Medial prefrontal and neostriatal lesions disrupt performance in an operant delayed alternation task in rats

Behavioural Brain Research 106 (1999) 13 – 28 www.elsevier.com/locate/bbr

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Medial prefrontal and neostriatal lesions disrupt performance in an operant delayed alternation task in rats Stephen B. Dunnett *, Falguni Nathwani, Peter J. Brasted MRC Cambridge Centre for Brain Repair and Department of Experimental Psychology, Uni6ersity of Cambridge, Downing Street, Cambridge CB2 2PY, UK Received 6 January 1999; received in revised form 20 May 1999; accepted 20 May 1999

Abstract An operant version of the classical delayed alternation task is presented and applied to evaluate the effects of bilateral prefrontal and striatal lesions in rats. Retractable levers in a conventional operant chamber control discrete trial opportunities for making sequential choice responses to the two sides, and the rats are required to maintain repeated nose poke responses to a central panel during the delay interval, which is randomly varied. The operant task provides measures of the speed and accuracy of response alternation and side bias; analysis at different delay intervals provides an index of the memory demands of accurate performance; and analysis of accuracy depending on the response on preceding trials provides measures of proactive interference and perseveration. Following pretraining in the task contingencies, both striatal and prefrontal lesions induced profound deficits in task accuracy, with no change in side bias and only small changes in movement times. The deficit in the prefrontal lesion group recovered more rapidly, neither group showed any change in sensitivity to proactive interference, while the rats with striatal lesions alone exhibited an increased tendency to perseverate incorrect responses on either side. We conclude that the operant delayed alternation task should assist analysis of fronto-striatal function in rats as well as be useful for the analysis of strategies for fronto-striatal repair. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Prefrontal cortex; Neostriatum; Frontostriatal systems; Delayed alternation; Operant tests; Rats

1. Introduction Ever since Jacobsen’s classic studies in the 1930s [35,36], spatial delayed response and spatial delayed alternation tasks have provided classic measures of prefrontal cortex dysfunction in monkeys [3,6,14,31,37,42,43] and in rats [10,25,38,39,62,67,68]. Based on the anatomical association of prefrontal cortex and anterior striatum, Rosvold first proposed that the neostriatum is a major subcortical nucleus in a prefrontal circuit for the control of cortical function, and subsequent studies have demonstrated that striatal lesions in rats and monkeys can mimic many of the deficits observed after prefrontal lesions [11,12,14,55– * Corresponding author. Tel.: +44-1223-331160; fax: +44-1223331174. E-mail address: [email protected] (S.B. Dunnett)

58]. This system is now conceptualised as one of the major parallel cortical-basal ganglia loops within the forebrain comprising a ‘prefrontal’ circuit involved in processing essential cognitive functions associated with the prefrontal cortex [1,2]. Consequently, delayed alternation tasks have been among the most popular to assess cognitive aspects of striatal function [48] and a series of studies have confirmed deficits on this classic prefrontal test after lesions in the medial striatum of rats [13,34,44,47,49,52,67] and other species [3,14,47]. Previous studies of delayed alternation function in rats have primarily been based on training in various types of maze apparatus, including T-maze, Y-maze, Grice box and swimming pools. Such tests are easy to establish but training is labour intensive and does not facilitate the systematic collection of other aspects of performance (such as the speed of responding) or a systematic variation of delay intervals. These latter

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functions are more readily obtained in operant tasks. However, although both free operant schedules [23,40,60] and other discrete trial operant tasks [4,15,16,41] have been used to asses the effects of striatal lesions, operant versions of delayed alternation tasks have been less successful [44]. Specifically, in this latter study, Mogensen and colleagues employed an operant chamber with two fixed keys; with the time window for making an alternation response signalled by flashing lights. Under these circumstances both striatal and intact rats learned over 10 – 20 sessions to alternate their responses during the signalled trials in the operant task, primarily by adopting strong positional mediating responses, even though the same lesioned animals exhibited marked deficits when trained to alternate in a conventional T-maze [44]. In previous studies to develop operant spatial delayed matching and non-matching to position tasks (DMTP and DNMTP, respectively) in rats, we have used retractable levers in an operant chamber to designate discrete trials when an animal may make choice responses to one of two levers located on either side of a central food well. We reduced the rats’ opportunities for making simple lateralised positional mediating responses by requiring them to press the central panel (covering the food well) repeatedly during the delay interval [19,21]. By making presentation of the levers contingent upon a panel press response on a variable interval schedule, a stable high rate of responding at the central panel is assured throughout the interval [21,29]. This task has been widely used to evaluate the effects of cortical and subcortical lesions, various pharmacological treatments and ageing on memory and other aspects of cognitive function [8,9,19,20,22,27,50,59,61]. However, whereas lesions or drug infusions into medial prefrontal cortex of rats produce reliable impairments in DMTP/DNMTP [5,7,20,26,33,54], striatal lesions produce rather modest deficits that recover rapidly [16,17]. This study was therefore designed to adapt the DNMTP task from a delayed non-matching contingency (in which a response to a single sample lever is followed by a choice response between two levers on each trial, akin to the pair-trial alternation protocol in a T-maze [53]) to an ‘operant delayed alternation’ task, requiring alternation between two choice levers on successive trials in the operant chamber, akin to the classic delayed alternation contingency employed in rodent maze tasks.

