A number of clinical investigations and postmortem brain studies have provided evidence that excessive corticotropin-releasing hormone (CRH) secretion and neurotransmission is involved in the pathophysiology of depressive illness, and several studies have suggested that the hyperactivity in CRH neurotransmission extends beyond the hypothalamus involving several extra-hypothalamic brain regions. The present study was designed to test the hypothesis that CRH levels are increased in specific brainstem regions of suicide victims with a diagnosis of major depression. Frozen tissue sections of the pons containing the locus coeruleus and caudal raphe nuclei from 11 matched pairs of depressed suicide and control male subjects were processed for radioimmunocytochemistry using a primary antiserum to CRH and a I-IgG secondary antibody. The optical density corresponding to the level of CRH-immunoreactivity (IR) was quantified in specific pontine regions from the film autoradiographic images. The level of CRH-IR was increased by 30% in the locus coeruleus, 39% in the median raphe and 45% in the caudal dorsal raphe in the depressed suicide subjects compared to controls. No difference in CRH-IR was found in the dorsal tegmentum or medial parabrachial nucleus between the subject groups. These findings reveal that CRH-IR levels are specifically increased in norepinephrine- and serotonin-containing pontine nuclei of depressed suicide men, and thus they are consistent with the hypothesis that CRH neurotransmission is elevated in extra-hypothalamic brain regions of depressed subjects.
Corticotropin-releasing hormone (CRH) is the primary chemical regulator mediating the stress-induced activation of the hypothalamic–pituitary–adrenal (HPA) axis stimulating the secretion of adrenocorticotropin hormone (ACTH) from the anterior pituitary which in turn stimulates cortisol release from the adrenal gland. Hyperactivity of the HPA axis resulting in increased circulating cortisol levels is one of the most consistent biological alterations reported in depressed patients.1,2,3,4,5 Several lines of evidence support the theory that excessive secretion of CRH is involved in the hypercortisolism in depressed patients, and that the anatomical sites of CRH hypersecretion include the hypothalamus and possibly other extra-hypothalamic brain regions. For example, a blunted ACTH response to exogenous CRH administration,6,7,8 increased CRH cerebrospinal fluid (CSF) levels,9,10,11 increased numbers of CRH-immunoreactive12 and CRH mRNA-containing paraventricular hypothalamic neurons13 and decreased binding sites of CRH receptors in the frontal cortex of depressed subjects14 represent some of the biochemical changes that support the hypothesis of hyperactivity of CRH function in depression. However, other studies have failed to document changes in CSF CRH levels,15,16,17 brain tissue levels of CRH18,19 or CRH receptor binding sites in the cortex of depressed subjects19,20 and thus do not support the CRH hyperactivity hypothesis of depression. With the exception of the reports documenting alterations in CRH substrates in the paraventricular nucleus of the hypothalamus and in the frontal cortex, there has been no additional direct evidence for CRH hyperactivity in other brain regions of depressed subjects.
In addition to the hormonal role of CRH, a variety of rodent studies have provided evidence supporting a neurotransmitter role for CRH in the central nervous system. In particular, both electrophysiological and biochemical studies have shown that CRH is involved in mediating the effects of stress on norepinephrine neurotransmission specifically at the level of the noradrenergic neurons in the locus coeruleus (LC).21,22,23 Furthermore, recent electrophysiological studies have found that CRH has both excitatory and inhibitory effects on serotonin neurons in the dorsal raphe nucleus.24,25,26 Given the evidence from animal studies supporting a functional role of CRH on LC and raphe neurons, it is interesting that both clinical and postmortem brain studies have documented alterations in various noradrenergic27,28,29 and serotonergic30,31,32 parameters in depressed subjects. Perhaps CRH has a similar functional role in the human brain and may contribute to the alterations in the monoamine systems in depressed patients? Indeed, recent immunocytochemical studies have shown that several monoaminergic nuclei in the human brainstem are innervated by CRH-containing axons including the locus coeruleus and raphe nuclei.33,34 Given the evidence for alterations in CRH, norepinephrine and serotonin neurotransmission in depressive illness together with the anatomical data suggesting an interaction of CRH on LC and raphe neurons in the human brainstem, we sought to test the hypothesis that the concentration of CRH immunoreactivity is increased in specific pontine nuclei in subjects with major depression who committed suicide.
