1

Inhibitory effects of beta-amyloid on the nicotinic receptors which

stimulate glutamate release in rat hippocampus: the glial contribution

Alessia Salamonea,1, Elisa Murab,1, Stefania Zappettinia, Massimo Grillia, Guendalina Oliveroa,

Stefania Predab, Stefano Govonib, Mario Marchia,c,*

a Section of Pharmacology and Toxicology, Department of Pharmacy, University of Genoa, Genoa,

Italy

b Department of Drug Sciences, Centre of Excellence in Applied Biology, University of Pavia,

Pavia, Italy

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

* Corresponding author. Section of Pharmacology and Toxicology, Department of Pharmacy,

University of Genoa, Genoa, Italy. Tel: +39 010 3532657; Fax:+39 010 3993360

E-mail address: marchi@pharmatox.unige.it (M. Marchi)

1 These authors contributed equally to this work.

This work was published by Elsevier in European journal of pharmacology 11/2013; DOI:10.1016/j.ejphar.2013.11.011

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ABSTRACT

_______________________________________________________________________________

We investigated on the neuronal nicotinic acetylcholine receptor subtypes involved in the

cholinergic control of in vivo hippocampal glutamate (GLU), aspartate (ASP) and inhibitory γ-aminobutyric acid (GABA) overflow. We also investigated on the possible contribution of nicotinic

acetylcholine receptors subtypes present on astrocytes in the regulation of the three neurotransmitter

amino acids overflow using hippocampal gliosomes and on the effects of beta-amyloid (Aβ) 1-40

on the nicotinic control of amino acid neurotransmitter release. Nicotine was able to enhance the in

vivo overflow of the three amino acids being more potent in stimulating GLU overflow. The α7

selective agonist PHA543613 induced an overflow very similar to that of nicotine. The α4β2

selective agonist 5IA85380 was significantly less potent in inducing GLU overflow while the

overflow of ASP and GABA were almost inconsistent. Aβ1-40 inhibited the neurotransmitter

overflow stimulated by PHA543613 but not the one evoked by 5IA85380. In hippocampal

gliosomes nicotine elicited selectively GLU overflow which was also evoked by 5IA85380 and by

the α7 selective agonist choline. Nicotine- and choline-induced glutamate overflow in gliosomes

was inhibited by Aβ1-40. In conclusion nicotine administration in vivo elicits hippocampal GLU

release mostly through α7 nicotinic acetylcholine receptors likely present both on neurons and

astrocytes. Aβ inhibitory effect on the nicotinic-control of GLU release seems to depend primarily

to the inhibition of α7 nicotinic acetylcholine receptors functional responses.

Keywords: β-amyloid; aminoacid release; α7 nicotinic receptors; α4β2 nicotinic receptors;

microdialysis; isolated nerve endings

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1. Introduction

It is well established that the cholinergic system modulates the function of glutamatergic

system in several brain areas mainly through the activation of neuronal nicotinic acetylcholine

receptor subtypes. These receptors are associated to permeability to Ca2+ thereby facilitating events

such regulation of second messenger cascades (Khiroug et al., 2003), cell survival (Mechawar et al.,

2004) and apoptosis (Berger et al., 1998) as well as the direct stimulation glutamate (GLU) release

(Toth, 1996; Fedele et al., 1998; Marchi et al., 2002). Moreover, specific intracellular mechanisms

have been also shown to be involved in the nicotinic modulation of GLU release (Lambe et al.,

2003; Dickinson et al., 2007; Zappettini et al., 2010). This modulatory effect may also be relevant

given that, in the hippocampus, glutamatergic and cholinergic systems have undoubtedly a

fundamental role in the mechanisms of learning and memory (Picciotto et al., 1995; Levin and

Simon, 1998; Giacobini, 2003; Errico et al., 2011; Parri et al., 2011) and both systems undergo age-

and pathology-associated changes such as attention and memory (Woodruff-Pak and Gould, 2002;

Mattson, 2008)..

