Acute stress induces an aberrant increase of presynaptic release of glutamate and cellular activation in the hippocampus of BDNFVal/Met mice

Abstract Stressful life events are considered major risk factors for the development of several psychiatric disorders, though people differentially cope with stress. The reasons for this are still largely unknown but could be accounted for by individual genetic variants, previous life events, or the kind of stressors. The human brain‐derived neurotrophic factor (BDNF) Val66Met variant, which was found to impair intracellular trafficking and activity‐dependent secretion of BDNF, has been associated with increased susceptibility to develop several neuropsychiatric disorders, although there is still some controversial evidence. On the other hand, acute stress has been consistently demonstrated to promote the release of glutamate in cortico‐limbic regions and altered glutamatergic transmission has been reported in psychiatric disorders. However, it is not known if the BDNF Val66Met single‐nucleotide polymorphism (SNP) affects the stress‐induced presynaptic glutamate release. In this study, we exposed adult male BDNFVal/Val and BDNFVal/Met knock‐in mice to 30 min of acute restraint stress. Plasma corticosterone levels, glutamate release, protein, and gene expression in the hippocampus were analyzed immediately after the end of the stress session. Acute restraint stress similarly increased plasma corticosterone levels and nuclear glucocorticoid receptor levels and phosphorylation in both BDNFVal/Val and BDNFVal/Met mice. However, acute restraint stress induced higher increases in hippocampal presynaptic release of glutamate, phosphorylation of cAMP‐response element binding protein (CREB), and levels of the immediate early gene c‐fos of BDNFVal/Met compared to BFNFVal/Val mice. Moreover, acute restraint stress selectively increased phosphorylation levels of synapsin I at Ser9 and at Ser603 in BDNFVal/Val and BDNFVal/Met mice, respectively. In conclusion, we report here that the BDNF Val66Met SNP knock‐in mice display an altered response to acute restraint stress in terms of hippocampal glutamate release, CREB phosphorylation, and neuronal activation, compared to wild‐type animals. Taken together, these results could partially explain the enhanced vulnerability to stressful events of Met carriers reported in both preclinical and clinical studies.


