Introduction

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Chlorpyrifos (CPF) is a high-volume organophosphate pesticide with multiple uses in the control of agricultural and household pests (USEPA, 1994). The widespread use of CPF raises the likelihood of inadvertent exposure to the pesticide in segments of the population involving either short-term high-level exposure (e.g., accidental spills) or long-term low-level exposure (e.g., in office buildings or homes treated with it). Reports of human organophosphate exposures have included a variety of signs (e.g., salivation, tremor, nausea and muscle cramps and weakness) that are closely linked to cholinergic activation due to cholinesterase inhibition (Jeyaratnam and Maroni, 1994). The cholinergic nervous system has long been known to play an important role in learning and memory in both humans and laboratory animals (e.g., Deutch, 1962; Drachman, 1977; D'Mello, 1992; Hasselmo and Bower, 1993; Lydon, 1995). In this regard it is interesting to note growing evidence of an association between organophosphates and alterations in higher central nervous system functions involved in learning and memory (Stephens et al., 1995; Steenland et al., 1994; Rosenstock et al., 1991; Savage et al., 1988; see also review by Annau, 1992).

Research in laboratory animals has found a number of effects produced by CPF. Signs of cholinergic overstimulation are common in rats (Pope et al., 1991; Moser, 1995), with neonate rats being more sensitive than adults (Pope et al., 1991). CPF also reduces motor activity, as do a number of other cholinesterase inhibitors (e.g., Crofton et al., 1991; Moser, 1995; Padilla et al., 1996). In contrast to the shorter duration of action of oral delivery, s.c. injection of rats with CPF produces a long-lasting inhibition of cholinesterase activity in blood and brain (Pope et al., 1991), and a persistent enhancement of the increasing effect of scopolamine on motor activity (Pope et al., 1992). In addition, Bushnell et al. (1993) found decreases in accuracy and increased response latencies in rats performing under a delayed matching task after s.c. CPF administration.

Given the association between the cholinergic nervous system and learning and memory (e.g., Deutch, 1962; Drachman, 1977; Lydon, 1995), CPF may be expected to have adverse effects on learning in rats. We therefore determined the effects of acute and repeated oral exposures to CPF in rats using a RA paradigm. RA procedures are operant learning preparations in which subjects acquire new sequences of responses during each experimental session (Boren and Devine, 1968; Cohn and Paule, 1995). Experimental organisms must learn on a daily basis which sequence of responses produces reinforcement. RA is ideally suited for long-term studies of learning, because learning can be determined repeatedly in individual organisms. It has been used extensively with a variety of species (including humans) to investigate the effects of a number of drugs and environmental chemicals including pesticides on learning (Anger and Setzer, 1979; Higgins, et al., 1989; see Thompson and Moerschbaecher, 1979; Cohn and Paule, 1995; and Cohn et al., 1996, for reviews). The strength with which accurate conclusions can be drawn regarding a treatment's effect on learning can be greatly enhanced by inclusion of a P component in which the response sequence remains constant across sessions. Under these conditions, a treatment affecting learning would be expected to selectively decrease learning accuracy while leaving performance accuracy relatively intact. This experiment determined the effect of acute and repeated CPF in rats using a multiple RA and P paradigm.

	Materials and Methods

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Animals

Subjects were 16 adult male Long-Evans rats maintained at approximately 300 g body weight via daily food restriction (Purina Rat Chow, St. Louis, MO). The rats were housed individually in a temperature- and humidity controlled vivarium maintained on a 12-hr light-dark schedule (lights at 0600 hr). Water was available ad libitum in the home cage. Sessions were conducted Monday through Friday between 0800 and 1000 hr.

