Michael E. Rhodes

Environmental enrichment lowers stress-responsive hormones in singly housed male and female rats

Structural and social aspects of an environmental system can influence the physiology and behavior of animals occupying that system. This study examined the physiological effects of environmental enrichment (EE) with Kong Toys and Nestlets on stress-responsive hormones of the hypothalamic-pituitary-adrenal (HPA) axis under basal and mild stress conditions in singly housed, jugular vein-cannulated, male and female rats. Animals of both sexes housed with EE had significantly lower baseline adrenocorticotropic hormone (ACTH) and corticosterone (CORT) concentrations compared to those housed without EE. ACTH responses to the mild stress of saline injection were significantly lower in female rats housed with EE. Interaction with the Kong Toys and Nestlets appears to have provided the rats with a diversion from monotonous cage life, resulting in lower HPA axis activity before and after mild stress. These results are important because low, stable baselines are essential for accurately discerning pharmacological and other influences on the HPA axis.

Male-female differences in rat hypothalamic-pituitary-adrenal axis responses to nicotine stimulation

Hypothalamic-pituitary-adrenal (HPA) axis responsiveness differs physiologically and pharmacologically between the sexes (sexual diergism). Central nicotinic receptors modulate this endocrine axis. Previous studies have established that nicotine (NIC) stimulates the HPA axis; however, only male animals have been used. We have demonstrated that plasma arginine vasopressin (AVP) and adrenocorticotropic hormone (ACTH) concentrations showed greater responsiveness in male than in female rats pretreated with scopolamine (SCOP), a muscarinic antagonist, followed by physostigmine (PHYSO), an acetylcholinesterase inhibitor. These results suggest that the SCOP + PHYSO effects may have resulted from an indirect nicotinic effect caused by increased synaptic acetylcholine with simultaneous muscarinic antagonism. In the present study, we investigated nicotinic cholinergic influences on HPA axis activity in male and female rats by administering NIC (0, 0.03, 0.1, 0.3, or 0.5 mg/kg) and determining plasma AVP, ACTH, and corticosterone (CORT) responses. Male rats had a significantly greater, dose-related AVP response to NIC than did females. In contrast, female rats had significantly greater, dose-related ACTH and CORT responses to NIC than did males. Hormone responses following NIC were similar to hormone responses following SCOP + PHYSO. These results suggest nicotinic receptors influence the HPA axis differentially in male and female rats.

Hypothalamo-pituitary-adrenal cortical responses to low-dose physostigmine and arginine vasopressin administration: sex differences between major depressives and matched control subjects.

Of heuristic value in understanding the neurochemistry of major depression is whether the hypothalamo-pituitary-adrenocortical (HPA) axis hyperactivity that occurs in this illness can be related to putative neurotransmitter dysfunction(s). Cholinergic neurotransmission stimulates hypothalamic corticotropin releasing hormone (CRH) and arginine vasopressin (AVP) secretion, both of which stimulate pituitary corticotropin (ACTH) secretion, but whether the HPA axis in humans is activated only by doses of cholinergic agonists that produce noxious side effects remains controversial. To test the hypothesis of increased cholinergic sensitivity in major depression, physostigmine (PHYSO), a reversible cholinesterase inhibitor, was administered to patients and control subjects at a dose that elevated plasma ACTH, cortisol, and AVP concentrations but produced few or no side effects. Exogenous AVP also was administered to determine if it would augment the effect of low-dose PHYSO on the HPA axis. Twelve premenopausal or estrogen-replaced female major depressives, 12 individually matched female control subjects, eight male major depressives, and eight matched male control subjects underwent four test sessions 5-7 days apart: PHYSO (8 microg/kg IV), AVP (0.08 U/kg IM), PHYSO + AVP, and saline control. Serial blood samples were taken before and after pharmacologic challenge and analyzed for ACTH1-39, cortisol, and AVP. Estradiol and testosterone were also measured at each test session. PHYSO (8 microg/kg) significantly increased plasma ACTH, cortisol, and AVP, while producing no side effects in approximately half the subjects and predominantly mild side effects in the other half. These hormone increases following PHYSO occurred primarily in the female depressives and the male control subjects and were not significantly related to the presence or absence of side effects. The greater the ACTH and AVP responses to PHYSO, the stronger their correlation, suggesting that AVP may have been acting as a secretagogue for ACTH. Administered AVP significantly increased the secretion of ACTH in the patients and control subjects to a similar degree, and AVP given after PHYSO did not augment the HPA axis response to a greater degree in the depressives than in the control subjects. Plasma estradiol and testosterone were within the normal range for all four groups of subjects and were not significantly related to their HPA axis hormone responses. The study results support the hypothesis of heightened cholinergic sensitivity in premenopausal female, but not in male, patients with major depression. The low dose of PHYSO used may represent a useful paradigm for central cholinergic stimulation of the HPA axis.