| INTRODUCTION
Stress is a physiological response to challenging or threatening environments that stimulates body adaptation and increases the chances of survival (McEwen, 2007). The physiological response to acute stress implies the rapid activation of the hypothalamic-pituitary-adrenal (HPA) axis, leading to the release in the bloodstream of glucocorticoids, and corticosterone in rodents (de Kloet et al., 1998;Mourtzi et al., 2021). Nevertheless, stressful life events impact memory and cognition and are considered the major environmental risk factors in the onset of neuropsychiatric disorders, while alterations of HPA axis activity have been reported in depressed patients (Ceruso et al., 2020). However, the capability to cope with stress is diverse among individuals, which may depend on different factors such as age, gender, previous experiences, and genetic background (Cathomas et al., 2019;McEwen et al., 2015;Sanacora et al., 2022).
Brain-derived neurotrophic factor (BDNF), the most abundant neurotrophin expressed in the adult brain, is one of the essential mediators of synapse formation, function, and plasticity in the mammalian brain and has been implicated in the pathophysiology of various stress-related disorders as well as in the action of therapeutic drugs (Bramham & Messaoudi, 2005;Edelmann et al., 2014). Accordingly, several preclinical animal models have clearly shown that stress reduces BDNF expression, especially in the hippocampus (HPC) and prefrontal cortex, while both pharmacological and environmental therapeutical approaches can enhance BDNF expression in these same regions (Björkholm & Monteggia, 2016;Castrén & Monteggia, 2021;Nestler et al., 2002;Notaras & van den Buuse, 2020).
A functional single-nucleotide polymorphism (SNP) has been identified in the human BDNF gene, which leads to the substitution of a valine with a methionine at the codon 66 (BDNF Val66Met;rs6265). This SNP is common in humans, with an allele frequency of 20%-30% in Caucasians, and is recognized as a contributing factor in morphological/functional brain variability (Miranda et al., 2019;Notaras et al., 2015;Tsai, 2018). The BDNF Val66Met SNP reduces the expression and the activity-dependent release of BDNF and has been correlated in humans with a smaller hippocampal volume, memory impairments, increased predisposition to develop neuropsychiatric and neurodegenerative disorders, and altered vulnerability to stress (Chen et al., 2006;Egan et al., 2003;Hosang et al., 2014).
In line with this evidence, BDNF Met/Met knock-in mice show a reduction in hippocampal volume, deficits in hippocampal-dependent memory, and an anxious-like phenotype (Chen et al., 2006). BDNF Val/Met mice also have alterations in hippocampal gene expression involved in dendritic and spine remodeling (Mallei et al., 2018), reduced response to the beneficial effects of antidepressants and physical exercise (Bath et al., 2012;Ieraci et al., 2020), and enhanced susceptibility to stress exposure (Sandrini et al., 2018;Yu et al., 2012). However, the mechanisms underlying the enhanced vulnerability to stress is still largely unknown.
Alteration of the glutamatergic system is considered a key mechanism of stress-related neuropsychiatric disorders (Duman et al., 2019;Sanacora et al., 2012Sanacora et al., , 2022. Clinical studies have consistently reported in patients with mood and anxiety disorders a volume reduction in brain areas enriched with glutamatergic neurons, such as the HPC and prefrontal cortex (Belleau et al., 2019).
Consistently, several studies in animal models of depression have shown that stress promotes dendritic atrophy, a decrease in synapse number, and altered connectivity in the HPC and prefrontal cortex (McEwen et al., 2016;Musazzi et al., 2015;Woo et al., 2021). Moreover, acute stress or corticosterone administration enhances the glutamate release in the HPC and prefrontal cortex of rodents Sanacora et al., 2012). Interestingly, BDNF is critical for the maturation of glutamatergic neurons, regulates glutamate release, and influences excitatory synapse formation (Carvalho et al., 2008), while the BDNF Val66Met SNP impairs glutamatergic transmission and synaptic plasticity in the mouse HPC (Ninan et al., 2010). However, it is not known if the BDNF Val66Met SNP may regulate the release of glutamate induced by acute stress. This work is aimed at investigating whether the presence of the BDNF Val66Met SNP can influence the presynaptic glutamate release induced by acute restraint stress in the HPC of mice and to studying possible related molecular mechanisms and changes.

| Animals
Male BDNF Val/Val and BDNF Val/Met mice (3-4 months old) were used in the study (Chen et al., 2006) N 349/2015-PR). All efforts were made to minimize animal distress and to reduce the number of animals used in this study.

| Restraint stress protocol
BDNF Val/Val and BDNF Val/Met mice were randomly divided into control (CNT) and acute restraint stress groups. For acute restraint stress, mice were individually restrained for 30 min in well-ventilated 50-ml polypropylene centrifuged tube (Ieraci et al., 2015). Acute restraint stress was performed during the morning period. CNT mice were left undisturbed in their home cages. Animals were killed by decapitation immediately after acute restraint stress protocol. The HPC was dissected on ice and right/left areas were pooled for purification of synaptosomes for glutamate release experiments, or randomly assigned to the purification of subcellular fractions for Western blot analyses or RNA extractions. Glutamate release and biochemistry/molecular analyses were conducted blinded.

| Corticosterone serum levels measurement
For plasma preparation, trunk blood was collected on an ice-cooled tube containing Ethylenediaminetetraacetic acid 0.5 M pH 8.00, separated by centrifugation, and stored at −80°C. Plasma corticosterone levels were measured using a commercial kit (Corticosterone ELISA kit; Enzo Life Sciences) (Ieraci et al., 2015).