Behavioral Apparatus

Behavioral sessions were conducted in operant chambers (Coulbourn Instruments, Inc., Lehigh Valley, PA) housed in sound-attenuating enclosures (Ralph Gerbrands Co., Arlington, MA) ventilated by a fan. Each chamber was equipped with three response levers, arranged horizontally with the lever closest to the left front side of the chamber designated L (left), the middle C (center) and the rightmost R (right). A pellet trough, into which 45-mg food pellets (P.J. Noyes Co. Inc., Lancaster, NH) were dispensed, was located below the middle lever. The trough contained a cue light that briefly flashed with food-pellet delivery. A Sonalert tone generator was situated above the right lever, and a white noise generator continuously masked extraneous sounds. A set of LED triple-cue lamps was located above each lever. A houselight was situated in the upper left corner of the front panel. Experimental contingencies and data collection were executed by a Digital Equipment Corporation (Maynard, MA) PDP 11/73 computer programmed with the SKED-11 system (Snapper et al., 1982).

Response Shaping

Rats were first trained to respond on each of the three response levers. Reinforcement was programed for each response on a specific lever on three successive overnight sessions. Rats were considered successfully shaped when they earned 80 of 100 possible reinforcers during a session. Shaping generally did not require more than three sessions for each rat to complete, and ensured comparable reinforcement histories on all three levers prior to implementation of the RA training procedures.

Repeated Acquisition

Baseline conditions. Rats were ultimately required to make four responses on three levers in a specific sequence for food reinforcement (e.g., LRCR, CRCL). The sequence changed every session, and was therefore the RA sequence. The four-response sequences met several criteria (see third column of table 1). Each lever was used at least once and no more than twice within a sequence. Sequences requiring repetitive responses (e.g., LLRC) were omitted to preclude a history of reinforced response perseveration. In addition, sequences were ordered such that no lever occupied the same serial position within a sequence from the preceding session. The RA sequence alternated with a P sequence twice within a session. The P sequence remained the same across sessions (CLRL). The two presentations of the RA and P components are hereafter referred to as RA1, P1, RA2 and P2. Houselight illumination signaled the beginning of a session. If the session began with a P component, the triple cue lamps above each lever were also illuminated.

Schedule parameters. Rats were required to complete the correct sequence without error for reinforcer delivery. Each error immediately initiated a 2-sec timeout with the houselight (and all other cue lights) off. Responses during the timeout extended it until 2 sec elapsed without a response, after which rats were required to restart the sequence. Correct responses were signaled by brief presentation of the tone stimulus. Correct completion of a sequence produced the tone, a flash of the pellet-trough light, an audible click of the pellet dispenser and food delivery. P components were signaled by illuminating the bank of cue lights above each lever (turned off during timeouts). The RA component was signaled by the absence of the cue lights. Each component lasted until either 25 reinforcers had been delivered or 15 min elapsed, whichever came first, resulting in a maximum session duration of 1 hr. The initial component (RA or P) alternated across sessions. Rats continued on this schedule until stable baselines of acquisition were obtained, at which point experimental manipulations were implemented.

Training criteria. Training involved a stepwise progression, beginning with a two-response sequence and culminating with a four-response sequence (table 1). During the initial training phase (sequence length of two), the criterion for advancement was obtaining 80 of 100 food pellets in eight of 10 consecutive sessions. Upon reaching this criterion, sequence length increased to three. Rats were maintained on three-member sequences until obtaining 80 of 100 possible pellets in four of five consecutive sessions. Sequence length then increased to four until at least 80 pellets were earned during four of five sessions. After completion of this phase, rats were placed on a multiple RA and P schedule in which the RA sequence changed each session, while the P sequence remained constant across sessions. Training lasted from 3 to 4 mo.

Experiment I: Acute CPF administration

CPF (ChemService, West Chester, PA) was dissolved in corn oil vehicle and either vehicle, 12.5, 25.0 37.5 or 50.0 mg/kg was administered by oral gavage (1 ml/kg) once per week (on Wednesdays). Two determinations were made at each dose level. Rats usually received different doses on a given day and sessions began approximately 3 hr post-administration. Sessions always began with the P component when CPF or vehicle was administered.

