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Systemic Administration of Atipamezole, aSelective Antagonist of Alpha-2 Adrenoceptors,

Facilitates Behavioural Activity but does notInuence Short-term or Long-term Memory in

Trimethyltin-intoxicated and Control Rats

M. NIITTYKOSKI a, R. LAPPALAINEN a, J. JOLKKONEN a, A. HAAPALINNA b,P. RIEKKINEN SR a , c AND J. SIRVIO a ,*

a A.I. Virtanen Institute, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland bOrion Corporation, OrionPharma, P.O. Box 425, FIN-20101 Turku, Finland

c Department of Neuroscience and Neurology, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland

NIITTYKOSKI M., R. LAPPALAINEN, J. JOLKKONEN, A. HAAPALINNA, P. RIEKKINEN Sr and J. SIRVIO . Systemicadministration of atipamezole, a selective antagonist of alpha-2 adrenoceptors, facilitates behavioural activity but does not inuenceshort-term or long-term memory in trimethyltin-intoxicated and control rats . NEUROSCI BIOBEHAV REV 22(6) 735750, 1998.The present study used trimethyltin (TMT)-intoxicated rats as a model for the behavioural syndrome seen after neuronal damage to thelimbic system. Behavioural assessments indicated increased locomotor activity and reduced number of groomings in an open-arena task

in TMT-intoxicated (6.6 mg/kg as a free base) rats, as has been found previously. A novel ndingwas the severe decit in swimming to avisible platform in the water maze task, with reduced swimming speed at the beginning of the training period. During the reacquisitionphase of a radial arm maze task, TMT-intoxicated rats made more short-term and long-term memory errors, and their behaviouralactivity was increased in comparison with controls. The administration of atipamezole (300 mg/kg), a selective antagonist of a 2-adrenoceptors, enhanced locomotor activity compared to saline-treated rats, but these effects did not differ between the TMT group andtheir controls. Atipamezole did not enhance short-term or long-term memory in either TMT or control groups. Taken together, thepresent data indicate that TMT intoxication is a model for global dementia rather than for a specic loss of relational memory. Previousstudies on the neurochemical effects of TMT and the alleviation or prevention of neurotoxicity of TMT are reviewed. 1998 ElsevierScience Ltd. All rights reserved

Alzheimers disease a 2-adrenoceptors Hippocampus Hyperactivity Memory Rat Schizophrenia Trimethyltin

INTRODUCTION

THE HIPPOCAMPUS has reciprocal, multisynaptic con-nections with the cerebral cortex, and is considered to beinvolved in certain forms of associative learning andmemory, especially the encoding phase of memory pro-cessing (101). In addition, the hippocampus and its outputarea, the subiculum, send projections to the nucleus accum-bens, which may be important in sensori-motor control(63,119). Lesions of hippocampal formation and associateddysfunctions are known to occur, e.g., in Alzheimersdisease (AD) (47,87,94,99,125) and schizophrenia (4,81).

Intoxication with trimethyltin (TMT) leads to profoundbehavioural and cognitive decits in both humans (33) and

experimental animals (30). In rats, TMT induces the degen-eration of pyramidal neurons in the hippocampus andcortical areas (pyriform cortex, entorhinal cortex, sub-iculum) connected to the hippocampus, but there is alsoneuronal loss in the association areas (7,13,19,20). Further-more, behavioural studies have shown increased locomotoractivity, disruption in self-grooming and learning decits inTMT-intoxicated rats (2,18,22,31,41,58,93,103,123). TMTintoxication impairs the acquisition of water maze and Bielmaze (water avoidance) tasks as well as HebbWilliamsmaze and radial arm maze performance (2,31,41,48,7274,93,103,113,123). In addition, TMT intoxication pro-duces decits in passive avoidance retention, but not inthe acquisition of the passive avoidance response

Neuroscience and Biobehavioral Reviews, Vol. 22, No. 6, pp. 735750, 19981998 Elsevier Science Ltd. All rights reserved

Printed in Great Britain0149-7634/98 $32.00 + .00

PII: S0149-7634(98)00002-5

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735

* To whom correspondence should be addressed. Tel.: 358-(0)17-162086; fax: 358-(0)17-163030; e-mail: jouni.sirvio@uku.


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(31,32,54,73,74,112). Furthermore, decits in acquisition of active avoidance at the beginning of training have beenreported (31). However, one study reported that there wereno differences between controls and TMT-intoxicated rats(using a 7 mg/kg TMT base) in either passive avoidance or

active avoidance learning, although intertrial activity was

elevated and extinction response was increased by TMT(41). Moreover, TMT has been shown to produce effects onoperant behaviour, since TMT-intoxicated rats had higherlever pressing rates under a xed-ratio schedule of foodpresentation (102) and TMT impaired the performance of

differential reinforcement at low response rates in an

TABLE 1The neurochemical effects of TMT

Rat strain TMT dose, administration route Effects, time from exposure Reference

LAC:P (m) 10 mg/kg TMT-Cl, i.g . GLU cd 1d; hip 2 d (12)F344 (m) 5.25 mg/kg TMT-Cl, s.c . GLU hip, 1 w; frctx st (117)WI 8 mg/kg TMT, p.o . GLU hip 4 w (49)LAC:P (m) 10 mg/kg TMT-Cl, i.g . GLU binding hip cd sep ent, 2 d (12)WI (m) 8 mg/kg TMT-Cl, i.p . GLU uptake CA1 sub, 13 w (66)WI (m) 3 3 mg/kg /once a week TMT-Cl, i.p . GLU uptake CA1 sub, 45 w after rst injection (66)LAC:P (m) 10 mg/kg TMT-Cl, i.g . GABA hip, 2 d (12)F344 (m) 5.25 mg/kg TMT-Cl, s.c . GABA hip frctx 1 w; st (117)SD (m) 67 mg/kg TMT-Cl, i.p . GABA hip 22 d, am (31)LE (m f) 14 1 mg/kg/alternate days TMT-base, i.p . GABA hip 52 d after rst injection; brst cer

hyp st frctx(55)

LAC:P (m) 10 mg/kg TMT-Cl, i.g . GABA A binding hip cd, 2 d (12)WI 8 mg/kg TMT, p.o . ACh hip 4 w (49)LE (m f) 14 1 mg/kg/alternate days TMT-base, i.p . ACh Ch hyp st cortex hip, 52 d after rst

injection(55)

WI (m) 7 mg/kg TMT-base, p.o . AChE # DG, 46 w (41)LE (m) 6 mg/kg TMT-base, p.o. AChE # DG, 120130 d (120)SD (m) 9 mg/kg TMT-OH, p.o . ChAT CA3 7 d; CA1 (48)LE (m) 6 mg/kg TMT-base, p.o . ChAT DG 360 d; CA1 760 d (15)WI (m) 3 3 mg/kg /once a week TMT-Cl, i.p . ChAT CA1 sub, 5 w after rst injection (66)

