Impairments in social functioning are in the core of clinical symptoms characterizing many neuropsychiatric disorders, ranging from social aversion in Autism Spectrum Disorder (ASD) and schizophrenia to an unusual hyper-social phenotype observed in Williams-Syndrome (Barak and Feng, 2016). One of the proposed pathophysiological mechanisms contributing to social withdrawal is the disruption of the balance between excitation and inhibition (E/I balance), often associated with hyper-glutamatergic and/or hypo-GABAergic function at large-scale neuronal circuits (Fatemi et al., 2009; Sohal and Rubenstein, 2019). At the single-neuron level, different factors contribute to E/I imbalance including aberrant synaptic transmission and plasticity arising from altered neurotransmitter release (Takada et al., 2015), impaired expression of excitatory (Catts et al., 2016) or inhibitory postsynaptic receptors (Fatemi et al., 2009) and/or their scaffolding proteins (Coley and Gao, 2019). Such alterations cause changes in circuit excitability in key cortical and subcortical regions, contributing to social withdrawal (Zikopoulos and Barbas, 2013).

The potential involvement of N-methyl-D-aspartate receptors (NMDAR) hypofunction in the manifestation of social withdrawal phenotype has been emphasized in several studies both in rodents and teleosts, since sub-chronic (+)-MK-801 treatment, a non-competitive NMDAR antagonist, decreased social interaction in adult rats (Matsuoka et al., 2005) and zebrafish (Perdikaris and Dermon, 2022). Also, the selective knockout of NMDARs in parvalbumin interneurons resulted in decreased sociability in mice (Saunders et al., 2013), further highlighting NMDA receptors’ importance in regulating social behavior. Indeed, NMDARs located on fast-spiking GABAergic interneurons have a role of E/I imbalance in NMDA hypofunction models of social withdrawal, leading to excessive glutamate release and hyperstimulation of downstream cortical and sub-cortical networks (Homayoun and Moghaddam, 2007). Consistent with this view, it was reported increased ratio of E/I activity, in a serine racemase knockout model of NMDAR hypofunction (Jami et al., 2021). However, further studies are necessary for better understanding the pathophysiological mechanisms implicated in the synaptic dysfunction and contribute to aberrant network activity observed in neuropsychiatric disorders characterized by impairments in social functioning.

There is increasing evidence from clinical data reporting a significant link between impaired social function and anxiety, since individuals with high-functioning autism exhibited higher levels of anxiety compared to healthy control subjects (Top et al., 2016). Similarly, animal studies support the above relationship as various ASD mouse models (Peça et al., 2011; Chao et al., 2018) and MK-801-treated zebrafish (Perdikaris and Dermon, 2022) exhibit both decreased sociability as well as increased anxiety levels. Moreover, experimental manipulations that facilitate social behaviors tend to diminish anxiety levels, suggesting that certain subsets of these two pathologies may arise from common neural mechanisms. Since the disturbance of E/I balance in various brain regions can also promote anxiety-like behavior (Yuen et al., 2012; Ghosal et al., 2020; Yu et al., 2020), it is worth investigating the specific synaptic mechanisms that contribute to E/I imbalance after NMDAR hypofunction, linking social withdrawal with anxiety-like behavior.

Previous studies have demonstrated that altered inflammatory responses are associated with various neuropsychiatric disorders including anxiety and ASD (El-Ansary and Al-Ayadhi, 2014; Masi et al., 2017; Zheng et al., 2021), whereas also the levels of both pro-inflammatory and anti-inflammatory cytokines were associated with symptoms severity (Ashwood et al., 2011; Masi et al., 2017). Interestingly, interleukin 1β (IL-1β) has been shown to induce anxiety-like behavior in mice by interacting with endocannabinoid binding receptors (Rossi et al., 2012) as well as to be implicated in the etiology of ASD by contributing to the E/I imbalance (El-Ansary and Al-Ayadhi, 2014). Also, the implication of IL-1β in influencing E/I balance is further supported by the manifestation of social withdrawal phenotype following the activation of IL-1 receptor 1 (IL-1R1), located on glutamatergic neurons in hippocampus (DiSabato et al., 2021). While evidence supports that altered cytokine profile contributes to social withdrawal and anxiety-like phenotype, by regulating synaptic structure and function (Pozzi et al., 2018), further research is required to better understand the relationship between E/I imbalance and excessive inflammatory responses in the above comorbidities.

