Introduction ORIGINAL INVESTIGATION. N. G. Muggleton A. P. Bowditch H. S. Crofts E. A. M. Scott P. C. Pearce

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1 Psychopharmacology (2003) 166: DOI /s ORIGINAL INVESTIGATION N. G. Muggleton A. P. Bowditch H. S. Crofts E. A. M. Scott P. C. Pearce Assessment of a combination of physostigmine and scopolamine as pretreatment against the behavioural effects of organophosphates in the common marmoset (Callithrix jacchus) Received: 10 March 2002 / Accepted: 22 October 2002 / Published online: 13 February 2003 Springer-Verlag 2003 N. G. Muggleton H. S. Crofts Psychopharmacology Unit, School of Medical Sciences, University Walk, Bristol, BS8 1TD, UK A. P. Bowditch E. A. M. Scott P. C. Pearce DSTL Biomedical Sciences, Porton Down, Salisbury, Wiltshire, SP4 0JQ, UK N. G. Muggleton () ) Department of Experimental Psychology, University of Oxford, South Parks Road, Oxford, OX1 3UD, UK neil.muggleton@psy.ox.ac.uk H. S. Crofts Department of Experimental Psychology, University of Cambridge, Downing Street, Cambridge, CB2 3EB, UK Abstract Rationale: There is a requirement to ensure that UK armed forces are provided with the best possible medical countermeasures to prevent or mitigate the effects of exposure to nerve agents. When pretreatments are under consideration, it is of particular importance to ensure that they do not in themselves give rise to adverse effects and do not exacerbate the effects of agent exposure. Objectives: The present study was designed to address these considerations for a combination of physostigmine and scopolamine as a potential pretreatment regimen. Methods: Common marmosets were trained to perform a two-choice discrimination serial reversal task, and baseline data were collected. Subjects received a dose of either soman or sarin after 2 weeks of pretreatment with either saline or physostigmine and scopolamine via miniosmotic pump. Results: No effects of physostigmine and scopolamine were seen on task accuracy or response rates. Neither accuracy of reversal performance nor number of responses made were significantly changed by administration of either soman or sarin subsequent to pretreatment with physostigmine/scopolamine. In the groups pretreated with saline, performance of the behavioural task, in terms of responses made, was virtually abolished on the day the OP was administered, but a significant increase in accuracy of performance was seen over the 2- to 14-day period following administration. Conclusions: A combination of physostigmine and scopolamine, which is known to protect against nerve-agent lethality, offers protection against the effects of soman and sarin on behavioural performance, as measured by a discrimination reversal task. The improved performance observed following nerve agent requires further investigation. Keywords Organophosphate Marmoset Discrimination Reversal Soman Sarin Physostigmine Scopolamine Pretreatment Introduction Nerve agents continue to pose a threat to UK armed forces and there is, in consequence, a requirement to develop medical countermeasures that are effective, acceptable and practicable. These organophosphate (OP) compounds cause rapid and irreversible inhibition of acetylcholinesterase (AChE), leading to an increase in acetylcholine (ACh) at both central and peripheral nerve synapses, potentially resulting in incapacitation and death. Currently, UK armed forces are issued with a nerve agent pretreatment set (NAPS) containing a reversible cholinesterase inhibitor, the quaternary carbamate pyridostigmine bromide (PB), in tablet form (30 g to be taken once every 8 h). When supported by post-poisoning therapy, PB pretreatment has been shown to provide protection against the lethal effects of soman in both guinea-pigs (Leadbeater et al. 1985) and non-human primates (Dirnhuber et al.1979) but was found to be less effective at preventing incapacitation. Comparative studies have demonstrated the relative benefits of the tertiary carbamate physostigmine as pretreatment, which, due to its ability to penetrate the central nervous system (CNS), is better able to protect against the incapacitating effects of OP exposure (Harris et al. 1991). Inclusion of the cholinergic antagonist scopolamine with the physostigmine pretreatment confers additional benefits, protecting against both lethality and incapacitation in guinea-pigs (Leadbeater et al. 1985; Harris et al. 1991; Lennox et al.

