Here we tested the hypothesis that stress-induced alterations in Neurogranin (Ng) synthesis and/ or utilization might underlie stress-related depression and anxiety. Rats were randomly divided into five conditions: chronic swim stress (CS), acute swim stress (AS), and three control groups. The CS group was exposed to daily swim stress (5 min/day) for 14 consecutive days, the AS group received a single swim stress, and control groups were maintained in a stress-free condition. Both before and after swim stress, rats were tested for body weight gain, open-field locomotor activity, and saccharine preference. Ng and phospho-Ng (P-Ng) levels in the hippocampus and prefrontal cortex were determined by Western blot analysis. Compared to controls, CS animals displayed significantly decreased body weight gain, ambulation, and saccharine intake, and increased grooming behavior. CS animals had decreased Ng levels in the hippocampus and prefrontal cortex. In CS animals, Ng levels were positively correlated with saccharine intake and ambulation, and inversely correlated with grooming behavior. Compared to controls, AS increased immobility behavior and P-Ng and Ng levels in the hippocampus and prefrontal cortex. In AS animals, immobility behavior was positively correlated with the P-Ng in the prefrontal cortex. Thus, CS and AS produced opposing effects on Ng and P-Ng levels in the hippocampus and prefrontal cortex. Low Ng levels in the hippocampus were associated with anhedonic behavior in CS animals, whereas high P-Ng levels in the prefrontal cortex were associated with anxiety-like behavior in AS animals. Thus, Ng dysfunction might contribute to the neural mechanisms underlying stress-induced depression and anxiety.
One important issue in stress research is to understand how stress signals in the brain result in behavioral disorders. Neurotransmission, kinase-dependent postsynaptic signal transduction, and synaptic plasticity all have been implicated in mediating behavioral responses to stress [
Located postsynaptically, Neurogranin (Ng) is a calcium (Ca2+) calmodulin (CaM) binding protein and protein kinase C (PKC) substrate. Ng is highly expressed in neurons within the prefrontal cortex, hippocampus, and amygdala: brain regions likely involved in stress and emotion responses [
Enhanced phosphorylation of Ng can facilitate N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) [
The role of Ng in stress and stress-related behavioral changes, however, is not clearly defined. In experimental animals, acute sleep deprivation decreased Ng levels in the cerebral cortex, but did not change Ng levels in the hippocampus [
In the present study, we examined the effects of either single or repeated swim stress on behavior and Ng levels in rats. A variety of behavioral parameters (i.e., grooming, exploratory events, immobility, and saccharine intake) were assessed using open field locomotor activity and saccharine preference tests. We hypothesized: 1) CS significantly induces depression-like behaviors, while AS induce anxiety-related behaviors; 2) CS and AS may produce opposite effects on brain levels of Ng and P-Ng in prefrontal cortex and hippocampus, with decrement in the former situation and enhancement in the latter; 3) Changes in Ng and P-Ng levels in the selected brain regions would be strongly associated with the stress-related behavioral changes. The forced swim stress test (i.e., a paradigm of behavioral despair; FST) is a putative animal model of depression. Consequently, the FST is one of the most frequently used methods for investigating antidepressant potential [
Male Sprague-Dawley rats weighing 276 to 338 g at the beginning of the experiment were obtained from Wei Tong Li Hua Lab Animal Center (Beijing, China). Rats were individually housed in cages (25 × 25 × 15 cm 3, L × W × H) in a temperature and humidity controlled (22˚C ± 2˚C; relative humidity of 50%) facility on a 12-h light cycle (lights on 08:00 h). To minimize the stressful effects of handling, rats were acclimated to the laboratory and gently handled daily (3 min/day) for seven days prior to testing. Food and water were provided ad libitum at all times except during the saccharine preference test. Fifty rats were randomly assigned to one of five groups (n = 10/group): chronic swim stress (CS), acute swim stress (AS), control 1 (C1), control 2 (C2), and control 3 (C3). Rats in the CS group were forced to swim individually for 5 min per day for 14 consecutive days. Rats in the AS group were forced to swim individually for 5 min on a single occasion. Rats in the C1 group received behavioral tests but no swimming stress during chronic swimming stress period. Rats in the C2 group received behavioral tests but no swimming stress during acute swimming stress period. Rats in the C3 group remained experimentally naive to control for the possible effects of behavioral testing on Ng levels. Rats in the CS, AS, C1, and C2 groups were tested for open-field locomotor activity and saccharine preference both before (baseline) and after (stress effects) swim stress. All experiments were performed in a sound-shielded room under identical conditions. The International Review Board of the Institute of Psychology, Chinese Academy of Sciences, approved all experimental procedures. All the behavioral procedures in a time line were shown in
According to previously described methods [
Rats were weighed on the 1st and 7th day of handling, and on the 1st, 7th, and 14th day of swimming stress. Body weight after handling (CWT), the first 7th day of swimming stress (SWT7), and 14th day of swimming stress (SWT14) were recorded.