Recent studies in vivo and in vitro have shown that beta-amyloid (Aß) was able to modulate

the function of neuronal nicotinic acetylcholine receptor subtypes (Dougherty et al., 2003; Liu and

Wu, 2006; Puzzo et al., 2008; Tong et al., 2011; Lilja et al., 2011; Ni et al., 2013). Moreover, non-

neurotoxic Aβ 1-40 concentrations were able to modulate the nicotine-evoked release of both

excitatory GLU and aspartate (ASP) and inhibitory γ-aminobutyric acid (GABA) and glycine (Mura

et al., 2010, 2012; Zappettini et al., 2012). It is important to recall that GLU is essential to memory

formation so its function might be pivotal to Alzheimer disease (AD) progression (Revett et al.,

2013).

The relevance of the activation of different neuronal nicotinic acetylcholine receptor

subtypes which stimulate in vivo the release of GLU has been so far poorly investigated.

Interestingly, nicotinic acetylcholine receptors are present in several non-neuronal cells including

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astrocytes (Sharma and Vijayaraghavan, 2001; Lim and Kim, 2003; Patti et al., 2007) and therefore

part of the GLU which is released in vivo might result also from these cells (Santello and Volterra,

2009; Santello et al., 2011). In the present study we have first comparatively investigated, in vivo,

the stimulatory effects of two selective α4β2 and α7 neuronal nicotinic acetylcholine receptor

agonists on the overflow of endogenous GLU, GABA and ASP and the modulatory effects of the

Aβ peptide 1-40 on the function of these neuronal nicotinic acetylcholine receptor subtypes. We

have then studied the effects of selective neuronal nicotinic acetylcholine receptors agonists and Aβ

peptide on amino acid release using hippocampal gliosomes an in vitro preparation originating

from adult astrocytes (Stigliani et al., 2006; Patti et al., 2007; Milanese et al., 2010; Matos et al.,

2012a,b).

From our results we can conclude that the administration in vivo of nicotine and of two

selective α4β2 and α7 nicotinic acetylcholine receptor agonists elicits a significant release of GLU,

ASP and GABA in the rat hippocampus. The inhibitory effect of Aβ on the modulation of the three

amino acid release in vivo seems to depend primarily on the interaction with the α7 nicotinic

acetylcholine receptors. Moreover, both α7 and α4β2 nicotinic acetylcholine receptors are present

on rat hippocampal gliosomes and modulate the release of GLU but not that of ASP and GABA and

Aβ is able to inhibit only the α7 but not the α4β2 nicotinic acetylcholine receptor subtype.

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2. Materials and methods

2.1. Animals and brain tissue preparation

Adult male Wistar rats (200–250 g, Harlan, Udine) were used for both in vivo experiments

and as brain tissue source for in vitro experiments. Animals were housed at constant temperature

(22 ± 1°C) and relative humidity (50%) under a regular light–dark schedule (light 7 a.m.–7 p.m.).

The in vitro experimental procedures were approved by the Ethical Committee of the Pharmacology

and Toxicology Section, Department of Pharmacy, in accordance with the European legislation

(European Communities Council Directive of 24 November 1986, 86/609/EEC) and were approved

by Italian legislation on animal experimentation (Decreto Ministeriale number 124/2003-A). The in

vivo protocol was approved by Ethical Committee of Pavia’s University (session of October 11th

2011, minutes 3/2011) according to international regulations for the care and treatment of laboratory

animals, to the Italian Act (D.L. n. 116, GU, suppl 40, 18 February, 1992) and to EEC Council

Directive (86/609, OJ L 358, 1, 12 December, 1987). All efforts were made to minimize animal

suffering and to use the minimal number of animals necessary to produce reliable results

2.2. In vivo experiments

2.2.1. Microdialysis probe implantation

Rats were anesthetized with Equithesin 3 ml/kg (pentobarbital 9.7 g, chloral hydrate 42.5 g,