2 1992) and rhesus monkeys (von Bredow et al. 1991) as well as preventing the neurotransmitter changes produced by such poisoning in the guinea-pig (Fosbraey et al. 1990). For both operational reasons and to address duty of care issues it is of critical importance that, when administering pretreatment compounds or combinations of compounds, they are not in themselves incapacitating. The in-service pretreatment of PB is lipophobic and, as such, is not readily able to cross the blood brain barrier. The candidate replacement pretreatment comprises two drugs with central effects and therefore the possibility of exacerbation of nerve-agent effects due to the presence of a centrally acting cholinomimetic requires investigation. Cholinergic antagonists are known to produce impairments in aspects of memory function, such as disrupting short-term visual recognition memory in humans (Robbins et al. 1997) as well as causing cognitive deficits in experimental animals, atropine and scopolamine being the most commonly used pharmacological interventions to simulate memory impairments (Blokland 1996). At doses higher than those proposed for incorporation in pretreatment regimens, scopolamine has been shown to affect performance on a range of cognitive tasks in rhesus monkeys (Aigner et al. 1987; Taffe et al.1999), and Ridley et al. (1984) showed that administration of scopolamine resulted in both impaired acquisition of object reward associations and impairments in transfer of that association to long-term memory in marmosets. Such object discrimination impairments in marmosets have been shown to be reversible by cholinergic agonists, such as arecoline (Carey et al. 1992). Additionally, various lesions of the marmoset central cholinergic system have also been shown to affect behavioural performance. Ibotenic acid lesions of the basal forebrain in marmosets, causing decreased activity of choline acetyltransferase (ChAT), the enzyme responsible for ACh production in nerve terminals, has been shown to impair ability to perform rule reversals of two-choice discrimination tasks (Ridley et al. 1985; Roberts et al. 1990). The Ridley et al. (1985) study showed that a low dose of scopolamine, albeit still higher than likely pretreatment doses, which did not affect controls, increased the impairments in lesioned subjects. The present study employed a discrimination reversal paradigm presented on a touch-sensitive screen, similar to that used by Roberts et al. (1992), which was presented to marmosets in their home room (Crofts et al. 1995; Crofts 1998). Reversal learning requires subjects, having been trained to associate one of a pair of stimuli with reward, to switch responding to the previously unrewarded stimulus. This is thought to be a two-stage process where a subject has to first inhibit responding to the previously rewarded stimulus then form a new stimulus reward association. Performance can be affected by problems with one or both of these processes. Scopolamine may be expected to disrupt performance on this task, in a manner similar to that seen following basal forebrain lesions, possibly by disruption of formation of an association with the stimulus rewarded following a rule change. Ridley et al. (1984) found impairments in discrimination learning with cholinergic blockade. Cholinergic agonists might be expected to improve performance by an opposite mechanism, although it is conceivable that overprocessing of stimulus attributes could lead to impaired performance (Sarter 1994). The relative doses of the compounds will affect the likelihood of effects being seen. The present study aimed to determine whether continuous administration of a combination of physostigmine and scopolamine over 14 days: (a) gives rise to significant changes in performance of a serial discrimination reversal task and (b) protects against effects of the OP compounds sarin or soman on such a task. As protection against nerve agent lethality has previously been shown (Dirnhuber et al. 1979; Leadbeater et al. 1985), the doses of nerve agent for the present study were selected on the basis that they would be expected to induce minor transient signs of poisoning (and so be mildly incapacitating) but not result in loss of posture or death. This would therefore allow the efficacy of the pretreatment regime under study to protect against behavioural incapacitation to be assessed. Materials and methods All procedures were carried out under a project licence granted under the United Kingdom Animals (Scientific Procedures) Act Animals Subjects Eighteen experimentally naive adult common marmosets (Callithrix jacchus) were used (eight males and ten females), aged between 1 year and 6 years and weighing g at the start of the study. All animals were bred on site at Dstl Porton Down. Outside periods of training or testing, animals were housed either singly or in mixed sex pairs, in which the males were vasectomised, in two linked H72 W47 D60-cm cage units per animal. During test sessions, which took place between 0900 hours and 1300 hours, Monday to Friday, all animals were housed separately in one of the single cage units from their home cage. The home room was lit on a 12-h/12-h light/dark cycle with 30-min dawn and dusk periods and daylight levels of lux at 1 m above the floor. Room temperature was maintained at 25C. Animals were fed 20 g primate pellets (Special Diet Services, Witham, Essex, UK) each day with a supplement of fruit (orange) on days when testing took place. In addition 1 2 tablespoons of forage mix consisting of Rice Krispies, raisins and sunflower seeds were given daily in the animals forage boxes prior to behavioural testing (approximately 0815 hours). On non-experimental days, this diet was supplemented with apple and banana. Animals had free access to drinking water at all times. Behavioural methods Apparatus 213 The behavioural testing apparatus (CeNeS Limited, Cambridgeshire, UK) consisted of a high-resolution colour video display unit (VDU) fitted with an Intasolve Microvitec touchsensitive screen. An IBM compatible personal computer (PC)

