Short-Term Irisin Treatment Enhanced Neurotrophin Expression Differently in the Hippocampus and the Prefrontal Cortex of Young Mice

As a result of physical exercise, muscle releases multiple exerkines, such as “irisin”, which is thought to induce pro-cognitive and antidepressant effects. We recently demonstrated in young healthy mice the mitigation of depressive behaviors induced by consecutive 5 day irisin administration. To understand which molecular mechanisms might be involved in such effect, we here studied, in a group of mice previously submitted to a behavioral test of depression, the gene expression of neurotrophins and cytokines in the hippocampus and prefrontal cortex (PFC), two brain areas frequently investigated in the depression pathogenesis. We found significantly increased mRNA levels of nerve growth factor (NGF) and fibroblast growth factor 2 (FGF-2) in the hippocampus and brain-derived growth factor (BDNF) in the PFC. We did not detect a difference in the mRNA levels of interleukin 6 (IL-6) and IL-1β in both brain regions. Except for BDNF in the PFC, two-way ANOVA analysis did not reveal sex differences in the expression of the tested genes. Overall, our data evidenced a site-specific cerebral modulation of neurotrophins induced by irisin treatment in the hippocampus and the PFC, contributing to the search for new antidepressant treatments targeted at single depressive events with short-term protocols.


Introduction
Physical exercise is widely recognized as a powerful nonpharmacological therapeutic tool by displaying marked health benefits in patients with pathologies involving the cardiovascular, locomotor, respiratory, nervous, and immune systems [1]. Furthermore, irrefutable evidence has proven the effectiveness of regular physical activity in reducing the risk factors associated with various chronic diseases [2]. Several biomolecules released by both muscle and non-muscle tissues in response to physical exercise have been implicated in the molecular pathways underlying the beneficial effects of exercise [3]. Recently, the term "exerkines" has been coined to globally refer to those humoral factors that are directly secreted lative dose used in [24], we administered irisin subcutaneously for 5 days consecutively (100 µg/kg/daily) in young healthy female and male mice, and we investigated the effects of irisin on the depressive-and anxiety-like behaviors, as well as memory performance. Of note, we showed that irisin exerted both a significant antidepressant and an anxiolytic effect without obvious sex differences [26].
Here, we aim to shed light on the molecular pathways underlying the intriguing findings regarding irisin's antidepressant effect by examining the expression of selected genes in cerebral areas considered to be the most implicated in MDD, i.e., the hippocampus and the PFC [27][28][29]. Therefore, in a group of mice already submitted to a behavioral test of depression [26], we examined the expression of some neurotrophic/growth factors associated with the pathogenesis of depressive disorders, such as BDNF, IGF-1, nerve growth factor (NGF), and fibroblast growth factor 2 (FGF-2) [30][31][32][33]. Furthermore, to complete our investigations, we considered irisin's effect on chronic neuroinflammation, a well-known risk factor for depression [19,34]. For this purpose, we evaluated the expression of a selection of pro-and anti-inflammatory cytokines, i.e., IL-6, IL-1β, IL-10, IL-4, and interleukin-1 receptor antagonist (IL-1ra).