MgSO4 21.3 g for 1L, 10% ethanol, 40% propylene glycol v/v) administered intraperitoneally and

placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA). The skin was

shaved, disinfected, and cut with a sterile scalpel to expose the skull. A hole was drilled to allow the

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implantation of the probe into the brain parenchyma. The probe was implanted in the hippocampus

(CA1/CA2 regions; AP -5.8 mm, ML ± 5.0 mm from bregma and DV−8.0mm from dura) according

to the Paxinos and Watson atlas (1986), and secured to the skull with one stainless steel screw and

dental cement. All in vivo experiments were performed using microdialysis probes, made in our

laboratory according to the original method described by Di Chiara (1990) (EmophanBellco

Artificial OR-internal diameter 200 µm, cut-off 40 KDa; Bellco, Mirandola, Modena, Italy), with a

nominal active length of 5mm. Finally, the skin was sutured, and the rats were allowed to recover

from anaesthesia for at least 24 h before the neurotransmitter release study. Previous

immunohistochemical analysis has shown that the administration of Aβ1-40 through the dialysis

probe at the concentrations tested in vivo (1 µM and 10 µM) allowed the delivery of the peptide

within the hippocampus. Despite the fact that we do not know the exact amounts of Aβ reaching the

tissue, there was a visible positive correlation between the concentration administered and the signal

of Aβ immunoreactivity in the tissue. Moreover, immunohistochemical analysis shows that no

evident signs of apoptosis were observed within the area of amyloid diffusion as shown by Hoechst

staining (Mura et al., 2012).

2.2.2. Microdialysis samples collection

Microdialysis experiments were performed on conscious freely moving rats. On the day of

the experiments (24 hrs after the surgical procedure), the probe was perfused with artificial CSF

containing 145 mM NaCl, 3.0 mM KCl, 1.26 mM CaCl2, 1.0 mM MgCl2, 1.4 mM Na2HPO4,

buffered at pH 7.2–7.4 and filtered through a Millipore 0.2 µm pore membrane. In all experiments,

the microdialysis membrane was allowed to stabilize for 1 h at the flow rate of 4 µl/min, without

collecting samples. At the end of the stabilization period, three samples were collected to evaluate

baseline release of GLU, ASP and GABA and then the specific treatment started. All treatments

were administered by manually switching syringes and tubing connections to allow drugs diluted in

artificial CSF to flow through the probes. Tubing switches were performed taking care to maintain

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constant flow rates and collection volumes. Both basal and treatment samples were collected every

20 min in 100 µl Eppendorf tubes. The flow rate of 4 µl/min was maintained using a 1000 µl

syringe (Hamilton) and a microinjection pump (CMA/100, CMA/Microdialysis AB). In vitro

recovery of the probe for GLU was 15.21 ± 0.42 (Mura et al., 2012). Each rat was used for only one

microdialysis session. At the end of each experiment animals were sacrificed by guillotine, rat

brains were removed and the position of the microdialysis probe was verified by histological

procedures, slicing the tissues by a cryostat microtome (LEICA CM 1510). Only data from rats in

which probe tracks were exactly located in the target area were used for statistical analysis.

2.3. In vitro experiments

2.3.1.Experiments of release

Rats were killed by decapitation and the hippocampus rapidly removed at 0–4°C. Purified

gliosomes were prepared essentially as described by Nakamura et al. (1993) with minor

modifications (Stigliani et al., 2006). The tissue was homogenized in 10 volumes of 0.32 M

sucrose, buffered to pH 7.4 with Tris (final concentration 0.01 M) using a glass Teflon tissue

grinder (clearance 0.24 mm). The homogenate was centrifuged at 1000 g for 5 min, to remove

nuclei and cellular debris, and the supernatant was gently stratified on a discontinuous Percoll

gradient (2, 6, 10 and 20% (v/ v) in Tris-buffered sucrose) and centrifuged at 33,500 g for 5 min.