3 214 connected to the VDU generated the discriminanda, in addition to controlling contingencies, recording response position, latency and accuracy. The touch screen was installed on a moveable platform to allow positioning adjacent to a rigid cage extension (dimensions L30 W17 H18 cm) attached to the front of the marmosets home cage. The mesh of this cage extension was cm, sufficient to allow the subjects to easily reach through and touch the VDU. Task reward for correct responses consisted of cold bananaflavoured milkshake (Nesquik, NestlØ), delivered via a licking spout which was positioned centrally with respect to the touch screen. A noise generator, which produced a 4-kHz, 60-dB tone, was positioned to the left of the VDU and sounded for 1 s to indicate the availability of reward. Licking at the metal spout was recorded by the interruption of a photocell beam across the spout, which in turn triggered a peristaltic pump to deliver the reward via the spout. Drugs Physostigmine salicylate (BDH) and scopolamine hydrobromide (Sigma) were diluted using 0.9% saline to give osmotic pump delivery rates of mg kg day -1 and 27 mg kg day Ÿ1, respectively. Isopropyl methylphosphonofluoridate (sarin) and 1,2,2-trimethylpropylmethylphosphonofluoridate (soman) were synthesised at Porton Down to 95% purity and were stored as 5.0 mg/ml solutions in isopropanol. Immediately prior to administration, both compounds were diluted to mg kg Ÿ1 for sarin and 4.5 mg kg Ÿ1 for soman using 0.9% saline in an injection volume of no more than 0.5 ml. These sublethal doses were selected on the basis of previous studies (D Mello and Duffy 1985) and were expected to be equipotent. Touch screen and discrimination training Animals were trained to respond to the touch screen for banana milkshake reward using methods described elsewhere (Pearce et al. 1998). Initially, animals were trained to perform a series of twochoice discrimination and reversal tasks involving simple stimulus pairs (blue filled shapes or white lines) and compound stimulus pairs (lines superimposed on shapes). Each animal had to complete a discrimination of each type for both a pair of shape stimuli and a pair of line stimuli. Stimuli were presented simultaneously and randomly on either side of the screen and, in the case of compound stimuli, line stimuli were superimposed over the shapes such that the configurations presented were randomised (Roberts et al. 1988; Pearce et al. 1998; Crofts et al. 1999). Once a response to either stimulus was made, both disappeared from the screen. A correct response resulted in the sounding of the 1-s tone, signalling the availability of reinforcement for 5 s (approximately 0.1 ml). Following an incorrect response, instead a 1-s time out was instigated. A criterion of 18 correct responses in 20 successive trials was used to determine that the discrimination had been learned. All trials were separated by an inter-trial interval of 3 s. Training sessions lasted for 15 min or 60 trials, whichever was reached first. The test sequence described below was presented on completion of training. These training stages served to ensure animals had been exposed to a broad range of discrimination tasks prior to entering the main part of the study. Test sequence The test sequence consisted of a series of compound reversals in which all the animals were required to respond to the shape element of the compound stimuli. No line relevant discriminations were used as it has previously been shown that the effects of basal forebrain lesions in the marmoset (which reduced cholinergic markers) on repeated line discrimination reversals differed from the effects on shape reversals (Roberts et al. 1992). Following attainment of criterion (18 of 20 correct as during training) on an initial simple discrimination (SD) between a pair of shapes, a compound discrimination (CD) followed in which a pair of lines was superimposed on the shapes and subjects were required to continue responding to the stimulus rewarded in the SD. On each occasion that the criterion was attained, the reward contingencies of the two shape stimuli were reversed such that a series of compound reversals was presented. A single pair of stimuli was used with counterbalancing for initial correct shape at the start of the test sequence to allow for any effect of an initial bias towards one particular stimulus. Subjects were required to complete at least ten reversals before entering the study, although most subjects performed more than this. This meant that minimal effects due to individual stimuli would be expected due to the well practised nature of the task. Two sessions each of 15-min duration or consisting of 60 trials each were presented each day, and subsequent sessions began with the last discrimination reached, but not passed, during the prior testing session. Study design The study phase of the experiment was divided into three blocks, a control phase (days 0 12), a pump phase (days 13 25) and a dose phase (days 26 37). Successive discrimination reversals were presented Monday Friday throughout the study phase with the exception of the day of pump