Locus Coeruleus Noradrenergic Modulation of Diurnal Corticosterone, Stress Reactivity, and Cardiovascular Homeostasis in Male Rats.

INTRODUCTION: Activation of the locus coeruleus-noradrenergic (LC-NA) system during awakening is associated with an increase in plasma corticosterone and cardiovascular tone. These studies evaluate the role of the LC in this corticosterone and cardiovascular response. METHODS: Male rats, on day 0, were treated intraperitoneally with either DSP4 (50 mg/kg body weight) (DSP), an LC-NA specific neurotoxin, or normal saline (SAL). On day 10, animals were surgically prepared with jugular vein (hypothalamic-pituitary-adrenal [HPA] axis) or carotid artery (hemodynamics) catheters and experiments performed on day 14. HPA axis activity, diurnally (circadian) and after stress (transient hemorrhage [14 mL/kg body weight] or air puff-startle), and basal and post-hemorrhage hemodynamics were evaluated. On day 16, brain regions from a subset of rats were dissected for norepinephrine and corticotropin-releasing factor (CRF) assay. RESULTS: In DSP rats compared to SAL rats, (1) regional brain norepinephrine was decreased, but there was no change in median eminence or olfactory bulb CRF content; (2) during HPA axis acrophase, the plasma corticosterone response was blunted; (3) after hemorrhage and air puff-startle, the plasma adrenocorticotropic hormone response was attenuated, whereas the corticosterone response was dependent on stressor category; (4) under basal conditions, hemodynamic measures exhibited altered blood flow dynamics and systemic vasodilation; and (5) after hemorrhage, hemodynamics exhibited asynchronous responses. CONCLUSION: LC-NA modulation of diurnal and stress-induced HPA axis reactivity occurs via distinct neurocircuits. The integrity of the LC-NA system is important to maintain blood flow dynamics. The importance of increases in plasma corticosterone at acrophase to maintain short- and long-term cardiovascular homeostasis is discussed.

The CRF1 receptor antagonist R121919 attenuates the neuroendocrine and behavioral effects of precipitated lorazepam withdrawal.

RATIONALE: Corticotropin-releasing factor (CRF) is the primary physiologic regulator of the hypothalamic-pituitary-adrenal (HPA) axis and serves to globally coordinate the mammalian stress response. Hyperactivity of central nervous system CRF neurotransmission, acting primarily via the CRF(1) receptor, has been strongly implicated in the pathophysiology of depression and anxiety. Furthermore, there is evidence of enhanced CRF transcription, release, and neuronal activity after the administration of and withdrawal from several drugs of abuse, including cannabis, cocaine, ethanol, and morphine. Treatment with CRF antagonists has been demonstrated to reduce the severity of certain drug withdrawal symptoms, implicating a specific role for activation of CRF neurons in mediating the anxiogenic and stress-like reactions observed after abrupt drug discontinuation. OBJECTIVES/METHODS: To extend these findings, we investigated whether pretreatment with the selective CRF(1) receptor antagonist R121919 decreases the behavioral and neuroendocrine activation observed after the precipitation of benzodiazepine (BZ) withdrawal in BZ-dependent rats. RESULTS: Pretreatment with R121919 attenuated the subsequent HPA axis activation, behavioral measures of anxiety, and expression of the CRF gene in the paraventricular nucleus of the hypothalamus, as measured by CRF heteronuclear RNA, which occurs after flumazenil-precipitation of withdrawal from the BZ, lorazepam. CONCLUSIONS: These results indicate that the activation of CRF neuronal systems may be a common neurobiological mechanism in withdrawal from drugs of abuse and moreover, that the CRF(1) receptor subtype plays a major role in mediating the effects of CRF on neuroendocrine and behavioral responses during BZ withdrawal. Therefore, CRF(1) receptor antagonists may be of therapeutic utility in the treatment of drug withdrawal syndromes.

Combined pre- and postnatal environmental enrichment programs the HPA axis differentially in male and female rats.