2. Materials and methods

2.1. Subjects The subjects were 30 young adult male rats of the hooded Lister strain (Charles River, UK). They were

housed in groups of 4–5 rats per cage under a reverse 12 h/12 h light/dark cycle (lights off at 09:30 h). During behavioural testing, the animals were maintained on a food deprivation regime, fed 14–17 g lab chow per day so as to maintain : 90% of free-feeding body weight. All experimental testing was conducted during the dark phase of the cycle, with food given 1–4 h after the completion of daily testing. Water was available ad libitum throughout the experiment. The experiment was conducted in compliance with the UK Animals (Scientific Procedures) Act 1986.

2.2. Apparatus Behavioural tests were conducted in a bank of eight operant chambers (Paul Fray, Cambridge, UK). Each chamber had two retractable response levers, positioned one either side of a central food well covered with a clear Perspex® panel hinged at the top. Food pellets (45 mg; Sandown Scientific, UK) could be delivered to the food well and retrieved by pushing back on the hinged flap. There were five lights in the chamber, a ceiling light that was illuminated during time out and the intertrial intervals, a panel light that signalled occasions when a panel response was required by the task contingencies, and three stimulus lights over the food well and each lever which were not used during this task. Each operant chamber was housed in a sound attenuating chamber and controlled by on-line connection to an Archimedes microcomputer running the Arachnid control language [30].

2.3. Delayed alternation task The delayed alternation task involves a modification of previous operant delayed alternation paradigms [18,44] with the following changes: (i) retractable levers define when successive choice responses may be made; (ii) the rats are required to press at the central panel during the delay intervals between responses in order to obtain presentation of the retracted levers. This served to position the animals mid-way between the two response locations (following the procedure previously adopted in an operant delayed matching/non-matching task [21]); and (iii) the delay interval is randomly varied between trials to provide an additional index of forgetting from short-term memory once the task contingencies had been accurately learned, and also to maintain a high rate of panel responding during the delay [21]. The contingencies of the full delayed alternation task are illustrated in Fig. 1. After the animals have been placed into the operant chambers, the start of the experimental session is signalled by the house light being turned off, and the panel light turned on. As soon as the rat presses the panel, the panel light is extinguished and both levers are presented simultaneously.

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Fig. 1. Schematic illustration of (A) the front panel of the operant chamber, and (B) the series of contingencies at each stage of a trial in the delayed alternation task.

When the rat presses one or other lever, both levers are retracted, a pellet is delivered to the food well. After 3 s, the start of the next trial is signalled by the panel light being switched on. The trial delay is randomly selected by the computer (5, 10, 15 or 20 s in the standard version of the task) and the variable interval delay timer started. A panel press prior to completion of the scheduled delay has no consequences, whereas the first panel press made after completion of the delay interval results in extinction of the panel light and simultaneous presentation of both levers into the chamber. (As confirmed in Section 3, under this second-order variable interval contingency, panel pressing is maintained at a high response rate, which serves to keep the animal in a central location during the delay interval [21]). On presentation of the two levers into the chamber, the rat is then free to respond to either lever and must makes a choice: 1. A response to the lever opposite to that pressed on the previous trial is ‘correct’; both levers are re-

tracted and a food pellet is delivered to the food well. Collection of the food pellet is again detected by the rat pressing back the panel. There is 3 s intertrial interval (ITI) for the rat to consume the pellet between the lever press and the start of the next trial, which is signalled by the panel light being illuminated. 2. A response to the lever on the same side as that pressed on the previous trial constitutes an ‘error’; both levers are retracted, and the house light is turned on to signal a 3 s ‘time out’ interval (TOI). At the end of this 3 s interval, the house light is turned off and the next trial commences. Each session is of 40 min duration. On each trial, the following parameters are recorded: the numbers of nose pokes during the delay interval, the latency of the last panel press after completion of the interval, the side and latency of the lever press response, and the latency to collect the delivered food pellet if the response is correct.