Open field locomotor activity testing was performed on day 8 after handling (baseline, four groups), and on the 1st (AS and C2 group) and 14th day (CS and C1 group) after swimming stress between 08:00 h and 12:00 h in a 180 cm diameter round arena with 50 cm high walls. A dim light (40 W) was used in the open field testing room to decrease the likelihood that the test would be aversive for the rats. Individual rats were placed near the wall of the chamber and the following variables were recorded by an automatic infrared behavioral analysis system (Etho Vision, Noldus Information Technology b.v., Netherlands): ambulation (distance traveled), number of rearing events (standing on the hind legs), number of grooming events (rubbing or licking of the body), number of exploratory events (entering the center zone of the open field), and immobility (motionless posture for 5 seconds). Data were collected for 5 min and analyzed by a computer-based system. At the end of each test, animals were removed and returned to their home cages.
Saccharine preference test were performed on day 8 after handling (baseline, four groups), and on the 1st (AS and C2 group) and 14th day (CS and C1 group) after swimming stress. Rats were water deprived overnight (20:00 h to 08:00 h) the day before saccharine preference testing. From that point onward, rats were exposed for 3 h each day to two bottles: one bottle contained tap water and the other bottle contained a 1% saccharine solution. Preference testing was performed 8 h into the light phase on 4 consecutive days. Total saccharine, water, and fluid (saccharine + water) intake was calculated for the sums of four days of testing. A chronic stress-in- duced reduction of saccharine consumption is considered a measure of anhedonia and we used this definition here. Saccharine and water bottle positions in the cages were alternated daily. At the end of preference testing, rats were returned to ad libitum water access.
Phospho-specific Ng (P-Ng; mouse polyclonal; 1:1000) and Ng primary antibodies (rabbit monoclonal; 1:1000) were obtained from Upstate Biotechnology (Lake Placid, NY) and β-actin primary antibody (mouse monoclonal; 1:1000) was obtained from Sigma-Aldrich (St. Louis, MO). Horseradish peroxidase (HRP)-labeled goat anti- rabbit and HRP-labeled goat anti-mouse secondary antibodies were obtained from Sigma-Aldrich. Nitrocellulose blotting membranes (0.2 μm), polyacrylamide gels, and buffers also were obtained from Sigma-Aldrich. Bicinchoninic acid (BCA) protein assay and enhanced chemiluminescence (ECL) reagents were obtained from Pierce Biotechnology (Rockford, IL). The Gel DocTMXR System and Quantity One 1D analysis software were purchased from Bio-Rad (Hercules, CA).
Immediately after the first swim stress and behavioral tests (day 12 after handling), rats in the AS, C2, and C3 groups were decapitated on day 16 after handling (or the following day after the second saccharine preference test is over), whereas rats in the CS and C1 groups were decapitated after the final swim stress and behavioral tests on day 29 after handling (or the following day after the second saccharine preference test is over). Brains were rapidly removed on ice. The brain was placed in a stainless steel brain matrix and the prefrontal cortex and whole body of hippocampus were removed according to a brain atlas [
Tissue samples were homogenized in 20 volumes of buffer ( 50 mM Tris-Cl, 2 mM EDTA, 2 mM EGTA, 0.05 mM okadaic acid, 1 μM sodium vanidate, 5 μg/ml pepstatin A, and 0.5% Nonidet P-40, pH 7.5) and used for protein and immunoblot analyses [
Proteins were separated by SDS-polyacrylamide gel electrophoresis in a 15% denaturing gel and then transferred to NC using an electroblotting transfer system. Blots were incubated with blocking buffer [10% nonfat dry milk in Tris-buffered saline containing 0.5% Tween-20 (TBST)] for 1 h at room temperature, followed by three 10-min washes in TBST. Blots were then incubated with P-Ng primary antibody 16 - 18 hours at 4˚C, followed by three 10-min washes in TBST. P-Ng labeled blots were then incubated with goat anti-rabbit secondary antibody for 1 h at room temperature. Following secondary application, blots were washed three times for 10 min each in TBST, treated with ECL reagents, and exposed to film. After determination of P-Ng immunoreactivity, blots were stripped of antibodies by a 10-min incubation at 50˚C with stripping buffer (50 mM DTT, 3% SDS, and 62.5 mM Tris-HCl, pH 6.8) [
The intensity of protein bands was determined using Quantity One 1D analysis software. The intensities of P-Ng, Ng, and β-actin all were within the linear range of sensitivity of the scanner. β-actin was used as an internal standard. All P-Ng and Ng blots were normalized to β-actin to correct for small differences in protein loading [
Statistical analyses were performed using the “Statistical Package for Social Sciences” option of SPSS, version 17.0 for Windows (Chicago, IL). All data are presented as mean values ± S.E.M. The control and swim stress groups were compared using the Mann-Whitney U test for 2 samples and the Kruskal-Wallis one-way ANOVA for k samples. The relationship of proteins and behavioral responses was determined using Pearson correlation analysis. The level of statistical significance was set at p < 0.05.
