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Stress is a major risk factor for numerous neuropsychiatric diseases. However, susceptibility to stress, and the qualitative nature of stress effects on behavior, differs markedly among individuals. This is partly due to the moderating influence of genetic factors. Inbred mouse strains provide a relatively stable and restricted range of genetic and environmental variability that is valuable for disentangling gene x stress interactions. Here, we screened a panel of inbred strains for anxiety- and depression-related phenotypes at baseline (trait) and following exposure to repeated restraint. Two strains, DBA/2J and C57BL/6J, differed in trait and restraint-induced anxiety-related behavior (dark/light exploration, elevated plus-maze). Gene expression analysis of amygdala, medial prefrontal cortex and hippocampus revealed divergent expression in DBA/2J and C57BL/6J both at baseline and after repeated restraint. Restraint produced strain-dependent expression alterations in various genes including glutamate receptors (e.g., Grin1, Grik1). To elucidate neuronal correlates of these strain differences, we performed ex vivo analysis of glutamate excitatory neurotransmission in amygdala principal neurons. Repeated restraint augmented amygdala excitatory postsynaptic signaling and altered metaplasticity (temporal summation of NMDAR currents) in DBA/2J but not C57BL/6J. Furthermore, we found that the C57BL/6J-like changes in anxiety-related behavior after restraint were absent in null mutants lacking the modulatory NMDA receptor subunit Grin2a, but not the AMPA receptor subunit Gria1. Grin2a null mutants exhibited significant (~30%) loss of dendritic spines on amygdala principal neurons under non-restraint conditions. Collectively, our data support a model in which genetic variation in glutamatergic neuroplasticity in corticolimbic circuitry underlies phenotypic variation in responsivity to stress.
While stress is a known risk factor for various neuropsychiatric disorders, ranging from mood and anxiety disorders to schizophrenia and attention deficit hyperactivity disorder (ADHD), individuals differ greatly in their susceptibility to stress. Moreover, the manner in which stress manifests phenotypically varies considerably across individuals; even similar stressors can increase risk for different neuropsychiatric states in different people (Yehuda and LeDoux, 2007). This variation in the impact of stress is likely to be, in some measure, under the modulating influence of genetic factors (Caspi et al., 2010).
Despite being the subject of enormous research efforts, genetic influences and their effects on the neurobiology of stress and emotion-mediating circuits are not yet fully understood. This is in part due to the complexity associated with myriad genetic and environmental factors in human populations. As an alternative approach, rodents provide important model systems for studying the pathophysiology of stress-related neuropsychiatric disease (Cryan and Holmes, 2005). Of considerable value in this regard are isogenic inbred mouse strains.
A panel of different mouse strains represents a significant but restricted degree of genetic diversity in which environmental variance can be carefully controlled. Previous studies have found differences in various ‘emotion-related’ phenotypes across inbred strains. This includes marked variation in anxiety-like behavior, although differences between specific strains appear to be contingent upon the behavioral task employed, consistent with the significant heterogeneity of rodent anxiety tasks (Turri et al., 2001; Ponder et al., 2007; Brigman et al., 2008a; Milner and Crabbe, 2008). Earlier work also indicates that inbred strains differ in their response to stress as measured by various neural, neuroendocrine and behavioral endpoints. For example, acute stress typically produces heightened anxiety-like behavior and hypothalamic-pituitary-adrenal (HPA)-axis activation in some strains (e.g., BALB/cJ, DBA/2J), but less so in others (e.g., C57BL/6J) (Belzung and Griebel, 2001; Jacobson and Cryan, 2007; Millstein and Holmes, 2007). Relatively few studies have examined strain differences in response to repeated stress (e.g., Pothion et al., 2004; Anisman and Matheson, 2005; Mineur et al., 2006). Such studies are of critical relevance to human stress-related disorders, which are commonly associated with a history of repeated trauma (Berton and Nestler, 2006; Yehuda and LeDoux, 2007).
Here, we first surveyed a panel of seven inbred strains for anxiety- and depression-related behaviors, and HPA-axis phenotypes at baseline and following repeated restraint stress. Subsequent analysis focused on two inbred strains (C57BL/6J and DBA/2J) exhibiting divergent trait and stress-induced anxiety-like phenotypes in the dark/light exploration test (and confirmed in the elevated plus-maze). To identify genes associated with this strain x stress interaction, we performed gene expression analysis in amygdala, ventromedial prefrontal cortex (vmPFC) and hippocampus. Prominent amongst the expression changes were glutamatergic genes. Therefore, we next tested for strain differences in stress-induced alterations in amygdala NMDAR-mediated excitatory neurotransmission and metaplasticity (temporal summation of NMDAR currents). To establish a causative link between glutamate function and stress effects on anxiety-related behavior, we tested whether stress effects were altered by deletion of either NMDAR NR2A or AMPAR GluR1 subunits. Because stress-induced increases in dendritic length and spine density in BLA neurons is posited to be a neuronal correlate of changes in behavior (Vyas et al., 2006), we also quantified BLA neuronal dendritic morphology and spine density in NR2A −/− mice.
The initial strain survey comprised 129S1/SvImJ (129S1), A/J, BALB/cJ, BALB/cByJ, C57BL/6J, DBA/2J, and FVB/NJ. These were selected on the basis of 1) their frequent use in behavioral neuroscience and as genetic backgrounds for mouse mutant lines, 2) inclusion as “group A” priority strains in the Mouse Phenome Project, an international effort to provide the biomedical research community with phenotypic data on the most commonly used mouse strains (www.jax.org/phenome), 3) prior studies demonstrating differential trait fear-, anxiety- and depression-related and stress-sensitivity phenotypes (see Introduction), and 4) their use as parental strains in several sets of recombinant inbred strains, including the AXB, BXA, CXB, and BXD sets (see www.genenetwork.org).