implantation. On the days preceding pump implantation and OP administration, subjects were required to complete a reversal such that a new one was presented on the first day of each of the three phases. As such, on days immediately prior to the start of the pump and dose phases, sessions were set to terminate upon completion of a stage if it was considered unlikely that another stage could be completed prior to the start of the next phase of the experiment. Such animals were not tested for any remaining sessions of that phase. Animals received an injection (0.2 saline, i.m.) on the first day of behavioural testing of the control phase (day 0) and of the pump phase (day 14), to act as procedural controls for the injection of sarin or soman on day 26. Animals were assigned to experimental groups pretreated with either physostigmine and scopolamine (n=10) or with saline (n=8). Of these 18 subjects, 10 received either soman (n=5 saline pretreated, n=5 physostigmine/scopolamine pretreated) and 8 received sarin (n=3 saline pretreated, n=5 physostigmine scopolamine). Where possible subjects were balanced for gender, housing (single or pair) and reversal performance immediately prior to commencement of the baseline period. Osmotic pump implantation Osmotic pumps (type 2002, Alzet Corp., CA, USA) were implanted subcutaneously in each subject on day 13 to allow simultaneous administration of either physostigmine and scopolamine or saline alone. The delivery rate of the pumps was 0.48 ml h Ÿ1 and the pumps in the saline group were filled with 0.9% saline solution. The pumps used delivered their contents over a 14-day period. Pumps were filled and implanted under sterile conditions. Animals were anaesthetised with a combination of medazolam [10 mg kg Ÿ1 i.m.; (Roche) and ketamine (10 mg kg Ÿ1 i.m.; Ketalar (Parke-Davis)]. A small area of hair was shaved from the dorsal surface, the skin was prepared for surgery and a single small incision was made to one side of the midline just posterior to the shoulder blades. The skin was separated from the underlying fascia/ muscle layer to make a pocket, into which the pump was inserted. No behavioural testing took place on the day of pump implantation. Control and OP injections Injections of 0.5 ml kg Ÿ1 0.9% saline i.m. were given on day 0 and day 14, 1 h prior to testing. These injections acted as procedural controls for the OP injection, which took place 1 h prior to

4 behavioural testing on day 26. Following OP administration, behaviour was monitored and any cholinergic side effects were noted. OP doses were administered in a volume similar to the saline control injections. Statistical analysis Rate of responding Analysis was conducted using the SPSS for Windows package. The number of responses made on the task each day was analysed using multivariate analysis of variance (MANOVA) with dependant variables of the change in number of responses from the mean baseline value for each day of testing and with factors of pump type (saline or physostigmine/scopolamine) and dose (sarin or soman). SPSS also provided an output of univariate ANOVA effects, which assessed the effects of pump type and dose for each day. Days included in the analysis were those where all subjects completed full behavioural sessions. Days where such data were not available included weekends (when no testing occurred) and the day of pump implantation. Additionally, days at the end of experimental phases were omitted if any subjects had passed a rule reversal and were not being tested because it was judged unlikely they would complete another before the start of the next phase. Bartlett s test was used to check for homogeneity of variance and no data transformation was necessary. Post-hoc analysis was carried out on the mean differences between groups for each group and for each day subsequent to administration using the method of least significant difference at a confidence interval of 95%. Serial reversal performance Reversal performance was analysed on the basis of the number of errors made before the criterion of 18 of 20 correct successive responses was achieved for each reversal. The mean number of errors made per reversal in each phase (control, pump and dose) was calculated for each subject and these values were analysed. A mixed-model ANOVA and was carried out on the mean errors made per reversal during the control, pump and dose phases, with one between-subject factor of pump type (physostigmine/ scopolamine or saline) and one within-subject factor of experimental phase (control, pump, dose). Analysis was carried out separately on those groups receiving sarin and soman. Mean errors per reversal was used as a measure because of the different number of reversals performed by each subject during the three experimental stages which precluded the time-based approach used for analysis of response rates. Examination of the residuals for normality using Chi square at a probability level of 0.05 showed mean error distribution to be normal. Bartlett s test was used to check for homogeneity of variance and no data transformation was necessary. Post-hoc tests were carried out using the method of least significant difference at a confidence interval of 95%. Response latencies were not analysed as these showed a large degree of variation both within and between subjects, possibly reflecting that, in a home cage test environment, animals may often not be facing the screen when the stimuli first appear (Crofts 1998). Results Behavioural observations following OP dose Animals were observed continually for the first 30 min subsequent to OP administration, then