Journal of Neuroendocrinology

For over 100 years, thyroid hormones have been known to be essential for neonatal neurodevelopment but whether they are required by the foetal brain remains a matter of controversy. For decades, the prevailing view was that thyroid hormones are not necessary until after birth because circulating levels in the foetus are very low and the placenta forms an efficient barrier to their transfer from the mother. Clinical observations of good neurological outcome following early treatment of congenital hypothyroidism were used to support the view that thyroid hormones are not required early in neurodevelopment. Nevertheless, the issue remained contentious because of findings that the severity of foetal neurological deficit due to maternal iodine deficiency correlated with the degree of maternal thyroxine (T4) deficiency. Furthermore, neurological damage in these cases could be prevented by correction of maternal T4 deficiency before mid‐gestation. This observation led to the opposing view, supported by epidemiological studies of neurological cretinism, that maternal thyroid hormones are important and necessary for early foetal neurodevelopment. It is now clear that thyroid hormones are essential for both foetal and post‐natal neurodevelopment and for the regulation of neuropsychological function in children and adults. In recent years, this controversial subject has progressed very rapidly following remarkable progress in understanding of the molecular mechanisms of thyroid hormone action. This article reviews the contributions of molecular biology and genetics to our new understanding of the physiological effects of thyroid hormones on neurodevelopment and in the adult brain.

Immediate‐early genes (IEG) are powerful tools for identifying activated neurosecretory neurones and extended circuits that affect neuroendocrine functions. The generally acknowledged scenario is when cells became activated, IEGs expressed and IEG‐encoded transcription factors affect target gene expression. However, there are several examples in which: (i) neuronal activation occurs without induction of IEGs; (ii) IEG induction is not related to challenge‐induced neuropeptide expression; and (iii) markers of neuronal activation are not expressed in chronically activated neurones. In spite of these limitations, the use of c‐Fos and other regulatory‐ or effector transcription factors as markers of neuronal activation will continue to be an extremely powerful technique. Recently‐developed models, including transgenic mice expressing different marker genes under the regulation of IEG promoters, will help to monitor neuronal activity in vivo or ex vivo and to reveal connection between activated neurones. Furthermore, combinations between novel imaging techniques, such as magnetic resonance and IEG‐based mapping strategies, will open new means with which to study functional activity in the neurosecretory systems.

Some environmental contaminants interact with hormones and may exert adverse consequences as a result of their actions as endocrine disrupting chemicals (EDCs). Exposure in people is typically a result of contamination of the food chain, inhalation of contaminated house dust or occupational exposure. EDCs include pesticides and herbicides (such as dichlorodiphenyl trichloroethane or its metabolites), methoxychlor, biocides, heat stabilisers and chemical catalysts (such as tributyltin), plastic contaminants (e.g. bisphenol A), pharmaceuticals (i.e. diethylstilbestrol; 17α‐ethinylestradiol) or dietary components (such as phytoestrogens). The goal of this review is to address the sources, effects and actions of EDCs, with an emphasis on topics discussed at the International Congress on Steroids and the Nervous System. EDCs may alter reproductively‐relevant or nonreproductive, sexually‐dimorphic behaviours. In addition, EDCs may have significant effects on neurodevelopmental processes, influencing the morphology of sexually‐dimorphic cerebral circuits. Exposure to EDCs is more dangerous if it occurs during specific ‘critical periods’ of life, such as intrauterine, perinatal, juvenile or puberty periods, when organisms are more sensitive to hormonal disruption, compared to other periods. However, exposure to EDCs in adulthood can also alter physiology. Several EDCs are xenoestrogens, which can alter serum lipid concentrations or metabolism enzymes that are necessary for converting cholesterol to steroid hormones. This can ultimately alter the production of oestradiol and/or other steroids. Finally, many EDCs may have actions via (or independent of) classic actions at cognate steroid receptors. EDCs may have effects through numerous other substrates, such as the aryl hydrocarbon receptor, the peroxisome proliferator‐activated receptor and the retinoid X receptor, signal transduction pathways, calcium influx and/or neurotransmitter receptors. Thus, EDCs, from varied sources, may have organisational effects during development and/or activational effects in adulthood that influence sexually‐dimorphic, reproductively‐relevant processes or other functions, by mimicking, antagonising or altering steroidal actions.

