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Keywords:

  • BDNF;
  • behavior;
  • immune;
  • metabolism;
  • social defeat;
  • stress;
  • TrkB

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

Accumulating evidences underlie the importance of the interplay between environmental and genetic factors in contributing to the risk to develop mental illness. Brain-derived neurotrophic factor (BDNF) and its Tyrosine receptor kinase B (TrkB) receptor play a fundamental contribution to brain development and plastic adaptations to life events. In the present study, the potential for the BDNF/TrkB contribution in increasing vulnerability to negative social experiences was assessed by subjecting TrkB.T1 overexpressing mice to a chronic social defeat model. TrkB.T1 mice overexpress the dominant-negative truncated splice variant of TrkB receptor leading to decreased BDNF signaling. After repeated social defeat, mice were assessed in a longitudinal study for behavioral, physiological, endocrine and immune responses potentially related to psychiatric endophenotypes. TrkB.T1 overexpression corresponded to smaller changes in metabolic parameters such as body weight, food intake, feed efficiency and peripheral ghrelin levels compared with wild-type (wt) littermates following social defeat. Interestingly, 4 weeks after the last defeat, TrkB.T1 overexpressing mice exhibited more consistent social avoidance effects than what observed in wt subjects. Finally, previously unreported effects of TrkB mutations could be observed on lymphoid organ weight and on peripheral immune biomarker levels, such as interleukin-1α and regulated on activation, normal, T-cell expressed, and secreted (RANTES), thus suggesting a systemic role of BDNF signaling in immune function. In conclusion, the present data support a contribution of TrkB to stress vulnerability that, given the established role of TrkB in the response to antidepressant treatment, calls for further studies addressing the link between stress susceptibility and variability in drug efficacy.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin extensively involved in synaptic plasticity and in dynamic phenomena occurring in the brain such as neuronal maturation, dendritic remodeling and formation of synaptic contacts (Schinder & Poo 2000). Binding of BDNF to its high affinity receptor TrkB initiates a cascade of events leading to modulation of three major intracellular signaling pathways, the phosphoinosite-3-kinase/Akt, the phospholipase C and the extracellular signal-regulated kinase–mitogen-activated kinase pathways (Shaltiel et al. 2007). These events are directly involved in the mode of action of psychotropic drugs (Chen & Manji 2006; Picchini et al. 2004; Rantamäki et al. 2007), suggesting that different psychiatric conditions may derive from impairments in BDNF/TrkB signaling. An impressive amount of data supports this hypothesis: decreased BDNF and TrkB expression has been found in suicide subjects (Dwivedi et al. 2003); a functional single nucleotide BDNF polymorphism in humans has been associated to different mental disorders (Gratacos et al. 2007) and to vulnerability to the depressogenic effects of early life stress (Gatt et al. 2009); BDNF has been shown to play a major role in the mood-improving actions of antidepressants (Chen et al. 2001; Nibuya et al. 1995); finally, TrkB receptors need to be present in hippocampal neural progenitor cells for the neurogenic and behavioral actions of antidepressant treatments (Li et al. 2008).

To assess the functional role of BDNF/TrkB signaling in phenotypes related to mood disturbances, TrkB.T1 dominant-negative overexpressing mice showing reduced TrkB brain signaling (Saarelainen et al. 2000, 2003) were tested in a chronic social defeat model. The social defeat model is particularly appropriate due to the essential role of central BDNF in regulating the response to social stress in the mesolimbic dopamine pathway and to its link to sustained hippocampal epigenetic regulation, both of which mediate the long-term neural and behavioral plasticity observed in response to antidepressant treatment (Berton et al. 2006; Tsankova et al. 2006, 2007). This model offers the possibility to assess the importance of a ‘gene × environment' interaction, as stress exposure might unveil a greater vulnerability to adult social stress in TrkB.T1 mutant mice. Interestingly, using a similar social stress model, increased stress susceptibility was observed in mice carrying a partial genetic deficiency in the serotonin transporter (SERT) (Bartolomucci et al. 2010). In line with these findings, clinical data from human subjects suggest a link between a functional polymorphism in the promoter region of SERT and increased risk of developing depression following life stress experience (Caspi et al. 2003). A common single gene polymorphism (val66met) interfering with BDNF activity-dependent secretion has been described (Egan et al. 2003) and is considered potentially relevant for susceptibility to psychiatric disorders (Gratacos et al. 2007). Interestingly, for the same polymorphism an interaction between early life stress and cognitive and brain structure parameters was shown, leading to more severe depressive symptoms (Gatt et al. 2009). Therefore, the TrkB.T1 mice were chronically defeated to evaluate the potential increased vulnerability of deficient TrkB signaling to social stress in behavioral, physiological, endocrine and immune parameters relevant to psychiatric endophenotypes.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

Animals

The generation and characterization of mice carrying TrkB.T1 overexpression under the neuron-specific Thy.1 promoter (TrkB.T1 mice) has been previously described by Saarelainen et al. (2000).