at 15-min intervals until 3 h after dosing. Irrespective of pretreatment, both groups exhibited mild signs of cholinergic poisoning following administration of sarin. Typically these included transient salivation, ataxia, piloerection and tail arching. Onset of signs took 5 10 min in the physostigmine/scopolamine-pretreated group compared with min in the saline-pretreated group. Signs in the physostigmine/scopolamine group appeared to be milder in nature and were no longer evident min following OP administration relative to min in the salinepretreated group. The pattern and time course of clinical signs following administration of soman were similar to those seen for sarin, although signs were more marked (more profuse salivation and a higher degree of ataxia). As with sarin, signs were faster in onset, milder in nature and disappeared more quickly in the physostigmine/scopolaminepretreated group than the saline pump group. Effects on responses per session Figure 1 shows responses per session for each of the four groups. The mean number of responses per session made throughout the control period of the study was in the range of 45 56, out of a possible maximum of 60 responses. No significant differences were seen between the physostigmine/scopolamine and saline pumps prior to OP administration (all univariate ANOVA effects for pump type P>0.05 for the days of the pump period, e.g. day-1 post-pump, F 1,14 =0.030, P=0.865; Fig. 1a). Comparison of data following administration of either sarin or soman showed a significant effect of pump type on the day of administration (F 3,14 =11.073, P=0.001) and the following day (F 3,14 =3.636, P=0.040; Fig. 1b). No significant differences were seen on subsequent days of the pump phase. Post-hoc analysis showed that both sarin and soman subjects pretreated with saline made significantly fewer responses than those equivalent groups pretreated with physostigmine and scopolamine on the day of sarin/soman administration (sarin: mean difference=50.7, limits 19.7, P<0.01 and soman: mean difference =36.2, limits 21.7, P<0.01). There were no significant differences between the groups on the following day. There were also no significant differences between the effects of sarin and soman on vehiclepretreated subjects following OP administration, suggesting that the OP-induced reduction in responses was equivalent for both compounds. In the case of physostigmine/scopolamine-pretreated subjects, those receiving soman made fewer responses than those receiving sarin on both the day of dosing (mean difference =27.6, limits 25.7, P<0.05) and the following day (mean difference =19.4, limits 6.3, P<0.05). Effects on serial reversal performance 215 A significant effect of experimental stage (baseline, pump, OP dose) was seen for the group of subjects receiving sarin (F 2,14 =9.19, P=0.0028) and the group receiving soman (F 2,16 =4.05, P=0.0376).

5 216 Fig. 1a, b Mean responses made per session SEM (maximum 60). Days not shown are weekends (where no testing occurred) or days prior to the start of the pump or dose blocks which contained fewer trials if a subject passed a stage (to allow the subsequent block to be started on a new reversal): see Methods. Additionally testing did not occur on day 13 (day of pump implant). a Effects of pump implantation (data grouped for subjects irrespective of whether subsequently receiving sarin or soman). b Effects of sarin or soman administration following saline or physostigmine/scopolamine pump

6 217 Fig. 2a, b Performance on the behavioural task. The mean errors made per reversal were calculated for each subject for each phase of the experiment. These were then averaged across subjects to obtain the values illustrated. Such an approach was necessary due to the differing rates at which animals completed stages: see Methods. a Effects of saline and physostigmine/scopolamine pumps ( SEM) combined irrespective of subsequent OP received. b Effects of (i) sarin or (ii) soman following saline or physostigmine/scopolamine pump ( 95% C.I. from post-hoc analyses). *P<0.05 relative to both baseline and pump periods. In both saline pump groups, all subjects made fewer errors per reversal following OP administration. This was not the case for subjects implanted with physostigmine/scopolamine pumps

7 218 Effects of pre-treatment pump implantation Post-hoc comparison showed that the type of pump that was implanted (saline or the physostigmine/scopolamine) had no significant effect on reversal performance when compared with baseline (all P values >0.05). In addition, the effects of the type of pump implanted did not differ between the subjects that were subsequently to receive sarin and those that were to receive soman (all P values >0.05). Effects of subsequent OP administration Post-hoc analysis revealed that after sarin administration, subjects implanted with the saline pump made significantly fewer errors in the dose phase than both their own baseline performance (mean difference = 17.6, limits 9.84, P<0.05) and the preceding saline pump phase (mean difference 14.1, limits 9.84, P<0.05) (Fig. 2b, part i). In subjects implanted with the physostigmine/scopolamine pump, however, there was no significant change in the number of errors made following sarin administration, relative both to baseline (mean difference =7.46, limits 9.36, P>0.05) and the preceding physostigmine/scopolamine pump administration phase (mean difference =4.24, limits 9.36, P>0.05). A similar pattern was seen in the subjects receiving soman (Fig. 2b, part ii). Following soman administration, subjects implanted with the