The zebrafish (Danio rerio) due to its well characterized repertoire of social and anxiety-like behavior and the fact that a high proportion of disease-related genes in humans possess at least one zebrafish orthologue (Howe et al., 2013), provide an attractive model to investigate the pathophysiological mechanisms that contribute to social deficits and anxiety-like behavior. Decision-making processes that are related to social behavior are regulated by the social-decision making network (SDMN), an evolutionary conserved network consisting of two interconnected circuits, the social behavior network and the mesolimbic reward system (O'Connell and Hofmann, 2011). This network is highly conserved between mammals and zebrafish sharing high structural and circuit homology thus giving the opportunity to investigate and identify specific neural circuits characterized by altered E/I balance, associating aberrant region activity with impairments in social function and anxiety-like behavior.

In the current study we used a zebrafish model displaying NMDAR hypofunction, after sub-chronic MK-801 administration in order to assess the possible influences of impaired glutamatergic and GABAergic neurotransmission in the manifestation of social withdrawal and anxiety-like behavior. Also, in order to associate impaired synaptic plasticity across the SDMN with the comorbidity of social impairment and increased anxiety levels, we analyzed the expression of IL-1β in different nodes within the SDMN to link altered cytokine profile with excitatory/inhibitory ratio. Moreover, for the first time in zebrafish, we questioned the involvement of endocannabinoid receptor 1 (CB1R), as an additional mechanism contributing to the neurochemical profile, based on the crucial role of endocannabinoid signaling in regulating both excitatory and inhibitory neurotransmission (Chevaleyre et al., 2006).

The present study advanced previous evidence (Perdikaris and Dermon, 2022) on MK-801-treated zebrafish characterized by impaired social communication together with increased anxiety levels, a common comorbid psychiatric symptom in individuals diagnosed with several neuropsychiatric disorders, such as ASD and schizophrenia (Top et al., 2016; Hall, 2017). Additionally, at the molecular level these behavioral defects were accompanied by altered expression levels of different proteins associated with glutamatergic and GABAergic neurotransmission, thus implying the possible presence of E/I imbalance at synaptic or circuit level in MK-801-treated fish. Such imbalance is suggested to represent a common pathophysiological mechanism implicated in the etiology of social deficits (Gao and Penzes, 2015). Importantly, the dysregulated GABAergic and glutamatergic neurotransmission was accompanied by increased telencephalic expression of CB1R protein levels and increased density of IL-1β⁺ cells in specific forebrain and midbrain nuclei, also highlighting the presence of endocannabinoid and immune system dysregulation. Τhis is of particular significance since several studies, both in humans as well as in rodents have reported alterations in endocannabinoid signaling as well as immune dysfunction in various conditions characterized by social impairment (Careaga et al., 2017; Zou et al., 2021; Robinson-Agramonte et al., 2022), thus further validating this zebrafish pharmacological model as a useful model for studying the mechanisms involved in social dysfunction.

MK-801-treated zebrafish displayed robust social deficits as indicated by the decreased sociability index. More specifically, MK-801-treated fish did not show any characteristic preference for the social zone compared to the non-social compartment, supporting previous findings of impaired social interaction in rodents and zebrafish after acute and sub-chronic MK-801 administration (Rung et al., 2005; Gururajan et al., 2012; Zimmermann et al., 2016; Perdikaris and Dermon, 2022). Although deficits in social communication commonly co-occur with increased anxiety, a possible common neural mechanism remains largely unclear, with studies reporting that the “basolateral amygdala-ventral hippocampus” circuitry is involved in regulating both behaviors (Felix-Ortiz and Tye, 2014). Similarly, MK-801-treated zebrafish displayed increased anxiety levels as indicated by their increased thigmotaxis index in the OFT, a well-validated index of fear and anxiety in zebrafish (Maximino et al., 2010b). Moreover, the increased latency of MK-801-treated fish to enter the central compartment as well as the decreased center entries serve as further indicators of their centrophobic behavior. Previous studies are consistent with this view, since NMDA receptor hypofunction after MK-801 administration, caused heightened anxiety state in male rats, as indicated by increased thigmotactic behavior (Feinstein and Kritzer, 2013) or the presence of hypoactivity and decreased exploratory behavior (Latysheva and Rayevsky, 2003) in the OFT.