All mice were males obtained from The Jackson Laboratory (Bar Harbor, ME) (as in Millstein et al., 2006) to reduce a potential source (i.e., supplier) of genetic and behavioral variation. Mice were aged 8–9 wks at the start of the study, housed 2/cage (by strain and stress condition), with cages placed side-by-side in a temperature- (72 ± 5°F) and humidity- (45 ± 15%) controlled vivarium under a 12 hr light/dark cycle (lights on 0600 h). Testing was conducted in a manner counterbalanced for strain and stress condition. The number of mice used is given in the figure legends. All experimental procedures were approved by the NIAAA Animal Care and Use Committee and followed the NIH guidelines outlined in ‘Using Animals in Intramural Research’ and the local Animal Care and Use Committees.
Ten days of immobilization in ‘immobilization bags’ produces significant alterations in dendritic arborization and/or spine density in the vmPFC, basolateral amygdala (BLA) and CA3 region of the hippocampus in rats and mice (see Holmes and Wellman, 2009; Roozendaal et al., 2009). We adopted a modified version of this protocol in which mice were placed in ventilated 50 mL Falcon tubes for 2 hr/day (1000 – 1200 hr) for 10 consecutive days. We reasoned that restraint in tubes would be a less severe stressor than restraint in immobilization bags and would therefore allow us to better detect differential sensitivity to restraint across strains than a severe stressor that might cause profound changes (i.e., ‘floor effect’) in all strains. Non-restrained mice remained in the home-cage (Vyas et al., 2002).
Twenty-four hr after the final stress, mice were tested for anxiety-like behavior using the light/dark exploration test. We employed this task rather than other commonly used tests for anxiety-like behavior for a number of reasons. First, under baseline conditions in our laboratory, C57BL/6J typically display lesser anxiety-like behavior in this test [~25% time out of shelter (e.g., this study)] than in the elevated plus-maze [~10% open arm time (e.g., Norcross et al., 2008)]. Therefore, it seemed less likely that a high baseline anxiety-like behavior would preclude us from detecting stress-induced increases in anxiety in this test. We conducted a relatively long (15 min) test in order to capture the most anxiety-sensitive period during the first 5 min and dissociate this from general changes in locomotion, as measured by behavior during the final 5 min. A second reason is that the light/dark exploration test has previously demonstrated utility as an assay for uncovering gene expression differences underlying mouse strain differences in basal anxiety-like behavior (Hovatta et al., 2005).
Mice were placed in an opaque black Plexiglas shelter (39 x 13 x 16 cm) with a 13 x 8 cm aperture at floor level that opened onto a large white Plexiglas square arena (39 x 39 x 35 cm) illuminated to ~90 lux. This apparatus is a 2/3 light vs. 1/3 dark design as used in the original validated formulation of the task (Crawley, 1981) rather than half light vs. half dark design we have used in some previous studies and found to be relatively insensitive to strain differences likely due to its less ‘stressful’ nature. Latency to first exit the shelter, the number of shelter exits (defined as all 4 paws out of the shelter) and time spent out of the shelter in the open field over a 15 min session was recorded by an observer using Hindsight (Hindsight, Services, Wokingham, UK). To dissociate the anxiety-related and general exploratory phases of the test session, data were separately analyzed during the first 5 min and last 5 min, respectively. The effects of strain and stress were analyzed using 2-factor analysis of variance (ANOVA) followed by Newman Keuls post hoc tests. Given the high number of strains tested and the resultant reduction of power in our analysis, we conducted planned post hoc comparisons of strain and/or stress effects in the presence of significant ANOVA main effects regardless of whether a significant strain x stress interaction effect was also found. Statistical significance for this and all other analyses was p≤.05.
In order to test whether trait and/or restraint-induced changes in anxiety-related behavior observed in DBA/2J and C57BL/6J in the light/dark exploration test (see Results) extended to another test for anxiety-like behavior, we assessed baseline and post-restraint behavior in these two strains in the elevated plus-maze. The apparatus consisted of 2 open arms (30 x 5 cm; 90 lux) and 2 closed arms (30 x 5 x 15 cm; 20 lux) extending from a 5 x 5 cm central area and elevated 47 cm from the ground (San Diego Instruments, San Diego, CA, USA), as previously described (Hefner and Holmes, 2007). The walls were made from black ABS plastic and the floor from white ABS plastic. A 0.5 cm raised lip around the perimeter of the open arms prevented mice from falling off the maze. The mouse was placed in the center facing an open arm and allowed to explore the apparatus for 6 min. Time spent in the open arms, and entries into the open and closed arms, and head-dipping (exploratory movement of head/shoulders over the sides of the open arms: Holmes and Rodgers, 2003) was recorded by an observer using Hindsight. The mouse was adjudged to be in an arm when all 4 paws were in an arm. Planned t-test comparisons were conducted to examine strain differences and stress effects within each strain.
We evaluated whether strain differences in stress-responsivity extended to depression-related phenotypes. As a systemic marker of the efficacy of repeated restraint as a stressor, we measured restraint-induced reductions in body weight in the 7 inbred strains (Willner et al., 1996; Pothion et al., 2004; Krishnan et al., 2007; Shansky et al., 2009). Changes in body weight over the 10-day restraint period were compared between restrained and non-restrained groups and analyzed using 2-factor (strain x restraint) analysis of variance (ANOVA) followed by Newman Keuls post hoc tests.
We used the forced swim test (FST) to measure depression-related behavior (Porsolt et al., 1977; Cryan and Holmes, 2005), conducting this assay 24 hr after the light/dark exploration test. Mice were gently lowered into a transparent Plexiglas cylinder (20 cm diameter) filled halfway with water (24 ± 1°C) for a 6 min session, as described (Boyce-Rustay and Holmes, 2006). The presence/absence of immobility (cessation of limb movements except minor involuntary movements of the hind limbs) was scored using an instantaneous sampling technique every 5 sec during min 2–6 and expressed as percent of total observations. The effects of strain and restraint were analyzed using 2-factor ANOVA followed by Newman Keuls post hoc tests.