Experimental environmental enrichment (EE) is usually applied in adulthood or immediately after weaning, with robust effects on physiology and behaviour. To investigate the effects of EE earlier in life, female rats were maintained under moderate enrichment during pregnancy and, together with their pups, during lactation until weaning. A separate group of dams housed under standard conditions during pregnancy and lactation served as controls. Dams housed under EE exhibited fewer nursing episodes and were off the nest more often, but the frequency of pup licking was not affected on postnatal days 3-5. EE effects on hypothalamus-pituitary-adrenal (HPA) axis responses to an acute stressor were determined in adult male and female offspring with and without previous exposure to the chronic stressor of constant light. In female offspring, chronic stress significantly increased basal corticosterone (CORT) levels, but not if rats had been exposed to early EE. Furthermore, while control females exposed to chronic stress showed a greatly reduced adrenocorticotropin (ACTH) response to an acute stressor, EE females did not display this desensitization. There was no significant effect of EE on basal ACTH and CORT levels in adult male offspring, nor did it alter their response to acute stress. Maternal licking frequency was moderately but significantly correlated with net corticosterone increases in response to acute stress, the direction of the correlation crucially depending on the offspring's sex and stress conditions. This study shows that EE during pregnancy and lactation has long-lasting effects on reactivity to acute and chronic stress in offspring and that these effects are dependent on the offspring's sex but not greatly on early postpartum maternal behaviour.

Long-term consequences of neonatal rearing on central corticotropin-releasing factor systems in adult male rat offspring.

In a series of studies on the long-term consequences of neonatal rearing, we compared hypothalamic and extrahypothalamic central corticotropin-releasing factor (CRF) systems in male rats reared under conditions of animal facility rearing, nonhandling (HMS0), handling with brief maternal separation for 15 min (HMS15), or handling with moderate maternal separation for 180 min (HMS180) daily from postnatal days 2-14. CRF-like immunoreactivity (CRFir) was elevated in lumbar cerebrospinal fluid of adult HMS180 and HMS0 rats relative to the other groups. In the paraventricular nucleus, central nucleus of the amygdala, bed nucleus of the stria terminalis, and locus coeruleus, CRFir and CRF mRNA levels were significantly elevated in HMS0 and HMS180 rats. Neonatal maternal separation was associated with regionally specific alterations in CRF receptor type 1 (CRF1) mRNA density in HMS180 rats. No rearing-associated differences in CRF2alpha binding were apparent in either the lateral septum or the ventromedial hypothalamus. These findings indicate that early rearing conditions can permanently alter the developmental set-point of central CRF systems, and potentially influence the expression of behavioral and endocrine responses to stress throughout life, thereby providing a possible neurobiological substrate for the relationship between early life events and increased vulnerability for hypothalamic-pituitary-adrenal axis and coping skill alterations and the frequency of mood disorders in patients with a history of such experiences.

Early life experience alters behavior during social defeat: focus on serotonergic systems.

Early life experience can have prolonged effects on neuroendocrine, autonomic, and behavioral responses to stress. The objective of this study was to investigate the effects of early life experience on behavior during social defeat, as well as on associated functional cellular responses in serotonergic and non-serotonergic neurons within the dorsal raphe nucleus, a structure which plays an important role in modulation of stress-related physiology and behavior. Male Long Evans rat pups were exposed to either normal animal facility rearing or 15 min or 180 min of maternal separation from postnatal days 2-14. As adults, these rats were exposed to a social defeat protocol. Differences in behavior were seen among the early life treatment groups during social defeat; rats exposed to 180 min of maternal separation from postnatal days 2-14 displayed more passive-submissive behaviors and less proactive coping behaviors. Analysis of the distribution of tryptophan hydroxylase and c-Fos-like immunoreactivity in control rats exposed to a novel cage and rats exposed to social defeat revealed that, independent of the early life experience, rats exposed to social defeat showed an increase in the number of c-Fos-like immunoreactive nuclei in serotonergic neurons in the middle and caudal parts of the dorsal dorsal raphe nucleus and caudal part of the ventral dorsal raphe nucleus, regions known to contain serotonergic neurons projecting to central autonomic and emotional motor control systems. This is the first study to show that the dorsomedial part of the mid-rostrocaudal dorsal raphe nucleus is engaged by a naturalistic stressor and supports the hypothesis that early life experience alters behavioral coping strategies during social conflict; furthermore, this study is consistent with the hypothesis that topographically organized subpopulations of serotonergic neurons principally within the mid-rostrocaudal and caudal part of the dorsal dorsal raphe nucleus modulate stress-related physiological and behavioral responses.