Previous studies from this laboratory have shown that progesterone (PROG) treatment in ovariectomized rats produces an anti‐anxiety response similar to that observed after the administration of prototypical anxiolytic benzodiazepine (BDZ) compounds. The PROG‐induced anxiolytic response was highly correlated with an increased level of 3α‐hydroxy‐5α‐pregnan‐20‐one (allopregnanolone) in the blood and brain, and was also associated with a facilitation of GABA‐stimulated chloride ion (Cl⁻) influx in cortical synaptoneurosomes. This correlative evidence suggested that the anxiolytic effect of PROG was a result of its in vivo reduction to the neuroactive steroid, allopregnanolone. In this report, a series of studies was conducted to determine the mechanism(s) by which PROG alters behavior in animal models of anxiety. In the first experiment, ovariectomized rats were injected with PROG (1 mg/0.2 ml, SC) 4 h prior to a test in the elevated plus‐maze. Some animals also received an injection of picrotoxin (0.75 mg/kg, IP), a GABA_A receptor‐gated Cl⁻ channel antagonist, whereas other animals were pretreated with RU 38486 (5 mg/0.2 ml, SC), a progestin receptor antagonist. PROG elicited anxiolytic behavior in the plus‐maze, an effect that was blocked by picrotoxin administration. Pretreatment with RU 38486 was not effective in altering PROG‐induced anxiolytic behavior in the plus‐maze. In a second experiment, the effect of PROG on behavior in the plus‐maze was determined in the presence of N,N‐diethyl‐4‐methyl‐3‐oxo‐4‐aza‐5α‐androstane‐17β‐carboxamide (4‐MA; 10 mg/0.2 ml, SC), a 5α‐reductase inhibitor. The enzyme inhibitor was potent in preventing the anxiolytic effect observed in the plus‐maze after PROG administration. In the defensive burying paradigm, PROG treatment also produced anxiolysis by reducing the duration of burying behavior, and this effect was prevented by 4‐MA pretreatment, but not by RU 38486 administration. After the completion of the behavioral assays, analysis of blood allopregnanolone levels revealed a marked increase in PROG‐treated females. The PROG‐induced elevation in circulating allopregnanolone was blocked by pretreatment with 4‐MA. In cortical synaptoneurosomes, the sensitivity (inverse of the EC₅₀) and the maximal response (Emax) in GABA‐stimulated ³⁶Cl⁻ uptake were increased in PROG‐treated females. The potentiation of PROG on both of these neurochemical measures was not observed in animals pretreated with 4‐MA. Together, these studies provide evidence that the anxiolytic effect of PROG is not associated with an intracellular steroid receptor that initiates genomic‐mediated responses. The evidence is consistent with a nongenomic mechanism whereby PROG is metabolized to allopregnanolone, a neuroactive steroid that potentiates GABA_A receptor‐mediated responses.

Enhanced corticosterone release by female compared to male rats under basal and stress conditions is well documented. The demonstration that gonadectomy enhances stress‐induced corticosterone secretion in male rats, but reduces such levels in female rats, suggests a causal association between gonadal steroids and corticosterone release. The present study examined the corticosterone profile of sham gonadectomized and gonadectomized female and male rats under basal and stress conditions. An automated sampling system collected blood from each freely moving, unanaesthetized rat every 10 min (i) over a 24‐h period; (ii) following noise stress; and (iii) following an immune‐mediated stress (lipopolysaccharide, LPS). Plasma was analysed for corticosterone content using radioimmunoassay. Castration resulted in a significant increase in basal corticosterone release compared to the sham‐castrated male rats. Pulsar analysis revealed a significant two‐fold increase in the number of corticosterone pulses over 24 h. Corticosterone increases in response to noise stress and to LPS injection were enhanced following castration. Conversely, ovariectomy resulted in a two‐fold reduction in the number of corticosterone pulses as well as the stress response compared to sham‐ovariectomized female rats. Arginine vasopressin (AVP), corticotrophin‐releasing hormone (CRH) and glucocorticoid receptor mRNAs in the paraventricular nucleus and pro‐opiomelanocortin (POMC) mRNA in the anterior pituitary were analysed post‐LPS administration by in situ hybridization. Significantly higher values were found for AVP, CRH and POMC mRNAs examined for sham females and castrated males compared to sham males and ovariectomized females. This study confirms previous reports concerning the influence of gonadal factors in regulating HPA axis activity and stress responsiveness. The present results extend these observations to the regulation of the dynamic pattern of corticosterone release under basal conditions and suggests that this alteration in pulsatility is important for the differences in stress responsiveness when comparing males and females.

We investigated the effects of gonadal hormone replacement on the pulsatile parameters underlying basal circadian corticosterone secretion in castrated male and ovariectomized female rats using an automated sampling system. Blood was collected from freely moving, unanaesthetized rats every 10 min over a 24‐h period and sampling was continued during a noise stress and after lipopolysaccharide (LPS) administration. Castrated male rats had markedly higher corticosterone levels than intact controls. This was reflected by increased number and frequency of pulses in addition to an increase in the pulse height and amplitude under both basal circadian and stress conditions. Hormone replacement with either testosterone or dihydrotestosterone returned these corticosterone levels and circadian profile to those found in intact males, confirming an androgen‐mediated effect. Ovariectomized females had significantly lower basal and stress‐induced corticosterone levels with lower frequency and amplitude of corticosterone pulses than intact females. 17β‐oestradiol replacement returned basal levels, pulsatile measurements and stress‐induced corticosterone levels to those found in intact females. Three hours post‐LPS administration, castrated males demonstrated significantly higher values of parvocellular paraventricular nucleus (PVN) arginine vasopressin and corticotrophin‐releasing factor and anterior pituitary pro‐opiomelanocortin mRNA while ovariectomized females showed significantly lower levels of all three transcripts compared to intact controls. PVN glucocorticoid receptor mRNA levels 3 h post‐LPS administration were significantly decreased in castrated males and significantly increased in ovariectomized female rats. Replacement of gonadal steroids resulted in a return to the levels found in intact controls after LPS. Gonadal steroid replacement is sufficient to reverse changes in the pulsatile characteristics of corticosterone release after gonadectomy. In addition, gonadal steroid replacement reverses stress‐induced alterations in hypothalamic‐pituitary‐adrenal (HPA) activity. These data demonstrate a major contribution of gonadal steroids to the regulation of HPA axis activity and to the pulsatile characteristics of corticosterone release.

