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\begin{document}

\title{Brief exposure to enriched environment rapidly shapes the glutamate
synapse in the rat brain: a metaplastic fingerprint}



\author[1]{Nicholas Pintori}%
\author[1]{Alessandro Piva}%
\author[2]{Francesca Mottarlini}%
\author[2]{Fernando Castillo Díaz}%
\author[2]{Coralie Maggi}%
\author[2]{Lucia Caffino}%
\author[2]{Fabio Fumagalli}%
\author[1]{Christian Chiamulera}%
\affil[1]{University of Verona}%
\affil[2]{University of Milan}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Environmental enrichment (EE) has been shown to produce beneficial
effects in addiction disorders; however, due to its configurational
complexity, the underlying mechanisms are not yet fully elucidated.
Recent evidence suggests that EE, acting as a metaplastic agent, may
affect glutamatergic mechanisms underlying appetitive memory and, in
turn, modulate reward-seeking behaviors: here, we have investigated such
possibility following a brief EE exposure. Adult male Sprague-Dawley
rats were exposed to EE for 22h and the expression of critical elements
of the glutamate synapse was measured 2h after the end of EE in the
medial prefrontal cortex (mPFC), nucleus accumbens (NAc), and
hippocampus (Hipp) brain areas, which are critical for reward and
memory. We focused our investigation on the expression of NMDA and AMPA
receptor subunits, their scaffolding proteins SAP102 and SAP97,
vesicular and membrane glutamate transporters vGluT1 and GLT-1, and
critical structural components such as proteins involved in morphology
and function of glutamatergic synapses, PSD95 and Arc/Arg3.1. Our
findings demonstrate that a brief EE exposure induces metaplastic
changes in glutamatergic mPFC, NAc, and Hipp. Such changes are
area-specific and involve postsynaptic NMDA/AMPA receptor subunit
composition, as well as changes in the expression of their main
scaffolding proteins, thus influencing the retention of such receptors
at synaptic sites. Our data indicate that brief EE exposure is
sufficient to dynamically modulate the glutamatergic synapses in
mPFC-NAc-Hipp circuits, which may modulate rewarding and memory
processes.%
\end{abstract}%



\sloppy


\textbf{Title}

\textbf{Brief exposure to enriched environment rapidly shapes the
glutamate synapse in the rat brain: a metaplastic fingerprint}

\selectlanguage{ngerman}\textbf{Nicholas Pintori \textsuperscript{1,3}, Alessandro
Piva\textsuperscript{1}, Francesca Mottarlini \textsuperscript{2},
Fernando Castillo Díaz \textsuperscript{2}, Coralie Maggi
\textsuperscript{2}, Lucia Caffino \textsuperscript{2}, Fabio
Fumagalli\textsuperscript{2\#}, Cristiano Chiamulera
\textsuperscript{1\#}}

\textbf{Short title: Glutamate synaptic changes induced by brief EE}

\textbf{Affiliations}

\textsuperscript{1}Section of Pharmacology, Department of Diagnostic \&
Public Health, University of Verona, Verona, Italy

\textsuperscript{2}Department of Pharmacological and Biomolecular
Science `Rodolfo Paoletti', University of Milan, Via Balzaretti 9,
20133, Milan, Italy

\textsuperscript{3}Current Affiliation: Department of Biomedical
Sciences, University of Cagliari, Cittadella Universitaria di
Monserrato, 09042, Monserrato, Cagliari, Italy

\textsuperscript{\#}Cristiano Chiamulera and Fabio Fumagalli share the
seniorship

\textbf{Corresponding author}

Nicholas Pintori, Section of Pharmacology, University of Verona,
Policlinico `GB Rossi', P.le Scuro 10, 37134, Verona, Italy.

Email:\emph{nicholas.pintori@univr.it}

Phone: +39045 8027223

\textbf{Acknowledgements:} Not applicable



\textbf{Abstract}

Environmental enrichment (EE) has been shown to produce beneficial
effects in addiction disorders; however, due to its configurational
complexity, the underlying mechanisms are not yet fully elucidated.
Recent evidence suggests that EE, acting as a metaplastic agent, may
affect glutamatergic mechanisms underlying appetitive memory and, in
turn, modulate reward-seeking behaviors: here, we have investigated such
possibility following a brief EE exposure.

Adult male Sprague-Dawley rats were exposed to EE for 22h and the
expression of critical elements of the glutamate synapse was measured 2h
after the end of EE in the medial prefrontal cortex (mPFC), nucleus
accumbens (NAc), and hippocampus (Hipp) brain areas, which are critical
for reward and memory. We focused our investigation on the expression of
NMDA and AMPA receptor subunits, their scaffolding proteins SAP102 and
SAP97, vesicular and membrane glutamate transporters vGluT1 and GLT-1,
and critical structural components such as proteins involved in
morphology and function of glutamatergic synapses, PSD95 and Arc/Arg3.1.

Our findings demonstrate that a brief EE exposure induces metaplastic
changes in glutamatergic mPFC, NAc, and Hipp. Such changes are
area-specific and involve postsynaptic NMDA/AMPA receptor subunit
composition, as well as changes in the expression of their main
scaffolding proteins, thus influencing the retention of such receptors
at synaptic sites.

Our data indicate that brief EE exposure is sufficient to dynamically
modulate the glutamatergic synapses in mPFC-NAc-Hipp circuits, which may
modulate rewarding and memory processes.

