caffeine and an adenosine a 2a receptor antagonist prevent memory impairment and synaptotoxicity in...

,

*Center for Neurosciences of Coimbra, Institute of Biochemistry, Faculty of Medicine, University of Coimbra, Portugal

�Departamento de Bioquımica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil

�Pharmazeutische Chemie I, Pharmazeutisches Institut, University of Bonn, Germany

During development, the brain undergoes marked plasticchanges which format its wiring and determine its potentialfor future experience-dependent plastic changes, i.e. thedeveloping brain expresses a marked metaplasticity (Abrahamand Bear 1996). An abnormal modification of firing of thedeveloping brain, as occurring during convulsions, causesminor immediate modifications but has potential detrimentalconsequences later in life (Stafstrom 2002; Holmes 2005). Infact, in animal models, a single convulsive period early in lifecauses delayed memory deficits in adulthood (Stafstrom2002; Holmes 2005). For instance, the administration ofkainate, which models temporal lobe epilepsy in adultrodents, causes minor immediate morphological or functionalmodifications in pups (Stafstrom 2002; Holmes 2005), buttriggers an impairments of spatial memory when animals are80–100 days old (Lynch et al. 2000; Sayin et al. 2004). The

mechanisms underlying this metaplasticity are not resolved(Holmes 2005). But more important than understanding themechanisms of convulsions-induced delayed memory defi-cits is the possibility of devising novel strategies to preserve

Received September 7, 2009; revised manuscript received October 22,2009; accepted October 26, 2009.Address correspondence and reprint requests to Rodrigo A. Cunha,

Center for Neuroscience of Coimbra, Institute of Biochemistry, Facultyof Medicine, University of Coimbra, 3004-504 Coimbra, Portugal.E-mail: [email protected] used: A1R, A1 receptor; A2AR, A2A receptor; BSA,

bovine serum albumin; DPCPX, 1,3-dipropyl-8-cyclopentylxanthine; IR,immunoreactivity; SCH58261, 5-amino-7-2-phenylethyl.-2-2-furyl-pyr-azolo[4,3-ex-1,2,4-triazolo-1,5-9pyrimidine; vGAT, vesicular GABAtransporter; vGluT1 and vGluT2, glutamate transporters types 1 and 2;XAC, 8-{4-[(2-aminoethyl)amino]carbonylmethyl-oxyphenyl}xanthine.

Abstract

Seizures early in life cause long-term behavioral modifica-

tions, namely long-term memory deficits in experimental ani-

mals. Since caffeine and adenosine A2A receptor (A2AR)

antagonists prevent memory deficits in adult animals, we now

investigated if they also prevented the long-term memory

deficits caused by a convulsive period early in life. Adminis-

tration of kainate (KA, 2 mg/kg) to 7-days-old (P7) rats caused

a single period of self-extinguishable convulsions which lead

to a poorer memory performance in the Y-maze only when

rats were older than 90 days, without modification of loco-

motion or anxiety-like behavior in the elevated-plus maze. In

accordance with the relationship between synaptotoxicity and

memory dysfunction, the hippocampus of these adult rats

treated with kainate at P7 displayed a lower density of syn-

aptic proteins such as SNAP-25 and syntaxin (but not

synaptophysin), as well as vesicular glutamate transporters

type 1 (but not vesicular GABA transporters), with no changes

in PSD-95, NMDA receptor subunits (NR1, NR2A, NR2B) or

a-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor

subunits (GluR1, GluR2) compared with controls. Caffeine

(1 g/L) or the A2AR antagonist, KW6002 (3 mg/kg) applied in

the drinking water from P21 onwards, prevented these

memory deficits in P90 rats treated with KA at P7, as well as

the accompanying synaptotoxicity. These results show that a

single convulsive episode in early life causes a delayed

memory deficit in adulthood accompanied by a glutamatergic

synaptotoxicity that was prevented by caffeine or adenosine

A2AR antagonists.

Keywords: A2A adenosine receptor, astrogliosis, caffeine,

convulsions, memory, synaptotoxicity.

J. Neurochem. (2010) 112, 453–462.

JOURNAL OF NEUROCHEMISTRY | 2010 | 112 | 453–462 doi: 10.1111/j.1471-4159.2009.06465.x

� 2009 The AuthorsJournal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2010) 112, 453–462 453

memory, which may also offer some additional insights intokey mechanistic processes.