Materials and methods
All procedures in our study were approved by the University of Pittsburgh's Institutional Review Board for Biomedical Research. Human brain specimens were obtained in the course of routine autopsies conducted at the Allegheny County Coroner's Office, Pittsburgh, PA, after obtaining consent from a surviving family member. A total of 11 subjects who died by suicide and had a diagnosis of depression were each matched with one control subject for sex and as closely as possible for age and postmortem interval (Table 1). Of the pairs, 10 were also matched for race. For each subject, a consensus DSM-IV diagnosis was made by an independent panel of experienced clinicians after reviewing medical records and the results of structured interviews conducted with family members of the deceased. Of the suicide subjects, 10 had a diagnosis of major depression, and one had a depressive disorder NOS. Three of the depressed subjects also had an Axis II diagnosis of obsessive–compulsive personality disorder and one depressed subject had an Axis II diagnosis of paranoid personality disorder. Three subjects with depression had a prior history of alcohol abuse and one depressed subject had a history of opioid abuse, but these disorders were in remission in all four subjects at the time of death. The mean (±SD) age at the onset of depression was 50.9±17.3 years, and the average duration of illness was 4.8±6.2 years. Eight of the 11 depressed subjects had a family history of depression. Neuropathological condition of each brain specimen was evaluated by macroscopic and microscopic examination. Thioflavin-S staining revealed a few senile plaques in subjects 598, 600, 685, 699 and 795 but clinical and neuropathological criteria to establish a diagnosis of Alzheimer's disease were not met. Toxicological screens were positive for chlordiazepoxide for one depressed subject (598) and CO for two other depressed subjects (614, 628). Also, three depressed subjects (598, 689, 698) were receiving antidepressants at the time of death and two of these depressed subjects (689, 698) were treated with antipsychotics at the time of death. Three depressed subjects (614, 596, 613) had been off antidepressant medication for 0.6, 1.8 and 7.1 years, respectively, and one subject (613) off antipsychotic medication for 7.6 years. The other five depressed subjects had no documented history of antidepressant medication.
Tissue preparation and radioimmunocytochemistry
Upon removal of the brain from the cranium, the cerebellum was removed and the brainstem was separated by a transverse cut at the rostral border of the superior colliculi. The brainstems were then cut into midbrain/pons and caudal medulla blocks, immediately frozen in isopentane and then stored at −80°C. The frozen midbrain/pontine blocks were sectioned transversely at 20 μm, sections were thaw-mounted onto gelatin-coated microscope slides and every 10th tissue section stained for Nissl substance with cresyl violet. The slides were stored at −80°C until processed.
Slide-mounted tissue sections of the pons were selected for assay based on the anatomical distribution of CRH-immunoreactive (IR) axons previously reported in the human brainstem.33 The radioimmunocytochemical assay was performed as previously described35 with some modifications. Four tissue sections were selected at 200 μm intervals from each case and placed into 4% paraformaldehyde for approximately 17 h. The sections were then rinsed twice for 15 min in PBS containing 0.4% Triton X-100 and then incubated for 60 min in PBS with 1.5% normal goat, 0.5% normal human serum and 0.4% Triton X-100 to reduce background.