Short-Term Irisin Injections Differently Increased the Expression of Neurotrophic/Growth Factors in the Hippocampus and the Prefrontal Cortex (PFC)
In our recent work, we found that once-daily irisin injections (100 µg/kg) for 5 consecutive days decreased the depressive-and anxiety-like behaviors in young healthy female and male mice [26]. In line with these findings, in this study, with the aim of assessing the impact of short-term irisin administration on the expression of the most well-known neutrophic/growth factors associated with depression [30][31][32][33], we evaluated the gene expression of Bdnf, Igf-1, Ngf, and Fgf-2 in hippocampal and PFC tissues. We noticed that irisin injections significantly increased Ngf and Fgf-2 expression in the hippocampus of irisintreated mice (p < 0.05), while no differences were observed for Bdnf and Igf-1 (p = 0.433 and p = 0.554, respectively) ( Figure 1).
The Food and Drug Administration (FDA) suggests the short-term use of new nonselective serotonin reuptake inhibitor (non-SSRI) antidepressants to treat single episodes of depression that often occur in children and adolescent [25]. Thus, in our previous work, we proposed a short-term irisin treatment schedule. Maintaining the same total cumulative dose used in [24], we administered irisin subcutaneously for 5 days consecutively (100 μg/kg/daily) in young healthy female and male mice, and we investigated the effects of irisin on the depressive-and anxiety-like behaviors, as well as memory performance. Of note, we showed that irisin exerted both a significant antidepressant and an anxiolytic effect without obvious sex differences [26].
Here, we aim to shed light on the molecular pathways underlying the intriguing findings regarding irisin's antidepressant effect by examining the expression of selected genes in cerebral areas considered to be the most implicated in MDD, i.e., the hippocampus and the PFC [27][28][29]. Therefore, in a group of mice already submitted to a behavioral test of depression [26], we examined the expression of some neurotrophic/growth factors associated with the pathogenesis of depressive disorders, such as BDNF, IGF-1, nerve growth factor (NGF), and fibroblast growth factor 2 (FGF-2) [30][31][32][33]. Furthermore, to complete our investigations, we considered irisin's effect on chronic neuroinflammation, a wellknown risk factor for depression [19,34]. For this purpose, we evaluated the expression of a selection of pro-and anti-inflammatory cytokines, i.e., IL-6, IL-1β, IL-10, IL-4, and interleukin-1 receptor antagonist (IL-1ra).

Short-Term Irisin Injections Differently Increased the Expression of Neurotrophic/Growth Factors in the Hippocampus and the Prefrontal Cortex (PFC)
In our recent work, we found that once-daily irisin injections (100 μg/kg) for 5 consecutive days decreased the depressive-and anxiety-like behaviors in young healthy female and male mice [26]. In line with these findings, in this study, with the aim of assessing the impact of short-term irisin administration on the expression of the most wellknown neutrophic/growth factors associated with depression [30][31][32][33], we evaluated the gene expression of Bdnf, Igf-1, Ngf, and Fgf-2 in hippocampal and PFC tissues. We noticed that irisin injections significantly increased Ngf and Fgf-2 expression in the hippocampus of irisin-treated mice (p < 0.05), while no differences were observed for Bdnf and Igf-1 (p = 0.433 and p = 0.554, respectively) ( Figure 1).  Igf-1 (d) was assessed by qRT-PCR in female (n = 7) and male (n = 5) mice for each treatment group. The Shapiro-Wilk test was followed by Student's t test for all data except for Ngf ; * p < 0.05. Data are presented as box-and-whisker plots with median and interquartile ranges, from max to min, with all data points shown.
In PFC tissue, the short-term irisin injections modulated the expression of the abovementioned genes in a different manner compared to the hippocampus. Indeed, we noticed a considerable increase in BDNF mRNA levels (p < 0.0001) and a value near to significance for Fgf-2 (p = 0.059) in irisin-treated mice compared to controls. We found no differences for either Ngf or Igf-1 (p = 0.765 and p = 0.643, respectively) ( Figure 2).
Igf-1 (d) was assessed by qRT-PCR in female (n = 7) and male (n = 5) mice for each treatment group. The Shapiro-Wilk test was followed by Student's t test for all data except for Ngf; * p < 0.05. Data are presented as box-and-whisker plots with median and interquartile ranges, from max to min, with all data points shown.
In PFC tissue, the short-term irisin injections modulated the expression of the abovementioned genes in a different manner compared to the hippocampus. Indeed, we noticed a considerable increase in BDNF mRNA levels (p < 0.0001) and a value near to significance for Fgf-2 (p = 0.059) in irisin-treated mice compared to controls. We found no differences for either Ngf or Igf-1 (p = 0.765 and p = 0.643, respectively) ( Figure 2). was assessed by qRT-PCR in female (n = 7) and male (n = 5) mice for each treatment group. The Shapiro-Wilk test was followed by Student's t-test for all data; **** p < 0.0001. Data are presented as box-and-whisker plots with median and interquartile ranges, from max to min, with all data points shown.