In order to have a better understanding on zebrafish anxiety state, we analyzed their behavior in the DLT, a well-established paradigm for estimating anxiety-levels based on a conflict between their preference for protected areas and their innate motivation to explore new environments (Maximino et al., 2010a). Our results indicated that MK-801-treated fish spent increased time in the light compartment compared to control group, as indicated by the dark–light index. Although, most of the studies have interpreted this behavior as anxiolytic (Lau et al., 2011; Duarte et al., 2019; Fontana et al., 2022) other findings characterize dark-avoidance behavior as an anxiety-like response (Champagne et al., 2010). The latter may appear paradoxical, but one must consider that zebrafish are diurnal species and unlike nocturnal rodents, moving to a lit area may be preferable in order to avoid predators (Ziv et al., 2013). It should also be noted that our control group displayed a characteristic dark preference/light avoidance behavior similar to other studies reporting the innate preference of zebrafish for dark vs. light environments (Maximino et al., 2010a). In an attempt to carefully elucidate this, we propose that the observed light preference of MK-801-treated zebrafish, accompanied by thigmotactic behavior, may indicate an adaptive risk assessment behavior, an anxiety-related response. Risk assessment behavior is based on individual’s inability to determine if there is a threatening situation or environment and subsequent HPA axis activation (Blanchard et al., 1990; Blanchard, 2018). In agreement, a previous study suggested that the exploration of white compartment in the DLT is primarily driven by fear/anxiety and not novelty, due to the absence of intrasession habituation (Maximino et al., 2010a). Also, the anxiolytic properties of isoflavones on zebrafish were demonstrated in another study, after decreasing their thigmotactic behavior in the light compartment (de Melo et al., 2020). Increased number of risk-assessment behaviors were also observed in BTBR mice, an animal model of ASD also characterized by impaired sociability and high anxiety (Chao et al., 2018). In line with the above, adult zebrafish that were subjected to social isolation during their early-life stages displayed a characteristic dark avoidance behavior that could not be interpreted as an anxiolytic response, suggesting the inability to develop stress-associated arousal state (Varga et al., 2020). Our perspective that MK-801-treated zebrafish are characterized by a heightened anxiety-state, is further reinforced by their diminished weight gain, a response that is reported in rodents and zebrafish after prolonged stress exposure (Riaz et al., 2015; Fulcher et al., 2017). In support, MK-801-treated zebrafish were previously shown to exhibit aspects of anxiety-like behavior in the novel tank test (Perdikaris and Dermon, 2022). Although new behavioral endpoints and may provide additional information about the behavioral phenotype of MK-801 treated fish, our data support the presence of a robust social withdrawal and anxiogenic profile, characterizing many neuropsychiatric disorders.

E/I imbalance is considered an important pathophysiological mechanism contributing to social withdrawal behavior (Rubenstein and Merzenich, 2003; Gao and Penzes, 2015). Increased evidence highlights the presence of glutamatergic dysfunction in the development of many neuropsychiatric disorders, characterized by social impairments and increased anxiety (Nasir et al., 2020; Nisar et al., 2022). In support, the imbalance of Ε/Ι in synaptic or circuit levels has been reported in several NMDA receptor hypofunction models (Flores-Barrera et al., 2020; Jami et al., 2021). In the present study, western blot results demonstrated alterations in brain neurochemistry regarding the function and stabilization of excitatory and inhibitory synapses, possibly contributing to alterations in E/I balance. Specifically, the protein expression levels of mGluR5 were significantly increased in the telencephalon and midbrain of MK-801-treated fish whereas PSD-95 protein expression levels were significantly decreased in both regions. In agreement, the pivotal role of mGluR5 signaling in social withdrawn behavior has been previously introduced in zebrafish (Perdikaris and Dermon, 2022). Studies using ASD-associated Shank3 knockout mice, that exhibit social deficits, demonstrated disrupted ability of hippocampal neurons to express mGluR1 and mGluR5, and impaired mGluR-dependent long-term depression (LTD; Lee et al., 2019), as well as increased mGluR5 expression levels in hippocampus, thalamus, and amygdala (Cai et al., 2019). On the other hand, there is increasing evidence that the mGluR5 agonists or the mGluR5 positive allosteric modulator, enhanced social interaction by normalizing NMDA receptor function in Shank2 mutant mice (Won et al., 2012), thus implying that deviation of mGluR5 activity in either direction leads to social impairments. Our results further validate the increased mGluR5 expression in telencephalon and midbrain of MK-801-treated fish, possibly indicating mGluR5 hyperactivity and that administration of mGluR5 antagonists may have beneficial effects in restoring social function.

