Failure to Mount Adaptive Responses to Stress Results in Dysregulation and Cell Death in the Midbrain Raphe

Abstract

Stress is a common trigger in affective disorder onset, yet the mechanism and predisposing factors of vulnerability remain unknown. Effective disease prevention requires a critical balance of responses within the serotonergic raphe nucleus, including a coordination of corticotropin-releasing factor (CRF) actions at both of its receptors, CRF receptor-1 and -2. Mice deficient in CRF receptor-2 (R2KO) were utilized as a model of maladaptive stress responsivity to examine the physiological and molecular markers of stress dysregulation within the raphe in the absence of this receptor. Following chronic stress, R2KO mice failed to display the robust stress-mediated adaptations characteristic of control mice, including elevations in tryptophan hydroxylase-2 and CRF receptor-1 expression and concordant increases in behavioral arousal. As a further indication of failed homeostatic mechanisms, R2KO mice displayed indices of cell death in the raphe following stress exposure with elevations in pro-apoptotic factors but a failure to mount adaptive increases in anti-apoptotic factors found in control mice. In vitro electrophysiological characterization of the specific influence of CRF on the raphe revealed both basal differences and a failure to respond to CRF administration in R2KO mice. These results support a requirement for homeostatic maintenance in response to stress in the raphe where dysregulation may be a critical predictor of affective disorder onset.

Introduction

Affective disorders involve a failure in orchestration of multiple neurotransmitter systems and brain regions, and an increased sensitivity to the deleterious effects of stress (Keller, 2005; Berton and Nestler, 2006). The present deficiency in treating these disorders underscores the need to elucidate those experiences that precipitate disease onset, including that of prolonged stress exposure (Arborelius et al., 1999; Holsboer, 2000). Whereas stress normally elicits a recruitment of adaptive and compensatory mechanisms required in homeostasis, an inability to mount such responses results in an increased burden on cellular systems that sensitizes an individual towards affective disorders, exposing the brain to potentially toxic effects of physiological dysregulation (McEwen, 2003).

Serotonin (5-HT) plays a critical role in the regulation of mood and in the etiology and treatment of affective disorders for which the mechanism is still not fully understood. In the raphe, stress increases gene expression related to 5-HT production (Chamas et al., 2004), suggesting downstream 5-HT elevations in raphe target regions important in coordinating adaptive responses. Alterations in excitatory and inhibitory drives on raphe neurons, as well as 5-HT increases in the raphe and to its output regions, can be modulated by corticotropin-releasing factor (CRF) acting on its two receptors, CRF receptor-1 (CRFR1) and -2 (CRFR2) (Kirby et al., 2000; Daugherty et al., 2001; Valentino et al., 2001; Kirby et al., 2003; Roche et al., 2003; Tan et al., 2004; Keeney et al., 2006). The coordination of these receptors is critical in regulation of homeostatic maintenance (Weisstaub et al., 2006; Kang et al., 2007). These regulatory events indicate stress mediation of not only the limbic and forebrain circuits critical in affect and arousal, but also in the cytoarchitectural stability of the raphe itself.

In order to determine the consequences of inappropriate stress responsivity and to differentiate between homeostatic maintenance and dysregulation within the raphe and the role of CRF pathways in these responses, we utilized a mouse deficient for CRFR2 (R2KO). R2KO mice display maladaptive physiological and behavioral responses to stress perturbations (Bale et al., 2000; Bale and Vale, 2003). We hypothesized that a failure to produce these adaptive responses to stress may be due in part to basal differences in cellular characteristics and CRF responses, that would be predisposing towards dysregulation in the raphe and affective disorder onset. To examine this, we exposed wild type (WT) and R2KO mice to chronic variable stress for a period of 4 weeks and examined molecular and cellular outcomes in the midbrain raphe nuclei compared to nonstressed control littermates. Ultimately, as many of the interactions critical in stress response and affect begin at the cellular level in raphe neurons receiving CRF input during stress exposure, application of CRF directly onto isolated 5-HT cells in the raphe nuclei during whole-cell electrophysiological investigation was also employed to determine how deficiency of CRFR2 may precipitate a dysregulated state.

Materials and Methods

Animals

CRFR2-deficient (R2KO) mice were generated on a mixed C57Bl/6:129J background as previously described (Bale et al., 2000). Male wild type (WT) and R2KO littermates (N = 40 - 42) were housed under a 12:12 light/dark cycle (lights on 0700 h), with food and water available ad libitum. All studies were done according to experimental protocols approved by the University of Pennsylvania Institutional Animal Care and Use Committee, and all procedures were conducted in accordance with institutional guidelines.

Stress Exposure

In order to determine the maladaptive responses of stress-sensitive R2KO mice, a model of chronic variable stress was employed. Stressors were utilized that would not directly perturb thermogenic, metabolic, or pain pathways. WT and R2KO adult male littermates (N = 5-7) were individually housed for 1 week prior to stress onset. Mice were then presented with one stressor per day at random times and in a randomized order for 4 weeks. Stressors included: overnight wet cage bedding (60 ml H₂O); housing in the dirty cage of a non-littermate male (overnight); 36 hrs constant light; overnight exposure to novel objects in cages (glass marbles,); 60 min predator odor exposure (Fox odor (Acros Organics, NJ) diluted 1:10,000), 15 min of restraint stress, and overnight novel noise using a white noise generator (Brookstone). Mice were sacrificed 48 hours following the final stressor. For all experimental assays, stressed WT and R2KO mice were compared to control groups comprised of unstressed, singly housed littermates who were exposed only to normal cage changing and to the weekly HPA assessment over the 4 week test period.

HPA Axis Assessment

To ensure that mice continued to respond to daily stressors over 4 weeks, corticosterone levels in WT and R2KO mice (N = 6-7) were examined following 15 minute restraint stress administered each week of the chronic stress exposure paradigm.

