caffeine potentiates the release of gaba mediated by nmda receptor activation: involvement of a1...

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Please cite this article in press as: Ferreira DDP et al. Caffeine potentiates the release of GABA mediated by NMDA receptor activation: Involvement of

A1 adenosine receptors. Neuroscience (2014), http://dx.doi.org/10.1016/j.neuroscience.2014.09.060

NSC 15734 No. of Pages 8

8 October 2014

Neuroscience xxx (2014) xxx–xxx

CAFFEINE POTENTIATES THE RELEASE OF GABA MEDIATEDBY NMDA RECEPTOR ACTIVATION: INVOLVEMENT OF A1 ADENOSINERECEPTORS

D. D. P. FERREIRA, a B. STUTZ, b F. G. DE MELLO, b

R. A. M. REIS b* AND R. C. C. KUBRUSLY a

a Laboratorio de Neurofarmacologia, Departamento de Fisiologia

e Farmacologia, Programa de Pos-graduacao em

Neurociencias, Universidade Federal Fluminense, Niteroi, Brazil

b Laboratorio de Neuroquımica, Instituto de Biofısica Carlos Chagas

Filho, Universidade Federal de Rio de Janeiro, Rio de Janeiro, Brazil

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Abstract—Caffeine, a methylated derivative of xanthine and

widely consumed psychoactive substance, acts in several

targets in the nervous system. We investigated its role in ret-

inal explants of chick embryo analyzing the role of puriner-

gic receptors in [3H]-GABA release induced by D-aspartate

(D-asp). D-Asp increases GABA-release 4.5-fold when com-

pared to basal levels from 13-day-old chick embryo retina

explants. Caffeine 500 lM elevated D-asp-induced GABA

release in 60%. The release was inhibited in the presence

of NNC-711, a GABA transporter-1 (GAT-1) blocker or by

MK-801, an N-methyl-D-aspartate receptor (NMDAR) antago-

nist. Caffeine did not modify [3H]-GABA uptake carried out

for 5, 10, 30 and 60 min and did not increase the release of

D-asp or glutamate at basal or stimulated conditions. The

caffeine effect was mimicked by the adenosine A1 receptor

antagonist DPCPX and by the adenylyl cyclase (AC) activa-

tor forskolin. It was also blocked by the protein kinase A

(PKA) inhibitor H-89, tyrosine kinase inhibitor genistein or

by the src family kinase (SFK) inhibitor PP1. Forskolin-

stimulated cyclic adenosine monophosphate (cAMP) levels

were reduced in the presence of the A1 receptor agonist

CHA. Western blot analysis revealed that 500 lM caffeine

increased phosphoGluN2B expression levels in approxi-

mately 60% when compared to total GluN2B levels in embry-

onic E13 retina. The GluN2B subunit-containing NMDAR

antagonist ifenprodil inhibited the caffeine effect. Our

results suggest that caffeine potentiates D-asp-induced

GABA release, which is mediated by GAT-1, via inhibition

of adenosine A1 receptor and activation of the PKA pathway.

Regulation of NMDAR by phosphorylation of GluN2B

http://dx.doi.org/10.1016/j.neuroscience.2014.09.0600306-4522/� 2014 Published by Elsevier Ltd. on behalf of IBRO.

*Corresponding author. Tel: +55-21-39386594.

E-mail addresses: [email protected] (D. D. P. Ferreira), [email protected] (B. Stutz), [email protected] (F. G. de Mello), [email protected] (R. A. M. Reis), [email protected] (R. C. C. Kubrusly).Abbreviations: AC, adenylyl cyclase; BSA, bovine serum albumin;cAMP, cyclic adenosine monophosphate; CNS, central nervoussystem; D-asp, D-aspartate; E#, embryonic day #; EDTA,ethylenediamine tetraacetic acid; GABA, gamma-aminobutyric acid;GAT, GABA transporter; GluN1, GluN2A, GluN2B, subunits of NMDAreceptor; HPLC, high-performance liquid chromatography; MEM,minimum essential medium; NMDAR, N-methyl-D-aspartate receptor;PKA, protein kinase A; SFK, src family kinase; TCA, trichloroaceticacid.

