1

Exposure to an enriched environment selectively increases the

functional response of the presynaptic NMDA receptors which

modulate noradrenaline release in mouse hippocampus

Massimo Grilli,* Stefania Zappettini,* Alessio Zanardi,† Federica Lagomarsino,* Anna

Pittaluga,* Michele Zoli† and Mario Marchi*,‡,¶

*Department of Experimental Medicine, Section of Pharmacology and Toxicology, University

of Genoa, Italy

†Department of Biomedical Sciences, Section of Physiology, University of Modena and

Reggio Emilia, Modena, Italy

‡Center of Excellence for Biomedical Research, University of Genoa, Italy

¶National Institute of Neuroscience, University of Genoa, Italy

Address correspondence and reprint requests to Dr. Mario Marchi, Dipartimento di

Medicina Sperimentale, Sezione di Farmacologia e Tossicologia, Università di Genova, Viale

Cembrano 4, 16148 Genova, Italy. E-mail: marchi@pharmatox.unige.it

Abbreviations used: AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole propionate;

EE, enriched environment; KO, knock out; NET, norepinephrine transporter; SE, standard

environment

The definitive version is available at www.blackwell-synergy.com. Journal of Neurochemistry 10/2009; 110(5):1598-606. DOI:10.1111/j.1471-4159.2009.06265.x

2

Abstract

We evaluated the impact of environmental training on the functions of presynaptic

glutamatergic NMDA and α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and

nicotinic receptors expressed by hippocampal noradrenergic nerve terminals. Synaptosomes

isolated from the hippocampi of mice housed in enriched (EE) or standard (SE) environment

were labelled with [3H]noradrenaline ([3H]NA) and tritium release was monitored during

exposure in superfusion to NMDA, AMPA, epibatidine or high K+. NMDA -evoked [3H]NA

release from EE hippocampal synaptosomes was significantly higher than that from SE

synaptosomes, while the [3H]NA overflow elicited by 100 µM AMPA, 1 µM epibatidine or

(9, 15, 25 mM) KCl was unchanged. In EE mice, the apparent affinity of NMDA or glycine

was unmodified, while the efficacy was significantly augmented. Sensitivity to non-selective

or subtype-selective NMDA receptor antagonists (MK-801, ifenprodil and Zn2+ ions) was not

modified in EE. Finally, the analysis of NMDA receptor subunit mRNA expression in

noradrenergic cell bodies of the Locus Coeruleus (LC) showed that NR1, NR2A, NR2B and

NR2D subunits were unchanged, while NR2C decreased significantly in EE mice as

compared to SE mice. Functional upregulation of the presynaptic NMDA receptors

modulating NA release might contribute to the improved learning and memory found in

animals exposed to an EE.

Keywords: enriched environment, noradrenaline release, presynaptic NMDA receptors,

presynaptic AMPA receptors, nicotinic receptors

Running title: Enriched environment and noradrenaline release

3

It is well known that brains of animals exposed to an enriched environment (EE) has profound

effects on central nervous system (CNS) structure and neurochemistry (Diamond et al. 1966;

Gagne et al. 1998; Duffy et al. 2001; Frick et al. 2003; Aguilar Valles et al. 2007; Costa et

al., 2008) as well as on mRNA expression of a variety of genes (Torasdotter et al.1998;

Dahlqvist et al. 1999; Li et al. 2007; Andin et al. 2007, Del Arco et al. 2007; Segovia et al.

2008). Enriched environment regulates excitability, synaptic transmission and LTP in the

hippocampus of freely moving animals (Duffy et al. 2001; Irvine and Abraham 2005). It has

also been reported that EE treatment enhances memory, restores impaired hippocampal

synaptic plasticity and cognitive deficits induced by prenatal chronic stress and protects from

memory loss (Rampon and Tsien 2000; Mohammed et al. 2002; Frick et al. 2003; Cao et al.

2008; Segovia et al. 2008). However the underlying neurobiological and neurochemical

mechanisms which could be involved in these effects remain poorly investigated.