The CRH primary antiserum (rabbit anti-CRH (human, rat), Pennisula Laboratories, San Carlos, CA, USA) was preadsorbed with natural melanin as previously described.33 The concentration of the CRH primary antiserum used on the tissue sections from the subject pairs was determined from a dilution curve constructed from control brainstem tissue sections that were incubated with varying concentrations of the primary antibody (1:1000–1:20 000), processed for radioimmunocytochemistry and quantified using densitometry. The 1:5000 concentration of primary antibody was chosen for the experiments because this antibody concentration produced the best signal-to-noise ratio from the autoradiographic images. Tissue sections from each matched subject pair were incubated at 4°C for 16–18 h with the primary antiserum to CRH (1:5000) diluted in PBS containing 0.4% Triton X-100, 3.0% goat serum and 0.05% bovine serum albumin. The sections were then washed three times for 5 min in 0.4% Triton X-100 in PBS and then incubated with [125I]-labeled secondary antibody (goat anti-rabbit IgG, F(ab′)2 fragment; NEN, Boston, MA, USA) using a dilution of 1:200 (0.5 μCi/ml) in 0.4% Triton X-100 in PBS for 120 min at room temperature. Following this incubation, the sections were washed for 15 min in 0.4% Triton X-100 in PBS followed by two 15 min washes in PBS. Two additional sections from each case were included to assess nonspecific binding of the [125I]-IgG and were processed as described above except that the primary antiserum was not included in the buffer. The slides were then dipped into 70% ethanol, dried and co-exposed with iodinated plastic standards to β-max Hyperfilm (Amersham, Arlington Heights, IL, USA) for 24–36 h. Exposure time was adjusted so that the resultant autoradiographic signal was within the optimum range of film sensitivity.
Quantitative image analysis
The quantification of the film autoradiographic images was accomplished using a personal computer-based image analysis system (the Microcomputer Imaging Device, MCID; M5) from Imaging Research, Inc. (St. Catherines, Ontario, Canada). Optical density values quantified from the iodinated standards (Amersham) were entered with their corresponding radioactivity values (nCi/mg protein) into a calibration table, and the relationship between tissue radioactivity and optical density determined using the MCID software. Each assayed tissue section was subsequently stained with cresyl violet. An image of the Nissl-stained section was digitized and used as an overlay on the autoradiographic image to allow specific pontine nuclei to be outlined. Based on the regional distribution of CRH-IR axons in the human pons,33 the cellular boundaries of the LC, medial parabrachial nucleus (MPB), dorsal tegmental nucleus (DTg), caudal nucleus of the dorsal raphe (cDR) and the median raphe (MR) were individually outlined on the Nissl-stained image. The optical density, corresponding to CRH-IR, was then quantified within each outlined region from the autoradiographic image. CRH-IR values (nCi/mg) for each pontine nucleus were derived by interpolation from the standard curve. Nonspecific binding of the secondary antibody to the tissue sections was measured from each pontine region for each case. The background values were averaged across the two sections, and this value was subtracted from each data point. The nonspecific binding averaged 18% of the total binding across all subjects. The CRH-IR value per sampled pontine nucleus was then adjusted for the sampling area by dividing the CRH-IR value by the scan area (pixel units) of the outlined region.
The data were analyzed using a repeated measures analysis of variance. The within factors were the subject pairing (control and depressed subjects) and the pontine regions (LC, MPB, DTg, cDR and MR). Each of the variables of interest were analyzed separately. Following a statistically significant omnibus test, Scheffe's post hoc procedure was performed to examine pairwise differences. In addition, multiple regression models were used to investigate the independent and concomitant effects of age, postmortem interval, age at onset and the duration of illness on the dependent variable of CRH-IR levels for each pontine region. A Mann–Whitney U test was used to examine the mean differences in CRH-IR levels in the pontine regions between depressed subjects receiving antidepressants and those depressed subjects with no known history of antidepressant medication. All statistical assumptions were met, and the statistical significance was defined as P≤0.05.
The quantitative autoradiographic measurements of CRH-IR among the pontine nuclei from the control subjects revealed that the DTg contained the highest concentration of CRH-IR, this was followed in rank order by the MPB>LC=cDR>MR (Figure 1).
