity of the corticotrophs in response to cocaine. However, prior iv injection of polyclonal antibodies against VP did not measurably alter cocaine-induced ACTH secretion, while in the same experiment, immunoneutralization of endogenous CRF completely blocked the stimulatory action of cocaine. Taken together, these results show that CRF of PVN origin represents the major modulator of ACTH released in response to cocaine, while VP does not appear to play an important role.

Endotoxin stimulates nitric oxide production in the paraventricular nucleus of the hypothalamus through nitric oxide synthase I: correlation with hypothalamic-pituitary-adrenal axis activation.

Nitric oxide (NO) is an unstable gas that is produced in brain tissues involved in the control of the activity of the hypothalamus-pituitary-adrenal (HPA) axis. Transcripts for constitutive neuronal NO synthase (NOS I), one of the enzymes responsible for NO formation in the brain, is up-regulated by systemic endotoxin [lipopolysaccharide (LPS)] injection. However, this change is delayed compared with LPS induced-ACTH release, which makes it difficult to determine whether it is functionally important for the hormonal response. To obtain a more resolutive time course of the NO response, we first measured NO in microdialysates of the paraventricular (PVN) nucleus of the hypothalamus. The iv injection of 100 microg/kg LPS induced a rapid and short-lived increase in concentrations of this gas, which corresponded to the initiation of the ACTH response. LPS-induced Ca2+-dependent NOS activity in the PVN as well as the number of PVN cells expressing citrulline (a compound produced stoichiometrically with NO) also increased significantly over a time course that corresponded to ACTH and corticosterone release. Finally, blockade of NO production with the arginine derivative Nomega-nitro-L-argininemethylester (L-NAME; 50 mg/kg, sc), which attenuated the ACTH response to LPS, virtually abolished basal NOS activity in the PVN, as well as anterior and neurointermediate lobes of the pituitary, and prevented the appearance of citrulline in the PVN of rats injected with LPS. Collectively, these results show that LPS-induced activation of the HPA axis correlates with the activation of the PVN NOergic system, and supports a stimulatory role for NO in the modulation of the HPA axis in response to immune challenges.