Evidence of compromised glutamatergic neurotransmission is further supported by the downregulation of PSD-95 in the telencephalon and midbrain of MK-801-treated zebrafish. Prior studies demonstrated that PSD-95 is involved in the molecular organization of the PSD (Chen et al., 2011) and synapse stabilization by regulating the recruitment and trafficking of NMDARs and AMPARs in the postsynaptic membrane (Chen et al., 2015). Indeed, disruption of PSD-95 expression is reported in various neuropathologies that are characterized by social impairment (Matosin et al., 2016; Coley and Gao, 2018) as well as in anxiety-related responses (Feyder et al., 2010; Wang et al., 2020). More specifically, a significant decrease in PSD-95 mRNA and protein expression levels was reported in prefrontal cortex and CA1 hippocampal region in postmortem brain samples from schizophrenic patients (Ohnuma et al., 2000; Matosin et al., 2016). We suggest that the decreased expression of PSD-95 in telencephalon and midbrain of MΚ-801-treated fish observed here, may be related to an overall reduction of NMDAR and/or AMPAR in the postsynaptic membranes, leading to aberrant PSD synaptic activity.

Increasing evidence supports that dysfunction of inhibitory neurotransmission is implicated in the pathophysiology of several neuropsychiatric disorders characterized by social impairment and anxiety (Fogaça and Duman, 2019; Zhao et al., 2021). To assess whether MK-801-treated zebrafish were characterized by GABAergic system dysregulation, we analyzed the expression of GAD1 (44 kDa), an enzymatically active isoform of GAD1 that catalyzes the conversion of L-glutamic acid into the neurotransmitter GABA (Trifonov et al., 2014). Although previous studies reported decreased mRNA and protein expression levels of GAD2 and GAD1 in cerebellum and parietal cortex in autistic individuals (Fatemi et al., 2002; Yip et al., 2007) or reduced GABA_A receptor binding in hippocampus and cingulate cortex (Blatt et al., 2001; Oblak et al., 2009), in the present study the protein expression levels of GAD1 (44 kDa) were significantly increased in the telencephalon of MK-801-treated fish. Our results, despite the fact that they do not appear to be in the same direction with previous studies reporting suppressed GABAergic inhibition in ASD or schizophrenia (Banerjee et al., 2013; Xu and Wong, 2018), highlight a more complex picture since enhanced glutamatergic activation and GABAergic inhibition in different microcircuits in specific telencephalic regions of MK-801-treated fish may contribute to social impairments and increased anxiety. In agreement with our results, previous studies reported increased GABAergic activity in striatum of rodent ASD and/or fragile X syndrome model (Centonze et al., 2008; Horder et al., 2018), a finding that is indicative of the heterogeneity of mechanisms that contribute to social deficits. It is possible that the increased expression of GAD1 (44 kDa) in the telencephalon of MK-801-treated fish serves as a compensatory mechanism to increased mGluR5 signaling. Indeed, a high GAD1 and mGluR5 co-expression pattern was observed in various SDMN nodes, such as Dm, Vv, PPa, PPp/PM and Hv, supporting the hypothesis that glutamate may influence the typical GABAergic firing pattern, via mGluR5 activation. In support, GAD1 expression is known to be modulated by neuronal activity (Patz et al., 2003; Lau and Murthy, 2012), therefore the observed upregulation of GAD1 protein expression, may represent a homeostatic mechanism to counteract the increased excitability of projection neurons and thus control network activity. While the present study cannot elucidate whether MK-801 administration does indeed contribute to E/I imbalance, we hypothesize that NMDAR hypoactivity in GABAergic interneurons and/or glutamatergic projection neurons (Bygrave et al., 2019) may lead to a compensatory increase in mGluR5 and GAD1 expression and possibly to changes in circuit excitability. Although, previous studies emphasize the role of NMDAR hypofunction specifically on PV⁺ interneurons in reduced sociability (Saunders et al., 2013; Cao et al., 2022), the contribution of mGluR5 and GAD1 co-expressing cells within nodes of SDMN may underlie the comorbidity between social deficits and anxiety-like behaviors, observed in the present study.

