Food-Restriction Lowers the Acoustic Startle Response in both Male and Female Rats, and, in Combination with Acute Ghrelin Injection, Abolishes the Expression of Fear-Potentiated Startle in Male Rats.

Food restriction has been reported to reduce anxiety-like behaviour in male rats, whereas the effects of food restriction on anxiety in female rats are less clear. Ghrelin is a peptide hormone produced and secreted in the stomach that stimulates food intake and is considered to play a role in reward and emotional responses such as fear expression. Under food restriction, endogenous ghrelin levels increase. In the present study, we examined the effect of moderate food restriction (80% of ad libitum fed weight), with or without an acute application of a small dose of exogenous ghrelin intended to cause an immediate hunger response, on the expression of the acoustic startle reflex (ASR). This was carried out under basal conditions (baseline ASR to 90- and 95-dB noise bursts), and in the presence of a light cue associated with a mild foot-shock, as measured by fear-potentiated startle, which compares the proportional change in ASR in the presence of the conditioned stimulus. The results obtained show that food-restriction reduces basal ASR in both male and female rats, apart from any concomitant change in motor activity, suggesting that food-restriction reduces anxiety levels in both sexes. In addition, the data show that food-restriction reduces fear-potentiated startle in male but not female rats. Acute ghrelin injection, prior to fear-potentiated startle testing, eliminates the expression of fear-potentiated startle in food-restricted male rats alone, suggesting a role for ghrelin in the reduction of fear expression in food-restricted male rats. These data imply that, although food-restriction decreases anxiety in both sexes, learned fear responses remain intact after food-restriction in female but not male rats.

Pituitary adenylate cyclase-activating polypeptide (PACAP) in the bed nucleus of the stria terminalis (BNST) increases corticosterone in male and female rats.

Single nucleotide polymorphisms (SNP) in the genes for pituitary adenylate cyclase-activating polypeptide (PACAP) and the PAC1 receptor have been associated with several psychiatric disorders whose etiology has been associated with stressor exposure and/or dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis. In rats, exposure to repeated variate stress has been shown to increase PACAP and its cognate PAC1 receptor expression in the bed nucleus of the stria terminalis (BNST), a brain region implicated in anxiety and depression-related behaviors as well as the regulation of HPA axis activity. We have argued that changes in BNST PACAP signaling may mediate the changes in emotional behavior and dysregulation of the HPA axis associated with anxiety and mood disorders. The current set of studies was designed to determine whether BNST PACAP infusion leads to activation of the HPA axis as determined by increases in plasma corticosterone. We observed an increase in plasma corticosterone levels 30min following BNST PACAP38 infusion in male and female rats, which was independent of estradiol (E2) treatment in females, and we found that plasma corticosterone levels were increased at both 30min and 60min, but returned to baseline levels 4h following the highest dose. PACAP38 infusion into the lateral ventricles immediately above the BNST did not alter plasma corticosterone level, and the increased plasma corticosterone following BNST PACAP was not blocked by BNST corticotropin releasing hormone (CRH) receptor antagonism. These results support others suggesting that BNST PACAP plays a key role in regulating stress responses.

Continuous expression of corticotropin-releasing factor in the central nucleus of the amygdala emulates the dysregulation of the stress and reproductive axes.

An increase in corticotropin-releasing factor (CRF) is a putative factor in the pathophysiology of stress-related disorders. As CRF expression in the central nucleus of the amygdala (CeA) is important in adaptation to chronic stress, we hypothesized that unrestrained synthesis of CRF in CeA would mimic the consequences of chronic stress exposure and cause dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, increase emotionality and disrupt reproduction. To test this hypothesis, we used a lentiviral vector to increase CRF-expression site specifically in CeA of female rats. Increased synthesis of CRF in CeA amplified CRF and arginine vasopressin peptide concentration in the paraventricular nucleus of the hypothalamus, and decreased glucocorticoid negative feedback, both markers associated with the pathophysiology of depression. In addition, continuous expression of CRF in CeA also increased the acoustic startle response and depressive-like behavior in the forced swim test. Protein levels of gonadotropin-releasing hormone in the medial preoptic area were significantly reduced by continuous expression of CRF in CeA and this was associated with a lengthening of estrous cycles. Finally, sexual motivation but not sexual receptivity was significantly attenuated by continuous CRF synthesis in ovariectomized estradiol-progesterone-primed females. These data indicate that unrestrained CRF synthesis in CeA produces a dysregulation of the HPA axis, as well as many of the behavioral, physiological and reproductive consequences associated with stress-related disorders.Molecular Psychiatry (2009) 14, 37-50; doi:10.1038/mp.2008.91; published online 12 August 2008.