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subunit by a SFK may also be involved in the effect

promoted by caffeine. � 2014 Published by Elsevier Ltd.

on behalf of IBRO.

Key words: adenosine receptors, caffeine, GABA, retina,

NMDA receptor.

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INTRODUCTION

Caffeine is a methylated derivative of xanthine and is

considered the most widely consumed psychoactive

substance in the world (Ogawa and Ueki, 2007). Caffeine

stimulates motor activity (Ferre, 2008), modulates onset

and quality of sleep (Diaz-Munoz and Salin-Pascual,

2010), improves attention/vigilance, increases memory

retention (Cunha and Agostinho, 2010) and is also a cog-

nitive enhancer (Daly, 2007). Many studies have reported

a potential therapeutical role for caffeine in several neuro-

degenerative disorders, including Parkinson and Alzhei-

mer diseases (Arendash and Cao, 2010; Marques et al.,

2011). Pharmacological mechanisms underlying caffeine

effects are primarily via a nonselective antagonism of

adenosine receptors, with A1 and A2A receptors as prefer-

ential targets (Fredholm et al., 1999; Ferre, 2008).

The embryonic retina has been used for the past

40 years as a model for development and neurochemical

signaling, since the major neurotransmitter systems are

present in the cellular components of this tissue. Among

these, dopamine, adenosine, c-aminobutyric acid

(GABA) and glutamate predominate as major

transmitters (Reis et al., 2007). Adenosine is a purine

nucleoside present in all cells. This neuromodulator has

many roles in the nervous system, including neuroprotec-

tion, synapse development and modulation of neurotrans-

mitter circuitry in the developing nervous system (Ferreira

and Paes-de-Carvalho, 2001; Paes-de-Carvalho et al.,

2003; Fredholm, 2010). Adenosine is able to modulate

synaptic transmission through activation of four distinct

G protein-coupled adenosine receptors (A1R, A2AR,

A2BR, A3R) (Paes-De-Carvalho, 2002). A1Rs are classi-

cally involved with the inhibition of neurotransmitter

release, whereas A2ARs facilitate it. A1R activation inhibits

adenylyl cyclase (AC), whereas A2AR activates this

enzyme (Ribeiro et al., 2002; Pearson et al., 2003), lead-

ing to a decrease and increase in cyclic adenosine mono-

http://dx.doi.org/10.1016/j.neuroscience.2014.09.060

mailto:[email protected]









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2 D. D. P. Ferreira et al. / Neuroscience xxx (2014) xxx–xxx


8 October 2014

phosphate (cAMP) levels, respectively. Cytosolic cAMP

activates protein kinase A (PKA) (van Calker et al., 1979).

GABA is the main inhibitory neurotransmitter in the

central nervous system (CNS), including the retina,

where it is located on horizontal and amacrine cells

(Frederick, 1987; Hokoc et al., 1990). Aspartate is the

endogenous selective N-methyl-D-aspartate receptor

(NMDAR) agonist in the embryonic chick retina

(Kubrusly et al., 1998). Aspartate induces GABA release

in a calcium-independent manner, through reversal of

the plasma membrane GABA transporter (GAT) in a

NMDAR-dependent fashion (do Nascimento and de

Mello, 1985; Ferreira et al., 1994; Do Nascimento et al.,

1998).

Since caffeine mainly interacts with adenosine

receptors and as these proteins are highly found in the

retina, we decided to explore how this xanthine could

regulate GABA output in the embryonic retinal circuit. In

this report, we investigated whether acute caffeine

exposure to embryonic avian retina is able to modulate

GABA release induced by D-asp. Our aim was to

investigate the influence of the non-selective antagonist

of A1R and A2AR caffeine upon the GABA circuitry in the

developing avian retina.

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EXPERIMENTAL PROCEDURES

Animals

Fertilized White Leghorn chicken eggs were obtained

from a local hatchery. Embryos were staged according

to Hamburger and Hamilton (1951) and were killed by

instantaneous decapitation. Retinas from 13-day-old

chick embryos (E13) were dissected out from other ocular

tissues in a Ca2+- and Mg2+-free solution and maintained

in saline for different experimental procedures. All animal

use and experiments were performed in compliance with

the recommendations of the Brazilian Society for Neuro-

science and Behavior (SBNeC), which is in accordance

with the U.S. National Institutes of Health Guide for the

Care and Use of Laboratory Animals. The authors declare

that all experiments received a formal approval from the

Committee on Animal Research and Ethics of The Fed-

eral University of Rio de Janeiro (protocol # IBCCF035).