LE (m) 7 mg/kg TMT-base, p.o . ChAT DG CA1 100120 d; cd (17)LE (m) 6 mg/kg TMT-base, p.o . M2 binding CA1 1 d; CA3 3 d (16)LE (m) 6 mg/kg TMT-base, p.o . M1 M 2 binding CA1 CA3c, 1 w (16)LE (m) 6 mg/kg TMT-base, p.o . M1 binding CA1 CA3, 2 w (16)SD (m) 8 mg/kg TMT-Cl, i.p . M1 M 2 binding sites hip, 22 d (31)SD (m) 8 mg/kg TMT-Cl, i.p . M1 M 2 binding hip 25 d (71)F344 (m) 5.25 mg/kg TMT-Cl, s.c . 5-HT frctx 1 w; hip st (117)SD (m) 8 mg/kg TMT-Cl, i.p . 5-HT hip frctx st cer, 1 w (3)LE (m) 7 mg/kg TMT-base, i.g . 5-HT am/py 1 w; st olf sep hip (26)SD (m) 68 mg/kg TMT-Cl, i.p . 5-HT hyp; 7 mg/kg hip; am midbr (31)SD (m) 8 mg/kg TMT-Cl, i.p . 5-HT hip st 12 w; cer; frctx (3)LAC:P (m) 10 mg/kg TMT-Cl, i.g . 5-HIAA cd 12 d; hip (12)F344 (m) 5.25 mg/kg TMT-Cl, s.c . 5-HIAA hip frctx st, 1 w (117)SD (m) 8 mg/kg TMT-Cl, i.p . 5-HIAA hip 12 w; frctx st cer (3)SD (m) 68 mg/kg TMT-Cl, i.p . 5-HIAA hip hyp am midbr (31)LE (m) 7 mg/kg TMT-base, i.g . 5-HIAA/5-HT st nac sep am/py hip olf, 1 w (26)LE (m) 7 mg/kg TMT-Cl, i.g . 5-HIAA/5-HT st sep 24 w; frctx (25)LAC:P (m) 10 mg/kg TMT-Cl, i.g . NA hip, 12 d (12)SD (m) 8 mg/kg TMT-Cl, i.p . NA hip frctx 1 w; st cer (3)SD (m) 67 mg/kg TMT-Cl, i.p . NA am; hip midbr hyp (31)LE (m f) 14 1 mg/kg/alternate days TMT-OH, i.p . NA brst cer, 52 d after rst injection (55)SD (m) 8 mg/kg TMT-Cl, i.p . NA hip 12 w; cer; frctx st (3)SD (m) 68 mg/kg TMT-Cl, i.p . b-adrenergic binding cerebral cortex, 22 d (31)LE (m) 6 mg/kg TMT-base, p.o . b-adrenergic binding am/py frctx, 4 w (59)F344 (m) 5.25 mg/kg TMT-Cl, s.c . DA DOPAC frctx st, 1 w (117)LE (m) 7 mg/kg TMT-base, i.g . DA DOPAC nac 1 w; st sep am/py olf (26)SD (m) 8 mg/kg TMT-Cl, i.p . DA nac, 2 w (3)LE (m) 7 mg/kg TMT-Cl, i.g . DA nac 23 w; st frctx (25)SD (m) 68 mg/kg TMT-Cl, i.p . DA hip hyp am midbr (31)LE (m f) 14 1 mg/kg/alternate days TMT-OH, i.p . DA st 52 d after rst injection; brst (55)LE (m) 7 mg/kg TMT-base, i.g . DOPAC/DA st nac sep am/py olf, 1 w (26)LE (m) 7 mg /kg TMT-Cl, i.g . DOPAC/DA st nac frctx, 24 w (25)LE (m) 7 mg/kg TMT-base, i.g . DA receptor binding st 7 d (26)

Abbreviations: increased; decreased; no effect; # histological staining; autoradiographical method; ACh acetylcholine; AChE acetylcholinesterase; am amygdala; brst brain stem; cd caudate; cer cerebellum; Ch choline; ChAT choline acetyltransferase; d day; DA dopamine; DG dentate gyrus; DOPAC 3,4-dihydroxyphenylacetic acid; ent entorhinal cortex; f female; frctx frontal cortex; F344 Fischer-344;GABA g-aminobutyric acid; GLU glutamate; hip hippocampus; 5-HIAA 5-hydroxyindoleacetic acid; 5-HT serotonin; hyp hypothalamus; i.g. intragastrically; i.p . intaperitoneally; LE LongEvans; m male; M muscarinic; midbr midbrain; NA noradrenaline; nac nucleusaccumbens; olf olfactory tubercle; p.o. per os; py pyriform cortex; s.c . subcutaneosly; SD SpragueDawley; sep septum; st striatum; sub subiculum; w week; WI Wistar.

736 NIITTYKOSKI ET AL.


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operant schedule (123). These anatomical and behaviouralndings have made TMT-intoxicated rats an attractivemodel for degenerative diseases such as AD, the mostcommon cause of dementia (121).

It is not understood how TMT can cause a selectivedestruction of neurons, but it has been suggested that anexcess activation of the N-methyl-D-aspartate (NMDA)type of glutamate receptors is involved (5). In TMTintoxication the uptake of glutamate is reduced (Table 1),and in vitro studies have shown that its efux has beenelevated (24,67). Both effects may lead to overactivation of glutamate receptors. The selectivity of damage might be dueto a protein named stannin, which is expressed in TMT-sensitive neurons, e.g. in the hippocampus and entorhinalcortex (108), and this protein may also be involved in theapoptosis induced by TMT (107).

TMT intoxication may induce other characteristics of ADthan simply nerve cell apoptosis. The expression of a formof amyloid precursor protein (APP) called APP-751 mRNAwas increased in CA1, but APP-695 mRNA levels wereunchanged in the rat hippocampus 25 days after TMT intox-

ication (75). It has been suggested that there is a correlationbetween the increase in the APP-751/APP-695 mRNA ratioand the number of senile plaques in the hippocampus inAlzheimers patients (see (75)). Furthermore, a similarlyincreased ratio has been reported in nucleus basalis of behaviourally impaired aged rats (see (75)). However,following TMT intoxication, rats do not develop amyloidplaques (or neurobrillary tangles) (121).

Gamma-amino butyric acid (GABA)-containing inter-neurons play an important role in the regulation of the excit-ability of principal cells, such as pyramidal neurons, in theforebrain. TMT intoxication can affect GABA-mediatedneurotransmission (Table 1). The level of GABA wasincreased and GABA A receptor binding was decreased inthe hippocampus soon after a large dose of TMT, whereasthe levels of GABA were reduced after smaller doses andlonger follow-up times (Table 1).