Chronic neuropeptide Y Y5 receptor stimulation suppresses reproduction in virgin female and lactating rats.

Continuous infusion of neuropeptide Y (NPY) disrupts cyclicity and delays the onset of puberty in female rats indicating that NPY can suppress reproduction. Central application of NPY also reliably increases food intake in rats. States with heavy demands on energy resources where reproduction is also inhibited, such as lactation, are similarly accompanied by elevations in central NPY expression. In previous studies, we have shown that, compared to lactating rats fed ad libitum, food-restricted lactating rats exhibit a longer period of lactational diestrus that is correlated with increased central NPY expression. These studies link NPY to the inhibition of reproduction that is mediated by low availability of energy resources. Here, we examine the effect of chronic 7-day infusion of the mixed Y1/Y4/Y5 agonist (Leu31, Pro34) NPY and selective agonists to the Y2 (NPY13-36) and Y5 (D-Trp32 NPY and D-Trp34 NPY) receptors on food intake and the oestrous cycle of virgin female rats. We also investigated the effect of chronic infusion from day 8-15 postpartum (pp) of D-Trp32 NPY and D-Trp34 NPY on food-intake and the length of lactational diestrus in lactating rats fed ad libitum. In virgin females, infusion of (Leu31, Pro34) NPY and both the Y5 agonists lengthened the period between consecutive oestrus days while the Y2 agonist NPY13-36 was without effect. Selective Y5 receptor activation alone caused an increase in food intake in virgin females. In lactating females, D-Trp32 NPY extended the length of lactational diestrus, while D-Trp34 NPY had no effect on this parameter. These data suggest that Y5 receptor activation suppresses the reproductive axis in both virgin and lactating rats and that Y5 receptor activation enhances food-intake in virgin females.

Y1 receptor activation is involved in the effect of exogenous neuropeptide Y on pup growth and the early termination of lactational diestrus in the postpartum rat.

The effect of chronic administration of exogenous neuropeptide Y (NPY) and specific NPY receptor agonists and antagonists on reproductive function was examined in lactating rats. As previously demonstrated in our laboratory, chronic (7-day) intracerebroventricular (i.c.v.) NPY infusion (6 microg/day) from days 8-15 postpartum (pp) caused a significant decrease in milk production and an early termination of lactational diestrus. Similar application of the mixed Y1/Y4/Y5 receptor agonist (Leu31, Pro34) NPY (at 3, 6 and 9 microg/day) reproduced the effect of chronic NPY infusion on milk production in a dose-independent manner. Consistent with this effect, the potent Y1 antagonist/Y4 agonist, 1229U91, given concomitantly with NPY eliminated the decline in milk production. The Y2 receptor agonist, NPY13-36, had no effect on milk production at any of the doses used. Length of lactational diestrus was reduced following administration of the Y2 agonist at 18 microg/day but not at 9 microg or 27 microg/day whereas (Leu31, Pro34) NPY infusion had no effect on this parameter at any of the doses used. However, the group that was treated with NPY plus 1229U91 exhibited the usual length of lactational diestrus, indicating that there is at least some Y1 involvement in the effects of NPY on lactational infertility. To test the possibility that the effects of NPY infusion are mediated through changes in circulating prolactin and progesterone, plasma concentrations of these hormones were measured on day 15 pp in NPY-, (Leu31, Pro34) NPY- and vehicle-treated females. NPY-infused females had lower plasma prolactin concentrations than vehicle-infused dams but progesterone concentrations were similar across groups. Overall, these data indicate that chronic exogenous NPY-infusion in lactating females disrupts milk production and shortens lactational diestrus, most likely through reducing prolactin secretion, and that this effect is mediated via Y1 receptor activity.

Hypothalamic and limbic expression of CRF and vasopressin during lactation: implications for the control of ACTH secretion and stress hyporesponsiveness.

