Pergamon

Neuroscience and Biobehavioral Reviews, Vol. 22, No. 6, pp. 735750, 1998 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0149-7634/98 $32.00 + .00

PII: S0149-7634(98)00002-5

Systemic Administration of Atipamezole, a Selective Antagonist of Alpha-2 Adrenoceptors, Facilitates Behavioural Activity but does not Inuence Short-term or Long-term Memory in Trimethyltin-intoxicated and Control Rats
M. NIITTYKOSKI a, R. LAPPALAINEN a, J. JOLKKONEN a, A. HAAPALINNA b, a ,* P. RIEKKINEN SR a , c AND J. SIRVIO
A.I. Virtanen Institute, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland b Orion 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
. Systemic NIITTYKOSKI M., R. LAPPALAINEN, J. JOLKKONEN, A. HAAPALINNA, P. RIEKKINEN Sr and J. SIRVIO administration of atipamezole, a selective antagonist of alpha-2 adrenoceptors, facilitates behavioural activity but does not inuence short-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 the limbic 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 nding was the severe decit in swimming to a visible platform in the water maze task, with reduced swimming speed at the beginning of the training period. During the reacquisition phase of a radial arm maze task, TMT-intoxicated rats made more short-term and long-term memory errors, and their behavioural activity was increased in comparison with controls. The administration of atipamezole (300 mg/kg), a selective antagonist of a 2adrenoceptors, enhanced locomotor activity compared to saline-treated rats, but these effects did not differ between the TMT group and their controls. Atipamezole did not enhance short-term or long-term memory in either TMT or control groups. Taken together, the present data indicate that TMT intoxication is a model for global dementia rather than for a specic loss of relational memory. Previous studies on the neurochemical effects of TMT and the alleviation or prevention of neurotoxicity of TMT are reviewed. 1998 Elsevier Science Ltd. All rights reserved Alzheimers disease a 2-adrenoceptors Hippocampus Hyperactivity Memory Rat Schizophrenia Trimethyltin
a

INTRODUCTION

THE HIPPOCAMPUS has reciprocal, multisynaptic connections with the cerebral cortex, and is considered to be involved in certain forms of associative learning and memory, especially the encoding phase of memory processing (101). In addition, the hippocampus and its output area, the subiculum, send projections to the nucleus accumbens, which may be important in sensori-motor control (63,119). Lesions of hippocampal formation and associated dysfunctions are known to occur, e.g., in Alzheimers disease (AD) (47,87,94,99,125) and schizophrenia (4,81). Intoxication with trimethyltin (TMT) leads to profound behavioural and cognitive decits in both humans (33) and

experimental animals (30). In rats, TMT induces the degeneration of pyramidal neurons in the hippocampus and cortical areas (pyriform cortex, entorhinal cortex, subiculum) connected to the hippocampus, but there is also neuronal loss in the association areas (7,13,19,20). Furthermore, behavioural studies have shown increased locomotor activity, disruption in self-grooming and learning decits in TMT-intoxicated rats (2,18,22,31,41,58,93,103,123). TMT intoxication impairs the acquisition of water maze and Biel maze (water avoidance) tasks as well as HebbWilliams maze and radial arm maze performance (2,31,41,48,72 74,93,103,113,123). In addition, TMT intoxication produces decits in passive avoidance retention, but not in the acquisition of the passive avoidance response

* 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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736 TABLE 1
The neurochemical effects of TMT Rat strain LAC:P (m) F344 (m) WI LAC:P (m) WI (m) WI (m) LAC:P (m) F344 (m) SD (m) LE (m f) LAC:P (m) WI LE (m f) WI (m) LE (m) SD (m) LE (m) WI (m) LE (m) LE (m) LE (m) LE (m) SD (m) SD (m) F344 (m) SD (m) LE (m) SD (m) SD (m) LAC:P (m) F344 (m) SD (m) SD (m) LE (m) LE (m) LAC:P (m) SD (m) SD (m) LE (m f) SD (m) SD (m) LE (m) F344 (m) LE (m) SD (m) LE (m) SD (m) LE (m f) LE (m) LE (m) LE (m) TMT dose, administration route 10 mg/kg TMT-Cl, i.g. 5.25 mg/kg TMT-Cl, s.c. 8 mg/kg TMT, p.o. 10 mg/kg TMT-Cl, i.g. 8 mg/kg TMT-Cl, i.p. 3 3 mg/kg /once a week TMT-Cl, i.p. 10 mg/kg TMT-Cl, i.g. 5.25 mg/kg TMT-Cl, s.c. 67 mg/kg TMT-Cl, i.p. 14 1 mg/kg/alternate days TMT-base, i.p. 10 mg/kg TMT-Cl, i.g. 8 mg/kg TMT, p.o. 14 1 mg/kg/alternate days TMT-base, i.p. 7 mg/kg TMT-base, p.o. 6 mg/kg TMT-base, p.o. 9 mg/kg TMT-OH, p.o. 6 mg/kg TMT-base, p.o. 3 3 mg/kg /once a week TMT-Cl, i.p. 7 mg/kg TMT-base, p.o. 6 mg/kg TMT-base, p.o. 6 mg/kg TMT-base, p.o. 6 mg/kg TMT-base, p.o. 8 mg/kg TMT-Cl, i.p. 8 mg/kg TMT-Cl, i.p. 5.25 mg/kg TMT-Cl, s.c. 8 mg/kg TMT-Cl, i.p. 7 mg/kg TMT-base, i.g. 68 mg/kg TMT-Cl, i.p. 8 mg/kg TMT-Cl, i.p. 10 mg/kg TMT-Cl, i.g. 5.25 mg/kg TMT-Cl, s.c. 8 mg/kg TMT-Cl, i.p. 68 mg/kg TMT-Cl, i.p. 7 mg/kg TMT-base, i.g. 7 mg/kg TMT-Cl, i.g. 10 mg/kg TMT-Cl, i.g. 8 mg/kg TMT-Cl, i.p. 67 mg/kg TMT-Cl, i.p. 14 1 mg/kg/alternate days TMT-OH, i.p. 8 mg/kg TMT-Cl, i.p. 68 mg/kg TMT-Cl, i.p. 6 mg/kg TMT-base, p.o. 5.25 mg/kg TMT-Cl, s.c. 7 mg/kg TMT-base, i.g. 8 mg/kg TMT-Cl, i.p. 7 mg/kg TMT-Cl, i.g. 68 mg/kg TMT-Cl, i.p. 14 1 mg/kg/alternate days TMT-OH, i.p. 7 mg/kg TMT-base, i.g. 7 mg /kg TMT-Cl, i.g. 7 mg/kg TMT-base, i.g. Effects, time from exposure