To assess HPA-axis activation induced by swim stress, mice were returned to the home cage after FST and 30 min later sacrificed via rapid cervical dislocation and decapitation to collect trunk blood samples for serum corticosterone analysis. An additional set of experimentally-naive mice was sacrificed at the same time to provide a baseline measure of corticosterone. Samples were taken between 1000 and 1200 hr. Blood samples were centrifuged at 13,000 rpm for 30 sec. Serum was extracted and assayed for total corticosterone (bound and free) using the Coat-a-Count RIA TKRC1 kit (limit of detection: 5.7 ng/ml; Diagnostic Products Corp, Los Angeles) as described (Boyce-Rustay et al., 2007). The effects of strain and restraint were analyzed using 2-factor ANOVA followed by Newman Keuls post hoc tests.
Microarray assays were conducted on tissue from 3 key corticolimbic regions mediating stress and anxiety (amygdala, vmPFC, hippocampus) in DBA/2J and C57BL/6J at baseline and after stress. After removal, brains were stored in RNAlater (Ambion, Austin, TX). The vmPFC, amygdala (principally the basolateral nucleus) and whole hippocampus, were microdissected within 48 hr of brain collection. The brain was placed in a coronal matrix and sectioned 1.4–2.4 mm from Bregma to obtain the vmPFC (tissue medial to the forceps minor, mainly comprising the infralimbic and prelimbic cortices), and 1.0–2.0 mm caudal to Bregma to obtain the amygdala and hippocampus. The amygdala was visualized under a dark-field microscope and dissected using the external and internal capsules as a guide to obtain the basolateral nucleus (although we cannot exclude some inclusion of tissue from the central nucleus and striatum). The whole hippocampus was then dissected.
Tissue was immediately frozen and stored at −80°C. Samples from each mouse were stored and analyzed separately (i.e., no pooling). Total RNA was isolated using RNAqueous Micro kit (Ambion, Austin, TX). RNA purity and concentration was evaluated with a spectrophotometer using a 260/280 nm absorbance ratio, and RNA quality was checked using Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). Samples were processed according to the manufacturer’s instructions and hybridized onto Illumina Mouse–6.1 arrays (Illumina, Inc, San Diego, CA). Samples from each strain, stress condition and brain region were balanced across arrays on a slide to avoid batch confounds. Three biological replicates for each brain region, strain, and treatment group were performed. For each of the 2 strains, there were 2 stress conditions and 3 brain regions (=total of 36 arrays). The number of arrays used meant that we did not perform technical replicates. Instead, we relied upon RT-PCR confirmation to validate specific expression differences (and then physiological and gene mutant experiments to establish specific functional links).
Raw microarray data were normalized using rank invariant and background subtraction protocols provided by the Illumina BeadStation software suite. We log-transformed the expression values and stabilized the variance of each array. We extensively re-annotated these probes on the array and the improved annotations were incorporated into the data analysis for the current study. The custom annotation for the Illumina Sentrix MouseWG-6 v1.1 is available at www.genenetwork.org/share/. The Illumina array contains over 46,000 probes and these were initially filtered by expression: i.e., expression signals below background in half or more of the samples were excluded – resulting in 25,908 probes for analyses. A false discovery rate was not applied because this proved overly stringent to detect effects of stress and therefore likely produced false negatives. Instead we applied a combined criterion for true positive expression differences of a fold-difference ≥1.3 and a statistically reliable difference (p<.01, t-test). Baseline strain effects were defined as gene expression values in non-stressed C57BL/6J compared to values in non-stressed DBA/2J. Stress effects were defined as gene expression values in stressed vs. non-stressed mice of the same strain. Functional categorization and enrichment analysis of these genes was done using DAVID (http://david.abcc.ncifcrf.gov).
To verify gene expression differences, we performed quantitative RT-PCR analysis on 15 genes that met the criteria of statistical reliability (p≤01) and 1.3-fold or greater gene expression difference. Eight genes (Gal, Atp1a2, Bdnf, Nr4a2, Chrna4, Comt, Drd1a, Rgs2) have previously been linked to anxiety- and stress-related behaviors (for references, see Discussion). Four genes (Grik1, Grin1, Gria1, Homer1) are mediators of glutamate neurotransmission and neural plasticity (for references, see Discussion). Three genes (Per1, Per2, Dbp) are circadian genes (for references, see Discussion).
cDNA was synthesized using a first strand cDNA kit (GE Healthcare, Piscataway, NJ) according to manufacturer’s protocol. Primers for RT-PCR were designed based on available sequences using the ProbeFinder software (Roche Diagnostics, Indianapolis, IN). The ProbeFinder software combines a suitable Universal Library probe (Roche Diagnostics, Indianapolis, IN) with a set of gene-specific PCR primer pairs. All primer pairs were checked by an in silico PCR algorithm that searches the relevant genome and transcriptome and flags any possible mis-priming sites that could lead to non-specific amplification. The assays were chosen to be as close as possible to the Illumina probe targets, and to span exons so as to avoid amplification of trace genomic DNA. To assess amplification efficiency, standard dilution curves were generated for all genes. Low efficiency assays were excluded. Two mL of cDNA was added to a PCR reaction mix containing 0.2 mL of forward and reverse primers (20 mM), 0.1 mL of probe (10 mM), 5 ml of 2X LC480 master mix (Roche) and 2.7 ml of DNAse free water. PCR was carried out using Roche LightCycler 480 system (Roche).
PCR amplification for each gene was conducted in technical duplicates and 3 biological replicates. The threshold cycle (Ct) of technical duplicates was then averaged. All expression values were normalized to the expression of cyclophilin D, as this gene showed no expression difference among the different RNA samples. Relative differences in RNA abundance among the different strains and treatment groups were then determined by comparing the normalized Ct values (Livak and Schmittgen, 2001). Upon determining polarity (upregulation or downregulation) of the effect of strain or stress, 1-tailed Student t-tests were used to test for statistical significance.