The layer between 2 and 6% Percoll (gliosomal fraction) was collected, washed by centrifugation

and resuspended in physiological medium containing 125 mM NaCl, 3 mM KCl, 1.2 mM MgSO4,

1.2 mM CaCl2, 1 mM NaH2PO4, 22 mM NaHCO3 and 10 mM glucose, pH 7.2–7.4 (aeration with

95% O2 and 5% CO2). Gliosomes were incubated at 37°C for 15 min and at the end of the

incubation period, identical portions of the gliosomal suspension was layered on microporous filters

at the bottom of parallel superfusion chambers thermostated at 37°C (Raiteri and Raiteri, 2000;

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Superfusion System, Ugo Basile, Comerio, Varese, Italy). Gliosomes were superfused at 1 ml/min

with standard physiological medium as previously described. The system was first equilibrated

during 36.5 min of superfusion; subsequently, four consecutive 90 s fractions of superfusate were

collected and the endogenous GLU, ASP and GABA content were measured by high performance

liquid chromatography as below described. Gliosomes were exposed to agonists for 90 s starting

from the second fraction collected (t = 38 min), with antagonists being added 8 min before agonists.

The evoked overflow was calculated by subtracting the corresponding basal release from each

fraction and was expressed as pmol/mg of synaptosomal proteins.

2.4 Endogenous amino acids determination

In both dialysates and fractions collected from gliosomes in superfusion levels of

endogenous GABA, GLU and ASP were measured by HPLC analysis following precolumn

derivatization with o-phthalaldehyde and resolution through a C18-reverse phase chromatographic

column (1064.6 mm, 3 mm; Chrompack, Middleburg, The Netherlands) coupled with fluorometric

detection (excitation wavelength 350 nm; emission wavelength 450 nm). Homoserine was used as

internal standard. Buffers and gradient program were prepared and executed as follows: solvent A,

0.1 M sodium acetate (pH 5.8)/methanol, 80:20; solvent B, 0.1 M sodium acetate (pH

5.8)/methanol, 20:80; solvent C, sodium acetate (pH 6.0)/methanol, 80:20; gradient program, 100%

C for 4 min from the initiation of the program; 90% A and 10% B in 1 min; 42% A and 58% B in

14 min; 100% B in 1 min; isocratic flow 2 min; 100% C in 3 min; flow rate 0.9 ml/min.

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2.5. Statistical analysis

2.5.1. In vivo experiments

Values were expressed either as amount of GLU measured in the dialysate (pmol/80 µL) or

as area under the curve (AUC), evaluating the cumulative release over time. AUC was used as a

measure of treatment exposure and was calculated, for each animal, using GraphPad Prism (version

4.03 GraphPad Software, San Diego, CA, USA), defining as baseline of the area the basal value

(average concentration of three consecutive samples immediately preceding the drug dose).

D’Agostino-Pearson Omnibus Test (GraphPad Prism, version 4.03, GraphPad Software, San Diego,

CA, USA) and Grubb’s Test (GraphPadQuickCalcs, online calculator for scientists at

http://www.graphpad.com/quickcalcs/, GraphPad Software, San Diego, CA, USA) were used as

preliminary tests in order to evaluate whether data were sampled from a Gaussian distribution and

to detect outliers respectively. All outliers were excluded from the analysis. Data were then

analyzed by analysis of variance (one- or two-way ANOVA) followed, when significant, by an

appropriate post hoc comparison test. Data were considered significant for P < 0.05. The reported

data are expressed as means ± S.E.M. The number of animals used for each experiment is reported

in the legend to figures.

2.5.2. In vitro experiments

Multiple comparisons were performed with one-way ANOVA followed by an appropriate

post hoc test (Dunnett’s and Bonferroni). Data were considered significant for P < 0.05, at least.