NIITTYKOSKI ET AL.

Reference (12) (117) (49) (12) (66) (66) (12) (117) (31) (55) (12) (49) (55) (41) (120) (48) (15) (66) (17) (16) (16) (16) (31) (71) (117) (3) (26) (31) (3) (12) (117) (3) (31) (26) (25) (12) (3) (31) (55) (3) (31) (59) (117) (26) (3) (25) (31) (55) (26) (25) (26)

GLU cd 1d; hip 2 d GLU hip, 1 w; frctx st GLU hip 4 w GLU binding hip cd sep ent, 2 d GLU uptake CA1 sub, 13 w GLU uptake CA1 sub, 45 w after rst injection GABA hip, 2 d GABA hip frctx 1 w; st GABA hip 22 d, am GABA hip 52 d after rst injection; brst cer hyp st frctx GABA A binding hip cd, 2 d ACh hip 4 w ACh Ch hyp st cortex hip, 52 d after rst injection AChE # DG, 46 w AChE # DG, 120130 d ChAT CA3 7 d; CA1 ChAT DG 360 d; CA1 760 d ChAT CA1 sub, 5 w after rst injection ChAT DG CA1 100120 d; cd M 2 binding CA1 1 d; CA3 3 d M 1 M 2 binding CA1 CA3c, 1 w M 1 binding CA1 CA3, 2 w M 1 M 2 binding sites hip, 22 d M 1 M 2 binding hip 25 d 5-HT frctx 1 w; hip st 5-HT hip frctx st cer, 1 w 5-HT am/py 1 w; st olf sep hip 5-HT hyp; 7 mg/kg hip; am midbr 5-HT hip st 12 w; cer; frctx 5-HIAA cd 12 d; hip 5-HIAA hip frctx st, 1 w 5-HIAA hip 12 w; frctx st cer 5-HIAA hip hyp am midbr 5-HIAA/5-HT st nac sep am/py hip olf, 1 w 5-HIAA/5-HT st sep 24 w; frctx NA hip, 12 d NA hip frctx 1 w; st cer NA am; hip midbr hyp NA brst cer, 52 d after rst injection NA hip 12 w; cer; frctx st b-adrenergic binding cerebral cortex, 22 d b-adrenergic binding am/py frctx, 4 w DA DOPAC frctx st, 1 w DA DOPAC nac 1 w; st sep am/py olf DA nac, 2 w DA nac 23 w; st frctx DA hip hyp am midbr DA st 52 d after rst injection; brst DOPAC/DA st nac sep am/py olf, 1 w DOPAC/DA st nac frctx, 24 w DA receptor binding st 7 d

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 nucleus accumbens; 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.

(31,32,54,73,74,112). Furthermore, decits in acquisition of active avoidance at the beginning of training have been reported (31). However, one study reported that there were no 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 on operant behaviour, since TMT-intoxicated rats had higher lever pressing rates under a xed-ratio schedule of food presentation (102) and TMT impaired the performance of differential reinforcement at low response rates in an

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737

operant schedule (123). These anatomical and behavioural ndings have made TMT-intoxicated rats an attractive model for degenerative diseases such as AD, the most common cause of dementia (121). It is not understood how TMT can cause a selective destruction of neurons, but it has been suggested that an excess activation of the N-methyl-D-aspartate (NMDA) type of glutamate receptors is involved (5). In TMT intoxication the uptake of glutamate is reduced (Table 1), and in vitro studies have shown that its efux has been elevated (24,67). Both effects may lead to overactivation of glutamate receptors. The selectivity of damage might be due to a protein named stannin, which is expressed in TMTsensitive neurons, e.g. in the hippocampus and entorhinal cortex (108), and this protein may also be involved in the apoptosis induced by TMT (107). TMT intoxication may induce other characteristics of AD than simply nerve cell apoptosis. The expression of a form of amyloid precursor protein (APP) called APP-751 mRNA was increased in CA1, but APP-695 mRNA levels were unchanged in the rat hippocampus 25 days after TMT intoxication (75). It has been suggested that there is a correlation between the increase in the APP-751/APP-695 mRNA ratio and the number of senile plaques in the hippocampus in Alzheimers patients (see (75)). Furthermore, a similarly increased ratio has been reported in nucleus basalis of behaviourally impaired aged rats (see (75)). However, following TMT intoxication, rats do not develop amyloid plaques (or neurobrillary tangles) (121). Gamma-amino butyric acid (GABA)-containing interneurons play an important role in the regulation of the excitability of principal cells, such as pyramidal neurons, in the forebrain. TMT intoxication can affect GABA-mediated neurotransmission (Table 1). The level of GABA was increased and GABA A receptor binding was decreased in the hippocampus soon after a large dose of TMT, whereas the levels of GABA were reduced after smaller doses and longer follow-up times (Table 1). Different functional types of GABAergic interneurons have been classied according to their neuropeptides and calcium-binding proteins (35). Recently, the effects of TMT on different hippocampal calcium-binding protein-positive neurons were studied with immunocytochemistry (37,38). Three weeks after TMT intoxication the number of calbindincontaining neurons was decreased, especially in the CA1 eld, but parvalbumin- and calretinin-containing interneurons were preserved (37,38). These ndings indicate that TMT intoxication affects one type of dendritic inhibitory cell, but not perisomatic inhibitory cells or interneuronselective inhibitory cells. Several neuropeptides are colocalized with GABA in interneurons (35). Recently, the effects of TMT on neuropeptides were studied (48,109). The expression of neuropeptide Y mRNA was increased in hilar interneurons and dentate granule cells 5 days after TMT intoxication, whereas the number of somatostatin mRNAcontaining neurons was decreased 16 days after the intoxication (109). In addition, the neuropeptide Y content was elevated in the entorhinal cortex 7 days after TMT intoxication (48). Neuropeptides, such as somatostatin, can also be compromised in AD (34). There are many studies into the effects of TMT intoxication on modulatory neurotransmitters such as acetylcholine and the monoamines (Table 1). TMT intoxication produces

