Biphasic effect of chronic postnatal caffeine treatment on cortical epileptic afterdischarges during ontogeny in rats

Biphasic effect of chronic postnatal caffeine treatment on cortical epileptic afterdischarges during ontogeny in rats
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  Research Report  Biphasic effect of chronic postnatal caffeine treatment oncortical epileptic afterdischarges during ontogeny in rats  Jana Tchekalarova a,b, ⁎  , Hana Kubová b,1 , Pavel Mare š  b,1 a Institute of Physiology, Acad. G. Bonchev Str., Bl. 23, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria b Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic A R T I C L E I N F O A B S T R A C T  Article history: Accepted 17 January 2006Available online 3 March 2006EEG and motor phenomena elicited by stimulation of sensorimotor cortex were used tostudy the effects of chronic postnatal administration of caffeine (10 and 20 mg/kg, s.c. fromP7 to P11) in rats. Rhythmic electrical stimulation was applied to 12-, 18-, 25- and 67-day-oldratswithimplantedelectrodes.Animalswiththehigherdoseofcaffeineexhibitedincreasedthresholds for elicitation of stimulation-bound movements, spike-and-waveafterdischarges (ADs) and clonic seizures accompanying these ADs at the age of 12 daysand decreased duration of spike-and-wave ADs at postnatal days (P) 18 and 25. In contrast,chronic administration of the lower dose of caffeine resulted in a proconvulsant effectexpressedasasignificantprolongationofspike-and-waveADsinP12,P18andP25groupsaswell as of the second  “ limbic ”  type of ADs (significant only in P12 and P25). The biphasicaction of chronic postnatal caffeine treatment was transient and was no longer present in67-day-old rats. Our results demonstrate that early postnatal caffeine exposure results ineither pro- or anticonvulsant effect during brain maturation in relation to the dose used.Caffeine is a mixed adenosine receptor antagonist, therefore its effects could be due to adifferent action on adenosine receptor subtypes; an additional mechanism of action cannotbe excluded.© 2006 Elsevier B.V. All rights reserved. Keywords: CaffeineDevelopmentEpileptic afterdischargeSensorimotor cortexRat  Abbreviations: ADs, spike-and-waveafterdischarges 1. Introduction Caffeinerepresentsthemostcommoncentralnervoussystemstimulant. The mechanism underlying its stimulatory effectsinvolves blockade of central adenosine A 1  and A 2A  receptors;endogenous adenosine exerts tonic inhibitory effect byreducing transmitter release from excitatory terminals (Dra-gunow, 1988) and by modulating intrinsic membrane proper-ties (Haas and Greene, 1984). The proconvulsant action of adenosine antagonists such as caffeine, aminophylline andothermethylxanthineswasdescribedinadult(Fileetal.,1988)as well as young rats (Berná š ková and Mare š , 2000; Trommer et al., 1989). These effects of methylxanthines in immaturerats appeared at lower doses than in adult animals (Berná š -kováand Mare š , 2000; Mare š  et al., 1994;Trommeret al., 1989).High doses of caffeine efficiently augmented cortical, hippo-campal and amygdala afterdischarges (Dragunow and God-dard, 1984; Koryntová et al., 2002) as well as electroshockseizures (Francis and Fochtmann, 1994) in adult rats. Theprolongation of afterdischarges is in agreement with aputative role of adenosine system in the mechanism of seizure termination (Dunwiddie, 1999). B R A I N R E S E A R C H 1 0 8 2 ( 2 0 0 6 ) 4 3  –  4 9 ⁎  Corresponding author.  Laboratory of Experimental Psychopharmacology, Institute of Physiology, Acad. G. Bonchev Str., Bl. 23, BulgarianAcademy of Sciences, Sofia 1113, Bulgaria. Fax: +359 2 8719 109.E-mail addresses: (J. Tchekalarova), (P. Mare š ). 