2.6. Preparation of beta-amyloid solutions

We used only Aβ 1–40 peptide in our experiments, for two main reasons. First,

physiologically the 40-aminoacid long peptide is the most abundant form (Pearson and Peers,

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2006). Second, Aβ1–42 has been reported to aggregate faster than Aβ1–40 and thus it is considered

as the most neurotoxic species. With the aim of exploring new effects of Aβ other than the

neurotoxic ones, we chose to avoid this potentially confounding element. In the case of both in vivo

and in vitro experiments, synthetic human Aβ1–40 was dissolved in aCSF at a concentration of 100

µM (stock solution). Then, this solution was filtered through a Millipore 0.2 mm pore membrane

and stocked in small aliquots at -80° C. Working solutions were freshly prepared by diluting an

aliquot of Aβ1–40 stock solution at the final concentrations (10 µM, 1 µM, or 100 nM Aβ1–40 for

in vivo experiments, 100 nM, 10 nM, 1 nM, or 100 pM for in vitro analysis), just before the

administration.

It is well known that the most likely explanation for the many discrepancies observed

between reports using Aβ preparations may be attributable to differences in the aggregation state of

the Aβ used. As far as the characteristics of the Aβ peptides delivered through the dialysis probe we

have previously shown, by western blot procedure, that we administered at least predominantly, Aβ

monomers although we cannot completely exclude that small amounts of Aβ oligomers are also

present and may participate to produce the observed effects (Mura et al., 2012). In regard to the in

vitro Aβ preparations, since we did not observe aggregation at the concentrations and the timing (up

to 40 min) analyzed in vivo, we also do not expect to observe aggregation at the lower

concentrations and shorter times used in vitro in light of the fact that aggregation is a concentration

and time-dependent process.

2.7. Chemicals

Beta-amyloid (1-40; 40-1), Percoll®, choline, dimethyl sulfoxide, nicotine hydrogen tartrate

salt (Sigma-Aldrich, St Louis, MO, USA); 5IA85380, PHA543613, (Tocris Bioscience, Bristol,

UK); all salts used for the preparation of aCSF (NaCl, KCl, CaCl2, MgCl2, Na2HPO4) and for

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Equithesin (MgSO4) were purchased at Merck KGaA, Darmstadt, Germany; chloral hydrate,

ethanol 96% and propylene glycol were used for the preparation of Equithesin and were obtained at

VWR BDH Prolabo, Belgium.

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3. Results

Figure 1a shows the stimulatory effect of the in vivo administration of nicotine and two

nicotinic acetylcholine receptor agonists, PHA543613 and 5-IA85380. In our experimental

conditions, 40 minutes-long administration of 50 mM nicotine was able to greatly enhance the

overflow of all the three amino acids being more potent in stimulating GLU overflow. Figure 1a

shows that the α7 agonist PHA543613 induced a significant release of amino acid. Conversely, the

amount of GLU elicited by the α4β2 nicotinic acetylcholine receptor agonist 5IA85380 was

significantly lower comparable to that elicited by nicotine (-70 %) while the overflow of ASP and

GABA was almost inconsistent. The nicotine evoked aminoacid release was strongly inhibited in

presence of the α7 antagonist MLA (10 µM) (-66 % GLU; -51 % ASP; -94% GABA). The

possibility that Aβ may differentially inhibit the two nicotinic acetylcholine receptors subtypes

which control amino acid release has been investigated. In order to verify this point we have studied

the effects of Aβ1-40 on the release elicited by the two different agonists PHA543613 and

5IA85380. Figure 1b shows that the PHA-evoked overflow of endogenous GLU, ASP and GABA

was significantly inhibited by Aβ 1-40 used at 10 µM concentration. Aβ 1-40 at 1 µM concentration

was still able to decrease ASP and GABA overflow but was ineffective on GLU overflow. Aβ1-40

(10 µM) did not modify the basal release of endogenous GLU, ASP and GABA (data not shown).

