The adipose tissue-derived hormone leptin regulates body weight homeostasis by decreasing food intake and increasing energy expenditure. The weight-reducing action of leptin is thought to be mediated primarily by signal transduction through the leptin receptor (LR) in the hypothalamus. We have used immunohistochemistry to localize LR-immunoreactive (LR-IR) cells in the rat brain using an antiserum against a portion of the intracellular domain of LR that is common to all LR isoforms. The antiserum recognized the short and long isoforms of LR in transfected hematopoietic BaF3 cells. To examine the chemical nature of target cells for leptin, direct double-labeling immunofluorescence histochemistry was applied. The results show extensive distribution of LR-like immunoreactivity (LR-LI) in the brain with positively stained cells present, e.g., in the choroid plexus, cerebral cortex, hippocampus, thalamus, and hypothalamus. In the hypothalamus, strongly LR-IR neurons were present in the supraoptic nucleus (SON) and paraventricular nucleus (PVN), periventricular nucleus, arcuate nucleus, and lateral hypothalamus. Weaker LR-IR neurons were also demonstrated in the lateral and medial preoptic nuclei, suprachiasmatic nucleus, ventromedial and dorsomedial nuclei, and tuberomammillary nucleus. Confocal laser scanning microscopy showed LR-LI in the periphery of individual cells. In magnocellular neurons of the SON and PVN, LR-LI was demonstrated in vasopressin- and oxytocin-containing neurons. In parvocellular neurons of the PVN, LR-LI was demonstrated in many corticotropin-releasing hormone-containing neurons. LR-IR neurons were mainly seen in the ventromedial aspect of the arcuate nucleus, where LR-LI co-localized with neuropeptide Y. In the ventrolateral part of the arcuate nucleus, LR-LI was present in many large adrenocorticotropic hormone-IR proopiomelanocortin-containing neurons and in a few galanin-, neurotensin-, and growth hormone-releasing hormone-containing neurons. In the dorsomedial arcuate nucleus, few tyrosine hydroxylase (dopamine)-containing neurons were seen to have LR-LI. Melanin-concentrating hormone-containing neurons in the lateral hypothalamus had LR-LI. Based on the immunohistochemical results, possible interactions of leptin with brain mechanisms are discussed.

The role of the neuropeptide corticotropin-releasing factor (CRF) in mediating the behavioral effects of ethanol withdrawal in the rat was examined using the elevated plus-maze test. In Experiment 1, CRF (0.5 microgram ICV) reduced the percentage of time spent on the open arms of the elevated plus-maze, consistent with an "anxiogenic-like" effect. CRF also reduced the total number of arm entries, indicating a reduction in general activity. Low doses (5 and 25 micrograms ICV) of the CRF antagonist, alpha-helical CRF produced no behavioral effects in the elevated plus-maze, while a higher dose (50 micrograms ICV) elicited CRF-like activity. In experiment 2, rats were maintained for 2-3 weeks on a liquid diet containing ethanol (8.5-11.5% v/v) or sucrose. Eight hours after withdrawal from the ethanol diet rats displayed "anxiogenic-like" responses as well as a reduction in general activity in the elevated plus-maze compared with rats withdrawn from control diet. Alpha-helical CRF significantly antagonized the "anxiogenic-like" effects of ethanol withdrawal in the plus-maze. General activity and physical signs of ethanol withdrawal such as tail stiffness, body tremor and ventromedial distal flexion were unaffected by alpha-helical CRF. Blood Alcohol Levels (BALs) determined immediately after removal of the ethanol diet showed no group differences in ethanol consumption. These results suggest that increased activity of central CRF systems may mediate the anxiogenic effects of ethanol withdrawal.