Statistical analyses. Analyses were conducted using the BMDP statistical software package (BMDP Statistical Software, Inc., Los Angeles, CA). In all analyses, an alpha-level of 0.05 was used to define statistical significance. RANOVA were conducted with CPF as a within-subjects variable for each component. The aim of these analyses was to determine whether there was a main effect of CPF on overall accuracy (defined as the number of responses on the correct lever in the proper order, and in correct relationship to timeout periods/total number of responses × 100%) or on overall response rate (total lever presses/session length). Separate analyses were carried out for RA1, RA2, P1 and P2.

Experiment II: Repeated CPF Administration

Two weeks after experiment I, the rats were randomly divided into two groups and administered either vehicle (n = 8) or 25 mg/kg CPF (n = 8) by gavage 3 hr before each session. Two CPF-treated rats died after approximately 1 wk. Dosing then ceased although behavioral sessions continued for two more weeks. One control rat was then randomly reassigned to the CPF group, resulting in seven rats in each group. Daily dosing with 12.5 mg/kg CPF or vehicle then began without further loss of animals. Dosing (5 days/wk) continued for 8 wk. Other details were as in experiment I.

Statistical analyses. Data from the 40 sessions of dosing were collapsed into five blocks of eight sessions. "Block" therefore was a within-group factor, although CPF was now a between-groups factor. Analytical procedures and details were otherwise as in experiment I with the exception of response rate. Mean response rate was calculated for each rat from the last five baseline sessions beginning with an RA component. When repeated CPF exposure began, rate data from sessions that began with the RA component were divided by that mean. Rate data from sessions that began with the P component were similarly transformed.

Cholinesterase inhibition. On completion of repeated dosing, two blood samples (1 wk apart) were obtained via orbital bleeding after each rat was placed under light CO₂ anesthesia. The first sample was taken 24 hr after administration of vehicle or 12.5 mg/kg CPF. The second sample was taken 45 min after vehicle or CPF. Blood was allowed to clot at room temperature and then centrifuged (12,500 × g for 5 min) to separate the serum, which was removed and kept on ice until analysis (within 1 hr). Serum cholinesterase activity was analyzed radiometrically (Johnson and Russell, 1975) using acetylcholine as substrate. Ten ml of serum were incubated for 3 minutes at 26°C with 90 ml acetylcholine iodide (final concentration = 1.2 mM) spiked with [³H]acetylcholine iodide (specific activity = 5 mCi/mmol; Du Pont-NEN, Boston MA), 0.1 mCi/assay tube. Counting efficiency (as determined by an external standard) was approximately 33%. Preliminary assays established incubation times and tissue concentrations that yielded linear rates of hydrolysis. Data were analyzed via one-way analyses of variance (one-tailed tests of significance).

Postrepeated CPF challenges. After assessment of cholinesterase inhibition, control sessions were conducted for an additional 2 wk. Thereafter, rats in both groups were administered 12.5 mg/kg CPF and tested. This acute CPF challenge was repeated after an additional 2 wk of control sessions.

	Results

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Baseline Characteristics

Under baseline conditions, overall RA accuracy was lower than P accuracy. RA accuracy increased reliably within RA1 and to a lesser extent in RA2. P accuracy also showed a slight within-component increase. Response rates were invariably higher in the first session component (RA or P), but thereafter reached a steady level in subsequent components.

Experiment I: Acute CPF Administration

Overall accuracy and response rate. CPF significantly decreased overall accuracy and response rate in all components (fig. 1). Post-hoc contrast analyses of accuracy data indicated that the minimum dose producing a significant effect was 25.0 mg/kg in RA1 and P1. The minimum effective dose was 12.5 mg/kg in RA2 and P2. CPF also significantly decreased response rate in all four components; the minimal effective dose for RA1 and P2 was 25 mg/kg, and 12.5 mg/kg for P1 and RA2.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Effects of acute CPF on mean ± SEM accuracy (top) and response rate (bottom). During vehicle control sessions, mean response rate data were 19.0 ± 1.7, 6.9 ± 0.9, 35.4 ± 3.0 and 7.1 ± 0.7, responses per minute for the RA1, RA2, P1 and P2 components, respectively.