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In this experiment, all rats were trained on the operant delayed alternation contingency prior to any lesion surgery. Rats are trained on the task as follows. The food deprivation regime was introduced, all rats were initially familiarised with the reward pellets given in their home cage. They were then first introduced to the operant chambers for one 15 min session with the house light off, the levers retracted, the panel light on and :50 pellets in the magazine; all rats ate at least some of the food pellets. The rats were then taught to respond to the two levers in alternation with the introduction of delays. In the first session, a panel press causes just one lever to be presented, which alternates between the two sides on successive trials. The lever stays out until a single lever press results in food delivery with no delay restriction. The session continued until 50 panel and lever presses were completed within one session of 25 min. In the second session, both levers were available on each trial, and the rats were trained to alternate with very short delays [1, 2, 3 or 4 s, 1 s ITI and 1 s TOI]. This session continued until 50 trials were completed within 30 min. In subsequent sessions, the rats were trained on the full alternation contingency using successively longer delays ([1, 2, 3,4 s; 1 s ITI and TOI], [1, 4, 8, 12 s; 1 s ITI and TOI], [2, 5, 10, 15 s; 2.5 s ITI and TOI] up to the standard set of task parameters [5, 10, 15, 20 s; 3 s ITI and TOI]) in 40 min sessions. Training continued on each set of delays until a criterion of 60% correct in a 40 min session was achieved, and the rats then progressed to the next set of delays. A correction procedure was used during task acquisition whereby, if the rat made three incorrect responses in a row (i.e. four successive responses on the same side), only the opposite lever was presented and the animal was forced to make a correct response to the ignored lever before continuing with normal trials.

2.4. Test procedure All 30 rats were initially trained on the delayed alternation task to the 60% criterion on the standard task parameters [5, 10, 15 and 20 s delays; 3 s ITI, 3 s TOI]. On reaching this criterion, each rat was then tested over ten sessions without the correction procedure, to establish the ‘pre-lesion’ baseline level of performance. The animals were allocated to groups, matched for pre-surgical baseline performance to receive prefrontal lesions (n =8), sham prefrontal lesions (n =8); striatal lesions (n =10); or sham striatal lesions (n= 4). All animals were allowed 5 days recovery following surgery, then 2 days to re-establish the food deprivation regime, followed by ‘post-lesion’ retesting over ten sessions on the standard task parameters. Finally, a series of two sessions of ‘probe trials’ were undertaken in which the delays were reduced [0.25, 1, 5 and 10 s, 1s

ITI, 1s TOI] in order to probe whether observed lesion deficits would be equally apparent when the memory load was reduced. Animals were perfused for histology : 6 weeks after surgery.

2.5. Surgery All experimental surgery was conducted with the rats mounted in a Kopf stereotaxic frame under gaseous anaesthesia using 1–3% halothane in a mixture of oxygen and nitrous oxide. Prefrontal lesions were made by aspiration. The overlying bone was removed from 0.5 to 2.0 mm either side of the midline and from bregma forward to the rostral pole of the brain. The midline neocortex was aspirated rostral to the genu of the corpus callosum from the dorsal lip to just above the olfactory bulb, as detailed elsewhere [25]. Sham prefrontal lesions were made by removal of the bone alone. Striatal lesions were made by stereotaxic injection of the excitotoxin quinolinic acid. A total volume of 0.7 ml 0.09 M quinolinic acid (Sigma, St. Louis, MO) dissolved in 0.1 M phosphate buffer pH= 7.4 was infused in 4× 0.175 ml aliquots into the anterior medial neostriatum on each side at AP= 0.2 and 1.2 mm anterior to bregma, L= 9 2.0 mm lateral to the midline and V= 4.0 and 5.0 mm vertical below dura, with the incisor bar set 2.3 mm below the interaural line. Each infusion was delivered over 2 min via a 30 gauge stainless steel cannula connected by polyethylene tubing to a 10 ml Hamilton glass microsyringe mounted in a Harvard microdrive pump, with a further 2 min allowed for diffusion before retraction of the cannula. Sham striatal lesions were made by infusion of phosphate-buffer alone. At the end of surgery, the wounds were cleaned and sutured. All animals were given tetracycline antibiotic and paracetamol analgesia via the drinking water for 24 h. No further post-operative care was required.

