International Journal of Molecular Sciences Article Acute Stress Induces Different Changes on the Expression of BDNF and trkB in the Mesocorticolimbic System of Two Lines of Rats Differing in Their Response to Stressors Maria Pina Serra 1,† , Francesco Sanna 2,† , Marianna Boi 1 , Laura Poddighe 1 , Lorenzo Secci 1 , Marcello Trucas 1 , Alberto Fernández-Teruel 3 , Maria Giuseppa Corda 2 , Osvaldo Giorgi 2,‡ and Marina Quartu 1, *,‡ 1 Department of Biomedical Sciences, Section of Cytomorphology, University of Cagliari, Cittadella Universitaria di Monserrato, 09042 Monserrato, Italy 2 Department of Life and Environmental Sciences, Section of Pharmaceutical, Pharmacological and Nutraceutical Sciences, University of Cagliari, Cittadella Universitaria di Monserrato, 09042 Monserrato, Italy 3 Medical Psychology Unit, Department of Psychiatry and Forensic Medicine & Institute of Neurosciences, School of Medicine, Autonomous University of Barcelona, Bellaterra, 08193 Barcelona, Spain * Correspondence: quartu@unica.it; Tel.: +39-070-675-4084 † These authors contributed equally to this work (joint first authors). ‡ These authors contributed equally to this work (joint senior authors). Abstract: The present work was undertaken to investigate the effects of acute forced swimming (FS) on the levels of brain-derived neurotrophic factor (BDNF) and tyrosine kinase receptor B (trkB) pro- teins in: the ventral tegmental area (VTA); the nucleus accumbens (Acb) shell and core compartments; Citation: Serra, M.P.; Sanna, F.; Boi, and the anterior cingulate (ACg), prelimbic (PL) and infralimbic (IL) territories of the prefrontal M.; Poddighe, L.; Secci, L.; Trucas, M.; cortex of genetic models of vulnerability (RLA, Roman low-avoidance rats) and resistance (RHA, Fernández-Teruel, A.; Corda, M.G.; Roman high-avoidance rats) to stress-induced depression. We report for the first time that FS induced Giorgi, O.; Quartu, M. Acute Stress Induces Different Changes on the very rapid and distinct changes in the levels of BDNF and trkB proteins in different areas of the Expression of BDNF and trkB in the mesocorticolimbic system of RHA and RLA rats. Thus, (1) in the VTA and Acb core, FS elicited a Mesocorticolimbic System of Two significant increase of both BDNF- and trkB-LI in RHA but not RLA rats, whereas in the Acb shell no Lines of Rats Differing in Their significant changes in BDNF- and trkB-LI across the line and treatment were observed; (2) in RLA rats, Response to Stressors. Int. J. Mol. Sci. the basal levels of BDNF-LI in the IL/PL cortex and of trkB-LI in the ACg cortex were markedly lower 2022, 23, 14995. https://doi.org/ than those of RHA rats; moreover, BDNF- and trkB-LI in the IL/PL and ACg cortex were increased by 10.3390/ijms232314995 FS in RLA rats but decreased in their RHA counterparts. These results provide compelling evidence Academic Editor: Alberto that the genetic background influences the effects of stress on BDNF/trkB signaling and support the Pérez-Mediavilla view that the same stressor may impact differently on the expression of BDNF in discrete brain areas. Received: 4 November 2022 Keywords: stress; depression; forced swimming; BDNF; trkB; Roman high- and low-avoidance rats; Accepted: 25 November 2022 nucleus accumbens; prefrontal cortex; ventral tegmental area Published: 30 November 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. Despite significant advances over the last decades, the etiopathology of depression is still poorly understood. Several hypotheses have been proposed to account for the overall pathophysiological state or core symptoms of depression, based on the dysfunction of Copyright: © 2022 by the authors. monoamine neurotransmission [1], the HPA axis [2] or the neuroimmune system [3]. An- Licensee MDPI, Basel, Switzerland. other hypothesis posits that the susceptibility to depression results from dysfunction of the This article is an open access article mechanisms underlying the plasticity of neuronal networks [4,5] and the abnormal expres- distributed under the terms and sion of genes that encode neurotrophic factors such as the brain-derived neurotrophic factor conditions of the Creative Commons (BDNF) and its tyrosine kinase receptor B (trkB) in neural circuits which are modulated Attribution (CC BY) license (https:// by monoaminergic