Within-session analyses. Fifteen responses within each component were deemed the minimum necessary for a particular session(s) to be included in the analyses, or five responses per bin. Substantially decreased responding at 50 mg/kg necessitated exclusion of these data from the analyses. One rat's data were also excluded as it failed to respond sufficiently at doses greater than 12.5 mg/kg. Figure 2 shows accuracy within each component under control conditions and after acute CPF. Significant main effects of bin were obtained for each component, confirming that acquisition of the appropriate sequence did occur. Small but significant increases in accuracy were also obtained for the P components under control conditions. CPF produced marginally significant main effects in the RA1 (P = .076) and RA2 (P = .078) components, and highly significant effects in the two P components. The bin x CPF interaction was significant in the RA1 component, marginal in the RA2 component (P = .079), and insignificant in either of the P components.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Mean ± S.E.M. accuracy changes within each component produced by acute CPF. Each component was divided into three equal time-based bins (see "Materials and Methods").

Microanalyses of response patterns: Initial response correct. Substantially decreased responding after 50 mg/kg CPF necessitated exclusion of these data from the microanalyses of response patterns. Given a correct response on the lever corresponding to the first member of a sequence, CPF did not affect the probability of responding correctly on the second member of the sequence. There was, however, a significant but non-dose-dependent alteration in perseverative responses (pressing the first member of the sequence again). CPF produced a 28% decrease in the likelihood of correctly proceeding from the second to the third member of the sequence (C2). This decrease in correct responding was accompanied by a 55% dose-dependent increase in perseverative responding (P2). Significant alterations in skipping errors were also observed but were not dose-dependent. CPF failed to affect the likelihood of correctly proceeding from the third to the fourth member of the sequence (C3). Significant (or nearly so) alterations were observed in the occurrence of perseverative (P3, marginal) and skipping (S3) errors; however, these were not consistently related to dose. Once the animals completed the sequence, CPF significantly decreased the likelihood of correctly beginning the next sequence by 20% at 25 mg/kg and by 10% at 37.5 mg/kg (C4). This decline in accuracy was offset by a 38% increase in perseverative responding (P4).

Microanalyses of response patterns: Initial response incorrect. The multiple RA and P procedure required subjects to restart a sequence after an error. CPF consistently reduced the probability of this occurring. After an incorrect response on the lever corresponding to the first member of the sequence (XR1), there was a 21% decrease in correctly restarting the sequence. There was a 37% decrease in correct restarts after an incorrect response on the second member of the sequence (XR2). CPF produced significant alterations in XR3 responding, but not in a dose-dependent manner. After an error on the lever corresponding to the fourth member of the sequence (XR4), CPF significantly decreased the likelihood of correctly starting the next sequence.

Experiment II: Repeated CPF Administration

Overall accuracy and response rate. Initially, repeated exposure to 12.5 mg/kg CPF significantly reduced accuracy during both the RA and P components. Figure 3 presents the results for each of the four components, and shows that P accuracy gradually recovered to near-control levels by the final block of eight sessions. Accuracy during the RA components, however, remained decreased relative to control animals; neither the main effects of Block nor the CPF x Block interaction was significant. The inset in Figure 3 depicts RA1 data across the eight sessions of Block 1, and illustrates the gradual emergence of CPF effects with repeated exposure to 12.5 mg/kg that were not observed following acute exposures in experiment I. The recovery of performance accuracy is illustrated by significant main effects of Block and the Block x CPF interaction for P1 and P2 components, while comparable analyses failed to achieve significance in the RA1 and RA2 components.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Mean ± S.E.M. accuracy by block in each component during repeated CPF administration. B, Baseline accuracy (mean of final 10 sessions before repeated dosing). T1, First 12.5-mg/kg CPF challenge 2 wk after completion of repeated dosing. T2, Second 12.5-mg/kg CPF challenge 4 wk after completion of repeated dosing. Inset, Mean ± S.E.M. RA1 accuracy in each of the sessions comprising block 1.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Mean ± S.E.M. response rate by block in each component during repeated CPF administration. Baseline responses rate for vehicle controls were 23.3 ± 1.8, 8.8 ± 0.6, 22.4 ± 3.7 and 6.6 ± 0.6 responses per minute for the RA1, RA2, P1 and P2 components, respectively. Baseline response rates for the CPF group were 24.9 ± 2.7, 8.8 ± 0.6. 20.1 ± 3.6 and 6.0 ± 0.5 responses per minute for the RA1, RA2, P1 and P2 components, respectively. T1, First 12.5 mg/kg CPF challenge 2 wk after completion of repeated dosing. T2, Second 12.5 mg/kg CPF challenge 4 wk after completion of repeated dosing.