2.6. Histology At the completion of all behavioural testing, all animals were perfused transcardially under terminal barbiturate anaesthesia (1 ml Euthatal) with 100 ml phosphate-buffered saline (PBS) and 250 ml 4% paraformaldehyde in PBS. The brains were removed, post-fixed for 4 h then transferred to 20% sucrose until they sank. Sections were cut through the rostral half of the brain at 60 mm on a freezing sledge microtome and two series of 1:6 sections were mounted on glass slides for processing with the conventional cresyl violet Nissl stain and for acetylcholinesterase activity by the thiocholine reaction using 0.01% ethoproprazine to inhibit non-specific esterases and 0.25% silver nitrate to enhance the sulphide reaction product.

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Sections were examined and photographed under standard light microscopy, and the extent of each lesion traced by projection in a drawing tube attached to a Leica Laborlux microscope. The extent of the striatal lesions was quantified by calculating the residual volume of the anterior striatum rostral to the level where the anterior commisure crossed the midline, based on tracing outlines of the striatum dorsal to the anterior commisure in successive sections, measuring the areas and summing with a correction for section thickness and frequency.

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ally small and confined to the target area in the medial segment of the neostriatum, but did spread in most cases at least into the mid portions of the anterior striatum (Figs. 3 and 4). The striatal lesions induced an approximately 20% decline in volume of the anterior striatum (striatal shams, 22.479 1.02 mm3, striatal lesions, 18.119 0.71 mm3, t24 = 3.11, PB0.01). In the majority of cases there was no overt damage observed outside the neostriatum, although in individual cases a limited amount of additional damage was seen to extend to the medial septum or dorsal parts of the nucleus accumbens.

2.7. Analysis 3.2. Task acquisition At no stage did behavioural performance differ between the sham prefrontal and sham striatal groups, which are therefore combined for analysis into a single sham control group (n = 12). All behavioural data were analysed by multifactorial analysis of variance, with groups as a single between — subject factor, and stage (pre- vs. post-lesion), delays and previous trial (correct or incorrect) as within — subject factors as required. Post hoc comparisons between groups were made, where appropriate, using Newman – Keuls tests. Histological data are analysed descriptively and by comparing striatal volumes using Student’s t-test between the striatal lesion and striatal sham groups.

3. Results

3.1. Histology Photomicrographs of representative control, prefrontal and striatal lesions are shown in Fig. 2. The prefrontal lesions were effective in removing the rostral parts of the medial wall of the prefrontal and anterior cingulate cortex (Fig. 2B,C). In the majority of cases, the floor of the lesion extended ventrally to the dorsal surface of the olfactory bulb, although in the smallest lesions the ventral-most part of the medial wall neocortex was spared (Fig. 2C and 3). In no case did the lesion extend posteriorly to the genu of the corpus callosum. Striatal lesions were most easily delineated at higher magnification in Nissl-stained sections, in which there is a total loss of medium-sized neurons, sparing of the scattered large neurons, and a modest proliferation of small glial cells (Fig. 2F,G). At lower magnification, the injection needle track is easily delineated, even in control brain (Fig. 2D), and a localised increase in gliosis is seen at the site of lesion, surrounded by a paler staining associated with the loss of striatal neurons, and a modest enlargement of the lateral ventricles (Fig. 2E). The extent of the lesions was confirmed by loss of acetylcholinesterase staining in the striatal neuropil over the same area (Fig. 2H,I). The lesions were gener-

All rats rapidly acquired the delayed alternation contingency over : 20 days of training. Although the progression criterion was only 60% at each stage of training, once they had passed this level at the standard set of task parameters, the rats achieved \ 75% correct within the next five sessions which were then used as the pre-lesion baseline for allocating rats into groups (see Fig. 5A). The animals exhibited remarkably low levels of side bias (responses to the preferred side/total responses; see Fig. 5B), indicating that the 25% of trials on which they were making errors occurred approximately equally on both sides, and cannot be attributable to a primary deficit in response bias to one side. During the pre-lesion period, a number of other measures of performance were collected. Thus, all rats exhibited a reliable rate of panel pressing, averaging : 17 presses per trial, but analysed in more detail by delay duration below. The movement time to make the choice response to the lever was stable at about 2 s latencies from initiation of the panel press that triggered extension of the levers to the lever press itself (Fig. 5C).The latency to move back to the panel to collect the reward pellet after a correct choice response was relatively stable at approximately 0.75 s (Fig. 5D).