Different functional types of GABAergic interneuronshave been classied according to their neuropeptides andcalcium-binding proteins (35). Recently, the effects of TMTon different hippocampal calcium-binding protein-positiveneurons were studied with immunocytochemistry (37,38).Three weeks after TMTintoxication the number of calbindin-containing neurons was decreased, especially in the CA1eld, but parvalbumin- and calretinin-containing inter-neurons were preserved (37,38). These ndings indicatethat TMT intoxication affects one type of dendritic inhibi-

tory cell, but not perisomatic inhibitory cells or interneuron-selective inhibitory cells. Several neuropeptides are co-localized with GABA in interneurons (35). Recently, theeffects of TMT on neuropeptides were studied (48,109). Theexpression of neuropeptide Y mRNA was increased in hilarinterneurons and dentate granule cells 5 days after TMTintoxication, whereas the number of somatostatin mRNA-containing neurons was decreased 16 days after the intox-ication (109). In addition, the neuropeptide Y content waselevated in the entorhinal cortex 7 days after TMT intoxica-tion (48). Neuropeptides, such as somatostatin, can also becompromised in AD (34).

There are many studies into the effects of TMT intoxica-tion on modulatory neurotransmitters such as acetylcholine

and the monoamines (Table 1). TMT intoxication produces

axonal sprouting within the cholinergic septohippocampalsystem (e.g. (15,17)). This sprouting is an intriguing nding,since it parallels the consequences of the lesion of theentorhinal cortex, and is also a characteristic nding inAD (see (121)). However, the cholinergic neurons of thebasal forebrain are not appreciably damaged by TMT (121),whereas there is a profound neocortical cholinergic decit inAD (34). However TMT does cause a loss of M 1 and M 2muscarinic receptor subtypes in both the hippocampus andneocortex (Table 1, (71)).

Several reports have described the effects of TMT on theserotonergic system. The levels of serotonin were decreasedand those of its metabolite, 5-HIAA, were elevated, indicat-ing that the utilization of serotonin is increased in severalbrain areas including the striatum, septum and hippocampus(Table 1). The levels of noradrenaline may be reduced(Table 1). The effects of TMT on the metabolites of noradrenaline were investigated in one study, but thechanges were not statistically signicant (12). However,the b -adrenergic receptor ligand binding was increased 4weeks after intoxication (59). This could be an adaptive

process following a reduction in levels of noradrenaline.Interestingly, Andersson et al . (3) found that serotonin andnoradrenaline levels had recovered in the hippocampuswhen measured 12 weeks after TMT intoxication. Thelevels of dopamine were reduced in nucleus accumbens,but TMT intoxication had no effect on the utilization of dopamine (i.e. the ratio of DOPAC to DA) (Table 1). Thelevels of serotonin and noradrenaline were decreased in thetemporal and frontal cortex in AD, but the concentration of dopamine remained unchanged (34). The effects of TMTintoxication on the histaminergic system are not known,though this transmitter is compromised in AD (1).

In the neocortex and hippocampus, synaptic transmissionand plasticity are state dependent (6,10,44); see (56,118).One candidate neurotransmitter associated with this mod-ulation is noradrenaline. Both inhibitory interneurons andprincipal neurons can be targets for noradrenergic synapticterminals in the hippocampus (36,61). In vitro and in vivophysiological experiments have shown that noradrenalinecan enhance the excitability of principal neurons in theforebrain (43,52,65,77,115). Furthermore, noradrenalinecan enhance the induction of long-term potentiation pro-duced by high frequency stimulation in the synapses of mossy bres on CA3 pyramidal neurons (45). Noradrenalinealso modulates the long-term potentiation of mbria-CA3synapses (50) as well as the activity of neocortical neurones(11).

The release of noradrenaline is regulated by a 2-adrenergicautoreceptors which are located both in the soma and in thepresynaptic terminals of the noradrenergic neurons (see(23)). The activation of those receptors inhibits the releaseof noradrenaline, and therefore antagonists of a 2-adreno-ceptors promote the release of noradrenaline in the brain(92,106). Recently, studies have been undertaken to inves-tigate the inuence of a 2-adrenergic agents on cognition inanimals and humans (23). The systemic administration of idazoxan or atipamezole, selective antagonists of a 2-adrenoceptors, has been found to enhance the excitabilityof granular neurons to the stimulation of the perforant path(90,126). The same doses were found to improve inter-mediate-term retention in the radial arm maze task (126) and

memory retrieval in the linear arm maze task (91). In

a 2-ANTAGONIST AND HIPPOCAMPAL FUNCTION 737


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addition, atipamezole improved the choice accuracy of ratsin an attentional task when the intensity of the visual stimuliwas reduced (96). It could be speculated that the adminis-tration of a drug would direct the attention of rats towardspertinent cues during the sampling phase of the radial armmaze task, improving their intermediate-term retention orretrieval of learned material.

Since our goal is to develop symptomatic treatment fordementia, it should be noted that none of the above-mentioned studies investigated the inuence of a 2-antago-nists on the performance of cognitively impaired rats suchas those with lesions in the hippocampal formation andrelated brain areas, which leads to the isolation of the hippo-campus from the neocortex. Therefore, the present studyinvestigated whether a selective antagonist of a 2-adreno-ceptors, atipamezole, could facilitate short-term or long-term memory in TMT-intoxicated rats, which exhibitneuronal damage in the limbic system. These memory func-tions were assessed with the radial arm maze task, using theversion in which working memory-type errors (revisits toarms) and reference memory-type errors (visits to non-

baited arms) can be investigated. In addition to potentialfacilitation of neuronal transmission and plasticity by theincreased activity of noradrenergic neurons (65,82,84,89),the enhancement in the release of acetylcholine in theneocortex by atipamezole (105) could be benecial inTMT rats, because these animals have reduced levels of muscarinic receptor binding in the cortex (71). The possibleinuences of atipamezole on hyperactivity and the disrup-tion of grooming behaviour in TMT-intoxicated rats werealso considered to be valuable preclinical information, sincethe treatment of non-cognitive symptoms of AD, such aswandering and disruption of personal grooming, can be of great importance in the everyday life of patients. Therefore,the performance of TMT-intoxicated rats was tested in theopen-eld task. Moreover, the inuence of atipamezole onhyperactivity produced by TMT was considered to be of special interest, since the symptoms of TMT intoxicationresemble the behavioural syndrome induced by MK-801, anon-competitive antagonist of N-methyl-D-aspartate recep-tors (9,42,80,124), a drug used as a model for psychosis(76), and it has recently been suggested that some atypicalneuroleptics can act as antagonists at a 2-adrenoceptors (70).Interestingly, atipamezole was found to reduce scopolamine-induced hyperactivity in rats (69). Before the drug trialswere started, the performance of the TMT group was alsotested in the water maze task which was used to assesssensori-motor control (by examining the ability of rats to

swim to a visible platform).

MATERIALS AND METHODS

Animals

Male Bkl Wistar rats (National Animal Center, Univer-sity of Kuopio, n 40) were used (3 months old at thebeginning of the experiment). Animals were housed andmaintained on a 12:12 h lightdark cycle, and the roomwas controlled for temperature (20 2C) and humidity (55 10%). All testing was conducted during the light part of the cycle. Except for two rats used for weight controls, therats (n 38) were housed singly, and their weight wasreduced by gradually decreasing their amount of daily food

(R36, Lactamin, Stockholm, Sweden); subsequently theywere maintained at approximately 8085% of their free-feeding body weight by limiting their food access to 1416 g per day throughout the duration of radial arm mazetests. Behavioral testing was initiated after 1 week of restricted access to food. Water was available ad libitum .