\textbf{Keywords}

Environmental Enrichment, glutamatergic synapses, metaplasticity,
reward, learning, memory

\textbf{1 Introduction}

Environmental enrichment (EE) has been shown to produce beneficial and
therapeutic effects in several preclinical models of central nervous
system disorders. Due to a combination of social, cognitive, and
sensorimotor stimulations, EE leads to a variety of positive effects,
ranging from cellular and molecular (e.g., neurogenesis, changes in the
expression of genes and receptors subunits, neurotransmitters and BDNF
levels) to cognitive and behavioral changes (e.g., enhanced learning and
memory performance, altered emotional state), in both health and disease
models (Falkenberg \emph{et al.} , 1992; Kempermann \emph{et al.} ,
1997; Moser \emph{et al.} , 1997; Chapillon \emph{et al.} , 1999; Young
\emph{et al.} , 1999; Rampon \emph{et al.} , 2000a; Rampon \emph{et al.}
, 2000b; van Praag \emph{et al.} , 2000; Roy \emph{et al.} , 2001; Tang
\emph{et al.} , 2001; Lee \emph{et al.} , 2003; Benaroya-Milshtein
\emph{et al.} , 2004; Leggio \emph{et al.} , 2005).

EE-induced neuroplasticity is also involved in the protective and
curative effects of addiction disorders, with experimental evidence
suggesting stress-related mechanisms as possible targets of the
anti-addictive effect of EE (Solinas \emph{et al.} , 2010;
Crofton\emph{et al.} , 2015). However, given the configurational
complexity of environmental stimulation, the underlying mechanisms still
need further investigation.

EE exposure duration and components (i.e., social, sensorimotor,
cognitive) seems a critical parameter for its effects. Indeed, although
chronic and acute exposure to a complex EE (with social, cognitive, and
sensory-motor components) reduces drug/sucrose-taking and -seeking
behaviors (Grimm \emph{et al.} , 2008; Solinas \emph{et al.} , 2008;
Grimm\emph{et al.} , 2013; Grimm \emph{et al.} , 2016; Margetts-Smith
\emph{et al.} , 2021), we however recently demonstrated that a brief EE
exposure in rats (22 hours, without motor and social components)
potentiates conditioned context (Cx)-induced sucrose-seeking (a
phenomenon called\emph{renewal} ) and Cx-memory reconsolidation after
reactivation (Pintori \emph{et al.} , 2022a).

Thus, the wide variety and complexity of EE features (e.g., length of
exposure, location, type of stimulation, and many others) limited the
translation into clinical practice so far. Therefore, understanding the
mechanisms underlying acute brief EE manipulation could be useful to
develop new and innovative configurations of EE - with translational
value and feasibility - to improve existing therapeutic approaches
against addiction and addiction-related CNS disorders.

Based on data about EE-induced enhancement of memory and learning
abilities {[}13,26, 87{]}, we suggested that this brief EE exposure may
act as a \emph{proactive interference agent} , influencing subsequent
memory processes (i.e., renewal, Cx-memory reactivation/reconsolidation)
(Pintori \emph{et al.} , 2022a). From a neurobiological point of view,
this proactive action might potentially act as a \emph{metaplastic
effect} (defined as ``the plasticity of synaptic plasticity'' (Abraham
\& Bear, 1996)), as recently suggested by electrophysiological evidence
(Eckert \& Abraham, 2013; Schmidt \emph{et al.} , 2013;
Chiamulera\emph{et al.} , 2020). Importantly, the ability of EE to
affect long-term potentiation (LTP) and long-term depression (LTD) seems
related to alterations of glutamatergic signaling particularly at the
level of NMDA and AMPA receptors (Duffy \emph{et al.} , 2001; Foster \&
Dumas, 2001; Li\emph{et al.} , 2006; Thomas \emph{et al.} , 2008; Eckert
\emph{et al.} , 2010).

In the present paper, we aim to investigate whether brief EE exposure
influences the homeostasis of the glutamate synapse that could be
correlated to the behavioral effects previously reported
(Pintori\emph{et al.} , 2022a). To this end, adult male Sprague-Dawley
rats were exposed to EE for 22h, and two hours after the end of EE
exposure we analyzed the expression of glutamate determinants such as:
vesicular glutamate transporter (vGluT1), which participates in the
regulation of presynaptic glutamate release and glial glutamate
transporter (GLT-1), which is responsible for glutamate reuptake,
together with the expression of the main subunits of NMDA (GluN1,
GluN2A, GluN2B) and AMPA (GluA1 and GluA2) receptors and their related
scaffolding protein SAP102 (synapse-associated protein 102), SAP97
(synapse-associated protein 97) and GRIP (glutamate receptor interacting
protein). Further, we set out to explore the potential structural
effects of brief EE exposure by measuring the expression levels of the
integral protein of the glutamate synapse post-synaptic density protein
95 (PSD95) and the cytoskeletal protein activity-regulated
cytoskeleton-associated protein (Arc/Arg3.1). These analyses were
performed in the medial prefrontal cortex (mPFC), nucleus accumbens
(NAc), and hippocampus (Hipp), both in the whole homogenate, which
provides information essentially about translational changes, as well as
in the post-synaptic density (PSD), which, instead, informs primarily
about the synaptic localization and composition of critical determinants
of the glutamatergic synapse.

\section*{2 Material and methods}

{\label{material-and-methods}}

\subsection*{}

{\label{section}}

\subsection*{2.1 Animals}

{\label{animals}}

Twelve adult male Sprague-Dawley rats (Charles River, Italy) 275-300g
were single-housed in temperature and humidity-controlled environment
(19\selectlanguage{english}-23 degC, 60 +- 20 \%) on a 12-h light/dark cycle, with light ON at 7:30
pm. Water and food were available \emph{ad libitum} . All animal care
and experimental procedures are reported in compliance with the European
Union regulations and Directive 2010/63/EU and were approved by the
ethical committee (OPBA) of the University of Verona and by the Ministry
of Health (authorization n. 780/2019-PR).