As shown in Figure 1 and Figure 2, the mean level of CRH-IR in the depressed subjects was significantly increased by 30% in the LC (F1,20=9.0, P=0.007), by 45% in the cDR (F1,20=5.4, P=0.03) and by 39% in the MR (F1,20=7.6, P=0.012) relative to the control subjects. The mean level of CRH-IR was not significantly different in the MPB (F1,20=1.8, P=0.19) or DTg (F1,20=3.2, P=0.09) of the depressed subjects compared to controls. The difference in CRH-IR between subject groups in the LC, cDR and MR could not be attributed to the effects of age or PMI. Comparison of individual matched pairs revealed that the mean CRH-IR level was increased in the LC in eight of the 11 depressed subjects and increased in the cDR and MR in nine of the 11 depressed subjects compared to the matched control subjects (Figure 3). A significant positive correlation was found between the magnitude of the increase in CRH-IR levels in the LC and the age at onset of depression (r=0.68, P=0.02), but no significant relationships were found in the cDR or MR (cDR: r=0.33, P=0.32; MR: r=0.41, P=0.21) nor were there any significant relationships between the increase in CRH-IR in the three pontine regions of the depressed subjects and the duration of illness (LC: r=−0.40, P=0.23; cDR: r=0.32, P=0.34; MR: r=−0.01, P=0.99).
The potential confounding effects of current or past antidepressant medication on CRH-IR levels in the LC, cDR and MR was examined in the depressed subject group. Three of the depressed subjects (598, 689, 698) were receiving antidepressants at the time of death. The remaining eight depressed subjects had no documented history that they were currently receiving antidepressants at the time of death. The mean (SD) increases in CRH-IR within pairs were 4.68 (22.2) and 10.6 (11.6) for the LC, 8.69 (21.6) and 13.8 (15.1) for the cDR, and 3.06 (1.8) and 4.96 (4.88) for the MR for the depressed subjects on and off antidepressants at the time of death, respectively. However, the difference between these groups did not reach statistical significance for any pontine region (LC: M–WU=8.0, P=0.50, cDR: M–WU=9.0, P=0.63, MR: M–WU=8.0, P=0.50).
Representative immunoautoradiographic images of CRH-IR concentrations in the specific pontine nuclei of a control (551) and depressed (613) subject pair are shown in Figure 4. The elevated immunoautoradiographic signal, corresponding to CRH-IR, is revealed by the increased density of yellow and orange pixels in the LC, MR and cDR regions in the depressed subject compared to the matched control.
The present study found that the concentration of CRH-IR was increased in the LC, cDR and MR of depressed suicide men, but was unchanged in the adjacent pontine structures of the MPB and DTg compared to matched control subjects. These results are consistent with the hypothesis that CRH is hypersecreted in the brain of individuals with major depression, and they support the theory that the hyperactivity in CRH neurotransmission extends beyond the hypothalamus and occurs in other brain regions of depressed subjects.
Within the last 15 years, a considerable amount of data has accumulated from studies measuring various indices of CRH function in depressed subjects (for review see Mitchell36). The majority of these studies measured CRH levels from the CSF of depressed patients, and although several reports found elevated CSF CRH levels in the depressed subjects,9,10,11 a number of other studies failed to replicate these findings.15,16,17 The inconsistency among these studies may be attributable to differences in methodology, study design and subject characteristics. For example, in several of the studies that found no change in CRH levels between subject groups, the depressed group was not uniform and had included individuals with major depression as well as bipolar depressed subjects. Given that Berrettini et al37 previously found no change in CSF CRH levels in 41 bipolar outpatients compared to controls, it is possible that any increase in CRH in the major depressives may have been masked by the inclusion of bipolar subjects in these studies.