One emerging candidate to manage memory impairmentunder different noxious conditions is caffeine. Caffeine is themost widely consumed psychoactive substance and alleviatescognitive impairment in both humans and animals, namelyupon aging or upon Alzheimer’s disease (Cunha 2008;Takahashi et al. 2008). Furthermore, caffeine also affordsprotection upon CNS injury (Cunha 2005; Chen et al. 2007).The only known molecular targets of caffeine at non-toxicdoses are adenosine receptors, mainly A1 and A2A receptors(A2AR) (Fredholm et al. 2005). A2AR blockade seems to beprimarily involved in these neuroprotective effects ofcaffeine since the prevention by caffeine of memory deficitsis mimicked by antagonists of A2AR but not A1R (Predigeret al. 2005; Dall’Igna et al. 2007).

In the present study, we investigated if chronic treatmentwith caffeine or with a selective A2AR antagonist couldprevent the memory deficits found in adult animals that wereexposed to an episode of convulsions early in life. Sincememory deficits in different neuropsychiatric conditions areaccompanied by dysfunction and loss of synapses (Colemanet al. 2004; Silva et al. 2007), we also investigated if aconvulsive episode early in life caused a modification ofsynaptic markers in adulthood while testing if there was aparticular modification of pre- and post-synaptic markers ofglutamatergic synapses.

Materials and methods

AnimalsWistar rats (Charles-River, Barcelona, Spain) were maintained under

controlled environment (23 ± 2�C, 12 h-light/dark cycle, free access

to food and water) and used according to EU guidelines (86/609/

EEC) with care to minimize the number of animals and their

suffering.

Kainate-induced convulsion and drug administrationMale young pups (7 days old, P7), bred at the Center’s animal house

by trained caretakers, were separated from their dams. A single

convulsive episode was induced by intra-peritoneal administration

of kainic acid (KA) 2 mg/kg (Lynch et al. 2000; Sayin et al. 2004)prepared in saline solution. Control littermates were injected with

saline solution. After spontaneous termination of convulsions,

within 3 h after their onset, KA-injected and control rats were

returned to their dams.

Caffeine was added to the drinking water using a dose (1 g/L)

previously found to afford neuroprotection (Duarte et al. 2009).

Caffeine was introduced at P21 (weaning day) and continuously

supplied until rats were killed. Non-treated rats (KA- or saline-

treated) drank water only. Weight and water/caffeine consumption

were continuously monitored, which allowed us to estimate the daily

amount of caffeine consumption as 69 ± 3 mg/kg/day (consumed in

a continuous intermittent mode) without correction for spillage

(estimated to be ca. 5%). Measurement of serum caffeine concen-

tration was done by reverse-phase HPLC using 40% (v/v) methanol/

water as eluent (flow rate of 0.8 mL/min) with UV detection at

257 nm. The average caffeine intake was similar in both KA- and

saline-treated rats through the treatment period, which displayed

similar serum caffeine concentrations (21.4 ± 2.8 lM, n = 6) at the

time of killing. This corresponds to the plasma concentration

reached by consumption of 5–6 cups of coffee in humans (e.g. Lelo

et al. 1986).Chronic intake of the selective A2AR antagonist, KW6002

(istradefylline, see Kase et al. 2003), also begun at P21. Everyday,

a 1 mg/L KW6002 suspension (prepared as previously described,

see Hockemeyer et al. 2004) in a vehicle solution (0.9% saline and

0.4% methylcellulose) was prepared and the adequate volume of the

suspension was added to the drinking water to obtain the desired

dose of KW6002 (3 mg/kg). This solution was left for a period of

2.5 h to ensure that rats consumed an amount of 3 mg/kg/day of

KW6002 (again with a spillage error of ca. 5%). Non-treated rats

(KA- or saline-treated) drank water supplemented with vehicle

solution.