1 Fax: +420 2 41062488.0006-8993/$  –  see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.brainres.2006.01.067 available at  Long-term treatment with methylxanthines such as caf-feineledtoadaptiveresponsesthatareusuallyoppositetotheacute drug effects under normal and pathological conditions( Jacobson et al., 1996a). Chronic treatment with methyl-xanthines resulted in decreased susceptibility to a variety of convulsants with different mechanisms of action (Georgiev etal.,1993;Johanssonetal.,1996b;VonLubitzetal.,1994).Onlyafew studies considered late consequences of repeated neona-tal treatment with caffeine on brain excitability (Guillet, 1995).Similarly to results on adult rodents, caffeine exposure during brainmaturationattenuatestheresponsesindifferentseizuremodels(Guillet,1995)suggestingthateffectsofcaffeinecannotberelatedtoanyspecificmechanismofseizuresbutrepresenta more general phenomenon. The decreased seizure suscep-tibility as a result of chronic administration of caffeine (atdoses of 15 – 20 mg/kg for 5 days) both during adulthood anddevelopment correlates with an increase in adenosine A 1 receptor density in specific brain structures (Daval et al., 1989;Guillet and Kellogg, 1991; Marangos et al., 1984). It waspostulated that up-regulation of A 2A  receptors in the striatumcould also take part in the adaptive effects of long-termcaffeine treatment in adult rodents ( Johansson et al., 1996b;Traversaetal., 1994). Nevertheless,thefunctionalchangesarenot always accompanied by an alteration in the number of adenosine receptors and/or modification in the receptor affinity (Nehlig et al., 1992).Previous data on the effects of caffeine on biochemical andbehavioral parameters varied with respect to both the expo-sure schedule (the duration and magnitude of caffeineadministration) and the age of testing (Concannon et al.,1983; Guillet,1995;Sobotkaet al., 1979). That couldexplainthediscrepancy among published data. Recently, we have dem-onstrated that neonatal exposure to caffeine causes transientimpairment of motor skills early after the end of caffeinetreatment and that this effect disappeared completely whentheratsgrewolder(Tchekalarovaetal.,2005).Furthermore,thedevelopmental period of caffeine exposure was crucial for thealterations in locomotor activity during ontogenesis. Dailyadministration of caffeine between postnatal days 7 and 11resulted in an increased activity in open field not only inimmature but also in postpubertal 60-day-old rats (Tchekalar-ova et al., 2005). This finding could indicate a generallyincreased excitability of the central nervous system, thereforewe started to study seizure susceptibility in animals with thisadministration schedule.We decided to start with a model of cortical epilepticafterdischarges. This model was described in detail in our laboratory (Mare š et al., 2002) andwasused fordevelopmentalpharmacologicalstudies(Berná š kováandMare š ,2000).Rhyth-mic electrical stimulation of the sensorimotor cortex in freelymoving rats offers an opportunity to study four differentphenomena:(1)movementsaccompanyingstimulationduetoa direct activation of the motor system; (2) cortical epilepticafterdischarges(ADs)withspike-and-waverhythmintheEEG,probably of the thalamocortical srcin; (3) clonic seizures of head and forelimb muscles accompanying ADs, indicating aspreadofepilepticactivityintothemotorsystem;(4)changeof spike-and-wave ADs into EEG and behavioral pattern charac-teristic for limbic stimulation (high-amplitude delta wavesand fast spikes in the EEG and behavioral automatisms), i.e. atransition of epileptic activity into the limbic structures(Koryntová et al., 1997; Kubová et al., 1996).Previously, acute effects of another methylxanthine ami-nophylline on the abovementioned four phenomena werestudied at different levels of brain maturation (Berná š kováand Mare š , 2000). The aim of the present study was to find outif repeated neonatal exposure of rat pups to caffeine causeslong lasting adaptive responses to the generation of phenom-ena elicited by electrical stimulation of the sensorimotor cortex. For this purpose, animals were injected with caffeine(10 or 20 mg/kg) and/or saline during postnatal days 7 – 11 andthen epileptic ADs were elicited at four different developmen-tal stages (12-, 18-, 25- and 67-day-old rats). 