| Preparation of purified synaptosomes and neurotransmitter release experiments
Purification and superfusion of synaptic terminals (synaptosomes) were performed as previously reported (Musazzi et al., 2017). Synaptosomes were freshly prepared from homogenized HPC (homogenization buffer: 0.32 M sucrose, buffered at pH 7.4 with Tris-HCl) of 8-12 animals/ group, by centrifugation, and incubated at 37°C for 15 min in standard physiological medium (140 mM NaCl, 3 mM KCl, 1.2 mM MgSO 4 , 1.2 mM CaCl 2 , 1.2 mM NaH 2 PO 4 , 5 mM NaHCO 3 , 10 mM glucose, sample was calculated as a fractional rate × 100 (percentage of the total synaptosomal neurotransmitter content at the beginning of the respective sample collection). The stimulus-evoked neurotransmitter overflow was estimated by subtracting the transmitter content of the two 3-min fractions representing the basal release from that in the 6-min fraction collected during and after the stimulating pulse.
Radioactivity was determined in each sample collected and in the superfused filters by liquid scintillation counting. Tritium released in each sample was calculated as a fractional rate × 100 (percentage of the total synaptosomal neurotransmitter content at the beginning of the respective sample collection).

| Preparation of subcellular fractions and western blot analysis
HPC was homogenized 1:10 (w/v) by a loose-fitting Potter in homogenization buffer (0.28 M sucrose buffered at pH 7.4 with Tris, containing phosphatase inhibitors (Thermo-Fisher Scientific) and 2 ml/ml of protease inhibitor cocktail [Sigma-Aldrich]). Total homogenates were centrifuged for 5 min at 1,000g, and the resulting pellets, enriched in nuclei (P1), were resuspended in lysis buffer (120 mM NaCl, 20 mM HEPES pH 7.4, 0.1 mM EGTA, 0.1 mM DTT, protease and phosphatase inhibitors as in HB). Synaptosomes were prepared by centrifugation as above and synaptic membrane fraction was prepared by centrifugation as described previously (Musazzi et al., 2010;Treccani et al., 2014).

| RNA isolation and reverse transcription
RNA isolation and reverse transcription were performed as previously described (Ieraci, Barbieri, et al., 2020). Briefly, total RNA from the HPC was extracted using the Direct-zol RNA MiniPrep (Zymo Research, purchased by Euroclone) according to the manufacturer's instructions and quantified by absorption at 260 nm measured by UV spectrophotometry (NanoVue, GE Healthcare Europe GmbH). Complementary DNA was synthetized using the iScript kit (Bio-Rad) according to the manufacturer's instructions.

| Statistical analysis
Statistical analysis of the data was carried out using GraphPad Prism6   (Song & Augustine, 2015) and has been previously shown to take part in the molecular processes involved in the increase of glutamate release induced by acute stress (Treccani et al., 2014).
Concerning synapsin I phosphorylation at both Ser 9 and Ser 603 , we found a significant effect of stress × genotype interaction (Ser 9 :  (Figure 3a,b,c). Post hoc analysis revealed that acute restraint stress selectively increased the phosphorylation levels of synapsin I at Ser 9 in BDNF Val/Val (p < 0.05) but not in BDNF Val/Met mice, in which however phosphorylation levels at Ser 9 were higher in control compared to control BDNF Val/Val mice (p < 0.05) and were not further increased by stress (Figure 3a,b).
Instead, synapsin I phosphorylation at Ser 603 was unchanged between the two genotypes in basal conditions, but acute restraint F I G U R E 1 Acute restraint stress activated the hypothalamus-pituitary-adrenal axis in both BDNF Val/Val and BDNF Val/Met mice. (a) Corticosterone plasma levels were measured immediately after the 30 min of acute restraint stress. Data are reported as mean ± SEM (n = 9-11 mice/group). Kruskal-Wallis test followed by the Dunn's multiple comparison test. ****p < 0.0001. (b-e) Western blot analysis of the hippocampal nuclear fraction of total MR (c), phosphorylated GR (p-GR) (d) and total GR (e). (b) Representative western blot images of total MR, total GR, p-GR, and β-actin. Densitometric analysis was obtained as a ratio of MR/β-actin (c), p-GR/GR (d) and GR/β-actin (e). Data are reported as mean ± SEM (n = 7-10 mice/group). Kruskal-Wallis test followed by the Dunn's multiple comparison test (a, d) and two-way ANOVA followed by Newman-Keuls multiple comparison test (c, e). *p < 0.05; ***p < 0.001. ANOVA, analysis of variance; A.U., arbitrary unit; BDNF, brain-derived neurotrophic factor; GR, glucocorticoid receptor; MR, mineralocorticoid receptor; SEM, standard error of the mean.

