Regional Differences in Brain-Derived Neurotrophic Factor Levels and Dendritic Spine Density Confer Resilience to Inescapable Stress

Background: In the learned helplessness (LH) paradigm, approximately 35% of rats are resilient to inescapable stress. Methods: The roles of brain-derived neurotrophic factor (BDNF) and dendritic spine density in the brain regions of LH (susceptible) and non-LH rats (resilient) were examined. Western blot analysis and Golgi staining were performed. Results: BDNF levels in the medial prefrontal cortex, CA3, and dentate gyrus (DG) were significantly lower in the LH group than in the control and non-LH groups, whereas BDNF levels in the nucleus accumbens (NAc) in the LH group but not the non-LH group were significantly higher than those in the control group. Furthermore, spine density in the prelimbic cortex, CA3, and DG was significantly lower in the LH group than in the control and non-LH groups, although spine density in the NAc was significantly higher in the LH group than in the control and non-LH groups. Conclusions: The results suggest that regional differences in BDNF levels and spine density in rat brain may contribute to resilience to inescapable stress.


Introduction
Humans display wide variability in their responses to psychological stress. Accumulating evidence suggests that resilience is mediated by adaptive changes in several neural circuits involving numerous neurotransmitter and molecular pathways (Feder et al., 2009). Although the understanding of the molecular mechanisms underlying resilience can facilitate the development of therapeutic drugs for stress-related mental disorders, including depression, the precise mechanisms underlying stress resilience remain unknown.
Brain-derived neurotrophic factor (BDNF) plays a key role in the pathophysiology of stress-related mental disorders such as depression, and in the therapeutic mechanism of antidepressants (Nestler et al., 2002;Hashimoto et al., 2004, Duman andMonteggia, 2006;Hashimoto, 2010Hashimoto, , 2013Duman and Li, 2012;Lindholm and Castrén, 2014). A single infusion of BDNF into the dentate gyrus (DG) and CA3 pyramidal cell layers of the hippocampus exerted a long-lasting antidepressant effect in rat learned helplessness (LH) models of depression (Shirayama et al., 2002). Furthermore, we reported that inflammation caused a reduction of BDNF levels in the CA3 region and DG of the hippocampus and prefrontal cortex (PFC), whereas inflammation increased BDNF levels in the nucleus accumbens (NAc), resulting in depression-like behaviors in rodents (Zhang et al., 2015). Krishnan et al. (2007) reported that BDNF in the ventral tegmental area (VTA)-NAc pathway plays a crucial role in resilience to social defeat stress. Moreover, studies using post-mortem brain samples uncovered increased BDNF levels in the NAc and decreased BDNF levels in the hippocampi of depressed patients (Dwivedi et al., 2003;Karege et al., 2005;Krishnan et al., 2007). Taken together, BDNF acts within the VTA-NAc pathway, inducing a depression-like phenotype (Nestler and Carlezon, 2006;Krishnan et al., 2007;Zhang et al., 2015), whereas it produces antidepressant-like effects in the hippocampus and PFC (Nestler et al., 2002;Shirayama et al., 2002;Duman and Monteggia, 2006;Zhang et al., 2015). However, there are no reports related to the relationship between BDNF levels in various brain regions and stress resilience in animal models of depression.
In the present study, we examined the role of BDNF in stress resilience using a rat LH model of depression. Furthermore, we examined whether dendritic spine density in the brain regions of LH (susceptible) and non-LH (resilient) rats were altered, because changes in dendritic spine density in the PFC and hippocampus are believed to contribute to the neurobiology of depression (McEwen, 2007).

Animals
Male Sprague-Dawley rats (200-230 g; 7 weeks old, Charles River Japan, Co.) were used. The animals were housed under a 12 h light/dark cycle with free access to food and water. The procedures of this animal experiment were approved by the Chiba University Institutional Animal Care and Use Committee.