We next tested for BLA glutamate function at the neuronal level by measuring NMDAR-mediated excitatory postsynaptic currents (eEPSCs) using ex vivo whole-cell voltage clamp recordings. Mice were subjected to repeated restraint (as above) and then, 24 hr after the final restraint, sacrificed via rapid decapitation under isoflurane anesthesia, along with a set of non-restrained mice. Brains were quickly removed and placed in ice-cold sucrose-artificial cerebrospinal fluid (ACSF = 194 mM sucrose, 20 mM NaCl, 4.4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 1.2 mM NaH2PO4, 10.0 mM glucose, and 26.0 mM NaHCO3 saturated with 95% O2/5% CO2). Three hundred μm slices were sectioned on a vibratome and transferred to a submerged recording chamber and perfused with heated (26°, unless otherwise stated), oxygenated ACSF at a rate of ~2 mL/min. Additional slices were stored in a heated (~28°C) and oxygenated (95% O2–5% CO2) holding chamber containing ‘normal’ ACSF (=124 mM NaCl, 4.4 mM KCl, 2 mM CaCl2, 1.2 mM MgSO4, 1 mM NaH2PO4, 10.0 mM glucose, and 26.0 mM NaHCO3) for later use. Slices equilibrated in normal ACSF for 1 hr before recording and were then submerged in the recording chamber (Warner Instruments, Hamden, CT). Neurons of the BLA were directly visualized with infrared video microscopy. Recording electrodes (3–6 MΩ) were pulled on a Flaming-Brown Micropipette Puller (Sutter Instruments, Novato, CA), using thin-walled borosilicate glass capillaries. Recording electrodes were filled with 135 mM Cs+-gluconate, 5 mM NaCl, 10 mM HEPES, 0.6 mM EGTA, 4 mM ATP, 0.4 mM GTP, and 290–295 mM mOsmol. Signals were acquired via a Multiclamp 700B amplifier (Axon Instruments, Sunnyvale, CA) and digitized and analyzed via pClamp 9.2 software (Axon Instruments).
NMDAR-mediated excitatory postsynaptic currents (eEPSCs) were evoked with bipolar ni-chrome stimulating electrodes placed locally within the BLA 100–500 μm dorsal from the recorded neuron. Electrical stimulation (5–40 V, 100–150 μs duration) was applied at 0.2 Hz unless otherwise stated. NMDAR-mediated eEPSCs were pharmacologically isolated from γ-aminobutyric acid type A receptor (GABAA-R)- and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR)-mediated currents by adding 25 μM picrotoxin and 10 mM NBQX respectively, and recording at a holding potential of +40 mV. Input resistance, holding current and series resistance were monitored continuously during recording. Experiments in which changes in series resistance were >20% were excluded from the data analysis.
Twenty traces (5 min) were averaged to obtain a baseline eEPSC. eEPSC decay was fitted with 2 exponentials (tau 1 and tau 2) using Clampfit 9.2 software (Axon Instruments) from trace normalized average traces. To directly compare decay times between experimental conditions, the 2 decay time components, tau 1 and tau 2, were combined into a weighted time constant, tw, using the equation: tw= (tau1*a1)+( tau2*a2), where a1 and a2 are the relative amplitudes of the 2 exponential components. Next, to evaluate temporal summation of eEPSCs (Philpot et al., 2001), we delivered a train of 10 pulses at the stated frequency. These data were analyzed by averaging traces from the same frequency, normalizing the averaged trace to the peak of the first eEPSC pulse and then measuring the normalized peak amplitude at the subsequent 9 pulses. Statistical analyses were performed using Microsoft Excel, Graphpad Prism and Microcal Origin. The effects of strain and stress on weighted tau were analyzed using 2-factor ANOVA. The effects of strain, stress and pulse number at each stimulation-intensity were analyzed using 3-factor ANOVA, with repeated measures for pulse number, followed by Newman Keuls post hoc tests.
Our gene expression analysis found that stress altered amygdala expression of glutamate receptors, including NMDA NR1 (Grin1), in C57BL/6J not DBA/2J. Constitutive null mutation of NR1 subunit is lethal. NR2A is a modulatory NMDAR receptor subunit highly expressed in the amygdala and previously linked to anxiety-like behavior (Boyce-Rustay and Holmes, 2006; Cryan and Dev, 2007). We tested whether NR2A was necessary for stress-induced changes in anxiety-like behavior (as above) in constitutive NR2A (Grin2a) null mutants on a C57BL/6J genetic background. Grin2a null mutants were generated and backcrossed to C57BL/6J congenicity as previously described (Sakimura et al., 1995; Brigman et al., 2008b). Grin2a −/− and Grin2a +/+ littermates were subjected to repeated stress and tested in the light/dark exploration test 24 hr later, as above.
While C57BL/6J had higher hippocampal expression of AMPA GluR1 than DBA/2J under non-stressed conditions, this receptor was not altered by stress. We tested whether GluR1 was unnecessary for stress-induced changes in anxiety-like behavior (as above) in constitutive GluR1 (Gria1) null mutant mice on a C57BL/6J genetic background. Gria1 null mutants were generated and backcrossed to C57BL/6J congenicity as previously described (Zamanillo et al., 1999; Wiedholz et al., 2008). Gria1 −/− and Gria1 +/+ littermates were subjected to repeated stress and tested in the light/dark exploration test 24 hr later, as above. This was the only experiment in which we used both males and females (due to low availability) – statistical analyses found no effect of genotype and no interaction between sex and genotype or stress.
Test naïve mice were overdosed with urethane and transcardially perfused with saline. Brains were removed and processed for Golgi histology using a modification of Glaser and Van der Loos’ Golgi stain (Glaser and Van der Loos, 1981) as described previously (Wellman et al., 2007). Coronal sections were cut at 160 μm on a sliding microtome (American Optical 860). Free-floating sections were then alkalinized, developed, fixed, dehydrated, mounted, and coverslipped.