2. Results PostnataltreatmentwitheitherdoseofcaffeinefromP7toP11did not influence spontaneous electrocorticogram character-ized by beta waves with low amplitude. Stimulus-boundmovements, spike-and-wave ADs and clonic seizures accom-panying ADs were registered after stimulation of the somato-sensorycortexinallanimals(Fig.1). Transitionintothemixedtype of ADs failed to appear in a part of rats (see Section 2.2). 2.1. Threshold intensities Developmental profiles of thresholds for all four phenomenainduced by stimulation were similar in controls and twoexperimental groups. Lower threshold intensities were re-quired in 18- and 25-day-old rats to provoke stimulus-boundmovements, spike-and-wave ADs and clonic seizures accom-panyingADscomparedtotheyoungestandoldestgroups(Fig. Fig. 1  –  EEG recordings of cortical afterdischarges (ADs) fromleft sensorimotor region of an 18-day-old rat. Individualrecordings from top to bottom: responses to stimulationintensities from 0.2 to 8 mA. Left part: last 8 s beforestimulation and first 2 s of stimulation; right part: last 2 s of stimulation and 38 s after the end of stimulation.Spike-and-wave type of AD appeared at the stimulationintensity 1.8 mA, transition into the mixed type of AD at the8-mA intensity. Time mark 5 s, amplitude calibration 1 mV. 44   B R A I N R E S E A R C H 1 0 8 2 ( 2 0 0 6 ) 4 3  –  4 9  2). ANOVA showed for these three phenomena a main Ageeffect [ F 3,112  = 11.1,  P  b  0.0113;  F 3,112  = 13.6,  P  b  0.035 and F 3,112  = 20.3,  P  b  0.0001, respectively] but neither the overallPretreatment nor the Pretreatment × Age interaction wassignificant. Threshold intensities necessary for elicitation of limbic type of ADs decreased till P25 and then increased againin control as well as both experimental groups.Significantly higher current intensities had to be applied toelicit stimulus-bound movements, spike-and-wave ADs andclonic seizures accompanying these ADs in 12-day-old ratpups treated with the higher dose of caffeine than in controlsiblings(PosthocTukey'stest:* P b 0.03)(Fig.2).Incontrast,thethreshold intensities necessary for elicitation of all four phenomena in 18-, 25- and 67-day-old animals were notsignificantlychangedbyearlychronicadministrationofeither dose of caffeine (Fig. 2). 2.2. Incidence of mixed type of ADs The incidence of the mixed (limbic) type of ADs in controlsmarkedly increased with age (Table 1). It was not significantlyinfluenced by either dose of caffeine at P12, P25 and P67.Surprisingly, the lower dose of caffeine resulted in a nearlycomplete suppression of the mixed (limbic) type of ADs in 18-day-old rats (this type of ADs was registered in only one out of 12 rats,  P  b  0.02 in comparison to controls). 2.3. Duration of ADs Analysis of variance demonstrated for the duration of spike-and-wave ADs elicited by threshold intensity of stimulationoverallAgeeffect[ F 3,94 =6.89, P b 0.001]aswellasPretreatmenteffect[ F 2,94 =9.56, P b 0.001].Age×Pretreatmentinteractionwasevident only for the duration of spike-and-wave ADs induced Fig.2  –  Effectsoflong-termtreatment withcaffeine(10and20mg/kg,s.c.)duringP7 – P11onthresholdintensities(mean±SEM)for elicitation of: stimulus-bound movements (upper left), spike-and-wave ADs (upper right), clonic seizures accompanyingADs(lowerleft)andmixedtype ofADs(lowerright).Abscissa: individualagegroups;ordinates:currentintensitiesof stimuli inmA.Individualcolumns — seeinset,10and20mg/kgmeananimalsreceivingcaffeineinthementioneddoseatpostnataldays7to11.Asterisksdenoteasignificantdifferenceincomparisonwithcontrols; ○ meansasignificantdifferenceincomparisonwiththe second caffeine group.Table 1  –  Incidence of mixed types of ADs Drug P12 P18 P25 P67 Controls 4/11 6/10 7/10 12/13Caffeine 10 mg/kg 6/10 1/13* 14/14 6/8Caffeine 20 mg/kg 5/10 6/13 12/12 7/8The incidence of mixed types of ADs following stimulation of somatosensory cortical area expressed as a number of ratsexhibiting the phenomenon/number of animals in the group.Rows represent individual groups in the experiment. Columnrepresents ages of stimulated animals. Asterisk indicates thesignificant difference in comparison with the controls (Fisher'sexact test). 