Interestingly the 5IA85380 (10 nM)-evoked in vivo overflow of GLU from rat hippocampus was

not affected by Aβ1-40 at all concentration used (Fig. 1c). Based on the in vivo data we then

analyzed the effects of Aβ1-40 on the administration of nicotine, choline (Ch) and 5IA85380,

known to act selectively on the α7 and α4β2 nicotinic acetylcholine receptor subtypes respectively

(Mukhin et al., 2000; Uteshev et al., 2003; Dickinson et al., 2007; Zappettini et al., 2010), on the

overflow of GLU, ASP and GABA from rat hippocampal gliosomes in vitro. Figure 2 shows that

nicotine, in a concentration dependent manner elicited a significant overflow of endogenous GLU

but not of ASP and GABA. This overflow was comparable to that produced by 9 mM KCl. A

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higher KCl concentration (15 mM) produced a significant increase of GLU release (406 ± 32). Also

Ch (1 mM) and 5-IA85380 hydrochloride (5IA85380; 10 nM) evoked a significant overflow of

GLU (104 ± 23; 118 ± 15) from hippocampal gliosomes demonstrating the involvement of two

nicotinic acetylcholine receptor subtypes. The Ch (1 mM)-and 5IA85380 (10 nM)-evoked GLU

overflow was significantly Ca2+-dependent (data not shown).

Both nicotine- and Ch-evoked GLU overflow were significantly inhibited by Aβ1-40 at 100

nM but not at 10 nM concentrations (-58%; -80 %; Fig. 3a, b). Interestingly, the 5IA85380 (10

nM)-evoked GLU overflow was unaffected in presence of Aβ1-40 (100 nM and 10 nM). Aβ1-40

(100 nM) did not modify the basal release of endogenous GLU (data not shown).

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4. Discussion

As previously mentioned there are several in vitro studies that demonstrate the existence in

hippocampus of α7 and α4β2 nicotinic acetylcholine receptor or both which modulate the release of

GLU (Bancila et al., 2009; Zappettini et al., 2011; Lozada et al., 2012). Huang et al. (2010) in an

iontophoretic study showed that some selective α7 nicotinic acetylcholine receptor agonists mimic

the effect of nicotine suggesting that the α7 nicotinic acetylcholine receptor is the major nicotinic

acetylcholine receptor subtype involved in evoking the excitation of CA3 neurons in rat

hippocampus. Using in vivo microdialysis technique it has been also reported that both GLU and

GABA are released upon activation α7 nicotinic acetylcholine receptors in rat striatum (Campos et

al., 2010). To the best of our knowledge, this is the first study that demonstrates the ability of α7

and of non-α7 nicotinic acetylcholine receptor agonists to directly modulate GLU, ASP and GABA

release in rat hippocampus in vivo. Our data showed that both α7 and α4β2 nicotinic acetylcholine

receptors are able to stimulate, in vivo, the release of GLU, ASP and GABA but the α7 nicotinic

acetylcholine receptors have a quantitatively predominant role. The nicotine-evoked aminoacid

release was indeed quantitatively relevant compared to that evoked by a selective α7 nicotinic

acetylcholine receptor agonist PHA543613 and strongly reduced in presence of MLA an inhibitor

of α7 nicotinic acetylcholine receptor. Moreover, the GLU and ASP release evoked in presence of

5IA85380, used at a concentration that normally produce the maximal effect (Livingstone et al.,

2009), was very modest (or inconsistent as in the case of GABA release). Of course, the hypothesis

that the administration of higher doses might also activate nicotinic acetylcholine receptor subtypes

different from those reported should not be excluded.

Quite interestingly the finding that the activation of α7 nicotinic acetylcholine receptor is

predominant in the in vivo release of GLU may explain also that the inhibitory effect of Aβ, in vivo,

seems to be exclusively due to the inhibition of the α7 nicotinic acetylcholine receptor. Indeed, as

mentioned before the stimulatory effect of 5IA85380, although modest, was not altered in presence

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of Aβ while the PHA543613-evoked GLU release was strongly and specifically reduced. This result

is also in agreement with the ineffectiveness of Aβ in inhibiting the 5IA85380-evoked GLU release

from gliosomes. It has to be also mentioned that Aβ was also unable to inhibit the in vivo release of

GLY elicited by α4β2 nicotinic acetylcholine receptors agonist while it exerted an inhibition of both

nicotine- and PHA543613-stimulated GLY release (Zappettini et al., 2012).