All efforts were done to minimize the number of animals

used and their suffering.
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Drugs

1,3,7-Trimethylxanthine (caffeine), N6-cyclohexylade-

nosine (CHA), 8-cyclopentyl-1,3-dipropylxanthine

(DPCPX), SCH 58261, PP1, (+)-MK-801 hydrogen

maleate (MK-801), Ifenprodil (+)-tartrate salt (ifenprodil),

genistein, forskolin, 4-(3-butoxy-4-methoxybenzyl)

imidazolidin-2-one (Ro 20–1724) and H-89 dihydro-

chloride hydrate (H-89) were obtained from Sigma–

Aldrich (St Louis, MO, USA). NNC-711 was purchased

from Tocris Bioscience (Minneapolis, MN, USA).

[3H]-GABA (specific activity 35 Ci/mmol) was obtained

from PerkinElmer (Waltham, MA, USA). All other

reagents were of analytical grade.

Please cite this article in press as: Ferreira DDP et al. Caffeine potentiates the r

A1 adenosine receptors. Neuroscience (2014), http://dx.doi.org/10.1016/j.neuro

[3H]-GABA uptake assay

Retinae (2 mg protein/mL) were placed in wells containing

1 mL of modified Hank’s solution without magnesium ions

(by replacing MgCl2 for EDTA) at 37 �C and incubated

with 0.1 lCi [3H]-GABA for different periods of time.

Caffeine 500 lM was added when specified together

with [3H]-GABA for the same amount of time. The

transport was stopped after the determined time with

successive washings with 1 mL of ice-cold modified

Hank’s solution followed by rapid freezing. The amount

of [3H]-GABA taken up by the retina cells was quantified

by liquid scintillation counting and the results were

normalized by protein quantification by the method of

Lowry et al. (1951) using bovine serum albumin (BSA)

as a standard.

[3H]-GABA release

The protocol was basically performed as previously

described (do Nascimento and de Mello, 1985). Briefly,

retinas were incubated with 1 mL minimum essential

medium (MEM) containing 0.1 lCi [3H]-GABA for 60 min

at 37 �C. Non-incorporated radioactive GABA was

removed by successive washings with modified Hank’s

solution. Afterward, the retinas were transferred to 1-mL

cups and superfused with medium at a rate of 0.5 mL/

10 min at 37 �C. The basal superfusion medium contained

the modified Hank’s solution and, when specified, drugs

of study. The stimulated superfusion medium was similar

to the basal one with the exception of D-asp added as a

stimulus for GABA release. After the superfusion, the

radioactivity released in the medium was quantified by

liquid scintillation and the remaining radioactivity in the

cells was counted after cell disruption with distilled water

followed by successive freeze–thaw cycles. The amount

of [3H]-GABA released was plotted as the percentage of

the total radioactivity taken up by the cells.

Cyclic AMP assay

Retinal tissue was incubated with MEM supplemented

with 0.5 mM Ro 20-1724 for 10 min. Afterward, DPCPX,

caffeine, forskolin and/or CHA were added to the

medium at the indicated final concentration, and

the samples were incubated for 60 min at 37 �C. The

reaction was stopped by adding trichloroacetic acid

(TCA) to a final concentration of 10%. The cAMP was

purified and assayed as described previously (Gilman,

1970; Matsuzawa and Nirenberg, 1975; de Mello et al.,

1982). The protein was measured by the method of

Lowry et al. (1951) using BSA as standard.

High-performance liquid chromatography (HPLC)

Endogenous aspartate and glutamate levels were

measured by HPLC as previously described (Stutz

et al., 2011). Explants were treated with caffeine for

20 min and were then washed 3 times with saline solution.