We have shown previously that footshock stress and priming injections of cocaine reinstate cocaine seeking in rats after prolonged drug-free periods (Erb et al., 1996). Here we examined the role of brain corticotropin-releasing factor (CRF) and the adrenal hormone corticosterone in stress- and cocaine-induced reinstatement of cocaine seeking in rats. The ability of footshock stress and priming injections of cocaine to induce relapse to cocaine seeking was studied after intracerebroventricular infusions of the CRF receptor antagonist D-Phe CRF12-41, after adrenalectomy, and after adrenalectomy with corticosterone replacement. Rats were allowed to self-administer cocaine (1.0 mg/kg/infusion, i.v) for 3 hr daily for 10-14 d and were then placed on an extinction schedule during which saline was substituted for cocaine. Tests for reinstatement were given after intermittent footshock (10 min; 0.5 mA) and after priming injections of saline and cocaine (20 mg/kg, i.p.). Footshock reinstated cocaine seeking in both intact animals and animals with corticosterone replacement but not in adrenalectomized animals. The CRF receptor antagonist D-Phe CRF12-41 blocked footshock-induced reinstatement at all doses tested in both intact animals and animals with corticosterone replacement. Reinstatement by priming injections of cocaine was only minimally attenuated by adrenalectomy and by pretreatment with D-Phe CRF12-41. These data suggest that brain CRF plays a critical role in stress-induced, but only a modulatory role in cocaine-induced, reinstatement of cocaine seeking. Furthermore, the data show that although reinstatement of cocaine seeking by footshock stress requires minimal, basal, levels of corticosterone, stress-induced increases in corticosterone do not play a role in this effect.

BACKGROUND

Despite prolonged abstinence, prior drug dependence is accompanied by lasting changes in physiology, psychosocial functioning and vulnerability to relapse. One proposed mechanism for these alterations is dysregulation of corticotropin-releasing factor (CRF) neurocircuitry.

OBJECTIVE

To determine regional brain CRF content and HPA-axis activity during protracted cocaine and ethanol withdrawal in dependent rats.

METHODS

To study protracted ethanol withdrawal, rats ( n=22) were fed a nutritionally complete, ethanol (10% v/v) liquid diet for 3-4 weeks. Controls ( n=12) were pair-fed an isocaloric, ethanol-free formulation. To study protracted cocaine withdrawal, rats ( n=23) self-administered cocaine (0.25 mg/infusion; FR-5) daily for 3 weeks during 3-h sessions and subsequently were allowed to self-administer cocaine during two 12-h "binge" sessions. Controls ( n=6) received yoked saline infusions. Regional brain CRF-like-immunoreactivity (CRF-LI), plasma ACTH-LI and CORT-LI levels were determined from 1 day to 6 weeks post-withdrawal.

RESULTS

Both ethanol- and cocaine-withdrawn rats initially exhibited reduced CRF-LI content in the amygdala followed by a progressive increase culminating in elevated levels 6 weeks post-withdrawal. Ethanol-withdrawn rats also initially had reduced hippocampal, frontal cortical and hypothalamic CRF-LI levels and time-dependent reductions in basal CORT levels. Cocaine-withdrawn rats showed time-dependent elevations in frontal cortical CRF-LI and basal CORT levels.

CONCLUSIONS

Protracted withdrawal from ethanol or cocaine is associated with altered limbic CRF-LI and circulating CORT levels beyond the detoxification stage. The delayed nature of some changes suggests that they may not represent residual effects of acute withdrawal, but rather emerging manifestations of a separate process, such as allostatic load.

In four independent studies, sex differences in cortisol responses to psychological stress were investigated in healthy adolescents and adults (total n = 153). Public speaking and mental arithmetic in front of an audience (Studies 1-3) reliably induced increases in free cortisol levels in both sexes with 2- to 4-fold increases above baseline levels. Mean cortisol responses were 1.5- to 2-fold higher in men compared with women. In Study 3, cortisol profiles were additionally investigated after human corticotropin-releasing hormone (h-CRH) and bicycle ergometry until exhaustion. Here, both sexes showed very similar adrenocortical responses. Furthermore, men showed elevated cortisol levels in anticipation of the psychological stress situation without actually having to perform the tasks (Study 4). Under this condition cortisol concentration was unchanged or decreased in women. From these data we conclude that the observed sex difference does not reflect an overall lower responsiveness of the female adrenal cortex. Although these studies do not provide conclusive data, we suggest sex differences in cognitive and/or emotional responses to distressing psychosocial situations which in turn may influence cortisol secretion.