Behavioral analysisBehavioral experiments were conducted between 9:00 AM and

4:00 PM (light phase). Locomotor and exploratory behavior were

monitored in an open-field apparatus, as previously described

(Dall’Igna et al. 2007), when rats were 28, 58, and 88 days old. On

the subsequent days (29, 59, and 89 days), rats were submitted to

the elevated-plus maze task to evaluate their anxiety status (Kaster

et al. 2004). Finally, on days 30, 60, and 90, rats performed a

memory task in a Y-maze apparatus with an inter-trial interval (ITI)

of 2 min and with a 2 h ITI 1 week later. We used an adapted

version of the Y-maze designed to measure spatial recognition

memory (Dellu et al. 1997). The three arms of the Y-maze were

randomly designated: start arm, in which rats started to explore

(always open), novel arm, which was blocked during the first trial,

but open during the second trial, and other arm (always open). The

Y-maze task consisted of two trials separated by an ITI to assess

response to novelty (2 min ITI) and spatial recognition memory (2 h

ITI) (Dellu et al. 1997). During the first trial (training, 5 min), rats

were allowed to explore only two arms (start and other arm), with

the third arm (novel arm) closed. For the second trial (after ITI), the

rat was placed back in the same starting arm, with free access to all

three arms for 5 min. The number of entries in each arm was

determined and data expressed as percentage of total entries during

the 5 min (time spent in each arm was also counted yielding similar

results but is not displayed for clarity of Figures).

Western blot analysisAfter all behavior tests, some rats were deeply anesthetized under

halothane atmosphere before being killed by decapitation for

preparing Percoll-purified hippocampal synaptosomes as described

(Rebola et al. 2005; Canas et al. 2009). Western blot analysis was

carried out in these synaptosomal membranes as previously described

(Rebola et al. 2005; Canas et al. 2009), except for glial fibrillaryacidic protein (GFAP) which was evaluated in total hippocampal

membranes (Rebola et al. 2005). The antibodies were against SNAP-25 (1 : 5000 dilution; from Sigma, Sintra, Portugal), syntaxin

(1 : 5000, Sigma), synaptophysin (1 : 2000, Sigma), vesicular

glutamate transporters type 1 (vGluT1, 1 : 5000, Chemicon,

Journal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2010) 112, 453–462� 2009 The Authors

454 | G. P. Cognato et al.

PG-Hitec, Lisbon, Portugal), vesicular GABA transporter (vGAT,

1 : 1000, Calbiochem, VWR, Lisbon, Portugal), post-synaptic

density 95 kDa protein (PSD-95, 1 : 20 000, Upstate Biotechology,

PG-Hitec, Lisbon, Portugal), NMDA receptor subunit 1 (NR1,

1 : 400, Chemicon), NMDA receptor subunit 2A (NR2A, 1 : 800,

Chemicon), NMDA receptor subunit 2B (NR2B, 1 : 200, BD

Biosciences, PG-Hitec, Lisbon, Portugal), a-amino-3-hydroxy-5-

methylisoxazole-4-propionate (AMPA) receptor subunit 1 (GluR1,

1 : 400, Upstate Biotechnology), AMPA receptor subunit 2 (GluR2,

1 : 400, Chemicon) or GFAP (1 : 1000, Cell Signalling, Izasa,

Lisbon, Portugal). Re-probing quantifying a-tubulin (1 : 10 000,

Sigma) immunoreactivity confirmed that similar amounts of protein

were applied to the gels (Rebola et al. 2005; Canas et al. 2009). We

also confirmed, by loading different amounts of protein in the same

gel, that wewere working under non-saturating conditions enabling to

effectively probe changes in the density of each tested protein (Rebola

et al. 2005; Canas et al. 2009).

Membrane binding assaysBinding assays were carried out as previously described (Cunha

et al. 2006) in membranes from hippocampal synaptosomes using

supra-maximal concentrations (6 nM) of selective antagonists of

either A1R (3H-1,3-dipropyl-8-cyclopentylxanthine, 3H DPCPX;

specific activity of 109.0 Ci/mmol; from DuPont NEN, ILC, Lisbon,

Portugal) or A2AR (3H-7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyr-

azolo-[4,3-e]-1,2,4-triazolol[1,5c]pyrimidine, 3H-SCH58261; spe-

cific activity of 77 Ci/mmol; prepared by Amersham and offered

by Dr E. Ongini, Shering-Plough, Italy).

Immunocytochemistry in purified nerve terminalImmunocytochemical analysis of purified hippocampal nerve

terminals was performed as previously described (Rodrigues

et al. 2008). The platted nerve terminals were labeled with goat

anti-adenosine A2AR (1 : 500; Santa Cruz Biotechnology, Heidel-

berg, Germany) or with rabbit anti-adenosine A1R antibody

(1 : 400; from Affinity Bioreagents, PG-Hitec, Lisbon, Portugal),

together with mouse anti-synaptophysin antibody (1 : 200; Sigma)

and visualized in a Zeiss Axiovert 200 inverted fluorescence

microscope equipped with a CCD camera and analyzed with

ImageJ 1.37v (NIH, Bethesda, MD, USA). Each coverslip (three

per animal) was analyzed by counting five different fields and in

each field a total amount of at least 100 individualized nerve

terminals.