The full-length TrkB.T1 complementary DNA was tagged N-terminally with an eight amino acid FLAG peptide as previously described (Haapasalo et al. 1999) and inserted into murine Thy 1.2 expression cassette, which directs transgene expression to postnatal neurons (Aigner et al. 1995; Ingraham & Evans 1986). Transgenic mice were generated by pronucleus injection of this construct into embryos from C57BL/6J (Charles River BL/6J - JAX Strain: stock number 000664; Charles River, L’Arbresle Cedex, France) female mated with C57BL/6J males. Heterozygous descendants were repeatedly backcrossed onto the C57BL/6J genetic background for 10 generations. Heterozygous subjects and their respective littermates were used in all studies.

To genotype the mice, genomic DNA was extracted from tail tips using a mouse tail extraction kit (Tepnel Research Products and Services, Manchester, UK) according to the manufacturer's instructions. The DNA was used in polymerase chain reactions (PCRs) using standard reagents (Qiagen, Crawley, UK) and gene-specific primers (5′-CGACTACAAAGACGACGATGAC-3′ and 5′-GTTCTCTGGGTCAATGCTG-3′). Thirty cycles of 94°C (30 seconds), 68°C (30 seconds) and 72°C (30 seconds) were used to generate amplicons of 134 bp that were resolved on a 2% agarose gel. Heterozygous and wild-type (wt) offspring were weaned at postnatal day 21, segregated and group-housed by genotype and sex (3–5/cage).

Adult (∼2 months old and weighing 18–20 g at the beginning of the experiments) heterozygous male mice (TrkB.T1, n = 31) and their wt (n = 30) littermates were used in all of the experiments. Mice were housed in 42.5 × 26.6 × 18.5 cm polycarbonate cages under constant temperature (21 ± 2°C) and a 12 h/12 h light/dark cycle (dark phase: 1800 h–0600 h). Food and water were available ad libitum.

All experimental procedures were carried out in accordance with Italian law (Legislative Decree no. 116, 27 January 1992), which acknowledges the European Directive 86/609/EEC and were fully compliant with the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) and GlaxoSmithKline policy on the care and use of laboratory animals and codes of practice.

General experimental design

A baseline phenotypization was carried out in 11 TrkB.T1 and 10 wt mice. For the social stress experiment, adult male mice were subjected to a repeated social defeat procedure (defeated n = 10/genotype; control n = 10/genotype ), followed by a long-term assessment of behavioral, physiological and biochemical responses relevant to stress and depressive/anxiety-like states.

Baseline phenotypization experiment

Mice were housed in the Comprehensive Lab Animal Monitoring System (CLAMS; Columbus Instruments, Columbus, OH, USA) that consists of eight individual live-in cages for automated, noninvasive data collection. Animals were allowed to acclimatize for 8 h before starting data collection during the following 60 h (two light and three dark periods). Body weights were also determined immediately before and after testing.

Calorimetric monitoring

Each cage is an indirect open circuit calorimeter that provides measures of oxygen consumption and carbon dioxide production. The system compared oxygen and carbon dioxide gas concentrations by volume at the inlet and outlet ports of the cage chamber through which ambient air flows at a constant rate (0.60 l/min). The difference in concentration between the two ports and the flow information was used to calculate oxygen consumption (ml/kg/h), carbon dioxide production (ml/kg/h) and respiratory exchange ratio (RER). Heat production (kcal/h) was also estimated with standard formulas using oxygen consumption and the RER.

Activity

An array of infrared beams (2.5 cm inter-beam distance) surrounded each cage. Ambulatory activity was defined as a movement producing sequential horizontal beam breaks of different beams. Activity was measured continuously and recorded with intervals of 1 h as number of beam breaks.

Social defeat stress experiment

Repeated social defeat procedure

CD-1 male mice (Charles River, Calco, Italy), selected on the basis of their attack latency consistency (shorter than 30 seconds on three consecutive screening tests), were used as aggressive residents. For the social defeat stress, subject mice were introduced into the home-cage (42.5 × 26.6 × 18.5 cm) of an unfamiliar CD-1 resident mouse for 10-min full interaction. During this exposure, all subject mice showed signs of subordination (i.e. sideways or upright submissive postures, withdrawal, fleeing, lying on its back or freezing). The subject mouse (defeated) was then separated from the aggressive resident by introducing into the resident home-cage a perforated plexiglass divider to allow sensory contact. The mice were housed in this way for the next 24 h, with food and water provided ad libitum. This procedure was repeated during 10 consecutive days but always with a new resident mouse. Control mice were housed in pairs, separated by the perforated plexiglass divider and handled daily.