Previous data show that glutamatergic pathways in the hippocampus are implicated in

cognitive functions and memory processes as well as in the neuronal plasticity (Lee et al.

2003). Indeed, hippocampal CA1 region specific inactivation of the NMDA receptor NR1

subunit gene induces memory impairment (Li et al. 2007), while transgenic mice with NR2B

subunit overexpression show better learning ability and memory performance (Tang et al.

1999). It is also well known that the noradrenergic system plays a crucial role in mediating

changes in the ability to concentrate and in memory performance in mouse (McGaugh 1989).

Interestingly the involvement of the noradrenergic system in the effects of EE has been

explored in the past (Brenner et al, 1983; O’Shea et al. 1983; Mohammed et al. 1986). As far

as the glutamatergic system quantitative analysis revealed changes in the mRNA levels for

glutamatergic α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors and in

NMDA and AMPA receptor expression in some brain areas of animals grown in EE, although

these findings are somewhat contradictory (Foster et al. 1996; Gagne et al. 1998; Tang 2001;

4

Naka et al. 2005; Wood et al. 2005; Andin et al. 2007). Moreover, the effects of EE on the

function of the glutamatergic AMPA and NMDA receptors, have not yet been studied in these

animals.

To extend the knowledge on the effect of EE on the glutamatergic and noradrenergic

neurotransmission in a brain region which is very important for learning and memory, we

studied the functional changes to presynaptic NMDA and AMPA receptors which modulate

noradrenaline (NA) release from isolated nerve endings of hippocampus from young mice

grown in an EE and compared those results to those from animals grown in a standard

environment (SE). We have also studied the mRNA levels of the NMDA receptor subunits in

the locus coeruleus (LC) of the same animals.

The results indicate that exposure to an EE induces a selective upregulation of

functional NMDA receptors located presynaptically on hippocampal noradrenergic terminals,

and a decreased expression of the mRNA encoding for the NR2C subunit in noradrenergic

cell bodies in the locus coeruleus.

5

Materials and methods

Animals

Female C57BL/6J mice were housed at constant temperature (22 ± 1°C) and relative humidity

(50%) under a regular light–dark schedule (lights 7 AM - 7 PM). Food and water were freely

available. The animals were killed by decapitation and the brain areas rapidly removed at 0–

4°C. The experimental procedures were approved by the Ethical Committee of the

Pharmacology and Toxicology Section, Department of Experimental Medicine, in accordance

with the European legislation (European Communities Council Directive of 24 November

1986, 86/609/EEC). Using animals, all efforts were made to minimize animal suffering and to

use only the number of animals necessary to produce reliable results.

Environmental enrichment

Three-week old female mice were either housed in either a standard environment (SE) or in

an EE. We used only female mice since male C57BL/6 mice housed in an EE often develop

aggressive behaviors which can lead to severe injuries or death of some of the mice (Young et

al. 1999). Animals exposed to SE were housed in group in standard cages (30 x 16 x 11 cm).

Animals exposed to an EE were housed together in a group of 10 in one of two large cages

(36 x 54 x 19 cm or 45 x 25 x 22 cm with a labyrinth) containing an assortment of objects,

including climbing ladders, running wheels, balls, plastic and wood objects suspended from

the ceiling, paper, cardboard boxes, and nesting material. Toys were changed every 2–3 days,

while the bedding was changed every week. We did not add any additional material to SE

cages. In both SE and EE cages, bedding consisted of sawdust. The animals were kept in the

enriched or standard environment for 2 months before the start of tests. Since training mice

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started from 3 weeks of age it is also important to consider an increased plasticity of the

brains of post-weaning animals.

Brain tissue preparation

Crude synaptosomes of hippocampus were prepared as previously described (Risso et al.