Stress Paradigm (LH Model)
The LH paradigm was created as reported previously (Shirayama et al., 2002;Muneoka et al., 2013). To create the LH paradigm, rats are initially exposed to uncontrollable stress. When the rat is later placed in a situation in which shock is controllable (escapable), it both fails to acquire the escape responses and often makes no effort to escape the shock. LH behavioral tests were performed using the Gemini Avoidance System (San Diego Instruments). This apparatus was divided into two compartments by a retractable door. On days 1 and 2, rats were subjected to 30 inescapable electric foot shocks (0.65 mA, 30 s duration, at random intervals averaging 18-42 s; Figure 1A). On day 3, a two-way conditioned avoidance test was performed as a post-shock test to determine whether the rats would exhibit the predicted escape deficits ( Figure 1A). This screening session consisted of 30 trials in which electric foot shocks (0.65 mA, 6 s duration, at random intervals averaging 30 s) were preceded by a 3 s conditioned stimulus tone that remained active until the shock was terminated. The numbers of escape failures and the latency to escape in each trial were recorded by the Gemini Avoidance System. Rats with more than 25 escape failures in the 30 trials were regarded as having achieved the criterion for depression (susceptible). Approximately 65% of the rats met this criterion. Rats with fewer than 25 failures did not meet the criterion and were defined as non-LH rats (resilient; Muneoka et al., 2013). On day 8, animals were decapitated ( Figure 1A). The left and right brain hemispheres were used for Western blot analysis and Golgi staining, respectively.

Western Blot Analysis
The medial PFC (mPFC), orbitofrontal cortex (OFC), NAc, striatum, CA1 and CA3 regions, and DG of the hippocampus were rapidly dissected on ice and stored at −80°C until biochemical analysis. Protein concentrations were determined using a bicinchoninic acid method assay kit (Bio-Rad). Next, samples were centrifuged at 3000 × g at 4°C for 5 min to obtain the supernatants. Protein was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 10% minigels (Mini-PROTEAN TGX Precast Gel; Bio-Rad). The proteins were then transferred onto polyvinylidene difluoride membranes using a Trans Blot Mini Cell (Bio-Rad). After blocking with 2% bovine serum albumin in phosphate buffer saline + 0.1% Tween-20 for 1 h at room temperature, membranes were incubated with rat anti-BDNF (H-117, 1:200; Santa Cruz Biotechnology, Inc.) overnight at 4°C. Subsequently, membranes were incubated for 1 h at room temperature with a secondary antibody. Bands were detected using enhanced chemiluminescence plus the Western Blotting Detection system (GE Healthcare Bioscience). Images were captured with a Fuji LAS3000-mini imaging system (Fujifilm) and then the band intensity was analyzed.

Golgi Staining
Golgi staining was performed using the FD Rapid GolgiStain Kit (FD Neuro Technologies, Inc.) according to our previous study (Zhang et al., 2015). Briefly, rats were deeply anesthetized using CO 2 vapor from dry ice, and the left brain hemisphere was removed from the skull and immersed in the impregnation solution, consisting of equal volumes of Solutions A and B, for 2 weeks in the dark. Then, brain tissues were transferred to Solution C and stored in fresh solution at 4°C for 1 week. Coronal brain sections (100 μm thickness) were cut on a cryostat (3050S; Leica Microsystems AG) with the chamber temperature set at −20°C. Each section was mounted in Solution C on saline-coated microscope slides and then dried naturally at room temperature. Dendrites within the prelimbic cortex, infralimbic cortex, OFC, NAc (shell and core), striatum, CA1 and CA3 regions, and DG of the hippocampus were imaged using a 100× objective with a Keyence BZ-9000 Generation II microscope. The atlas of the rat brain given by Paxinos and Watson (1998) was used to determine these brain regions. For spine density measurements, all clearly evaluable areas containing 50-100 μm of secondary dendrites from each imaged neuron were used. We chose an area approximately 200 μm from the soma. Three neurons per section and three sections per animal were analyzed.