Clinical and preclinical studies have gathered substantial evidence that alterations of the stress hormone system play a major, causal role in the development of depression. In this review article, a summary of studies sustaining that view is given and data are presented which demonstrate that depression is associated with an impairment of corticosteroid receptor function that gives rise to an excessive release of neurohormones to which a number of signs and symptoms characteristic of depression can be attributed. The studies referred to in the following unanimously support the concept of an antidepressant mechanism of action that exerts its effects beyond the cell membrane receptors of biogenic amines and particularly includes the improvement of corticosteroid receptor function. When activated by ligands, corticosteroid receptors act as transcription factors in correspondence with numerous other transcription factors already known to be activated by antidepressants. Furthermore, the potential of drugs that interfere more directly with stress hormone regulation, such as corticosteroid receptor antagonists and corticotropin-releasing hormone receptor antagonists, is discussed.

Psycho-neuro-endocrine-immune modulation through the brain-gut axis likely has a key role in the pathogenesis of inflammatory bowel disease (IBD). The brain-gut axis involves interactions among the neural components, including (1) the autonomic nervous system, (2) the central nervous system, (3) the stress system (hypothalamic-pituitary-adrenal axis), (4) the (gastrointestinal) corticotropin-releasing factor system, and (5) the intestinal response (including the intestinal barrier, the luminal microbiota, and the intestinal immune response). Animal models suggest that the cholinergic anti-inflammatory pathway through an anti-tumor necrosis factor effect of the efferent vagus nerve could be a therapeutic target in IBD through a pharmacologic, nutritional, or neurostimulation approach. In addition, the psychophysiological vulnerability of patients with IBD, secondary to the potential presence of any mood disorders, distress, increased perceived stress, or maladaptive coping strategies, underscores the psychological needs of patients with IBD. Clinicians need to address these issues with patients because there is emerging evidence that stress or other negative psychological attributes may have an effect on the disease course. Future research may include exploration of markers of brain-gut interactions, including serum/salivary cortisol (as a marker of the hypothalamic-pituitary-adrenal axis), heart rate variability (as a marker of the sympathovagal balance), or brain imaging studies. The widespread use and potential impact of complementary and alternative medicine and the positive response to placebo (in clinical trials) is further evidence that exploring other psycho-interventions may be important therapeutic adjuncts to the conventional therapeutic approach in IBD.

There is considerable evidence to suggest that adverse early-life experiences have a profound effect on the developing brain. Neurobiological changes that occur in response to untoward early-life stress can lead to lifelong psychiatric sequelae. Children who are exposed to sexual or physical abuse or the death of a parent are at higher risk for development of depressive and anxiety disorders later in life. Preclinical and clinical studies have shown that repeated early-life stress leads to alterations in central neurobiological systems, particularly in the corticotropin-releasing factor system, leading to increased responsiveness to stress. Clearly, exposure to early-life stressors leads to neurobiological changes that increase the risk of psychopathology in both children and adults. Identification of the neurobiological substrates that are affected by adverse experiences in early life should lead to the development of more effective treatments for these disorders. The preclinical and clinical studies evaluating the consequences of early-life stress are reviewed.