TCA was added to the supernatants to a final concentra-

tion of 5%. After centrifugation at 12,000�g for 10 min,

supernatants were filtered and injected into reverse

phase column LC-18 column (4.6 � 250 mm, Tosoh

elease of GABA mediated by NMDA receptor activation: Involvement of

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D. D. P. Ferreira et al. / Neuroscience xxx (2014) xxx–xxx 3


8 October 2014

Bioscience). HPLC parameters were described else-

where (Hashimoto et al., 1992). Pellets were resus-

pended in 0.1 M NaOH and protein was assayed by the

BCA method (Pierce).

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Fig. 1. D-Aspartate-induced GABA release in E13 explants is

mediated by GAT-1. GABA release is 4.5-fold higher in D-aspartate-

stimulated condition (D-Asp = 9.95 ± 0.24, n= 6) when compared

to basal levels (Basal = 2.21 ± 0.05, n= 6). NNC-711 (10 lM), a

GAT-1 blocker, inhibited D-aspartate-mediated GABA release

(NNC= 2.42 ± 0.33, n= 3). Statistical analysis compared D-aspar-

tate-stimulated columns to stimulated control.

Western blotting

Samples of retinas from E13 were homogenized with

lysis buffer (Tris 62.5 mM, 2% SDS). Protein

concentration was assayed by the BCA method (Pierce)

using BSA as standard. Samples were diluted in buffer

composed of 10% glycerol (v/v), 1% b-mercaptoethanol,

5% DTT, 2% SDS, 0.002% bromophenol blue and

62.5 mM Tris–HCl and boiled for 5 min. Samples

containing 30 lg of protein were electrophoresed in

10.5% SDS–PAGE and transferred to PVDF

membranes (ECL-Hybond). Membranes were incubated

overnight with anti-pGluN2B antibody (1:1000) or anti-

GluN2B antibody (1:1000), rinsed and incubated with

peroxidase-conjugated secondary antibody 1:5000 anti-

rabbit (pGluN2B), anti-mouse 1:5000 (GluN2B).

Labeling was detected with Luminata kit (Millipore).

Images were obtained by ChemiDoc MP (BIO-RAD)

and band intensities were quantified using the software

Image Lab 5.1 (BIO-RAD).

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Statistics

Statistical analysis was performed (GraphPad Prism)

using two-tailed Student’s t test when two conditions

were compared and a one-way ANOVA followed by

Bonferroni post hoc test for multiple comparisons. Data

are expressed as mean ± standard error of the mean

(SEM). For all tests, P 6 0.05 was considered to be

statistically significant.

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RESULTS

Caffeine potentiates D-asp-induced GABA release

It is known that GABA is released upon aspartate

stimulation in mixed retinal cell culture in a transporter-

mediated process (Do Nascimento et al., 1998;

Kubrusly et al., 1998). We asked if the same would occur

in the embryonic retina explants at day 13 (E13). As

shown in Fig. 1, 500 lM D-asp stimulated 4.5-fold the

release of [3H]-GABA when compared to basal levels.

This process was mediated by GAT type 1 transporter

(GAT-1) since D-asp-induced GABA release was com-

pletely inhibited by the addition of 10 lM NNC-711, a

selective blocker of GAT-1 (Fig. 1). In order to verify

whether adenosine receptors could modulate GABA

release induced by D-asp, we used caffeine. We

performed a dose–response curve for caffeine in -

D-aspstimulated GABA release with 10, 100 and

500 lM. As observed, 10 lM caffeine had no effect, while

100 lM and 500 lM increased stimulated GABA release

(Fig. 2a). As shown, 500 lM caffeine had the most prom-

inent effect by increasing in 60% D-asp-mediated GABA

release (Fig. 2a, b). However, basal release of GABA



was unaffected by caffeine (Fig. 2b). Addition of 10 lMMK-801, a non-competitive antagonist of NMDAR, com-

pletely blocked D-asp-stimulated GABA release, even in

the presence of caffeine (Fig. 2b).

We performed GABA uptake assays to investigate if

the effect of caffeine was on GAT-1 activity. As shown

in Fig. 3, different periods of incubation with caffeine (5,

10, 30, 60 min) did not modify GABA uptake. In

addition, HPLC analysis showed that caffeine did not

increase the release of either aspartate or glutamate,

when both basal and aspartate-stimulated effluxes were

considered (Table 1).