axonal sprouting within the cholinergic septohippocampal system (e.g. (15,17)). This sprouting is an intriguing nding, since it parallels the consequences of the lesion of the entorhinal cortex, and is also a characteristic nding in AD (see (121)). However, the cholinergic neurons of the basal forebrain are not appreciably damaged by TMT (121), whereas there is a profound neocortical cholinergic decit in AD (34). However TMT does cause a loss of M 1 and M 2 muscarinic receptor subtypes in both the hippocampus and neocortex (Table 1, (71)). Several reports have described the effects of TMT on the serotonergic system. The levels of serotonin were decreased and those of its metabolite, 5-HIAA, were elevated, indicating that the utilization of serotonin is increased in several brain 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 the changes were not statistically signicant (12). However, the b-adrenergic receptor ligand binding was increased 4 weeks after intoxication (59). This could be an adaptive process following a reduction in levels of noradrenaline. Interestingly, Andersson et al. (3) found that serotonin and noradrenaline levels had recovered in the hippocampus when measured 12 weeks after TMT intoxication. The levels 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). The levels of serotonin and noradrenaline were decreased in the temporal and frontal cortex in AD, but the concentration of dopamine remained unchanged (34). The effects of TMT intoxication on the histaminergic system are not known, though this transmitter is compromised in AD (1). In the neocortex and hippocampus, synaptic transmission and plasticity are state dependent (6,10,44); see (56,118). One candidate neurotransmitter associated with this modulation is noradrenaline. Both inhibitory interneurons and principal neurons can be targets for noradrenergic synaptic terminals in the hippocampus (36,61). In vitro and in vivo physiological experiments have shown that noradrenaline can enhance the excitability of principal neurons in the forebrain (43,52,65,77,115). Furthermore, noradrenaline can enhance the induction of long-term potentiation produced by high frequency stimulation in the synapses of mossy bres on CA3 pyramidal neurons (45). Noradrenaline also modulates the long-term potentiation of mbria-CA3 synapses (50) as well as the activity of neocortical neurones (11). The release of noradrenaline is regulated by a 2-adrenergic autoreceptors which are located both in the soma and in the presynaptic terminals of the noradrenergic neurons (see (23)). The activation of those receptors inhibits the release of noradrenaline, and therefore antagonists of a 2-adrenoceptors promote the release of noradrenaline in the brain (92,106). Recently, studies have been undertaken to investigate the inuence of a 2-adrenergic agents on cognition in animals and humans (23). The systemic administration of idazoxan or atipamezole, selective antagonists of a 2adrenoceptors, has been found to enhance the excitability of granular neurons to the stimulation of the perforant path (90,126). The same doses were found to improve intermediate-term retention in the radial arm maze task (126) and memory retrieval in the linear arm maze task (91). In

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addition, atipamezole improved the choice accuracy of rats in an attentional task when the intensity of the visual stimuli was reduced (96). It could be speculated that the administration of a drug would direct the attention of rats towards pertinent cues during the sampling phase of the radial arm maze task, improving their intermediate-term retention or retrieval of learned material. Since our goal is to develop symptomatic treatment for dementia, it should be noted that none of the abovementioned studies investigated the inuence of a 2-antagonists on the performance of cognitively impaired rats such as those with lesions in the hippocampal formation and related brain areas, which leads to the isolation of the hippocampus from the neocortex. Therefore, the present study investigated whether a selective antagonist of a 2-adrenoceptors, atipamezole, could facilitate short-term or longterm memory in TMT-intoxicated rats, which exhibit neuronal damage in the limbic system. These memory functions were assessed with the radial arm maze task, using the version in which working memory-type errors (revisits to arms) and reference memory-type errors (visits to nonbaited arms) can be investigated. In addition to potential facilitation of neuronal transmission and plasticity by the increased activity of noradrenergic neurons (65,82,84,89), the enhancement in the release of acetylcholine in the neocortex by atipamezole (105) could be benecial in TMT rats, because these animals have reduced levels of muscarinic receptor binding in the cortex (71). The possible inuences of atipamezole on hyperactivity and the disruption of grooming behaviour in TMT-intoxicated rats were also considered to be valuable preclinical information, since the treatment of non-cognitive symptoms of AD, such as wandering 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 the open-eld task. Moreover, the inuence of atipamezole on hyperactivity produced by TMT was considered to be of special interest, since the symptoms of TMT intoxication resemble the behavioural syndrome induced by MK-801, a non-competitive antagonist of N-methyl-D-aspartate receptors (9,42,80,124), a drug used as a model for psychosis (76), and it has recently been suggested that some atypical neuroleptics can act as antagonists at a 2-adrenoceptors (70). Interestingly, atipamezole was found to reduce scopolamineinduced hyperactivity in rats (69). Before the drug trials were started, the performance of the TMT group was also tested in the water maze task which was used to assess sensori-motor control (by examining the ability of rats to swim to a visible platform).
MATERIALS AND METHODS