Within-session analyses. Analyses of within-session accuracy changes confirmed that learning occurred, and suggested recovery of P but not RA accuracy across sessions of repeated CPF exposure. A comparison of panels RA1 and RA2 of figure 5 demonstrates that most learning occurred during the first RA component. In control rats, large increases in accuracy were obtained in RA1 across all five blocks of sessions although a much smaller increase occurred in the RA2 component. CPF-treated animals demonstrated little or no change either within RA components or across Blocks. Analyses of RA1 data yielded significant main effects of CPF and bin, as well as a CPF x bin interaction, confirming that CPF decreased the rate of acquisition within that component. Analyses of RA2 data also produced significant main effects of CPF and bin, while the CPF x bin interaction failed to achieve significance. No effects involving the variable Block achieved significance during the RA components.

View larger version (64K): [in this window] [in a new window]   Fig. 5.   Mean accuracy changes within each component by block of sessions for the control (black bars) and CPF (white bars) groups throughout repeated CPF administration. Each component block was divided into three equal time-based bins, thus each bar represents data for 1/3 of a component. Errors bars were omitted for clarity.

Microanalyses of response patterns: Initial response correct. As the analyses of overall accuracy produced no significant main effects of Block during the RA components, data were collapsed across block, resulting in one-way analysis of variance for each of the response pairs. Effects of CPF on response pairs beginning with a correct response were not particularly striking. The probability of correctly proceeding from the first to the second member of the sequence significantly decreased by 10%. In concert with this decline, skipping errors increased significantly by 20%, although perseverative errors remained unaffected. Although correct procession from the second to the third member in the sequence was unaffected by CPF, a 26% increase in skipping errors was significant. No significant alterations in response patterning were observed in proceeding from the third to fourth member of the sequence. However, on completion of a sequence, the likelihood of correctly proceeding to the first member of the next sequence decreased significantly by 14%, and was accompanied by significant increases in both perseverative (35%) and skipping (36%) errors.

Microanalyses of response patterns: Initial response incorrect. Response pairs beginning with an error were affected more consistently by CPF exposure than were those beginning with a correct response. No significant changes in response patterns were observed when the error was an incorrect response on the lever corresponding to the first member of the sequence. However, CPF significantly reduced the likelihood of correctly restarting a sequence by approximately 21% when the rats incorrectly responded on the lever corresponding to the second, third or fourth members of the sequence. Accompanying these changes were significant increases in responses in the "Other" category averaging 27% for XO2, XO3 and XO4 pairs. No significant changes in response perseveration were obtained in these analyses.

Cholinesterase Inhibition

After repeated CPF administration, a single administration of 12.5 mg/kg produced 90% inhibition of cholinesterase activity 3 hr postexposure and 84% inhibition 24 hr postexposure. Figure 6 presents individual data for both time points. For the 3-hr postexposure samples, mean cholinesterase levels (±S.E.M.) were 303.4 ± 16.2 nmol/min/ml serum in control rats, and 29.5 ± 1.4 in CPF-treated rats. For the 24 hr postexposure samples, mean cholinesterase levels were 306.1 ± 19.6 nmol/min/ml serum in controls, and 48.4 ± 5.6 in CPF-treated rats.