2004a) with some minor modifications. Briefly, the tissue was homogenized in 40 volumes of

0.32 M sucrose, buffered to pH 7.4 with phosphate (final concentration 0.01 M). The

homogenate was centrifuged at 1000 g for 5 min, to remove nuclei and cellular debris, and

crude synaptosomes were isolated from the supernatant by centrifugation at 12 000 g for 20

min. The synaptosomal pellet was then resuspended in physiological medium having the

following composition (mM): 128 NaCl, 2.4 KCl, 3.2 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25

HEPES, pH 7.5 and 10 glucose, pH 7.2-7.4. In release experiments, synaptosomes were

incubated 15 min at 37°C with [3H]NA (final concentration 0.05 µM). Labelling with [3H]NA

was performed in presence of 6-nitroquipazine (final concentration 0.03 µM) to avoid false

labelling of serotonergic terminals. We have performed some experiments also in presence of

desipramine (0.1 µM). The results show that in presence of desipramine the [3H]NA uptake

was almost totally inhibited (SE 91 % controls 8.92 ± 1.89 pCi/mg tissue; in presence of

desipramine 0.8 ± 0.31 pCi/mg tissue; EE 84 % controls 9.83 ± 2.21 pCi/mg tissue; in

presence of desipramine 1.57 ± 0.56 pCi/mg tissue).

Release experiments from synaptosomes

Identical portions of the synaptosomal suspension were then layered on microporous filters at

the bottom of parallel superfusion chambers thermostated at 37°C (Superfusion System, Ugo

Basile, Comerio, Varese, Italy) and then superfused (Raiteri and Raiteri 2000). Superfusion

(0.5 mL/min) was started with standard physiological solution. Starting from the 37 min of

7

superfusion, six consecutive 1-min fractions were collected; Agonists were present in the

medium starting from the second fraction collected and antagonists were added 8 min before

agonists. When NMDA-evoked release was studied the physiological solution was replaced at

t = 20 min with a magnesium free medium. Samples collected and superfused synaptosomes

were then counted for radioactivity. The amount of tritium released into each superfusate

fraction was expressed as a percentage of the total tissue content at the start of the fraction

collected. The [3H]NA evoked overflow was calculated by subtracting the corresponding

basal release to each fraction.

When K+-evoked release was studied, synaptosomes were first superfused with

standard medium for 36 min, then the following consecutive samples were collected: basal

release (b1; 3 min), K+-evoked release (S; 6 min), and basal release after depolarization (b2; 3

min). Synaptosomes were exposed to the depolarizing stimuli (9-12-15 mM KCl) for 90 s

starting at the end of the b1. Samples collected and superfused synaptosomes were then

counted for radioactivity. The amount of tritium released into each fraction was expressed as

a percentage of the total tissue content at the start of the fraction collected. The K+-evoked

overflow was calculated by subtracting the basal release (b1 + b2) from the K+-evoked

release (S).

Experiments of catecholamine uptake

[3H]NA uptake was studied according to the following procedure. The synaptosomal pellets

were resuspended in a standard medium. Aliquots of standard synaptosomal suspension were

incubated with [3H]NA (0.05 µM) for 15 min. At the end of the incubation the synaptosomes

were rapidly filtered on Millipore filters washed with two 5 ml aliquots of medium. Each

filter removed, scintillation fluid was added and vials were counted for radioactivity.

8

In situ hybridization

Following an analysis for mRNA secondary structure using GCG Sequence Analysis

Software 7.1, oligodeoxynucleotide sequences were chosen in unique regions of the mouse

NR1 and NR2A, B, C, D subunit and norepinephrine transporter (NET) mRNAs. The probe

characteristics are reported in Table I. Specificity controls included the demonstration that 1)

2 or more probes for each mRNA give identical labelling pattern, 2) probes with the same

base composition but different sequence do not give the specific labelling pattern. The

oligonucleotide probes were labelled at the 3' end using 33P-dATP (PerkinElmer,

NEG312H250UC) and terminal deoxynucleotidyl transferase (Roche) following the

specifications of the manufacturer to a specific activity of 100-300 KBcq/pmol. The labelled

probes were separated from unincorporated 33P-dATP using ProbeQuant™ G50 Micro

columns (GE Healthcare), precipitated in ethanol and resuspended in distilled water

containing 50 mM dithiothreitol.