Exposure to stress during early development causes long‐lasting alterations in behaviour and hypothalamic pituitary adrenal (HPA) axis activity, including increased expression of corticotrophin‐releasing hormone (CRH). To determine whether early‐life stress causes epigenetic changes in the CRH promoter leading to increased CRH transcription, 8‐week old female and male rats, subjected to maternal deprivation (MD) between days 2 and 13 post‐birth, were studied for HPA axis responses to stress and CRH promoter methylation in the hypothalamic paraventricular nucleus (PVN) and central nucleus of the amygdala (CeA). Plasma corticosterone and PVN CRH heteronuclear (hn)RNA responses to acute restraint stress were higher in MD rats of both sexes. DNA methylation analysis of the CRH promoter revealed a significantly lower percentage of methylation in two CpGs preceding (CpG1) and inside (CpG2) the cyclic AMP‐response element (CRE) at −230 bp in the CRH promoter in the PVN but not the CeA of MD rats. Gel‐shift assays, using nuclear proteins from forskolin‐treated hypothalamic 4B cells and CRH promoter CRE oligonucleotides, unmethylated or methylated at CpG1, revealed a strong band that was supershifted by phospho‐cAMP response element‐binding antibody. This band was 50% weaker using oligonucleotides methylated at CpG2 (intra‐CRE), or methylated at both CpG1 and CpG2. These findings demonstrate that HPA axis hypersensitivity caused by neonatal stress causes long‐lasting enhanced CRH transcriptional activity in the PVN of both sexes. Hypomethylation of the CRH promoter CRE, a region critical for CRH transcriptional activation, could serve as a mechanism for the increased transcriptional responses to stress observed in MD rats.

The present experiments were conducted to test the hypothesis that septal arginine vasotocin (AVT) and vasoactive intestinal polypeptide (VIP) modulate directed song (a courtship behaviour) and aggression in male zebra finches (Taeniopygia guttata). Subjects were surgically fitted with a guide cannula directed at the septum. Following recovery they were tested for aggression and directed song following infusions of AVT, its antagonist (anti‐vasopressin, AVP), and saline volume control. Infusion of the AVT antagonist significantly reduced all three aggressive behaviours measured (pecks, beak fences and chases); and AVT infusion significantly facilitated beak fencing. Vasoactive intestinal polypeptide treatment significantly reduced pecking. No treatment produced a change in directed song. Comparison with findings in mammals suggests that modulation of aggression by septal AVT (or AVP) is evolutionarily conserved in vertebrates, but modulation of aggression by VIP has not previously been reported for any vertebrate.

The hypothalamic‐pituitary‐adrenal axis is hyporesponsive to stress in late pregnancy, exemplified as reduced adrenocorticotropic hormone (ACTH) and corticosterone responses to restraint, but the mechanisms are unknown. We investigated forward drive and negative feedback upon the hypothalamic‐pituitary‐adrenal axis in pregnant rats. Corticotropin‐releasing hormone (CRH) and vasopressin mRNA expression in the parvocellular paraventricular nucleus and mineralocorticoid and glucocorticoid receptor expression in the paraventricular nucleus and hippocampus were quantified with in situ hybridization. Because it can enhance the corticosterone negative feedback signal, 11β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1) bioactivity in these brain regions and anterior pituitary was measured in vitro, and ACTH and corticosterone stress responses were measured after intracerebroventricular glycyrrhetinic acid, an 11β‐HSD inhibitor. Changes in corticosterone feedback on ACTH secretion were examined after pharmacological adrenalectomy by metyrapone and aminoglutethimide. Parvocellular paraventricular nucleus CRH mRNA content was reduced on day 21 and the CRH mRNA : vasopressin mRNA ratio was unaltered, indicating decreased production of both CRH and vasopressin. An increase in glucocorticoid receptor mRNA expression in the dentate gyrus (mineralocorticoid receptor mRNA expression was unaltered) and increased 11β‐HSD1 activity in the paraventricular nucleus and anterior pituitary suggest an increase in slow negative feedback mechanisms in pregnancy, but glycyrrhetinic acid did not modify the stress response. After metyrapone/aminoglutethimide treatment, corticosterone decreased ACTH secretion more slowly in pregnancy, indicating a decrease in rapid feedback sensitivity. Thus, reduced forward drive rather than increased effectiveness of glucocorticoid negative feedback may underlie stress hyporesponsiveness of the hypothalamic‐pituitary‐adrenal axis in pregnancy.