\subsection*{2.2 Brief Environmental Enrichment
exposure}

{\label{brief-environmental-enrichment-exposure}}

After two weeks of acclimatisation period in the animal facility, half
of the rats were exposed for 22 h to Environmental Enrichment (EE
group). EE consisted of a novel housing cage (35.6 x 48.5 x 21.8 cm,
Optirat Gen II, Animal Care System), where rats were single-housed
(i.e., no social component) with various objects (toys with different
materials, shapes, and colours, i.e., plastic balls and ladders, wood
bricks) for sensory stimulation. The second half of the rats did not
receive Environmental Enrichment exposure (NoEE group).

\subsection*{2.3 Western Blot Assays}

{\label{western-blot-assays}}

Two hours after the end of EE exposure, rats were sacrificed, the brains
were rapidly removed, and following the brain atlas of Paxinos and
Watson (Paxinos \& Watson, 2007), 1-mm thick slices containing the
medial prefrontal cortex (mPFC, bregma +3.20 mm), the nucleus accumbens
(NAc, bregma +1.70 mm), and the hippocampus (Hipp, bregma -3.30 mm) were
dissected by using a 1-mm Coronal Brain Matrix (SouthPointe Surgical
Supply, Florida, USA), frozen on dry ice and stored at \selectlanguage{english}-80 degC. After the
dissection of brain areas, proteins were isolated and analyzed in the
whole homogenate and PSD as previously described with minor
modifications (Piva \emph{et al.} , 2020). Briefly, mPFC, NAc, and Hipp
were homogenized in a Teflon-glass potter in cold 0.32 M sucrose buffer
pH 7.4 containing 1 mM HEPES, 1 mM MgCl\textsubscript{2}, 1 mM
NaHCO\textsubscript{3,} and 0.1 mM PMSF, in presence of commercial
cocktails of protease (Roche, Monza, Italy) and phosphatase
(Sigma-Aldrich, Milan, Italy). An aliquot of each homogenate was then
sonicated and stored at -20degC. The remaining homogenate was centrifuged
at 800 g for 5 min; the obtained supernatant was then centrifuged at
13000 g for 15 min, obtaining a pellet. This pellet was resuspended in a
buffer containing 75 mM KCl and 1\% Triton X-100 and centrifuged at
100000 g for 1 h. The resulting supernatant, referred as Triton X-100
soluble fraction (TSF, extra-synaptic fraction), was stored at -20degC;
the pellet, referred as PSD or Triton X-100 insoluble fraction (TIF,
post-synaptic density), was homogenized in a glass--glass potter in 20
mM HEPES, protease and phosphatase inhibitors and stored at -20degC in
presence of glycerol 30\%. Total proteins have been measured in the
homogenate and TIF fractions according to the Bradford Protein Assay
procedure (Bio-Rad, Milan, Italy), using bovine serum albumin as
calibration standard.

Western blots were run as previously described (Mottarlini \emph{et al.}
, 2022). Briefly, equal amounts of proteins of the homogenate (10 \selectlanguage{greek}μ\selectlanguage{english}g)
and TIF fraction (8 \selectlanguage{greek}μ\selectlanguage{english}g) were run on a sodium dodecyl sulfate-8\%
polyacrylamide gel under reducing conditions and then
electrophoretically transferred onto nitrocellulose membranes (GE
Healthcare, Milan, Italy). Blots were blocked for 1 h at room
temperature with I-Block solution (Life Technologies Italia, Italy) in
TBS 0.1\% Tween-20 buffer and incubated with antibodies against the
proteins of interest. The conditions of the primary antibodies were the
following: anti-vGLUT-1 (1:1000, Cell Signaling Technology Inc. RRID:
AB\_2797887), anti-GLT-1 (1:5000, AbCam. RRID: AB\_1566262), anti-GluN1
(1:1000, Cell Signaling Technology Inc. RRID: AB\_659874), anti-GluN2B
(1:1000, Cell Signaling Technology Inc; RRID: AB\_1264223), anti-GluN2A
(1:1000, Cell Signaling Technology Inc. RRID: AB\_2112295), anti-SAP102
(1:1000, AbCam; RRID: AB\_1860292), anti-GluA1 (1:1000, Cell Signaling
Technology Inc. RRID: AB\_2732897), anti-GluA2 (1:2000, Cell Signaling
Technology Inc. RRID: AB\_10622024), anti-SAP97 (1:1000, AbCam),
anti-PSD95 (1:2000, Cell Signaling Technology Inc. RRID: AB\_2292883),
anti-GRIP (1:1000, Synaptic System. RRID: AB\_887728), anti-Arc/Arg3.1
(1:500, BD Transduction Lab. RRID: AB\_399886), and anti-\selectlanguage{greek}β-\selectlanguage{english}Actin
(1:10000, Sigma-Aldrich. RRID: AB\_476744). Results were standardized
using \selectlanguage{greek}β-\selectlanguage{english}actin as the control protein, which was detected by evaluating
the band density at 43 kDa. Each set of proteins shown in Figures 1-2-3
was run in the same WB assay, thus only one band of \selectlanguage{greek}β-\selectlanguage{english}Actin is
presented. Immunocomplexes were visualized by chemiluminescence using
the Chemidoc MP Imaging System (Bio-Rad Laboratories). Gels were run 3
times each and the results represent the average from 3 different
western blots, averaged and normalized by using a specific correction
factor (Caffino \emph{et al.} , 2020). Cropped immunoblots are reported
in Supplementary Information (SI) (Fig.S1-6).

\subsection*{2.4 Statistical analysis}

{\label{statistical-analysis}}

All the numerical data are given as mean \selectlanguage{ngerman}± SEM. Data were tested for
normal distribution using Shapiro--Wilk's test. Data were collected in
individual animals (independent determinations) and molecular changes
produced by Environmental Enrichment exposure were analyzed separately
for each brain area using unpaired Student's t-test. Non-parametric test
(i.e., Mann-Whitney U-test) was chosen when data were found not to be
normally distributed, while t-test with Welch's correction was chosen
when there was significant variance in homogeneity. Differences were
considered significant at p \textless{} 0.05. All analyses were
performed using the GraphPad software package (Prism, version 9;
GraphPad, San Diego, California, USA).