Immunohistochemistry analysisThe preparation of brain sections was carried out as previously

described (Cunha et al. 2006) and always analyzed by three

independent researchers. The general neuronal morphology was

evaluated with Cresyl Violet staining of Nissl bodies, as previously

described (Lopes et al. 2003). Hippocampal astrocytes were

evaluated by immunohistochemical detection of GFAP, a marker

of activated astrocytes (Pekny and Nilsson 2005). Blocked brain

sections were incubated for 72 h at 4�C with Cy3-conjugated anti-

GFAP mouse monoclonal antibody (from Sigma; 1 : 1000 dilution

containing 0.2% Triton X-100 and 10% goat serum). After washing,

sections were dehydrated and cleared in xylol and mounted on slides

using Vectaschield H-1400 mounting medium (Vector Laboratories,

Batista Marques, Lisbon, Portugal). The numbers of immuno-

staining-positive cells in each hippocampal region was averaged

from four hippocampal sections per rat.

Statistical analysisData are mean ± SEM of n animals. Significance (p < 0.05) was

assessed by one-way Student’s t-test or by a one-way ANOVA

followed by Bonferroni’s test to compare data of the four

experimental groups.

Results

A single episode of convulsions in early life causes memoryimpairment in adult ratsKainate (KA, 2 mg/kg, i.p.) caused a convulsive-like activityin 7-day-old pups (P7); this consisted of hyperactive‘bicycling’ movements of all extremities with opisthonicarching of the back and tonic limbic extension, occurringwith a fast onset (ca. 10 min), lasting 80–210 min, anddisappearing spontaneously, as previously reported (Lynchet al. 2000; Sayin et al. 2004).

We then tested if this single convulsive episode at P7modified behavior later in adolescence and adulthood. AtP30 or P60, KA-injected rats (at P7) displayed a behaviorprofile indistinguishable from that of saline-injected rats (Fig.1, third and second rows). However, at P90, KA-injected ratsdisplayed significantly (p < 0.05) poorer performance in aspatial memory version of the Y-maze test compared withsaline-injected rats (Fig. 1, top left panel, whereas locomo-tion and anxiogenic-like behavior were not significantlydifferent (p > 0.05) from saline-injected rats (Fig. 1, firstrow).

Caffeine and A2AR blockade prevent convulsions-inducedmemory impairmentTo test if consumption of caffeine or selective A2ARantagonist prevented this delayed memory deficit (onlyobservable at P90, but not at P30 or P60) caused by aconvulsive episode early in life, rats injected with either KA-or saline at P7 were divided into two groups: one group wasexposed to caffeine (1 g/L) or to the selective A2ARantagonist, KW6002 (3 mg/kg, see Kase et al. 2003),whereas the non-treated group only drank water from P21(weaning) onwards.

Caffeine consumption prevented memory impairment ofKA-treated rats at P90, whereas caffeine was devoid of effectin saline-injected rats (Fig. 2, upper left panel). Furthermore,locomotion and anxiety behaviors were similar in all fourgroups (Fig. 2, upper panels).

Likewise, memory impairment displayed at P90 by KA-treated rats was abrogated by KW6002 consumption fromP21 onwards (Fig. 2, bottom left panel), whereas KW6002did not modify behavior of saline-injected rats. Also,locomotion and anxiety behaviors were similar (p > 0.05)in all four groups (Fig. 2b, bottom panels).

� 2009 The AuthorsJournal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2010) 112, 453–462

Caffeine prevents memory impairment on convulsions | 455

Fig. 1 Rats exposed to a kainate-induced convulsive period early in

life display selective memory impairment in adulthood, but not in late

adolescence or early adulthood, without modification of locomotion,

exploratory or anxiogenic behavior. Pups with 7 days of age received

an i.p. injection of either saline (open bars) or kainate (2 mg/kg, black

bars), which caused a period of convulsions; rats were then behaviorly

analyzed when adolescent (P30), young adults (P60) and adults (P90-

100). It was found that a period of convulsions in early life selectively

affected spatial memory and only in adult P90 rats, without modifica-

tion of spatial learning at other ages, nor modification of locomotion or

anxiety. Data are means ± SEM of n = 6–7 rats per group; * p < 0.05

using a Student’s t-test.