The central amygdala (CeA) plays a role in the relationship among stress, corticotropin-releasing factor (CRF), and alcohol abuse. In whole-cell recordings, both CRF and ethanol enhanced gamma-aminobutyric acid-mediated (GABAergic) neurotransmission in CeA neurons from wild-type and CRF2 receptor knockout mice, but not CRF1 receptor knockout mice. CRF1 (but not CRF2) receptor antagonists blocked both CRF and ethanol effects in wild-type mice. These data indicate that CRF1 receptors mediate ethanol enhancement of GABAergic synaptic transmission in the CeA, and they suggest a cellular mechanism underlying involvement of CRF in ethanol's behavioral and motivational effects.

The nucleotide sequence of cloned cDNA for preproenkephalin from bovine adrenal medulla indicates that the precursor protein contains four copies of Met-enkephalin and one copy each of Leu-enkephalin, Met-enkephalin-Arg6-Phe7 and Met-enkephalin-Arg6-Gly7-Leu8, a previously undetected opioid peptide. The enkephalin and extended enkephalin sequences are each bounded by paired basic amino acid residues. Preproenkephalin may represent a multi-hormone precursor, like the corticotropin-beta-lipotropin precursor.

This article reviews the mechanisms believed to mediate stress-induced inhibition of reproductive functions and the anatomical sites at which these effects take place. Particular emphasis is placed on the potential modulating role of hormones or neurotransmitters released during stress. At the level of the gonads, adrenal corticoids, pro-opiomelanocortin (POMC)-like peptides, and corticotropin-releasing factor (CRF) are reported to interfere with the stimulatory action of gonadotropins on sex steroid-producing cells. Increased circulating corticosteroid levels may also decrease pituitary responsiveness to GnRH. There is, however, increasing evidence that these mechanisms are primarily involved in mediating the effects of prolonged stress, but not those of an acute stimulus. In contrast, a variety of hormones or neurotransmitters, including CRF, POMC peptides, and biogenic amines act within the brain to mediate the inhibitory influence of both acute and prolonged stresses on reproductive function.

The mechanism by which pharmacologic administration of the hormone FGF21 increases energy expenditure to cause weight loss in obese animals is unknown. Here we report that FGF21 acts centrally to exert its effects on energy expenditure and body weight in obese mice. Using tissue-specific knockout mice, we show that βKlotho, the obligate coreceptor for FGF21, is required in the nervous system for these effects. FGF21 stimulates sympathetic nerve activity to brown adipose tissue through a mechanism that depends on the neuropeptide corticotropin-releasing factor. Our findings provide an unexpected mechanistic explanation for the strong pharmacologic effects of FGF21 on energy expenditure and weight loss in obese animals.

Cytokines mediate and control immune and inflammatory responses. Complex interactions exist between cytokines, inflammation and the adaptive responses in maintaining homeostasis, health, and well-being. Like the stress response, the inflammatory reaction is crucial for survival and is meant to be tailored to the stimulus and time. A full-fledged systemic inflammatory reaction results in stimulation of four major programs: the acute-phase reaction, the sickness syndrome, the pain program, and the stress response, mediated by the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system. Common human diseases such as atopy/allergy, autoimmunity, chronic infections and sepsis are characterized by a dysregulation of the pro- versus anti-inflammatory and T helper (Th)1 versus Th2 cytokine balance. Recent evidence also indicates the involvement of pro-inflammatory cytokines in the pathogenesis of atherosclerosis and major depression, and conditions such as visceral-type obesity, metabolic syndrome and sleep disturbances. During inflammation, the activation of the stress system, through induction of a Th2 shift, protects the organism from systemic 'overshooting' with Th1/pro-inflammatory cytokines. Under certain conditions, however, stress hormones may actually facilitate inflammation through induction of interleukin (IL)-1, IL-6, IL-8, IL-18, tumor necrosis factor-alpha and C-reactive protein production and through activation of the corticotropin-releasing hormone/substance P-histamine axis. Thus, a dysfunctional neuroendocrine-immune interface associated with abnormalities of the 'systemic anti-inflammatory feedback' and/or 'hyperactivity' of the local pro-inflammatory factors may play a role in the pathogenesis of atopic/allergic and autoimmune diseases, obesity, depression, and atherosclerosis. These abnormalities and the failure of the adaptive systems to resolve inflammation affect the well-being of the individual, including behavioral parameters, quality of life and sleep, as well as indices of metabolic and cardiovascular health. These hypotheses require further investigation, but the answers should provide critical insights into mechanisms underlying a variety of common human immune-related diseases.

