Louis D. Van de Kar

Neuroendocrine pharmacology of stress.

Exposure to hostile conditions initiates responses organized to enhance the probability of survival. These coordinated responses, known as stress responses, are composed of alterations in behavior, autonomic function and the secretion of multiple hormones. The activation of the renin-angiotensin system and the hypothalamic-pituitary-adrenocortical axis plays a pivotal role in the stress response. Neuroendocrine components activated by stressors include the increased secretion of epinephrine and norepinephrine from the sympathetic nervous system and adrenal medulla, the release of corticotropin-releasing factor (CRF) and vasopressin from parvicellular neurons into the portal circulation, and seconds later, the secretion of pituitary adrenocorticotropin (ACTH), leading to secretion of glucocorticoids by the adrenal gland. Corticotropin-releasing factor coordinates the endocrine, autonomic, behavioral and immune responses to stress and also acts as a neurotransmitter or neuromodulator in the amygdala, dorsal raphe nucleus, hippocampus and locus coeruleus, to integrate brain multi-system responses to stress. This review discussed the role of classical mediators of the stress response, such as corticotropin-releasing factor, vasopressin, serotonin (5-hydroxytryptamine or 5-HT) and catecholamines. Also discussed are the roles of other neuropeptides/neuromodulators involved in the stress response that have previously received little attention, such as substance P, vasoactive intestinal polypeptide, neuropeptide Y and cholecystokinin. Anxiolytic drugs of the benzodiazepine class and other drugs that affect catecholamine, GABA(A), histamine and serotonin receptors have been used to attenuate the neuroendocrine response to stressors. The neuroendocrine information for these drugs is still incomplete; however, they are a new class of potential antidepressant and anxiolytic drugs that offer new therapeutic approaches to treating anxiety disorders. The studies described in this review suggest that multiple brain mechanisms are responsible for the regulation of each hormone and that not all hormones are regulated by the same neural circuits. In particular, the renin-angiotensin system seems to be regulated by different brain mechanisms than the hypothalamic-pituitary-adrenal system. This could be an important survival mechanism to ensure that dysfunction of one neurotransmitter system will not endanger the appropriate secretion of hormones during exposure to adverse conditions. The measurement of several hormones to examine the mechanisms underlying the stress response and the effects of drugs and lesions on these responses can provide insight into the nature and location of brain circuits and neurotransmitter receptors involved in anxiety and stress.

Long-term fluoxetine, but not desipramine, inhibits the ACTH and oxytocin responses to the 5-HT1A agonist, 8-OH-DPAT, in male rats

The present studies determined whether serotonin 5-HT1A receptor-mediated function is modified by chronic exposure to antidepressants. Hormone responses to the 5-HT1A agonist, 8-OH-DPAT, were evaluated after long-term exposure to two antidepressants, the 5-HT uptake blocker, fluoxetine, and the norepinephrine uptake blocker, desipramine (DMI). In addition, the density and affinity of 5-HT1A receptors in the hypothalamus and cerebral cortex were measured. Male rats received fluoxetine (10 mg/kg i.p.), DMI (5 mg/kg i.p.) or saline injections once daily for 21 days. 8-OH-DPAT (0-500 micrograms/kg s.c.) was administered 18 h after the final antidepressant injection and 15 min before sacrifice. 8-OH-DPAT significantly increased plasma ACTH, corticosterone, oxytocin and prolactin, but not renin or vasopressin concentrations. Chronic injections of fluoxetine inhibited the ACTH, corticosterone and oxytocin responses to 8-OH-DPAT, suggesting reduced 5-HT1A receptor function. In contrast, chronic DMI did not alter the hormone responses to 8-OH-DPAT. The density and affinity of 5-HT1A receptors in the frontal cortex or hypothalamus were not altered by either fluoxetine or DMI. To verify that the observed effects require prolonged exposure to fluoxetine, rats received a single injection of fluoxetine (10 mg/kg, i.p.), 3 h before 8-OH-DPAT (0-500 micrograms/kg s.c.). Acute fluoxetine did not reduce any of the hormone responses to 8-OH-DPAT. In conclusion, the results suggest that chronic, but not acute, exposure to fluoxetine decreases 5-HT1A receptor function. This effect is not seen in rats chronically exposed to DMI.(ABSTRACT TRUNCATED AT 250 WORDS)

Ibotenic Acid Lesions in the Bed Nucleus of the Stria terminalis Attenuate Conditioned Stress-Induced Increases in Prolactin, ACTH and Corticosterone