Short-Term Irisin Administration Did Not Affect the Expression of Il-6 and Il-1β in Either the Hippocampus or the PFC
Extensive clinical studies have shown the role of the immune system in depression; in fact, immune dysfunctions that alter the balance between proinflammatory and antiinflammatory cytokines have been related to depressive disorders [35]. In accordance with this "cytokine hypothesis of depression", we examined how short-term irisin injections may affect the cytokine profile in the hippocampus and the PFC by analyzing the expression of pro-(IL-6 and IL-1β) and anti-inflammatory (IL-4, IL-10, and IL-1ra) cytokines majorly involved in MDD. In the hippocampus, we observed no differences for the IL-6 and IL-1β mRNA levels between the irisin-treated mice and controls (p = 0.476 and p = 0.549, respectively) ( Figure 3). Igf-1 (d) was assessed by qRT-PCR in female (n = 7) and male (n = 5) mice for each treatment group. The Shapiro-Wilk test was followed by Student's t-test for all data; **** p < 0.0001. Data are presented as box-and-whisker plots with median and interquartile ranges, from max to min, with all data points shown.

Short-Term Irisin Administration Did Not Affect the Expression of Il-6 and Il-1β in Either the Hippocampus or the PFC
Extensive clinical studies have shown the role of the immune system in depression; in fact, immune dysfunctions that alter the balance between proinflammatory and antiinflammatory cytokines have been related to depressive disorders [35]. In accordance with this "cytokine hypothesis of depression", we examined how short-term irisin injections may affect the cytokine profile in the hippocampus and the PFC by analyzing the expression of pro-(IL-6 and IL-1β) and anti-inflammatory (IL-4, IL-10, and IL-1ra) cytokines majorly involved in MDD. In the hippocampus, we observed no differences for the IL-6 and IL-1β mRNA levels between the irisin-treated mice and controls (p = 0.476 and p = 0.549, respectively) ( Figure 3).
In the PFC, we found a slight increase in Il-6 expression in irisin-treated mice that nonetheless did not reach statistical significance (p = 0.081), while no differences were noticed for Il-1β (p = 0.409) ( Figure 4). The gene expression of Il-6 (a), and Il-1β (b) was evaluated by qRT-PCR in female (n = 7) and male (n = 5) mice for each treatment group. The Shapiro-Wilk test was followed by Student's t test for Il-6 data analysis and by the Mann-Whitney test for Il-1β. Data are presented as box-and-whisker plots with median and interquartile ranges, from max to min, with all data points shown.
In the PFC, we found a slight increase in Il-6 expression in irisin-treated mice that nonetheless did not reach statistical significance (p = 0.081), while no differences were noticed for Il-1β (p = 0.409) ( Figure 4). The gene expression of Il-6 (a) and Il-1β (b) was evaluated by qRT-PCR in female (n = 7) and male (n = 5) mice for each treatment group. The Shapiro-Wilk test was followed by Student's t test for Il-6 data analysis and by the Mann-Whitney test for Il-1β. Data are presented as box-and-whisker plots with median and interquartile ranges, from max to min, with all data points shown.
As far as the expression of anti-inflammatory cytokines is concerned, we observed that their mRNA levels were too low for accurate analysis to allow comparisons between the experimental groups. It can be hypothesized that anti-inflammatory cytokines are expressed only under conditions of memory impairment. Therefore, their expression was undetectable in young and healthy mice, and the behavioral paradigm of depression (forced swim test) was not effective in increasing their expression levels.