(R36, Lactamin, Stockholm, Sweden); subsequently they were maintained at approximately 8085% of their freefeeding body weight by limiting their food access to 14 16 g per day throughout the duration of radial arm maze tests. Behavioral testing was initiated after 1 week of restricted access to food. Water was available ad libitum. Pharmaceutical agents Trimethyltin chloride (TMT, Research Biochemicals Inc., USA) was dissolved in saline and injected intraperitoneally (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 free base) have been used to produce the behavioural syndrome of TMT intoxication in rats as well as to study the distribution of neuronal degeneration induced by TMT (e.g. (7,31,41,123)). Atipamezole (4-(2-ethyl-2,3-dihydro-1H-inden-2-yl)-1Himidazole) is a highly selective and specic a 2-adrenoceptor antagonist (92,111). In receptor binding studies, atipamezole is reported to have about 100 times higher afnity for a 2-adrenoceptors and an a 2/a 1 selectivity ratio over 100 times higher than that of idazoxan and yohimbine. In studies with isolated organs, atipamezole is a more potent a 2-adrenoceptor antagonist and has about 200 times higher relative a 2/a 1 blocking ratio than idazoxan (111). Atipamezole has an almost equal afnity for the different a 2adrenoceptor subtypes (88). Atipamezole penetrates rapidly into the brain (8), causing a dose-dependent increase in the release of central noradrenaline and serotonin (92). In the present study, atipamezole hydrochloride (Orion Pharma, Turku, Finland) was dissolved in saline and injected subcutaneously, 300 mg/kg (1.0 ml/kg). Behavioural experiments Radial arm maze task The 10-arm maze apparatus has been described previously (80,83). The rats were habituated to handling and then to the maze. In the rst day of maze habituation, food pellets (45 mg Precision pellets, Bio-Serv, NJ, USA) were in the centre of the arena and all arms were closed. In the second session, the rat had access to one arm, which was baited. The rats were allowed to explore the maze for 10 min on each testing session. Gradually, more arms were opened, and food pellets were located only at the end of every baited arm. Finally, the rats were able to search for 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 locations in the 10-arm maze. The pattern of baited arms was the same from day to day but could vary between animals (ve different congurations) in order to reduce the aid of olfactory cues left by a previous rat. The rat was put into the maze with every door open. When the rat had succeeded in obtaining all the food pellets, it was returned to its home cage. Maximum testing time was 10 min. The maze was cleaned between animals. A correct choice was recognized when the rat visited a food well of the baited arm. Long-term memory errors were assessed by the number of entries to non-baited arms, and short-term memory errors were assessed by the number of entries to previously visited arms during a trial. The number of arm entries per minute was taken as a

Animals Male Bkl Wistar rats (National Animal Center, University of Kuopio, n 40) were used (3 months old at the beginning of the experiment). Animals were housed and maintained on a 12:12 h lightdark cycle, and the room was 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, the rats (n 38) were housed singly, and their weight was reduced by gradually decreasing their amount of daily food

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

measure of locomotor activity. The number of correct choices before the rst error (a visit to an empty arm) was also calculated. The training was continued until rats showed a consistent choice accuracy above the chance levels (e.g. approximately 1.6 correct choices before the rst error, for which the chance level is 0.8). TMT administration After this initial training phase in the radial arm maze task, the rats were given a free-feeding period of 12 days and were then treated with TMT chloride (8 mg/kg, n 22) or saline (1 ml/kg, n 16). The rats were allowed to recover for 28 days. The rats were given food pellets, which were made as palatable as possible, inside their cages. If a rat refused to eat these pellets, it was provided with some chocolate, apple or banana according to its preference. The rats were given also saline intraperitoneally. In spite of this post-intoxication treatment, seven TMT-treated rats died 522 days after the administration of the toxin. The weight of the surviving rats was 91%, 83% and 91% of saline-treated rats on days 3, 10 and 24, respectively, after TMT administration. The TMT-intoxicated rats exhibited the signs of TMT syndrome, such as hyperreactivity, resistance 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 previously (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 TMT intoxication. The animals were placed in the open arena for a 9 min period (3 3 min sessions interrupted by a 10 s period to load the computer). The oor and walls of the open arena 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, the number of groomings and the number of fecal boli were counted 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 the non-spatial version (navigation cued by the visibility of the platform, the position of which was also altered trial by trial in order to minimize the contribution of spatial memory). However, the hidden platform version was not assessed, because TMT-intoxicated rats were severely impaired even when the platform was visible. The water maze apparatus has been described previously (69,86). The swim paths were monitored by a video camera linked to a computer through an image analyzer (HVS Image, UK). The computer software (made by HVS Image) calculated total 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 TMT intoxication. In the habituation trial, the rats were allowed to swim for 90 s in the pool, which did not contain the platform. Two days later, the rats were trained to nd a visible platform (top 1.5 cm above the water line), which was located in the south-west quadrant during the rst part of

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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 TMT administration. The results on the ambulation, rearings and groomings are expressed as group means SEM for each interday trial.