Body weight, food intake and feed efficiency parameters

Animals were weighed 3 days before the start of the experiment to allow a balanced distribution between groups. Body weight and food intake measures were taken at multiple time points during the 10-day social defeat stress procedure (Fig. 1). On experimental days 1–10, mice were weighed immediately before being exposed to the social defeat procedure. Additional body weight measures were taken during the weekly change of the home-cage and at the end of the experimental procedure. Food intake was assessed daily during the social defeat procedure (days 1–10) and daily, Monday to Friday, from experimental day 11 to 40; chow was removed from the food hopper, weighed and replaced. To minimize food spill, only food pellets weighing more than 5 g were used for replacing the amount of chow available in the food hopper.

image

Figure 1. Experimental procedure.

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The feed efficiency index was calculated as total body mass gained (g)/cumulative food intake (g) either at the end of the social defeat phase or the single-housing phase.

Social avoidance test

Four weeks following the end of the social defeat (Fig. 1), defeated (n = 10/strain ) and control (n = 10/strain ) mice were placed individually in a 45 × 45 cm arena with an empty wire-mesh cage (10 × 4.5 cm) located at one end, and their movement was tracked for 2.5 min (‘no aggressor’ phase), followed by 2.5 min in the presence of a confined unfamiliar aggressor, represented by one of the resident CD-1 male mice that was introduced into the wire-mesh cage (‘aggressor’ phase) (Berton et al. 2006). Between the two sessions, the subject mouse was removed from the arena and placed back into its home-cage for approximately 1 min. The procedure was performed under red light and video-recordings were performed using a video-camera equipped with infrared filter. The duration of the subject's presence in the ‘interaction zone’ (defined as the 8-cm wide area surrounding the wire-mesh cage) was obtained using the automated video-tracking system based on the Ethovision XT software (Noldus Information Technology, Wageningen, the Netherlands).

BDNF hippocampal levels, peripheral biomarker sampling and internal organ weight

Two days following the social avoidance test, a time was chosen for animals to normalize biomarkers of immediate stress response after the social avoidance test, mice were killed by rapid decapitation for brain and trunk blood collection between 1000 and 1300 h (Fig. 1). During autopsy, both hippocampi and internal organs such as testis, seminal vesicles, spleen, adrenal glands and thymus were dissected and weighed. Organ weight was analyzed as relative weight (i.e. absolute organ weight/body weight).

Hi ppocampal BDNF protein analysis

Hippocampal BDNF levels were measured using two-site enzyme-linked immunosorbent assay (ELISA) essentially as described in Karpova et al. (2010). Briefly, the samples were homogenized in NP++ buffer [137 mm NaCl, 20 mm Tris, 1% NP-40, 10% Glycerol, 48 mm NaF, 2× complete inhibitor mix (Roche-Applied Science, Indianapolis, IN, USA) and 2 mm Na3VO4], incubated on ice approximately 15 min and cold-centrifugated at 16 000 g for 15 min. The resulting supernatant was briefly acidified (1n HCl, 15 min, room temperature), neutralized (1n HCl) and pipetted (170 µl) together with POD-conjugated BDNF secondary antibodies (30 µl) into Maxisorb® ELISA plate (Thermofischer Scientific, Roskilde, Denmark) pre-blocked and pre-coated with primary BDNF antibodies. Next morning the wells were extensively washed with PBS-T and POD substrate (BM Blue; Roche-Applied Science, Indianapolis, IN, USA) added to the wells according to manufacturer's instructions. The reaction was stopped within 10–15 min by adding 50 µl of 1n H2SO4 and the absorbance was immediately measured with Victor® ELISA plate reader (PerkinElmer, Waltham, MA, USA). BDNF protein levels in the samples were calculated based on BDNF standard curve (R2 = 1.00) and the values were further normalized against the total protein content of samples (measured using Bio-Rad DC kit; Bio-Rad Laboratories, Hercules, CA, USA).

Blood sampling

Trunk blood was collected in Microtainer BD K2EDTA tubes (Becton Dickinson Italia, Milan, Italy) with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and a DPPIV protease inhibitor (Millipore, Billerica, MA, USA). After 10-min centrifugation at 1800 g, 4°C, plasma was collected, split into aliquots and stored at −80°C.

Plasma metabolic hormone, cytokine and chemokine levels

Analytes were measured with two Milliplex kits (Millipore, Billerica, MA, USA) using the Luminex technology in a Bio-Plex instrument (Bio-Rad, Hercules, CA, USA), a technology that simultaneously measures concentrations of multiple analytes. Active amylin, active ghrelin (a-ghrelin), total gastric inhibitory polypeptide (GIP), insulin, leptin, total polypeptide YY (PYY) and pancreatic polypeptide (PP) were determined with the Mouse Gut Hormone Panel kit (Millipore) (Mouse Gut Hormone Panel kit inter-assay precision percentage: <23%; intra-assay precision percentage: <7%; amylin assay sensitivity: 7 pg/ml; a-ghrelin assay sensitivity: 2 pg/ml; GIP assay sensitivity: 1 pg/ml; insulin assay sensitivity: 25 pg/ml; leptin assay sensitivity: 22 pg/ml; PYY assay sensitivity: 16 pg/ml; PP assay sensitivity: 17 pg/ml). Interleukin (IL)-1α, IL-1β, IL-6, IL-12p(40), IL-12p(70), eotaxin and RANTES levels were assessed with the Mouse Cytokine/Chemokine Panel I kit (Millipore) (Mouse Cytokine/Chemokine Panel I kit inter-assay precision percentage: 4.2–21.2%; intra-assay precision percentage: 3–22.6%; assay sensitivity: 3.2 pg/ml).