saline pump made significantly fewer errors than both their own baseline (mean difference =4.83, limits 4.38, P<0.05) and preceding saline pump phase (mean difference =5.52, limits 4.38, P<0.05). For the subjects implanted with the physostigmine/scopolamine pump, there were no significant changes in the number of errors made following soman administration relative to baseline (mean difference =4.70, limits 7.47, P>0.05) or the pump phase (mean difference =3.44, limits 7.47, P>0.05). To investigate whether the improvement in performance seen following OP administration reflected a reduction in persistent responding to the incorrect stimulus immediately after the rule had changed (perseveration) or more rapid learning of the currently relevant stimulus reward association, the pattern of errors following a rule change was investigated in the subjects implanted with saline pumps. Following the introduction of a reversal, the median number of errors made prior to the first correct response were counted for each subject for the control, pump and dose phases. These values were analysed using repeated-measures ANOVA with a withinsubject factor of experimental stage (control, pump and dose) and between-subject factors of pump type (saline or physostigmine/scopolamine) and of dose type (sarin or soman). There was no significant within-subject main effect of phase (F 1,14 =1.46, P>0.1) nor were there significant between-subject effects of pump (F 1,14 =2.78, P>0.1) or dose (F 1,14 =1.032, P>0.1). The number of perseverative errors made was found to be low for all conditions, with means for the phases in the different groups lying between 1.95 and 3.5 errors. Discussion The continuous administration of a combination of physostigmine and scopolamine over a period of 14 days had no effect on the performance of marmosets on a serial discrimination reversal task. The combination also provided protection against both the immediate and shortterm effects of OP administration. Pretreatment with physostigmine/scopolamine was seen, in the case of sarin, to prevent and, in the case of soman, to substantially ameliorate the marked reduction in responses which was seen following the administration of these OP compounds in the absence of this pretreatment. In addition, the severity of the clinical signs of poisoning observed following OP administration was also ameliorated by the pretreatment. Pretreatment with physostigmine/scopolamine did not produce impairments on either motivation to perform the task (as measured by number of responses made) or on serial reversal task accuracy when compared with baseline performance. When administered alone, however, both compounds, albeit at higher doses, have previously been shown to disrupt performance on a variety of cognitive tasks. Scopolamine has been reported to impair aspects of memory function, such as encoding of new information into long-term memory (Ridley et al. 1984) and working memory performance (Rusted and Warburton 1988), as well as attentional performance on tasks such as a five-choice serial reaction time task (Mirza and Stolerman 2000). Physostigmine has been shown to result in improved performance on tests of both discrimination learning (Warburton and Brown 1972) and perceptual function (Wetherell 1992). The absence of effects on performance when these two compounds were administered in combination in the current study could be due either to the antagonistic pharmacological actions of scopolamine and physostigmine or the relatively low levels at which these compounds were administered in comparison with the studies mentioned above. The fact that the concentration of scopolamine used here has been shown to be equivalent to an acute dose of mg kg Ÿ1 (Scott et al. 1994), which is considerably lower than doses which have been shown to affect marmoset cognitive performance (e.g mg kg Ÿ1 ; Harder et al. 1998), suggests that the latter explanation is more likely. In addition, other behaviours sensitive to cholinergic disruption beyond the scope of this study, such as sleep or physiological function, may be affected by the physostigmine/scopolamine combination investigated. Mild clinical signs of poisoning following OP administration were noted in all marmosets regardless of pretreatment status, but signs were more rapid in onset, milder in nature and of shorter duration in the physostigmine/scopolamine pretreated subjects. This would suggest the occurrence of a transient cholinergic hyperstimulation

8 of sufficient degree to elicit symptoms due to AChE inhibition resulting from the presence of both OP and physostigmine. This quickly resolved through a mechanism likely to involve both metabolism of unbound OP and dissociation of the physostigmine enzyme complex and, thus, increased AChE function. It is possible that the presence of scopolamine during this period minimises the effects of the crisis due to its anticholinergic actions. Previous studies in the guinea-pig have illustrated effective protection when physostigmine is administered with scopolamine (Wetherell 1994) and the protection afforded by such a regime is better than when physostigmine is administered alone. The combined physostigmine/scopolamine pretreatment protected against the marked change in response levels that were seen 1 h after OP administration in the saline-pretreated animals, where no responses were made by those administered soman and only a few responses by those administered sarin. The reductions in numbers of responses observed following OP exposure were prevented by pretreatment with physostigmine/scopolamine in animals exposed