Analysis of BLA pyramidal neurons was restricted to locations between 0.8 and 2.0 mm posterior to Bregma. Within this region, BLA is readily identified in Golgi-stained material, as the external capsule branches into 2 smaller fiber tracts that define the dorsal, medial and lateral borders of the BLA. Likewise, axon fibers clearly delineate basal amygdala from BLA. Pyramidal neurons within BLA were defined by the presence of a distinct, single apical dendrite, 2 or more basilar dendritic trees extending from the base of the soma, and dendritic spines. Neurons selected for reconstruction were located in the middle third of the section, did not have truncated branches, and were not obscured by neighboring neurons and glia, with dendrites that were easily discriminable by focusing through the depth of the tissue. Within each region examined, 10 neurons were drawn for each mouse. Neurons were drawn at 600X and morphology was quantified in 3 dimensions using a computer-based neuron tracing system (Neurolucida, MBF Bioscience, Williston, VT) with the experimenter blind to genotype. Total length and number of dendrites, as well as the length and number of terminal branches, were measured. To assess differences in the amount and location of dendritic material, a 3-dimensional version of a Sholl analysis (Larkman, 1991) was performed in which the number of intersections of dendrites with 10-μm concentric spheres centered on the soma was measured.
Spines were counted on dendritic branches from 10 neurons per mouse. Spines were counted on first- through fourth-order branches, as these make up approximately 90 percent of the dendritic arbor of BLA pyramidal neurons. For each neuron, 1 dendritic tree containing at least one third-order branch was chosen. One to two branches at each order were drawn and spines counted at 1000X using a computer-based neuron tracing system (Neurolucida, MBF Bioscience, Williston, VT). Branches sampled averaged 10.49 ± 0.65, 45.48 ± 2.16, 57.15 ± 3.56, and 62.23 ± 3.30 μm for first- through fourth-order dendrites. Spines were identified based on morphological criteria for “mushroom” and “thin” spines (Peters and Kaiserman-Abramof, 1970): only protrusions perpendicular to the dendritic shaft and possessing a clear neck and bulbous head were counted. Because spine density varies with branch order, the lengths of dendritic segments were recorded, and spine densities (spines per 10 μm) for each branch order were calculated separately.
The effect of genotype on dendrite length and number was analyzed using t-tests. The effects of genotype x distance from soma on amount and location of dendritic material, and the effects of genotype x branch order on dendritic spine density, were analyzed using 2-factor ANOVA with repeated measures for distance and branch order, respectively.
In the light/dark exploration test, there was a significant effect of strain (F6,102=31.73, p<.01), but no main effect of stress or a strain x stress interaction, for time out of the shelter. Despite the absence of an interaction effect, our a priori hypothesis that strains would differ in their response to stress led us to perform post hoc comparisons between stressed and non-stressed groups. DBA/2J spent significantly less time out of the shelter than non-stressed controls (Figure 1A). Conversely, stressed C57BL/6J spent significantly more time out of the shelter than non-stressed C57BL/6J (Figure 1A). Under non-stressed conditions, 129S1, A/J, BALB/cByJ, and BALB/cJ spent significantly less time, and FVB/NJ spent more time, out of the shelter than C57BL/6J.
There was also a significant strain x restraint stress interaction (F6,102=3.21, p<.01) for shelter exits during the first 5 min. Post hoc analysis showed that stressed DBA/2J and FVB/NJ made significantly fewer exits than non-stressed DBA/2J and FVB/NJ controls, while stressed C57BL/6J made significantly more exits than C57BL/6J controls (Figure 1B). 129S1, A/J, BALB/cByJ, and BALB/cJ showed no change in shelter exits after stress - likely due to low shelter exits under non-stressed conditions; where all strains except FVB/NJ made significantly fewer exits than C57BL/6J.
There was a significant effect of strain (F6,102=19.96, p<.01) but not stress, and no strain x stress interaction for latency to first exit the shelter. Planned post hoc comparisons showed that C57BL/6J had a significantly shorter latency to exit than non-stressed counterparts, except FVB/NJ and DBA/2J (Figure 1C). Non-stressed 129S1, A/J, BALB/cByJ, and BALB/cJ were significantly slower to exit the shelter than non-stressed C57BL/6J.
Stress did not affect any measure during the last 5 min of the test. There were significant strain effects for shelter exits (F6,102=35.02, p<.01) and time out of the shelter (F6,102=52.01, p<.01). On both measures, non-stressed 129S1, A/J, BALB/cByJ, and BALB/cJ had lower scores than non-stressed C57BL/6J (data not shown).
In the elevated plus-maze, stressed C57BL/6J mice spent significantly more time in the open arms (non-stressed C57BL/6J=6.9 ±1.4 mean ±SEM, stressed C57BL/6J=14.2 ±3.0; non-stressed DBA/2J=3.3 ±1.1; stressed DBA/2J=4.3 ±1.1; t=2.33, df=18, p<.05, n=8–11/strain/stress) and made significantly more open arm entries (non-stressed C57BL/6J=3.3 ±0.6, stressed C57BL/6J=5.2 ±0.7; non-stressed DBA/2J=1.8 ±0.5; stressed DBA/2J=2.4 ±0.4; t=2.11, df=18, p<.05) and head-dips (non-stressed C57BL/6J=17.6 ±1.9, stressed C57BL/6J=25.1 ±2.4; non-stressed DBA/2J=5.8 ±1.0; stressed DBA/2J=6.1 ±1.1; t=2.46, df=18, p<.05) than non-stressed C57BL/6J, while stress did not affect any of these behaviors in DBA/2J. Open arm time (t=3.15, df=35, p<.01), open arm entries (t=3.41, df=35, p<.01) and head-dips (t=7.66, df=35, p<.01) were all significantly lower in DBA/2J than C57BL/6J, irrespective of stress. There was a non-significant trend for more closed arm entries in stressed relative to non-stressed mice of both strains (non-stressed C57BL/6J=16.4 ±0.9, stressed C57BL/6J=19.6 ±1.4; non-stressed DBA/2J=13.0 ±0.8; stressed DBA/2J=15.4 ±1.2). Finally, there was a significant effect of stress (F1,32=40.81, p<.01), but not genotype and no interaction, for body weight in this cohort. Stress significantly reduced body weight regardless of strain (non-stressed C57BL/6J=0.9 ±0.2 g mean ±SEM, stressed C57BL/6J=−0.6 ±0.1; non-stressed DBA/2J=0.6 ±0.3; stressed DBA/2J=−1.1 ±0.3).