Fig. 2 Consumption of caffeine or of the selective adenosine A2A

receptor antagonist KW6002 beginning in adolescence prevents the

memory impairment present in adult rats that were exposed to a con-

vulsive period early in their life. Pups with 7 days of age received an i.p.

injection of either saline (open bars) or kainate (2 mg/kg, filled bars),

which caused a period of convulsions. From day 21 onwards each of

these two groups of rats were further sub-divided into two groups, one

drinking water (black bars) and the other (gray bars) drinking either

caffeine (1 g/L, upper row of panels) or the A2A receptor antagonist

KW6002 (3 mg/kg, bottom row of panels). Rats were then behaviorly

analyzed when adults, between 90 and 100 days of age. It was found

that a period of convulsions in early life selectively affected spatial

memory and this deficit was abrogated by consumption of either caf-

feine or KW6002. Data are means ± SEM of n = 7–9 rats per group;

*p < 0.05 using a one-way ANOVA followed by Bonferroni’s test.



Impact of convulsions early in life on the set-up ofadenosine receptors in adulthoodWe next investigated the status of hippocampal adenosinereceptors of KA-treated rats at P90. Binding of the A2ARantagonist 3H-SCH58261 was increased by 65.3 ± 4.2%(p < 0.05; Fig. 3b), whereas binding of the A1R antagonist3H-DPCPXwas decreased by 24.0 ± 2.4% inP90 rats injected

with KA at P7 compared with saline-injected rats (p < 0.05;Fig. 3a). Furthermore, the percentage of synaptophysin-immunopositive terminals endowed with A1R immunoreac-tivity was similar (Fig. 3d), whereas the number ofsynaptophysin-immunopositive terminals endowed withA2AR immunoreactivity increased in P90 rats injected withKA at P7 compared with saline-injected rats (Fig. 3c and e).

(d) (e)

(c)

(b)(a)

Fig. 3 Adult rats that were exposed to a convulsive period in early life

display an increased density of synaptic adenosine A2A receptors and

a decreased density of synaptic adenosine A1 receptors. Panels a and

b compare the binding density (presented as specific binding) of a

supra-maximal concentration (6 nM) of a selective antagonist of

adenosine A1 receptors (3H-DPCPX, a) or of a supra-maximal con-

centration (6 nM) of the selective antagonist of adenosine A2A

receptors (3H-SCH58261, b) to membranes of hippocampal synapto-

somes of adult (P90) rats that were either injected with saline (open

bars) or with 2 mg/kg of kainate at P7 (filled bars) to trigger a period of

convulsions. Panel c shows immunocytochemical double labeling of

synaptophysin (a synaptic marker, left photographs) and of A2A

receptors (right photographs) in hippocampal purified nerve terminals

from adult (P100) rats that were either injected with saline (top pho-

tographs) or with 2 mg/kg of kainate at P7 (bottom photographs) to

trigger a period of convulsions. The average quantification of the

number of nerve terminals endowed with A2A receptors is displayed in

panel e, whereas panel d shows the number or nerve terminals en-

dowed with A1 receptors in hippocampal purified nerve terminals of

adult (P100) rats that were either injected with saline (open bars) or

with 2 mg/kg of kainate at P7 (filled bars) to trigger a period of con-

vulsions. Data are mean ± SEM of n = 4 rats per group. *p < 0.05

using a Student’s t-test.



This indicates that KA-induced convulsions at P7 led to anincrease of nerve terminals endowed with A2AR, whereas thenumber of nerve terminals endowed with A1R was preserved;however, the density of A1R per terminal might havedecreased, since receptor binding analysis revealed a globaldecrease of A1R in hippocampal synaptosomal membranes(Fig. 3a).