(a) (b)
F I G U R E 2 Acute restraint stress induced a higher increase of presynaptic glutamate release in the hippocampus of BDNF Val/Met than in BDNF Val/Val mice. (a) Basal glutamate release from hippocampal synaptosomes in superfusion. Data are reported as mean ± SEM (n = 4-9 mice/ group). Kruskal-Wallis test followed by Dunn's multiple comparison test. ***p < 0.001. (b) 15 mM KCl-evoked glutamate release from hippocampal synaptosomes in superfusion. The net depolarization-evoked overflow was calculated by subtracting transmitter content of the basal outflow. Data are reported as mean ± SEM (n = 4-8 mice/group). Kruskal-Wallis test followed by Dunn's multiple comparison test. *p < 0. 01. BDNF, brain-derived neurotrophic factor; SEM, standard error of the mean. % = % overflow.

| Acute restraint stress induced a higher increase of CREB phosphorylation in the HPC of BDNF Val/Met compared to BDNF Val/Val mice
Having found selective glutamatergic alterations induced by acute restraint stress in the two genotypes, we next assessed whether this was accompanied by changes in intracellular signaling. In particular, we analyzed the nuclear expression and phosphorylation levels of the transcription factor CREB, one of the major regulators of neurotrophic responses, which is also known to be activated by glutamatergic stimulation and after acute stress (Ieraci et al., 2015;Mao et al., 2004;Nasca et al., 2017).