Pharmaceutical agents

Trimethyltin chloride (TMT, Research BiochemicalsInc., USA) was dissolved in saline and injected intraperi-toneally (i.p.), 8 mg/kg (i.e. 6.6 mg/kg as a free base).Previously, doses between 6 and 7 mg/kg (as the freebase) have been used to produce the behavioural syndromeof TMT intoxication in rats as well as to study the distribu-tion of neuronal degeneration induced by TMT (e.g.(7,31,41,123)).

Atipamezole (4-(2-ethyl-2,3-dihydro-1H-inden-2-yl)-1H-imidazole) is a highly selective and specic a 2-adrenocep-tor antagonist (92,111). In receptor binding studies,atipamezole is reported to have about 100 times higherafnity for a 2-adrenoceptors and an a 2 / a 1 selectivity ratioover 100 times higher than that of idazoxan and yohimbine.In studies with isolated organs, atipamezole is a more potenta 2-adrenoceptor antagonist and has about 200 times higherrelative a 2 / a 1 blocking ratio than idazoxan (111). Atipame-zole has an almost equal afnity for the different a 2-adrenoceptor subtypes (88). Atipamezole penetrates rapidlyinto the brain (8), causing a dose-dependent increase in therelease of central noradrenaline and serotonin (92). In thepresent study, atipamezole hydrochloride (Orion Pharma,Turku, Finland) was dissolved in saline and injected sub-cutaneously, 300 mg/kg (1.0 ml/kg).

Behavioural experiments

Radial arm maze task The 10-arm maze apparatus has been described pre-

viously (80,83). The rats were habituated to handling andthen to the maze. In the rst day of maze habituation, foodpellets (45 mg Precision pellets, Bio-Serv, NJ, USA) werein the centre of the arena and all arms were closed. In thesecond session, the rat had access to one arm, which wasbaited. The rats were allowed to explore the maze for10 min on each testing session. Gradually, more armswere opened, and food pellets were located only at theend of every baited arm. Finally, the rats were able to searchfor food pellets in the maze with all the arms open.

In order to assess both short-term and long-term memory,a rat was trained to obtain food pellets from only ve loca-tions in the 10-arm maze. The pattern of baited arms was thesame from day to day but could vary between animals (vedifferent congurations) in order to reduce the aid of olfac-tory cues left by a previous rat. The rat was put into the mazewith every door open. When the rat had succeeded in obtain-ing all the food pellets, it was returned to its home cage.Maximum testing time was 10 min. The maze was cleanedbetween animals. A correct choice was recognized when therat visited a food well of the baited arm. Long-term memoryerrors were assessed by the number of entries to non-baitedarms, and short-term memory errors were assessed by thenumber of entries to previously visited arms during a trial.The number of arm entries per minute was taken as a



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measure of locomotor activity. The number of correctchoices before the rst error (a visit to an empty arm) wasalso calculated. The training was continued until ratsshowed a consistent choice accuracy above the chancelevels (e.g. approximately 1.6 correct choices before therst error, for which the chance level is 0.8).

TMT administrationAfter this initial training phase in the radial arm maze

task, the rats were given a free-feeding period of 12 daysand were then treated with TMT chloride (8 mg/kg, n 22)or saline (1 ml/kg, n 16). The rats were allowed to recoverfor 28 days. The rats were given food pellets, which weremade as palatable as possible, inside their cages. If a ratrefused to eat these pellets, it was provided with somechocolate, apple or banana according to its preference.

The rats were given also saline intraperitoneally. In spiteof this post-intoxication treatment, seven TMT-treated ratsdied 522 days after the administration of the toxin. Theweight of the surviving rats was 91%, 83% and 91% of saline-treated rats on days 3, 10 and 24, respectively, afterTMT administration. The TMT-intoxicated rats exhibitedthe signs of TMT syndrome, such as hyperreactivity, resis-tance to handling and tail mutilation (30).

The open-arena test To assess their exploratory behaviour, the rats were tested

in an open arena. The apparatus has been described pre-viously (69). Before the start of testing in the open arena,the rats were habituated to the open arena testing board and

environment for 3 min each day for three days.

The open arena test was performed 31 days after TMTintoxication. The animals were placed in the open arena fora 9 min period (3 3 min sessions interrupted by a 10 speriod to load the computer). The oor and walls of the openarena were cleaned between animals. Locomotor activity,expressed by the path length, was recorded for each animal.The number of times the rat reared up on its hind legs, thenumber of groomings and the number of fecal boli werecounted by the experimenter.

The water maze task This task was aimed to assess spatial memory (navigation

cued by distal cues) and non-mnemonic factors using thenon-spatial version (navigation cued by the visibility of theplatform, the position of which was also altered trial by trialin order to minimize the contribution of spatial memory).

However, the hidden platform version was not assessed,because TMT-intoxicated rats were severely impairedeven when the platform was visible. The water mazeapparatus has been described previously (69,86). Theswim paths were monitored by a video camera linked to acomputer through an image analyzer (HVS Image, UK).The computer software (made by HVS Image) calculatedtotal distance and time swum as well as the percentage of time spent in each quadrant and annuli.

The water maze testing was initiated 5 weeks after TMTintoxication. In the habituation trial, the rats were allowed toswim for 90 s in the pool, which did not contain the plat-form. Two days later, the rats were trained to nd a visibleplatform (top 1.5 cm above the water line), which was

located in the south-west quadrant during the rst part of

FIG. 1. Schedule of the study. The numbers represent the days from TMT administration.



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training. Training was continued for 2 days (4 trials/day,

maximum duration 60 s, 10 s reinforcement on the platform).In the second part of the training, the visible platform wasmoved to a different quadrant after each trial. Training wascontinued for 4 days (2 trials/day, maximum duration 60 s,10 s reinforcement on the platform). If the rat failed to ndthe platform within 60 s, it was put onto it for 10 s.

Reacquisition of the radial arm maze task and open-arenatestings after vehicle or atipamezole injections

After water maze testing, the rats were returned torestricted feeding to reduce the weight to approximately85% of weight controls. Then (68 days after the previoustesting in the radial arm maze) the rats were retrained for theradial arm maze task for 5 days. During the last three days,all the rats were injected with saline (1 ml/kg, s.c.) tohabituate them to the injections.

The TMT-intoxicated rats and their controls were dividedrandomly to groups to be treated with either atipamezolehydrochloride (300 mg/kg, s.c., TMT: n 8 and controls:n 8) or saline (TMT: n 7 and controls: n 8) beforedaily testing either in the radial arm maze task or in theopen-arena task (see schedule in Fig. 1). One TMT-intoxicated rat treated with atipamezole died on the rstday of testing (about 1 h after atipamezole injection). 1

Food intakeIn addition, food intake was measured. The animals were

given more food than they normally consumed and theamount of food was calculated 24 h later. The baselinemeasurements were done 8 and 4 days before TMT intoxi-cation, when the rats were free-feeding. To assess the effectof TMT intoxication, the food intake was measured 8 daysafter the TMT intoxication. The nal measurement investi-gated the effect of atipamezole on food intake (98 days afterTMT administration, following the last test in the radial armmaze).