3.3. Post operati6e performance Post-operatively, both striatal and prefrontal lesions induced a marked deficit in accuracy of delayed alternation performance to chance levels (Fig. 5A; F2,27 = 61.18, PB 0.001). In spite of this marked deficit, there was no change in side bias in either lesion group (Fig. 5B); although the striatal lesioned rats had a slightly higher bias to their preferred side (55 vs. 53% in the other two groups), this was not due to a change following lesion but was already apparent prior to the lesion and reflects an incomplete matching of the animals to groups (which was done on the basis of matching for accuracy, not bias). The lesion rats were also unaffected in the rate of panel pressing during the delay intervals

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Fig. 2. Photomicrographs of the prefrontal and striatal lesions visualised using Nissl (A – G) and acetylcholinesterase (H,I) stains. (A) Prefrontal cortex of a control animal. (B) Aspirative cavity in the rostral prefrontal medial wall neocortex. (C) Aspirative cavity in the anterior cingulate medial wall neocortex. (D,H) Intact striatum from a control animal, in which the needle tracks can be visualised bilaterally in the Nissl stained section. (E,I) Excitotoxic lesion of the medial striatum, in which the needle track can be seen in the medial striatum on the left side and a modest gliosis at the site of the lesion on the right side in the Nissl stain, and loss of AChE activity over the area of cell loss. (F) High magnification of the normal striatum, indicating numerous medium sized neurons and several scattered large neurons (arrows). (G) High magnification of the lesioned striatum, indicating a complete loss of medium sized neurons but apparent sparing of the scattered large neurons (arrows), and numerous small glial cell profiles. Sixty micrometers thick sections. Scale bar for panels F,G = 50 mm.

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Fig. 3. Camera lucida tracings of the extent of tissue removal after aspiration of the medial prefrontal cortex in animals with (A) the smallest, (B) the median, and (C) the largest lesion size. Tracings are based on 60 mm Nissl stained sections at 360 mm intervals.

(see below, Fig. 6B). However there was a modest increase in the movement times in both lesion groups (Fig. 5C, lesion, F2,27 =8.95, P B0.001), which was independent of delay (delay× lesion, F6,81 =0.97, n.s.),

and the striatal lesion group took significant longer than the other two groups in the time to collect reward after a correct response (Fig. 5D; F2,27 = 6.41, PB 0.001).

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Fig. 4. Camera lucida tracings of the extent of the striatal lesions in animals with (A) the smallest, (B) the median, and (C) the largest lesion size. Tracings are based on 60 mm Nissl stained sections at 360 mm intervals.

3.4. Probe trial performance On the probe trials, the delays and intertrial intervals were reduced. Both lesion groups exhibited improve-

ments in response accuracy over their initial post-lesion levels, which was more marked in the prefrontal than the striatal group, although they still remained impaired with respect to control performance (Fig. 5A; F2,27 =

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Fig. 5. Performance of the three experimental groups on key parameters of performance in the delayed alternation task, evaluated in blocks of 10 sessions, pre-lesion and post-lesion with the standard set of delay intervals and on a further two sessions during the probe trials with shorter delay intervals (see text): (A) percent accuracy of choice performance; (B) percent bias to the more preferred side; (C) movement time to execute the lateralised choice response; and (D) collect time to return to the central panel to collect food reward (on correct trials only). Vertical bars indicate mean9 SEM of each parameter, group and stage. Horizontal dashed lines in panel A indicate chance performance. Horizontal dotted lines indicate pre-lesion control performance as a baseline to facilitate comparison of group performance post-lesion.

bias (Fig. 5B), nor in the rate of panel pressing during the delay intervals (although as a result of the shorter delays, the total numbers of presses made were lower, see below, Fig. 6C). The increased movement time had recovered to control levels in the prefrontal lesion animals but remained significantly lengthened in the striatal lesion group (Fig. 5C; F2,27 =3.83, P B0.01), although this deficit was small in comparison to the magnitude of the disruption in response accuracy. The differences between groups in the latencies to collect reward were no longer significant (Fig. 5D).

3.5. Analysis by delays Several aspects of performance need to be analysed according to the delay interval. First, the rate of panel pressing during the delays remained relatively constant, so that the number of panel presses was proportional to the length of each delay interval. This is shown for the three stages of testing in Fig. 6. In the pre-lesion stage of testing, all animals responded to the panel at a consistent rate of :1.2 – 1.5 presses/s that remained the same at short and long delays (Fig. 6A). This relatively high rate of responding suggests that the rats maintained a consistent level of central panel pressing during the delay intervals. Neither lesion produced any significant difference in rates of panel pressing either during the post-lesion or the probe tests (post-lesion, Fig. 6B,