There was a significant strain x stress interaction for change in body weight over the 10 day restraint period (F6,123=3.87, p<.01). Post hoc tests showed that, with the exception of 129S1, all strains had a significant reduction in body weight after restraint, as compared to the weight gain over the same period in non-stressed mice (Figure 2A).
There was a significant effect of strain (F6,98=58.24, p<.01) and stress (F1,98=5.61, p<.05) but no strain x stress interaction for percent immobility in the FST. Planned post hoc analysis found that stressed BALB/cByJ showed significantly less immobility than non-stress BALB/cByJ counterparts, but no other strain showed a change in behavior after stress (Figure 2B). Under baseline conditions, DBA/2J and FVB/NJ showed significantly less immobility than C57BL/6J (Figure 2B).
There was a significant stress x strain interaction (F6,152=4.82, p<.01) for corticosterone levels. Post hoc analysis showed that, regardless of whether mice had been repeatedly restrained, swim stress significantly increased corticosterone in all strains as compared to a non-swim/non-restrained baseline group (Figure 2C). Moreover, swim stress produced significantly higher corticosterone levels in restrained C57BL/6J and DBA/2J than in non-restrained counterparts. A maximal (‘ceiling’) response under non-restrained conditions may have prevented detection of similar increases in restrained A/J, BALB/cJ and BALB/cByJ. However, contrasting with all other strains, restraint actually produced a blunted corticosterone response to swim stress in 129S1.
Non-stressed DBA/2J and C57BL/6J differentially expressed 1786 probes (1524 known genes) in amygdala, 2540 probes (2154 known genes) in hippocampus, and 1712 probes (1491 known genes) in the vmPFC. A large proportion of the same genes was differentially expressed between strains in either 2 or all 3 regions (Figure 3A). For full list see Supplemental Spreadsheet 1.
Analyzing the effect of stress as a function of strain, stress produced a similar number of upregulations and downregulations in the amygdala of DBA/2J and C57BL/6J (Figure 3B). Stress caused twice as many changes in the C57BL/6J than DBA/2J hippocampus (mainly upregulations). C57BL/6J showed more downregulations than upregulations in vmPFC after stress, while DBA/2J showed the opposite pattern. For all 3 regions, very few (~2%) of the stress-induced expression changes in C57BL/6J and DBA/2J involved the same genes.
Functional classification of stress-sensitive genes revealed enrichment in synaptic plasticity (e.g., glutamate receptors) and ion transport genes in C57BL/6J, and enrichment in genes related to nervous system development, programmed cell death, and myelination in DBA/2J (Supplemental Spreadsheet).
RT-PCR confirmed significantly lower expression of hippocampal Gria1 and vmPFC Chrna4 and Rgs2, and higher expression of hippocampal Bdnf and prefrontal Comt, in non-stressed C57BL/6J as compared to non-stressed DBA/2J (all p<.05 by t-test) (Figure 3C). In C57BL/6J, stress significantly reduced amygdala expression of Grik1 and Gal, and hippocampal Nr4a2, and increased expression of amygdala Grin1 and vmPFC Chrna4, Drd1a and Per2. In DBA/2J, stress significantly increased amygdala Per1, hippocampal Dbp and prefrontal Per2, and decreased hippocampal Homer1. Array showed higher basal expression and stress-induced upregulation of Atp1a2 in C57BL/6J, while RT-PCR showed the reverse effect, likely due to differentially spliced variants.
NMDAR-mediated eEPSC decay time (as measured by weighted tau) was significantly affected by strain (F1,30=23.67, p<.05) but not stress, reflecting significantly longer decay time in DBA/2J than C57BL/6J, irrespective of stress (non-stressed C57BL/6J=97.1 ±5.7, stressed C57BL/6J=104.7 ±12.5; non-stressed DBA/2J=181.6 ±24.3; stressed DBA/2J=172.2 ±17.2; n=6–12/strain/stress).
There was a significant pulse x strain x stress interaction for normalized eEPSC amplitude at all 3 stimulation frequencies: 10 Hz (F8,240=2.32, p<.05), 20 Hz (F8,240=3.61, p<.01) and 40 Hz (F8,240=2.31, p<.05). Post hoc analysis showed that eEPSC amplitude at the lower (10 Hz) stimulation frequency was unaltered by stress in C57BL/6J, but was significantly increased by stress in DBA/2J at later pulses (i.e., 6, 8–10) (Figure 4A,B). After 20 Hz stimulation, stress significantly increased amplitudes (pulses 7–10) in DBA/2J, but not C57BL/6J (Figure 4C,D). Amplitudes were not significantly altered by stress after 40 Hz stimulation, probably due to saturation (Figure 4E,F). Under non-stressed conditions, amplitude was significantly lower in DBA/2J than C57BL/6J after 10 Hz (pulses 5–10) and 20 Hz (pulses 8–10) but not 40 Hz.