Histology

Some rats from each group (two from salinesaline,one from salineatipamezole, two from TMTsaline, onefrom TMTatipamezole) were processed for histology.The rats were anaesthetized with MEBUNAT (sodiumpentobarbital, 100 mg/kg) before perfusion. They weretranscardially perfused with 0.9% saline followed by xa-tive containing 4% paraformaldehyde, 0.05% glutaralde-hyde and 0.2% picric acid in 0.1 m phosphate buffer(pH 7.4). After perfusion, the brains were removed fromthe skull, postxed in the perfusion xative for 2 h andplaced in 20% glycerol in 0.02 m potassium phosphatebuffered saline (pH 7.4) for 22 h. Then the brains werefrozen on dry ice.

Frozen sections (30 mm) were cut with a sliding micro-tome in the coronal plane and stored in 10% formalin in0.1 m phosphate buffer (pH 7.4). The overall neuronaldamage caused by TMT was estimated from Nissl stainedsections. In addition, the cortical shrinkage was measuredusing an image analysis system (Imaging Research Inc.)combined with a DAGE MTI CCD-72 series camera(DAGE.MTI) at two anteroposterior levels: AP 1.60 mmand 3.80 mm from the Bregma (79).

Statistical analysis of dataThe group effect (controls vs TMT-intoxicated rats),

treatment effect (saline vs atipamezole), testing effect(three 3 min periods) and their interactions in the data on

the open-arena task (distance, number of rearings andgroomings) were analysed using ANOVA for repeatedmeasurements. The group effect in the number of fecalboli was analysed using ANOVA. The group effect, testingeffect (training days) and their interactions in the data onwater maze performance were analysed with ANOVA forrepeated measurements. The group effect in the data onthe retention of a radial arm maze task was also analysedusing ANOVA. The group effect, treatment effect, testingeffect (training blocks) and their interactions in the dataon reacquisition of a radial arm maze task were analysedusing ANOVA for repeated measurements. The datafrom two or three consecutive days were combined toblocks of trials. The feeding data were analysed withANOVA.

1 The water maze task was not used as an outcome measure, becauseatipamezole reduces the speed of swimming; atipamezole ( 300 mg/kg)treated rats put into a water tank oat for some time ( 10 s) before theybegin to swim (our unpublished ndings).

FIG. 2. The performance of saline-treated control ( n 16) and TMT-intoxicated ( n 15) rats in the open arena task assessed 31 days after TMTadministration. The results on the ambulation, rearings and groomings are expressed as group means SEM for each interday trial.



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RESULTS

Behavioural testings before atipamezole treatment

Open-arena test The TMT group travelled a longer path length than their

controls (a signicant group effect, F (1, 29) 5.57, p 0.05), and this difference between the groups in the pathlength increased with time within the testing session (asignicant interaction between the group and testing effect,F (2, 58) 4.85, p 0.05) as shown in Fig. 2. The numberof rearings did not differ between controls and TMT group(F (1, 29) 0.23, p 0.1), but there was a signicantinteraction between group and testing effects ( F (2, 58) 4.77, p 0.05). The number of groomings was decreased inTMT-intoxicated rats ( F (1, 29) 22.92, p 0.001),whereas the number of fecal boli produced by TMT-intoxi-cated rats (6.1 1.1, mean SEM) was signicantlyincreased in comparison with controls (0.3 0.3)(F (1, 29) 27.29, p 0.001).

The water maze task with a visible platform

In the habituation trial, TMT-intoxicated rats swam less

than their controls (a signicant group effect ( F (1, 29) 22.92, p 0.001) in total distance (in metres, mean SEM); controls: 21.3 0.7 (n 16) and TMT: 14.0 0.1(n 15)).

In the swim-to-platform test (platform kept in a constantposition), TMT-intoxicated rats had signicantly longerescape latencies ( F (1, 29) 400.3, p 0.001) and distances(F 109.7, p 0.001) than their controls, and theirswimming speed was slower than that of the controls ( F 16.3, p 0.001), as shown in Fig. 3A.

In another swim-to-platform test (platform moved to adifferent location in each trial), TMT-intoxicated rats hadsignicantly increased escape latencies ( F (1, 29) 418.2, p 0.001) and distances ( F 189.1, p 0.001). Swim-ming speed did not differ between controls and TMT-intoxicated rats (a non-signicant group effect ( F 0.2, p 0.1) (Fig. 3B)).

Retention of radial arm maze task During the retesting phase of the radial arm maze task,

TMT-intoxicated rats ( n 15) made more re-entries to thevisited arms (11.17 1.54) than controls ( n 16) (5.63

0.95, mean SEM; a signicant group effect, F (1, 29)

FIG. 3. The performance of saline-treated control and TMT-intoxicated rats in the water maze task using a visible platform in a constant position (A) andvariable position (B). The group means SEM for escape latency and distance as well as swimming speed are presented for each training day.



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9.64, p 0.01). Importantly, both groups performed closeto a chance level in memory scores (e.g. 4.5 for the numberof the visits to the non-baited arms and 0.8 for the number of correct choices before the rst error) even though thesescores were signicantly better after the acquisition phase of this task before vehicle or TMT administration. This indi-cates that even the controls had forgotten the task before thetests were performed with atipamezole.

The effects of atipamezole treatment on the performance of rats

Reacquisition of radial arm maze task TMT-intoxicated rats made less correct choices before

the rst error (F (1, 26) 16.45, p 0.001), visited moreincorrect arms ( F 8.08, p 0.01) and made markedlymore re-entries to visited arms than controls ( F 148.66, p 0.001) during the reacquisition phase, as shown inFig. 4. Atipamezole treatment did not inuence these para-meters in either control or TMT groups ( p 0.1 for allparameters and their interaction with the group effect, i.e.TMT vs controls) (Fig. 4).

TMT-intoxicated rats made more arm entries in a giventime than control rats (a signicant group effect, F (1, 26) 22.72, p 0.001) (Fig. 4). Atipamezole increased this kindof activity (a signicant treatment effect, F (1, 26) 8.30, p 0.01), but its inuence on behavioural activity did notdiffer between controls and TMT-intoxicated rats (a non-signicant interaction between treatment and group effects,F (1, 26) 0.02, p 0.1). The inuence of atipamezole onbehavioural activity became clearer as the testing pro-gressed (a signicant interaction between treatment effectand trial block, F (7, 182) 3.13, p 0.01) (Fig. 4).