Frozen brains were cut at the cryostat (14 µm thick sections) at level –5.3 mm from Bregma,

thaw mounted on Super-Frost Plus slides and stored at -80°C for 1-3 days. The procedure was

carried out according to Zoli et al. (1995). Probes were applied at a concentration of 2000-

3000 Bcq/30 µL/section (corresponding to around 15 fmol/section). The two oligonucleotide

probes of each NR subunit were combined to increase the intensity of the hybridization

signal. In order to minimise experimental variability, all sections to be compared were run in

parallel, using the same solutions. The slides were exposed for 21 days to BioMax MR film

(Kodak). The semiquantitative evaluation of film autoradiograms was performed according to a

previously published microdensitometric method (Zoli et al. 1990) using an automatic image

analyzer (KS300, Zeiss Kontron, Munich, Germany). Non-specific labelling was assessed in

adjacent sections incubated with an oligonucleotide of same length and GC content but unrelated

sequence.

9

Statistical analysis

Multiple comparisons were performed with two-way ANOVA followed by a Bonferroni post

hoc test. In situ hybridization data were compared by means of Mann-Whitney U-test. Data

were considered significant for p < 0.05 at least.

Chemicals

1-[7,8–3H]Noradrenaline (specific activity 39 Ci/mmol) was obtained from Amersham

Radiochemical Centre (Amersham, UK). NMDA, AMPA, ifenprodil and MK801 were

obtained from Tocris Bioscience (Bristol, UK). Glycine, 6-nitroquipazine and epibatidine

were purchased from Sigma Chemical Co (St Louis, MO, USA).

10

Results

Effects of NMDA, AMPA or epibatidine on the [3H]NA release from mouse isolated nerve

endings

When hippocampal synaptosomes prepared from mice grown in SE and prelabelled with

[3H]NA were exposed in superfusion to varying concentrations (1-300 µM) of NMDA (in

Mg2+-free medium, containing 1 µM glycine), the glutamatergic agonist was found to increase

the release of the radioactive tracer in a concentration-dependent manner. The apparent EC50

value of NMDA amounted to 14.9 ± 2.36 µM, the maximal overflow of [3H]NA being

obtained in presence of 100 µM NMDA (Fig. 1A). The concentration-dependent effects of

NMDA (10-100 and 300 µM) on the [3H]NA release was significantly higher in EE mice

when compared with SE animals (+14%, +29%, +28%) while the apparent EC50 were almost

similar (EE = 12.3 ± 2.27 µM). Figure 1B illustrates the time-course of the NMDA (100 µM)-

evoked [3H]NA release from hippocampal synaptosomes of EE and SE animals. The EE did

not modify the basal release, while the NMDA (100 µM)-evoked release was significantly

increased at min 40 and 41 of superfusion with respect to that in SE mice.

Figure 2 illustrates the [3H]NA overflow evoked by NMDA(100 µM) / glycine (1

µM), AMPA (100 µM), and epibatidine (1 µM) from hippocampal synaptosomes of EE and

SE animals. The [3H]NA overflow evoked by AMPA and epibatidine in SE animals were

quantitatively less pronounced (-33%; -84%) when compared with that evoked by NMDA and

were unaffected in the EE mice.

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Effects of MK801, Zn2+ and ifenprodil on [3H]NA release evoked by NMDA from mouse

hippocampal nerve endings

Figure 3 shows that the [3H]NA overflow evoked by NMDA (100 µM) from hippocampal

synaptosomes of SE mice was almost completely antagonized by the non-selective

antagonists MK801 (1 µM; -83%) and partially antagonized (-30%) by ifenprodil (1 µM, a

selective antagonist for the NR2B-containing receptor) while the presence of Zn2+ (0.01 µM,

a selective antagonist for the NR2A-containing receptor) did not affect the NMDA-evoked

overflow (-4%). A similar pattern was found when studying NMDA-evoked [3H]NA overflow

from hippocampal synaptosomes of EE mice. Figure 3 shows that EE did not change the

inhibitory effect of MK801, or ifenprodil (-83%; -34% respectively) in mouse hippocampal

synaptosomes, and that Zn2+ did not become effective (-4%).