45 B R A I N R E S E A R C H 1 0 8 2 ( 2 0 0 6 ) 4 3  –  4 9  by the threshold intensity [ F 6,94  = 2.89,  P  b  0.015]. Post hocanalysis showed biphasic effects of caffeine injected during P7 – P11. Thus, the lower dose (10 mg/kg) led to a markedprolongation of spike-and-wave ADs during development, 12-, 18- and 25-day-old pups ( P  b  0.001). Furthermore, thisdose of caffeine tended to prolong the duration of mixed typeof ADs. Due to a high variability of data, the level of statisticalsignificance was reached only in 12- and 25-day-old rats( P  b  0.05) (Fig. 3). In contrast, the higher dose of caffeineshortened the duration of spike-and-wave ADs in P18 and P25( P  b  0.046) (Fig. 3). Results for ADs elicited by two timesthresholdstimulationweresimilar.Nosignificantchangewasfound in 67-day-old rats. 3. Discussion The proconvulsant effect of the lower dose of caffeine ad-ministered from P7 to P11 was apparent in two measures  –  amarked prolongation of both spike-and-wave and mixed typeof ADs in P12, P18, P25 and P12 and P25 rats, respectively. Theincrease in seizure susceptibility as a result of postnatalexposure to low doses of caffeine appears to be rather unexpected. Because these effects were detected as early as1 day after cessation of drug treatment, they could be due toresidual caffeine and/or modifications of adenosine release or receptor system. To our knowledge, caffeine's pharmacoki-netic profile varied among species and it is affected by doseand age (Bonati and Garattini, 1988). So far, all pharmacoki-netic data are derived from humans and there are no studiesin immature rats at disposal. In spite of missing data for developing rats, a pharmacokinetic explanation for theincreased brain excitability demonstrated in 12-day-old ratstreated with the lower dose of caffeine during P7 – P11 seemsimprobable. The higher dose of caffeine had the oppositeeffectinthesameagegroup — itspeaksstrictlyagainsttheroleof residual plasma caffeine. Since the duration of limbic typeof ADs was significantly increased with low-dose caffeine atP25, a common mechanism might be in force for the delayedenhanced brain excitability as a consequence of neonatalcaffeine exposure.There is strong evidence that the effects of caffeine at thelow concentrations achieved in our experimental scheduleresult by the antagonism of endogenous adenosine actions(for review see: [25]). Recently, Guillet and Kellogg (1991) reported that P12 rat pups had disrupted developmentalpattern of sensitivity to an acute challenge with adenosinereceptor ligands as a result of caffeine exposure at P2 – P6 atdoses of 15 – 20 mg/kg/day. Because caffeine and other methylxanthines antagonize both adenosine A 1  and A 2 receptor subtypes with nearly equal affinities (with Ki valuesof 29 and 48  μ M, respectively), it is reasonable to assume thatthese receptor subtypes are the most likely targets. In the rat,the appearance of the adenosine A 1  receptors is gradual andarea-specific (Guillet and Kellogg, 1991). Adult receptor densities were reached in the brain stem and hypothalamusin P14 while in the hippocampus, cerebellum and cortex inP21 – P28. Neonatal exposure to caffeine accelerate the devel-opment of adenosine A 1  receptors in rats with attainment of adult densities of receptors at earlier ages which correlateswith altered age-related behavioral development (Guillet andKellogg, 1991). Surprisingly, we found that the low dose of caffeine