\section*{3 Results}

{\label{results}}

\subsection*{3.1 Effects of brief EE exposure on medial prefrontal cortex
protein
levels}

{\label{effects-of-brief-ee-exposure-on-medial-prefrontal-cortex-protein-levels}}

Western blot analysis of the NMDA receptors in the PSD of the mPFC after
EE exposure showed increased expression of the GluN2A subunit
(t\textsubscript{(10)} = 4.86, p \textless{} 0.001, +45\%) with no
changes for the other subunits GluN1 and GluN2B. This result led to an
increased GluN2A/GluN2B ratio (t\textsubscript{(10)} = 2.44, p
\textless{} 0.05, +20\%). The analysis of the expression of the
scaffolding proteins related to NMDA receptors revealed an increase of
SAP102 after EE exposure (t\textsubscript{(10)} = 2.39, p \textless{}
0.05, +22\%) with no changes for PSD95. The analysis of AMPA receptors
showed that EE exposure decreased GluA1 subunit expression
(t\textsubscript{(10)} = 2.83, p \textless{} 0.05, -22\%) while
increasing GluA2 levels (t\textsubscript{(10)} = 3.34, p \textless{}
0.01, +25\%), a result that led to a significant decrease of GluA1/GluA2
ratio (t\textsubscript{(10)} = 7.47, p \textless{} 0.0001, -38\%).
Considering the scaffolding proteins related to AMPA receptors, both
SAP97 (t\textsubscript{(10)} = 2.60, p \textless{} 0.05, +31\%) and GRIP
(U = 0, p \textless{} 0.01, +18\%) protein levels significantly
increased after EE exposure. Finally, we found Arc/Arg3.1 significantly
increased in the mPFC of the EE group as compared to the NoEE group
(t\textsubscript{(10)} = 3.27, p \textless{} 0.01, +30\%) (Fig. 1a).

The analysis of the whole homogenate showed a significant increase of
vGluT1 (t\textsubscript{(10)} = 2.49, p \textless{} 0.05, +22\%) and
GLT-1 expression (t\textsubscript{(10)} = 2.57, p \textless{} 0.05,
+16\%) as compared to NoEE group. In line with the results of the
post-synaptic density, only the expression of the GluN2A subunit of NMDA
receptors was significantly increased (t\textsubscript{(10)} = 2.83, p
\textless{} 0.05, +20\%), an effect that was accompanied by SAP102
up-regulation (t\textsubscript{(10)} = 2.96, p \textless{} 0.05, +22\%)
and, at variance from the PSD, by PSD95 up-regulation
(t\textsubscript{(10)} = 4.82, p \textless{} 0.001, +20\%) as compared
to NoEE group. Analyzing AMPA receptors, the pattern of expression was
quite different from the PSD with increased expression of GluA1 subunit
(t\textsubscript{(10)} = 3.53 p \textless{} 0.01, +22\%) and no effects
on GluA2 subunit whereas the related scaffolding proteins showed
significantly increased expression {[}SAP97 (t\textsubscript{(10)} =
3.90, p \textless{} 0.01, +21\%) and GRIP (U = 2, p \textless{} 0.01,
+15\%){]}, in line with data in the PSD. Finally, no changes in
Arc/Arg3.1 expression were observed when compared to the NoEE group
(Fig. 1b).

\subsection*{3.2 Effects of brief EE exposure on nucleus accumbens
protein
levels}

{\label{effects-of-brief-ee-exposure-on-nucleus-accumbens-protein-levels}}

Western blot analysis in the PSD of the NAc after EE exposure revealed
an overall downregulation of NMDA receptor subunits as compared to NoEE
group (GluN1: t\textsubscript{(10)} = 2.77, p \textless{} 0.05, -8\%;
GluN2A: t\textsubscript{(10)} = 3.44, p \textless{} 0.01, -19\%; GluN2B:
t\textsubscript{(10)} = 2.68, p \textless{} 0.05, -13\%); accordingly,
the GluN2A/GluN2B ratio was not changed in the EE group. The analysis of
the NMDA-related scaffolding proteins revealed a significant decrease of
PSD95 after EE exposure (t\textsubscript{(10)} = 6.48, p \textless{}
0.0001, -33\%), with no changes in SAP102 expression. Analysis of AMPA
receptors shows a significant reduction of GluA2 levels after EE
exposure (t\textsubscript{(10)} = 4.95, p \textless{} 0.001, -24\%),
with no GluA1 alterations. This result led to an increased GluA1/GluA2
ratio (t\textsubscript{(6.19)} = 2.95, p \textless{} 0.05, +22\%). The
analysis of AMPA-related scaffolding proteins showed a significant
increase of SAP97 (t\textsubscript{(10)} = 3.17, p \textless{} 0.01,
+10\%) and a reduction of GRIP expression (U = 5, p \textless{} 0.05,
-8\%) after EE exposure. Finally, Arc/Arg3.1 expression was
significantly decreased in the EE group as compared to the NoEE group
(t\textsubscript{(6.37)} = 3.91, p \textless{} 0.01, -33\%) (Fig. 2a).

When analyzing the whole homogenate, we found no changes in vGluT1
levels while a significant increase of GLT-1 expression as compared to
the NoEE group (t\textsubscript{(5.33)} = 2.56, p \textless{} 0.05,
+24\%). With respect to NMDA receptors and related scaffolding proteins,
no changes were observed except for a significant decrease of PSD95
(t\textsubscript{(10)} = 2.62, p \textless{} 0.05, -13\%). Similarly, no
changes were observed for AMPARs and related scaffolding proteins.
Finally, Arc/Arg3.1 protein levels were significantly decreased as
compared to the NoEE group (t\textsubscript{(10)} = 2.57, p \textless{}
0.05, -17\%) (Fig. 2b).