Chronic maternal stress inhibits the capacity to up-regulate placental 11beta-hydroxysteroid dehydrogenase type 2 activity.

This study investigated the effects of acute and chronic restraint stress during the third week of pregnancy on placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) activity in rats. Acute exposure to stress on gestational day 20 immediately up-regulated placental 11beta-HSD2 activity by 160%, while chronic stress from day 14 to day 19 of pregnancy did not significantly alter basal 11beta-HSD2 activity. However, the latter reduced the capacity to up-regulate placental 11beta-HSD2 activity in the face of an acute stressor by 90%. Thus, immediate up-regulation of 11beta-HSD2, the feto-placental barrier to maternal corticosteroids, may protect the fetus against stress-induced high levels of maternal corticosteroids, but exposure to chronic stress greatly diminishes this protection.

Differential neuroendocrine responses to chronic variable stress in adult Long Evans rats exposed to handling-maternal separation as neonates.

Burgeoning evidence supports a preeminent role for early- and late-life stressors in the development of physio- and psychopathology. Handling-maternal separation (HMS) in neonatal Long Evans hooded rats leads to stable phenotypes ranging from resilient to vulnerable to later stressor exposure. Handling with 180 min of maternal separation yields a phenotype of stress hyper-responsiveness associated with facilitation of regional CRF neurocircuits and glucocorticoid resistance. This study assessed whether or not prolonged HMS (180 min/day, HMS180) on post-natal days 2-14 sensitizes the adult limbic hypothalamo-pituitary-adrenal (LHPA) axis to chronic variable stress (CS) compared to brief HMS (15 min/day, HMS15). We examined regional mRNA densities of corticotropin-releasing factor (CRF), its receptor CRF1, glucocorticoid receptor (GR), and mineralocorticoid receptor (MR); regional CRF1 and CRF2alpha binding, and pituitary-adrenal responses to an acute air-puff startle (APS) stressor in four groups: HMS15, nonstressed; HMS15, stressed; HMS180, nonstressed; HMS180, stressed. As expected we observed exaggerated pituitary-adrenal responses to APS, increased regional CRF mRNA density, decreased regional CRF1 binding, and decreased cortical GR mRNA density in nonstressed HMS180 vs. HMS15 animals. However, in contrast to our hypothesis, CS decreased pituitary-adrenal reactivity and central amygdala CRF mRNA density in HMS180 rats, while increasing cortical GR mRNA density and CRF1 binding. CS had no effect on the pituitary-adrenal response to APS in HMS15 rats, despite tripling hypothalamic paraventricular CRF mRNA density. The data suggest that many effects of prolonged HMS are reversible in adulthood by CS, while the neuroendocrine adaptations imbued by brief HMS are sufficiently stable to restrain pituitary-adrenal stress responses even following CS.

Age-related alterations of hypothalamic-pituitary-adrenal axis function in male Fischer 344 rats.

Aging is frequently associated with changes in physiological and cognitive processes. Among these changes is a distinct dysregulation of the hypothalamic-pituitary-adrenal axis. In the current experiments, aspects of hypothalamic-pituitary-adrenal axis function were compared in young (3- to 4-month-old) and aged (21- to 24-month-old) Fisher 344/N male rats. Basal ACTH and corticosterone levels during the circadian trough were elevated in aged compared to young rats. During the evening peak of the circadian cycle, plasma ACTH levels in the young and aged rats were comparable; however, aged rats had significantly lower corticosterone levels than young rats. Stimulus-induced secretion of pituitary-adrenal hormones was attenuated in aged rats. The ACTH response to hemorrhage in the aged group was only 45 +/- 3% of the hemorrhage response in young rats. Pituitary responsiveness to an iv CRF challenge was 58 +/- 6% of that in the young population. These changes were associated with a 38 +/- 5% loss of anterior pituitary CRF receptor number in the aged population. Changes in the hypothalamic regulation of pituitary-adrenal function were also evident in the aged rats. Hypophysial-portal blood concentrations of CRF were significantly greater in aged (56 +/- 6 pM) compared to young rats (37 +/- 4 pM; P < 0.02, by two-tailed unpaired t test; n = 8/group), whereas portal levels of arginine vasopressin were significantly reduced in aged (0.56 +/- 0.01 nM) compared to young rats (0.89 +/- 0.01 nM; P < 0.01, by two-tailed unpaired t test; mean +/- SEM; n = 8/group). Portal CRF responses to hemorrhage were significantly (P < 0.01) greater in aged rats, whereas hemorrhage-stimulated increases in portal AVP levels were significantly (P < 0.01) reduced in the aged group compared to those in the young rats. Finally, regional assay of CRF content demonstrated significant reductions in the median eminence and frontal cortex of aged rats compared to young rats, whereas in situ hybridization analysis failed to reveal age-related differences in paraventricular CRF mRNA levels. Overall, these observations are consonant with the hypothesis that senescence is associated with hypothalamic CRF hypersecretion and a consequent down-regulation of corticotrope CRF receptor.