Statistical Analysis
The data have been presented as the mean ± standard error of the mean. Analysis was performed using PASW Statistics 20 (formerly SPSS statistics; SPSS). Comparisons between groups were performed by a one-way analysis of variance (ANOVA), followed by a post hoc least significant difference test. A value of p < 0.05 was considered statistically significant. Post hoc analyses demonstrated that the BDNF levels in the mPFC in the LH group were significantly lower than those in the control (p < 0.001) and non-LH groups (p < 0.01) and that the BDNF levels in the mPFC were also significantly lower in the non-LH group (p < 0.05) than in the control group ( Figure 1B). BDNF levels in the OFC were significantly lower in the LH group (p < 0.05) than in the control group, whereas BDNF levels in the OFC were not significantly different between the non-LH and control groups ( Figure 1C). In contrast, BDNF levels in the NAc were significantly higher (p < 0.05) in the LH group than in the control group, whereas BDNF levels in the NAc in the non-LH group were not altered ( Figure 1D). Furthermore, there were no differences in BDNF levels in the striatum and CA1 region among protein in the mPFC (medial prefrontal cortex), OFC (orbitofrontal cortex), NAc (nucleus accumbens), striatum, CA1, CA3, and DG (dentate gyrus) of hippocampi in the control (n = 6), LH (n = 7), and non-LH (n = 7) groups was performed. Data are shown as mean ± standard error of the mean. *p < 0.05, ***p < 0.001 compared to control group. # p < 0.05, ## p < 0.01 compared to LH group. the three groups ( Figure 1E and F). BDNF levels in the CA3 region and DG were significantly lower in the LH group (p < 0.05) than in the control and non-LH groups ( Figure 1G and H). Notably, the levels of BDNF in the DG were significantly higher in the non-LH group (p < 0.05) than in the control group ( Figure 1H).

Spine Density in the Brain Regions of LH and Non-LH Rats
One-way ANOVA was performed to analyze the results of spine density in the prelimbic cortex [F (2, 20) = 14.35, p = 0.0001], Figure 2. Spine densities in the brain regions of control, learned helplessness (LH), and non-LH groups. Golgi staining in the rat brains of control (n = 6), LH (n = 7), and non-LH (n= 7) groups was performed. Spine density in the prelimbic cortex, infralimbic cortex, OFC (orbitofrontal cortex), nucleus accumbens (NAc)-shell, NAc-core, striatum, CA1, CA3, and DG (dentate gyrus) of the hippocampus was measured. Data are shown as mean ± standard error of the mean. *p < 0.05, **p < 0.01, ***p < 0.001 compared to control group. # p < 0.05, ## p < 0.01 compared to LH group. The bar is 10 μm. infralimbic cortex [F (2, 18)  Post hoc analyses demonstrated that the spine density in the prelimbic cortex was significantly lower in the LH group than in the control (p < 0.001) and non-LH groups (p < 0.05) and that spine density in the prelimbic cortex group was also significantly lower in the non-LH group (p < 0.05) than in the control group (Figure 2A). The spine density in the infralimbic cortex in the LH group was not different from that of the control group, but spine density in the infralimbic cortex was significantly higher in the non-LH group (p < 0.05) than in the control group and LH group ( Figure 2B). Spine density in the OFC was significantly lower in the LH (p < 0.01) and non-LH groups (p < 0.05) than in the control group ( Figure 2C). Conversely, the spine density in the NAc (shell and core) was significantly higher in the LH group (p < 0.01) than in the control group, but was not altered in the non-LH group ( Figure 2D and E). Furthermore, there were no differences in the spine density of the striatum among the three groups ( Figure 2F). The spine density in the CA1 region was significantly lower in the non-LH group than in the control (p < 0.01) and LH groups (p < 0.05), although the spine density in the CA1 region in the LH group was not altered ( Figure 2G). The spine density in the CA3 region and DG of LH rats was significantly (p < 0.05) lower than in the control and non-LH group ( Figure 2H and I).