to sarin and substantially ameliorated in animals exposed to soman. In addition, physostigmine/ scopolamine pretreatment prevented changes in task accuracy observed after administration of either sarin or soman following vehicle pre-treatment. In those animals pretreated with saline, irrespective of which OP was administered, there was a significant increase in the accuracy of reversal performance. Although performance may be expected to improve over repeated reversals (Cotterman et al. 1956; Mackintosh 1962), this is unlikely to account for the results obtained here. First, all animals performed a series of reversals (at least ten) before commencing the baseline phase and therefore would not be expected to show major levels of improvement over subsequent reversals. Second, the pattern of results does not support the idea that a learning effect may account for the significant differences seen. If learning accounted for the results seen, then improvements might be expected in the dose phase relative to control phase in all groups. This was not the case, with the improvements in performance occurring in the dose phase relative to both the control and pump phases specifically in subjects implanted with saline pumps. This pattern suggests the improvements were due to OP administration rather than a learning effect. Analysis of errors immediately following the initiation of a reversal showed no evidence of a change in the number of perseverative responses made by the subjects that showed improved reversal learning following OP administration. This suggests that the reduction in the number of errors made was due instead to more rapid acquisition of the newly relevant stimulus reward association following a rule reversal. In the current study, all subjects made few perseverative errors, probably because of the high degree of training prior to the start of the experimental phases. In future studies, assessment of perseveration in less-experienced subjects may provide a 219 fuller analysis of the effects of OP compounds on perseveration. Results obtained in this study show that AChE inhibition by an irreversible inhibitor (either soman or sarin) produced an initial behavioural impairment, in terms of rate of responding and, subsequently, an improvement of around 20%, in reversal performance, following administration in normal (saline pump) animals. Results from investigations of other AChE inhibitors, such as physostigmine, have shown improvements in behavioural measures, but such findings have often been inconsistent (Muir 1997) with impairments following administration commonly ascribed to adverse peripheral effects. Although the doses of the two OP compounds used here initially produced peripheral effects and caused an impairment in terms of the number of responses made, the irreversible nature of the AChE inhibition caused by these compounds meant that the levels of enzyme inhibition would have remained high once peripheral effects were no longer evident. Thus, with the slow rate of recovery from such inhibition [1 3% per day, which approximates the rate of red blood cell turnover; Pearce et al. (1999)] subjects pretreated with saline would have had relatively high levels of inhibition following sarin or soman in the absence of peripheral effects. This is in contrast with physostigmine, where, due to its short half life, producing high inhibition levels without peripheral effects would be difficult. As it has been suggested that there is a role for the cholinergic system in modulating the signal to noise ratio in attentional processing (Sarter 1994), the improved performance seen here is most likely due to an increased ability to switch between two competing stimulus attributes as a result of sustained elevation of cholinergic neurotransmission. It is unlikely that the side effects initially observed confounded this effect in the present study as they may have been expected to interfere with performance rather than cause improvement on the task presented. Clearly the improvement on the measure of performance analysed here does not rule out a decrement in other measures. Sarter (1994) suggested that a hyperactive cholinergic system may indiscriminately amplify the detection and processing of available stimuli, possibly resulting in the attribution of behavioural significance to irrelevant stimuli. The improvement seen here might not be of functional benefit and, as such, may represent an impairment. For example, changing of a hypothesis at a quicker rate may be disadvantageous in situations where reward occurs in only a proportion of trials. This could be viewed as being more akin to real-life situations where decisions are often based on incomplete information. Further investigation would be needed to clarify this possibility. Thus, this study has illustrated that physostigmine/ scopolamine offers protection against behavioural effects resulting from exposure to the OPs soman and sarin, while in itself causing no decrements in the performance of a behavioural task. It has also illustrated that, while