In addition, CB1Rs expression in zebrafish SDMN and their possible involvement in the manifestation of social withdrawal and anxiety-like behavior, was addressed in the present study, taking into account that the endocannabinoid system regulates excitatory and inhibitory synaptic transmission by mediating short- and long-term of plasticity at glutamatergic and GABAergic synapses (Chevaleyre et al., 2006). Indeed, evidence is provided on the presence of disrupted endocannabinoid signaling as indicated by the increased protein expression levels of CB1R in the telencephalon of MK-801-treated zebrafish, in agreement to previous findings reporting molecular alterations in components of endocannabinoid signaling in several psychiatric conditions characterized by social impairment (Zou et al., 2021) as well as in anxiety disorders (Maldonado et al., 2022). The increased protein expression levels of CB1R in the telencephalon of MK-801-treated fish may serve as homeostatic mechanism to decreased endocannabinoid signaling, as previously described in ASD children and VPΑ-treated rats (Aran et al., 2019; Zou et al., 2021). Interestingly, a previous study reported that Fmr1 knockout mice, that are characterized by excessive mGluR5 signaling (Mody et al., 2021), displayed diminished 2-arachidonoyl-sn-glycerol (2AG)-dependent LTD at excitatory synapses of mouse forebrain (Jung et al., 2012). Moreover, the endocannabinoid system controls the activity of NMDARs (Sánchez-Blázquez et al., 2014) and as a consequence altered endocannabinoid signaling may cause excessive NMDAR hypofunction. Importantly, in the present study, the localization of CB1Rs in astroglial cells within forebrain regions belonging to the SDMN raises the possibility that may influence astrocyte activity, thus regulating the Ca²⁺-dependent gliotransmitter release and in turn modulating neuronal excitability, as an active part of the “tripartite synapse” concept (Perea et al., 2009). A recent study by Wang et al., supports the above assumptions since impaired Ca²⁺ signaling in astrocytes resulted in diminished release of ATP and contributed to ASD-like behavior (Wang et al., 2021). Considering the above, the identification of specific neural circuits characterized by aberrant E/I balance as well as altered endocannabinoid signaling may provide important synaptic endpoints of impaired neurotransmission in order to link social deficits with increased anxiety levels.

Importantly, the presence of excessive IL-1β expression in MK-801-treated fish, as indicated by the increased number of IL-1β⁺ cells colocalized with NeuN or GFAP, within the SDMN is of particular significance. In contrast to the neuronal or astroglial phenotype of IL-1β⁺ cells, only a small proportion expressed OX-42 in Dm and SRF, suggesting a minor contribution of activated microglia in the enhanced IL-1β expression within the SDMN. The contribution of neutrophils and monocytes also cannot be excluded since they also express OX-42 (Jeong et al., 2013). In agreement, excessive neuroinflammation is an element of neuropathology across many neuropsychiatric disorders characterized by social impairment and increased anxiety (El-Ansary and Al-Ayadhi, 2014; Müller et al., 2015). More specifically, IL-1β has been reported to be elevated in plasma of children and adults with ASD (Ashwood et al., 2011; Masi et al., 2017) as well as in the brain of schizophrenic patients (Söderlund et al., 2009) whereas enhanced IL-1β expression has also been implicated in anxiety-like responses in rodents (Rossi et al., 2012) and zebrafish (Song et al., 2018). Our results support the view of a dysregulated proinflammatory state in MK-801 treated fish, that is mediated by neurons and astrocytes and are consistent with a previous study reporting increased IL-1β immunoreactivity in glia cells and neurons, in structures of limbic system under stress conditions (Badowska-Szalewska et al., 2009). Similarly, increased IL-1β expression, specifically in neuronal populations of hippocampal CA1 region, striatum and paraventricular nucleus were reported in mice subjected to repeated immobilization stress (Kwon et al., 2008). Several studies have begun to investigate the non-immunological role of IL-1β, indicating that IL-1β regulates neuronal plasticity by impairing LTP in several regions of hippocampus and regulating neurotransmitter release (Merali et al., 1997; Hoshino et al., 2017) or affecting synapse structure and function, by decreasing PSD-95 expression and frequency of miniature excitatory postsynaptic currents (mEPSCs), through a MeCP2 dependent mechanism (Tomasoni et al., 2017). There is also increased evidence that excessive IL-1β disrupts the E/I balance, both by enhancing glutamatergic transmission (Mandolesi et al., 2013) or by delaying excitatory to inhibitory switch of GABA in the offspring of a maternal immune activation mouse model (Corradini et al., 2018). Therefore it is important to further link neural circuits displaying aberrant activity, with excessive IL-1β expression within SDMN regions, in MK-801-treated fish. Toward that direction, Dm a putative homologous to the mammalian basolateral amygdala (BLA) and PM, a part of the preoptic area, are key nodes in the SDMN (O'Connell and Hofmann, 2011), regulate both social behavior (Bickart et al., 2014; Nunes et al., 2021) as well as fear and anxiety-related responses (Lal et al., 2018; Chuang et al., 2021), so it is logical to considered as targets of great importance. The enhanced expression of IL-1β may alter synaptic plasticity in these brain regions, contributing to social impairments and anxiety-like behavior, observed in MK-801-treated fish. Similarly, it was recently shown that excessive inflammatory response induced anxiety and depressive-like behavior by increasing the excitability of BLA glutamatergic neurons (Zheng et al., 2021). Consistent with this interpretation, excessive IL-1β production in Dm was positively correlated with thigmotactic behavior, providing further evidence about the implication of this region in regulating anxiety responses. Moreover, heightened inflammatory responses in SRF, a region implicated in regulation of arousal states (Faraguna et al., 2019), may contribute to the behavioral profile of MK-801-treated fish, since abnormally elevated arousal is associated with stress and anxiety (Pfaff et al., 2007). Indeed, IL-1β expression in SRF was positively correlated with social withdrawal and anxiety-like behavior, supporting the notion that SRF may be part of a common network regulating both behaviors.