resemble the proconvulsant effects of acutelyadministered high doses of caffeine (Dragunow and Goddard,1984; Koryntová et al., 2002). Because the activation of adenosine A 1  and A 2A  receptors have opposing actions(Londos et al., 1980), it is important also to consider whichreceptor subtype might be affected by caffeine exposureduring development. Adenosine A 2A  receptors are present inthe forebrain during the postnatal period, when caffeinecould modulate development of seizure threshold ( Johanssonet al., 1996b). The results from adult rat hippocampusshowed an interaction between co-expressed and co-local-ized A 2A  and A 1  receptors, providing a mixture of excitatoryand inhibitory modulation of neuronal excitability (Cunha etal., 1994). The apparent divergence in the effects of the twodoses of caffeine on the susceptibility to seizures might bedue to disturbed balance between A 1  and A 2  receptors inbrain areas associated with seizure phenomena. Fig. 3  –  Effects of long-term treatment with caffeine (10 and20 mg/kg, s.c.) during P7 – P11 on the average duration (±SEM)of afterdischarges. From top to bottom: spike-and-wave ADselicited by threshold current intensity, elicited by intensityequal to two times threshold and mixed type of ADs elicited by threshold intensities at P12, P18, P25 and P67. Abscissa:12-, 18-, 25- and 67-day-old rats; ordinates: duration of afterdischarges in seconds. Details as in Fig. 2. 46  B R A I N R E S E A R C H 1 0 8 2 ( 2 0 0 6 ) 4 3  –  4 9  Since the duration of mixed type of ADs was significantlyincreased with low-dose caffeine at P25, it could be suggestedthat the prolongation of the ADs present at this age could bedue to facilitation of transition into this type of ADs. Incontrast, 18-day-old rats exposed to the lower dose of caffeinefrom P7 to P11 exhibited very limitedincidence of mixedtypesof ADs. In accordance, Guillet and Kellogg (1991) found thatadenosine A 1  receptor binding in cortex from 18-day-oldcaffeine-exposed rat pups was significantly lower than incortex from 14-, 21- or 28-day-old caffeine-exposed pups aswell as from 18-day-old control pups. In the present study,failure of our 18-day-old caffeine-exposed rats to exhibitmixed type of ADs might signify that this age is crucial for the development of the connection between thalamocorticalcircuits and limbic structures.The anticonvulsant effect observed in rat pups exposed tothe higher dose of caffeine (higher thresholds in P12 andshorter duration of ADs in P18 and 25) is in accordance withliterary data showing that chronically applied methyl-xanthines decreased the seizure susceptibility in adult rats(Georgiev et al., 1993). However, little attention has been paidto the effect on seizure susceptibility during ontogenesis as aconsequence of early pre- and/or postnatal administration of caffeine. The proconvulsant effect observed in our study wastransient and evident as early as 1 day after the end of caffeine exposure (in 12-day-old rats). Different age of administration as well as examination can explain adifference between our findings in immature rats and dataof  Guillet (1995) demonstrating a decrease in seizure suscep-tibility at later ages.The increased threshold for movements elicited by stim-ulation, spike-and-wave ADs and accompanying clonic sei-zures elicited by cortical stimulation in 12-day-old ratsexposed to the higher dose of caffeine might representadaptive changes in adenosine modulatory system. In addi-tion, 18- and 25-day-old rats showed attenuated seizureresponses expressed as a decreased duration of spike-and-wave ADs. Because the threshold for these phenomena wasnot modified either in P18 or P25 like in P12, we could suggestthat different adaptive mechanisms underlie the responsesduring development. Our data means that 12-day-old pupsexposed to the higher dose of caffeine had decreasedexcitability of motor as well as thalamocortical