Statistical analysis

Statistical analyses were conducted using Statistica V8 (Statsoft, Inc. Tulsa, OK, USA). For all data, distribution was checked for satisfying analysis of variance (anova) assumptions and, when appropriate, data were log transformed (ghrelin, GIP, IL-1α, IL-1β, IL-6 and IL-12p70).

Data from the baseline phenotypization experiment were analyzed by one-way anova with genotype (TrkB.T1 and wt) as between-subject variables.

For the social defeat experiment, body weight gain data were analyzed as differences from baseline values. Two-way anova with genotype (TrkB.T1 and wt) and social defeat stress (defeated vs. control) as between-subject variables was performed on the body weight gain at the end of the social defeats (1–10 days), as well as on the cumulative amount of food consumed during the 10-day social defeat. anova for repeated measures, with genotype (TrkB.T1 and wt) and social defeat stress (defeated vs. control) as between-subject variables, and time as within-subject variable (weeks 1–4), was performed on the differences between each of the weekly body weight measurements (1, 2, 3, and 4 weeks) and the value at the end of the social defeat (day 10). Similarly, anova for repeated measures, with genotype (TrkB.T1 and wt) and social defeat stress as between-subject variables, and time as within-subject variable, was performed on the total weekly food intake values, recorded from week 1 to 4 following the end of the social defeat. Significant differences due to main effects were followed by multiple group comparisons using Tukey's HSD test. Feed efficiency data were analyzed by means of two-way anova with genotype (TrkB.T1 and wt) and social defeat stress (defeated vs. control) as between-subject variables, for values recorded both at the end of the social defeat and the single-housing phases. One subject within the control TrkB.T1 group was excluded from the analysis of these parameters due to a technical issue preventing the assessment of food intake and body weight.

Social avoidance data were analyzed by repeated measures anovas with genotype (TrkB.T1 and wt) and social defeat stress (defeated vs. control) as between-subject variables, and test phase (aggressor vs. no aggressor) as within-subject variable, followed by Holm corrected planned comparisons.

Data from internal organs, metabolic hormones and inflammation biomarker levels were analyzed by means of two-way anova, with genotype (TrkB.T1 and wt) and social defeat stress (defeated vs. control) as between-subject variables.

All results are expressed as mean ± standard error of raw data (SEM). For all the data the statistical significance was set at P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

Basal phenotypization experiment

None of the considered parameters varied significantly between TrkB.T1 and wt mice (Table 1).

Table 1.  Basal phenotype
ParameterwtTrkB.T1
  1. Values represent group mean ± SEM recorded during 60 h in CLAMS.

  2. TrkB.T1 = transgenic mice over-expressing TrkB.T1 receptor; wt, wild-type.

Oxygen consumption (ml/kg/h)5.845 ± 0.1695.663 ± 0.167
Carbon dioxide production (ml/kg/h)5.426 ± 0.1055.433 ± 0.159
Respiratory exchange ratio0.961 ± 0.0261.195 ± 0.231
Heat production (kcal/h)0.448 ± 0.1400.428 ± 0.140
Distance traveled (cm)10130.3 ± 951.77571.7 ± 906.2
Initial body weight (g)30.673 ± 0.74329.14 ± 0.616
Final body weight (g)29.100 ± 0.44227.75 ± 0.847

Social defeat stress experiment

Body weight, food intake and feed efficiency parameters

Body weight. At the end of social defeat, in general TrkB.T1 mice gained significantly less body weight compared with wt (F1,31 = 13.96, P < 0.001) while defeated subjects gained significantly more body weight than controls (F1,31 = 33.00, P < 0.001). wt defeated subject body weight gain values were significantly greater than wt controls (P < 0.001) as well as TrkB.T1 defeated mice (P < 0.001); no differences were observed within TrkB.T1 mice (Table 2).

Table 2.  Metabolic parameters
ParameterwtTrkB.T1
ControlDefeatedControlDefeated
  1. Values represent group mean ± SEM. Feed efficiency was calculated as body mass gained (g) / cumulative food intake (g). Measures were taken either at the end of the 10-day social defeat (days 1–10) or following 4 weeks of single housing (days 10–38). N = 8–10/group. Data were analyzed with two-way anova followed by Tukey's HSD post hoc test.

  2. TrkB.T1, transgenic mice over-expressing TrkB.T1 receptor; wt, wild-type mice.

  3. Significant genotype effect (anova).

  4. Significant social defeat stress effect (anova).

  5. §Significant genotype × social defeat stress interaction (anova).

  6. Tukey's HSD post hoc test:

  7. * P < 0.05 vs. control;

  8. ** P < 0.01 vs. control;

  9. ## P < 0.01 vs. wt.