Hypothalamic-pituitary-adrenal axis function in the Zucker obese rat.

The development of many endocrine, metabolic, and behavioral abnormalities characteristic of genetically obese Zucker rats is dependent upon the presence of glucocorticoids, the secretion of which is regulated by a neuroendocrine cascade initiated by hypothalamic release of CRF. Recent reports have inferred alterations in central CRF tone as a putative factor contributing to dysregulation of the pituitary-adrenal axis and of metabolic processes in this phenotype. In the current study the hypothalamic CRF system in Zucker lean (FA/?) and obese (fa/fa) phenotypes was functionally evaluated. Neither the stalk median eminence content of CRF or arginine vasopressin (AVP) nor hypothalamic levels of CRF or AVP mRNA differed in the lean and obese phenotypes. No phenotypic differences were observed in either basal or stimulated CRF release from hypothalamic tissue obtained from lean and obese rats. Furthermore, in intact rats the magnitude of pituitary-adrenal responses to various stressors was also similar between phenotypes. However, secretion of CRF and AVP into the hypophysial-portal circulation of obese rats was, respectively, 73% and 35% lower than that of the lean rats. Adrenalectomy was associated with a 3-fold elevation of hypophysial-portal CRF levels in obese rats compared to intact controls. Corticosterone infusion was more effective in suppressing portal CRF levels in adrenalectomized obese compared to adrenalectomized lean rats. Finally, neither CRF receptor number and affinity nor the magnitude of pituitary-adrenal responses to rat CRF challenge (1 micrograms, iv) differed between Zucker phenotypes. These observations lead us to infer that rats of the obese phenotype exhibit reduced hypothalamic CRF tone due to dysregulation of the HPA axis at a site proximal to the hypophysiotropic CRF system that mediates glucocorticoid feedback regulation.

Long-term adaptations in glucocorticoid receptor and mineralocorticoid receptor mRNA and negative feedback on the hypothalamo-pituitary-adrenal axis following neonatal maternal separation.

BACKGROUND: Maternally separated rats exhibit exaggerated hypothalamic-pituitary-adrenal responses to an acute stressor but normal diurnal trough functioning. We hypothesized that maternally separated rats experience adequate proactive glucocorticoid negative feedback but deficient "reactive" negative feedback, contributing to prolonged hypothalamic-pituitary-adrenal stress responses. METHODS: We measured plasma adrenocorticotropic hormone and corticosterone concentrations following an acute stressor or 6 to 8 hours after dexamethasone administration in adult rats previously exposed to daily handling-maternal separation for 15 minutes (HMS15) or 180 minutes (HMS180) during postnatal days 2 to 14. We also examined regional mineralocorticoid receptor and glucocorticoid receptor messenger RNA density in these two groups. RESULTS: HMS180 rats appeared to escape dexamethasone suppression of plasma adrenocorticotropic hormone and corticosterone faster than their HMS15 counterparts (p <.01). In situ hybridization analysis revealed increased hippocampal mineralocorticoid receptor messenger RNA density (p <.05) with decreased cortical (p <.05) and hippocampal (p <.05) glucocorticoid receptor messenger RNA density in HMS180 versus HMS15 animals. CONCLUSIONS: These results are consistent with the hypothesis that in rats exposed to moderate neonatal handling-maternal separation, enhanced proactive feedback maintains the hypothalamic-pituitary-adrenal axis during the diurnal trough, while decreased reactive feedback contributes to prolonged responsiveness of the hypothalamic-pituitary-adrenal axis following an acute stressor.