system.Shortening of ADs seen in 18- and 25-day-old pups might beassociated with accelerated adaptive adenosine-mediatedmechanisms involved in the termination of the dischargeactivity like in adult rats (Handforth and Treiman, 1994). Theexact reason for the observed delayed biphasic effects of long-term treatment with caffeine is still not very well understoodbut participation of different mechanisms in caffeine actioncannot be excluded. Caffeine can influence many neurotrans-mittersystemsincludingnoradrenaline,dopamine,serotonin,acetylcholine, glutamate and GABA ( Jacobson et al., 1996a).In conclusion, model of cortically induced seizures dem-onstrated that early postnatal exposure to caffeine exertseither pro- or anticonvulsant action during brain maturationin relation to the dose. Considered as a mixed adenosinereceptor antagonist, caffeine effects could be due to diverseactivity on adenosine receptor subtypes or to additionalmechanisms of action. 4. Experimental procedures 4.1. Subjects The experiments were carried out on male Wistar rats. Litterswere culled to 8 pups at postnatal day 1 (postnatal day 0 is theday of birth). Rats were kept under controlled temperature(22±1°C)andhumidity(50 – 60%)witha12/12hlight/darkcycle(lights on at 6 a.m.). The animals determined for examinationin adulthood were weaned on postnatal day 28 (P28) and thenhoused in stable social groups. Food and water were freelyavailable (with the exception of the test period). The projectwas approved by the Animal Care and Use Committee inagreement with Animal Protection Law of the Czech Republic(fully compatible with European Community Council direc-tives 86/609/EEC). 4.2. Caffeine treatment  Ratpupsineachlitterwererandomlyassignedtoacontrolandtwoexperimentalgroups.Eachexperimentalandcontrolgroupconsistedofatleast10animalsandcontainedpupsfromfouror fivelitters.TheadministrationstartedatP7.Animalsassignedto experimental groups were injected with caffeine (Sigma, St.Louis,MO,#C0750,10mg/kgor20mg/kg,s.c.,respectively)inavolume of 1 ml/kg body weight. Control rats received saline(1 ml/kg).Injections were repeated daily for 5 days. 4.3. Surgery Surgery was performed in 12-, 18-, 25- and 60-day-old rats.Surgical preparation was different in pups and adult rats. Ratpups were anesthetized with ether and trephine openingswere made by a razor blade. Flat silver electrodes (connectedwith light flexible wires) were put epidurally over the rightsensorimotor cortical area (two electrodes for stimulation atcoordinates AP  − 1 and +1, L 2 mm in relation to bregma),recording electrodes over the left sensorimotor cortical area(AP 0, L 2 mm), left parietal area and occipital (visual) areas of both hemispheres. Coordinates for parietal and occipitalelectrodes were recalculated from those for adult rats, i.e. AP3, L 3 and AP 6, L 4, respectively, according to bregma – lambdadistance.An indifferentelectrode was put intothe nasal bone,groundingelectrodeintotheoccipitalbone.Allelectrodeswerefixed to the skull by fast curing dental acrylic. The surgerylasted approximately 10 min. Then the anesthesia wasinterrupted and the animals were allowed to recover for atleast 1 h. The rats 12 and 18 days old were maintained in thetemperatureof34°C(i.e.thetemperatureofthenest).Aftertherecovery period, righting and placing reflexes were examinedandtheanimalswereoffered5%sucrosesolutionnotonlyasanutrient but also to check the suckling reflex. Only then theexperiment started.Sixty-day-old rats were operated under pentobarbitalanesthesia (Nembutal® Abbott, 40 mg/kg, i.p.). The silver ballstimulation electrodes were implanted at coordinates AP  − 1and +1, L 2.5 mm over the right hemisphere. Flat silver recording electrodes were again located epidurally at coordi-natesAP0,L2.5andAP3,L3mmoverthelefthemisphereand 47 B R A I N R E S E A R C H 1 0 8 2 ( 2 0 0 6 ) 4 3  –  4 9
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