Stress induces the release of the peptide corticotropin-releasing factor (CRF) into the ventral tegmental area (VTA), and also increases dopamine levels in brain regions receiving dense VTA input. Therefore, stress may activate the mesolimbic dopamine system in part through the actions of CRF in the VTA. Here, we explored the mechanism by which CRF affects VTA dopamine neuron firing. Using patch-clamp recordings from brain slices we first determined that the presence of I(h) is an excellent predictor of dopamine content in mice. We next showed that CRF dose-dependently increased VTA dopamine neuron firing, which was prevented by antagonism of the CRF receptor-1 (CRF-R1), and was mimicked by CRF-R1 agonists. Inhibition of the phospholipase C (PLC)-protein kinase C (PKC) signalling pathway, but not the cAMP-protein kinase A (PKA) signalling pathway, prevented the increase in dopamine neuron firing by CRF. Furthermore, the effect of CRF on VTA dopamine neurons was not attenuated by blockade of I(A), I(K(Ca)) or I(Kir), but was completely eliminated by inhibition of I(h). Although cAMP-dependent modulation of I(h) through changes in the voltage dependence of activation is well established, we surprisingly found that CRF, through a PKC-dependent mechanism, enhanced I(h) independent of changes in the voltage dependence of activation. Thus, our results demonstrated that CRF acted on the CRF-R1 to stimulate the PLC-PKC signalling pathway, which in turn enhanced I(h) to increase VTA dopamine neuron firing. These findings provide a cellular mechanism of the interaction between CRF and dopamine, which can be involved in promoting the avoidance of threatening stimuli, the pursuit of appetitive behaviours, as well as various psychiatric conditions.

Naturally occurring variations in maternal care in early postnatal life are associated with the development of individual differences in behavioral and hypothalamic-pituitary-adrenal responses to stress in the rat. These effects appear to be mediated by the influence of maternal licking/grooming on the development of central systems that serve to activate (corticotropin-releasing factor) or inhibit (gamma-aminobutyric acid) the expression of behavioral and endocrine responses to stress through effects on forebrain noradrenergic systems. Importantly, individual differences in maternal care are transmitted from mother to daughter, providing a mechanism for the behavioral transmission of individual differences in stress reactivity across generations.

The differential modulation of learning and anxiety by corticotropin-releasing factor (CRF) through CRF receptor subtypes 1 (CRFR1) and 2 (CRFR2) is demonstrated. As learning paradigm, context- and tone-dependent fear conditioning of the mouse was used. Injection of CRF into the dorsal hippocampus before training enhanced learning through CRFR1 as demonstrated by the finding that this effect was prevented by the local injection of the unselective CRFR antagonist astressin, but not by the CRFR2-specific antagonist antisauvagine-30 (anti-Svg-30). In contrast, injection of CRF into the lateral intermediate septum impaired learning through CRFR2, as demonstrated by the ability of antisauvagine-30 to block this effect. When antisauvagine-30 was injected alone into the lateral intermediate septum, learning was enhanced. Such tonic control of learning was not observed when astressin or antisauvagine-30 was injected into the dorsal hippocampus. Injection of CRF after the training into the dorsal hippocampus and the lateral intermediate septum also enhanced and impaired learning, respectively. Thus, it was indicated that CRF acted on memory consolidation. It was concluded that the observed effects reflected changes of associative learning and not arousal, attention, or motivation. Although a dose of 20 pmol human/rat CRF was sufficient to affect learning significantly, a fivefold higher dose was required to induce anxiety by injection into the septum. Immobilization for 1 hr generated a stress response that included the induction of anxiety through septal CRFR2 and the subsequent enhancement of learning through hippocampal CRFR1. The involvement of either receptor subtype was demonstrated by region-specific injections of astressin and antisauvagine-30.