Foster litters prevent hypothalamic-pituitary-adrenal axis sensitization mediated by neonatal maternal separation.

Neonatal maternal separation of rat pups has been shown to produce long-term increases in hypothalamic-pituitary-adrenal (HPA) axis responsiveness, elevated levels of hypothalamic corticotropin releasing factor (CRF) mRNA in the hypothalamic paraventricular nucleus (PVN), and enhanced anxiety-like behavior. These effects appear to be at least partially mediated by subtle disruptions in the quality of maternal-pup interactions. This hypothesis was tested by providing half the dams with foster litters during the maternal separation paradigm, so that in those litters, only the pups and not the dams were experiencing a period of separation. The separation protocol took place daily from PND2-14 for either 15 min (HMS15, handled) or 180 min (HMS180, maternal separation). During the period of separation dams were either transferred to adjacent cages without any pups present (HMS15, HMS180) or to cages containing an age-matched foster litter (HMS15F, HMS180F). As adults, the HMS180 progeny exhibited the expected increased expression of CRF mRNA in the PVN, stress hyper-responsiveness to airpuff startle and evidence of impaired feedback both in the CORT response, as well as in response to the dexamethasone suppression test. The HMS180F rats, however, appeared to be resistant to these effects of maternal separation as they demonstrated CRF mRNA levels intermediate between HMS15 and HMS180 rats. Their stress responses and feedback regulation of the HPA axis was comparable to that of the HMS15 rats. GR mRNA was elevated in the cortex of HMS180F rats. Overall, these studies support the thesis that the long-term effects of neonatal maternal separation may largely result from alterations in the quality of maternal care rather than from direct effects of the separation per se on the pups.

Endocrine and behavioral effects of airpuff-startle in rats.

Adult male rats chronically implanted with cannulae in the jugular vein were used to characterize the endocrine and behavioral consequences of airpuff-startle. In the first series of experiments, resting animals subjected to three blocks of airpuff (blocks of three airpuffs each with each block separated by 1 min) showed a 10-fold increase in plasma adrenocorticotropin (ACTH) and corticosterone levels, indicating a significant but moderate activation of the hypothalamo-pituitary-adrenal (HPA) axis when compared with the untreated controls (n = 5 each). In the second series of experiments, monitoring of anxiety-related behavior in the defensive withdrawal paradigm revealed a significant increase in anxiety induced by airpuff-startle application compared with the untreated controls (n = 10 each). This behavioral effect, however, was not correlated with plasma hormone levels, as blood samples taken immediately before and 12 min after exposure to airpuff-startle and exposure to the defensive withdrawal paradigm showed a significant rise in ACTH and corticosterone in both groups. In summary, the findings of the present study indicate the airpuff-startle (1) is a potent stimulus in the activation of the HPA axis, and (2) increases anxiety-related behavior as measured in the defensive withdrawal paradigm. Furthermore, the results of our study support the hypothesis that peripheral endocrine parameters that are used to measure activation of the HPA axis do not necessarily correlate with behavioral data obtained in tests with are thought to measure anxiety.

Reduced noradrenergic tone to the hypothalamic paraventricular nucleus contributes to the stress hyporesponsiveness of lactation.