\subsection*{3.3 Effects of brief EE exposure on hippocampal protein
levels}

{\label{effects-of-brief-ee-exposure-on-hippocampal-protein-levels}}

Western blot analysis in the PSD of the Hipp after EE exposure showed no
changes in the main NMDA subunit GluN1 whereas the expression of GluN2A
and GluN2B were, respectively, significantly decreased
(t\textsubscript{(10)} = 3.33, p \textless{} 0.01, -27\%) and increased
(t\textsubscript{(10)} = 2.56, p \textless{} 0.05, +13\%). This result
led to a significant reduction of the GluN2A/GluN2B ratio
(t\textsubscript{(10)} = 4.64, p \textless{} 0.001, -36\%). The analysis
of the NMDA-related scaffolding proteins after EE exposure revealed a
significant decrease of the scaffolding protein SAP102
(t\textsubscript{(10)} = 2.28, p \textless{} 0.05, -15\%), with no
changes for PSD95. At variance from NMDA receptors, no changes in the
AMPARs subunits GluA1 and GluA2 expression were observed after EE
exposure, with the consequence of an unaltered GluA1/GluA2 ratio.
However, we found SAP97 and GRIP levels significantly decreased in the
EE group as compared to the NoEE group (t\textsubscript{(10)} = 2.52, p
\textless{} 0.05, -17\%; t\textsubscript{(10)} = 3.39, p \textless{}
0.01, -11\%; respectively). Finally, Arc/Arg3.1 significantly increased
after EE exposure (t\textsubscript{(10)} = 3.57, p \textless{} 0.01,
+12\%) (Fig. 3a).

Examining the whole homogenate, vGluT1 levels were decreased
(t\textsubscript{(10)} = 3.45, p \textless{} 0.01, -24\%) with no
changes for GLT-1 as compared to NoEE group. Notably, the expression of
NMDA receptors was not altered after EE exposure, except for a
significant decrease for both SAP102 (t\textsubscript{(10)} = 3.17, p
\textless{} 0.01, -19\%) and PSD95 (t\textsubscript{(10)} = 3.07, p
\textless{} 0.05, -11\%). Differently from NMDA receptors, the GluA2
subunit of AMPA receptors was significantly increased
(t\textsubscript{(10)} = 2.56, p \textless{} 0.05, +27\%) while no GluA1
changes were observed. The analysis of AMPA-related scaffolding proteins
showed a slight but significant increase of SAP97 as compared to the
NoEE group (t\textsubscript{(10)} = 2.58, p \textless{} 0.05, +14\%).
Finally, Arc/Arg3.1 expression was not altered after EE exposure (Fig.
3b).

\textbf{4 Discussion}

In the present study, we investigated the effects of brief EE exposure
on glutamatergic transmission to understand whether potential
metaplastic effects underlie EE modulation of sucrose-seeking behavior
previously reported (Pintori \emph{et al.} , 2022a). Our findings reveal
that 22 hours of EE exposure differently affect glutamate homeostasis in
the brain areas involved in reward and reward-related memory. These
results demonstrate that a brief EE exposure is sufficient to cause
different adaptive mechanisms that may influence glutamate receptor
availability at the synapse; such mechanisms are peculiar for each brain
region.

\textbf{4.1 Effects of brief EE exposure in the medial prefrontal
cortex}

In the mPFC, brief EE exposure selectively increased GluN2A levels,
leading to a significant increase in GluN2A/GluN2B ratio. The analysis
of the AMPA receptor, instead, revealed a decrease of GluA1 and an
increase of GluA2 subunits expression leading to a decrease of
GluA1/GluA2 ratio. This suggests that brief EE exposure alters the
composition of these receptors which might contribute to changes in the
homeostasis of the glutamate system.

According to the literature, GluN2A/GluN2B ratio change correlates with
modifications of the threshold for LTP and LTD (Chiamulera \emph{et al.}
, 2020). For instance, while lack of sensory experiences, such as light
deprivation, decreases the GluN2A/GluN2B ratio, restoring visual
experience rapidly increases the GluN2A/GluN2B ratio, raising the
LTP/LTD threshold in the visual cortex (Quinlan \emph{et al.} , 1999;
Philpot \emph{et al.} , 2003). This bidirectional change of

LTP/LTD threshold induced by rapid modification of NMDAR subunits
composition has been proposed as a demonstration of metaplasticity
(Kirkwood \emph{et al.} , 1996; Philpot \emph{et al.} , 2003).
Similarly, we hypothesize that brief EE may cause an increase in LTP/LTD
threshold, by inducing a rapid switch of NMDARs with a higher proportion
of GluN2A subunits, thus reducing Ca\textsuperscript{2+} influx.

In line with an `overstimulation hypothesis', these AMPA/NMDA receptors
changes are coupled with an increase of the related scaffolding proteins
levels both in the PSD fraction (SAP102, SAP97, GRIP) and in the whole
cortical homogenate (SAP102, SAP97, GRIP, PSD95). Particularly, it is
interesting to note that while GluA1 expression is reduced in the PSD,
the expression of the specific scaffolding protein of this receptor,
SAP97, is significantly elevated, presumably as an adaptive mechanism to
anchor more tightly this receptor at the membrane. Considering their
role in the formation, trafficking, and stabilization of NMDARs and
AMPARs at excitatory synapses (Kim \emph{et al.} , 2005; Vickers
\emph{et al.} , 2006), these EE-induced scaffolding proteins changes
could be involved in increased membrane stability of new glutamate
receptors configuration induced by EE exposure. Consistently with this
hypothesis, Arc/Arg3.1 increased in the PSD after EE exposure, an
observation in line with its involvement in a simultaneous strengthening
of stimulated synapses and maintaining weakness at non-potentiated
synapses through AMPARs trafficking modulation (Zhang \& Bramham, 2021).
Finally, the overstimulation hypothesis is further accounted for by the
up-regulation of vGluT1, which promotes glutamate release (Li \emph{et
al.} , 2020), and that may be partly countered by the increased levels
of GLT-1, which is chiefly responsible for glutamate reuptake (Shigeri
\emph{et al.} , 2004).