In NR2A null mutants, there were significant genotype x stress interactions for shelter exits (F1,37=6.08, p<.05) and time out of the shelter (F1,37=14.12, p<.01) during the first 5 min. Post hoc comparisons showed that non-stressed −/− exhibited a trend for more time out of the shelter than +/+, consistent with data in other assays (Boyce-Rustay and Holmes, 2006). Stress produced a significant, C57BL/6J-like, increase in shelter exits and time out of the shelter in +/+, but had no effects on exits and significantly decreased time out of the shelter in −/−, as compared to non-stressed −/− (Figure 5A,B). There was a trend for shorter latency to exit after stress, but no main effects or interaction (Figure 5C). There was a significant effect of stress (F1,29=64.83, p<.01), but not genotype and no interaction, for body weight. Stress significantly reduced body weight regardless of genotype (Figure 5D).
In GluR1 null mutants, there was a significant effect of stress but no stress x genotype interaction for time out of the shelter (F1,37=9.13, p<.01) and shelter exits (F1,37=9.77, p<.01). Stress significantly increased both measures irrespective of genotype (Table 1). −/− made significantly more shelter exits than +/+ (main genotype effect: F1,37=8.04, p<.01). Neither stress nor genotype affected latency to exit the shelter. There was a significant effect of stress (F1,37=18.12, p<.01), but not genotype and no interaction, for body weight. Stress significantly reduced body weight regardless of genotype (Table 1).
There was a significant effect of genotype (F1,12=4.90, p<.05) and branch order (F3,12=94.10, p<.05), but no interaction, for spine density. −/− had a significantly (~30%) lower spine density than +/+ regardless of branch order (Figure 6A,B). Overall amount of dendritic material, dendritic branch number and length, either in all branches or terminal branches only, was unaffected by genotype (Figure 6C–F).
The current data demonstrate significant variation in basal and stress-induced anxiety-related behavior in different inbred mouse strains. This variation correlated with divergent corticolimbic gene expression and excitatory glutamatergic signaling in the amygdala. Null mutation of the modulatory NMDAR NR2A subunit was sufficient to reverse stress-induced changes in behavior.
Our strain survey revealed that DBA/2J had higher basal anxiety-like behavior than C57BL/6J in two separate behavioral assays (light/dark exploration test, elevated plus-maze), as previously found in some but not all studies (e.g., Bouwknecht and Paylor, 2002; Anisman and Matheson, 2005; Hovatta et al., 2005; DuBois et al., 2006; Mineur et al., 2006; Millstein and Holmes, 2007; Cohen et al., 2008; Schweizer et al., 2009). DBA/2J responded to repeated restraint stress with further increase in anxiety-like behavior (i.e., reduced exploration of aversive areas) in the light/dark exploration test, but not the elevated plus-maze (possibly due to a baseline ‘floor effect). C57BL/6J responded to stress in the opposite manner (i.e., increased exploration of the open areas) in both tests. One explanation is that DBA/2J is ‘susceptible’ to this stressor, while C57BL/6J is ‘resilient.’ However, a more circumscribed but potentially more accurate interpretation is that both strains react strongly to this particular stress regime, but differ in the manner in which the response manifests behaviorally. Thus, DBA/2J may develop a classic ‘passive’ anxiety-like suppression of approach behavior, while C57BL/6J may exhibit more of an ‘active’ response to stress. This could reflect an increased panic-like escape drive or manic-like reaction to stress in C57BL/6J, rather than a decrease in anxiety-like behavior. Indeed, previous studies have found that C57BL/6J (and DBA/2J) exhibit strong anxiety-like reactions to certain other stressors (e.g., acute predator odor exposure) (Cohen et al., 2008). The nature of the behavioral response to repeated restraint in both strains could be parsed further by testing its reversibility by drug classes with efficacy in normalizing different diseases states (e.g., anti-anxiety, anti-panic, anti-manic).
The differential response to stress in anxiety-related measures between C57BL/6J and DBA/2J did not generalize to measures of depression-related behavior. Repeated restraint produced an equivalent loss of body weight and sensitization of the corticosterone response to forced swim stress in DBA/2J and C57BL/6J, but had no effects on forced swim test depression-related behavior. Thus, anxiety-like behavior may be an especially sensitive readout of the effects of repeated restraint, as it is for other repeated stressors, in mice (Strekalova et al., 2004; Krishnan et al., 2007).
There is mounting evidence that genetically-driven variation in corticolimbic function underlies individual differences in anxiety and stress (Caspi et al., 2010). Genome-wide analyses have identified patterns of gene expression underlying strain (Hovatta et al., 2005) and strain-subpopulation (Krishnan et al., 2007) differences in trait and stress-induced alterations in anxiety. Gene expression in amygdala, vmPFC and hippocampus differed markedly at baseline between DBA/2J and C57BL/6J, with an approximately equivalent number of differences in each region (similar to the regional uniformity reported by Kerns et al., 2005). Previous studies have also reported major differential basal gene expression between the strains in various forebrain regions, including the PFC and striatum (Hovatta et al., 2005; Kerns et al., 2005; Korostynski et al., 2006; Grice et al., 2007). Some of the genes our analysis found to differ between strains have been previously reported to differ in other regions. For example, the higher expression of Comt we found in the vmPFC of C57BL/6J relative to DBA/2J has also been reported in the nucleus accumbens (Grice et al., 2007), and the differential expression and putative splice variation of Atp1a2 between these two strains has also been detected in the striatum (Korostynski et al., 2006).
We confirmed expression differences driven by strain- and/or or stress in sixteen genes. Eight have previously been linked to anxiety- and stress-related behaviors: Gal (galanin, e.g., Karlsson and Holmes, 2006), Atp1a2 (sodium pump alpha2 subunit, e.g., Ikeda et al., 2003), Bdnf (brain-derived nerve growth factor, e.g., Nestler et al., 2002), Nr4a2 (aka Nurr1, e.g., Rojas et al., 2007), Chrna4 (alpha 4 nicotinic receptor, e.g., Labarca et al., 2001), Comt (catechol-O-methyltransferase, e.g., Zubieta et al., 2003; Papaleo et al., 2008) Drd1a (dopamine D1 receptor, e.g., Hains and Arnsten, 2008), and Rgs2 (Yalcin et al., 2004). Another four genes are key components of glutamate-mediated neurotransmission and neural plasticity (Malenka and Bear, 2004; Szumlinski et al., 2004; Szumlinski et al., 2005): Grik1 (glutamate receptor, ionotropic, kainate 1), Grin1 (glutamate receptor, ionotropic, NMDA 1), Gria1 (glutamate receptor, ionotropic, AMPA 1), and Homer1 (Homer homolog 1). The final 3 genes we examined are circadian genes (the period homologs Per1, Per2, and their regulator Dbp, D-box binding protein), increasingly implicated circadian genes in stress-related neuropsychiatric diseases such as mania (McClung, 2007).