Sexual diergism in rat hypothalamic-pituitary-adrenal axis responses to cholinergic stimulation and antagonism.

The hypothalamic-pituitary-adrenal (HPA) axis has differential physiological activity in male and female animals (sexual diergism). Central cholinergic systems stimulate this endocrine axis. In the present study we investigated muscarinic and nicotinic cholinergic influences on HPA axis activity in male and female rats by pretreatment with selective cholinergic receptor antagonists followed by stimulation with physostigmine (PHYSO), an acetylcholinesterase inhibitor. Hormonal measures were plasma arginine vasopressin (AVP), adrenocorticotropic hormone (ACTH), and corticosterone (CORT). Male rats had significantly greater AVP and ACTH responses to PHYSO alone than did females. Scopolamine (SCOP) enhanced the AVP response to PHYSO to a greater extent in males than in females. In contrast, mecamylamine (MEC) enhanced the AVP response in females but decreased it in males. SCOP potentiated, and MEC inhibited, the stimulatory effect of PHYSO on ACTH in both sexes, but SCOP potentiation was greater in males, and MEC inhibition was greater in females. Absolute CORT increases following PHYSO were greater in females, but percent increases over baseline were greater in males. Similar to their effects on ACTH responses, MEC attenuated, and SCOP enhanced, CORT responses to PHYSO. These results suggest that cholinergic receptor subtypes may influence HPA axis activity differentially in male and female rats.

Influence of environmental enrichment on hypothalamic-pituitary-adrenal (HPA) responses to single-dose nicotine, continuous nicotine by osmotic mini-pumps, and nicotine withdrawal by mecamylamine in male and female rats.

In the present study, we determined the effects of environmental enrichment (EE; Kong Toys and Nestlets) on sexually diergic HPA axis responses to single-dose nicotine (NIC), single-dose NIC following continuous NIC administration for two weeks, and NIC withdrawal by single-dose mecamylamine (MEC) in male and female rats. Blood sampling occurred before and after MEC and NIC administrations for the determination of adrenocorticotropic hormone (ACTH) and corticosterone (CORT). Supporting and extending our previous findings, EE appeared to produce anxiolytic effects by reducing hormone responses: Male and female rats housed with EE had lower baseline ACTH and significantly lower HPA axis responses to the mild stress of saline (SAL) injection than did those housed without EE. The sexually diergic responses to single dose NIC, continuous NIC, and MEC-induced NIC withdrawal were reduced by EE in many male and female groups. ACTH responses to continuous NIC and MEC-induced NIC withdrawal were blunted to a greater extent in female EE groups than in male EE groups, suggesting that females are more sensitive to the anxiolytic effects of EE. Because EE lowered stress-responsive hormones of the HPA axis in most groups, EE may be a useful intervention for stress reduction in animal models of NIC addiction. As well, the effectiveness of EE in animal studies of NIC withdrawal may enlighten human studies addressing coping styles and tobacco cessation in men and women.