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 was moved to a different quadrant after each trial. Training was continued for 4 days (2 trials/day, maximum duration 60 s, 10 s reinforcement on the platform). If the rat failed to nd the platform within 60 s, it was put onto it for 10 s. Reacquisition of the radial arm maze task and open-arena testings after vehicle or atipamezole injections After water maze testing, the rats were returned to restricted feeding to reduce the weight to approximately 85% of weight controls. Then (68 days after the previous testing in the radial arm maze) the rats were retrained for the radial arm maze task for 5 days. During the last three days, all the rats were injected with saline (1 ml/kg, s.c.) to habituate them to the injections. The TMT-intoxicated rats and their controls were divided randomly to groups to be treated with either atipamezole hydrochloride (300 mg/kg, s.c., TMT: n 8 and controls: n 8) or saline (TMT: n 7 and controls: n 8) before daily testing either in the radial arm maze task or in the open-arena task (see schedule in Fig. 1). One TMTintoxicated rat treated with atipamezole died on the rst day of testing (about 1 h after atipamezole injection).1 Food intake In addition, food intake was measured. The animals were given more food than they normally consumed and the amount of food was calculated 24 h later. The baseline measurements were done 8 and 4 days before TMT intoxication, when the rats were free-feeding. To assess the effect of TMT intoxication, the food intake was measured 8 days after the TMT intoxication. The nal measurement investigated the effect of atipamezole on food intake (98 days after TMT administration, following the last test in the radial arm maze).

Histology Some rats from each group (two from salinesaline, one from salineatipamezole, two from TMTsaline, one from TMTatipamezole) were processed for histology. The rats were anaesthetized with MEBUNAT (sodium pentobarbital, 100 mg/kg) before perfusion. They were transcardially perfused with 0.9% saline followed by xative containing 4% paraformaldehyde, 0.05% glutaraldehyde and 0.2% picric acid in 0.1 m phosphate buffer (pH 7.4). After perfusion, the brains were removed from the skull, postxed in the perfusion xative for 2 h and placed in 20% glycerol in 0.02 m potassium phosphate buffered saline (pH 7.4) for 22 h. Then the brains were frozen on dry ice. Frozen sections (30 mm) were cut with a sliding microtome in the coronal plane and stored in 10% formalin in 0.1 m phosphate buffer (pH 7.4). The overall neuronal damage caused by TMT was estimated from Nissl stained sections. In addition, the cortical shrinkage was measured using 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 mm and 3.80 mm from the Bregma (79). Statistical analysis of data The 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 and groomings) were analysed using ANOVA for repeated measurements. The group effect in the number of fecal boli was analysed using ANOVA. The group effect, testing effect (training days) and their interactions in the data on water maze performance were analysed with ANOVA for repeated measurements. The group effect in the data on the retention of a radial arm maze task was also analysed using ANOVA. The group effect, treatment effect, testing effect (training blocks) and their interactions in the data on reacquisition of a radial arm maze task were analysed using ANOVA for repeated measurements. The data from two or three consecutive days were combined to blocks of trials. The feeding data were analysed with ANOVA.

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

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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) and variable position (B). The group means SEM for escape latency and distance as well as swimming speed are presented for each training day.

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 path length increased with time within the testing session (a signicant interaction between the group and testing effect, F(2, 58) 4.85, p 0.05) as shown in Fig. 2. The number of rearings did not differ between controls and TMT group (F(1, 29) 0.23, p 0.1), but there was a signicant interaction between group and testing effects (F(2, 58) 4.77, p 0.05). The number of groomings was decreased in TMT-intoxicated rats (F(1, 29) 22.92, p 0.001), whereas the number of fecal boli produced by TMT-intoxicated rats (6.1 1.1, mean SEM) was signicantly increased 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 constant position), TMT-intoxicated rats had signicantly longer escape latencies (F(1, 29) 400.3, p 0.001) and distances (F 109.7, p 0.001) than their controls, and their swimming 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 a different location in each trial), TMT-intoxicated rats had signicantly increased escape latencies (F(1, 29) 418.2, p 0.001) and distances (F 189.1, p 0.001). Swimming speed did not differ between controls and TMTintoxicated 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 the visited arms (11.17 1.54) than controls (n 16) (5.63 0.95, mean SEM; a signicant group effect, F(1, 29)

742

NIITTYKOSKI ET AL.

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), working memory 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: TMT atipamezole (TMT/ATI, n 7), TMTsaline (TMT/sal, n 7), salineatipamezole (sal/ATI, n 8), salinesaline (sal/sal, n 8).

9.64, p 0.01). Importantly, both groups performed close to a chance level in memory scores (e.g. 4.5 for the number of the visits to the non-baited arms and 0.8 for the number of correct choices before the rst error) even though these scores were signicantly better after the acquisition phase of this task before vehicle or TMT administration. This indicates that even the controls had forgotten the task before the tests 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 more incorrect arms (F 8.08, p 0.01) and made markedly more re-entries to visited arms than controls (F 148.66, p 0.001) during the reacquisition phase, as shown in Fig. 4. Atipamezole treatment did not inuence these parameters in either control or TMT groups (p 0.1 for all parameters and their interaction with the group effect, i.e. TMT vs controls) (Fig. 4). TMT-intoxicated rats made more arm entries in a given time than control rats (a signicant group effect, F(1, 26) 22.72, p 0.001) (Fig. 4). Atipamezole increased this kind of activity (a signicant treatment effect, F(1, 26) 8.30, p 0.01), but its inuence on behavioural activity did not differ between controls and TMT-intoxicated rats (a nonsignicant interaction between treatment and group effects, F(1, 26) 0.02, p 0.1). The inuence of atipamezole on behavioural activity became clearer as the testing progressed (a signicant interaction between treatment effect and 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 signicant group effect, F(1, 26) 68.82, p 0.001 in the third testing session), and this difference became clearer with time 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 slightly increased path length on the third testing, F(1, 26) 3.24, p 0.08 (whereas on the rst and second testings the treatment effect was not signicant, p 0.1), but this effect did not differ between the TMT group and their controls (a non-signicant interaction between treatment effect 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 became more prominent over time within each testing session (e.g. a signicant interaction between group effect and time effect in the third testing, F(2, 52) 3.58, p 0.05), since controls, but not TMT-intoxicated rats, made less rearings with time within a testing session. Atipamezole did not signicantly inuence the number of rearings in the TMT group or their controls (e.g. a non-signicant treatment effect, F 0.00, p 0.1, and interaction between treatment and group effects, F 0.44, p 0.1 in the third testing) (Fig. 5). The incidence of groomings was slightly lower in TMTintoxicated rats in comparison with their controls (a signicant group effect, F(1, 26) 7.22, p 0.05 in the third testing). The inuence of atipamezole and these interactions with the group effect was not signicant in any of the tests (p 0.1). The number of fecal boli was consistently higher in TMTintoxicated rats than in their controls, and atipamezole increased the number of fecal boli consistently in controls (Fig. 5). However, no signicant differences were found in any of the tests (p 0.05). Food intake Food intake during a 24 h period was equal in controls and TMT-intoxicated rats before TMT intoxication, as well as between controls and those TMT-intoxicated rats which survived when assessed 8 days after the administration of TMT (Fig. 6). When measured after the completion of the radial arm maze testing, including atipamezole treatments,