Convulsions early in life lead to features of synaptotoxicityin adulthoodWe next investigated if early-life convulsions-induced mem-ory impairment in adult rats was associated with synapticdegeneration, which has been implicated in Alzheimer’sdisease-associated memory impairment (Selkoe 2002; Cole-man et al. 2004). We found a reduced immunoreactivity ofSNAP-25 ()21.4 ± 3.7%, p < 0.05) and syntaxin ()20.2 ±3.0%, p < 0.05) but not of synaptophysin ()6.3 ± 3.0%,

p > 0.05) in hippocampal membranes of P90 rats treatedwith KA at P7 (Fig. 4a–c), suggesting the occurrence ofsynaptic degeneration. There was also a reduced immuno-reactivity of vGluT1 (-27.2 ± 3.3%, p < 0.05) but not ofvGAT (2.3 ± 2.6%, p > 0.05), suggesting the occurrence ofglutamatergic rather than GABAergic synaptic degeneration(Fig. 4d and e). In contrast, the immunoreactivity of PSD-95(Fig. 4f), NR1 (Fig. 4g), NR2A (Fig. 4h) or NR2B subunitsof NMDA receptors (Fig. 3i) and of GluR1 (Fig. 4j) orGluR2 subunits of AMPA receptors (Fig. 4k) was notmodified (p > 0.05).

Semi-quantitative immunohistochemistry of the astrocyticprotein marker GFAP (Pekny and Nilsson 2005) revealed asimilar (p > 0.05) density of GFAP-positive cells in thehippocampus of P90 rats injected with KA at P7, comparedwith saline-treated rats in the three hippocampal regions(Figs 5b and c), which was confirmed by western blot

(a)

(d)

(g) (h) (i) (j) (k)

(e) (f)

(b) (c)

Fig. 4 Adult rats that were exposed to a convulsive period in early life

display a decreased density of synaptic markers, in particular of

glutamatergic but not GABAergic terminals. Pups with 7 days of age

received an i.p. injection of either saline (open bars) or kainate (2 mg/

kg, black bars), which caused a period of convulsions. Rats were killed

as adults (P100) and synaptosomal membranes prepared from their

hippocampi for western blot analysis of the immunoreactivity (IR) of 3

pre-synaptic proteins, SNAP-25 (a) syntaxin (b) and synaptophysin

(c), markers of glutamatergic (vesicular glutamate transporter type 1,

vGluT1, d) and GABAergic nerve terminals (vesicular GABA

transporter, vGAT, e), markers of the post-synaptic density (PSD-95,

f) and different subunits of different glutamate ionotropic receptors

expected to be mainly located post-synaptically, such as NMDA

receptor subunit 1 (NR1, g), NMDA receptor subunit 2A (NR2A, h),

NMDA receptor subunit 2B (NR2B, i), AMPA receptor GluR1 subunit

(j), AMPA receptor GluR2 subunit (k). Data are means ± SEM of n = 4

rats per group, except data in panel j (n = 3) and the data in panel K

(which are individual data from two experiments); *p < 0.05 using a

Student’s t-test.



analysis (Fig. 5d), suggesting a lack of evident glialactivation.

Caffeine and A2AR antagonist prevent adulthoodsynaptotoxicity upon convulsions early in lifeWe next investigated if caffeine or KW6002 prevented thissynaptotoxicity at P90 upon KA administration at P7. Asillustrated in Fig. 6, the consumption of caffeine or KW6002from P21 onwards prevented the decreased density of SNAP-25 (p < 0.05) and of syntaxin (p < 0.05) in hippocampalmembranes of P90 rats that were subject to kainate-inducedconvulsion at P7; likewise, the decrease of vGluT1 was alsoprevented (p < 0.05) by both caffeine and KW6002 (Fig. 6),which were devoid of effects on vGAT density that wassimilar (p > 0.05) in all four groups (Fig. 6).

Discussion

The present results indicate that a single period ofKA-induced convulsions in early life (P7) triggers selective

memory deficits later, only in adulthood (P90), which areaccompanied by a loss of pre-synaptic markers, in particularof glutamatergic terminals, without modification of post-synaptic markers, astrogliosis or global organization ofprincipal neurons (Cresyl violet staining) in the hippocam-pus. Furthermore, chronic consumption of caffeine or of aselective A2AR antagonist prevents memory deficits andconcurrent synaptotoxicity present in adult rats that weresubject to a single period of convulsions early in life.