Short-Term Irisin Administration Modulated Gene Expression in a Sex-Independent Way in Both the Hippocampus and the PFC
To investigate the effect of short-term irisin treatment, sex, and their interactions on gene expression in the hippocampus and the PFC, we performed separate group comparisons according to treatment and sex by two-way ANOVA analysis. All the results of this analysis are included in Tables 1-3. For the hippocampus, we did not find any significant interaction of treatment and sex for all the analyzed genes. As far as the PFC tissue is concerned, two-way ANOVA analysis showed an interaction of sex and treatment for Bdnf and Ngf expression (p = 0.009 and p = 0.034, respectively). A post hoc test revealed that The gene expression of Il-6 (a), and Il-1β (b) was evaluated by qRT-PCR in female (n = 7) and male (n = 5) mice for each treatment group. The Shapiro-Wilk test was followed by Student's t test for Il-6 data analysis and by the Mann-Whitney test for Il-1β. Data are presented as box-and-whisker plots with median and interquartile ranges, from max to min, with all data points shown.  The gene expression of Il-6 (a), and Il-1β (b) was evaluated by qRT-PCR in female (n = 7) and male (n = 5) mice for each treatment group. The Shapiro-Wilk test was followed by Student's t test for Il-6 data analysis and by the Mann-Whitney test for Il-1β. Data are presented as box-and-whisker plots with median and interquartile ranges, from max to min, with all data points shown.
In the PFC, we found a slight increase in Il-6 expression in irisin-treated mice that nonetheless did not reach statistical significance (p = 0.081), while no differences were noticed for Il-1β (p = 0.409) ( Figure 4). Il-1β (b) was evaluated by qRT-PCR in female (n = 7) and male (n = 5) mice for each treatment group. The Shapiro-Wilk test was followed by Student's t test for Il-6 data analysis and by the Mann-Whitney test for Il-1β. Data are presented as box-and-whisker plots with median and interquartile ranges, from max to min, with all data points shown.
As far as the expression of anti-inflammatory cytokines is concerned, we observed that their mRNA levels were too low for accurate analysis to allow comparisons between the experimental groups. It can be hypothesized that anti-inflammatory cytokines are expressed only under conditions of memory impairment. Therefore, their expression was undetectable in young and healthy mice, and the behavioral paradigm of depression (forced swim test) was not effective in increasing their expression levels.

Short-Term Irisin Administration Modulated Gene Expression in a Sex-Independent Way in Both the Hippocampus and the PFC
To investigate the effect of short-term irisin treatment, sex, and their interactions on gene expression in the hippocampus and the PFC, we performed separate group comparisons according to treatment and sex by two-way ANOVA analysis. All the results of this analysis are included in Tables 1-3. For the hippocampus, we did not find any significant interaction of treatment and sex for all the analyzed genes. As far as the PFC tissue is concerned, two-way ANOVA analysis showed an interaction of sex and treatment for Bdnf and Ngf expression (p = 0.009 and p = 0.034, respectively). A post hoc test revealed that Il-1β (b) was evaluated by qRT-PCR in female (n = 7) and male (n = 5) mice for each treatment group. The Shapiro-Wilk test was followed by Student's t test for Il-6 data analysis and by the Mann-Whitney test for Il-1β. Data are presented as box-and-whisker plots with median and interquartile ranges, from max to min, with all data points shown.
As far as the expression of anti-inflammatory cytokines is concerned, we observed that their mRNA levels were too low for accurate analysis to allow comparisons between the experimental groups. It can be hypothesized that anti-inflammatory cytokines are expressed only under conditions of memory impairment. Therefore, their expression was undetectable in young and healthy mice, and the behavioral paradigm of depression (forced swim test) was not effective in increasing their expression levels.

Short-Term Irisin Administration Modulated Gene Expression in a Sex-Independent Way in Both the Hippocampus and the PFC
To investigate the effect of short-term irisin treatment, sex, and their interactions on gene expression in the hippocampus and the PFC, we performed separate group comparisons according to treatment and sex by two-way ANOVA analysis. All the results of this analysis are included in Tables 1-3. For the hippocampus, we did not find any significant interaction of treatment and sex for all the analyzed genes. As far as the PFC tissue is concerned, two-way ANOVA analysis showed an interaction of sex and treatment for Bdnf and Ngf expression (p = 0.009 and p = 0.034, respectively). A post hoc test revealed that female irisin-treated mice had higher BDNF mRNA levels compared to their control group (F = 8.99, DF:20, p < 0.0001) and male irisin-treated mice (F = 4.314, DF:20, p < 0.05). No differences were noticed among groups for Ngf expression ( Figure 5). Table 1. Summary of the statistical analysis for the factors treatment, sex, and treatment × sex by two-way ANOVA.