Lactation in mammals is accompanied by a marked decrease in stress responsiveness that we previously attributed, in part, to a reduction in noradrenergic (NA) innervation of hypothalamic paraventricular nucleus (PVN) neurons controlling neuroendocrine stress responses. In the present study, we compared in-vivo PVN catecholamine secretion by microdialysis between nonlactating and lactating females and tested the effects of NA alpha-1 and alpha-2 receptor antagonists (corynanthine and idazoxan, respectively) on the acute stress response of lactating and virgin female rats. To determine if PVN alpha-adrenoreceptor density, affinity, or synthesis, changes as a function of lactation, we performed receptor autoradiography, Scatchard analysis and in situ hybridization of alpha-adrenoreceptors. Densitometric analysis of the alpha-adrenoreceptors in the supraoptic nucleus (SON) was used to evaluate changes in magnocellular neurons. Endogenous PVN norepinephrine release under basal conditions was lower in lactating females than in females who had their pups removed for 2 days, and microdialysate concentrations of adrenaline and MHPG were attenuated in lactating females. Alpha-2 adrenoreceptor density in the PVN showed a significant decrease from lactation day 3 to lactation days 10-12 and a reduction to 40% of virgin controls on days 10-20 of lactation. A similar pattern was observed for the SON. The affinity of hypothalamic alpha-2 adrenoreceptors was reduced as a function of lactation. Alpha-1 adrenoreceptor density in the PVN and in the hypothalamus rose as a function of lactation, although the affinity of these receptors was not altered. In contrast, alpha-1D adrenoreceptor subtype mRNA expression in the PVN decreased in middle lactating females (day 10) compared to virgins. Intracerebroventricular (i.c.v.) application of idazoxan, significantly increased the ACTH response to swim stress in virgin females, but had the opposite effect in lactating females. In contrast, i.c.v. corynanthine treatment significantly decreased the ACTH response in virgins, but not in lactating females. Overall, these data suggest that the secretion of NA in the PVN is reduced during lactation, and that the ability of PVN parvocellular neurons to respond to changes in synaptic NA levels (i.e. after stress) is also altered.

Development of adult ethanol preference and anxiety as a consequence of neonatal maternal separation in Long Evans rats and reversal with antidepressant treatment.

RATIONALE: This study was based on the findings of a high comorbidity among anxiety and depression as well as with alcohol abuse. OBJECTIVE: To evaluate first exposure alcohol preference in a rodent model of moderate neonatal maternal separation. METHODS: Rat pups were exposed to either normal animal facility rearing (AFR) or 15 min (HMS15) or 180 min (HMS180) of maternal separation from postnatal days 2-14. The adult (>60 days) male Long Evans progeny was tested for pituitary-adrenal axis responsiveness to airpuff startle, anxiety-like behavior in the elevated plus maze, and alcohol preference using a two-bottle, free-choice test. RESULTS: In response to home cage airpuff startle, HMS180 rats displayed an elevation in the integrated adrenocorticotropic hormone and corticosterone responses. In addition, HMS180 rats spent less time in the open arms and more time in the closed arms in the elevated plus maze. HMS180 rats drank significantly less of a water-sucrose solution and significantly more of an ethanol-sucrose solution than AFR or HMS15 rats. No rearing group differences were observed in total fluid intake. The integrated corticosterone response to airpuff startle was highly correlated with ethanol consumption and there was a negative correlation between percentage of time spent in the open arms of the elevated plus maze and ethanol consumption. Treatment with the selective serotonin reuptake inhibitor paroxetine for 21 days eliminated differences in the elevated plus maze and HPA axis responsiveness, and significantly reduced the amount of ethanol consumed by the HMS180 rats, without affecting these parameters in the HMS15 rats. CONCLUSIONS: These observations suggest that this maternal separation paradigm is a good model to study the effects of early adverse experience on the development of alcohol preference and anxiety.

Sensitivity to glucocorticoid-mediated fast-feedback regulation of the hypothalamic-pituitary-adrenal axis is dependent upon stressor specific neurocircuitry.