| DISCUSSION
In this study, we showed that acute restraint stress, although leading to a similar activation of the HPA axis, increased depolarizationevoked presynaptic release of glutamate, phosphorylation of CREB, (f) F I G U R E 4 Acute restraint stress increased CREB phosphorylation and cFOS mostly in the hippocampus of BDNF Val/Met mice. (a-c) Western blot analysis of hippocampal nuclear fraction of phosphorylated CREB (b) and total CREB (c). (a) Representative western blot images of CREB, p-CREB, and β-actin. Densitometric analysis were obtained as ratio of p-CREB/CREB (b) and CREB/β-actin (c). Data are reported as mean ± SEM (n = 7-10 mice/group). Two-way ANOVA followed by Newman-Keuls multiple comparison test (b, d-f) and Kruskal-Wallis test followed by the Dunn's multiple comparison test (c). *p < 0.05; **p < 0.01; ****p < 0.0001. (d-f) qPCR analysis of hippocampal mRNA levels of c-Fos (d), Arc (e) and Bdnf (f). Data are reported as mean ± SEM (n = 6-10 mice/group). One-way ANOVA followed by Newman-Keuls multiple comparison test. **p < 0.01. ANOVA, analysis of variance; A.U., arbitrary unit; BDNF, brain-derived neurotrophic factor; CREB, cAMP-response element binding protein; F.C.; fold change; mRNA, messenger RNA; qPCR, quantitative real-time PCR; SEM, standard error of the mean. and levels of the immediate early gene c-Fos in the HPC of BDNF Val/Met compared to BFNF Val/Val mice. Moreover, acute restraint stress selectively increased the phosphorylation levels of synapsin I at Ser 9 and at Ser 603 in BFNF Val/Val and BDNF Val/Met mice, respectively.
Activation of the HPA axis represents a primary hormonal response to a homeostatic challenge. Temporal activation of the HPA axis depends on stress duration and type but, being essential for coping with the environment and increase of survival probability, the stress response is extremely fast in nature (Herman et al., 2016). Acute stress efficiently drives HPA stress response, and feedback mechanisms effectively terminate the response after the stressor subsides.
The overall effect of acute restraint stress on the HPA axis is well documented: corticotropin-releasing hormone, adrenocorticotropic hormone, and glucocorticoid secretion all increase significantly during the stressor period but returned to basal levels within an hour after stress. Previous evidence showed that corticosterone levels are remarkably increased after 30 min of restraint stress but rapidly go back to control levels within 120 min (Mcclennen et al., 1998).
Interestingly, it has also been reported that plasma corticosterone levels peaked 30 min after a single saline injection, a very mild acute stress and that this increase was higher in BDNF +/heterozygous mice compared to wild-type mice (Advani et al., 2009). This and other evidence suggest that BDNF may be involved in the regulation of the HPA axis response to stress (Naert et al., 2015;Numakawa et al., 2013). However, the possible role of the BDNF Val66Met SNP in the HPA reactivity and cortisol release after stress exposure is still controversial. While some human studies have reported a stronger cortisol response in BDNF Met/Met compared to Val/Val carriers (Schüle et al., 2006), others have found a greater rise in Val/Val compared to Met carriers (Alexander et al., 2010), or similar cortisol release among different genotypes (Hennings et al., 2019). Here, by using the knock-in BDNF Val66Met mice in a well-controlled laboratory environment, we found that acute restraint stress similarly rises corticosterone plasma levels in both BDNF Val/Val and BDNF Val/Met male mice. Interestingly, it was previously reported that, after seven consecutive days of restraint stress, the corticosterone release was greater in BDNF Val/Met compared to BDNF Val/Val mice (Yu et al., 2012).  (Adzic et al., 2009).
It has been reported that BDNF Val66Met SNP impairs NMDA receptor-dependent synaptic transmission (Ninan et al., 2010) and affects the stress response in the HPC (Notaras et al., 2016;Yu et al., 2012 (Turrigiano, 2012). An impairment of these mechanisms is likely to be involved in maladaptive stress response and increased psychopathological risk, while homeostatic synaptic plasticity has been implicated in antidepressant effects (Kavalali & Monteggia, 2020).
Similarly, an abnormal glutamatergic activation under stress conditions as observed for BDNF Val/Met mice could induce noxious effects and alter physiological glutamate transmission and plasticity (Musazzi et al., 2011;Popoli et al., 2012;Sanacora et al., 2022). Accordingly, the BDNF Val66Met SNP has been associated with impairments in hippocampal glutamatergic transmission (Ninan et al., 2010).  (Huttner & Greengard, 1979), this result is in line with alteration in presynaptic calcium homeostasis in BDNF Val/Met mice.
This in turn might lead to hyperexcitability and excitotoxic risk, in line with the higher susceptibility to behavioral consequences of chronic stress previously reported in BDNF Val/Met mice compared with BDNF Val/Val mice (Yu et al., 2012).
CREB is a nuclear transcription factor activated by phosphorylation, downstream of several intracellular cascades involved in neuronal activation and neuroplastic processes (Carlezon et al., 2005). Previous evidence consistently showed that acute stress increases CREB phosphorylation in the HPC (Alboni et al., 2011;Ieraci et al., 2015). Accordingly, we found that CREB phosphorylation was increased after acute restraint stress exposure in both BDNF Val/ Val and BDNF Val/Met mice, although the increase was higher in BDNF Val/Met compared to BDNF Val/Val mice. This suggests that the observed presynaptic glutamatergic dysregulation in BDNF Val/Met mice is accompanied by an abnormal cellular response. This hypothesis is confirmed by the expression of the immediate early gene c-Fos, which is considered a good marker of neuronal activity (Benito & Barco, 2015). On the other hand, we showed that in the HPC of wild-type BDNF Val/Val mice acute restraint stress did not promote any significant change in the mRNA levels of c-Fos. This result is consistent with previous work showing an increase of c-Fos only 140 min after the end of 30 min acute restraint stress (Alboni et al., 2011). No changes were instead measured in Arc and Bdnf expression.
In conclusion, we found that knock-in mice for the human BDNF