Open-arena task TMT-intoxicated rats had signicantly longer path

lengths than controls in every testing session (e.g. a signi-cant group effect, F (1, 26) 68.82, p 0.001 in the thirdtesting session), and this difference became clearer withtime within each test session (a signicant interaction

between group effect and time effects, F (2, 52) 11.2, p 0.001), as shown in Fig. 5. Atipamezole slightlyincreased path length on the third testing, F (1, 26) 3.24, p 0.08 (whereas on the rst and second testings thetreatment effect was not signicant, p 0.1), but thiseffect did not differ between the TMT group and theircontrols (a non-signicant interaction between treatmenteffect and group effect, F (1, 26) 1.34, p 0.1) (Fig. 5).

TMT-intoxicated rats made more rearings in all testings(e.g. a signicant group effect in the third testing, F (1, 26) 7.54, p 0.05). This difference between groups becamemore prominent over time within each testing session (e.g. asignicant interaction between group effect and time effectin the third testing, F (2, 52) 3.58, p 0.05), sincecontrols, but not TMT-intoxicated rats, made less rearingswith time within a testing session. Atipamezole did notsignicantly inuence the number of rearings in the TMTgroup or their controls (e.g. a non-signicant treatmenteffect, F 0.00, p 0.1, and interaction between treatmentand group effects, F 0.44, p 0.1 in the third testing)(Fig. 5).

The incidence of groomings was slightly lower in TMT-intoxicated rats in comparison with their controls (a signi-cant group effect, F (1, 26) 7.22, p 0.05 in the thirdtesting). The inuence of atipamezole and these interactionswith the group effect was not signicant in any of the

tests ( p

0.1).The number of fecal boli was consistently higher in TMT-intoxicated rats than in their controls, and atipamezoleincreased the number of fecal boli consistently in controls(Fig. 5). However, no signicant differences were found inany of the tests ( p 0.05).

Food intake

Food intake during a 24 h period was equal in controlsand TMT-intoxicated rats before TMT intoxication, as wellas between controls and those TMT-intoxicated rats whichsurvived when assessed 8 days after the administration of TMT (Fig. 6). When measured after the completion of theradial arm maze testing, including atipamezole treatments,

FIG. 4. The effects of saline and atipamezole (300 mg/kg) treatments on the performance of TMT-treated rats and their controls in the radial arm maze task.The results indicate the group means SEM across the trial blocks for the reference memory errors (the number of the entries to non-baited arms), workingmemory errors (the numbers of re-entries to either baited or non-baited arms) and locomotor activity (the number of arm entries in a minute). Groups: TMTatipamezole (TMT/ATI, n 7), TMTsaline (TMT/sal, n 7), salineatipamezole (sal/ATI, n 8), salinesaline (sal/sal, n 8).



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TMT-intoxicated rats consumed signicantly more foodthan controls ( F (1, 29) 6.82, p 0.02), and this groupdifference was not affected by treatment (saline vs atipa-mezole) ( F (1, 29) 0.18, p 0.1). In general, the amountof food consumed was higher than in the previous measure-ments, since the rats were on restricted feeding immediatelybefore this last testing point.

Histology

The gross inspection of Nissl-stained sections revealed asevere neuronal loss following systemic injection of TMT-chloride (8 mg/kg) at 112 days after intoxication. There wassome selectivity in the neuronal loss. The pyramidal

neurons in the hippocampal CA1 and CA3 area wereparticularly vulnerable (Fig. 7D). The neuronal loss was

also apparent in the cortical areas. Interestingly, the anteriorneocortex seemed to be less severely affected than theposterior neocortex, as judged by the shrinkage of thecortex (Fig. 7B and D). Posterior spreading of corticaldamage by TMT was also demonstrated by the almostcomplete disappearence of the entorhinal cortex.

DISCUSSION

Toxicity of TMT

In the present study, approximately 30% of rats exposedto TMT died within 22 days despite receiving postoperativecare. Dyer et al . (30) described mortality after TMT and

noted that heavier animals were more susceptible to TMTtoxicity than leaner animals; for example, 10% mortality

FIG. 5. The effects of saline and atipamezole (300 mg/kg) treatments on the performance of TMT-treated rats and their controls in the open-arena task. Theresults show the group means SEM on the length of path, the number of rearings, the number of groomings and the number of fecal boli.

FIG. 6. The amount of food consumed in 24 h ( SEM) in the baseline conditions, 8 days after TMT administration, and 98 days after TMT administration

following the last testing day in a radial arm maze. The wctrl group includes two weight control rats.



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was reported in rats weighing about 440 g treated with7 mg/kg TMT (as a free base (30)). Recently, Alessandriet al . (2) reported 40% mortality using the same dose but adifferent rat strain. The present death rate may be due to thehigh body weight (and age) of the animals being trained forthe radial arm maze task before TMT administration. How-ever, body weight prior to exposure did not differ betweenrats surviving (mean 417 g) and succumbing (mean 416 g).In addition, there are known to be differences between ratstrains in their sensitivity to the neurotoxic effects of TMT(21,64).

Histological examination of Nissl-stained sections at 16weeks after intoxication indicated a severe atrophy in the

hippocampusand the cerebral cortex. Moreover, the posteriorneocortex seemed to be moreseverelyaffected, which agreeswith a previous study (7), although that study had shorterfollow-up times than the present study. One study, using arelatively long follow-up time (13), employed a different kindof TMT administration schedule, but the effects of TMT onhippocampal andcortical shrinkage, as well as on enlargementof ventricles, were similar to those found in the present study.

Inuence of TMT intoxication on the performance of rats

The results from the open-arena and radial arm mazetasks indicate that TMT intoxication caused persistent

FIG. 7. Photomicrographs from Nissl-stained sections of a control (salinesaline-treated) rat (A and C) and a TMT-intoxicated, saline-treated rat (B and D) attwo rostrocaudal levels. Note the severe neuronal loss in the hippocampus and cortical areas and enlargement of ventricles in the TMT-intoxicated rat in theposterior level (D). Magnication: 12.8; scale bar: 1 mm.



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hyperactivity in the rats, which is in good agreement withprevious studies (see (57)).For example, Swartzwelder etal .(102) found a more than 3-fold increase over controls in theopen-eld activity of rats administered 7 mg/kg TMT-Clwhen they were tested 40 days after exposure. Morerecently, Earley et al . (31) found that 8 mg/kg, but not 6or 7 mg/kg, TMT-Cl increased ambulation approximately 2-fold when it was measured 21 days after exposure. Afterintoxication with 8 mg/kg TMT-Cl, the increase in ambula-tion was approximately 2-fold at one month and 3-fold attwo months after exposure. Hyperactivity may be a conse-quence of the damage to the hippocampus (60,68), possiblydue to decits in adaptive responses to novelty againstfamiliarity, and behavioural inhibition (51). Interestingly,the locomotor activity of TMT-intoxicated rats was less thanthat of controls in the habituation test of water maze. (Thus,in one environment TMT intoxication is associated withhyperactivity, whereas in another it reduces locomotoractivity.) The previous studies (31,41,93,123) did notreport data on the inuence of TMT intoxication on swim-ming speed (even though escape latency was used to express

the acquisition of the water maze task). The reducedlocomotor activity in a swimming pool could indicate anenhanced emotional response to novel environments. Itshould be noted that TMT-intoxicated rats produced morefecal boli in open-arena testing and this is considered as anindex of emotional response. It is evident that TMTintoxication affects also the amygdala, which is known tobe important in mediating emotions. In addition, the self-grooming of TMT-intoxicated rats was disrupted in theopen-arena task. These ndings are in good agreementwith previous studies (18,30).

The feeding data suggest that food restriction mayincrease the food intake of TMT-intoxicated rats morethan in the controls. Thus, the effect of TMT-intoxicationon food intake may be dependent on food restriction,because no effect of TMT was observed after eight daysof ad libitum feeding. However, another possibility is thatthe inuence of TMT on food intake depends on the post-intoxication time point. When the rats are receiving arestricted diet, the value of food could be higher in TMT-intoxicated rats. This could partly explain their increasedbehavioural activity in the radial arm maze task. In addition,it is known that the state of satiety can inuence a ratsperformance in the open-eld task (51).

Hyperactivity can result as a secondary consequence of the loss of spatial memory (114). Indeed, TMT-intoxicatedrats exhibited an increased number of errors, indicative of a

decit both in short-term and in long-term memory duringthe reacquisition phase of the radial arm maze task. Thendings on the reacquisition phase, especially the perse-verating performance of TMT-intoxicated rats, agree with aprevious study ((113), using TMT 6.0 mg/kg as a base),even though it should be recognized that the present studyinvestigated relative contributions of both reference andworking memory errors. Moreover, TMT-intoxicated ratsmade more working memory errors in an unbaited 6-armradial tunnel maze (2). Swartzwelder et al . (103) found aprofound decit akin to perseverative behaviour in rats after7.0 mg/kg TMT-Cl when they were tested in a series of HebbWilliams maze problems. The decit could be theresult of hippocampal lesion (78).

The disruption of olfactory memory (e.g. due to a

neuronal loss in the olfactory tubercle and pyriform cortex,(7,19)) could, at least partly, explain the increased perse-veration in the radial arm maze task and reduced habituationin the open-arena task, since it is possible that a rat uses itsown olfactory cues, in addition to allocentric visual andegocentric tactile cues, to track visited places. In addition,the decit in visual discrimination could be a factor con-tributing to their poor performance in the radial arm mazetask. Even though previous histological studies have indi-cated that the visual system is fairly well preserved in TMT-intoxicated rats (7,19), abnormalities in visual evokedpotentials have been found (29).

In fact, TMT-intoxicated rats were not impaired in theacquisition of lightdark discrimination task (49,122).Ishikawa et al . (49) reported that administration of 6 mg/ kg TMT did not affect the percent correct response duringthe acquisition or retention phase. In contrast, the rats given6 mg/kg TMT (as a free base) made more correct responsesin the reversed version of the same task (122). Thesendings were unexpected, since it is known that acquisitionof visual discrimination tasks may also be impaired after

surgical hippocampal lesions (see (122)).Interestingly, we found that TMT-intoxicated rats wereseverely impaired in the water maze task in the version thatshould not be dependent on spatial (relational) memory.Four previous studies (using TMT in a range of 67 mg/ kg as a free base) showed a clear impairment in theacquisition of hidden platform version (expressed asescape latency), but none of them reported a controlexperiment using a visible platform task (31,41,93,123).However, Hagan et al . (41) found that TMT-intoxicated(7 mg/kg as a free base per os ) and controls did not differ inthe visual discrimination version of the water maze task,even though it was more difcult for rats, whereas there wasa clear difference between the groups in the spatial versionof the water maze task. The lowest dose (5 mg/kg as a freebase) did not impair water maze learning (41). The dis-crepancies between the present results and those of Haganet al . on cued navigation could be related to the more severebrain damage in the present study.

It should be noted that blind rats are not profoundlyimpaired in the reference memory version of the Morriswater maze (53). Blind rats performed better than atropine-treated rats, although they were worse than controls. Inaddition, there was no clear difference between blind ratsand atropine-treated rats in a cued version of the watermaze. Furthermore, there were no differences betweencontrols, atropine-treated and blind rats in the straight

swim version of the water maze (53).One should consider possibilities other than primary dys-function in visual discrimination (decit in retino-geniculate-striate cortex transmission) in the interpretationof the present data on the water maze task. The behaviouraldecits of TMT-intoxicated rats found in the present studyresemble the behavioural abnormalities (hyperactivity,decits in grooming behaviour and in a swim-to-platformtask) observed in rats with p-chlorophenylalanine treatment(to deplete serotonin levels in the brain) combined with theadministration of a high dose of scopolamine, a muscarinicantagonist (110). As discussed by Vanderwolf, this syn-drome was akin to decits seen in decorticated animals, andit was more severe than the combined ablation of the

hippocampus and amygdala when this was assessed using



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the open-eld, swim-to-platform and Lashley III maze tests(28). It was postulated to result from a dysfunction in thecontrol of motor output by sensory cues, i.e behaviouralorganization, rather than loss of learning and memory, orprimary sensory dysfunction (110). Interestingly, it has beenreported that TMT-intoxicated rats have a modest reduction

in thelevelsof serotoninandmuscarinicbinding in thecerebralcortex (3,71,117), which could augment the functionalconsequences of neuronal loss in the cerebral cortex (7).

Taken as a whole, the present data suggest thatbehavioural dysfunctions (reduced habituation, reducedself-grooming, impairment in a swim-to-platform task,hyperactivity, impaired working and reference memory)induced by exposure to an intermediate to high dose of TMTappear to represent a model for global dementia rather thana specic decit in relational (declarative) memory. How-ever, the present data do not contradict the possibility thatexposure to a lower dose of TMT ( 6 mg/kg) could inducemorespecicassociationaldecitsdevoidof hyper-reactivity,especially when these are assessed with delay-dependentoperant tasks (14,22).

Inuence of atipamezole on the performance of control and TMT-intoxicated rats

The present results do not provide evidence that an a 2-antagonist, atipamezole, can improve mnemonic perfor-mance either in TMT-intoxicated or control rats. This lack

of efcacy in the controls cannot simply be attributed toinadequate dosing, because previous studies have indicatedthat the systemic administration of 300 mg/kg (used in thepresent study), but not 30 or 1000 mg/kg, atipamezoleenhanced the excitability of granular cells to the stimulationof the perforant path as reected in the increased size of thepopulation spike, this being unaccompanied by any signi-cant increase in the EPSP slope (126). In addition, this mosteffective dose of atipamezole, known to be sufcient toincrease the release of noradrenaline in the brain (92),improved the intermediate-term (6 h) retention of theradial arm maze task (delayed non-matching to positionversion) (126). Furthermore, 300 mg/kg atipamezoleimproved the acquisition of a linear arm maze task (40),and atipamezole (301000 mg/kg) improved the choice

TABLE 2The inuence of various drugs on the behavioural neurotoxicity of TMT

Rat strain TMT dose, administrationroute

Drug, administrationroute

Outcome measured Effect Reference

LE (m) 7 mg/kg TMT-Cl, p.o . d-amphetamine0.5 mg/kg, i.p .2.0 mg/kg4.0 mg/kgtested 5 weeks after TMT

open eld:activity score(crossing of grids)