Tail blood was collected at restraint onset (basal value) and at stress completion (stress). Plasma samples were centrifuged and stored at −80 °C until analysis. Corticosterone content was determined by radioimmunoassay (MP Biomedicals, Orangeburg, NY) using 3 μl of plasma and examined using a logarithmic scale.

At sacrifice, adrenal glands were collected, formalin fixed and embedded in paraffin (Bale et al., 2000). Sectioned tissue was hematoxylin and eosin stained. Images of stained tissue were collected using a Nikon DMZ800 microscope and a 10-bit QICam digital camera (QImaging). Adrenal cortex comparisons were measured from anatomically-matched slides using a 0.01μm micrometer and IPlabs software (BD Biosciences/Scanalytics, Rockville MD).

Gene Expression

To determine changes in gene expression related to CRF and 5-HT, in situ hybridization analysis in the raphe was used to examine tryptophan hydroxylase-2 (TPH2) and CRF receptor-1 (CRFR1). Following stress, whole brains were removed from WT and R2KO mice (N = 5-6), frozen, and coronally sectioned in three consecutive sets (20 μm) using a Leica CM3050S cryostat. Sections were post-fixed in 4% paraformaldehyde and processed as previously described (Teegarden and Bale, 2007). Briefly, atlas-matched tissue was hybridized with an antisense TPH2 (generous gift from Dr. Michael Bader, Max-Delbruck Ctr for Molecular Medicine, Berlin) or CRFR1 (previously described in (Bale et al., 2000)) probe. Slides were dipped in NTB liquid nuclear emulsion (Eastman Kodak, Rochester, NY) for 15-21 days, developed, counter-stained with hematoxylin (Fisher), and coverslipped.

The DR and MR were divided into anatomical subregions for analysis of mRNA-representative silver grains based on rostral, middle and caudal position (Bregma −4.16, −4.45, and −4.72 mm, respectively). The DR was divided into dorsomedial and ventromedial subregions (dmDR, vmDR) as well as lateral wing (lwDR) in caudal DR sections. Atlas-matched images were photographed at 20X magnification using a Nikon Eclipse E600 microscope and a 10-bit QICam digital camera (QImaging, British Columbia). Brightfield and darkfield images were captured to view nuclei and mRNA-representative silver grains, respectively. Analysis was accomplished using IPlabs software (BD Biosciences/Scanalytics, Rockville, MD). Cells were defined by contrast imaging of hematoxylin-stained nuclei. TPH2 or CRFR1 mRNA expressing cells (referred to as TPH2 or CRFR1-positive) and non-expressing cells (referred to as TPH2 or CRFR1-negative) were distinguished and counted by presence of nuclear silver grains within the cell perimeter. Total grain number was calculated as number of grains located within cell nuclei. For average grains per cell, total grain number was divided by number of cells containing grains.

Serotonin Transporter Autoradiography

In order to examine the effects of stress dysregulation in raphe output regions, 5-HT transporter (SERT) levels were analyzed in WT and R2KO mice using ³H-Citalopram autoradiography. Fresh frozen, atlas-matched brain sections were selected from the control and chronically stressed WT and R2KO mice used in gene expression analyses (N = 5-6) and immersed in incubation buffer containing: Tris pH 7.6 (50 mM), NaCl (120 mM), KCl (5 mM). Sections containing dorsal and ventral hippocampus and basolateral amygdala were then incubated at room temperature in buffer containing ³H-Citalopram (0.7 nM, Perkin Elmer, Boston, MA) for one hour. Slides were washed in incubation buffer twice for 10 min each, and dipped in mH2O. Slides were dried and apposed to Kodak Hyperfilm (Eastman Kodak, Rochester, NY) for 21 days. Atlas matched images were normalized to background and analyzed by region of interest measurement in the basolateral amygdala and dorsal and ventral dentate gyrus and CA3 hippocampus. Optical density measurements were made by IPlabs software (BD Biosciences/Scanalytics, Rockville, MD).

Behavioral Responses Following Chronic Stress

To determine whether observed differences in electrophysiology and stress-induced changes in gene expression were associated with maladaptive behavioral responses to stress, WT and R2KO behaviors were examined following the 4-week stress period. The effects of the 4-week chronic stress on WT and R2KO behaviors were examined in a separate set of mice (N = 6-7). After 4 weeks of stress exposure, behavioral tests were introduced as novel daily stressors. Unstressed WT and R2KO littermates were used as controls.

Elevated plus maze

Testing was executed as previously described (Teegarden and Bale, 2007). The 5-min test occurred during the light cycle between 11:00 and 2:00pm. Measurements were taken to ensure light intensity on the open arms was 6 lux and uniform throughout open arm regions. Analysis utilized AnyMaze software (Stoelting) to measure time and entries into open and closed arms and total distance traveled.

Light-Dark Box

Testing was performed as previously described (Bale et al., 2000). Light intensity was 5 lux in the dark compartment and 300 lux in the light compartment. Test duration was 10 min and occurred 2 hrs into the dark cycle. AnyMaze software was used to measure distance traveled, time spent in light and dark sides and light-dark transitions.

Examination of Regional Cell Loss

To assess whether chronic stress led to changes in cytoarchitecture, total cell number was calculated in each raphe subregion by counting hematoxylin-stained nuclei. Utilizing raphe sections from TPH2 and CRFR1 in situ assays, total cell number was calculated in representative slices of each raphe subregion using IPlabs software (BD Biosciences/Scanalytics, Rockville, MR). Data is presented as the average of samples within treatment groups.