View larger version (9K): [in this window] [in a new window]   Fig. 6.   Cholinesterase activity in individual rats assessed 3 and 24 hr after administration of vehicle (V) or 12.5 mg/kg CPF.

CPF Challenges

Results of the CPF challenges 2 wk (T1) after termination of repeated CPF are shown in figures 3 and 4. CPF (12.5 mg/kg) significantly reduced accuracy and response rate in rats in the vehicle group during both RA and P components. Rats in the CPF group exhibited accuracy levels during RA and P components that did not differ from those obtained during the final block of repeated dosing. Because RA accuracy during repeated CPF exposure was essentially at chance, it is unlikely the CPF challenge would produce significantly different results in rats that had received CPF repeatedly.

Results of the second CPF challenge, 2 wk (T2) after the first challenge, produced a different outcome (figs. 3 and 4). CPF administration produced inconsistent effects in vehicle-control rats. There were significant decreases in RA1 and P1 accuracy. Mean RA2 accuracy declined as well, but the large amount of variability between rats negated the significance of the effect. P2 accuracy was unaffected, except for a large increase in variability. CPF significantly reduced response rate in both RA components, and in P2, with no significant effect on P1 rate.

Rats that had received repeated CPF showed little difference in RA accuracy after the second CPF challenge from what was exhibited during the repeated exposure phase. These rats did, however, demonstrate a significant decline in accuracy during the P components, suggesting that the tolerance to repeated CPF exposure was disappearing. This effect was not, however, duplicated in analyses of response rate. It should be noted that no significant changes in accuracy occurred in the CPF-exposed group during the 2-wk periods between cessation of repeated exposure and each of the CPF challenges. A slight trend toward higher accuracy was observed during the RA2 component when a linear regression was calculated across all 20 sessions conducted from the end of repeated dosing to the conclusion of the investigation (P < .082). Because rats were sacrificed at this time it is not known whether rats in the CPF group would have continued this trend and eventually approached control levels of accuracy.

	Discussion

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Our study indicates that CPF administration produced selective deficits in RA accuracy. In experiment I, acute CPF-induced decreases in accuracy were significantly greater at 37.5 and 50 mg/kg in the RA components than the P components. In experiment II, repeated administration of a low dose (12.5 mg/kg) initially decreased response accuracy and rate in both the RA and P components. After approximately 8 wk of dosing, accuracy recovered to near-baseline levels in the unchanging P components while remaining impaired in the RA components. Two acute CPF challenges occurring at 2-wk intervals after cessation of repeated dosing indicated that selective tolerance to the behavioral effects of CPF lasted approximately one month beyond the final dose.

The effects of CPF on RA accuracy and response rate are consistent with those produced by other cholinergic compounds including the muscarinic antagonist scopolamine (Cohn et al., 1992; Cohn and Cory-Slachta, 1992; Peele and Baron, 1988; Yamamoto et al., 1990; Yatsurgi et al., 1989) and the carbamate pesticide carbaryl (Anger and Setzer, 1979; Cohn and MacPhail, 1994; Penetar, 1985). Further studies are warranted, using muscarinic and nicotinic agonists and antagonists, to determine the extent and nature of cholinergic-receptor involvement in the acquisition deficits produced by CPF and other cholinesterase-inhibiting chemicals (Lydon, 1995; Granon et al., 1995).

Microanalysis of response patterns showed that CPF produced inconsistent alterations in response pairs that began with a correct response. This differs from results of similar analyses of the muscarinic antagonist scopolamine, which consistently increased the percentage of skipping errors after a correct response (Cohn et al., 1992; Cohn and Cory-Slechta, 1992). Also unlike scopolamine, CPF produced consistent and selective effects on the pattern of response pairs after an initially incorrect response. CPF produced significant decreases in the likelihood of correctly restarting a sequence after an error; rather, in other words the rats tended to continue as though their initial response was correct. CPF also produced significant increases in time-out responding, even though each response prolonged the timeout. These changes in response patterns suggest a failure to attend to the stimuli associated with errors.