At the systems level, of the three brain regions analyzed, stress produced the most expression changes in the amygdala in both strains. This is consistent with the amygdala being the central node within the corticolimbic ‘stress’ network and its exquisite sensitivity to genetic factors and stress (LeDoux, 2000; Vyas et al., 2002; Hariri and Holmes, 2006; Yang et al., 2008). Critically however, stress affected an almost entirely different set of genes in DBA/2J than C57BL/6J (in the amygdala as well as vmPFC and hippocampus). For example, stress caused ubiquitous upregulation of circadian (e.g., period) genes in DBA/2J, but altered plasticity-related (e.g., glutamate receptor) genes in C57BL/6J. These data argue against a canonically common molecular ‘stress network’ activated to different degrees in the two strains but, instead, suggest the mobilization of largely autonomous gene networks. They are also in line with the divergent behavioral responses to stress in DBA/2J and C57BL/6J being qualitative, rather than simply quantitative, in nature.
Our expression analysis nominates various gene sets as correlates of the divergent behavioral response to stress in DBA/2J and C57BL/6J. In the current study, we chose to focus further on the glutamate system because this system mediates both corticolimbic excitability and plasticity. Stress augmented temporal summation of NMDAR-mediated currents in BLA neurons in DBA/2J but not C57BL/6J. This effect was evident specifically at sub-saturation stimulation frequencies and in the absence of altered NMDAR-mediated current duration. Temporal summation assays metaplasticity (‘the plasticity of synaptic plasticity’) (Philpot et al., 2001; Kash et al., 2009) and provides a surrogate measure of the excitatory neuronal response in the face of accumulating extracellular glutamate (Kullmann et al., 1996). Enhanced summation after stress in DBA/2J could reflect impaired regulation of glutamatergic neurotransmission at the presynaptic (reduced release by metabotropic glutamate or cannabinoid receptors), glial (reduced glutamate reuptake) or postsynaptic (increased spillover activation of perisynaptic/extrasynaptic NMDARs) levels.
Further studies will be needed to elucidate these mechanisms. Notwithstanding, the main conclusion is that the heightened anxiety-related response to stress in DBA/2J is associated with increased amygdala neuronal excitability. A similar association has been reported in other models, such as in rodents selectively bred for high anxiety-like behavior (Muigg et al., 2007). It was notable that these physiological changes occurred in this strain, while changes in amygdala expression of plasticity genes, including glutamate receptors, were evident in C57BL/6J. We hypothesize that these expression changes reflect the orchestration of neuroadaptations that serve to effectively ‘protect’ against amygdala hyperexcitability in C57BL/6J. The failure to recruit these mechanisms in DBA/2J could underlie the increased passive-like anxiety-like response to stress. This could potentially have implications for developing novel drug treatments for stress-related anxiety disorders that target the glutamate system to mitigate the development or expression of adverse reactions to stress.
Divergent mobilization of the glutamate system per se is unlikely to fully account for the differential strain responses to stress. However, supporting the importance of these particular changes, null mutation (on a C57BL/6J background) of one key modulatory component, the NMDAR NR2A subunit, was not only sufficient to prevent the C57BL/6J-like anxiety-related response to stress, but partially reversed the direction of these effects – i.e., producing a DBA/2J-like increase in anxiety-like behavior. Demonstrating the specificity of the contribution of NR2A, null mutation of another key glutamate receptor, AMPA GluR1 (also on a C57BL/6J background), did not mitigate the C57BL/6J-like response to stress. In addition, BLA dendritic spine density was significantly reduced in NR2A null mutants under basal conditions. This is noteworthy because increased BLA dendritic spine density (and length) is posited to be a neuronal correlate of stress-induced increases in anxiety-like behavior (in rats) (Vyas et al., 2006). Thus, current data raise the intriguing possibility that basal status of BLA spine density may determine the nature of the anxiety-related response (i.e., C57BL/6J-like or DBA/2J-like) to stress.
In summary, our initial strain survey identified two strains exhibiting divergent basal and stress-induced anxiety-related phenotypes, DBA/2J and C57BL/6J. This behavioral variation was associated with marked differences in the expression of corticolimbic genes expressed at baseline and in response to stress. Differential recruitment of glutamate plasticity genes characterized strain differences in gene expression. Metaplasticity of NMDAR-mediated neuronal amygdala signaling was impaired in the strain (DBA/2J) showing a passive anxiety-related response to stress, but not the strain (C57BL/6J) exhibiting an active behavioral response to stress and effective mobilization of plasticity genes in the amygdala. Finally, null mutation of NR2A (but not GluR1) was sufficient to prevent and partially reverse the active, C57BL/6J-like, anxiety-related response to stress, and caused a decrease of BLA dendritic spines. Our findings could have implications for elucidating the neural and genetic basis of individual differences in risk for stress-related neuropsychiatric disease.
Research supported by the NIAAA (Z01-AA000411) and NIMH (Z01-MH002784) Intramural Research Programs, and by NIDA, NIMH, and NIAAA HPG grant P20-DA 21131, NCRR BIRN grant U24 RR021760 (BIRN), and NIAAA INIA grants U01AA13499 and U24AA13513. We are grateful to Dr. Heather Cameron for help with radioimmunoassay.