The contribution of the bed nucleus of the stria terminalis (BST) to the expression of stress-induced increases in ACTH/corticosterone, prolactin and renin secretion was assessed. Neurons in the lateral part of the BST were destroyed with bilateral injections of the cell-selective neurotoxin ibotenic acid (1.5 micrograms in 0.1 microliter of solution per side). Two weeks later, the rats were stressed using an immobilization or conditioned stress paradigm. Rats with lesions in the lateral part of the BST showed attenuated ACTH and corticosterone responses to conditioned stress. Bilateral ablation of lateral BST significantly reduced the prolactin secretory response to conditioned stress. The same lesions had no effect upon plasma increases in renin that occur in response to conditioned stress. Also, destruction of neurons in the BST did not affect immobilization-induced increases in ACTH, corticosterone, prolactin or renin. Previous studies have demonstrated that ibotenic acid lesions in the central amygdala reduce corticosterone and renin response to conditioned stress. Thus, both the BST and central amygdala are important for the adrenocortical response to conditioned stress. Neurons in the central nucleus of the amygdala are part of the circuitry that mediates renin responses to conditioned stress. Neurons in the BST are important for the full expression of prolactin responses to conditioned stress. The neuronal circuitry and stressor specificity in the mediation of prolactin, renin and ACTH/corticosterone responses are discussed.

Differential Effects of Serotonin (5-HT1A and 5-HT2) Agonists and Antagonists on Renin and Corticosterone Secretion

The present study was designed to investigate the effect of distinct serotonin (5-HT1A and 5-HT2) agonists and antagonists on renin and corticosterone secretion. Low doses of the selective 5-HT1A agonists 8-hydroxy-2-(di-N-propylamino)tetralin (8-OH-DPAT) (5.0-500.0 micrograms/kg, i.p.) and ipsapirone (TVX Q 7821; 0.5-2.5 mg/kg, i.p.), and of the 5-hydroxytryptamine (5-HT) agonist MK-212 (2.0 mg/kg, i.p.), did not elevate plasma renin activity (PRA) and concentration (PRC) 30 min postinjection. Administration of a higher dose of MK-212 (10.0 mg/kg, i.p.) and of higher doses of ipsapirone (5.0-10.0 mg/kg, i.p.), as well as the 5-HT releaser, fenfluramine (5.0 mg/kg, i.p.), resulted in large increases in PRA and PRC. The effects of MK-212 and fenfluramine on PRA and PRC were blocked by pretreatment with the selective 5-HT2 antagonist, LY53857, in a dose-dependent (0.3-1.0 mg/kg, i.p.) manner. LY53857 (1.0 mg/kg, i.p.) by itself did not affect PRA or PRC. LY53857, furthermore, unmasked a renin-suppressive effect of MK-212, since injection of MK-212 (10.0 mg/kg, i.p.) following LY53857 administration led to a reduction in PRA and PRC. MK-212 (2.0 and 10.0 mg/kg), the high doses of 8-OH-DPAT (500.0 micrograms/kg), ipsapirone (1.0-10.0 mg/kg), and fenfluramine (5.0 mg/kg) all produced an increase in plasma corticosterone levels. The effects of MK-212 and fenfluramine on corticosterone were not inhibited by pretreatment with LY53857. These data suggest that 5-HT1A receptors do not play a role in the regulation of renin secretion, whereas stimulation of 5-HT2 receptors enhances renin release.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective serotonin reuptake inhibitors and neuroendocrine function.

Selective serotonin (5-HT) reuptake inhibitors (SSRIs) are effective drugs for the treatment of several neuropsychiatric disorders associated with reduced serotonergic function. Serotonergic neurons play an important role in the regulation of neuroendocrine function. This review will discuss the acute and chronic effects of SSRIs on neuroendocrine function. Acute administration of SSRIs increases the secretion of several hormones, but chronic treatment with SSRIs does not alter basal blood levels of hormones. However, adaptive changes are induced by long-term treatment with SSRIs in serotonergic, noradrenergic and peptidergic neural function. These adaptive changes, particularly in the function of specific post-synaptic receptor systems, can be examined from altered adrenocorticotrophic hormone (ACTH), cortisol, oxytocin, vasopressin, prolactin, growth hormone (GH) and renin responses to challenges with specific agonists. Neuroendocrine challenge tests both in experimental animals and in humans indicate that chronic SSRIs produce an increase in serotonergic terminal function, accompanied by desensitization of post-synaptic 5-HT1A receptor-mediated ACTH, cortisol, GH and oxytocin responses, and by supersensitivity of post-synaptic 5-HT2A (and/or 5-HT2C) receptor-mediated secretion of hormones. Chronic exposure to SSRIs does not alter the neuroendocrine stress-response and produces inconsistent changes in alpha2 adrenoceptor-mediated GH secretion. Overall, the effects of SSRIs on neuroendocrine function are dependent on adaptive changes in specific neurotransmitter systems that regulate the secretion of specific hormones.