Adenosine A1 receptor blockade leads to an increasein D-asp-induced [3H]-GABA release

We next investigated which adenosine receptor was

involved in the caffeine potentiation effect on D-asp-

induced GABA release. Addition of 100 nM DPCPX, an

A1R antagonist, increased in 120% the contents of

GABA released induced by D-asp (Fig. 4). On the other

hand, 100 nM SCH58261, a selective A2AR antagonist,

had no effect on stimulated GABA release (Fig. 4). This

result suggests that adenosine A1, but not A2A, receptor

blockade is involved with the caffeine effect on GABA

release induced by D-asp.

Since adenosine receptors can be coupled to the

signaling pathway of AC/cAMP/PKA, we evaluated if

forskolin, a direct activator of AC, could participate in

the effect promoted by caffeine. As shown, forskolin

(25 lM) increased in 88% D-asp-mediated GABA

release (Fig. 5). Indeed, blockade of PKA with 1 lMH-89 prevented the caffeine effect upon D-asp-induced

GABA release (Fig. 5). Corroborating the role of A1R in

this effect, 100 nM DPCPX, an A1R antagonist,

increased cAMP levels eightfold when compared to


science.2014.09.060


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Fig. 2. A. Caffeine increases D-aspartate-mediated GABA release in a concentration-dependent manner. D-Aspartate-mediated GABA release was

not different from control in the presence of 10 lM caffeine. Caffeine at 100 lM and 500 lM increased stimulated GABA release

(Control = 4.10 ± 0.46, n= 3; Caf 10 lM= 3.40 ± 1.46, n= 3; Caf 100 lM= 10.38 ± 0.59, n= 3; Caf 500 lM= 13.38 ± 2.63, n= 3). B.

Caffeine potentiates D-aspartate-induced GABA release, which is completely dependent of NMDAR. Caffeine (500 lM) increased D-aspartate-

mediated GABA release in 60% (Caf = 16.10 ± 2.29, n= 7). MK-801 (10 lM), a competitive NMDAR antagonist, blocked all stimulated GABA

release (MK801 = 3.18 ± 0.58, n= 3). Statistical analysis compared D-aspartate-stimulated columns to stimulated control.

Fig. 3. Caffeine did not modify [3H]-GABA uptake. GABA uptake in

the presence of caffeine 500 lM (red) was not significantly different

from the basal uptake (blue) in 5, 10, 30 and 60 min. (5 min:Ctrl =

96.14 ± 1.73; Caf = 101.4 ± 11.84; 10 min:Ctrl = 157 ± 1.97;

Caf = 156.7 ± 1.46; 30 min:Ctrl = 290.2 ± 58.58; Caf = 247.4 ±

29.65; 60 min:Ctrl = 312.1 ± 6.76; Caf = 313.3 ± 36.13; n= 2).Q6

Table 1. Caffeine did not modify the basal efflux of glutamate and

aspartate or the aspartate-stimulated release of glutamate

Control (fmol/mg) Caffeine (fmol/mg)

Basal release

Glutamate 14.29 ± 2.98 12.94 ± 2.61 ns

Aspartate 9.64 ± 1.45 8.78 ± 1.22 ns

Aspartate-stimulated release

Glutamate 15.40 ± 3.23 11.66 ± 3.31 ns

Fig. 4. Blockade of A1R potentiated D-aspartate-induced GABA

release, while blockade of A2AR had no effect. DPCPX (100 nM),

A1R antagonist, increased in 120% stimulated GABA release

(DPCPX= 21.95 ± 1.42, n= 4). SCH58261 (100 nM), A2AR antag-

onist, had no significant effect in stimulated GABA released levels

(SCH58261 = 12.27 ± 1.27, n= 3). Statistical analysis compared

D-aspartate-stimulated columns to stimulated control.



8 October 2014

basal levels, while 500 lM caffeine showed a slight

increase of twofold (Fig. 6a). Accordingly, 100 nM CHA,

an A1R agonist, decreased in 52% the forskolin-

stimulated cAMP levels (Fig. 6b).