Lactation is associated with physiological and behavioral changes that optimize conditions for development of the offspring. Although neuroendocrine and emotional stress responses are blunted, the central mechanisms involved are unclear. In addition to a reduction in stimulatory noradrenergic inputs to paraventricular nucleus (PVN) neurons, we demonstrate that lactation induces: (1) unique phenotypic changes in neuropeptide expression by hypothalamic PVN neurons (reduced expression of corticotropin-releasing factor (CRF) mRNA and increased expression of vasopressin mRNA in parvocellular PVN neurons); and (2) changes in pituitary sensitivity to CRF (reduced) and vasopressin (increased) as a consequence of differential CRF/vasopressin secretion into the hypophysial portal blood. Neurons in the bed nucleus of the stria terminalis (BNST) and the central amygdala (CeA) that are implicated in the control of the hypothalamopituitary-adrenal axis also display changes in lactation: expression of CRF mRNA in the CeA is reduced, consistent with the diminished responsiveness to acoustic startle observed in nursing mothers. In contrast, expression of CRF mRNA is increased in the dorsolateral portion of the BNST, probably because of the tonic increases in endogenous glucocorticoid production during this period. Using immuno-targeted lesions of CRF or vasopressin in the PVN of virgin females, we have shown that CRF neurons of the PVN send inhibitory projections to the dorsolateral portion of the BNST and stimulatory inputs to CRF neurons in the CeA. Thus, it is possible that lactation-induced changes in the activity of parvocellular PVN neurons might also modulate the expression of neuropeptides and neurotransmitters in the BNST and the amygdala.

Altered pituitary sensitivity to corticotropin-releasing factor and arginine vasopressin participates in the stress hyporesponsiveness of lactation in the rat.

The regulation of the activity of the hypothalamic-pituitary-adrenal (HPA) axis is modified during lactation, wherein a blunted stress-induced adrenocorticotropic hormone (ACTH) and glucocorticoid secretion is coupled with elevated basal secretion of these hormones. The involvement of pituitary modifications in lactation-induced stress hyporesponsiveness has yet to be established. In this study we tested the hypothesis that the pituitary sensitivity to corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) is altered in lactation in the rat. We examined the effect of exogenous CRF (0.1-5 microg/rat), AVP (0.01-0.5 microg/rat), and AVP (0.01-0.5 microg/rat)+CRF (0.1 microg/rat) on the ACTH response of virgin, mid-lactating (lactation day 10-12) females, as well as nursing females separated from their pups for 48 h. Additionally, to determine if changes in CRF- or AVP-receptor densities might mediate alterations in pituitary sensitivity, we compared pituitary CRF- and AVP-receptor binding by autoradiography in pregnant, mid-lactating, and virgin female rats. While both virgin and lactating female rats exhibited significant ACTH responses to CRF, the responses to the highest doses of CRF (2.0 and 5.0 microg/rat) were greater in virgin than in lactating females. Separation of the litter for 48 h partially restored pituitary responsiveness to 2.0 microg of CRF. Conversely, whereas lactating females displayed robust ACTH secretion following a high dose of AVP or following a combination of AVP+CRF, the response of virgin females was much smaller. These modifications in pituitary responsiveness were not accompanied by significant differences in pituitary CRF-and AVP-receptors levels between female groups. Our results demonstrate that a reduction in pituitary sensitivity to CRF, but not to AVP occur during lactation in the rat which mediates, at least in part, the stress hyporesponsiveness of lactation.

Lactation-induced reduction in rats' acoustic startle is associated with changes in noradrenergic neurotransmission.

The acoustic startle response (ASR) with or without fear conditioning was compared between cycling (CYC) and lactating (LACT) female rats. ASR sensitivity to changes in endogenous noradrenergic (NA) release was examined using the alpha-2 NA receptor drugs yohimbine and clonidine. Groups of CYC and LACT females were also tested in the open field. ASR was reduced in all LACT, compared with that in CYC females. Both groups exhibited a robust response to fear conditioning and unpotentiated ASR subsequent to conditioning was increased in LACT females. The lowest dose of yohimbine significantly increased ASR in LACT females, but not in CYC females. Clonidine reduced ASR in both groups of females, with a greater potency in CYC females. In the open field, LACT females displayed a shorter latency to emerge, less freezing behavior, and more entries into the field than did CYC females. The authors concluded that (a) LACT females are less anxious in a novel environment and that decreased anxiety can be efficiently counteracted by fear conditioning, and (b) changes in NA neurotransmission contribute to lactation-induced modifications in ASR.