Discussion
In the last decade, the myokine irisin emerged as an interesting neuroprotective factor in the muscle-brain crosstalk thanks to its ability to cross the blood brain barrier and to induce the cerebral expression of neurotrophins [9,36,37].
In our first study, we showed that, in healthy young male mice, treated weekly with irisin for 1 month, depressive-like behaviors were reduced [24]. In line with these results, we recently reported that short-term irisin treatment daily for 5 days also displayed antidepressant effects, as well as a reduction in anxiety-like behaviors, in young healthy female and male mice [26]. Herein, we investigated the molecular basis of the antidepressant effect of short-term irisin treatment analyzing the cerebral expression of the most well-known factors associated with MDD pathogenesis. This study was performed in a group of mice previously submitted to a behavioral paradigm of depression (i.e., forced swim test) displaying high mobility when treated with irisin compared to untreated control mice [26]. Cross-sectional studies using structural neuroimaging in patients with MDD and investigations in rodent models of depression demonstrated the presence of morphological (i.e., volume reduction, reduced synapse number, etc.) and molecular alterations in both the hippocampus and the PFC [19,27,28,38]; thus, we focused our attention on these specific cerebral regions.
The present study revealed that the recent antidepressant effect of irisin could be mediated by a site-specific action on the modulation of neurotrophins and growth factors at the level of the hippocampus and prefrontal cortex (PFC). Indeed, we found that irisin injection once daily for five consecutive days increased the expression of Ngf in the hippocampus and that of Bdnf in the PFC of irisin-treated mice. NGF and BDNF are two members of the neurotrophic factor family with crucial roles in the formation and plasticity of cerebral neural networks, and they are mainly involved in the pathophysiological mechanism of depression [30,32,39]. In fact, it was reported that depressed patients displayed reduced circulating NGF levels [40]. Regarding BDNF, several evidence found an abnormal decrease in its serum levels in MDD patients, thereby representing a biomarker of the pathogenesis of depression [41][42][43][44]. In addition, it was reported that chronic stress and depressive conditions reduced the expression of Bdnf in several brain regions, including the hippocampus and the PFC, in rodent models [30,45,46]. Interestingly, the site-specific modulation of the two neurotrophins induced by short-term irisin administration demonstrated here in the two brain regions is in agreement with studies of Lima-Filho and coworkers, who described a region-specific gene expression induced by antidepressant drugs in mice [47].
Conflicting literature data about the effect of irisin on Bdnf levels in the hippocampus have been reported. The unaltered Bdnf modulation in the hippocampus we found is in contrast with the upregulation demonstrated by other authors [9,36], but in line with the observations of Siteneski and colleagues [15], who found an irisin time-dependent modulation of Bdnf and other genes. In particular, the latter authors showed that Bdnf was downregulated in the hippocampus 1 h after irisin administration and upregulated after 6 h. Therefore, it might be speculated that the unaltered expression of Bdnf in the hippocampus we detected might have been due to the short time elapsed since the last irisin injection.
We also studied FGF-2, another neurotrophic factor able to regulate neurogenesis, synaptic formation, and growth [33]. FGF-2 has been implicated in stress-related mood disorders, such as depression, and its expression is reduced in the hippocampus of MDD patients [48][49][50][51]. In this work, we observed a significant increase in the mRNA levels of FGF-2 in irisin-treated mice in the hippocampus and the PFC, suggesting that, unlike the above-discussed neurotrophins, Fgf-2 did not display a cerebral site-specific increase. Of note, other studies reported the upregulation of Fgf-2 expression after antidepressant treatments [52,53]. For example, Maragnoli et al. observed higher levels of FGF-2 mRNA in the PFC of rats submitted to short-and long-term treatment with a combination of antidepressant drugs [52].
IGF-1 is a neutrotrophic factor that is expressed by different cells in the CNS [31]. In the synaptic plasticity process, IGF-1 is involved in the control of synapse formation, neurotransmitter release, and neuron excitation [54]. Considering these roles, as well as IGF-1's ability to suppress neuroinflammation [31], it has been hypothesized that the impairment of IGF-1 functions could be responsible for some clinical alterations observed in MDD patients, including cognitive compromission [31]. In line with this assumption, a reduced Igf-1 expression was described in the hippocampus and the PFC of prenatally stressed rats that exhibited depressive-like behaviors [55]. Unlike our previous study, in which we found Igf-1 upregulation in irisin-treated mice receiving a long-term intermittent administration [24], we observed here that short-term irisin treatment seemed to not involve the IGF-1 system as its expression did not change in either the hippocampus or the PFC.