The distribution of neural inputs to the paraventricular (PVH) and supraoptic (SO) nuclei from the regions of the A1, the A2, and the A6 (locus coeruleus) noradrenergic cell groups was investigated by using a plant lectin, Phaseolus vulgaris leucoagglutinin (PHA-L), as an anterogradely transported tracer. An immunofluorescence double-labeling procedure was used to determine the extent to which individual anterogradely labeled fibers and terminals in the PVH and the SO also displayed immunoreactive dopamine-beta-hydroxylase (DBH), a marker for catecholaminergic neurons. The results may be summarized as follows: (1) Projections from the A1 region were found primarily, and in some experiments almost exclusively, in those parts of the magnocellular division of the PVH and the SO known to contain vasopressinergic neurons. (2) Projections from the A2 region were distributed primarily throughout the parvicellular division of the PVH and were most dense in the dorsal medial part, a region known to contain a prominent population of corticotropin-releasing factor (CRF)-immunoreactive neurons. In addition, a less-dense projection to the magnocellular division of the PVH and to the SO was consistently found. (3) Fibers originating from the locus coeruleus were distributed almost exclusively to the parvicellular division of the PVH, with the most prominent input localized to the periventricular zone, a part of the PVH known to contain dopamine-, somatostatin-, and thyrotropin-releasing-hormone-containing neurons. We found no evidence for a projection from A6 to the SO. (4) The majority of fibers originating from the A1, the A2 or the A6 regions contained DBH immunoreactivity, although an appreciable number did not. These results suggest that each of the three brainstem noradrenergic cell groups that contribute to the innervation of the PVH and/or the SO is in a position to modulate the activity of anatomically and chemically distinct groups of neurosecretory neurons.

Clinical studies link disruption of the neuroendocrine stress system with alcoholism, but remaining unknown is whether functional differences in the hypothalamic-pituitary-adrenal (HPA) axis precede alcohol abuse and dependence or result from chronic exposure to this drug. Using an operant self-administration animal model of alcohol dependence and serial blood sampling, we show that longterm exposure to alcohol causes significant impairment of HPA function in adult male Wistar rats. Acute alcohol (voluntary self-administration or experimenter-administered) stimulated the release of corticosterone and its upstream regulator, adrenocorticotropic hormone, but chronic exposure sufficient to produce dependence led to a dampened neuroendocrine state. HPA responses to alcohol were most robust in 'low-responding' non-dependent animals (averaging < 0.2 mg/kg/session), intermediate in nondependent animals (averaging approximately 0.4 mg/kg/session), and most blunted in dependent animals (averaging approximately 1.0 mg/kg/session) following several weeks of daily 30-min self-administration sessions, suggesting that neuroendocrine tolerance can be initiated prior to dependence and relates to the amount of alcohol consumed. Decreased expression of corticotropin-releasing factor (CRF) mRNA expression in the paraventricular nucleus of the hypothalamus and reduced sensitivity of the pituitary to CRF may contribute to, but do not completely explain, neuroendocrine tolerance. The present results, combined with previous studies, suggest that multiple adaptations to stress regulatory systems may be brought about by excessive drinking, including a compromised hormonal response and a sensitized brain stress response that together contribute to dependence.