It is now well known that astrocytes can cross talk with neighboring neurons and are

involved in the active control of synaptic transmission and brain activity (Araque et al., 1999;

Haydon, 2001; Volterra and Meldolesi, 2005). They can exhibit an increase in Ca2+ response,

(Enkvist et al., 1989; Cornell-Bell et al., 1990; Pasti et al., 1995; Harris-White et al., 1998; Parri et

al., 2011) which produce as consequence the release of gliotransmitters, such as GLU. This event

can be observed also following stimulation by several compounds including nicotine (Dani et al.,

1992; Porter and McCarthy, 1996; Parpura and Haydon, 2000; Pasti et al., 2001; Bezzi et al., 2004;

Chen et al., 2005). Indeed, astrocytes in culture express nicotinic acetylcholine receptors,

particularly those containing α7 and α4β2 subunits (Sharma and Vijayaraghavan, 2001; Xiu et al.,

2005; Shen and Yakel, 2012) and it has been demonstrated that the stimulation of these receptors

modulates GLU uptake and GLU synthase activity in astrocytes (Lim and Kim, 2003).

In a previous paper Stigliani and colleagues (2006) characterized in detail a particular

particle called gliosomes which are an in vitro vital preparation originating from mature astrocytes

present in different brain areas. Gliosomes are able to respond to exogenous stimuli and present

some differences from their neuronal counterpart (synaptosomes). Furthermore, it is well known

that GLU is also released from astrocytes and the presence of nicotinic receptors stimulating GLU

release from cortical gliosomes in mice have been already demonstrated (Patti et al., 2007). We here

report that the cholinergic modulation of GLU release on rat hippocampal gliosomes was modulated

by both α7 and α4β2 nicotinic acetylcholine receptors. Interestingly, both receptors elicited

selectively the release of GLU but not that of ASP or GABA. We do not know their physiological

importance in the intact astrocytes since information in the literature regarding to the presence of

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nicotinic acetylcholine receptors on these cells are different depending the species considered, the

areas and the age of animals (Xiu et al., 2005; Yu et al., 2005). However, from our findings it is

possible to hypothesize that GLU release evoked, in vivo, by nicotine or nicotinic agonists, as

shown in Fig. 1, might originate also from nicotinic stimulation of astrocytes. Conversely, the

overflow of ASP and GABA seem to be exclusively from neuronal origin.

It is significant to note that Aβ selectively inhibits, in gliosomes, the GLU overflow evoked

by α7 nicotinic acetylcholine receptor subtypes but is inactive in inhibiting that evoked by α4β2

nicotinic acetylcholine receptor. This finding is very relevant because Aβ at the same concentration

used in this paper, was able to inhibit the 5IA85380-evoked GLU overflow from isolated nerve

endings (Mura et al., 2012). Therefore there is the possibility that the 5IA85380-sensitive nicotinic

acetylcholine receptors present on gliosomes are different from those present on synaptosomes.

Although the exact nature of the Aβ interaction with nicotinic acetylcholine receptor subtypes is so

far not well understood it is quite interestingly to note that the α7 nicotinic acetylcholine receptor

subtypes modulating the overflow of GLU from gliosomes (Fig. 2) and from synaptosomes (Mura

et al., 2012) were functionally inhibited in vitro apparently to a similar extent by Aβ concentrations

in the nanomolar range.