groups F2,27 = 1.48; probe, Fig. 6C, groups F2,27 =1.35; both n.s.), and critically there is no indication that the rate of responding declined as the delays lengthened which would suggest a breakdown in the maintenance of a central position by the rats throughout the delay intervals. Response accuracy also declined at progressively longer delay intervals (Fig. 7A, delays, F3,81 =50.87, PB 0.001). Specifically, during the pre-lesion baseline period, accuracy declined at a steady rate of :0.9%/s (Fig. 7A), which may provide an index of the rate of short-term forgetting. This pattern was retained by the control animals in the post-lesion period, whereas the disruption of performance in the two lesion groups was manifest by a reduction in choice accuracy to a constant level just above 50%, close to a chance level of performance at all delays (Fig. 7B; groups×delays, F6,81 = 3.48, PB 0.01). Note that although 50% is close to the chance level of random performance, if the animals had a strong position bias to one side or the others, performance in delayed alternation can fall to close to 0% accuracy-this did not happen. During the second phase of post-lesion probe tests, the slope is much less apparent, due to the abbreviated range of delays used (Fig. 7C; delays, F3,81 = 3.24, PB 0.05), and there were no significant differences in the slope of the accuracy function between groups (groups×delays, F6,81 = 0.73, n.s.). In particular, the apparent delay-

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dependent separation between prefrontal lesion and control animals, that might be considered suggestive of faster forgetting, is not significant, over the ranges of delays tested here.

3.6. Proacti6e interference and perse6eration In order to determine whether the errors in delayed alternation may have been attributable to proactive interference from responses on the preceding trials, accuracy data were subdivided in terms of whether the response had been correct or incorrect on the previous trial (Fig. 8). During the pre-lesion baseline there was no evidence for any proactive interference-all rats exhibited a similar level of performance when the previous trial had been incorrect (i.e. the preceding two responses were both opposite the side of the next correct response, reducing interference from the preced-

Fig. 7. Analysis of accuracy by delay intervals: (A) pre-lesion baselines; (B) post-lesion on the standard task parameters; and (C) post-lesion on the shorter delay intervals used in the probe trial. Symbols indicate mean 9 SEM of each group and condition.

Fig. 6. Analysis of panel presses by delay intervals: (A) Pre-lesion baselines; (B) Post-lesion on the standard task parameters; and (C) post-lesion on the shorter delay intervals used in the probe trial. Symbols indicate mean 9 SEM of each group and condition.

ing trial) and when it had been correct (i.e. the preceding two responses were on opposite sides, increasing the opportunity for interference) (previous trial, F1,27 = 0.81, n.s.). By contrast, whereas both the control and prefrontal lesion rats again exhibited no differences between previous correct and previous incorrect trials on the post-lesion tests, the striatal lesion group showed a significant difference (Fig. 8; groups× previous trial, F2,27 = 4.59, PB 0.05). Specifically, note that the rats with striatal lesions performed better when the previous trial was correct than when it was incorrect, which is opposite to the prediction that would arise from a consideration of proactive interference effects, but would be compatible with an enhanced tendency to perseverate. This effect had dissipated by the probe trials (previous trial, F1,27 = 0.36; group×previous trial, F2,27 = 1.82; both n.s.).

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In order to analyse perseveration in more detail on the post lesion trials, a further analysis was undertaken of the runs of repeated responses on the same side in the three groups. The mathematical basis of this analysis is outlined in Appendix A. As shown in Fig. 9A, the control rats exhibited a higher incidence of runs involving 0 repeats (C(0) =…LRL… or …RLR…) and a lower incidence of repetitions on the same side (e.g. …LRRL… or …RLLLR… etc). This difference is highly significant (Groups ×Run length, F20,270 =23.66, PB 0.001), but is fully predicted simply by the lower overall accuracy of the two lesion groups. Of greater interest, is the conditional probabilities of making a correct response after different length runs of repeated responses on the same side, analysed according to the principles defined in Appendix A and shown in Fig. 9B–D for the three groups. (Note that the formula for calculating the conditional probabilities involves division by the number of opportunities, which drops to very low numbers for repeat lengths \4, see Fig. 9A and Appendix A; consequently, the analysis of conditional probabilities is only presented for runs of 0 – 4 repeat responses on the same side before a correct

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alternation.) The analysis of variance indicated a significant Group× Run length interaction (F8,102 =2.83, PB 0.01). Inspection of Fig. 9B–D, followed by post hoc Newman–Keuls comparisons, confirmed that this was due to accuracy remaining relatively constant in both the control and the prefrontal lesion groups, close to the probability of a correct response overall, irrespective of runs of preceding perseverative responses. By contrast, in the striatal lesion group, the conditional probability of an accurate alternation declined the greater the number of previous repeats, i.e. the longer the perseverative run, the greater the chance of the animal continuing to make a further perseverative error.

4. Discussion In this study we use a novel operant version of the classical delayed alternation task for rats, which we have validated by comparing the effects of medial prefrontal and anteromedial striatal lesions.