UNLABELLED: Fos-protein immunoreactivity (Fos-IR) was used to identify neurocircuits potentially participating in the regulation of hypothalamic-pituitary-adrenal (HPA) axis sensitivity to glucocorticoid-mediated fast-feedback in rats exposed to the physical stressor, hemorrhage, or the psychological stressor, airpuff startle. Marked regional brain differences in the Fos-IR expression were observed in response to these stressors. Specifically, after hemorrhage, nuclear Fos-IR increased in the nucleus of the solitary tract and other brainstem regions known to regulate hemodynamic processes including the supraoptic nucleus, and the magnocellular division of hypothalamic paraventricular nucleus (PVN). In contrast, after airpuff startle Fos-IR increased in the dorsomedial and lateral hypothalamus as well as in the lateral septum. Thus, activation of brainstem neurocircuits predominated after hemorrhage whereas activation of forebrain neurocircuits predominated after airpuff startle. In other regions, the magnitude of stressor-induced Fos-IR expression varied in a region-specific manner. When stressor exposure was preceded by administration of corticosterone to achieve levels within the physiological range after stressors, HPA axis responses were suppressed in response to the airpuff startle but not to either a small or moderate hemorrhage. IN CONCLUSION: (1) fast-feedback mediated inhibition of HPA axis activity is critically dependent upon stressor modality; (2) this apparent selectivity is reflected by differences in the nature of the neurocircuitry mediating these stressors. It is suggested that determination of the central actions of glucocorticoids in mediating fast-feedback regulation of the HPA axis requires evaluation of the interactions between activated glucocorticoid receptors and intracellular signaling cascades evoked by convergent neuronal input.

Alterations of locus coeruleus noradrenergic activity in relation to pituitary secretion after hemorrhage in cats.

Noradrenergic (NA) activity in the cat ventral locus coeruleus (vLC), measured either by voltammetry or by push-pull perfusion, increased in response to hemorrhage. This stimulus also elicited plasma adrenocorticotropin (ACTH) and vasopressin responses. Increased vLC NA activity following the initial hemorrhage (InHem) persisted even after plasma hormone levels returned toward prestimulus values. Upon stimulus repetition, the peak increase in vLC NA activity was similar to that observed during InHem while the hormone responses were of greater magnitude (i.e. potentiated). Hence, it is suggested that the LC may exert a modulatory role in the hemodynamic control of hypothalamic-pituitary axis function.

Jugular vein catheterization for repeated blood sampling in the unrestrained conscious rat.

The ability to obtain repeated, low-stress blood samples from adult rats enables the design of complex experiments in which time course information or evaluation of repeated treatments is necessary. Furthermore, it reduces the number of animals necessary to acquire such information and, thus, facilitates compliance with the animal use 3Rs (reduction, refinement and replacement). To this end, a microsurgical technique to collect blood samples from the right atrium through a catheter (cannula) implanted into the right external jugular vein of adult rats is described. Rats tolerate this simple and efficient vascular access technique as evidenced by the absence of overt morbidity or abnormal behaviors. Blood is easily sampled while the rats reside in their home cages. Because the sample volume is replaced, repeated sampling is possible without compromising blood volume. Successful adoption of this procedure by other investigators will be aided by the photographic illustrations accompanying this detailed description of the procedure. Application of this technique to monitor temporal changes in plasma stress hormones during stressor paradigms as well as after behavioral and pharmacological challenges is discussed.

Long-term behavioral effects of repetitive pain in neonatal rat pups.

Human preterm neonates are subjected to repetitive pain during neonatal intensive care. We hypothesized that exposure to repetitive neonatal pain may cause permanent or long-term changes because of the developmental plasticity of the immature brain. Neonatal rat pups were stimulated one, two, or four times each day from P0 to P7 with either needle prick (noxious groups N1, N2, N4) or cotton tip rub (tactile groups T1, T2, T4). In groups N2, N4, T2, T4 stimuli were applied to separate paws at hourly intervals;each paw was stimulated only once a day. Identical rearing occurred from P7 to P22 days. Pain thresholds were measured on P16, P22, and P65 (hot-plate test), and testing for defensive withdrawal, alcohol preference, air-puff startle, and social discrimination tests occurred during adulthood. Adult rats were exposed to a hot plate at 62 degrees C for 20 s, then sacrificed and perfused at 0 and 30 min after exposure. Fos expression in the somatosensory cortex was measured by immunocytochemistry. Weight gain in the N2 group was greater than the T2 group on P16 (p < 0.05) and P22 (p < 0.005); no differences occurred in the other groups. Decreased pain latencies were noted in the N4 group [5.0 +/- 1.0 s vs. 6.2 +/- 1.4 s on P16 (p < 0.05); 3.9 +/- 0.5 s vs. 5.5 +/- 1.6 s on P22 (p < 0.005)], indicating effects of repetitive neonatal pain on subsequent development of the pain system. As adults, N4 group rats showed an increased preference for alcohol (55 +/- 18% vs. 32 +/- 21%; p = 0.004); increased latency in exploratory and defensive withdrawal behavior (p < 0.05); and a prolonged chemosensory memory in the social discrimination test (p < 0.05). No significant differences occurred in corticosterone and ACTH levels following air-puff startle or in pain thresholds at P65 between N4 and T4 groups. Fos expression at 30 min after hot-plate exposure was significantly greater in all areas of the somatosensory cortex in the T4 group compared with the N4 group (p < 0.05), whereas no differences occurred just after exposure. These data suggest that repetitive pain in neonatal rat pups may lead to an altered development of the pain system associated with decreased pain thresholds during development. Increased plasticity of the neonatal brain may allow these and other changes in brain development to increase their vulnerability to stress disorders and anxiety-mediated adult behavior. Similar behavioral changes have been observed during the later childhood of expreterm neonates who were exposed to prolonged periods of neonatal intensive care.