Recently, a number of meta-analysis results and animal studies suggested that the immune system dysregulation and the activation of the inflammatory response system are closely related to depressive disorders [30,[56][57][58]. In particular, it was shown that the levels of some inflammatory cytokines (i.e., IL-6, IL-1, TNF-α, etc.) were significantly increased in the serum of MDD patients [56,57] and in the brain of rodent models of depression [58]. Proinflammatory cytokine secretion can contribute to depression by impacting neurotransmitter levels, neurogenesis, neuroendocrine function, neuroplasticity, and depression-related neural networks [30,56,58]. As a result, some anti-inflammatory drugs have been shown to have antidepressant effects or increase the action of antidepressant drugs [30]. In the present study, we analyzed the gene expression of some proand anti-inflammatory cytokines and we noticed that there were no differences between the irisin-treated mice and the control group. These observations are in contrast with the results of our previous study in which we observed the effect of irisin in the modulation of different cytokines, such as IL-1β and IL-4 [24]. Collectively, these different findings regarding cytokines, together with the results of Igf-1 expression, lead us to hypothesize that the modulation of these factors might depend on several conditions such as the time of treatment, i.e., intermittent long-term [24] versus once-daily short-term, and the different brain areas (whole brain versus hippocampus and PFC tissues). However, the complexity of the cytokine system, including a plethora of mediators with multiple effects, requires further in-depth investigations.
Epidemiological findings point to a gender difference in the prevalence and incidence of MDD as depressive disorders affect women more frequently than men [59]. Female hormonal fluctuations and environmental/social factors, such as psychological stress, may be triggers for MDD development [59]. In light of possible different treatment response due to gender, we evaluated the effect of sex on gene expression. Consistently with the behavioral paradigm results [26], no significant differences were noticed between female and male for the analyzed genes except for Bdnf expression in the PFC. It is known that the brain expression profile of BDNF is regulated by several factors, including gonadal hormones, estrogens in women, and androgens in men [60]. Sex-related differences in BDNF signaling and function have been observed; indeed, estrogen regulates not only BDNF levels at the gene and protein level, but also those of its receptor in the brain [61][62][63][64], including the hippocampus and PFC [65]. Although some evidence suggests reduced BDNF levels in the PFC in depressed women [66], studies on the subject have been less thorough in the PFC, making the relationship between BDNF and estrogen levels in this part of the brain less clear to date. Similarly, the effects of androgens on BDNF levels have been less studied; hence, further studies on the involvement of sex hormones in the regulation of neurotrophin levels are needed.
Notably, our results showed that short-term irisin treatment markedly increased Bdnf expression in female mice compared to males. Collectively, the absence of sex differences, together with the greater response in female mice, is a promising result for desirable clinical applications in humans.
In a recent study by Kim and Leem, the effect of a daily irisin treatment (0.5 µg/kg/day) for seven consecutive days was investigated by evaluating the morphological changes in the hippocampus and the expression of astrocyte-secreted factors associated with neurogenesis [67]. However, to the best of our knowledge, our study is the first to elucidate the molecular mechanisms underlying short-term irisin administration in young mice showing a site-specific expression of neurotrophic factors mainly involved in neurogenesis and/or synaptic plasticity alterations associated with MDD. Noteworthily, our study adds knowledge to the search for new nonselective serotonin reuptake inhibitor (non-SSRI) antidepressant treatments in children and adolescents with MDD, as envisioned by the Food and Drug Administration (FDA) and new regulatory changes encouraging short-term acute efficacy studies [25]. Indeed, we showed here that the expression of neurotrophic factors, such as BDNF, NGF, and FGF-2, which are negatively affected in the pathogenesis of depressive disorders, can be enhanced by short-term treatment with irisin. It is well known that MDD, especially in children and adolescents, is characterized by a disorder that can be sudden and episodic or chronic and recurrent. In fact, great efforts are being made to implement effective pharmacotherapy for the short-term treatment of young people with MDD to understand how to treat a single episode of depression while avoiding the many side-effects of long-term therapies.