In addition to the in vivo studies measuring CSF CRH levels in depressed patients, several studies have examined various CRH parameters in postmortem samples of suicide and depressed subjects. Arató et al11 measured cisternal CSF CRH levels from individuals that committed suicide and found that CRH levels were significantly higher in the suicide group relative to the control group. Nemeroff et al14 measured CRH receptor binding sites in the frontal cortex of suicide victims and reported a decrease in the number of CRH receptor binding sites in the suicide subjects. However, it is important to note that the psychiatric diagnosis of the suicide victims in both these studies was not reported. Subsequent receptor binding studies failed to detect a decrease in CRH binding sites in frontal, motor or occipital cortices of subjects with major depression that died of natural causes or by suicide.19,20 The study by Leake et al19 also measured the concentration of CRH-IR in frontal and occipital cortices by radioimmunoassay, but found that the cortical levels of CRH did not differ between the depressed and control subject groups. This report confirmed an earlier study by this same group showing that CRH-IR levels did not differ in frontal, temporal, motor or parietal cortices between the depressed and control groups.18
The postmortem CRH data discussed above are conflicting and do not provide convincing evidence to support the theory of CRH hyperactivity in depression, but recent neuromorphological studies have revealed some intriguing CRH abnormalities in depressed subjects.12 Raadsheer et al12 using immunocytochemical and unbiased stereological techniques counted the number of CRH-IR neurons in the paraventricular nucleus of the hypothalamus (PVN) of depressed and control subjects. A four-fold increase in the number of PVN CRH-IR neurons was found in the depressed subjects: however, there was no change in the size of CRH cells or in PVN volume between the subject groups.12 A subsequent in situ hybridization study by this group conducted in the same brain specimens revealed an increase in the number of CRH mRNA-containing PVN cells in the depressed subjects relative to controls, but the mRNA concentration per cell was not changed between the subject groups. 13 There are some limitations of these two reports which include the small sample size of the depressed group (n=6), the diagnostic heterogeneity of the depressed subjects (three major depression, three bipolar depression) and the positive medication history in the last month of life for all the depressed subjects. Nonetheless, these findings have identified a specific brain nucleus exhibiting a cellular abnormality that may contribute to the excessive CRH neurotransmission in the brain of depressed subjects. Further studies in a larger, diagnostically homogeneous depressed group are warranted to confirm these morphological alterations.
The effects of antidepressants on CSF CRH concentrations in depressed patients have been examined by a number of studies. Most of the reports found that CSF CRH levels were decreased in the treated depressed patients,38,39,40 but one postmortem study found no change in cortical CRH levels between drug-treated and drug-free depressed suicide subjects.18 Since three depressed suicide subjects had a history of antidepressant use at the time of death, we examined whether current antidepressant use may have influenced the CRH concentrations in the pontine nuclei of our depressed suicide subjects. Although the depressed subjects with no known history of antidepressant treatment had larger mean increases in CRH levels in all three pontine regions than the depressed subjects receiving antidepressants at the time of death, the differences were not statistically significant.
Our findings reveal a localized increase in the concentration of CRH-IR in the LC, cDR and MR nuclei in the pons of depressed male subjects who have committed suicide. The increase in CRH-IR in these regions may correspond to an increased biosynthesis and transport of CRH to the nerve terminals and/or an increase in the density of CRH-IR axons innervating these pontine structures. One question that is raised from our observations; what is the origin of the CRH innervation of the human LC, cDR and MR? A number of tracing studies in nonhuman primates have shown that afferents to several midbrain and pontine structures including the DR, MR and LC originate from the dorsolateral and dorsomedial prefrontal cortices, anterior and posterior cingulate cortices, central nucleus of the amygdala and posterior hypothalamic regions.41,42,43,44,45,46 These findings are potentially interesting given that a number of these cortical and subcortical regions in the monkey have been reported to contain CRH-IR cell bodies.47,48,49 Unfortunately, one can only speculate on the origin of the CRH afferents to the LC and raphe nuclei in the human brain. However, given the anatomical tracing studies in monkeys, it is possible that biochemical or morphological alterations exist in CRH neurons located in the prefrontal cortex, amygdala or hypothalamus, which contribute to the elevated CRH levels in the terminal fields of the LC and raphe nuclei of depressed subjects.