4.1. The operant delayed alternation task

Fig. 8. Analysis of trial accuracy in terms of proactive interference from the response on the preceding trial. Accuracy is plotted for (A) trials in which the response on the preceding trial had been correct and hence the preceding two responses were on opposite sides, and (B) trials in which the response on the preceding trial had been correct and hence the preceding two responses were on the same sides. Vertical bars indicate mean9 SEM of each group and stage. Horizontal lines as in Fig. 5.

Several previous studies have utilised various types of delayed spatial alternation contingencies in operant chambers [18,32,46,63], including their use to evaluate prefrontal [64,65] or medial striatal [44] lesions. As in other delayed responses, several key parameters of the task contingency affect the ways in which rats learn to perform accurately [66]. The essential features of the present design are the use of retractable levers rather than some other signal to indicate the times within each discrete trial when a response is required, and the introduction of panel pressing during the delay interval in order to reduce overt mediating responses. The use of distinct variable delays within each session served to maintain panel responding at a stable high rate, characteristic of variable interval schedules [29]. As in other delayed response tasks, a key issue has been the extent to which the rats can solve the mnemonic component of the task by adopting mediating responses [7,28,51]. Indeed, when the task contingencies allow, rats will simply await at the location of the correct lever until it is available for response [44]. The introduction of another response during the delay interval can largely overcome the problem of blocking overt mediating responses [19,21,46,66] although this may never completely resolve the issue of the animals still employing some minor or covert mediating response [7,51]. In this study, we have used a nose poke at the central panel maintained on a second-order variable interval schedule which has been successful in ensuring a high response rate throughout the intervals. Critically, not only were the response rates high (with

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Fig. 9. Analysis of postoperative trial accuracy in terms of perseveration (for mathematical details and rationale, see Appendix A). (A) Number of runs of repeated responses on the same side (whether right or left) for run lengths of 0 (i.e. a correct alternation) or 1 – 10 perseverative responses on the same side (combined data from left and right sides) before making an alternation response to the other side (i.e. the parameter C(n), for n =0 … 10, as defined in Appendix A). (B–D) Conditional probabilities of a correct response after 0 … 4 perseverative responses on the same side (i.e. the parameter PC(n), for n= 0 … 4, as defined in Appendix A). Vertical bars indicate mean9 SEM of each group and repeat length. Horizontal bars indicate the means 9 SEM of the overall performance of each group across all trials, independent of repeat lengths.

the rats averaging 1.3 presses/s), but also responding was consistently maintained throughout the longer intervals, as manifest by the relative linearity of the relationship between panels responses and delay interval (shown in Fig. 6). Consequently, we do not believe that mediating responses provide a major strategy used by the rats for solving the delayed alternation rule in this configuration of the task. Nevertheless, it remains the case that mediating responses can never be fully excluded in any task in which the correct response is specified at the time of the previous sample or trial. This does not, however, preclude delayed alternation and matching/non-matching to position tasks providing a powerful index for identifying cognitive dysfunction.

Additional advantages of operant tasks over conventional maze studies are that they allow precise timing of responses, and collection of several parameters of performance [66]. The collection of data from a large number of trials not only allows analysis with relatively tight standard errors of the means of estimated parameters, but also allows systematic variation of controlling factors, such as by randomly varying the delay intervals to provide an index of rates of short-term forgetting [21,51]. We have utilised these factors advantageously in the present operant delayed alternation task, not only to analyse of delay effects between and within trials [45,46,64] but also to provide additional analyses of proactive interference and response perseveration

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based on the conditional probabilities of choice accuracy given alternative outcomes of the preceding trial(s). We have previously employed an analysis in terms of previous trial correct/incorrect to analyse proactive interference in the closely related DMTP task, in which modest proactive interference was observed in young animals using short intertrial intervals [24]. In that study, because the sample and choice response are clearly separated in the DMTP paired-trial task design, it was possible to identify the fact that proactive interference arises particularly from the responses made on previous trial(s) rather than the sample stimulus. This observation is particularly pertinent to the present situation because in the operant delayed alternation task there are no separate stimuli that identify each trial and the response interference is consequently even more likely to occur. However, in well trained rats, we saw little effect of proactive interference per se, the control animals performing at a similar level irrespective of whether the previous trial was correct or incorrect. Similarly the more detailed analysis of accuracy after perseverative runs of different lengths suggested that the influence of previous trials was minimal in the control animals, and only yielded clear effects when the consequences of specific lesions were considered.