The bHLH-PAS transcription factor SIM1 is expressed during the development of the hypothalamic-pituitary axis in three hypothalamic nuclei: the paraventricular nucleus (PVN), the anterior periventricular nucleus (aPV), and the supraoptic nucleus (SON). To investigate Sim1 function in the hypothalamus, we produced mice carrying a null allele of Sim1 by gene targeting. Homozygous mutant mice die shortly after birth. Histological analysis shows that the PVN and the SON of these mice are hypocellular. At least five distinct types of secretory neurons, identified by the expression of oxytocin, vasopressin, thyrotropin-releasing hormone, corticotropin-releasing hormone, and somatostatin, are absent in the mutant PVN, aPV, and SON. Moreover, we show that SIM1 controls the development of these secretory neurons at the final stages of their differentiation. A subset of these neuronal lineages in the PVN/SON are also missing in mice bearing a mutation in the POU transcription factor BRN2. We provide evidence that, during development of the Sim1 mutant hypothalamus, the prospective PVN/SON region fails to express Brn2. Our results strongly indicate that SIM1 functions upstream to maintain Brn2 expression, which in turn directs the terminal differentiation of specific neuroendocrine lineages within the PVN/SON.

The hypothalamic-pituitary-adrenal axis exerts profound, multilevel inhibitory effects on the female reproductive system. Corticotropin-releasing hormone (CRH) and CRH-induced proopiomelanocortin peptides inhibit hypothalamic gonadotropin-releasing hormone secretion, whereas glucocorticoids suppress pituitary luteinizing hormone and ovarian estrogen and progesterone secretion and render target tissues resistant to estradiol. The hypothalamic-pituitary-adrenal axis is thus responsible for the "hypothalamic" amenorrhea of stress, which is also seen in melancholic depression, malnutrition, eating disorders, chronic active alcoholism, chronic excessive exercise, and the hypogonadism of the Cushing syndrome. Conversely, estrogen directly stimulates the CRH gene promoter and the central noradrenergic system, which may explain adult women's slight hypercortisolism; preponderance of affective, anxiety, and eating disorders; and mood cycles and vulnerability to autoimmune and inflammatory disease, both of which follow estradiol fluctuations. Several components of the hypothalamic-pituitary-adrenal axis and their receptors are present in reproductive tissues as autacoid regulators. These include ovarian and endometrial CRH, which may participate in the inflammatory processes of the ovary (ovulation and luteolysis) and endometrium (blastocyst implantation and menstruation), and placental CRH, which may participate in the physiology of pregnancy and the timing of labor and delivery. The hypercortisolism of the latter half of pregnancy can be explained by high levels of placental CRH in plasma. This hypercortisolism causes a transient postpartum adrenal suppression that, together with estrogen withdrawal, may partly explain the depression and autoimmune phenomena of the postpartum period.

In a previous positron emission tomography (PET) study of major depression, we demonstrated that cerebral blood flow was increased in the left amygdala in unipolar depressives with familial pure depressive disease (FPDD) relative to healthy controls [J. Neurosci. 12 (1992) 3628.]. These measures were obtained from relatively low-resolution PET images using a stereotaxic method based upon skull X-ray landmarks. The current experiments aimed to replicate and extend these results using higher-resolution glucose metabolism images and magnetic resonance imaging (MRI)-based region-of-interest (ROI) analysis. The specificity of this finding to FPDD was also investigated by assessing depressed samples with bipolar disorder (BD-D) and depression spectrum disease (DSD). Finally, the relationship between amygdala metabolism and plasma cortisol levels obtained during the scanning procedure was assessed. Glucose metabolism was measured using PET and 18F-fluorodeoxyglucose (18FDG) in healthy control (n=12), FPDD (n=12), DSD (n=9) and BD-D (n=7) samples in the amygdala and the adjacent hippocampus. The left amygdala metabolism differed across groups (P<.001), being increased in both the FPDD and BD-D groups relative to the control group. The left amygdala metabolism was positively correlated with stressed plasma cortisol levels in both the unipolar (r=.69; P<.005) and the bipolar depressives (r=0.68;.1<P<.05). In contrast, neither significant main effects of diagnosis nor significant relationships with plasma cortisol were evident in post hoc analyses of metabolism in the right amygdala or the hippocampus. Preliminary assessment of BD subjects imaged during remission suggested that amygdala metabolism is also elevated in remitted subjects who are not taking mood-stabilizing drugs, but within the normal range in subjects taking mood stabilizers. These data confirm our previous finding that neurophysiological activity is abnormally increased in FPDD, and extend it to BD-D. These abnormalities were not accounted for by spilling in of radioactivity from the adjacent hippocampus. The correlation between left amygdala metabolism and stressed plasma cortisol levels may conceivably reflect either the effect of amygdala activity on <em>corticotropin</em>-releasing hormone (CRH) secretion or the effect of cortisol on amygdala function.