5. Conclusions

From our results we can therefore conclude that a) both α7 that α4β2 nicotinic acetylcholine

receptors are present on rat hippocampal gliosomes and they modulate the release of GLU but not

that of ASP and GABA; b) Aβ is able to selectively inhibit only the α7 but not the α4β2 nicotinic

acetylcholine receptor subtype present on gliosomes c) the administration of nicotine in vivo is able

to elicit GLU release mostly through the activation of α7 nicotinic acetylcholine receptors present

both on neurons and astrocytes; d) the nicotinic evoked release of ASP and GABA has exclusively a

neuronal origin. We can therefore foresee that in an integrated system, where cellular networks and

their functional relationships are completely preserved and several direct and indirect processes are

simultaneously taking place in neurons and astrocytes, the effect of Aβ on the cholinergic

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modulation of GLU function may mostly depend on the interaction of Aβ with the α7 nicotinic

acetylcholine receptors. This finding should be relevant considering that α7 nicotinic acetylcholine

receptors are strongly increased in astrocytes of patients with AD (Nagele et al., 2003) and an

increase in the proportion of astrocytes expressing α7 immunoreactivity was observed in AD

patients compared with age-matched controls (Teaktong et al., 2003).

Acknowledgements

This work was supported by from the Italian Ministero Università Ricerca to Prof. Mario Marchi

(Prot. N° 2009R7WCZS_003), by Compagnia di San Paolo, by University of Genoa ‘Progetto

Ricerca Ateneo’ and by project AROMA (ALCOTRA 2007-2013) by the European Community

Project PYRGI, project n° B51H10000000006 and by the project 7FP-KBBE BAMMBO and to

Prof. Govoni (MIUR 2009). This work is supported by a grant of the Alzheimer’s Association

(NIRG-11-205183) to Elisa Mura. We wish to thank Maura Agate for editorial assistance and Dr.

Silvia E. Smith, Ph.D. (University of Idaho, IBEST, School of Life Sciences) for reviewing the

manuscript.

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Legends to the Figures

Fig. 1. Panel a) shows the effects of the in vivo administration of nicotine (NIC), PHA543613

(PHA) and 5IA85380 on the endogenous overflows (AUC) of glutamate (GLU), aspartate (ASP)

and GABA from rat hippocampus. Data are mean ± S.E.M. of 4–12 individual rats for each

experimental group. ** P < 0.01 versus nicotine-evoked GLU overflow; # P < 0.05; ## P < 0.01

versus nicotine-evoked ASP overflow; ςς P < 0.01 versus PHA543613-evoked ASP overflow, ° P <

0.05; °° P < 0.01 versus nicotine-evoked GABA overflow, § P < 0.05 versus PHA543613-evoked

GABA overflow (One-way ANOVA followed by Bonferroni post hoc test). Panel b) shows the in

vivo effects of Aβ 1-40 on the PHA543613 (PHA)-evoked endogenous GLU, ASP and GABA

overflows from rat hippocampus. Data are mean ± S.E.M. of 4–12 individual rats for each

experimental group. òòò P < 0.001 versus PHA543613-evoked GLU overflow, § P < 0.05 versus

PHA543613-evoked ASP overflow; $ P < 0.05; $$P < 0.01 versus PHA543613-evoked GABA

overflow (One-way ANOVA followed by Dunnett's Multiple Comparison Test). Panel c) shows the

in vivo effects of Aβ 1-40 on the 5IA85380-evoked endogenous GLU overflow from rat

hippocampus. Data are mean ± S.E.M. of 4–12 individual rats for each experimental group.

Fig. 2. Effect of different concentrations of nicotine (NIC) on endogenous overflow of glutamate

(GLU), aspartate (ASP) and GABA and effect of KCl, 5IA85380 and Choline (Ch) on the

endogenous overflow of GLU from rat hippocampal gliosomes. Data are mean ± S.E.M. of 4

experiments run in triplicate. ## P < 0.01 versus NIC 10µM, *** P < 0.001 versus NIC 100µM

evoked endogenous GLU overflow (one-way ANOVA followed by Newman Keuls post hoc test).

26

Fig. 3. The figure shows the concentration dependent effect of Aβ 1–40 on the nicotine (NIC)-,

choline (Ch)- and 5IA85380- induced overflow of GLU, in rat hippocampal gliosomes (Panels a, b

and c, respectively). *** P < 0.001 vs. NIC and Ch (One-way ANOVA followed by Dunnett's

Multiple Comparison Test). Data are expressed as mean ± S.E.M. of 4 experiments run in triplicate.

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