While we did not extensively determine the phenotype of IL-1β + cells, our data regarding the localization of β₂-adrenoceptors (β₂-ARs) in the IL-1β expressing neurons or astrocytes, raise the possibility of a direct noradrenergic regulation of IL-1β production. Such noradrenergic influence has been reported via modulation of the cAMP cell levels (Bourne et al., 1974; Vizi, 1998). The induction of IL-1β by β₂-AR signaling has been previously observed in a mouse model of social defeat stress, where the increased expression of IL-1β mRNA in microglia cells was prevented by a β-ΑR antagonist (Wohleb et al., 2011). The above interpretation does not exclude the possibility that additional neurotransmission systems, such as glutamate, are implicated in excessive proinflammatory responses, since previous studies reported increased IL-1β production following prolonged neuronal activity (Del Rey et al., 2016). However, the present data combined with previous evidence on increased β₂-AR protein expression levels in the forebrain and midbrain of MK-801-treated zebrafish (Perdikaris and Dermon, 2022) support a possible contribution of noradrenergic neurotransmission in the excessive IL-1β expression. Nevertheless, further studies are needed in order to investigate the above assumption and associate it with the comorbidity between social deficits and increased anxiety levels.

In the present study the possible presence of dysregulated of excitatory and inhibitory neurotransmission is highlighted in forebrain and midbrain of a zebrafish pharmacological model, displaying robust social impairment and increased anxiety-like behavior. Specifically, glutamatergic dysfunction was positively associated with social withdrawal behavior whereas impaired GABAergic signaling was linked with anxiety-like behavior. In addition, altered endocannabinoid signaling accompanied the manifestation of these behaviors, suggesting the possible regulatory role of CB1Rs in astrocytes.

Moreover, the presence of excessive IL-1β expression in neurons and astroglia, within key nodes of the SDMN, as well as the colocalization with β₂-ARs, is suggested to serve as an additional pathophysiological mechanism contributing to social withdrawal and anxiety-like behavior. Evidence provided here using zebrafish, confirm and expand previous rodents’ studies providing an additional insight into the putative contribution of altered E/I balance and exaggerated proinflammatory response in the pathophysiology of social deficits and anxiety-like behavior.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

All experimental procedures were approved by the Ethics Committee of University of Patras and were in accordance with the European Communities council directive (86/609/EEC) for the care and use of laboratory animals. All efforts were made to minimize animal suffering and to reduce the number of animals used.

PP and CD conceived and designed experiments and wrote and reviewed the manuscript. PP performed the experiments and analyzed the data. All authors contributed to the article and approved the submitted version.

Part of the work was supported by the Hellenic Foundation for Research and Innovation (HFRI) scholarship to PP. The publication fees of this manuscript have been financed by the Research Council of the University of Patras.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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