Brain region-specific alterations of 5-HT2A and 5-HT2C receptors in serotonin transporter knockout mice

The aim of the present studies was to determine the effects of reduced or absent serotonin (5‐HT) transporters (5‐HTTs) on 5‐HT2A and 5‐HT2C receptors. The density of 5‐HT2C receptors was significantly increased in the amygdala and choroid plexus of 5‐HTT knockout mice. On the other hand, the density of 5‐HT2A receptors was significantly increased in the hypothalamus and septum, but reduced in the striatum, of 5‐HTT knockout mice. However, 5‐HT2A mRNA was not changed in any brain region measured. 5‐HT2C mRNA was significantly reduced in the choroid plexus and lateral habenula nucleus of these mice. The function of 5‐HT2A receptors was evaluated by hormonal responses to (+/–)‐1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane (DOI). Oxytocin, but not adrenocorticotrophic hormone or corticosterone, responses to DOI were significantly greater in 5‐HTT knockout mice. In addition, Gq and G11 proteins were not significantly changed in any brain region measured. The present results suggest that the constitutive alteration in the function of 5‐HTTs changes the density of 5‐HT2A and 5‐HT2C receptors in a brain region‐specific manner. These changes may not be mediated by alterations in their gene expression or in the level of Gq/11 proteins. The alterations in these receptors may be related to the altered behaviors of 5‐HTT knockout mice.

Estrogen desensitizes 5-HT1A receptors and reduces levels of Gz, Gi1 and Gi3 proteins in the hypothalamus

The present study investigated whether estrogen would desensitize hypothalamic serotonin(1A) (5-HT(1A)) receptors by examining the neuroendocrine response to 8-OH-DPAT, a 5-HT(1A) agonist. Rats were ovariectomized, allowed to recover for 5 days, then given 2 daily injections of estradiol benzoate or vehicle (10 microg/day, s.c.). Twenty-four hours after the second injection, rats were challenged with a sub-maximal dose of 8-OH-DPAT (50 microg/kg, sc) or saline 15 min prior to sacrifice. 8-OH-DPAT produced a significant increase in plasma oxytocin, ACTH and corticosterone levels in ovariectomized rats. While estrogen treatment for 2 days did not alter basal hormone levels, it did significantly reduce the magnitude of oxytocin, ACTH and corticosterone responses to 8-OH-DPAT. The reduction in hormone responses was accompanied by a significant reduction in hypothalamic levels of G(z), G(i1) and G(i3) proteins (by 50%, 30% and 50%, respectively). These findings suggest that a reduction in these G proteins may contribute to the mechanisms underlying estrogen-induced desensitization of 5-HT(1A) receptors. The desensitization of 5-HT(1A) receptors has been suggested to underlie the therapeutic effects of antidepressant 5-HT uptake inhibitors (SSRIs). Thus, the present results suggest that estrogen or estrogen-like substances in combination with SSRIs may prove effective in developing novel therapeutic strategies for neuropsychiatric disorders in women.

WAY-100635 inhibits 8-OH-DPAT-stimulated oxytocin, ACTH and corticosterone, but not prolactin secretion.

Previous studies suggest that the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) increases the secretion of oxytocin, adrenocorticotropic hormone (ACTH), corticosterone and prolactin but not renin. However, the lack of selective 5-HT1A receptor antagonists made it difficult to confirm that 5-HT1A receptors mediate the neuroendocrine responses to 8-OH-DPAT. This study investigated the effects of increasing doses of a selective 5-HT1A receptor antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexanecarboxamide (WAY-100635) on neuroendocrine responses induced by the 5-HT1A receptor agonist 8-OH-DPAT in adult male rats. 8-OH-DPAT, 500 microg/kg s.c., increased plasma levels of oxytocin (to 970% above basal levels); ACTH (to 1622% above basal levels), corticosterone (to 458% above basal levels) and prolactin (to 313% above basal levels), but not renin. The lowest dose of WAY-100635 (0.1 mg/kg s.c.) significantly inhibited the 8-OH-DPAT-induced increase in plasma oxytocin but not ACTH or corticosterone levels. At a dose of 1 mg/kg (s.c.), WAY-100635 completely blocked the oxytocin and ACTH responses and maximally inhibited the corticosterone response to 8-OH-DPAT, although corticosterone levels were still above basal. In contrast, the increase in prolactin secretion, induced by 8-OH-DPAT was not inhibited by any dose of WAY-100635. At the highest dose of WAY-100635 (10 mg/kg, s.c.), basal prolactin levels were markedly elevated (1550%) and administration of 8-OH-DPAT significantly elevated plasma renin concentration. Taken together, these data indicate that: (1) 8-OH-DPAT stimulates oxytocin, ACTH, and corticosterone but not prolactin secretion via activation of 5-HT1A receptors and (2) blockade of 5-HT1A receptors may unmask 8-OH-DPAT simulation of renin secretion via non-5-HT1A receptor mechanisms.