Cholinesterase inhibition in the central and/or peripheral nervous system is widely considered to be the target effect underlying the behavioral and physiological effects of CPF (e.g., Padilla et al., 1996). A partially overlapping range of doses (10-100 mg/kg, p.o.), for example, has recently been shown to produce a dose-related inhibition of whole-blood cholinesterase activity (Padilla et al., 1996). The degree of cholinesterase inhibition in the central nervous system after acute oral doses of CPF is currently unknown. However, Padilla et al. (1994) have shown a strong correlation between central cholinesterase inhibition and inhibition in peripheral tissues at the time of peak effect after a single s.c. administration of CPF. Cholinesterase inhibition in adult rats after s.c. CPF is persistent, lasting several weeks in some cases (Chakraborti et al., 1993; Pope et al., 1992; Bushnell et al., 1993; Padilla et al., 1994). Preliminary data indicate a considerably shorter duration of action for orally administered CPF (McDaniel et al., 1996). Correlative analyses of CPF-induced cholinesterase inhibition in central and peripheral tissues, after oral administration, would aid greatly in estimating the degree of inhibition in brain based on inhibition in tissues (viz., blood and its components) that are relatively accessible in trained behaving animals.

The initial decrease in RA and P accuracy emerged gradually over the first several administrations of CPF. The early time-course of effects is reminiscent of the gradual decrease in accuracy in delayed matching-to-sample in rats receiving repeated diisopropyl fluorophosphate (Bushnell et al., 1991)., an effect that is thought to be related to down-regulation of muscarinic cholinergic receptors (e.g., Overstreet et al., 1974). However, with continued treatment, tolerance developed completely to the CPF-induced decrease in P accuracy, although the decrease in RA accuracy persisted throughout dosing. Repeated (weekly) s.c. administration of CPF has been shown to persistently decrease accuracy in a delayed matching-to-sample procedure, as well as increase matching-response latency and nose-poke interresponse times during the delay intervals (Bushnell et al., 1994). No tolerance developed to these effects of CPF. Our results therefore are to our knowledge the first report of behavioral tolerance with repeated CPF administration.

Drawing strong conclusions about whether an experimental treatment produces a selective learning deficit requires several considerations. First, because learning involves the acquisition of behavior, the repeated acquisition of response sequences is justifiably a learning task. Second, a treatment-related decrease in learning could be due to the action of a chemical on motor, motivational, sensory or other neurobiological processes. An exclusionary strategy is therefore needed in studying chemical effects on learning in which alternative interpretations of a chemical's effect can be first ruled out. In our experiment inclusion of a P component, in which the reinforced response sequence remained invariant, was used to demonstrate a selective effect of CPF on acquisition. The P component involved the same response characteristics and the same reinforcer as in the RA component. The development of complete tolerance to CPF effects on P accuracy, at a time when the pesticide's effect on RA accuracy endured, argues strongly for a selective effect of CPF on acquisition.

There was, however, one important difference between the RA and P baselines, with response accuracy in the P component being consistently higher. An unequivocal demonstration of a selective CPF-induced acquisition deficit may therefore require equating accuracy in the two components, perhaps by arranging a longer sequence of responses in the P component. However, such a manipulation would create an imbalance in response requirements between the two components and introduce new complexities in interpreting CPF effects on acquisition.

A fundamental tenet of both pharmacology and toxicology is that chemicals have multiple actions. In behavioral pharmacology, the concept of drug-behavior interactions (Sidman, 1956) was advanced to highlight the multiplicity (and selectivity) of a drug's effects on behavior. The action of a drug depends on the drug and dose, as well as the conditions under which the behavior was acquired and maintained. A review of pesticide effects on schedule-controlled (free-operant) behavior indicated no prominent drug-behavior interactions for acutely delivered organophosphates and carbamates (MacPhail, 1985). However, our results along with other findings in the literature (Overstreet et al., 1974; Genovese et al., 1988; Bushnell et al., 1994) suggest that drug-behavior interactions may be a prominent feature of repeated exposure to cholinesterase inhibitors.