Taken together, these neuroplastic changes occurring at glutamatergic
synapses, induced by brief EE exposure, could reflect a limitation of
mPFC synaptic excitability (and in turn a reduction of its inhibitory
action on subcortical regions) by further stimuli (i.e.,
reward-associated stimuli) as a consequence to previous strong cortical
activation. In line with a reduction of cortical inhibitory activity, we
previously reported that brief EE exposure enhanced rather than
inhibited context-induced sucrose-seeking (Pintori \emph{et al.} ,
2022a). Electrophysiological experiments would be needed to better
understand the change of synaptic properties in the mPFC under EE
conditions.

\textbf{4.2 Effects of brief EE exposure in the Nucleus accumbens}

Brief EE exposure caused post-synaptic glutamatergic signaling changes
in the NAc that are opposite compared to mPFC. In particular, EE induced
an overall reduction of NMDARs subunits, both accessory (GluN2A, GluN2B)
and obligatory (GluN1), in the PSD. Notably, no changes were observed in
the expression of these subunits in the whole homogenate, suggesting
that receptor synthesis is not affected while receptor retention is
reduced as a consequence of brief EE exposure. This possibility is
corroborated by the fact that the expression of SAP102, the scaffolding
protein specific for NMDA receptors, is not increased to compensate for
reduced expression, thus leading to unstable synapses. These results
suggest that NMDA-mediated neurotransmission is depotentiated in the
NAc, evidence also strengthened by reduced expression of PSD95 that,
besides being a scaffolding protein, is an integral protein of the
glutamate synapse centrally involved in multiple aspects of synaptic
function (Vallejo \emph{et al.} , 2017). Moreover, since structural
changes of dendritic spines are sustained by PSD95 and Arc/Arg3.1
(Newpher \emph{et al.} , 2018), their reduced expression suggests that a
brief EE exposure might have reduced the density of dendritic spines
contributing to the toning down of the NMDA receptors-mediated glutamate
neurotransmission.

A different pattern can be observed for AMPA receptors. We found a
significant reduction only in the expression of the GluA2 subunit, thus
leading to an increase in GluA1/GluA2 ratio. This enhancement of
GluA2-lacking AMPA receptors reflects a higher
Ca\textsuperscript{2+}permeability AMPA-mediated in the excitatory
synapse, representing a metaplastic phenomenon, as it is established
that the up-regulation of calcium-permeable AMPARs (CP-AMPAR) is able to
change the threshold for different forms of plasticity (Liu \& Zukin,
2007). While CP-AMPARs are expressed at low levels under basal
conditions, their expression in the excitatory neurons increased in
response to neuronal activity and neuronal insults. Importantly,
CP-AMPARs in the NAc are involved in drug-seeking behaviors (see review
(Neuhofer \& Kalivas, 2018)). For instance, it has been demonstrated
that the increase of NAc CP-AMPARs plays a critical role in the
incubation of cocaine craving (Conrad\emph{et al.} , 2008; Wolf \&
Ferrario, 2010; Lee \emph{et al.} , 2013; Caffino \emph{et al.} , 2021).
These data show that even a brief EE exposure may influence, through
changes in accumbal glutamate homeostasis, salience attribution, and
behavioral response to reward-associated stimuli (i.e., cue, context),
ultimately driving maladaptive reward-related behaviors. In line with
this evidence, the enhancement of CP-AMPARs levels observed in the NAc
after brief EE exposure is consistent with the potentiation of
Cx-induced sucrose-seeking previously reported (Pintori \emph{et al.} ,
2022a). Of note, the excessive calcium influx via EE-induced increase of
CP-AMPARs can contribute to NMDA receptor inactivation, as previously
demonstrated (Legendre \emph{et al.} , 1993; Paoletti \emph{et al.} ,
2013; Sibarov \& Antonov, 2018), thus further reducing the postsynaptic
response.

We also found no changes in glutamate release as shown by the lack of
effect on vGluT1. In addition, EE exposure leads to increased GLT-1
levels presumably to limit glutamate spillover and to prevent activation
of extra-synaptic glutamate receptors that can impair synaptic
plasticity (Scimemi \emph{et al.} , 2009).

\textbf{4.3 Effects of brief EE exposure in the hippocampus}

The analysis of the Hipp revealed a different situation in comparison to
mPFC and NAc. Brief EE exposure only slightly altered glutamatergic
postsynaptic signaling in this brain area. The most relevant effect is
the reduced GluN2A/GluN2B ratio in the PSD, due to a decrease of GluN2A
and an increase of GluN2B receptor subunits expression together with a
reduction in SAP102 expression. This change of synaptic NMDA receptors
configuration may reflect a decrease of the LTP/LTD threshold, in line
with the reduced presynaptic glutamate release as evidenced by a
significant down-regulation of vGluT1. Our evidence is consistent with
the literature showing that EE exposure (both long- and short-term)
facilitates the LTP in CA1 (see review (Ohline \& Abraham, 2019)). It is
plausible that this EE-induced NMDARs hippocampal change may contribute
to an enhancement of learning and memory abilities, such as spatial
recognition. Moreover, GluN2B-containing receptors in the dorsal Hipp
are necessary for drug context-induced cocaine-seeking behavior
(Xie\emph{et al.} , 2013), as well as memory reconsolidation (Wells
\emph{et al.} , 2016), and relapse even after a long period of
abstinence from cocaine self-administration (Werner \emph{et al.} ,
2020). In line with this speculation, we previously reported that brief
EE exposure improves discrimination efficiency (an index of instrumental
learning and memory) and increases sucrose-seeking behavior during both
contextual-memory reactivation session and renewal test (Pintori
\emph{et al.} , 2022a), suggesting that the increased hippocampal
GluN2B-containing receptor expression might be the underlying mechanism
of such behaviors.