Neurochemical, endocrine and immunological responses to stress in young and old fischer 344 male rats

Two experiments were performed. In the first, a 20 min conditioned emotional response (CER) paradigm was used to compare the neurochemical, endocrine and immunological responses to stress of 7- and 22-month-old Fischer 344 (F344) male rats. In the second, corticosterone levels 20 min following ether stress, and regional brain type I and II corticosterone receptor densities were examined using 7- and 17.5-month-old F344 male rats. Dopamine (DA) metabolism in old nonstressed rats was significantly reduced in the medial frontal cortex, neostriatum, nucleus accumbens and hypothalamus, but not in the amygdala. The CER procedure, nevertheless, increased medial frontal cortical, nucleus accumbens and amygdaloid DA turnover in both the young and old rats. The young and old nonstressed rats did not evidence differences in norepinephrine (NE) and serotonin (5-HT) concentrations. However, stress resulted in a decrease in medial frontal cortical 5-hydroxyindoleacetic acid (5-HIAA) and hypothalamic 5-HT levels in old but not in young animals. These observations suggest age-related differences in the response of central NE and 5-HT systems to stress. Ether and the CER procedure led to exaggerated corticosterone responses in the old rats (17.5 and 22 month, respectively). Hippocampal type I but not type II corticosterone receptors were decreased by 47% in the 17.5-month-old rats. Thus, age-related changes in hippocampal corticosterone receptor types do not occur in unison, and the exacerbated corticosterone response to stress precedes the reported down-regulation of hippocampal type II corticosterone receptors in aged rats. Age-related changes were not observed in the concentrations of corticosterone receptors in other brain regions, or in the prolactin response to stress. The old rats, however, evidenced a reduction in the availability of the renin substrate, angiotensinogen, and in stress-induced renin secretion. Immune function was impaired in the old nonstressed rats, and further compromised by exposure to the CER procedure. In comparison to the young control rats, the old nonstressed rats showed an increased percentage of splenic large granular lymphocytes, reduced splenic natural killer cytotoxicity, and impaired Con-A-stimulated splenic T lymphocyte proliferation. Reductions in T splenic cell proliferation and natural killer cytotoxicity were observed in the young rats subjected to the CER paradigm, but not to the same extent as in the old rats. These observations indicate that aging male F344 rats evidence major alterations in basal central monoamine, endocrine and immune functions, and an increased sensitivity of these systems to stress.

Neuropharmacological Characterization of Serotoninergic Stimulation of Vasopressin Secretion in Conscious Rats

In this study we have evaluated a possible role for brain serotoninergic neurons in the regulation of vasopressin secretion using pharmacological methods. In order to accomplish this, we have developed a specific and sensitive vasopressin radioimmunoassay along with a highly reproducible plasma extraction protocol. These tools were used to evaluate the plasma vasopressin response to several pharmacological challenges in conscious rats. Treatment with the serotonin (5-HT) releaser p-chloroamphetamine caused a significant increase in plasma vasopressin concentration. This effect was blocked by posterior hypothalamic deafferentation which separates serotonin cell bodies in the midbrain from their nerve terminals in the hypothalamus. Administration of graded doses of several 5-HT agonists had no effect. However, treatment with MK212, a serotonin agonist with 5-HT1 + 5-HT2 activity, induced a significant increase in plasma vasopressin concentration. The effect of MK212 on plasma vasopressin was completely abolished by the selective 5-HT2 receptor blocker LY53857. These studies confirm and extend studies by others that provide pharmacological evidence for serotoninergic regulation of vasopressin secretion via a selective 5-HT2 receptor mechanism. The specific neuroanatomical site(s) where serotonin exerts this effect are unknown, and the physiological consequences of these studies remain to be established.