inputs [5,6]. A key tenet of this hypothesis is that the hippocampal creativecommons.org/licenses/by/ expression of BDNF is negatively modulated by stressors and positively modulated by 4.0/). chronic antidepressant treatments [4]; in addition, more recent studies have shown that Int. J. Mol. Sci. 2022, 23, 14995. https://doi.org/10.3390/ijms232314995 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022, 23, 14995 2 of 23 BDNF/trkB signaling and function in the mesocorticolimbic dopamine (DA) circuits also play a key role in the etiopathology of depression [6]. It is noteworthy, however, that the way by which BDNF is involved in the reactivity to stress in animal models and in the pathogenesis of depression has not yet been precisely established. Thus, the type or severity of stressors may impact differently on the expression of BDNF in discrete limbic brain areas involved in reward- and emotion-related behaviors. For instance, repeated social defeat stress increases both short-term BDNF expression in prefrontal cortical regions and delayed, prolonged BDNF expression in the medial amyg- dala (AMYG) and ventral tegmental area (VTA) [7]. On the other hand, the local infusion of BDNF in the hippocampus (HC) mimics the behavioral effects of antidepressants [8] but elicits depression-like behaviors if infused in the VTA [9]. This finding suggests that BDNF has an antidepressant effect in the HC but, in contrast, plays a pro-depressant role in the VTA, supporting the view that different molecular mechanisms and neuronal pathways are involved in the effects of BDNF in depression. Several animal models have been designed to investigate the impact of the inter- actions between genetic and aversive environmental factors on the neural substrates of depression. One of these models, the Roman High- (RHA) and Low-Avoidance (RLA) rats, were psychogenetically selected for rapid (RHA) vs. extremely poor (RLA) acqui- sition of active avoidance in a shuttle-box [10,11]. Subsequent studies have shown that rather than cognitive processes, emotional reactivity is the most prominent behavioral difference between the two lines, with RLA rats being more fearful/anxious and prone to developing stress-induced depression-like behavior than their RHA counterparts, who behave as proactive copers when exposed to stressors [12–14]. Interestingly, our recent studies using western blot and immunohistochemistry assays have shown that, under baseline conditions, the expression of BDNF and its receptor trkB is significantly lower in the HC of RLA rats compared with their RHA counterparts [15]. This result is in line with previous studies showing that the availability and/or responsiveness to BDNF are reduced in the HC of depressed patients and of experimental animals exposed to acute or chronic stressful conditions. Of note in this context, we have recently shown that, in RLA rats, an acute stressor such as forced swimming (FS) for 15 min elicits a marked decrease in the level of BDNF protein in the ventral HC (vHC) associated with a significant increase in the dorsal HC (dHC), whereas no significant changes in BDNF levels are observed in either hippocampal compartment of RHA rats [16]. Collectively, these results indicate that multiple factors can determine distinct alterations in the levels of BDNF, including the modality of the stressor, the brain area examined and the genetic background of the experimental subjects. Based on the above findings, the present work was undertaken to extend our previous studies in the HC of RLA and RHA rats by investigating the impact of FS on the expression of BDNF and its receptor trkB in the mesocorticolimbic system, using western blot analysis to evaluate their levels and immunohistochemical assays to study their tissue distribution. BDNF-like immunoreactivity (LI) and trkB-LI were measured in limbic areas including the VTA and the shell and core subregions of the nucleus accumbens (Acb), which play a key role in brain reward as well as in the prelimbic/infralimbic (PL/IL) and anterior cingulate (ACg) subregions of the prefrontal cortex (PFC), which is involved in the process of decision-making and shows functional alterations in depressed patients [17,18]. 