Differently from mPFC and NAc, EE did not change postsynaptic AMPA
receptor expression and subunit configuration in the Hipp, while
reducing the expression of AMPA receptor scaffolding proteins SAP97 and
GRIP in the PSD, suggesting reduced AMPA receptor retention at the
synaptic site. Together with a more mobile pool of GluN2B-containing
NMDA receptors (Groc \emph{et al.} , 2006) and supported by increased
Arc/Arg3.1 levels (Chowdhury \emph{et al.} , 2006), this effect might
contribute to altering the synaptic strength of the excitatory synapse
thus leading to a less stable hippocampal synapse.

These changes, together with the decreased GluN2A/GluN2B ratio, may
represent a `canonical' metaplastic mechanism to set up glutamatergic
synapses to respond more robustly to future stimulations/experiences,
and subsequence learning (Chiamulera \emph{et al.} , 2020).

\textbf{4. 4 Limitations}

The study owns some limitations such as, firstly, the focus on
ionotropic glutamate signaling and not, for instance, on the potential
role of metabotropic mGluR5 receptors as suggested by EE-induced mGluR5
enhancement of hippocampal LTP (Buschler \& Manahan-Vaughan, 2017).
Therefore, \emph{ad-hoc} molecular studies are needed on the entire
glutamatergic components, as well as on other neurochemical pathways
(e.g., BDNF, 5-HT) and brain regions (e.g., amygdala) relevant to
rewarding and learning. Further, we did not investigate the mechanism
underlying the expression change, whether due to changes in synthesis or
degradation. We measured the glutamatergic changes only at 2 hours after
the end of the brief EE exposure: we are aware that a series of
timepoint assessments may provide a better understanding. We are also
aware that only detecting protein expression cannot establish a causal
relationship and that~protein expression alone may not reflect
functional mechanisms. Our rats were kept isolated thus raising the
possibility that housing conditions may indeed influence the response of
the glutamatergic synapse. For instance, we recently demonstrated in
adult male mice that living environment (i.e., social vs isolation
housing) influences the efficacy of EE/fluoxetine interaction in
inhibiting relapse to sucrose (Pintori \emph{et al.} , 2022b). Finally,
we used only male rats in our study and, therefore, we do not know if
our findings can be extended to female rats.

\textbf{4.5 Conclusions}

Our findings demonstrate that a brief EE exposure dynamically modulates
the glutamatergic synapses in mPFC-NAc-Hipp circuitry, which play an
important role in reward and reward-related processes. Particularly,
brief EE exposure was able to alter postsynaptic NMDA/AMPA receptor
configurations, through changes in subunit composition of such
receptors, as well as changes in the expression of their main
scaffolding proteins.

An added value of our study derives from the fact that each investigated
region showed its specific modulation profile of the glutamate synapse.
Consistently with our previous behavioral findings (Pintori \emph{et
al.} , 2022a), this metaplastic fingerprint induced by EE supports the
hypothesis that brief EE exposure potentiates sucrose-seeking behaviors
affecting subsequent learning processes, such as renewal or Cx-memory
reactivation and reconsolidation. In line with this speculation, the
reduction of cortical excitability induced by EE experience, along with
a concomitant increase of Ca\textsuperscript{2+} permeability in the NAc
and the Hipp, may alter salience attribution and behavioral response to
reward-associated stimuli, thus enhancing sucrose-seeking behaviors when
re-exposed to sucrose-paired context (Fig. 4).

In conclusion, our data confirm the modulatory activity of brief EE
manipulation on glutamatergic synapses, which could pave the way to
develop innovative configurations of EE - with greater translational
value feasibility - to improve existing therapeutic approaches against
addiction and addiction-related CNS disorders.

\textbf{Author contributions} Cristiano Chiamulera: Conceptualization;
methodology; project administration; resources; writing. Nicholas
Pintori: Conceptualization; methodology; investigation; data curation;
formal analysis; visualization; writing. Fabio Fumagalli:
Conceptualization, methodology; writing. Alessandro Piva: investigation;
data curation; visualization. Francesca Mottarlini: Investigation; data
curation; formal analysis; visualization. Coralie Maggi: Investigation;
data curation; visualization. Lucia Caffino: Investigation; data
curation; formal analysis; visualization.

\textbf{Funding and disclosure} Research was supported by Fondazione
Cariverona (Italy) for the project ''\emph{The role of neurogenesis and
enriched environment in maladaptive memories'' and} by grants from MIUR
Progetto Eccellenza 2023-2027\emph{.} Nicholas Pintori was the recipient
of a fellowship from Fondazione Cariverona for the same project.
Francesca Mottarlini is the recipient of a fellowship from the
Zardi-Gori Foundation.

\textbf{Data Availability} The data sets generated during and/or
analyzed during the current study are not publicly available due to the
confidentiality restrictions of the funding body but are available from
the corresponding author on reasonable request.

\textbf{Ethics Approval} This study was approved by the ethical
committee (OPBA) of the University of Verona and by the Ministry of
Health (authorization n. 780/2019-PR) in compliance with the European
Union regulations and Directive 2010/63/EU.

\textbf{Conflict of Interest} The authors declare no competing
interests.