Body weight did not differ significantly between control and stress groups on the 7th day of handling; after the 1st day of swimming stress, there are no significant differences between AS, C2 and C3 group; after seven days of stress, however, CS rats showed less body weights than the C1 group (340.53 ± 10.10 g vs. 365.90 ± 7.26 g , respectively; z = −1.96, p < 0.05). This difference persisted after 14 days of stress, when body weights of CS rats were markedly less than those of the C1 group (333.47 ± 9.37 g vs. 376.53 ± 9.05 g , respectively; z = −2.77, p < 0.01;
Baseline behavioral parameters (ambulation, number of rearing, number of grooming, number of exploration, and immobility time) were not significantly different between C1, C2, AS and CS group [X2 = 3.51, p > 0.05; X2 = 2.49; p > 0.05; X2 = 1.93, p > 0.05; X2 = 1.40, p > 0.05; X2 = 3.33, p > 0.05] on the 7th day of handling. After the 1st day of swim stress, rats in the AS group showed increased immobility time compared to rats in the C2 group (35.15 ± 14.75 s vs. 3.00 ± 3.00 s, respectively; z = −2.10, p < 0.05;
After 14 days of stress, rats in the CS group showed less ambulation and more grooming behavior compared to rats in the C1 group (ambulation = 2221.86 ± 323.82 cm vs. 3230.57 ± 299.56 cm , respectively; z = −2.208, p < 0.05; number of grooming = 2.60 ± 0.31 vs. 1.33 ± 0.24, respectively; z = −2.66, p < 0.01;
Baseline total sum of saccharine intake (X2 = 2.19; p > 0.05), water intake (X2 = 0.08; p > 0.05) and total fluid intake (X2 = 0.32; p > 0.05) for four days during the test period did not differ significantly between control and stress groups after day 7 of handling. Similarly, saccharine (X2 = −1.50; p > 0.05), water (X2 = −0.23; p > 0.05), and total fluid intake (X2 = −0.57; p > 0.05) was not significantly different between the AS group and C2 group following the first swim stress. After 14 days of stress, rats in the CS group consumed less saccharine than rats in the C1 group (39.47 ± 7.48 vs. 59.48 ± 6.15 g, respectively; z = −2.27, p < 0.05). There were no significant differences in water or total fluid intake between the CS and C1 groups (water = 72.92 ± 7.60 g vs. 68.50 ± 7.75 g, respectively; z = −0.23, p > 0.05; total liquids = 112.39 ± 8.50 g vs. 127.98 ± 6.20 g, respectively; z = −1.36, p > 0.05;
On Western blot, P-Ng was detected as a 20 kDa band, Ng was detected as a 17 kDa band, and β-actin was detected as 42 kDa band [
In the hippocampus and prefrontal cortex, CS rats had less Ng levels than rats in the C1 group (hippocampus: 99.54% ± 7.82% vs. 137.10% ± 6.58%, respectively; z = −2.69, p < 0.01; prefrontal cortex: 74.55% ± 5.92% vs. 143.29% ± 15.31%, respectively; z = −2.68, p < 0.01). However, P-Ng levels were too weak to be detected in the hippocampus and prefrontal cortex of rats in the CS group (
In AS animals, P-Ng in the prefrontal cortex was positively correlated with immobility (r = 0.50, p < 0.05; Fig- ure 11). In CS animals, Ng in the hippocampus was positively correlated with saccharine intake (r = 0.53, p < 0.05) and ambulation (r = 0.57, p < 0.05). An inverse correlation was also found between Ng in the hippocampus and grooming (r = −0.64, p < 0.01;
Environmental factors, such as stress, can impact the neurobehavioral profile of an organism and precipitate a depression-like syndrome. Here we determined the effects of single or repeated forced swim stress on behavioral and neurobiological responses in the rat. Interestingly, we found that chronic and acute forced swim stress produce qualitatively different effects on behavior and brain Ng levels.