Corticotropin-releasing factor (CRF) and its related family members are implicated in stress-related disorders such as anxiety and depression. Recently, two new members of this neuropeptide family have been discovered in the brain: urocortin II (also known as stresscopin-related peptide) and urocortin III (also known as stresscopin). These urocortins are selective agonists for the CRF(2) receptor, show a distinct neuroanatomical localization and are involved in stress-coping responses such as anxiolysis. Thus, CRF, the urocortins and their receptors form an intricate network in the brain involved in the acute phase as well as the recovery phase of the stress response.

The concept of the "extended amygdala", developed and explored by Lennart Heimer, Jose de Olmos, George Alheid, and their collaborators, has had an enormous impact on the field of neuroscience and on our own work. Measuring fear-potentiated startle test using conditioned stimuli that vary in length we suggest that the central nucleus of the amygdala (CeA) and the lateral division of the bed nucleus of the stria terminalis (BNST(L)) are involved in short-term versus long-term fear responses we call phasic versus sustained fear, respectively. Outputs from the basolateral amygdala (BLA) activate the medial division of the CeA (CeA(M)) to very rapidly elicit phasic fear responses via CeA(M) projections to the hypothalamus and brainstem. The BLA also projects to the BNST(L), which together with other BNST(L) inputs from the lateral CeA (CeA(L)) initiate a slower developing, but sustained fear response, akin to anxiety. We hypothesize this occurs because the CeA(L) releases the peptide corticotropin releasing hormone (CRF) into the BNST(L) which facilitates the release of glutamate from BLA terminals. This activates the BNST(L) which projects to hypothalamic and brainstem areas similar to those innervated by the CeA(M) that mediate the specific signs of fear and anxiety. The generality of this idea is illustrated by selective studies looking at context conditioning, social defeat, drug withdrawal and stress induced reinstatement.

Corticotropin-releasing hormone (CRH) and GABA have been implicated in depression, and there is reason to believe that GABA may influence CRH functioning. The levels of CRH, and mRNA for CRH-binding protein, CRH1, and CRH2 receptors, as well as various GABA(A) receptor subunits (alpha1, alpha2, alpha3, alpha4, alpha5, delta, and gamma2), were determined in several frontal cortical brain regions of depressed suicide victims and nondepressed individuals who had not died by suicide. Relative to the comparison group, CRH levels were elevated in frontopolar and dorsomedial prefrontal cortex, but not in the ventrolateral prefrontal cortex of suicide victims. Conversely, using quantitative PCR analyses, it was observed that, in frontopolar cortex, mRNA for CRH1, but not CRH2, receptors were reduced in suicide brains, possibly secondary to the high levels of CRH activity. In addition, mRNA of the alpha1, alpha3, alpha4, and delta receptor subunits was reduced in the frontopolar region of suicide victims. Interestingly, a partial analysis of the GABA(A) receptor functional genome revealed high cross-correlations between subunit expression in cortical regions of nondepressed individuals, suggesting a high degree of coordinated gene regulation. However, in suicide brains, this regulation was perturbed, independent of overall subunit abundance. These findings raise the possibility that the CRH and GABA(A) receptor subunit changes, or the disturbed coordination between these GABA(A) receptor subunits, contribute to depression and/or suicidality or are secondary to the illness/distress associated with it.