Increased fat intake during lactation modifies hypothalamic-pituitary-adrenal responsiveness in developing rat pups: a possible role for leptin.

High fat feeding reportedly enhances hypothalamus-pituitary-adrenal (HPA) responses to stress in adult rats. The present study tested whether elevated fat intake during suckling could have short and/or long lasting consequences on HPA regulation in the offspring. Mothers were fed either a control (C; 5% fat) or high fat (HF; 20% fat) diet during the last week of gestation and throughout lactation. After weaning (day 21), pups from C and HF mothers were fed a chow diet. Offspring from both C- and HF-fed mothers were tested for ACTH and corticosterone responses to stress on postnatal days 10 and 35. We found that HF feeding produced higher lipid levels in the milk of HF compared with C lactating rat dams and that offspring of these mothers had significantly increased retroperitoneal fat pad weight and relative adipose mass on day 21 as well as elevated plasma leptin levels on days 10 and 21 of age. After weaning, pups from the HF mothers had lower plasma leptin levels than those from C mothers. Maternal dietary fat affected HPA responsiveness in the offspring in an age-related manner. Neonatal pups (day 10) from the HF mothers exhibited a reduction in the ACTH and corticosterone responses to ether stress. However, in 35-day-old offspring from HF-fed dams, stress-induced ACTH secretion was increased compared with that in pups from the C-fed mothers. These results demonstrate that maternal diet and increased fat intake through the milk are important regulators of HPA responsiveness in neonates and prepubertal rats. During neonatal life, the blunted stress responsiveness seen with elevated fat intake and the resulting high leptin levels might protect the pups from excessive HPA activation. After removal of the maternal dietary influence and reduced leptin levels, enhanced ACTH stress responses are observed as in adult rats fed a HF diet. Because of the inverse relationship between plasma levels of leptin and HPA responses in pups, the possibility exists that the effects of the HF diet on stress responsiveness are mediated by changes in leptin exposure during development.

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.

Noradrenergic facilitation of the adrenocorticotropin response to stress is absent during lactation in the rat.

During lactation, the regulation of the activity of the hypothalamic-pituitary-adrenal (HPA) axis is modified in that tonically elevated glucocorticoid secretion is observed together with blunted ACTH secretion following exposure to various stressors. Although decreased CRF mRNA levels have been reported in neurons of the paraventricular nucleus (PVN) which control ACTH secretion, the mechanisms underlying stress hyporesponsiveness during lactation are still largely unknown. In addition, lactation is associated with inhibition of reproductive functions and the involvement of the PVN neurons in this inhibition is unclear. In these studies, we tested the hypothesis that the effects of stimulatory noradrenergic afferents to the hypothalamic PVN are decreased during lactation, maintaining stress hyporesponsiveness. We also determined whether PVN noradrenergic afferents could modulate suckling-induced luteinizing hormone (LH) suppression. Virgin and lactating females, on day 2 of lactation, received either sham (SHAM) or 6-hydroxydopamine (6OH-DA) lesions over the PVN. Suppression of plasma LH secretion following a suckling test was determined on day 9 in ovariectomized females and plasma ACTH and corticosterone (B) responses to swim stress were determined on day 11 of lactation. In virgin females, 6OH-DA lesion caused a significant reduction in the ACTH and B responses to swim stress. In SHAM lactating females, plasma ACTH response to stress was blunted compared to SHAM virgins, but 6-OHDA lesion did not reduce ACTH levels further. Lesions in lactating females reduced basal LH secretion, although not significantly, but suckling did not further inhibit LH secretion as observed in SHAM lactating females. In all lesioned groups, PVN tyrosine hydroxylase (TH) immunoreactivity was reduced compared to SHAM rats. These results suggest that brainstem (nor)adrenergic inputs to the PVN act to facilitate ACTH stress response in virgin rats, while in lactating rats this facilitation is absent. In addition, (nor)adrenergic cells projecting to the PVN might also participate in the modulation of GnRH and LH secretion during suckling.