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\textbf{Figures and Legends}\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

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\textbf{Figure 1}\selectlanguage{english}
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\textbf{Figure 2}\selectlanguage{english}
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\textbf{Figure 3}

\textbf{Hosted file}

\verb`image4.emf`  available at \url{https://authorea.com/users/636788/articles/653488-brief-exposure-to-enriched-environment-rapidly-shapes-the-glutamate-synapse-in-the-rat-brain-a-metaplastic-fingerprint}

\textbf{Figure 4}

\textbf{Fig. 1. Effects of brief EE exposure on medial prefrontal cortex
protein levels.}

Quantification of vGluT1 and GLT-1, of NMDA receptor subunits, GluN1,
GluN2A, and GluN2B, of the AMPA receptor subunits, GluA1 and GluA2, of
scaffolding proteins, SAP102, SAP97, GRIP, and PSD95, and the
cytoskeletal marker Arc/Arg3.1 in the post-synaptic density
\textbf{(a)}and in the whole homogenate \textbf{(b)} of the medial
prefrontal cortex (mPFC) 2 hours after the end of EE exposure. In the
right insert, representative immunoblots are shown for vGluT1 (62 kDa),
GLT-1 (60 kDa), GluN1 (120 kDa), GluN2A (180 kDa), GluN2B (180 kDa),
SAP102 (102 kDa), GluA1 (108 kDa), GluA2 (108 kDa), SAP97 (97 kDa), GRIP
(130 kDa) Arc/Arg3.1 (55 kDa) and \selectlanguage{greek}β-\selectlanguage{english}actin (43 kDa) in the post-synaptic
density or the homogenate of mPFC. Data are shown as the mean + SEM and
are expressed as percentage of the NoEE group. NoEE = white columns; EE
= teal columns. N = 6 rats/group. \textsuperscript{*}p \textless{}
0.05,\textsuperscript{**}p \textless{} 0.01, \textsuperscript{***}p
\textless{} 0.001 vs. NoEE; unpaired two-tailed Student's t-test or
Mann-Whitney test.

\textbf{Fig. 2. Effects of brief EE exposure on nucleus accumbens
protein levels} \emph{.}

Quantification of vGluT1 and GLT1, of NMDA receptor subunits, GluN1,
GluN2A, and GluN2B, of the AMPA receptor subunits, GluA1 and GluA2, of
scaffolding proteins, SAP102, SAP97, GRIP, and PSD95, and the
cytoskeletal marker Arc/Arg3.1 in the post-synaptic density
\textbf{(a)}and in the whole homogenate \textbf{(b)} of nucleus
accumbens (NAc) 2 hours after the end of EE exposure. In the right
insert, representative immunoblots are shown for vGluT1 (62 kDa), GLT-1
(60 kDa), GluN1 (120 kDa), GluN2A (180 kDa), GluN2B (180 kDa), SAP102
(102 kDa), GluA1 (108 kDa), GluA2 (108 kDa), SAP97 (97 kDa), GRIP (130
kDa) Arc/Arg3.1 (55 kDa) and \selectlanguage{greek}β-\selectlanguage{english}actin (43 kDa) in the post-synaptic
density or the homogenate of NAc. Data are shown as the mean + SEM and
are expressed as percentage of the NoEE group. NoEE = white columns; EE
= teal columns. N = 6 rats/group. \textsuperscript{*}p \textless{} 0.05,
\textsuperscript{**}p \textless{} 0.01, \textsuperscript{***}p
\textless{} 0.001 vs. NoEE; unpaired two-tailed Student's t-test or
Mann-Whitney test.

\textbf{Fig. 3.} \textbf{Effects of brief EE exposure on hippocampus
protein levels.}

Quantification of vGluT1 and GLT-1, of NMDA receptor subunits, GluN1,
GluN2A, and GluN2B, of the AMPA receptor subunits, GluA1 and GluA2, of
scaffolding proteins, SAP102, SAP97, GRIP, and PSD95, and the
cytoskeletal marker Arc/Arg3.1 in the post-synaptic density
\textbf{(a)}and in the whole homogenate \textbf{(b)} of hippocampus
(Hipp) 2 hours after the end of EE exposure. In the right insert,
representative immunoblots are shown for vGluT1 (62 kDa), GLT-1 (60
kDa), GluN1 (120 kDa), GluN2A (180 kDa), GluN2B (180 kDa), SAP102 (102
kDa), GluA1 (108 kDa), GluA2 (108 kDa), SAP97 (97 kDa), GRIP (130 kDa)
Arc/Arg3.1 (55 kDa) and \selectlanguage{greek}β-\selectlanguage{english}actin (43 kDa) in the post-synaptic density or
the homogenate of Hipp. Data are shown as the mean + SEM and are
expressed as percentage of the NoEE group. NoEE = white columns; EE =
teal columns. N = 6 rats/group. \textsuperscript{*}p \textless{}
0.05,\textsuperscript{**}p \textless{} 0.01, \textsuperscript{***}p
\textless{} 0.001 vs. NoEE; unpaired two-tailed Student's t-test or
Mann-Whitney test.

\textbf{Fig. 4. Graphical representation of the main metaplastic changes
on glutamatergic mPFC-NAc-Hipp brain areas induced by brief EE
exposure.}

A brief EE exposure was able to alter postsynaptic NMDA/AMPA receptor
configurations, through changes in subunit composition of such
receptors, as well as changes in the expression of their main
scaffolding proteins (i.e., SAP102, SAP97). Importantly, the modulation
profile of glutamatergic synapses induced by brief EE exposure was
unique for each brain region investigated (mPFC, NAc, Hipp).
Abbreviations: |: increased expression; |: decreased expression; -: no
change of expression; GluA2, glutamate AMPA receptor subunit 2; GluN2B,
glutamate NMDA receptor subunit 2B; Hipp, hippocampus; mPFC, medial
prefrontal cortex; NAc, nucleus accumbens; SAP102, synapse-associated
protein 102; SAP97, synapse-associated protein 97.\selectlanguage{english}
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