Caffeine potentiation on D-asp-stimulated GABArelease involves NMDAR phosphorylation

It is known that NMDAR activity is regulated by

phosphorylation promoted by protein tyrosine kinases

and PKA (Ali and Salter, 2001; Wenthold et al., 2003).

Thus, 10 lM genistein, a non-specific tyrosine kinase

inhibitor was added to the superfusion medium with caf-

feine. As shown, genistein prevented the potentiating

effect of caffeine on D-asp-mediated GABA release

(Fig. 7). In addition, 3 lM PP1, a specific src family kinase

(SFK) inhibitor, also prevented the caffeine effect using

the same experimental conditions (Fig. 7). These results

indicate the participation of SFK in modulating the

caffeine effect in GABA release promoted by D-asp.


science.2014.09.060


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Fig. 5. Caffeine’s effect was mimicked by adenylyl cyclase activation

and blocked by PKA inhibition. Forskolin (FK, 25 lM) increased in

88% D-aspartate-mediated GABA release (FK = 18.7 6 ± 1.82,

n= 4). H-89 (1 lM), PKA inhibitor, blocked caffeine’s effect on

stimulated GABA release (H-89 = 11.58 ± 0.98, n= 4). Statistical

analysis compared D-aspartate-stimulated columns to stimulated

control.

Fig. 7. Inhibition of protein tyrosine kinase prevented caffeine’s effect

on D-aspartate-induced GABA release. Caffeine potentiation on

GABA release was blocked by genistein, a protein tyrosine kinase

inhibitor (Gen = 9.19 ± 0.31, n= 3), as well as by the SFK inhibitor

PP1 (3 lM) (PP1 = 9.76 ± 0.42, n= 3). Statistical analysis com-

pared D-aspartate-stimulated columns to stimulated control.



8 October 2014

NMDAR phosphorylation by SFK could occur in

GluN2A or GluN2B subunits (Kennedy and Manzerra,

2001; Chen and Roche, 2007). To address this possibility,

we used ifenprodil, an allosteric inhibitor of GluN2B-

containing NMDARs (Bhatt et al., 2013). Ifenprodil

(1–10 lM) reduced, in a concentration-dependent man-

ner, the effect of caffeine in the release of GABA induced

by aspartate (Fig. 8). Additionally, western blotting analy-

sis revealed that caffeine increased phosphoGluN2B

expression levels in approximately 60% when compared

to total GluN2B levels (Fig. 9).

Altogether, our data suggest that caffeine potentiates

D-asp-induced GABA release via inhibition of adenosine

A1 receptor. As a consequence of A1 receptor inhibition,

we observe increased cAMP production followed by

PKA activation. In addition, we also detect phosphory-

lation of a tyrosine residue of the GluN2B subunit after

Fig. 6. A. Caffeine and A1R antagonist increased cAMP levels in E13 reti

eightfold when compared to basal levels, while caffeine (500 lM) increase

n= 4; DPCPX= 835%± 222.1, n= 4;). B. A1R agonist decreased FK-stim

able to reduce about 50% the cAMP levels stimulated with FK (FK = 23

compared FK+ CHA to FK.



caffeine treatment. Our data also indicate that a

member of SFK participates in the caffeine effect

described above.

DISCUSSION

GABA is a central inhibitory amino acid transmitter

present mainly in amacrine and horizontal neurons in

the retina, and in Muller glia cells. It well characterized

the interaction between excitatory (vertical axis) and

inhibitory (horizontal axis) synapses in the retinal

circuitry (Barnstable, 1993), while other transmitter sys-

tems, such as adenosine or dopamine, fine tune the reg-

ulation of neural excitability (Reis et al., 2007). For

instance, L-glutamate-evoked GABA release is detected

very early during development, around E7/E8 in the chick

retina. We confirmed that D-asp induced GABA release in

retina explants similar to previous observations reported

nas. DPCPX (100 nM), A1R antagonist, increased cAMP production

was about twofold (Basal = 100% n= 3; Caffeine = 199.5%± 8.5,

ulated cAMP levels in E13 retinas. CHA (100nM), A1R agonist, was

17 ± 447, n= 3; CHA= 1094 ± 420, n= 2). Statistical analysis


science.2014.09.060


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Fig. 8. Inhibition of GluN2B-containing NMDAR reduced, in a

concentration-dependent manner, caffeine’s potentiation on

D-aspartate-mediated GABA release. Ifenprodil, GluN1/GluN2B

NMDAR antagonist, reduced the effect of caffeine on stimulated

GABA release at the concentration of 1 lM, and completely blocked

caffeine’s effect at 10 lM (IF 1 lM= 11.88 ± 0.72, n= 6; IF

10 lM= 8.78 ± 0.35; n= 6). Statistical analysis compared Ifenpro-

dil columns to D-Asp + Caf column.