Rat estrous cycle influences the sexual diergism of HPA axis stimulation by nicotine

We previously reported that female rats had significantly greater hypothalamic-pituitary-adrenal (HPA) axis responses to cholinergic stimulation by nicotine (NIC) than did male rats. Females in defined estrous cycle stages, however, were not studied because of sample size limitations. We further explored this finding by determining HPA axis responses to two doses of NIC in female rats (N = 101) during different estrous cycle stages, and in males (N = 69). NIC doses were: 0.3 mg/kg, which provided the greatest female-male difference in the earlier study, and 0.5 mg/kg, which stimulated the HPA axis similarly in the two sexes. Plasma AVP, ACTH, and corticosterone were measured. Proestrous and estrous females had higher ACTH responses to NIC (0.3 mg/kg) compared to metestrous and diestrous females, and compared to males. ACTH responses to NIC (0.5 mg/kg) were similar, regardless of estrous cycle stage or sex. Males had higher AVP responses to both NIC doses compared to females in all estrous cycle stages. Corticosterone responses followed the ACTH responses, except that females in all estrous stages started from a higher corticosterone baseline compared to males. These results are similar to our earlier findings across the estrous cycle with non-specific cholinergic stimulation by physostigmine and suggest that the nicotinic system contributes to the differential HPA axis responses to cholinergic challenge across the estrous cycle.

Estrous cycle influences on sexual diergism of HPA axis responses to cholinergic stimulation in rats.

Central cholinergic systems differentially modulate hypothalamic-pituitary-adrenal (HPA) axis activity in female and male animals (sexual diergism). We previously reported that male rats had significantly greater HPA axis responses to stimulation by physostigmine (PHYSO), an acetylcholinesterase (AChE) inhibitor, compared to females. Females in defined estrous cycle stages, however, were not studied because of sample size limitations. We, therefore, determined HPA axis responses to stimulation by PHYSO in females during all estrous cycle stages (n = 78), and in male rats (n = 75). Plasma arginine vasopressin (AVP), adrenocorticotropic hormone (ACTH), and corticosterone (CORT) were measured. Estrous cycle stage was determined by light microscopy of vaginal smears. Proestrous and estrous females had higher ACTH and CORT responses compared to metestrous and diestrous females. Males had higher ACTH and AVP responses compared to females in all cycle stages. CORT responses followed the ACTH responses, except that females started from a higher baseline in all estrous stages, compared to males. These results suggest that cholinergic regulation of the HPA axis differs among females across stages of the estrous cycle, as well as between males and females. These effects are likely due to differences in circulating sex steroids and their actions within the brain.

Sexually diergic hypothalamic-pituitary-adrenal (HPA) responses to single-dose nicotine, continuous nicotine infusion, and nicotine withdrawal by mecamylamine in rats

Hypothalamic-pituitary-adrenal (HPA) responses to single-dose nicotine (NIC) are sexually diergic: Female rats have higher adrenocorticotropic hormone (ACTH) and corticosterone (CORT) responses than do males. In the present study we determined HPA responses in male and female rats following single doses of NIC, a single-dose of NIC immediately following continuous NIC for two weeks, and NIC withdrawal by single-dose mecamylamine (MEC) following continuous NIC infusion for two weeks. Blood sampling occurred before and after MEC and NIC administrations for the determination of ACTH and CORT. In accordance with our previous findings, female ACTH and CORT responses to single-dose NIC were greater than male responses. This sex difference remained after single-dose NIC followed continuous NIC infusion, but HPA responses in both sexes were significantly lower in magnitude and duration than in the single-dose NIC alone groups. Sex differences also were observed following NIC withdrawal by MEC: the HPA responses to pretreatment with MEC were significantly higher in magnitude and duration in the continuous NIC groups than in the single-dose NIC groups. These results demonstrate that HPA responses to NIC are reduced and transient following continuous NIC infusion but are enhanced and sustained following NIC withdrawal by MEC after continuous NIC, suggesting that NIC habituation and withdrawal influence the stress responses in a diergic manner. These findings highlight the importance of sex differences in the effect of NIC on HPA axis activity and stress responsiveness, which may have implications for directing NIC-addiction treatment specifically towards men and women.