Discussion
In this study, we observed that BDNF levels in the mPFC, CA3 region, and DG of LH rats were significantly lower than those in control and non-LH rats, and that BDNF levels in the NAc of LH rats but not non-LH rats were significantly higher than those in control rats. Previously, Shirayama et al. (2002) reported that direct infusion of BDNF into the CA3 region and DG but not the CA1 region of the hippocampus resulted in antidepressant effects in the LH rat, supporting our findings of decreased BDNF levels in the hippocampi of LH rats. In contrast, BDNF levels in the DG but not in the CA3 region in non-LH rats were higher than those in control rats. BDNF in the DG plays an important role in modulating stress-induced dendritic changes and depression-like behaviors (Duman and Li, 2012;Zhang et al., 2015). In addition, Taliaz et al. (2012) reported that hippocampal BDNF expression plays a critical role in resilience to chronic mild stress. Although the reasons underlying higher BDNF levels in the DG of non-LH rats are currently unknown, it is possible that increased BDNF levels in the DG may partly contribute to stress resilience. The expression of BDNF mRNA and protein in brain regions is dependent on the time after chronic stress exposure (Marmigere et al., 2003;Naert et al., 2011). Therefore, BDNF levels and spine density in brain regions immediately after inescapable electric foot shock and with a longer recovery should be studied.
The VTA-NAc pathway and BDNF in the NAc play a key role in the pathophysiology of depression (Nestler and Carlezon, 2006;Shirayama and Chaki, 2006;Krishnan et al., 2007;Zhang et al., 2015). Recently, we reported that increased BDNF levels in the NAc may be implicated in inflammation-induced depression-like behavior and that the TrkB antagonist ANA-12 exerted antidepressant effects in this model (Zhang et al., 2015). In this study, we observed increased BDNF levels and spine densities in the NAcs of LH rats but not in those of non-LH rats. Therefore, it appears that a lack of changes in BDNF levels and spine densities in the NAc may contribute to stress resilience. Because of the key role of BDNF-TrkB signaling in depression (Nestler et al., 2002;Hashimoto et al., 2004;Duman and Monteggia, 2006;Hashimoto, 2010Hashimoto, , 2013Duman and Li, 2012;Lindholm and Castrén, 2014), regional differences in the alterations of BDNF levels in LH and non-LH rats may also contribute to resilience to inescapable stress.
Changes in dendritic length and spine density in the PFC and hippocampus contribute to the neurobiology of stress-related depression (McEwen, 2007;Magariños et al., 2011;Duman and Li, 2012). On tracking dendritic morphology, we observed differential changes in spine density in the mPFC (prelimbic cortex and infralimbic cortex), hippocampus (CA1 region, CA3 region, and DG), and NAc (shell and core) of LH and non-LH rats. Although the reasons underlying the differential regulation of spine densities in the two regions of the mPFC is currently unknown, prelimbic and infralimbic regions may play a role in the susceptibility and resilience to stress, respectively. The alterations in spine densities in the prelimbic cortex, CA3 region, DG, and NAc of LH rats observed in this study are similar to the findings observed in rodents with unpredictable chronic mild stress  and those with inflammation-induced depression (Zhang et al., 2015). In contrast, spine densities in the NAc (shell and core) and hippocampus (CA3 region and DG) were not altered in the non-LH rats. A two-photon imaging study revealed that BDNF and protein synthesis are crucial to the structural plasticity of single dendritic spines (Tanaka et al., 2008), thus linking changes in BDNF levels in the mPFC, CA3 region, DG, and NAc with altered spine densities in these regions. In addition, we observed significant correlations between BDNF levels and spine density in the DG (p = 0.044) and NAc-core (p = 0.002) of all three groups (data not shown). Because of the role of BDNF in synaptogenesis (Magariños et al., 2011;Duman and Li, 2012), the relationship between alterations in BDNF levels and alterations in spine morphology after inescapable electric foot shock are of great interest.
In conclusion, the results of this study suggest that differential alterations in BDNF levels in brain regions, including the mPFC, CA3 region, DG, and NAc, may contribute to resilience versus susceptibility in rats subjected to inescapable electric foot shock.