Q7



8 October 2014

in mixed retinal cell culture (Kubrusly et al., 1998). Indeed,

aspartate is considered to be a selective agonist of the

NMDAR in the chick retina (Kubrusly et al., 1998). Our

main results in the present work propose that caffeine

potentiates GABA release induced by D-asp through reg-

ulation of the NMDAR possibly via phosphorylation of

GluN2B subunit. Interestingly, caffeine itself did not mod-

ify GABA efflux at basal levels.

In our model, caffeine potentiated GABA release

induced by D-asp in a GluN2B-dependent manner, since

ifenprodil reduced the effect of caffeine in the D-asp-

evoked GABA release. Caffeine also interacts with

adenosine receptors, mainly A1R and A2AR (Fredholm

et al., 2005). Our data suggest that caffeine potentiates

D-asp-induced GABA release via inhibition of adenosine

A1R, activation of PKA and phosphorylation of a tyrosine

residue of the NMDAR-GluN2B subunit by a member of

SFK. The AC/PKA pathway seems also to be involved

Fig. 9. Caffeine increased phosphorylation of NMDAR-GluN2B. Expression

caffeine when compared to total GluN2B levels. (Control = 0.98 ± 0.02, n=



in the caffeine effect, since it is mimicked by forskolin

and blocked by H-89 (PKA inhibitor). Also, caffeine and

DPCPX produced an augmentation in cAMP production

over basal levels. PKA phosphorylates NMDAR at GluN1,

GluN2C and possibly GluN2A subunits and might regu-

late NMDAR through SFKs (Trepanier et al., 2012;

Rojas and Dingledine, 2013). It remains unclear whether

phosphorylation of the NMDAR might cause an increase

in GABA release, or how PKA may activate Fyn or other

members of SFK, which subsequently phosphorylates

the NMDAR-GluN2B subunit to promote the effect

observed with caffeine.

We observed that caffeine did not modify [3H]-D-asp

release in the presence or absence of magnesium ions

(data not shown). Caffeine also had no effect on the

basal levels of excitatory amino acids released, which

suggests that its effect on GABA release does not occur

by a feedback mechanism on the neural excitability.

GAT-1 and GAT-3 are the most abundantly expressed

subtypes in the CNS, with GAT-1 mainly on neurons,

while GAT-3 is found predominantly on glial cells. These

transporters are regulated by several mechanisms

(Madsen et al., 2010). We observed that GABA is

released through the reversion of the carrier GAT-1. Both

GAT-1 (Quick et al., 2004) and GluN2B subunit (Luo

et al., 2014) activities are subject to modulation by phos-

phorylation. However, in our model, phosphorylation inhi-

bition by genistein and PP1 only prevent GABA release

when D-asp stimulus is present and the receptor is acti-

vated. Genistein and PP1 did not modify basal GABA

release, suggesting that inhibition of tyrosine phosphory-

lation, which prevents the caffeine effect, might not occur

at the GABA carrier, but at the NMDAR.

The literature shows that phosphorylation of the

NMDAR by fyn increases NMDAR-mediated synaptic

currents in neurons (Salter and Kalia, 2004). Also, fyn

phosphorylation of GluN2B potentiates channel activity

(Thornton et al., 2004). In addition, other studies using

whole-cell recordings reported that endogenous tyrosine

phosphatase activity produces a calcium-independent

decline in NMDA currents (Wang et al., 1996). Our results

show a phosphorylation-mediated effect in the caffeine

effect on D-asp-induced GABA release. Accordingly, after

20 min of retinal explants incubation with caffeine, there is

levels of phosphoGluN2B were increased in the presence of 500 lM3; Caffeine = 1.56 ± 0,046. n= 3.)


science.2014.09.060


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8 October 2014

an increase in phosphorylation levels of the GluN2B

subunit. The total levels of GluN2B remained unaltered

following caffeine administration.