The effect of different concentrations of the NMDA receptor co-agonist glycine (3 –

1000 nM) on the NMDA(100 µM)-evoked [3H]DA overflow from hippocampal

synaptosomes in EE and SE mice is reported in Figure 4. The apparent EC50 of glycine did

not significantly change in the EE in comparison with the SE mice (0.52 ± 0.04 µM versus

0.46 ± 0.02 µM), although facilitation caused by high glycine (100-1000 nM) in EE

hippocampal synaptosomes was significantly more pronounced when compared to SE mice.

Effects of EE on the [3H]NA uptake and on the KCl-evoked [3H]NA overflow in mice

hippocampal nerve endings

To assess whether changes in the NMDA-induced release of [3H]NA in the EE mice were the

consequence of changes to the exocytotic machinery, we investigated if and to what extent the

EE can affect i) the release of [3H]NA caused by depolarizing stimulus (such as the transient

exposure of synaptosomes to different concentrations of KCl) and ii) the [3H]NA uptake.

12

Results in Figure 5 show that EE did not cause alterations of [3H]NA uptake nor it modified

the depolarization-evoked release elicited by high K+ when compared to the SE animals.

Effects of EE on the expression of NMDA receptor subunit mRNAs in the locus coeruleus

To assess whether the observed increase in NMDA-induced NA release in the hippocampus

of mice exposed to EE was due to changes in NMDA receptor subtypes, we performed an

analysis of NMDA receptor subunit mRNA expression in NA cell bodies of the LC. We

investigated the mRNA levels of NR1 and NR2A, B, C and D subunits (Fig. 6A,C). The

precise location of LC was assessed by labelling the mRNA of NET, a specific marker of

noradrenergic neurons, in adjacent sections (Fig. 6B,D). Whereas no significant difference in

the levels of NR1, NR2A, NR2B, and NR2D mRNA levels was observed between mice

exposed to SE and EE, a significant decrease by 33% in NR2C mRNA levels was detected in

EE mice as compared to SE mice (Mann-Whitney U test, Z=-2.84, p = 0.004, n: SE=5, EE=7)

(Fig. 7).

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Discussion

The central message of the present manuscript is that exposure to an EE produces significant

changes to the functions of glutamatergic NMDA receptors which presynaptically modulate

the release of NA in mouse hippocampus (Pittaluga and Raiteri 1990). Interestingly enough,

EE leaves unmodified the glutamatergic AMPA-preferring receptors (Pittaluga and Raiteri

1992, Pittaluga et al. 2006) and the epibatidine-sensitive nicotine receptors (Risso et al.

2004a) presynaptically located on the same hippocampal noradrenergic synaptosomal

subpopulation. Based on the present results, the NMDA-evoked release of NA from

hippocampal nerve endings of EE mice was significantly higher than that evoked from nerve

terminals of SE mice, while the AMPA-evoked release or the epibatidine-induced overflow of

tritiated amine was unmodified by behavioural training. It has been reported in the literature

that epibatidine-induced overflow of amine seems to be mediated by α3β2 or α3β4 nicotinic

ACh receptors (Sershen et al. 1997, Kulak et al. 2001, Azam and McIntosh 2006) which

therefore appear not to be modified by EE. However the possibility that the function of other

nicotinic receptor may be altered by EE can not be excluded (Zanardi et al. 2007)

It is known that activation of presynaptic NMDA, AMPA or nicotine receptors located on

noradrenergic terminals causes a local depolarization of synaptosomal membranes, due to the

positive charges flowing through the receptor-associated ionic channels, that in turn favours

the gating of voltage-sensitive calcium channels located nearby the ionic receptors and

external Ca2+-dependent vesicular release of neurotransmitters (Pittaluga and Raiteri 1992;

Malva et al. 1994; Sershen et al. 1997; Risso et al. 2004a; Pittaluga et al, 2006). Some may

argue that the modification in the NMDA-induced release of NA observed in EE mice could

depend on changes to the exocytotic machinery induced by the behavioural training. This

hypothesis, however, seems unlikely, since i) contrary to what is observed for NMDA-