9 220 performance was abolished immediately following OP in the absence of physostigmine/scopolamine pretreatment, subsequent improvement in this task was seen. The nature of the performance improvements attained following soman or sarin administration would also seem to warrant further investigation to ascertain whether they generalise to other tasks or impair different aspects of performance, thus more clearly elucidating the nature of any improvements. Acknowledgement This study was funded by the UK Ministry of Defence. References Aigner TG, Mitchell SJ, Aggleton JP, DeLong MR, Struble RG, Price DL, Wenk GL, Mishkin M (1987) Effects of scopolamine and physostigmine on recognition memory in monkeys with ibotenic-acid lesions of the nucleus basalis of Meynert. Psychopharmacology 92: Blokland A (1996) Acetylcholine: a neurotransmitter for learning and memory? Brain Res Rev 21: Carey GJ, Costall B, Domeney AM, Gerrard PA, Jones DN, Naylor RJ, Tyers MB (1992) Ondansetron and arecoline prevent scopolamine-induced cognitive deficits in the marmoset. Pharmacol Biochem Behav 42:75 83 Cotterman TE, Meyer DR, Wickens DD (1956) Discrimination reversal learning in marmosets. J Comp Physiol Psychol 49: Crofts HS (1998) Methodology for the repeated assessment of cognitive and physiological parameters within a home cage environment in the common marmoset (Callithrix jacchus): assessment of repeated attentional set shifting and the sleep electroencephalogram. Thesis, University of Bristol Crofts HS, Muggleton NG, Pearce PC, Nutt DJ, Scott EAM (1995) A home-cage system for neuropsychological testing in nonhuman primates. J Psychopharmacol Suppl 9:A12 Crofts HS, Muggleton NG, Bowditch AP, Pearce PC, Nutt DJ, Scott EA (1999) Home cage presentation of complex discrimination tasks to marmosets and rhesus monkeys. Lab Anim 33: Dirnhuber P, French MC, Green DM, Leadbeater L, Stratton JA (1979) The protection of primates against soman poisoning by pretreatment with pyridostigmine. J Pharm Pharmacol 31: D Mello GD, Duffy EA (1985) The acute toxicity of sarin in marmosets (Callithrix jacchus): a behavioral analysis. Fundam Appl Toxicol 5:S169 S174 Fosbraey P, Wetherell JR, French MC (1990) Neurotransmitter changes in guinea-pig brain regions following soman intoxication. J Neurochem 54:72 79 Harder JA, Baker HF, Ridley RM (1998) The role of the central cholinergic projections in cognition: implications of the effects of scopolamine on discrimination learning by monkeys. Brain Res Bull 45: Harris LW, Talbot BG, Lennox WJ, Anderson DR, Solana RP (1991) Physostigmine (alone and together with adjunct) pretreatment against soman, sarin, tabun and VX intoxication. Drug Chem Toxicol 14: Leadbeater L, Inns RH, Rylands JM (1985) Treatment of poisoning by soman. Fundam Appl Toxicol 5:S225 S231 Lennox WJ, Harris LW, Anderson DR, Solana RP, Murrow ML, Wade JV (1992) Successful pretreatment/therapy of soman, sarin and VX intoxication. Drug Chem Toxicol 15: Mackintosh NJ (1962) The effects of overtraining on a reversal and non reversal shift. J Comp Physiol Psychol 55: Mirza NR, Stolerman IP (2000) The role of nicotinic and muscarinic acetylcholine receptors in attention. Psychopharmacology 148: Muir JL (1997) Acetylcholine, aging, and Alzheimer s disease. Pharmacol Biochem Behav 56: Pearce PC, Crofts HS, Muggleton NG, Scott EAM (1998) Concurrent monitoring of EEG and performance in the common marmoset: a methodological approach. Physiol Behav 63: Pearce PC, Crofts HS, Muggleton NG, Ridout D, Scott EAM (1999) The effects of acutely administered low dose sarin on cognitive behaviour and the electroencephalogram in the common marmoset. J Psychopharmacol 13: Ridley RM, Bowes PM, Baker HF, Crow TJ (1984) An involvement of acetylcholine in object discrimination learning and memory in the marmoset. Neuropsychologia 22: Ridley RM, Baker HF, Drewett B, Johnson JA (1985) Effects of ibotenic acid lesions of the basal forebrain on serial reversal learning in marmosets. Psychopharmacology 86: Roberts AC, Robbins TW, Everitt BJ (1988) The effects of intradimensional and extradimensional shifts on visual discrimination learning in humans and non-human primates. Q J Exp Psychol B 40: Roberts AC, Robbins TW, Everitt BJ, Jones GH, Sirkia TE, Wilkinson J, Page K (1990) The effects of excitotoxic lesions of the basal forebrain on the acquisition, retention and serial reversal of visual discriminations in marmosets. Neuroscience 34: Roberts AC, Robbins TW, Everitt BJ, Muir JL (1992) A specific form of cognitive rigidity following excitotoxic lesions of the basal forebrain in marmosets. Neuroscience 47: Robbins TW, McAlonan G, Muir JL, Everitt BJ (1997) Cognitive enhancers in theory and practice: studies of the cholinergic hypothesis of cognitive deficits in Alzheimer s disease. Behav Brain Res 83:15 23 Rusted JM, Warburton DM (1988) The effects of scopolamine on working memory in healthy young volunteers. Psychopharmacology 96: Sarter M (1994) Neuronal mechanisms of the attentional dysfunctions in senile dementia and schizophrenia: two sides of the same coin? Psychopharmacology 114: Scott EAM, Hollands RN, Wetherall JR (1994) The current UK medical countermeasures for nerve agent poisoning; physostigmine and hyoscine as an alternative to pyridostigmine. In: Proceedings of the CB Medical Treatment Symposium, AC- Laboratorium Spiez, Switzerland, pp Taffe MA, Weed MR, Gold LH (1999) Scopolamine alters rhesus monkey performance on a novel neuropsychological test battery. Cogn Brain Res 8: von Bredow J, Corcoran K, Maitland G, Kaminskis A, Adams N, Wade J (1991) Efficacy evaluation of physostigmine and anticholinergic adjuncts as a pretreatment for nerve agent intoxication. Fundam Appl Toxicol 17: Warburton DM, Brown K (1972) The facilitation of discrimination performance by physostigmine sulphate. Psychopharmacologia 27: Wetherell A (1992) Effects of physostigmine on stimulus encoding in a memory-scanning task. Psychopharmacology 109: Wetherell JR (1994) Continuous administration of low dose rates of physostigmine and hyoscine to guinea-pigs prevents the toxicity and reduces the incapacitation produced by soman poisoning. J Pharm Pharmacol 46: