FKBP ligand homodimers can be used to activate signaling events inside cells and animals that have been engineered to express fusions between appropriate signaling domains and FKBP. However, use of these dimerizers in vivo is potentially limited by ligand binding to endogenous FKBP. We have designed ligands that bind specifically to a mutated FKBP over the wild-type protein by remodeling an FKBP-ligand interface to introduce a specificity binding pocket. A compound bearing an ethyl substituent in place of a carbonyl group exhibited sub-nanomolar affinity and 1,000-fold selectivity for a mutant FKBP with a compensating truncation of a phenylalanine residue. Structural and functional analysis of the new pocket showed that recognition is surprisingly relaxed, with the modified ligand only partially filling the engineered cavity. We incorporated the specificity pocket into a fusion protein containing FKBP and the intracellular domain of the Fas receptor. Cells expressing this modified chimeric protein potently underwent apoptosis in response to AP1903, a homodimer of the modified ligand, both in culture and when implanted into mice. Remodeled dimerizers such as AP1903 are ideal reagents for controlling the activities of cells that have been modified by gene therapy procedures, without interference from endogenous FKBP.

We reported previously that the neuropeptide oxytocin attenuates stress-induced hypothalamo-pituitary-adrenal (HPA) activity and anxiety behavior. This study sought to identify forebrain target sites through which oxytocin may mediate its anti-stress effects. Ovariectomized, estradiol-treated rats received intracerebroventricular infusions of oxytocin (1 or 10 ng/hr) or vasopressin (10 ng/hr), and the patterns of neuronal activation after restraint stress were determined by semiquantitative mapping of c-fos mRNA expression. Oxytocin administration significantly attenuated the release of ACTH and corticosterone and the increase in corticotropin-releasing factor mRNA expression in the hypothalamic paraventricular nucleus (PVN) in response to 30 min restraint. Restraint also induced the expression of c-fos mRNA in selective regions of the forebrain, including the PVN, paraventricular thalamic nucleus, habenula, medial amygdala, ventrolateral septum (LSV), most subfields of the dorsal and ventral hippocampus, and piriform and endopiriform cortices. In most cases, this level of gene expression was unaffected by concomitant administration of oxytocin. However, in the PVN, LSV, and throughout all subfields of the dorsal hippocampus, restraint evoked no detectable increase in c-fos mRNA in animals treated with either dose of oxytocin. Vasopressin had no effects on either HPA axis responses or neuronal activation in response to restraint, indicating that the effects were highly peptide selective. These data show that central oxytocin attenuates both the stress-induced neuroendocrine and molecular responses of the HPA axis and that the dorsal hippocampus, LSV, and PVN constitute an oxytocin-sensitive forebrain stress circuit.

The behavioural and endocrine responses to a 10 min white noise stress have been characterized in female virgin and undisturbed lactating Sprague-Dawley rats. Animals were continuously video-taped and frequent blood samples were collected using an automated sampling system. Noise stress caused hypothalamo-pituitary-adrenal (HPA) activation, as indicated by a rapid increase in plasma corticosterone and ACTH in the virgins: corticosterone concentrations peaked 20 min after initiation of the stress before declining rapidly back to basal concentrations. In contrast, noise stress had no significant effect on either plasma corticosterone or ACTH concentrations in the lactating animals. However, 72 h after weaning the corticosterone response of the ex-lactating rats was of comparable magnitude, but longer duration to that seen in the virgins. Plasma prolactin concentrations were significantly higher in the lactating animals and declined in response to the noise whereas, a transient but reproducible increase was seen in the virgin group. In situ hybridization revealed a significantly lower basal expression of CRF mRNA in the paraventricular nucleus of lactating rats as compared to the virgins, but noise stress had no further effect. Virgin animals showed behavioural responses to the stress, including an increase in the total activity, exploratory behaviours (rearing) and displacement behaviours (grooming). Lactating animals also showed behavioural responses to the noise, but their activities were principally directed towards the pups. These data show that although lactating rats showed normal behavioural reactivity to a psychological stress they showed no statistically significant activation of the HPA axis, suggesting a dissociation of behavioural and neuroendocrine responses to this mild stress.

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. 17beta-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.