In vivo dialysis setup with a loop injection valve facilitates retrodialysis studies.

INTRODUCTION: Retrodialysis, as used in neuropharmacological research, is a technique for in vivo delivery of neuroactive agents with concurrent monitoring of their effects on cellular activity with a separation between certain degree of spatial and temporal resolution. Typically, this is accomplished either by the use of a liquid-switch requiring multiple pumps, or by exchange of flow tubing requiring stopping and restarting dialysis. In the present study, we describe the use of a medium pressure injection valve for retrodialysis that overcomes these problems. METHODS: The valve was configured with a loop to deliver 20µL of solution, and artificial CSF flow from the pump to the probe was established via this device. The application of this setup was evaluated in urethane anesthetized adult male C57BL/6J mice prepared with a CMA 11 probe implanted in the ventral hippocampus. By switching between the load and inject positions, the loop was filled with escitalopram solution (0.3µM) and delivered at a rate of 1µL/min at the probe for retrodialysis. Escitalopram (2mg/kg BW) was administered subcutaneously for microdialysis studies. During these treatments, dialysate fractions were collected for the determination of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA). RESULTS: Irrespective of route of escitalopram administration, the pattern of dialysate 5-HT, and 5-HIAA response was comparable to that reported by other investigators. Accordingly, the in-line valve assembly did not compromise retrodialysis or microdialysis sampling. The manipulations to carry out retrodialysis using the valve setup are easy and simple. DISCUSSION: An in-line injection valve is a promising adaptation for retrodialysis studies and can be incorporated as a standard part of in vivo dialysis instrumentation.

Neuroendocrine homeostasis after vagus nerve stimulation in rats.

BACKGROUND: The vagus nerve is important in maintaining HPA axis and sympatho-adrenal system (SAS) homeostasis, however little is known about the effect of vagus nerve stimulation (VNS), as used therapeutically, on these functions. Accordingly, the effect of VNS on plasma indices of HPA axis (ACTH, corticosterone), and SAS (norepinephrine, epinephrine) function were evaluated in rats. METHODS: Male rats, on day-0 (D0), underwent surgeries for implantation of catheters into the right jugular vein and programmable (VNP) or non-programmable (VND) neurocybernetic devices encircling the left cervical vagus. On D7, after a blood sample, the device in VNP rats was programmed to deliver 500 µs width, 0.25 mA current pulses at 20 Hz ('on' 30s, 'off' 5 min) followed by timed blood samples during the next 90 min. In acute studies, VNS was stopped at 60 min and the rats were perfused at 90 min to evaluate neuronal Fos immunoreactivity (Fos-IR). In chronic studies, the probe remained active. In these rats, the HPA axis response to airpuff-startle stressor (D17) and anterior pituitary CRF-receptor binding (D26) were evaluated. RESULTS: During acute VNS, plasma indices of HPA axis and SAS activity, as well as Fos-IR activation pattern in brain regions known to increase after stress, were not different between VND and VNP rats. During chronic VNS, stress-induced HPA axis responses exhibited a tendency toward faster recovery to baseline in VNP rats. CONCLUSIONS: Therapeutic VNS is not a stressor and does not compromise HPA axis or SAS homeostasis. Chronic VNS may facilitate development of efficient feedback mechanisms.