14

induced NA release, the amine overflow caused by activation of AMPA or nicotinic receptors

was unmodified by EE and ii) the Ca2+-dependent, exocytotic-like release of NA provoked by

depolarization with different KCl concentrations was identical in synaptosomes from SE or

EE animals. While evaluating whether behavioural training could modify this functional

event, we also considered if EE can induce changes to NA uptake. Again, we were unable to

observe significant differences in the amount of [3H]NA uptake in EE mice when compared to

control, observations that lead us to exclude the occurrence of EE-induced adaptive changes

in noradrenergic terminals in term of vesicular uptake or exocytosis. Indeed, present

observations tally well with previous evidence that EE does not significantly alter NA

concentration in the hippocampus (Naka et al. 2002).

It should be remembered that AMPA-preferring receptors as well as epibatidine-

sensitive nicotinic receptors are known to colocalize with NMDA receptors and to exert a

permissive role on the NMDA-mediated releasing functions, as suggested by the observation

that activation of these receptors permits the functioning of NMDA receptors also in the

presence of physiological concentrations (~ 1.2 mM) of Mg2+ ions (Pittaluga and Raiteri

1992; Risso et al. 2004a), that usually impede NMDA-induced neurotransmitter release. In

both cases, receptor cross-talk involves local depolarization of plasma membrane and

phosphorylative processes that ultimately reduce sensitivity to the presence of Mg2+ ions. The

lack of changes to AMPA or nicotine–induced release of amine, however, allows us to

speculate that i) selective changes only occurred at the presynaptic NMDA receptor complex

and ii) that EE probably does not interfere with receptor-receptor-dependent mechanisms of

activation of NMDA receptors on noradrenergic terminals.

Similarly to what described for postsynaptic NMDA receptors, glutamate (or NMDA)

and glycine are co-agonists at presynaptic NMDA receptors, whose activation is assured by

the simultaneous binding of both ligands to the respective binding sites (Pittaluga and Raiteri

15

1990). In particular, based on the potency and efficacy of quinolinate, the endogenous ligand

at the glutamate binding site, NMDA receptors localized presynaptically on noradrenergic

terminals were proposed to belong to low affinity NMDA receptor subtype (Luccini et al.

2007). The possibility that the increase in the functional response by NMDA we observed

could be due to an increase in the affinity of the glutamate binding site seems however

unlikely, since the apparent EC50 of NMDA did not change in the hippocampal nerve endings

of EE mice in comparison with the SE mice. Similarly, we were also unable to demonstrate a

significant modification in the apparent affinity of glycine. Therefore, the increasing efficacy

of NMDA receptors appeared not to be accompanied by changes in the potency of the two

essential co-agonists, suggesting that alternative events such as an increase in the number or a

change in the subunit composition of NMDA receptors present in the synaptosomal

membranes may concur to the observed modification of NMDA-induced NA release at the

hippocampal level.

Hippocampal noradrenergic synaptosomes originate from varicosities of ascending

noradrenergic fibers of aminergic neurones located in the LC. We performed a

pharmacological study of the subunit composition of NMDA receptors located

presynaptically on noradrenergic terminals, although the study was limited by the lack of

selective ligands able to discriminate for all the different subunits (NR1, NR2A to D, NR3)

potentially involved in the receptor assembly. In particular, selective ligands for NR2C,

NR2D or NR3 subunits are not so far available, so that the involvement of these subunits in

receptor expression cannot be predicted by a functional perspective. Furthermore, the very

low percentage of the noradrenergic terminals when compared to entire synaptosomal

population isolated from mouse hippocampus does not allow to investigate the subunit

composition with biochemical or immunohistochemical approaches alternative to the

experimental approach here used here.

16

In previous functional studies, the pharmacological characterization of the NR

subunits involved in the assembly of the presynaptic NMDA receptors located on

noradrenergic terminals unveiled that NR2B, but not NR2A, subunit was involved (Pittaluga

et al. 2001). We found that ifenprodil, a selective NR2B antagonist, was equally active in

inhibiting NMDA receptor-induced NA release in EE and SE animals, while nM Zn2+, a

selective NR2A antagonist, was inactive in both cases. This suggests that behavioural training

did not significantly alter the proportion of NR2B subunits in NMDA receptors, and did

induce insertion of NR2A subunits in the same receptors.