As previously reported [
were exposed to 5-min of forced swim repeatedly for 14 days, their body weight gain was markedly less than that of control rats. This finding suggests that CS is a severe stressor and these rats were unable to physiological adapt to the situation. When compared to stress-naive rats, CS rats also demonstrated decreased ambulation and increased grooming in open field and decreased saccharine intake. In rats, a low preference for 1% saccharine, defined as hedonic deficit, is analogous to the core symptom of major depression in humans: namely, lack of pleasure [
In rats, exploratory behavior in a novel environment is considered a stress-coping behavior [
Compared to stress-naive animals, a single forced swim stress experience increased rats’ immobility in the open field test; this could be interpreted as a passive coping style [
Repeated exposure to swim stress for 14 days produced a marked reduction in Ng levels in the hippocampus and prefrontal cortex. Changes in Ng levels following repeated stress is in accordance with a previous study using chronic restraint stress [
P-Ng could not be detected in our CS animals. Qi and colleagues [
Interestingly, AS produced the opposite effect of CS on Ng levels. Compared to stress-naive animals, AS increased Ng and P-Ng levels in the hippocampus and prefrontal cortex. These findings are in accordance with a study by Shen et al. [
The exact reasons for differences in Ng levels between CS and AS animals are unclear. Interestingly, differences between the effects of acute and chronic stress also have been reported for physiological responses [
These forms of structural remodeling are mediated by glucocorticoid mechanisms working in concert with excitatory amino acids [
Chronic exposure to swim stress in rats induced depression-like behaviors and decreased Ng levels. Deceased Ng levels in the hippocampus were positively correlated with saccharine intake, suggesting a relationship between Ng with this depression-like behavior. We believe that inhibition of Ng synthesis might be one of the mechanisms underlying stress-related depression. The cascade of signaling events triggered by cAMP has been suggested to play a pivotal role in depression pathogenesis [
Decreased Ng levels were inversely correlated with grooming behavior, also suggesting a relationship between Ng and anxiety-like behavior. Previous studies support decreased Ng levels in anxiety-like behavior: Ng knockout mice exhibit anxiety-like tendencies in the light-dark exploration test and in time spent in center of an open field [
Acute stress (i.e., single forced swim stress) increased immobility and P-Ng and Ng levels. Further, P-Ng in the prefrontal cortex was positively correlated with immobility. Acute foot shock stress was found to increase immobility in open field and this effect could be attenuated by anxiolytic administration [
In animals, several behavioral parameters are considered signs of anxiety: increased immobility, increased grooming, and decreased ambulation [
Here we show that Ng may be the neurobiological substrate mediating the effects of stress on mood. To our knowledge, this is the first report demonstrating a relationship between Ng and stress-induced behavioral disorders. Although a targeted therapy might be difficult to apply clinically, our findings suggest that induction of Ng could play a pivotal role in depression therapy. The role of Ng in depression and anxiety symptomatology, however, remains to be elucidated.
Similar to previous studies, rats in our study were individually housed [
In summary, CS produced depression- and anxiety-like behaviors and decreased Ng levels in the hippocampus and prefrontal cortex. Ng levels in the hippocampus were correlated with both depression- and anxiety-like behaviors. AS induced anxiety-like behavior and increased P-Ng and Ng levels in the hippocampus and prefrontal cortex. P-Ng in the prefrontal cortex was correlated with anxiety-like behavior. Our data suggest that CS and AS differentially affect depression- and anxiety-like behaviors and Ng levels in rats. Ng dysfunction might contribute to the neural mechanisms underlying stress-induced depression and anxiety.
This research was supported by the research grants from the National Science Foundation of China (NSF30370482; NSF84201120), and a grant from the innovational project of the Chinese Academy of Sciences (KSCX 2-2-03 ).