Days 1–10
 Delta body weight†,‡ (g)−0.346 ± 0.1181.51 ± 0.317**−0.495 ± 0.1790.130 ± 0.306##
 Cumulative food intake†,‡ (g)49.980 ± 1.28756.697 ± 1.774*46.768 ± 1.01750.898 ± 3.124
 Feed efficiency†,‡−0.007 ± 0.0020.027 ± 0.006**−0.010 ± 0.004##0.001 ± 0.006##
Days 10–38
 Delta body weight‡,§, (g)1.332 ± 0.232−0.201 ± 0.406**0.474 ± 0.4320.856 ± 0.442
 Cumulative food intake†,‡ (g)102.519 ± 3.347108.783 ± 3.20398.117 ± 2.466104.847 ± 3.519
 Feed efficiency‡,§0.010 ± 0.002−0.003 ± 0.004**0.001 ± 0.0030.001 ± 0.005

At the end of the 4-week single-housing phase, social defeat stress significantly decreased body weight gain (F1,31 = 5.24, P < 0.05) and a significant interaction genotype × social defeat stress was found (F1,31 = 5.24, P < 0.01) because of a significant decrease in wt defeated compared with wt controls (P < 0.01).

Food intake. The total food intake during the 10-day social defeat was significantly decreased in TrkB.T1 compared with wt mice (F1,31 = 8.79, P < 0.01) and significantly increased in defeated compared with control subjects (F1,31 = 12.47, P < 0.001). While no differences were found within TrkB.T1 mice, wt defeated consumed significantly more food than wt controls (P < 0.05) (Table 2).

In general, the total food intake at the end of the single-housing phase was significantly lower in TrkB.T1 compared with wt subjects (F1,31 = 4.99, P < 0.05) and significantly increased in defeated subjects compared with controls (F1,31 = 10.86, P < 0.01).

Feed efficiency. The index value at the end of social defeat was significantly lower in TrkB.T1 compared with wt mice (F1,31 = 13.02, P < 0.01) and significantly higher in defeated compared with control subjects (F1,31 = 27.94, P < 0.001). Within wt, defeated subjects feed efficiency values were significantly higher compared with controls (P < 0.001); wt defeated mice values were also significantly higher than TrkB.T1 defeated mice (P < 0.01) (Table 2).

At the end of 4 weeks following the social defeat, in general defeated mouse feed efficiency values were found to be significantly lower than control (F1,31 = 4.66, P < 0.05) and a significant genotype × social defeat stress interaction was observed (F1,31 = 9.09, P < 0.01); no differences among groups were found except for significantly decreased values in wt defeated compared with wt control mice (P < 0.01).

Social avoidance test

In general, the occupation of the corners opposite to the interaction zone was significantly increased in defeated subjects (F1,32 = 9.14, P < 0.01) and a significant interaction test phase × social defeat stress was detected (F1,32 = 6.62, P < 0.05), due to the significantly longer duration observed in TrkB.T1 defeated subjects compared with respective controls during the ‘aggressor phase’ of the test (P < 0.05) (Fig. 2a).

image

Figure 2. Behavioral outcome of the social avoidance test conducted 4 weeks following the last social defeat in the presence of a confined aggressor. (a) Time spent by experimental subjects in the corners opposite to the cage holding the aggressor, for which significant effects of social defeat stress (F1,32 = 9.14, P < 0.01) and test phase × social defeat stress interaction (F1,32 = 6.62, P < 0.05) were found; (b) time spent by experimental subjects in proximity to the confined aggressor, for which significant effects of social defeat stress (F1,32 = 12.26, P < 0.01) and test phase × social defeat stress × genotype interaction (F1,32 = 4.05, P = 0.05) were found; (c) total distance traveled by experimental subjects, for which a significant effect of genotype was found (F1,32 = 5.59, P < 0.05). Data are represented as group averages ± SEM (histograms) alongside with the respective individual values (points). Data were analyzed with two-way anova followed by Holm corrected planned comparisons: *P < 0.05 and **P < 0.01, TrkB.T1 defeated versus TrkB.T1 control group.

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Coherently, the average time spent by subjects in the interaction zone was significantly increased by the aggressor presence compared with the ‘nonaggressor phase’ of the test (F1,32 = 8.01, P < 0.01–not shown ), and significantly decreased by social defeat stress (F1,32 = 9.01, P < 0.01) (Fig. 2b). Significant interactions between test phase and social defeat stress (F1,32 = 12.26, P < 0.01) and test phase × social defeat stress × genotype (F1,32 = 4.05, P = 0.05) were also found. Specifically, the time spent in the interaction zone during the ‘aggressor phase’ was significantly lower in TrkB.T1 defeated compared with TrkB.T1 control mice (P < 0.01).

The total distance traveled was significantly less during the ‘aggressor’ phase compared with the ‘nonaggressor’ phase (F1,32 = 37.61, P < 0.001–not shown ); overall, TrkB.T1 mice covered a significantly longer distance than wt mice (F1,32 = 5.59, P < 0.05), although no significant differences could be detected at individual group level (Fig. 2c).