a 2-ANTAGONIST AND HIPPOCAMPAL FUNCTION

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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. The results show the group means SEM on the length of path, the number of rearings, the number of groomings and the number of fecal boli.

TMT-intoxicated rats consumed signicantly more food than controls (F(1, 29) 6.82, p 0.02), and this group difference was not affected by treatment (saline vs atipamezole) (F(1, 29) 0.18, p 0.1). In general, the amount of food consumed was higher than in the previous measurements, since the rats were on restricted feeding immediately before this last testing point. Histology The gross inspection of Nissl-stained sections revealed a severe neuronal loss following systemic injection of TMTchloride (8 mg/kg) at 112 days after intoxication. There was some selectivity in the neuronal loss. The pyramidal neurons in the hippocampal CA1 and CA3 area were particularly vulnerable (Fig. 7D). The neuronal loss was

also apparent in the cortical areas. Interestingly, the anterior neocortex seemed to be less severely affected than the posterior neocortex, as judged by the shrinkage of the cortex (Fig. 7B and D). Posterior spreading of cortical damage by TMT was also demonstrated by the almost complete disappearence of the entorhinal cortex.
DISCUSSION

Toxicity of TMT In the present study, approximately 30% of rats exposed to TMT died within 22 days despite receiving postoperative care. Dyer et al. (30) described mortality after TMT and noted that heavier animals were more susceptible to TMT toxicity than leaner animals; for example, 10% mortality

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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NIITTYKOSKI ET AL.

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) at two rostrocaudal levels. Note the severe neuronal loss in the hippocampus and cortical areas and enlargement of ventricles in the TMT-intoxicated rat in the posterior level (D). Magnication: 12.8; scale bar: 1 mm.

was reported in rats weighing about 440 g treated with 7 mg/kg TMT (as a free base (30)). Recently, Alessandri et al. (2) reported 40% mortality using the same dose but a different rat strain. The present death rate may be due to the high body weight (and age) of the animals being trained for the radial arm maze task before TMT administration. However, body weight prior to exposure did not differ between rats surviving (mean 417 g) and succumbing (mean 416 g). In addition, there are known to be differences between rat strains in their sensitivity to the neurotoxic effects of TMT (21,64). Histological examination of Nissl-stained sections at 16 weeks after intoxication indicated a severe atrophy in the

hippocampus and the cerebral cortex. Moreover, the posterior neocortex seemed to be more severely affected, which agrees with a previous study (7), although that study had shorter follow-up times than the present study. One study, using a relatively long follow-up time (13), employed a different kind of TMT administration schedule, but the effects of TMT on hippocampal and cortical shrinkage, as well as on enlargement of 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 maze tasks indicate that TMT intoxication caused persistent

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hyperactivity in the rats, which is in good agreement with previous studies (see (57)). For example, Swartzwelder et al. (102) found a more than 3-fold increase over controls in the open-eld activity of rats administered 7 mg/kg TMT-Cl when they were tested 40 days after exposure. More recently, Earley et al. (31) found that 8 mg/kg, but not 6 or 7 mg/kg, TMT-Cl increased ambulation approximately 2fold when it was measured 21 days after exposure. After intoxication with 8 mg/kg TMT-Cl, the increase in ambulation was approximately 2-fold at one month and 3-fold at two months after exposure. Hyperactivity may be a consequence of the damage to the hippocampus (60,68), possibly due to decits in adaptive responses to novelty against familiarity, and behavioural inhibition (51). Interestingly, the locomotor activity of TMT-intoxicated rats was less than that of controls in the habituation test of water maze. (Thus, in one environment TMT intoxication is associated with hyperactivity, whereas in another it reduces locomotor activity.) The previous studies (31,41,93,123) did not report data on the inuence of TMT intoxication on swimming speed (even though escape latency was used to express the acquisition of the water maze task). The reduced locomotor activity in a swimming pool could indicate an enhanced emotional response to novel environments. It should be noted that TMT-intoxicated rats produced more fecal boli in open-arena testing and this is considered as an index of emotional response. It is evident that TMT intoxication affects also the amygdala, which is known to be important in mediating emotions. In addition, the selfgrooming of TMT-intoxicated rats was disrupted in the open-arena task. These ndings are in good agreement with previous studies (18,30). The feeding data suggest that food restriction may increase the food intake of TMT-intoxicated rats more than in the controls. Thus, the effect of TMT-intoxication on food intake may be dependent on food restriction, because no effect of TMT was observed after eight days of ad libitum feeding. However, another possibility is that the inuence of TMT on food intake depends on the postintoxication time point. When the rats are receiving a restricted diet, the value of food could be higher in TMTintoxicated rats. This could partly explain their increased behavioural activity in the radial arm maze task. In addition, it is known that the state of satiety can inuence a rats performance in the open-eld task (51). Hyperactivity can result as a secondary consequence of the loss of spatial memory (114). Indeed, TMT-intoxicated rats exhibited an increased number of errors, indicative of a decit both in short-term and in long-term memory during the reacquisition phase of the radial arm maze task. The ndings on the reacquisition phase, especially the perseverating performance of TMT-intoxicated rats, agree with a previous study ((113), using TMT 6.0 mg/kg as a base), even though it should be recognized that the present study investigated relative contributions of both reference and working memory errors. Moreover, TMT-intoxicated rats made more working memory errors in an unbaited 6-arm radial tunnel maze (2). Swartzwelder et al. (103) found a profound decit akin to perseverative behaviour in rats after 7.0 mg/kg TMT-Cl when they were tested in a series of HebbWilliams maze problems. The decit could be the result 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 perseveration in the radial arm maze task and reduced habituation in the open-arena task, since it is possible that a rat uses its own olfactory cues, in addition to allocentric visual and egocentric tactile cues, to track visited places. In addition, the decit in visual discrimination could be a factor contributing to their poor performance in the radial arm maze task. Even though previous histological studies have indicated that the visual system is fairly well preserved in TMTintoxicated rats (7,19), abnormalities in visual evoked potentials have been found (29). In fact, TMT-intoxicated rats were not impaired in the acquisition 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 during the acquisition or retention phase. In contrast, the rats given 6 mg/kg TMT (as a free base) made more correct responses in the reversed version of the same task (122). These ndings were unexpected, since it is known that acquisition of visual discrimination tasks may also be impaired after surgical hippocampal lesions (see (122)). Interestingly, we found that TMT-intoxicated rats were severely impaired in the water maze task in the version that should 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 the acquisition of hidden platform version (expressed as escape latency), but none of them reported a control experiment 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 in the visual discrimination version of the water maze task, even though it was more difcult for rats, whereas there was a clear difference between the groups in the spatial version of the water maze task. The lowest dose (5 mg/kg as a free base) did not impair water maze learning (41). The discrepancies between the present results and those of Hagan et al. on cued navigation could be related to the more severe brain damage in the present study. It should be noted that blind rats are not profoundly impaired in the reference memory version of the Morris water maze (53). Blind rats performed better than atropinetreated rats, although they were worse than controls. In addition, there was no clear difference between blind rats and atropine-treated rats in a cued version of the water maze. Furthermore, there were no differences between controls, atropine-treated and blind rats in the straight swim version of the water maze (53). One should consider possibilities other than primary dysfunction in visual discrimination (decit in retinogeniculate-striate cortex transmission) in the interpretation of the present data on the water maze task. The behavioural decits of TMT-intoxicated rats found in the present study resemble the behavioural abnormalities (hyperactivity, decits in grooming behaviour and in a swim-to-platform task) observed in rats with p-chlorophenylalanine treatment (to deplete serotonin levels in the brain) combined with the administration of a high dose of scopolamine, a muscarinic antagonist (110). As discussed by Vanderwolf, this syndrome was akin to decits seen in decorticated animals, and it was more severe than the combined ablation of the hippocampus and amygdala when this was assessed using

746 TABLE 2
The inuence of various drugs on the behavioural neurotoxicity of TMT Rat strain TMT dose, administration route LE (m) 7 mg/kg TMT-Cl, p.o. Drug, administration route d-amphetamine 0.5 mg/kg, i.p. 2.0 mg/kg 4.0 mg/kg tested 5 weeks after TMT Clonidine 5 mg/kg, i.p. 10 mg/kg, 20 mg/kg tested 1926 days after TMT DGAVP 1 7.5 mg/kg, s.c. tested 38 days after TMT Tacrine 3 mg/kg, i.p. started 1 week prior to TMT, post-behavioural testing administration THIP 2 3 mg/kg, i.p. started 1 week prior to TMT PCP 3 5 mg/kg, i.p. Ketamine 5 mg/kg ( ) SKF 10,047 5 mg/kg started 1 week prior to TMT JO 1784 1 mg/kg, s.c. 3 mg/kg started 1 week prior to TMT Outcome measured Effect

NIITTYKOSKI ET AL.

Reference

open eld: activity score (crossing of grids) hole board task: hole visits, crossing scores, rearings lever pressing in non-learners (a forward autoshaping task) PA, WM, LMA (started 3 weeks after TMT)

3 hyperactivity in TMT: 0.5 in TMT, in controls 2.0 in TMT and in controls 4.0 in TMT, in controls no effect in TMT, 5 in controls TMT 10 in controls, TMT 20 in controls, tendency in TMT no effects on horizontal activity and rearings TMT impaired acquisition, DGAVP improved learning in non-learners TMT impaired WM and PA, increased LMA; Tacrine improved WM outcome in TMT, tendency in PA and LMA outcomes TMT impaired WM and PA, increased LMA; THIP improved WM in TMT, no effects on PA and LMA outcomes PCP: improved WM, LMA outcomes , no effects on PA outcome; ketamine and SKF 10,047: no effects on any behavioural outcome TMT impaired PA and RAM, increased LMA; 1: improved RAM in TMT, in controls and on other outcomes 3: LMA and improved RAM and PA in TMT, in controls

(104)

LE (m)

6 mg/kg, TMT-base, p.o.

(58)

LE (m) SD (m)

6 mg/kg TMT-base, p.o. 8 mg/kg TMT-Cl, i.p.

(100) (73)

SD (m)

8 mg/kg TMT-Cl, i.p.

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

(72)

SD (m)

8 mg/kg TMT-Cl, i.p.

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

(32)

SD (m)

8 mg/kg TMT-Cl, i.p.

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

(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.

the open-eld, swim-to-platform and Lashley III maze tests (28). It was postulated to result from a dysfunction in the control of motor output by sensory cues, i.e behavioural organization, rather than loss of learning and memory, or primary sensory dysfunction (110). Interestingly, it has been reported that TMT-intoxicated rats have a modest reduction in the levels of serotonin and muscarinic binding in the cerebral cortex (3,71,117), which could augment the functional consequences of neuronal loss in the cerebral cortex (7). Taken as a whole, the present data suggest that behavioural dysfunctions (reduced habituation, reduced self-grooming, impairment in a swim-to-platform task, hyperactivity, impaired working and reference memory) induced by exposure to an intermediate to high dose of TMT appear to represent a model for global dementia rather than a specic decit in relational (declarative) memory. However, the present data do not contradict the possibility that exposure to a lower dose of TMT ( 6 mg/kg) could induce more specic associational decits devoid of hyper-reactivity, especially when these are assessed with delay-dependent operant 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 2antagonist, atipamezole, can improve mnemonic performance either in TMT-intoxicated or control rats. This lack of efcacy in the controls cannot simply be attributed to inadequate dosing, because previous studies have indicated that the systemic administration of 300 mg/kg (used in the present study), but not 30 or 1000 mg/kg, atipamezole enhanced the excitability of granular cells to the stimulation of the perforant path as reected in the increased size of the population spike, this being unaccompanied by any signicant increase in the EPSP slope (126). In addition, this most effective dose of atipamezole, known to be sufcient to increase the release of noradrenaline in the brain (92), improved the intermediate-term (6 h) retention of the radial arm maze task (delayed non-matching to position version) (126). Furthermore, 300 mg/kg atipamezole improved the acquisition of a linear arm maze task (40), and atipamezole (301000 mg/kg) improved the choice

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747

accuracy of rats in the attentional task when the intensity of the visual stimuli was reduced (96). However, it should be noted that the working memory, as assessed by the delayed non-matching to position task in an operant chamber, and reference memory, as assessed by the water maze task, were not improved by atipamezole (103000 mg/kg) in either adult or aged rats ((95,98) and unpublished data). In fact, atipamezole increased the behavioural activity of control and TMT-intoxicated rats. This agrees with previous ndings showing that acute administration of 3001000 mg/ kg increased premature responses (impulsivity) in an attentional 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 have been found to increase locomotor and exploratory activity in the open-arena task (27). The present data indicate that TMT intoxication does not inuence the mechanisms by which atipamezole activates these behavioural functions. The likely candidates, i.e. activation of the locus coeruleus noradrenergic neurons and subsequent indirect activation of dopaminergic neurons in the ventral tegmental area projecting to limbic neocortex and striatum (39), have not been demonstrated to be affected by TMT intoxication (at the dose used in the present study) (7). Recent data indicate that 300 mg/kg atipamezole could reduce scopolamine (0.5 mg/ kg)-induced ambulation in the open-arena task even though it slightly increased it when administered alone (69). Taken together, the present data suggest that atipamezole, a selective and specic antagonist of a 2-adrenoceptors, did not alleviate the behavioural disturbances in TMTintoxicated rats, but did enhance the behavioural activity in 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-intoxicated rats (Table 2). Swartzwelder et al. (104) found that TMT intoxication inuenced the doseresponse relationship of d-amphetamine on the locomotor activity of rats in the open-eld task. Those ndings are in line with the results of recent studies showing that hippocampal lesions sensitize rats to the hyperactivity induced by d-amphetamine (62,116). Since the doseresponse relationship of d-amphetamine is an inverted U-shape, Swartzwelder et al. found that the highest d-amphetamine dose reduced the locomotor activity of TMT rats whereas this dose increased the locomotor activity of controls. The authors made an interesting proposal that TMT rats could be considered as a model for attention decit hyperactivity disorder (ADHD). However, Messing et al. (58) found that TMT rats tended to be less sensitive to the sedative effects of clonidine, a partial agonist of a 2-adrenoceptors, which has been found to alleviate the symptoms of ADHD patients (46). The apparent discrepancy between the results of Swarztwelder et al. (104) and Messing et al. (58) could be due to a dosedependent inuence of TMT on noradrenergic neurons. The medium dose increases b-adrenergic receptor binding (possibly due to loss of afferent input), whereas lower and higher 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 vasopressin analogue improved the acquisition of a lever pressing task in the subgroup of TMT rats which were non-learners for the task. Another line of research has investigated how the neurotoxicity of TMT can be prevented by different kinds of compounds (Table 2). The results of OConnell, Earley and their colleagues indicate that the systemic administration of tacrine, PCP, THIP and JO 1784 can diminish the behavioural outcomes of TMT intoxication. Interestingly, the decits in the water maze task and radial radial arm maze were alleviated whereas both the locomotor hyperactivity and passive avoidance retention were prevented only with a higher dose of JO 1784. Neuroanatomical substrates for this behavioural selectivity (spatial learning vs locomotor activity) still have to be investigated. The mechanisms of neuroprotection could include the antagonism of NMDA receptors by tacrine and PCP. In addition, none of the studies has investigated whether post-intoxication treatment could improve the outcome, possibly by preventing the progressive degeneration induced by a single dose of TMT.
ACKNOWLEDGEMENTS

The authors thank Kirsi Puurunen for help in perfusions and Nanna Huuskonen for histological assistance. Ewen MacDonald is acknowledged for revising the language of the manuscript. REFERENCES
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