RT² Profiler Apoptosis PCR Array

To determine how genetic regulation of factors related to cell death might account for observed cell loss in the raphe, dissected dorsal raphe tissue was utilized in a PCR microarray focusing on gene families relevant to the induction and inhibition of apoptosis. Tissue punches of DR were taken from brains of chronically stressed WT and R2KO mice, as well as age-matched WT and R2KO littermate controls, from groups utilized in behavioral experiments (N = 4-6). 300 um punches were collected using a .75 mm circular punch (Ted Pella, Redding, CA). Following homogenization by TRIZOL (Invitrogen) and sonication, total RNA was isolated with chloroform and precipitated with isopropanol. cDNA was transcribed using a RT2 first strand synthesis kit (SuperArray Bioscience, Frederick, MD) and was analyzed using the mouse apoptosis PCR array and the RT2 SYBR green/Rox PCR master mix (APMM 012C and PA-012-24, respectively, SuperArray Bioscience, Frederick, MD) data were normalized using multiple housekeeping genes and analyzed by comparing 2^-ΔCt of the normalized sample.

Electrophysiological Characteristics of 5-HT and non-5-HT Cells

In order to investigate physiological differences in raphe cells of R2KO mice, electrophysiological recordings were used, employing both current and voltage clamp techniques. Whole cell recording techniques were used in the ventromedial dorsal raphe (vmDR), a subregion selected for its robust 5-HT content as previously described (Beck et al., 2004; Lemos et al., 2006). Male WT and R2KO mice (N = 19 and 15, respectively; 10-14 weeks) were taken from colony housing between 7:00 and 7:15 and habituated to the testing room for 90-120 min prior to dissection of raphe slices as previously described (Beck et al., 2004). Brains were sectioned with a Leica VT 1000S vibratome (Leica, Allendale NJ) filled with oxygenated artificial cerebrospinal fluid (aCSF) with sucrose replacing NaCl (124mM). Slices (200 μm thick) were incubated at 37°C in oxygenated aCSF for 60 min, then stored at room temperature until being transferred to a recording chamber superfused with aCSF at a rate of 1.5 ml/min. aCSF was composed of (in mM): NaCl (124); KCl (2.5); NaH₂PO₄ (1.25); MgSO₄ (2.0); CaCl₂ (2.5); dextrose (10); and NaHCO₃ (26). For recordings, electrode resistance was 3–6 MΩ when filled with an intracellular solution of (in mM) K-gluconate, 70; KCl, 70; NaCl, 2; Na phosphocreatine, 10; EGTA, 4; HEPES, 10; MgATP, 2; and Na2GTP, 0.3; with biocytin, 0.1%; pH 7.3.

Cellular characteristics were recorded using current clamp techniques and pClamp software (Molecular Devices, Sunnyvale, CA). Membrane potential, resistance, time constant, action potential (threshold, amplitude, duration) and afterhyperpolarization characteristics were measured for each cell. Cells were discarded if the series resistance of the electrode became ≥20 MΩ or if membrane potential fell below −45 mV. As an in vitro model of how stress-induced CRF release may modulate raphe cell physiology in a genotype-specific manner, the effects of CRF on mIPSC activity were analyzed. Cells were voltage clamped at −70mV and DNQX (20μM) and TTX (1μM) were added to isolate mIPSCs (Lemos et al., 2006). CRF (10nM) was added to superfusate and activity (frequency, amplitude) was recorded and analyzed using Minianalysis software (Synaptosoft, Decatur, GA).

Following recordings, slices were fixed in 4% PFA for immunohistochemical staining to identify tryptophan hydroxylase (TPH) content. Techniques employed fluorophores and mouse monoclonal anti-TPH as described (Beck et al., 2004; Lemos et al., 2006). Sections were incubated with mouse anti-TPH (1:500, Sigma), and visualized with AlexaFlour 488-conjugated donkey antimouse secondary (1:200; Invitrogen). Biocytin was visualized using streptavidin-conjugated AlexaFluor 633 (1:100, Invitrogen) and visualized using DMR fluorescence (OpenLab software, Improvision, Lexington, MA) and confocal DMIRE2 (Leica software) microscopy. Patched cells were identified as either 5-HT-positive or negative by overlapping TPH immunoreactivity with biocytin label.

Statistical Analyses

All analyses were conducted by an investigator blinded to genotype and treatment. Electrophysiological characteristics were compared using 2-way ANOVA by genotype and 5-HT content, and voltage clamp data was assessed using repeated measure ANOVA analysis. Corticosterone measurements were analyzed using 2-way repeated measures ANOVA examining genotype and stress by time point (0 or 15 minutes). For in situ hybridization, ANOVA was employed with factors for rostral-caudal axis, genotype, and stress exposure, and was followed by paired two-tail Student's T-test. Statistical analysis of behavioral data included 2-way ANOVA for stress and genotype. PCR microarray data was assessed by logistic regression where upregulation and downregulation were coded as nominal variables and effects of genotype were tested by a Chi-square test. Analysis was carried out with JMP statistical software (SAS).

Results

HPA Axis Assessment

To ensure a continued HPA axis response to daily stressors during the chronic stress period, corticosterone levels were examined following the 15 minute restraint stress each week of the study. Corticosterone levels at the onset of restraint (0 min) showed a significant interaction of stress exposure and genotype (F_(1,19) = 7.5, p < 0.01), where chronic stress exposure was associated with increased basal corticosterone in WT mice, but not in R2KO mice. Weekly acute restraint continued to induce a significant elevation in corticosterone throughout the chronic stress protocol for both genotypes (F_(1,19) = 68.9 to 323.3, p < 0.0001 Table 1). Interestingly, analysis of peak corticosterone levels displayed a similar effect, where R2KO mice not exposed to chronic stress displayed elevated corticosterone, and stress did not increase this level further as was observed in WT mice exposed to chronic stress (interaction of stress by genotype, F_(1,19) = 4.2, p < 0.05).

Table 1.

Serum corticosterone measurements in wild type (WT) and stress-sensitive (KO) mice showing continued stress response throughout the chronic stress paradigm (N = 6-7). Stressed mice (CVS) of each genotype were compared to unstressed littermates (CTRL). Samples taken prior to onset (Basal) and at completion (Stressed) of a 15 minute restraint stress. Values are expressed as mean ± SEM.

Corticosterone, ng/ml	Week 1	Week 2	Week 3	Week 4
Basal (0 min)
WT CTRL	26.8 +/− 3.5	16.0 +/− 2.0	22.1 +/− 8.5	28.1 +/− 4.2
KO CTRL	28.4 +/− 6.3	32.7 +/− 7.5	44.1 +/− 25.3	22.7 +/− 3.9
WT STRESS	42.7 +/− 13.5	25.4 +/− 2.9	36.7 +/− 12.5	37.1 +/− 14.3
KO STRESS	26.2 +/− 7.2	39.6 +/− 15.9	24.6 +/− 1.8	28.2 +/− 10.0
Stressed (15 min)
WT CTRL	128.9 +/− 18.6	118.7 +/− 10.7	155.4 +/− 8.5	155.7 +/− 16.4
KO CTRL	198.8 +/− 26.0	225 +/− 52.2	205.8 +/− 25.3	156.5 +/− 17.2
WT CVS	172.5 +/− 24.1	173.6 +/− 29.4	176.6 +/− 12.1	177.7 +/− 13.0
KO CVS	163.2 +/− 43.3	161.1 +/− 22.2	156.3 +/− 16.5	151 +/− 12.3

In order to further examine the potential peripheral effects of chronic mild stress exposure, adrenal cortex diameter was examined at the end of stress exposure. No significant differences in adrenal cortex size were observed between genotypes (F_(1,23) = 0.1, p = 0.76) or treatment groups (F_(1,23) = 0.8, p = 0.37). Mean values for groups were as follows: WT control = 2.85 mm ± 0.14, WT stress = 2.72mm ± 0.01, R2KO control = 2.81mm ± 0.10, R2KO stress = 2.80mm ± 0.029.

Gene Expression

To determine changes in gene expression related to corticotropin-releasing factor (CRF) and 5-HT, in situ analysis in the raphe was used to detect changes in tryptophan hydroxylase-2 (TPH2) and CRF receptor-1 (CRFR1).

TPH2

TPH2 expression was basally elevated in R2KO mice compared to WT mice in middle dorsomedial DR (dmDR) (F_(1,9) = 2.5, p < 0.05, Figure 1) and middle MR (F_(1,9) = 2.5, p < 0.05, Supplementary Table 2). These basal elevations were accompanied by increased grains per cell in R2KO mice in both regions (F_(1,9) = 2.7, p < 0.05, dmDR and F_(1,9) = 3.2, p < 0.05, MR).

Figure 1.

CRFR2-deficient mice (R2KO) fail to produce stress-mediated increases in tryptophan hydroxylase-2 (TPH2) and corticotropin-releasing factor receptor-1 (CRFR1) expression found in wild type (WT) mice. Representative images of TPH2 expression in middle dmDR (A) and CRFR1 expression in rostral vmDR (D) of WT and R2KO mice comparing non-stressed control (CTRL) and chronically stressed (STRESS) mice. Atlas image indicating region of analysis for TPH2 (B) and CRFR1 (E), adapted from Paxinos and Franklin (Paxinos and Franklin, 2003). Quantification of TPH2 in middle-caudal dmDR (C) and CRFR1 in rostral vmDR (F) (N = 5-6). R2KO mice showed a basal elevation of TPH2 that was not altered by stress. There was a main effect of stress for increasing TPH2 and CRFR1 expression in WT mice. Data are presented as mean ± SEM. *, significantly different from WT CTRL group, p < 0.05. Anatomical abbreviations: Aq, aqueduct; dmDR, dorsomedial dorsal raphe, aqueduct; lwDR, dorsal raphe lateral wings; vmDR, ventromedial dorsal raphe.

Stress did not significantly alter TPH2 silver grain count or grains per cell in either genotype in ventromedial DR (vmDR, F_(2,61) = 0.15, p < 0.49). In the combined analysis of middle and caudal dmDR, TPH2 expression was increased with stress in WT mice (F_(1,18) = 4.6, p < 0.05, Figure 1), Stress did not increase TPH2 in the R2KO mouse by this analysis (F_(1,18) = 0.5, p = 0.62). Analysis of MR by subregion displayed similar results, where stress-exposed mice displayed elevated grain expression (F_(1,60) = 5.3, p < 0.05) and grains per cell (F_(1,60) = 10.3, p < 0.01, Supplementary Table 2). Both increases were greatest in rostral and middle MR, and chiefly in stressed WT mice. Further detailed analysis of TPH2 expression and the number of cells expressing TPH2 by raphe subregion are found in supplementary Tables 1 and 2.

CRFR1

Analysis of unstressed WT and R2KO displayed no basal differences in grain count within the DR. In the MR, R2KO mice displayed lower CRFR1 expression (F_(1,9) = 3.1, p < 0.05) and grains per cell (F_(1,9) = 2.4, p < 0.05) within the caudal sections compared to WT mice (supplementary Table 4).

Comparison of stress effects on CRFR1 expression revealed increased CRFR1 grain count and CRFR1-positive cell number in the rostral vmDR and caudal dmDR of stressed WT mice (p < 0.05, Figure 1, Supplementary Table 3). R2KO mice displayed no change in CRFR1 grain count or CRFR1-positive cell number (Figure 1). Additional detailed analysis of stress effects on CRFR1 expression patterns and the numbers of cells expressing CRFR1 in each subregion is found in supplementary Tables 3 and 4.

Serotonin Transporter Autoradiography

Assessment of 5-HT transporter (SERT) levels in WT and R2KO mice by ³H-Citalopram autoradiography was conducted to determine effects of stress on 5-HT output brain regions. Genotypic differences in SERT were observed in multiple regions receiving raphe input. While data from ventral hippocampus showed no effect of genotype or stress exposure (Figure 2), a significant basal reduction in SERT density was observed in dorsal DG and CA3 in R2KO mice compared to WT mice (F_(1,21) = 6.9 and 9.2, respectively, p < 0.01, Figure 2). A main effect of stress was also observed wherein SERT levels in the dorsal hippocampus DG and CA3 increased for all groups (F_(1,21) = 8.6 and 9.8, respectively, p < 0.01, Figure 2). SERT levels in the basolateral amygdala were reduced in R2KO mice (F_(1,21) = 7.5, p < 0.05, Figure 2).

Figure 2.

CRFR2-deficient mice (R2KO) show basal reductions and stress-induced changes in serotonin transporter (SERT) levels in limbic brain regions compared to wild type (WT) mice. Significant genotypic differences in SERT levels were detected in the dorsal hippocampus (dHpC) and the basolateral amygdala (BLA). No genotypic differences or stress treatment effects on SERT levels were observed in ventral hippocampus (vHpc) (A) DG or (B) CA3. SERT levels in dHpC (C) DG and (D) CA3 increased across genotypes following chronic stress exposure. No stress effects on SERT levels were observed in the (E) BLA. Data are presented as mean ± SEM. *, significantly different from control, p < 0.05. ⌘, significantly different from WT, p < 0.05.

Behavioral Responses Following Chronic Stress

To determine whether observed genotypic differences in stress-induced gene expression were associated with maladaptive behavioral responses to stress, WT and R2KO behaviors were examined following the 4-week stress period. In the elevated plus maze, WT mice displayed trend for increased total activity as measured by total arm entries (F_(1,9) = 1.96, p = 0.08). Additionally, a significant increase in entries into and time spent in the open arms of the elevated plus maze was seen in WT mice following exposure to chronic stress (Stress × Genotype F_(3,20) = 2.6 and 2.1 respectively, p < 0.02). In the light-dark box, though data suggest WT mice spending more time in the more aversive light side of the test apparatus (Figure 3), these results did not reach significance.

Figure 3.

CRFR2-deficient mice (R2KO) fail to show stress-induced behavioral responses of elevated arousal following 4 weeks of chronic stress. A) Behavioral analysis in the elevated plus maze revealed a significant stress x genotype interaction in open arm entries and time in open arms (p = 0.02), and in total arm entries (p < 0.01), indicative of increased arousal state and vigilance detected in WT mice. *, significantly different from genotype-matched unstressed group, p < 0.05; **, p < 0.01. B) While similar trends for increased locomotion and arousal for WT mice were found in the light-dark box test following chronic stress, these comparisons did not reach significance. Data (mean ± SEM) is presented as percent difference of stressed WT and R2KO mice from unstressed controls.

R2KO mice showed no change in activity in the EPM following stress exposure. A significant interaction of stress and genotype was observed in total arm entries, where decreased entries were observed in stressed R2KO mice but not in stressed WT mice (Stress × Genotype F_(3,20) = 3.1, p < 0.01). Further analysis of this interaction effect revealed a significant decrease in total entries in R2KO mice (p < 0.05, Figure 3). No effects of stress were observed in R2KO mice in the light-dark box.

Examination of Regional Cell Loss

To assess whether chronic stress affected raphe cytoarchitecture, total cell numbers were calculated within raphe subregions. Stress-exposed R2KO mice displayed significant reductions in cell numbers relative to nonstressed R2KO mice in dmDR (F_(1,34) = 6.8, p < 0.01), vmDR (F_(1,34) = 8.4, p < 0.01), lateral wings (F_(1,34) = 2.8, p < 0.05), and median raphe (F_(1,34) = 7.1, p = 0.01) subregions (Figure 4). Stress did not promote a significant decrease in total cell numbers in WT mice (dmDR: F_(1,28) = 1.50, p = 0.15; vmDR: F_(1,28) = 0.60, p = 0.44; lateral wings: F_(1,28) = 1.23, p = 0.255; median raphe: F_(1,28) = 1.87, p = 0.07). Reductions in vmDR cell number were greatest in rostral and caudal regions (Figure 4) where they were associated with decreased TPH-negative cells (p < 0.01, Supplementary Table 1), and increased TPH-positive cells (p = 0.02). Similar effects were observed in dmDR and median raphe, where cell loss was accompanied by fewer TPH-negative cells (F_(1,61) = 18.0 and 10.2, respectively, p < 0.01), and greater TPH-positive cell number (F_(1,61) = 9.0 and 4.7, respectively, p < 0.01).

Figure 4.

Chronic stress induces significant subregion-specific cell loss in CRFR2-deficient mice (R2KO) throughout the raphe. Total cell number was calculated in WT and R2KO mice (N = 5-6) in the (A) rostral ventromedial dorsal raphe (vmDR), and (B) caudal subdivision of the median raphe (MR). Chronic stress significantly reduced cell numbers in R2KO mice in the vmDR and MR. *, significantly different from R2KO CTRL, p < 0.05. **, significantly different from R2KO CTRL, p < 0.01. (C) Analysis of cell numbers as measured across the entire dorsomedial dorsal raphe (dmDR) where there was a main effect of stress to promote cell loss (p < 0.01). (D) Representative images of hematoxylin-stained brain sections from the middle dmDR showing a decrease in cell number in stressed R2KO mice. Data are presented as mean ± SEM.

RT² Profiler Apoptosis PCR Array

To assess how regulation of factors related to cell death might account for observed cell loss in the raphe, dorsal raphe punches were analyzed by a focused PCR array comparing stress-mediated changes in pro- and anti-apoptotic genes. Following stress, a significant effect of genotype was observed by logistic regression analysis whereby WT mice showed a increase in both markers of apoptotic (Chi square = 13.7, p < 0.001) and anti-apoptotic signaling (Chi square = 26.6, p < 0.0001, Figure 5). In contrast, stressed R2KO mice showed elevations in pro-apoptotic markers, but failed to display increases in anti-apoptotic gene expression (Figure 5). A complete gene list and stress effects in WT and R2KO mice is shown in Supplementary Tables 6 and 7.

Figure 5.

CRFR2-deficient mice (R2KO) fail to increase anti-apoptotic gene expression, and display dysregulation of genes associated with cell death following chronic stress. Total RNA isolated from dorsal raphe punches from wild type (WT, A) and R2KO (B) mice exposed to chronic stress was analyzed using an RT² Profiler Apoptosis PCR Array. Analysis by logistic regression of pro- and anti-apoptotic genes revealed a genotype effect where R2KO mice failed to show the responses of upregulated anti-apoptotic factors seen in WT mice following stress (p < 0.0001). Genes related to apoptosis are highlighted in red. Genes associated with anti-apoptotic signaling are highlighted in yellow. Data are presented as fold difference from unstressed control, calculated from average ΔCT normalized to housekeeping genes (Beta actin, GAPDH, HPRT1, HSP90AB1).

Electrophysiological Characteristics of 5-HT and non-5-HT Cells

In order to investigate physiological differences in raphe cells of stress-sensitive CRFR2-deficient mice (R2KO), whole cell electrophysiological recordings were used, employing both current and voltage clamp techniques. A total of 52 current clamp recordings were analyzed from (27 WT from 19 mice, 25 R2KO from 15 mice). Cellular characteristics that were measured included resting membrane potential, resistance, time constant, afterhyperpolarization, and action potential amplitude, width and threshold (Figure 6, Table 2). While characterization of 5-HT-containing and non-5-HT cells (5-HT+ and 5-HT−, respectively) revealed several similar characteristics, resting membrane potential was significantly depolarized in R2KO cells relative to corresponding WT cells (F_(1,46) = 3.9, p = 0.03, Figure 6). There was also an increase in action potential duration among 5-HT+ cells across genotypes (F_(1,40) = 4.1, p = 0.04). This increase was most robust in 5-HT+ cells from R2KO mice (Table 2).

Figure 6.

Basal cellular physiology and CRF responses of the dorsal raphe are altered in CRFR2-deficient mice (R2KO). Whole cell current clamp recordings of serotonin (5-HT+, black bars) and non-5-HT (5-HT-, white bars) cells were examined across the rostral-caudal axis of the ventromedial dorsal raphe (vmDR) in wild type (WT; N = 8 and 19 for 5-HT and non-5-HT, respectively) and R2KO (N = 11 and 14 for 5-HT and non-5-HT, respectively) mice. A) Representative traces from each cell type recorded in the vmDR. Voltage traces in response to current injection and action potentials are displayed. B) Resting membrane potential of cells recorded across the rostral-caudal axis of the vmDR. Both 5-HT and non-5-HT cells in R2KO mice displayed depolarized potentials compared to corresponding WT cells (p < 0.05). C) Sample voltage clamp traces (−70mV holding potential) representing i) Basal IPSC activity, ii) DNQX (20μM) and TTX (1μM)-treated miniature IPSC (mIPSC) activity, and iii) mIPSC recordings after CRF administration. D) Effect of CRF administration on mIPSC event frequency. E) Change in mIPSC amplitude during CRF administration. ⌘, significantly different from WT non-5-HT group, p < 0.05. *, significant difference between WT and R2KO 5-HT groups, p = 0.01.

Table 2.

Active and passive cell characteristics in whole cell recordings of cells in ventromedial dorsal raphe (vmDR). Recordings were taken from serotonin (5-HT+) and non 5-HT (5-HT−) cells in wild type (WT) and stress-sensitive (KO) mice. Abbreviations are as follows: Vm, resting membrane potential; R, resistance; AHP, afterhyperpolarization; Amp, amplitude; AP, action potential. Values are expressed as mean ± SEM.

	Vm rest (mV) ‡	R (mW)	Tau (ms)	AHP Amp (mV)	AP Threshold (mV)	AP Amp (mV)	Peak AP Height	AP Duration (ms)*
Cell Type
WT 5-HT− (19)	−60.9 +/− 1.6	464.0 +/− 22.4	37.8 +/− 4.3	24.0 +/− 2.0	−24.2+/−3.3	48.5 +/− 2.3	21.0+/−2.2	1.5 +/− 0.07
WT 5-HT+ (8)	−61.3 +/− 2.8	447.1 +/− 53.9	39.1 +/− 4.3	26.2 +/− 0.7	−28.0 +/− 2.6	50.0 +/− 4.4	22.0+/−3.3	1.8 +/− 0.07
KO 5-HT− (14)	−54.9 +/− 1.5	475.0 +/− 25.7	42.7 +/− 2.5	26.1 +/− 1.0	−28.4 +/− 1.0	52.4 +/− 2.7	24.0+/−1.8	1.6 +/− 0.07
KO 5-HT+ (11)	−57.9 +/− 2.3	415.5 +/− 36.6	42.7 +/− 6.3	24.9 +/− 0.8	−28.0 +/− 1.2	50.0 +/− 3.3	20.1+/−3.0	2.1 +/− 0.36

^*significant difference across 5-HT+and 5-HT− cells, p ≤ 0.05.

^‡significant difference across genotypes, p ≤ 0.05.

Voltage clamp recordings of GABAergic inhibitory postsynaptic currents (IPSC) revealed several functional differences across cell types. The frequency of spontaneous IPSCs was reduced in 5-HT+ cells from R2KO mice (N = 7) as compared to 5-HT+ cells from WT mice (N = 7) (F_(1,8) = 2.5, p = 0.03, Figure 6). Upon addition of CRF (10 nM), the mIPSC frequency was increased in WT 5-HT+ cells but not in 5-HT+ cells from R2KO mice ((F_(1,8) = 3.4, p = 0.01, Figure 6). Significant differences were not observed in amplitude between groups, although repeated measures ANOVA detected a R2KO-specific decrease in mIPSC amplitude in the presence of CRF in 5-HT+ cells (F_(3,15) = 3.4, p = 0.04, Figure 6). No differences were observed in frequency and amplitude measurements between WT and R2KO non-5-HT cells (N = 15 and 10, respectively). Addition of bicucculine (20μM) eliminated mIPSC activity (data not shown).

Discussion

Maintenance of organismal homeostasis in response to stress insults is of primary importance in the prevention of affective disorders. Therefore, it is the manifestation of inappropriate stress responses that becomes a disease risk factor. To examine the consequences of stress dysregulation within the serotonergic raphe in the absence of CRFR2, we compared molecular and cellular outcomes in WT and R2KO mice following 4 weeks of chronic variable stress. HPA axis assessment throughout the chronic stress period revealed a continuous response to the 15 min restraint from both genotypes, confirming that mice did not habituate to the stressors during the 4-week exposure. In assessing these HPA effects during the chronic stress period, it was interesting to note that while R2KO control mice displayed a predicted heightened response to restraint compared to WT controls, chronic stress exposure elevated the restraint response of WT mice, eliminating this genotypic difference. These changes in corticosterone levels in response to restraint corresponded to genotypic differences we detected for TPH2 expression where WT mice significantly elevated TPH2 expression during chronic stress, and R2KO mice showed higher basal levels but failed to respond to chronic stress. As 5-HT is involved in stress-induced activation of the HPA axis, these results suggest that an absence of CRFR2 dysregulates the 5-HT system such that these mice are unable to mount further responses found in WT mice (Nakamura and Kurasawa, 2000; Millan et al., 2003; Lowry and Moore, 2006). No significant differences between genotypes or treatment groups were observed in histological analysis of the adrenal gland cortex, suggesting that the variable stress exposure was mild enough in nature to not produce a peripheral impact in either genotype.

We examined CRF and 5-HT related gene expression where analyses by raphe subregion revealed dynamic heterogeneity in basal and stress-activated expression of tryptophan hydroxylase-2 (TPH2). We found basally increased TPH2 expression in R2KO mice indicative of the increased sensitivity to stress in these mice. Chronic stress increased the number of TPH2-expressing cells in WT mice. However, R2KO mice failed to exhibit these adaptations during stress experience. The largest increase in TPH2 expression in the WT raphe was detected in middle and caudal dorsomedial DR (dmDR), areas along the rostral-caudal axis of this subregion that receive the greatest CRF input (Valentino et al., 2001; Roche et al., 2003).

The observed increases in TPH2 may serve to meet a higher 5-HT demand in raphe target regions necessary during stress for adaptive responses and restoration of systems to homeostatic baseline (Tan et al., 2004). The absence of such changes in TPH2 in R2KO mice further indicates a dysregulation that may incapacitate necessary behavioral coping responses. These results are of particular interest in light of recent work indicating topographically-specific changes in total tryptophan hydroxylase content within the DR of suicide patients (Boldrini et al., 2007).

The relevance of CRF receptor-1 (CRFR1) to stress and depression has been demonstrated (Contarino et al., 1999; Varga et al., 2001; Bale et al., 2002; Ducottet et al., 2003; Alonso et al., 2004; Bale and Vale, 2004; Kasahara et al., 2006). We examined CRFR1 expression levels following chronic stress as an important mediator of CRF-induced effects in the raphe. Again, R2KO mice showed evidence of basal differences within the raphe and failed to respond to the stress exposure. Increased CRFR1 expression by stress in WT mice was specific to rostral vmDR and caudal dmDR, areas receiving robust CRF innervation (Price et al., 1998; Lowry et al., 2000; Valentino et al., 2001; Lowry, 2002). These data present an important correlation within the raphe reflecting concordant changes related to CRF and 5-HT, as raphe regions displaying increased CRFR1 overlapped with the same areas showing stress-induced increase in TPH2 expression. However, as detected CRFR1 mRNA levels are relatively low, it is not currently known if all CRFR1 in the raphe is localized pre- or post-synaptically.

To examine possible effects of chronic stress on 5-HT output regions important in mediation of coping and affect (Vertes and Kocsis, 1994; Vertes et al., 1999), 5-HT transporter (SERT) density was examined. Interestingly, R2KO mice displayed a basal reduction in SERT levels in the basolateral amygdala and dorsal hippocampus, and an increase in response to stress in the dorsal hippocampus. As the dorsal hippocampus receives major inputs from the rostral MR (Vertes et al., 1999), a region where we detected increased TPH2 expression with stress only in WT mice, these findings support a possible further depletion of 5-HT in R2KO mice during stress due to both a failure to increase the rate-limiting enzyme TPH2 and a counterproductive increase in 5-HT reuptake via SERT.

As a functional output measure of behavioral responses in a novel stress-provoking environment, we compared WT and R2KO mice to nonstressed controls at the end of the chronic stress period. As predicted, chronic stress induced robust increases in arousal and exploratory behaviors in WT mice, distinguishing effects between acute and chronic stress impact on behavioral responses (Strekalova et al., 2005). Significant alterations in overall activity were found in WT mice with increased time and entries into the open arms and total arm entries in the plus maze and a similar trend for increasing transitions in the light-dark box. We hypothesize that these responses represent adaptive changes in arousal state and exploratory behavior as has been reported with other stress paradigms (Lowry and Moore, 2006; Teegarden and Bale, 2007). The observed increase in arousal correlates with TPH2 expression increases detected in WT mice following chronic stress. These changes in gene expression and behavior likely reflect an adaptive response to stress whereby 5-HT circuits are recruited to provide necessary coping stimuli (Nakamura and Kurasawa, 2000; Millan et al., 2003; Lowry and Moore, 2006; Bondi et al., 2007; Clark et al., 2007). Again, R2KO mice failed to produce a change in behavioral responses in these tests following stress exposure, which may be due to the lack of TPH2 response following chronic stress.

Raphe neurotransmission is dependent on an intricate and highly differentiated set of cell populations acting both within the raphe and in target output brain regions. Therefore, we examined total cell numbers throughout the DR and MR to determine the effect of chronic stress exposure in the raphe. Stress exposure produced a significant cell loss in R2KO mice in the vmDR and dmDR. Cell loss in the vmDR was largest in rostral sections where CRF input is greater (Valentino et al., 2001). The absence of significant cell loss in WT mice may suggest that CRFR2, hypothesized to function in restoring systems to homeostatic baseline (Bale et al., 2002), plays a critical role in maintaining healthy raphe physiology.

Ultimately, the determination of cell death or survival is dependent on a balance between pro- and anti-apoptotic signals. To elucidate the possible mechanism of cell loss following chronic stress in the absence of CRFR2, DR punches were analyzed by an apoptosis-focused PCR array in WT and R2KO mice following chronic stress. Results from this array revealed dramatic genotypic differences in directional regulation of apoptotic and anti-apoptotic genes. Following chronic stress exposure, WT mice displayed an upregulation of anti-apoptotic genes. R2KO mice, however, failed to produce this adaptive change in anti-apoptotic factors, while still displaying increased expression of pro-apoptotic genes, a pattern that may place the raphe at a particular susceptibility to cell death during chronic stress, and may ultimately increase the risk for affective disorder onset in stress-sensitive populations.

In the absence of CRFR2, mice displayed a complex phenotypic dysregulation of behavioral and gene expression measures in response to a chronic stress period. To determine whether these maladaptive responses may be associated with underlying genotypic differences in raphe cell physiology, we examined in vitro electrophysiological recordings in 5-HT and non 5-HT-containing cells in the ventromedial dorsal raphe (vmDR), a region selected due to its robust CRF innervation (Valentino et al., 2001). In basal recordings, both 5-HT-containing and non-5-HT cells from R2KO mice showed greater depolarization at rest compared to WT mice. Previous studies have examined the influence of CRF in stress-mediated 5-HT neurotransmission (Kirby et al., 2000; Thomas et al., 2003; Tan et al., 2004; Zhong and Yan, 2004; El Mansar et al., 2005). An absence of CRFR2 in the raphe, or in other key brain regions modulating effects in the raphe, likely contributed to the depolarized resting membrane potential observed in R2KO raphe cells. As such, this depolarization may become increasingly influential during stress exposure, priming raphe cells to be more sensitive to excitatory impulses (Lowry and Moore, 2006).

In order to examine the change in physiological responses of these neurons to stress in the absence of CRFR2, we utilized CRF application as a potential mechanism whereby stress could produce raphe dysregulation, potentially leading to cell death. In addition to basal differences between genotypes, alterations in GABA-mediated signaling were observed in cell recordings following direct CRF administration. A moderate dose of CRF caused a significant increase in firing frequency in WT mice, suggestive of increased presynaptic GABAergic activity specifically affecting 5-HT cells, in agreement with previous findings of CRF actions in the raphe (Kirby et al., 2000; Valentino and Commons, 2005). This effect was absent in recordings from R2KO mice. In addition, there was a significant decrease in mIPSC amplitude in 5-HT cells from R2KO mice. These findings suggest that CRFR2 is directly or indirectly involved in the appropriate physiological responses to CRF in the raphe, and may play an important role in altering 5-HT cell function. An involvement of CRFR1 in these genotypic differences cannot be ruled out.

Maladaptive responses to even mild stressors, including a failure to adapt appropriately, may be predictive of an elevated vulnerability to affective disorders. Under chronic stress conditions in an adaptive organism, a shift in CRF tone is a likely contributor to the observed elevations in CRFR1, TPH2, and anti-apoptotic factors found in the raphe, resulting in the induction of compensatory behavioral and physiological strategies and, ultimately, cell survival (see Figure 7). While these adaptive changes were multifaceted and robust in WT mice, analyses of R2KO mice revealed a consistent and categorical failure to mount the same homeostatic responses in the absence of CRFR2. The inability to produce these changes may result in maladaptive strategies and ultimately to the cell death in a brain region critical for regulation of affect.

Figure 7.

Summary of chronic stress effects on homeostatic increases in tryptophan hydroxylase-2 (TPH2) and corticotropin-releasing factor receptor-1 (CRFR1) expression in wild type (WT) and CRFR2-deficient (R2KO) mice. Dorsal and median raphe are displayed along their rostral-caudal axis, with symbols illustrating increased (↑) or decreased (↓) TPH2 and CRFR1 expression following chronic stress. A significant cell loss was observed in R2KO mice following stress in subfields that correspond with those that in WT mice showed stress-mediated increases in TPH2 and CRFR1 (i.e. middle to caudal dmDR and rostral vmDR). Trends for cell loss in these areas in WT mice did not reach significance. These results suggest that the inability to appropriately respond to stress may contribute to cytoarchitectural changes in key raphe subregions. Anatomical abbreviations are as follows: DR, dorsal raphe; MR, median raphe; dmDR, dorsomedial DR; vmDR, ventromedial DR; lwDR, DR lateral wings.