The first conclusion of this study was that a period ofconvulsions at P7 led to selective memory deficits, which isdelayed in time, appearing only in adulthood. We now showthat the spatial memory deficits were observed only at P90 asobserved by others (Lynch et al. 2000; Sayin et al. 2004),but are not evident in late adolescence (P30) or earlyadulthood (P60), an aspect which was not systematicallyaddressed in previous studies. The extrapolated relevance ofthese findings for humans is provocative: although it is stilldebatable if early-life convulsions are associated with poorermemory performance, this was only evaluated in late

(a)

(c) (d)

(b)

Fig. 5 Adult rats exposed to a convulsive period in early life do not

display modified astrocytic reactivity. Pups with 7 days of age received

an i.p. injection of either saline (open bars) or kainate (2 mg/kg, black

bars), which caused a period of convulsions. Rats were killed as adults

(P100) to prepare either brain sections or total hippocampal mem-

branes. Staining of the sections with cresyl violet (a) revealed a pattern

of cell body organization in the hippocampal formation similar in the

two groups. Immunohistochemical staining of the section for GFAP (an

astrocytic marker, b) revealed a similar number of stained elements

(quantified in c) with similar morphology in the three main hippocampal

regions, CA1 CA3 and dentate gyrus (DG). This lack of modification of

astrocytic density was confirmed by western blot analysis (d) of GFAP

immunoreactivity (IR) in total membranes from the two groups of rats.

Histochemical analysis was performed in three rats per group,

whereas the Western bots were carried out in membranes derived

from four rats per group. Data in the bar graphs are means ± SEM.



adolescence (Shinnar and Hauser 2002; Vingerhoets 2006).This leaves room to question whether deficits might onlybecome evident later in adulthood.

The second conclusion of this study is related to thepossible neurochemical traits underlying the observed mem-ory deficits. Adult animals subject to KA-induced convul-sions in early life display minor morphological modificationscompared with control animals (Lynch et al. 2000; Stafstrom2002; Holmes 2005; Cornejo et al. 2007), whereas theydisplay neurophysiological modifications, such as modifiedsynaptic plasticity (Lynch et al. 2000; Cornejo et al. 2007).Interestingly each group proposed different neurochemicalexplanations to interpret these neurophysiological changes,namely enhanced efficiency of inhibitory transmission(Lynch et al. 2000), enhanced efficiency of glutamatergicsynapses (Cornejo et al. 2007) or persistent astrogliosis(Somera-Molina et al. 2007). In the present study, we failedto find evidence for persistent hippocampal astrogliosis sinceneither GFAP density, nor the number of GFAP-labeledastrocytes, nor the morphology of GFAP-labeled elementswere modified in the hippocampus of rats exposed to early-life convulsions. However, we provide the first directdemonstration of pre-synaptic modifications accompanyingmemory deficits in adult rats that were subject to aconvulsive period in early life. Thus, we observed a decreaseof two synaptic markers, SNAP-25 and syntaxin. Thissynaptotoxicity has also been proposed to be a primary and

crucial feature responsible for memory impairment occurringin mild cognitive impairment (Scheff et al. 2007) andAlzheimer’s disease (Selkoe 2002; Coleman et al. 2004),since synaptotoxicity is the only morphological parameterthat correlates with memory impairment (Selkoe 2002;Coleman et al. 2004). Furthermore, features of synaptotox-icity are characteristic of several other brain conditions wherememory impairment is also present, such as aging (Canaset al. 2009), Huntington’s (Li et al. 2001) or prions’ diseases(Ferrer 2002), HIV infection (Garden et al. 2002) orschizophrenia (Glantz et al. 2006). Most interestingly, wefound a reduction of a marker of glutamatergic synapses(vGluT1), whereas a marker of GABAergic synapses (vGAT)was preserved, indicating selective changes of glutamatergicrather than GABAergic synapses. This is in remarkableagreement with recent results indicating that memory-relatedsynaptotoxicity might occur particularly in glutamatergicterminals, since the density of vesicular glutamate transport-ers was found to be decreased in cortical regions of memory-impairment individuals with Alzheimer’s disease (Kirvellet al. 2007), as well as in animal models of Alzheimer’sdisease (Bell et al. 2006; Minkeviciene et al. 2008). Thus, asoccurs for Alzheimer’s disease, it is likely that the reduceddensity of hippocampal synaptic proteins, particularly ofglutamatergic terminals, may contribute to the memoryimpairment found in adult rats that were exposed toconvulsions early in life. Interestingly, we mostly found