3 hyperactivity in TMT:0.5 in TMT, in controls2.0 in TMT and in controls4.0 in TMT, in controls

(104)

LE (m) 6 mg/kg, TMT-base, p.o . Clonidine5 mg/kg, i.p .10 mg/kg,20 mg/kgtested 1926 days afterTMT

hole board task:hole visits,crossing scores,rearings

no effect in TMT,5 in controls TMT10 in controls, TMT20 in controls, tendency inTMT no effects on horizontalactivity and rearings

(58)

LE (m) 6 mg/kg TMT-base, p.o . DGAVP 1

7.5 mg/kg, s.c .tested 38 days after TMT

lever pressing innon-learners (a forwardautoshaping task)

TMT impaired acquisition,DGAVP improved learningin non-learners

(100)

SD (m) 8 mg/kg TMT-Cl, i.p . Tacrine3 mg/kg, i.p .started 1 week prior to TMT,post-behavioural testingadministration

PA, WM, LMA(started 3 weeks after TMT)

TMT impaired WM and PA,increased LMA;Tacrine improved WMoutcome in TMT, tendencyin PA and LMA outcomes

(73)

SD (m) 8 mg/kg TMT-Cl, i.p . THIP2

3 mg/kg, i.p.started 1 week prior to TMT

LMA, WM, PA(started 3 weeks after TMT) TMT impaired WM and PA,increased LMA;THIP improved WM in TMT,no effects on PA and LMAoutcomes

(72)

SD (m) 8 mg/kg TMT-Cl, i.p . PCP 3

5 mg/kg, i.p.Ketamine5 mg/kg( ) SKF 10,0475 mg/kgstarted 1 week prior to TMT

LMA, PA, WM(started 3 weeks after TMT,WM and passive avoidanceafter withdrawal of drug)

PCP: improved WM, LMAoutcomes , no effects on PAoutcome;ketamine and SKF 10,047:no effects on any behaviouraloutcome

(32)

SD (m) 8 mg/kg TMT-Cl, i.p . JO 17841 mg/kg, s.c .3 mg/kgstarted 1 week prior to TMT

LMA, PA, RAM(started 3 weeks after TMT)

TMT impaired PA and RAM,increased LMA;1: improved RAM in TMT, in controls and on

other outcomes3: LMA and improvedRAM and PA in TMT, in controls

(74)

1 DGAVP desglycinamide-8-arginine vasopressin; 2 THIP gaboxadol; 3 PCP phencyclidine; increased; no effect; decreased; LE LongEvans; LMA locomotor activity in open eld; m male; PA passive avoidance retention; RAM radial arm maze; SD SpragueDawley;WM water maze acquisition.



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accuracy of rats in the attentional task when the intensity of the visual stimuli was reduced (96). However, it should benoted that the working memory, as assessed by the delayednon-matching to position task in an operant chamber, andreference memory, as assessed by the water maze task, werenot improved by atipamezole (103000 mg/kg) in eitheradult or aged rats ((95,98) and unpublished data).

In fact, atipamezole increased the behavioural activity of control and TMT-intoxicated rats. This agrees with previousndings showing that acute administration of 3001000 mg/ kg increased premature responses (impulsivity) in an atten-tional task and the time to obtain a cumulative period of 20 min immobility in electrophysiological recordings(85,96,97). In addition, other specic a 2-antagonists havebeen found to increase locomotor and exploratory activity inthe open-arena task (27). The present data indicate thatTMT intoxication does not inuence the mechanisms bywhich atipamezole activates these behavioural functions.The likely candidates, i.e. activation of the locus coeruleusnoradrenergic neurons and subsequent indirect activation of dopaminergic neurons in the ventral tegmental area project-

ing to limbic neocortex and striatum (39), have not beendemonstrated to be affected by TMT intoxication (at thedose used in the present study) (7). Recent data indicate that300 mg/kg atipamezole could reduce scopolamine (0.5 mg/ kg)-induced ambulation in the open-arena task even thoughit slightly increased it when administered alone (69).

Taken together, the present data suggest that atipamezole,a selective and specic antagonist of a 2-adrenoceptors, didnot alleviate the behavioural disturbances in TMT-intoxicated rats, but did enhance the behavioural activityin rats, at least when they were in a familiar environment.

Previous studies on the alleviation or prevention of

behavioral neurotoxicity of TMT Only a few studies have investigated the inuence of

drugs on the behavioural symptoms of TMT-intoxicatedrats (Table 2). Swartzwelder et al . (104) found that TMTintoxication inuenced the doseresponse relationship of d-amphetamine on the locomotor activity of rats in theopen-eld task. Those ndings are in line with the resultsof recent studies showing that hippocampal lesions sensitizerats to the hyperactivity induced by d-amphetamine(62,116). Since the doseresponse relationship of d-amphet-amine is an inverted U-shape, Swartzwelder et al . found thatthe highest d-amphetamine dose reduced the locomotoractivity of TMT rats whereas this dose increased the

locomotor activity of controls. The authors made an inter-esting proposal that TMT rats could be considered as amodel for attention decit hyperactivity disorder (ADHD).However, Messing et al . (58) found that TMT rats tended tobe less sensitive to the sedative effects of clonidine, a partialagonist of a 2-adrenoceptors, which has been found toalleviate the symptoms of ADHD patients (46). The appar-ent discrepancy between the results of Swarztwelder et al .(104) and Messing et al . (58) could be due to a dose-dependent inuence of TMT on noradrenergic neurons.The medium dose increases b -adrenergic receptor binding(possibly due to loss of afferent input), whereas lower andhigher doses do not share this effect (59).

One study has investigated the inuence of a drug on the

learning of TMT-intoxicated rats. Sparber et al . (100)

reported that the systemic administration of a vasopressinanalogue improved the acquisition of a lever pressing task inthe subgroup of TMT rats which were non-learners for thetask.

Another line of research has investigated how the neuro-toxicity of TMT can be prevented by different kinds of compounds (Table 2). The results of OConnell, Earleyand their colleagues indicate that the systemic administra-tion of tacrine, PCP, THIP and JO 1784 can diminish thebehavioural outcomes of TMT intoxication. Interestingly,the decits in the water maze task and radial radial armmaze were alleviated whereas both the locomotor hyper-activity and passive avoidance retention were preventedonly with a higher dose of JO 1784. Neuroanatomical sub-strates for this behavioural selectivity (spatial learning vslocomotor activity) still have to be investigated. Themechanisms of neuroprotection could include the antagon-ism of NMDA receptors by tacrine and PCP. In addition,none of the studies has investigated whether post-intoxica-tion treatment could improve the outcome, possibly by pre-venting the progressive degeneration induced by a single

dose of TMT.

ACKNOWLEDGEMENTS

The authors thank Kirsi Puurunen for help in perfusionsand Nanna Huuskonen for histological assistance. EwenMacDonald is acknowledged for revising the language of the manuscript.

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