We discarded a role of A2A receptor in caffeine effect

by using SCH 58261 at 100 nM. This concentration was

selected based on previous reports as it blocks A2A

receptors in culture (Matos et al., 2012), synaptosomes

(Marchi et al., 2002) and slices (Fontinha et al., 2008).

We also performed a dose–response curve with SCH

58261 as well. Micromolar concentrations produced an

increase in GABA release. However, we believe that at

higher concentrations the selectivity to A2A receptor is lost

and A1 receptor is then antagonized, thus increasing stim-

ulated GABA release.

It is well known that adenosine can modulate vesicular

neurotransmitter release through activation of its

receptors (Paes-De-Carvalho, 2002; Sebastiao and

Ribeiro, 2009). Here we show an indirect modulation of

adenosinergic signaling, in which the blockade of A1R

increases GABA release in a non-vesicular manner,

through reversion of GAT-1. The caffeine effect on

D-asp-induced GABA release seems to be mediated by

A1R. Forskolin also potentiated D-asp-induced GABA

release and H-89 prevented the caffeine effect. This could

be in accordance with previous results, which suggest

that the increase in stimulated GABA release is due to

the blockade of A1R, but also raises the possibility that

any increase in cAMP levels or PKA activation could

promote the same effect.

In the chick retina, the A1 receptor expression reaches

its maximal levels between E12 and E16, a stage with the

highest rate of synaptic formation (de Carvalho et al.,

1992). A2AR, expressed in E13 retina, begins to accumu-

late cAMP at around E14 (Paes de Carvalho and de

Mello, 1982). Caffeine, by inhibiting A1R, potentiates stim-

ulated GABA release in E13 retinas. Caffeine potentiates

this release, which is completely dependent on the

NMDAR regulation. Considering all this, it is possible that

caffeine during CNS development might impact the for-

mation, function and strength of the synapses.
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Conclusion

Our data suggest that caffeine potentiates D-asp-induced

GABA release, which is mediated through reversion of

GAT-1 and is dependent on NMDA receptors. Caffeine

seems to promote this effect by antagonizing adenosine

A1 receptor, which is functionally coupled to AC. cAMP

levels and PKA may also be involved in the caffeine

effect on stimulated GABA release. Additionally,

phosphorylation of the GluN2B-containing NMDAR by

SFK is likely to participate in the potentiation of D-asp-

induced GABA release by caffeine.

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CONTRIBUTORS

DDPF: Conception and design; Provision of study

material; Collection and/or assembly of data; Data

analysis and interpretation; Manuscript writing. BS:

Provision of study material; Collection and/or assembly

of data; Data analysis and interpretation; Manuscript

writing. FGM: Data analysis and interpretation;



Manuscript writing. RAMR: Provision of study material;

Collection and/or assembly of data, Manuscript writing,

Data analysis and interpretation. RCCK: Conception and

design; Provision of study material; Collection and/or

assembly of data; Data analysis and interpretation;

Manuscript writing; Final approval of manuscript;

Administrative support.

Acknowledgments—The authors are thankful to Luciano Ferreira

and Aurizete Bizerra for technical support. This work was sup-

ported by Coordenacao de Aperfeicoamento de Pessoal de Nıvel

Superior (CAPES), PROAP/CAPES, Fundacao Carlos Chagas

Filho de Amparo a Pesquisa do Estado do Rio de Janeiro

(FAPERJ), Conselho Nacional de Desenvolvimento Cientıfico e

Tecnologico (CNPq), Instituto Nacional de Ciencia e Tecnologia

(INCT-INNT), Pro-reitoria de Pesquisa e Pos-graduacao–Univer-

sidade Federal Fluminense (PROPPI/UFF). The institutions had

no further role in study design; in the collection, analysis and

interpretation of data; in the writing of the report; and in the deci-

sion to submit the paper for publication.

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(Accepted 26 September 2014)(Available online xxxx)


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