It is important to note that our study was conducted only in male subjects. Therefore, it is possible that a gender difference may exist in the integrity of CRH biosynthesis in depressed subjects, and it will be critical for future studies to determine whether the elevation in CRH in the pontine nuclei is also present in depressed female subjects.
Considerable evidence has accumulated demonstrating a functional role of CRH on LC neurons. Both electrophysiological and biochemical studies have shown that CRH increases the spontaneous discharge rate of LC neurons, increases norepinephrine release and increases tyrosine hydroxylase protein levels in the LC.21,23,50,51,52 Furthermore, depending on the type of stressor, the stress-induced activation of LC neurons appears to be mediated by CRH presumably released from terminals within the LC region.21,22,53 These findings are consistent with the elevation in CRH levels in the LC following stress.54 Recent studies have also provided evidence of a neurotransmitter role for CRH on serotonin neurons in the DR. For example, injection of CRH into the ventricles or directly into the DR of rats inhibited the discharge rate of serotonin neurons in the DR.24,25 However, Lowry et al,26 using an in vitro slice preparation, reported that CRH had no effect on the firing rate of serotonin neurons from control rats, but significantly increased the firing rate of serotonin neurons from rats previously exposed to repeated restraint stress. Taken together, these studies support a neurotransmitter role for CRH in regulating the activity of noradrenergic LC and serotonergic raphe neurons.
Numerous clinical and postmortem brain studies have documented abnormalities in various noradrenergic and serotonergic parameters in subjects with mood disorders. In particular, postmortem studies have reported alterations in norepinephrine transporter binding sites and tyrosine hydroxylase protein levels in the LC27,28 and in serotonin 1A receptor binding sites in the DR30,32 of depressed suicide subjects. Given our findings of elevated CRH-IR levels in the LC and raphe nuclei of depressed suicide subjects, it is conceivable that the excessive CRH neurotransmission in these regions contributes to the altered functioning of the noradrenergic and serotonergic systems in depression and suicidal behavior. Within this context, it is interesting that a recent open-label clinical trial explored the antidepressant potential of a CRH receptor antagonist.55 Zobel et al55 administered R121919, a water-soluble pyrrolopyrimidine with high affinity for CRH-R1 receptors, to 24 patients with a major depressive episode and found that patient- and clinician-rated depression and anxiety scores were significantly improved over the course of treatment. These preliminary findings highlight the importance of continued research exploring the therapeutic potential of CRH receptor antagonists for treating mood disorders as well as continued investigations into the cellular interactions of CRH on the monoamine systems.
Convergent lines of evidence support the hypothesis that CRH neurotransmission is over-active in the brain of individuals with major depression. The localized increase in CRH-IR concentrations in the LC and caudal raphe nuclei of depressed suicide subjects suggest that either a biochemical or a cellular alteration exists in the CRH neurons that innervate these pontine structures. It remains to be determined whether the elevated levels of CRH in the LC and raphe nuclei have a functional impact on norepinephrine and serotonin neurotransmission in subjects with major depression.
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This work was supported by USPHS Grants MH57011 and MH45156. The authors thank Drs Gretchen Haas, Carol Sue Johnston, Cameron Carter and Matcheri Keshavan for their participation in the diagnostic conferences.
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Austin, M., Janosky, J. & Murphy, H. Increased corticotropin-releasing hormone immunoreactivity in monoamine-containing pontine nuclei of depressed suicide men. Mol Psychiatry 8, 324–332 (2003). https://doi.org/10.1038/sj.mp.4001250
- corticotropin-releasing factor
- locus coeruleus
- mood disorders
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