2. Results 2.1. Behavioral Measures during FS In keeping with previous studies [16,19,20], the latency to the first immobility episode was significantly shorter in RLA vs. RHA rats. Moreover, the cumulative immobility time was more than two-fold longer in RLA vs. RHA rats. On the other hand, compared with their RLA counterparts, RHA rats spent significantly more time displaying active behaviors like swimming, climbing, and diving. Finally, RHA rats excreted fewer fecal boli than RLA rats (Table 1). Int. J. Mol. Sci. 2022, 23, 14995 3 of 23 Table 1. Behaviors displayed by RHA and RLA rats during the forced swimming session 1 . Behavioral Measures RHA RLA Immobility latency (s) 291 ± 45 124 ± 21 * Immobility time (s) 256 ± 19 538 ± 22 *** Swimming (s) 299 ± 28 211 ± 36 * Climbing (s) 286 ± 31 132 ± 12 *** Diving (s) 59 ± 8 19 ± 2 *** Fecal boli (n) 3±2 7±1* 1 Shown are the mean ± SEM of eight rats in each experimental group. * p < 0.05; *** p < 0.001 vs. the RHA group (two-tailed Student’s t test for independent samples). 2.2. Western Blot 2.2.1. BDNF Protein Levels Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEWThe antibody against BDNF recognized one protein band with a relative molecular 4 weight (mw) ∼ = 13 kD (Figures 1–3), in agreement with the reported mw of the monomeric form of the protein [21]. Figure 1. Western blot analysis of brain-derived neurotrophic factor (BDNF) protein levels in the Figure 1.tegmental ventral Western(VTA)blot analysis area of RHAof brain-derived neurotrophic and RLA rats under factor (BDNF) baseline conditions (CTRL) and protein after levels ventral a 15 min session of forced swimming (FS): (A) shown are representative samples from two rats; and a tegmental (VTA) area of RHA and RLA rats under baseline conditions (CTRL) 15 (B) min session ofanalysis densitometric forced ofswimming (FS): (A)band the BDNF/GAPDH shown are representative gray optical samples density (O.D.) ratios. from two ra Columns densitometric and bars denoteanalysis the meanof the BDNF/GAPDH ± S.E.M. of seven–eightband gray rats in eachoptical density experimental (O.D.) group. *: pratios. < 0.05 Column bars denote (Tukey’s postthe hocmean ± S.E.M. test or Sidak’s of seven–eight correction rats for multiple in each experimental group. *: p < 0.05 (Tu comparisons). post hoc test or Sidak’s correction for multiple comparisons). The statistical evaluation with two-way ANOVA (main factors: rat line and treatment, i.e., FS) of the densitometric values of BDNF-LI in tissue homogenates from the VTA revealed significant effects of line and treatment and also a significant line × treatment interaction (all p values < 0.05; Table 2); subsequent pairwise post-hoc contrasts showed that, in RHAs but not RLAs, FS increased the BDNF-LI by 70% over the respective control group (Figure 1). Figure 1. Western blot analysis of brain-derived neurotrophic factor (BDNF) protein levels in the ventral tegmental (VTA) area of RHA and RLA rats under baseline conditions (CTRL) and after a 15 min session of forced swimming (FS): (A) shown are representative samples from two rats; (B) densitometric analysis of the BDNF/GAPDH band gray optical density (O.D.) ratios. Columns and Int. J. Mol. Sci. 2022, 23, 14995 bars denote the mean ± S.E.M. of seven–eight rats in each experimental group. *: p < 0.05 (Tukey’s 4 of 23 post hoc test or Sidak’s correction for multiple comparisons). Figure 2. Western blot analysis of brain-derived neurotrophic factor (BDNF) protein levels in the Figure 2. Western blot analysis of brain-derived neurotrophic factor (BDNF) protein levels in the nucleus nucleus accumbens accumbens shellshell (A,B) (A,B) and and core core subregions subregions (C,D) (C,D) ofof RHA RHA andand RLA RLA rats rats under under baseline baseline t. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW conditions conditions(CTRL) (CTRL)andandafter a 15 after a min session 15 min of forced session swimming of forced (FS). (A,C) swimming (FS).shown (A,C) are representative shown are repre- 5 o samples sentativefrom two from samples rats; and two (B,D) densitometric rats; and analysis of (B,D) densitometric the BDNF/GAPDH analysis of the BDNF/GAPDHband gray optical band gray optical (O.D.) density densityratios. (O.D.)Columns ratios. Columns and bars and denotebars denote the ± S.E.M. mean the mean of± S.E.M. of eight eight rats in eachrats in each ex- experimental perimental group. *: p