Pituitary‐Adrenal Responses to Cholinergic Stimulation and Acute Mild Stress are Differentially Elevated in Male and Female M2 Muscarinic Receptor Knockout Mice

Adrenocorticotrophic hormone (ACTH) and corticosterone responses to cholinergic stimulation are greater in male rats and mice than in females. To explore the role of M2 muscarinic receptors in this sex difference, we administered the nonselective muscarinic agonist, oxotremorine, the acetylcholinesterase inhibitor, physostigmine, and saline (a mild stressor) to male and female M2 receptor knockout (KO) and wild‐type (WT) mice of the same genetic background. Because M2 receptors function primarily as presynaptic autoreceptors, we hypothesized that their absence in M2 KO mice would increase the sensitivity of hormone responses to cholinergic stimulation in these groups. Both male and female M2 KO mice were significantly more responsive to the stress of saline injection than were their WT counterparts. Oxotremorine and physostigmine increased ACTH and corticosterone in all four groups, but to a significantly greater degree in KO males compared to WT males, KO females, and WT females. The increase in ACTH also was significantly greater in WT males compared to WT females. By contrast, the increase in corticosterone was significantly more in females compared to males, independent of genotype. Following pretreatment with the nonselective muscarinic antagonist, scopolamine, ACTH and corticosterone responses to oxotremorine and to saline in the M2 KO mice were comparable with those of their WT counterparts. These findings suggest that the M2 muscarinic receptor subtype influences male and female pituitary‐adrenal responses following stimulation by both mild stress and cholinergic drugs. The M2 receptor appears to regulate ACTH responses to cholinergic stimulation in males but not in females; however, other muscarinic receptors may be involved because corticosterone responses were higher in females compared to males. Because ACTH and corticosterone responses were greater in male and female M2 KO mice, the M2 receptor appears to dampen the stress response.

Sexual diergism of hypothalamo-pituitary-adrenal cortical responses to low-dose physotigmine in elderly vs. young women and men

We previously demonstrated that the reversible cholinesterase inhibitor, physostigmine (PHYSO), administered to normal young adult women and men (average age 35 years) at a dose that produced few or no side effects, resulted in a sex difference (sexual diergism) in hypothalamo-pituitary-adrenal cortical (HPA) axis responses: Plasma ACTH1–39, cortisol, and arginine vasopressin (AVP) concentrations increased to a significantly greater extent in the men than in the women. To explore the effect of age on these sexually diergic hormone responses, in the present study we used the same dose of PHYSO (8 μg/kg IV) to stimulate ACTH1–39, cortisol, and AVP secretion in normal elderly, non-estrogen-replaced women and elderly men (average ages 73 years and 70 years, respectively). The subjects underwent three test sessions 5–7 days apart: PHYSO, saline control, and a second session of PHYSO. Serial blood samples were taken for hormone analyses before and after pharmacologic challenge.As with the previously studied younger subjects, PHYSO administration produced no side effects in about half the elderly subjects and mild side effects in the other half, with no significant female-male differences. The hormone responses were 2–5 fold greater in the elderly subjects than in the younger subjects, but in contrast to the younger subjects, the elderly men did not have significantly greater hormone responses to PHYSO administration than did the elderly women. The ACTH1–39 and AVP responses to PHYSO for the two sessions were significantly positively correlated in the men (+0.96, +0.91) but not in the women. None of the hormone responses was significantly correlated with the presence or absence of side effects in either group of subjects.These results indicate a greater sensitivity of the HPA axis to low-dose PHYSO, and a loss of overall sex differences in hormone responses, in elderly compared with younger subjects. The lack of a difference in side effects between the elderly women and men and the lack of significant correlations between presence or absence of side effects and hormone responses suggest that the increase in hormone responses with aging is due to correspondingly increased responsiveness of central cholinergic systems and/or the HPA axis, and not to a nonspecific stress response.