BDNF hippocampal levels

TrkB.T1 mice expressed significantly reduced protein levels of BDNF compared with wt littermates in the hippocampus (F1,32 = 9.61, P < 0.01) (Fig. 3). No effect of social defeat stress was highlighted.

image

Figure 3. BDNF protein levels in the hippocampus. Data are represented as group averages ± SEM (histograms). Data were analyzed with two-way ANOVA: **P < 0.01 genotype effect.

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Peripheral biomarkers and internal organ weight

Internal organs. Adrenal gland's size was significantly increased in defeated animals (F1,32 = 7.81, P < 0.01); spleen size was significantly decreased in TrkB.T1 mice (F1,32 = 5.62, P < 0.05); thymus weight tended to be decreased in TrkB.T1 subjects (F1,32 = 3.49, P = 0.07); seminal vesicle weight was significantly diminished by both genotype (F1,32 = 5.76, P < 0.05) and social defeat stress (F1,32 = 6.06, P < 0.05); testicle size tended to be increased in TrkB.T1 mice (F1,32 = 4.05, P = 0.05); abdominal fat amount was significantly increased in TrkB.T1 mice compared with wt (F1,32 = 5.87, P < 0.05) and significantly decreased in defeated subjects compared with controls (F1,32 = 10.04, P < 0.01); a significant interaction between genotype and social defeat stress was also highlighted (F1,32 = 4.67, P < 0.05): defeated wt mice abdominal fat was significantly diminished compared with wt controls (P < 0.01) as well as compared with TrkB.T1 defeated subjects (P < 0.05) (Table 3).

Table 3.  Internal organ relative weight (g/100 g body weight)
 wtTrkB.T1
 ControlDefeatedControlDefeated
  1. Values represent group mean ± SEM. Data were analyzed as relative values over 100 g body weight. Data were analyzed with two-way anova followed by Tukey's HSD post hoc test.

  2. TrkB.T1, transgenic mice over-expressing TrkB.T1 receptor; wt, wild-type mice.

  3. Significant genotype effect (anova).

  4. Significant social defeat stress effect (anova).

  5. §Significant genotype × social defeat stress interaction (anova).

  6. Tukey's HSD post hoc test: **P < 0.01 vs. control;°P < 0.05 vs. wt.

Adrenal glands0.027 ± 0.0020.032 ± 0.0040.030 ± 0.0040.038 ± 0.003
Spleen0.252 ± 0.0090.232 ± 0.0260.230 ± 0.0100.241 ± 0.007
Thymus0.177 ± 0.0150.166 ± 0.0210.160 ± 0.0130.158 ± 0.007
Seminal vesicles†,‡0.976 ± 0.0320.837 ± 0.0970.928 ± 0.0180.828 ± 0.019
Testicles0.835 ± 0.0130.741 ± 0.0810.873 ± 0.0210.847 ± 0.018
Abdominal fat †,‡,§1.384 ± 0.0760.934 ± 0.100**1.402 ± 0.0591.334 ± 0.085°

Plasma metabolic hormone, cytokine, and chemokine levels. Only a limited number of the assessed parameters were altered at the end of the experimental procedure (Table 4). In TrkB.T1 mice as a whole, active ghrelin levels significantly increased (F1,33 = 4.50, P < 0.05), while IL-1α levels significantly decreased (F1,32 = 4.67, P < 0.05) and RANTES levels tended to be reduced (F1,32 = 3.32, P = 0.08).

Table 4.  Plasma hormone levels (pg/ml)
 wtTrkB.T1
 ControlDefeatedControlDefeated
  1. Values represent group mean ± SEM. Data were analyzed with two-way anova.

  2. TrkB.T1, transgenic mice over-expressing TrkB.T1 receptor; wt, wild-type mice.

  3. *Significant genotype effect.

Metabolic hormones
 Amylin71.49 ± 9.5971.53 ± 14.3759.80 ± 12.80104.20 ± 21.03
 a-Ghrelin*20.41 ± 5.5020.21 ± 3.7827.52 ± 6.4029.01 ± 3.67
 GIP50.27 ± 16.8532.60 ± 9.3729.29 ± 11.9737.29 ± 11.98
 Insulin1690.27 ± 240.612353.08 ± 851.391335.76 ± 163.951550.99 ± 416.10
 Leptin1972.03 ± 278.111331.96 ± 140.371547.11 ± 167.641468.98 ± 212.69
 PYY34.67 ± 6.7438.30 ± 9.2228.68 ± 8.2346.24 ± 9.72
 PP25.98 ± 4.7130.02 ± 6.8036.93 ± 11.7720.80 ± 6.46
Cytokines
 IL-1α*28.49 ± 8.5729.40 ± 10.3812.18 ± 5.1111.99 ± 6.55
 IL-1β11.69 ± 5.0911.67 ± 3.9234.04 ± 28.1858.59 ± 15.50
 IL-64.47 ± 0.945.87 ± 1.7274.29 ± 37.1211.80 ± 4.80
 IL-12p4095.92 ± 52.1743.83 ± 7.2747.13 ± 9.3531.60 ± 5.60
 IL-12p7018.41 ± 10.1229.81 ± 15.5841.58 ± 24.3061.43 ± 44.76
 Eotaxin727.09 ± 69.98674.86 ± 48.42685.40 ± 28.96774.33 ± 76.00
 RANTES6.22 ± 0.525.80 ± 0.465.21 ± 0.514.79 ± 0.70