Animals
In this study, in order to explore sex-specific treatment responses, male (n = 10) and female (n = 14) black Swiss mice (C57BL/6) aged 22-25 weeks were used [26]. Animals were housed into six home cages (four per group) 2 weeks before the experiment starting time. Mice had free access to food and water (Harlan Teklad 2019, SDS, England), and they were maintained under 12 h light/dark cycle, controlled noise, and temperature (21 • C). All study procedures were previously approved by the Italian Health Authority (aut. n • 12/2022-PR, obtained on 12 January2022) and conducted in accordance with European Directive no. 2010/63 and with the Italian Regulatory system (D.L. vo n. 26, 4 March 2014). The animal welfare agency (OPBA) of the University of Bari approved all parts of this study concerning animal care and handling protocols.

Short-Term Irisin Administration
Mice were subcutaneously injected with recombinant irisin at 100 µg/kg (n = 12) or vehicle (0.9% saline solution sterilized by 0.22 µ filtration) (n = 12) once a day for a total of five consecutive days. Recombinant irisin was provided by Adipogen International (San Diego, CA, USA). Treatment was regularly executed at the same time each day. The concentration of irisin was chosen on the basis of previous studies performed by our research team [6,24].

Gene Expression Analysis by qRT-PCR Assays
As described in [26], after the last vehicle/irisin injection, a group of 24 mice consisting of vehicle-treated mice (n = 12) and irisin-treated mice (n = 12) were submitted to the FST. Each treatment group included n = 5 males and n = 7 females to assess sex differences. The FST was performed according to the procedure suggested by Porsolt and coworkers [68,69]. Briefly, mice were placed, one at a time, in a clear acrylic cylinder (diameter 13 cm, height 24.5 cm) filled with tap water (18 cm) at 23-25 • C. The immobility time was assessed during 6 min of observation. Mice were considered immobile when they stopped swimming and assumed a passive floating position.
After sacrifice, mouse brains were carefully dissected to isolate the hippocampus and the PFC. These brain tissues were harvested and stored at −80 • C until further analysis. The extraction of total RNA was executed using Trizol (Invitrogen, Carlsbad, CA, USA) according to standard protocols. To synthesize complementary DNA (cDNA), 1 µg of RNA in 20 µL of reaction mixture, i.e., iScript Reverse Transcription Supermix (Bio-Rad Laboratories, Hercules, CA, USA), was used. The expression of mRNAs was measured by qRT-PCR in a 10 µL reaction volume consisting of cDNA transcripts, primer pairs, and SsoFast EvaGreen Supermix (Bio-Rad Laboratories, Hercules, CA, USA). All primers were designed by Primer Blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/, accessed on 10 January 2023). Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was selected as the reference gene. The sequences for target and reference genes are included in Table 4. To assess the specificity of qRT-PCR products, a melt curve analysis was performed. All samples were assayed in triplicate and quantifications were normalized to Gapdh in each reaction.

Statistical Analysis
Data were presented as box-and-whisker plots with median and interquartile ranges, from max to min, with all data points shown, and analyzed using the GraphPad Prism statistical software release 7.0. Data normality was initially verified using the Shapiro-Wilk test. Statistically significant differences between vehicle (control) and irisin-treated mice were evaluated using Student's t-test or the Mann-Whitney test. Statistical significance was set at p < 0.05. To determine the effects of irisin treatment (first factor) and sex (second factor) on gene expression, two-way ANOVA was used. Main effects with a p-value <0.05 were considered statistically significant. A post hoc Tukey's multiple comparison test and Student's t-test were used to analyze the differences among the groups.

Conclusions
Recent studies have evidenced that several defects in the synaptic plasticity (i.e., dendritogenesis, neurogenesis, and axon branching) in some brain regions, including the hippocampus and the PFC, are due to the reduced expression of neurotrophic factors in MDD [39,50,70]. Our data demonstrated that short-term irisin treatment was effective in increasing the cerebral expression of neutrophins in the hippocampus and/or the PFC in young healthy mice previously submitted to a behavioral paradigm of depression. This observation is in line with recent studies evidencing that the neuroprotective effect of antidepressant drugs is mediated, at least in part, by the stimulation of crucial molecular factors involved in brain neurogenesis and/or neural plasticity in both the hippocampus and the PFC [19,71,72]. Taking into account the better behavioral performances of irisintreated mice in the depression test FST [26], we demonstrated here that exogenous irisin might be responsible for the antidepressant effect in both female and male mice receiving short-term irisin treatment. However, as we observed that the peripheral irisin increase significantly enhanced Fndc5 and Pgc-1 expression in both the hippocampus and the PFC [26], we cannot exclude that the activation of the FNDC5/irisin pathway in these regions could contribute to the antidepressant effect of irisin.