NMDA receptors are also located in plasma membranes of noradrenergic neurones in

LC, where they represent preferential targets of glutamate input. In mammalian brain, they

have been proposed to be predominantly composed of NR1 and NR2C subunits (Karolewicz

et al. 2005). NR2C mRNA expression was significantly reduced in the LC of EE mice when

compared to SE animals, suggesting that NMDA receptor composition in NA neurons could

be modified by enriched training at cell body and/or nerve terminal levels. Yet, the lack of

selective ligands towards NR2C subunit does not allow to directly assess whether receptors

containing NR2C subunits participate to NMDA-evoked release of NA in mouse

hippocampus, or whether their contribution to NA release is modified in EE mice. Notably,

NMDA receptors composed of NR1/NR2C subunits were shown to exhibit low conductance

and reduced sensitivity to Mg2+ blockade when compared to receptors composed of

NR1/NR2A or NR1/NR2B subunits (Daggett et al. 1998; Chen et al. 2006). A change in

subunit composition of presynaptic NMDA receptors with a reduced participation of NR2C

subunit would fit very well with the present observation of an increased efficacy of the

receptors to elicit NA release.

We have previously observed that animals chronically treated with nicotine show an

up-regulation of NMDA receptors stimulating [3H]NA release in hippocampus in a very

17

similar manner to that reported in this paper (Risso et al. 2004b). Several studies by our and

other groups indicate that cholinergic transmission is indeed increased in animals exposed to

EE (Park et al. 1992, Tees 1999, Degroot et al. 2005) and that β2* nAChRs mediate part of

morphological and functional effects of EE (Zanardi et al. 2007). That chronic treatment with

nicotine and EE share some molecular and cellular nAChR-dependent mechanisms, including

potentiation of NMDA-mediated NA release is a distinct possibility and will be the matter of

future investigation.

Whatever is the mechanism involved, a functional upregulation of the presynaptic

NMDA receptors which modulate NA release in mouse hippocampus represents a new

instance of neuroplastic changes induced by EE exposure that may contribute to the

improvements in attentional performance and memory found in these animals.

.

18

Acknowledgements

This work was supported by Italian MIUR2007 20072BTSR2 (MM, MZ) and

200728AA57_002 (AP), by Fondazione San Paolo di Torino (MM) and by University of

Genoa ‘Progetto Ricerca Ateneo’ (MM, AP). The authors wish to thank Mrs. Maura Agate for

editorial assistance and Silvia E. Smith (University of Utah) for reviewing the manuscript.

19

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25

Legend to the Figures

Fig. 1 (A) Concentration response curve of NMDA evoked [3H]NA overflow from

hippocampal synaptosomes prepared from SE or EE mice. Data are means ± SEM of five

experiments run in quadruplicate. *p < 0.05, ** p < 0.01 versus SE; °°° p < 0.001 versus

NMDA 1 µM in SE mice; ### p < 0.001 versus NMDA 1 µM in EE mice. Two Way

ANOVA followed by Bonferroni post hoc test. (B) time course of NMDA plus glycine (Gly)

evoked [3H]NA release from SE or EE mice (black line = permanent stimulation). Data are

means ± SEM of five experiments run in quadruplicate. * p < 0.05, ** p < 0.01 versus SE; °°°

p < 0.001 versus basal release in SE mice; ### p < 0.001 versus basal release in EE mice.

Two Way ANOVA followed by Bonferroni post hoc test.

Fig. 2 Effects of enriched environmental on NMDA, AMPA and epibatidine evoked [3H]NA

overflow from mouse hippocampal synaptosomes. Data are means ± SEM of five experiments

run in quadruplicate * p < 0.05 versus SE; # p < 0.05, ### p < 0.001 versus NMDA in SE

mice; ççç p < 0.001 versus NMDA in EE mice; °° p < 0.01 versus AMPA in SE mice; $$$ p <

0.001 versus AMPA in EE mice. Two Way ANOVA followed by Bonferroni post hoc test.