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

In the present study, we characterized the behavioral, physiological, endocrine and immune response to chronic psychosocial stress in mice overexpressing a truncated ‘dominant-negative’ isoform of the BDNF receptor TrkB.T1 that represents a natural splicing variant of the receptor lacking the kinase domain (Saarelainen et al. 2000). TrkB.T1 has been shown to be upregulated in pathological states such as human Alzheimer's disease (Ferrer et al. 1999), is involved in pain processing (Renn et al. 2009) and is promoted by stress and various environmental factors (Jeanneteau et al. 2008). Mutant mice utilized in the present study display an overexpression of TrkB.T1 isoform in the hippocampus, several cortical layers, thalamus and amygdale, leading to a decrease in TrkB signaling in several, but not all (e.g. striatum), areas of the brain (Saarelainen et al. 2000). Importantly, the Thy1 promoter targets the expression of TrkB.T1 transgene to postnatal neurons (expression starts at early postnatal days) (Saarelainen et al. 2000). As a result of this modification, an inhibition in the activation of the TrkB kinase, a decrease in BDNF signaling and in BDNF hippocampal levels (∼60%) was detected in TrkB.t1 mice, whereas no differences from wt could be observed, at least in our experimental conditions, in basal metabolic function and general activity.

Therefore, to analyze the interaction between genotype and environment, TrkB.T1 overexpressing mice were subjected to a chronic social stress procedure. Social defeat effects elicited robust effects on metabolic parameters, more pronouncedly and persistently in wt compared with TrkB.T1 mice that, in turn, exhibited a greater resistance to defeat-induced metabolic alterations. The present results in wt mice are in agreement with the growing data showing obesity inducing adaptations to defeat stress whose underlying mechanisms have only recently begun to be elucidated (Chuang et al. 2010), and that confer further translational relevance to the social defeat model. A mounting literature indicates that obesity is one of the most important environmental risk factors for developing mental disorders (Richardson et al. 2003; Vieweg et al. 2006), and recent epidemiological evidences support the comorbidity between depressive and metabolic disorders (Beydoun & Wang 2009; Chuang et al., 2010; Goldbacher et al., 2009; Golden et al., 2008; Ma & Xiao, 2009). A metabolic role is also played by BDNF and TrkB that are implicated in the neuroendocrine control of mammalian feeding behavior and energy homeostasis (Tsao et al. 2008; Wisse & Schwartz 2003). Mice (Xu et al. 2003) or humans (Yeo et al. 2004) with defective tyrosine kinase receptor TrkB display excessive appetite, reduced energy expenditure and obesity. The expression of BDNF in the ventromedial hypothalamus, an important component of the central nervous system (CNS) energy homeostasis circuitry, is reduced after food deprivation (Kernie et al. 2000; Xu et al. 2003); mice with genetically reduced BDNF expression also develop obesity and hyperphagic behavior (Fox & Byerly 2004; Kernie et al. 2000; Lyons et al. 1999; Unger et al. 2007). Although the mechanism by which TrkB regulates feeding behavior and weight is complex and not well understood, one explanation for the lack of overt hyperphagia and obesity phenotype in TrkB.T1 mice presently reported should take into consideration compensatory changes in CNS regions critical for feeding behavior and body weight homeostasis. Furthermore, the hyperphagic/obese phenotype produced by BDNF deficiency is age-dependent, as its occurrence is reported mostly in mature animals (above 5 months old) (Fox & Byerly 2004; Kernie et al. 2000; Lyons et al. 1999) wherein it has been related to a late reduction in serotonin levels (Rios et al. 2001). Nevertheless, an altered metabolic homeostasis in TrkB.T1 compared with wt mice is supported by the observed increased levels of active ghrelin, consistent with the proposed role of BDNF as a metabotrophin (Gomez-Pinilla et al. 2008). Ghrelin is secreted from the stomach generally in response to energy depletion related to decreased food intake (Ariyasu et al. 2001; Kojima et al. 1999) and, interestingly, it can also bind hippocampal receptors (Guan et al. 1997) with profound effects on hippocampal synaptic plasticity (Diano et al. 2006), thus linking metabolic and cognitive systems. Given the established cognitive deficits of TrkB.T1 mice (Saarelainen et al. 2000) and the altered ghrelin secretion observed in the present experiments, further studies should be performed to identify the mediators involved in the cross-talk between energy homeostasis and high brain functions.