Fig. 6 Consumption of caffeine or of the selective A2A receptor

antagonist KW6002 prevents the loss of synaptic markers, namely of

glutamatergic terminals, in the hippocampus of adult rats that were

exposed to a convulsive period in early life. Pups with 7 days of age

received an i.p. injection of either saline (bars with white background)

or kainate (2 mg/kg, bars with dark background), which caused a

period of convulsions. From day 21 onwards each of these two groups

of rats were further sub-divided into two groups, one drinking water

(white or black bars) and the other (grided bars) drinking either

caffeine (1 g/L, upper row of panels) or the A2A receptor antagonist

KW6002 (3 mg/kg, bottom row of panels). Rats were killed as adults

(P100) and synaptosomal membranes prepared from their hippocampi

for western blot analysis of the immunoreactivity (IR) of two pre-syn-

aptic proteins (SNAP-25 and syntaxin), a marker of glutamatergic

(vesicular glutamate transporter type 1, vGluT1) and a marker of

GABAergic nerve terminals (vesicular GABA transporter, vGAT). Data

are means ± SEM of n = 4 rats per group; *p < 0.05 using a one-way

ANOVA followed by Bonferroni’s test.



modifications of pre-synaptic markers of glutamatergicsynapses rather than changes in the density of differentsubunits of ionotropic glutamate receptor, in spite of the factthat the convulsive period was applied precisely at the timewhere the maturation of receptors in glutamatergic synapsesis taking place (reviewed in Jensen 2002).

The likely involvement of synaptotoxicity in the mecha-nism of memory impairment in adulthood caused by early-life convulsions is further supported by the main finding ofthe present work, i.e. that long-term caffeine consumptionprevented both memory impairments and loss of nerveterminal markers in the hippocampus of adult rats that wereexposed to convulsions early in life. This is paralleled by theability of caffeine to prevent memory deficits found in aging,in Alzheimer’s and Parkinson’s diseases and in attentiondeficit and hyperactivity disorders (Cunha 2008; Takahashiet al. 2008), suggesting that caffeine can interfere with keymechanisms of memory dysfunction (Cunha 2008). The onlyknown molecular targets for caffeine at non-toxic concen-trations are adenosine receptors, which are antagonized bycaffeine (Fredholm et al. 2005). The prevention of memorydeficits by caffeine likely results from A2AR antagonismsince the beneficial effects of caffeine on memory deficitsupon aging or Alzheimer’s disease are mimicked by selectiveA2AR antagonists (Prediger et al. 2005; Dall’Igna et al.2007). Accordingly, we now observed that the selectiveA2AR antagonist, KW6002, also prevented both memoryimpairment and loss of nerve terminal markers in thehippocampus of adult rats that were exposed to a convulsiveperiod in their early life. Interestingly, A2AR antagonism notonly abrogates memory dysfunction but also affords robustneuroprotection against brain damage (Cunha 2005; Chenet al. 2007). This further supports the involvement ofsynaptotoxicity in memory dysfunction since A2AR have apredominant synaptic localization in the hippocampus (Re-bola et al. 2005), where they control synaptotoxicity (Cunhaet al. 2006; Silva et al. 2007), which is the most evidentmorphological change found in the hippocampus of adult ratsthat were exposed to a convulsive period early in their life.These synaptic A2AR undergo a gain of function in noxiousbrain conditions (Cunha 2005; Fredholm et al. 2005).Accordingly, we now found an increase in the density ofsynaptic A2AR in the hippocampus of adult rats that wereexposed to a convulsive period early in their life. However,the mechanism by which A2AR control synaptotoxicity inglutamatergic synapses is still unclear, albeit the control ofNMDA receptors (Rebola et al. 2008), calcium loading(Goncalves et al. 1997) and synaptic mitochondria (Silvaet al. 2007) are likely candidates.

In conclusion, the present study shows that a convulsiveperiod early in life causes selective impairment of memoryperformance later in adulthood, which is accompanied by aloss of synaptic markers, in particular of glutamatergicterminals. Furthermore, chronic consumption of caffeine or

of A2AR antagonists starting in adolescence prevented thisdelayed memory deficit and accompanying synaptotoxicity.These observations widen the prophylactic interest incaffeine and A2AR antagonists to manage conditions asso-ciated with memory deterioration.

Acknowledgments

The authors are grateful to Paula M. Canas, Ana Patrıcia Simoes,

Carla G. Silva, Gabriele Ghisleni and Manuella P. Kaster for their

help in carrying out some of the experiments. GPC acknowledges

the support and help of Lisiane O. Porciuncula and Carla

Bonan. The dedicated and competent help of Alexandre Pires to

handle the animals is especially acknowledged. This work was

supported by FCT (PTDC/SAU-NEU/74318/2006) and was

made possible by a joint Portuguese-Brazilian grant (CAPES-

GRICES).

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