Fig. 3 Effects of multiple NMDA antagonists on NMDA evoked [3H]NA overflow from

hippocampal synaptosomes prepared from SE or EE mice. Data are means ± SEM of five

experiments run in quadruplicate. * p < 0.05, *** p < 0.001 versus NMDA in SE; # p < 0.05,

### p < 0.001 versus NMDA in EE mice. Two Way ANOVA followed by Bonferroni post

hoc test.

26

Fig. 4 Effect of different concentrations of glycine (Gly) on NMDA evoked [3H]NA overflow

from hippocampal synaptosomes prepared from SE or EE mice. Data are means ± SEM of

five experiments run in quadruplicate. * p < 0.05, ** p < 0.001 versus SE; ## p < 0.01, ### p

< 0.001 versus Gly 3 nM in SE mice; °° p < 0.01, °°° p < 0.001 versus Gly 3 nM in EE mice.

Two Way ANOVA followed by Bonferroni post hoc test.

Fig. 5 (A) Concentration-response curve of K+-evoked [3H]NA overflow from hippocampal

synaptosomes prepared from SE or EE mice. Data are means ± SEM of five experiments run

in quadruplicate. ### p < 0.001 versus KCl 9 mM in SE mice; ççç p < 0.001 versus KCl 9

mM in EE mice; °°° p < 0.001 versus KCl 15 mM in SE mice; $$$ p < 0.001 versus KCl 15

mM in EE mice. Two Way ANOVA followed by Bonferroni post hoc test. (B) [3H]NA

uptake from control or enriched mice. Data are means ± SEM of three experiments run in

quadruplicate.

Fig. 6 Bright-field microphotographs of autoradiograms showing NR2C subunit (A and C)

and NET (B and D) mRNAs in the pons of mice exposed to SE (A and B) or EE (C and D).

Images A and C report the boundaries of LC assessed by labelling for NET mRNA in

adjacent sections. Bregma level -5.3 mm.

Fig. 7 Semiquantitative analysis of NR1, NR2A, B, C and D subunit mRNA levels in the LC

of mice exposed to SE or EE. Means ± SEM are shown, n = 5 for SE and n = 7 for EE.

Statistical analysis according to Mann-Whitney U test, Z =-2.84, p = 0.004.

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Table 1 Sequences of the oligonucleotides used in the study. __________________________________________________________ Mouse NR1: sequence code NM008169 NR1-1 5’atgaggtcct cacacactga cagggccatc tgtatggcgt tgggc3’ NR1-2 5’agcttgttgt cccgcacagc ctggatggcc tcagctgcac tctca3’ Mouse NR2A: sequence code NM008170 NR2A-1 5’tgtctgcccg tagcagctgg ccttggcctc gggaatgtag gagaa3’ NR2A-2 5’gggcaggttt gagaggcagc ttctgcaatg cgtggagttc tgccg3’ Mouse NR2B: sequence code NM008171 NR2B-1 5’cagcaggctg gtccagttcc tgcagggagt tgtcctcgct gatgt3’ NR2B-2 5’tgcccgatac ggccaagacg gccaacacca accagaactt gggag3’ Mouse NR2C: sequence code NM010350 NR2C-1 5’gtgagtggct ggatctccag aggcaagtcc aggaagttct gcggg3’ NR2C-2 5’gccagattag gtactaacca cacgtgaccg ggtcccacca agcca3’ Mouse NR2D: sequence code NM008172 NR2D-1 5’gtcgctgagc ccagacacgg tgtccacgta ctcctcctgg atcat3’ NR2D-2 5’catgaaccag acgtagccag gcccagtgag accagcctct tctgc3’ Mouse NET: sequence code AY188506 NET-1 5’tggcaaggtc cacagcgaag cccaccacgg acagcaggaa atcaa3’ NET-2 5’aggagtgtcc gcagttggtc cagggcaggt tcaaggtgaa ggatg3’

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