Although the impairment in TrkB signaling induced in either TrkB.T1 and BDNF+/− mice (MacQueen et al. 2001; Monteggia et al. 2004; Saarelainen et al. 2003) does not lead to depressive-like phenotypes in basal conditions, TrkB.T1 defeated mice exhibited a very consistent social avoidance. Differential coping styles are reported following social defeat in mice (Krishnan et al. 2007); coherently, in the present study a dichotomic response was shown within defeated wt mice, although, because of the small sample size, experimental subjects could not be separated into resilient/susceptible subpopulations of meaningful size. On the other hand, a homogeneously aversive response (i.e. diminished time in interaction zone) was observed in defeated TrkB.T1 mice toward the confined aggressor. BDNF signaling has been repeatedly linked to defeat-induced social avoidance and particularly to a susceptible phenotype (Berton et al. 2006; Krishnan et al. 2007). BDNF levels in specific brain areas, such as the nucleus accumbens (NAc), have been linked to the development of susceptible vs. unsusceptible phenotypes. Bilateral intra-NAc infusions of BDNF enhanced susceptibility (Krishnan et al. 2007), whereas a knockdown of BDNF within the ventral tegmental area (VTA) was shown to prevent defeat-induced avoidance (Berton et al. 2006). These data are consistent with a model wherein susceptibility to a defeat-induced avoidant phenotype is caused by upregulation of VTA neuronal activity, which results in increased BDNF signaling within the NAc. The apparent discrepancy with our findings, showing a consistent avoidance induced in TrkB.T1 mice expressing diminished hippocampal BDNF levels, is resolved by taking into consideration the exquisite region-specificity of BDNF functional role in modulating depression-like behaviors (Wang et al. 2008). BDNF increases in hippocampus have been shown to attenuate depression-related phenotypes (Shirayama et al. 2002; Siuciak et al. 1997), whereas hippocampal TrkB.T1 overexpression or BDNF deletion may prevent the efficacy of antidepressant medications (Monteggia et al. 2004; Saarelainen et al. 2003). In agreement with the current results, increased hippocampal levels of BDNF have been recently linked to attenuated defeat-induced social avoidance in mice (Yan et al. 2010). The directionality of these effects and the related region-specificity are shared with other factors implicated in the phenomenology of depression (i.e. cAMP response element binding protein) (Nibuya et al. 1996; Pliakas et al. 2001). Together with the present data, these studies suggest a differential pathophysiology within the VTA-NAc than within the hippocampus.

The well-established consequences of defeat on rodent physiology were confirmed in the present study by persistently enlarged adrenal glands and decreased reproductive organ weight (Raab et al. 1985; Razzoli et al. 2010; Selye 1950; Van Kampen et al. 2002). Several abnormalities were also observed in TrkB.T1 internal organ anatomy, such as spleen and thymus that presented decreased sizes, whose mechanisms are unclear, given the CNS expression of the TrkB.T1 transgene. On the other hand, peripheral changes are observed following experimental disruptions of brain areas characterized by rich neurotrophic activity as in other preclinical models such as the olfactory bulbectomy (Connor et al. 2000; Komori et al. 2002). Similar peripheral alterations are also reported in clinically depressed patients (Weisse 1992), thus further corroborating the existence of a cross-talk between central and peripheral systems in stress-related disorders to explain the observed immunomodulatory effect observed in TrkB.T1 mice. It is noteworthy that BDNF is considered a stress-responsive intercellular messenger modifying hypothalamus–pituitary–adrenal axis activity, as suggested in depressed patients carriers of the Met/Met genotype (Derijk 2009; Schüle et al. 2006) and through its action on stress-related pathways it could exert a peripheral effect on immune function.

An imbalanced immune function due to TrkB.T1 transgene is also supported by the reported immune biomarker results. Only a limited number of the parameters were altered at the end of the experimental procedure, as IL-1α and RANTES levels were found to be decreased in mutant mice. Further studies should be aimed at assessing in a more dynamic fashion the immune system response of TrkB.T1 mice, or after subjecting them to systemic challenge, thus leading to a better understanding of the functional relevance of the highlighted immune alterations.

In conclusion, our results support a role of neurotrophin mediated signaling in contributing to the vulnerability to social stress; furthermore, a complex phenotype depending on the overexpression of TrkB.T1 receptor and the consequent decreased hippocampal BDNF has been highlighted jointly with peripheral alterations in parameters related to the metabolic and immune function that demand further investigation. In consideration of the involvement of BDNF/TrkB pathway in response to the currently available antidepressant treatments, such studies bear the potential to further advance the knowledge of the neuroplastic mechanisms linked to stress vulnerability and ultimately to the risk of psychiatric disorders and their comorbidities.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
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Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

The authors wish to thank Michela Andreoli, Francesco Borgo, and Claudio Righetti for their excellent technical assistance in the conducting of experiments and Dr Francesca Michielin for her valuable discussion with respect to the statistical analyses.