4.2. Effects of prefrontal and striatal lesions on operant delayed alternation The present operant task has demonstrated the hypothesised disruption of delayed alternation performance both in rats sustaining damage in the medial wall of prefrontal cortex and in rats with anteromedial lesions of the neostriatum. These results clearly support both Rosvold’s original prefrontal system hypothesis and modern formulations of functional corticostriatal connectivity in parallel loops, including the ‘prefrontal loop’ of particular interest in the present context. Following many classical demonstrations in various maze tasks [25,38,39,62,67,68], an operant delayed alternation task has been used previously to demonstrate deficits in both acquisition and retention after selective medial prefrontal lesions [64,65], and these observations are clearly replicated in this study. Although in their latter study, van Haaren and colleagues did use retractable levers, it is not clear from their account whether the animals could adopt a simple positional mediating response during the delay intervals [65], as has been found in other situations where no alternative responses are demanded during the interval [44]. Consequently it is hard to determine the precise nature of the lesion deficit in that study. Thus, the disruption of performance on such a task could represent spatial, mnemonic or executive dysfunctions, a response-related deficit, or even impairment of a mediation strategy.

25

Although our previous studies indicated clear delaydependent deficits in similar delayed matching and nonmatching to position tasks following prefrontal manipulations [20,26], which was taken to suggest a specific impairment in short-term memory, the present results did not yield a significant delay-dependent deficit in delayed alternation after similar lesions. In the first post-lesion block of testing, the lesioned rats were disrupted at all delay intervals. Then, at the shorter set of delays used in the probe trials, the deficit although partially recovered was still apparent at all delays and the group× delay interaction did not achieve significance. Consequently, there is no direct support for a specific memory deficit whereby rats with medial prefrontal lesions forget in the delayed alternation task faster than controls. Nevertheless, inspection of Fig. 7C does suggest a greater impairment at the 10 s than at the 1 s delay. Moreover, the range of delays used in both stages of the present tests were relatively narrow. Consequently, a delay-dependent contribution cannot be excluded until performance has been tested over a broader set of delays (e.g. from 1 … 20 s, or 1 … 30 s) within the same block of test sessions. Further experiments to evaluate this issue are currently in preparation. There is no evidence that the prefrontal lesion deficit in task accuracy is attributable to interference effects since like the controls they showed little sensitivity to proactive interference from previous trials. Nor are there any clear suggestions of motoric or mediating response deficits in the prefrontal lesion rats: they maintain the panel press response and complete as many trials with a similar rapidity as the controls, and they show no tendency to perseverate or bias responding to one side. Rather the deficit appears to be one of a disruption of the ability to retain the alternation contingency itself, suggesting a specific ‘executive’ deficit in selecting appropriate responses according to changing task demands. This appeared to show partial recovery, or relearning, over test sessions following the lesions, although again further experiments are suggested to analyse in detail the progression of recovery using the same range of delays at all time points after the lesion, and comparing groups with repeated sessions of training against other groups in which recovery may take place due to the passage of time alone. In line with Rosvold’s hypothesis, there are also numerous studies confirming that striatal lesions induced delayed alternation deficits in a variety of maze tasks. By contrast, striatal lesions failed to disrupt delayed alternation performance in the one previous study known to us in an operant task. In that study, the authors employed fixed levers, and the discrete trials were signalled by lights over the levers. The authors observed the rats to adopt strong positional mediating habits to maintain correct response alternation and

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they emphasised that this opportunity may account for why their striatal lesions disrupted alternation in a T maze but not in the operant version of the task. This interpretation is supported by the present results. Here, we show that when the intervening positional or lever pressing mediating responses are blocked, by a combination of using retractable levers and requiring panel press responses throughout the delay interval, then striatal lesions do indeed produce marked impairments on an operant delayed alternation task, in accord with the prefrontal systems hypothesis. Of particular interest in the present context is the nature of the striatal lesion deficit. Although both prefrontal and striatal lesions disrupted the ability of rats to perform the delayed alternation task, there were distinct differences in the nature of the deficit. Firstly, the prefrontal lesions showed a greater degree of recovery when tested with shorter delays on the probe trial. Although this may suggest differences in effect on relearning capacity, it could equally be due to differences in the completeness of lesions in the two structures and so cannot be resolved without further systematic experimentation. Of greater relevance is the fact that whereas the prefrontal lesion rats appeared to have a primarily executive deficit, the performance of rats with striatal lesions exhibited features indicative of a more response-related deficit. Although they showed no significant slowing in their rates of responding at the panel, the rats with striatal lesions were slowed in the execution of a lateralised response and in the times to collect food pellets following a correct lever press, and they showed a significantly greater tendency to perseverate, independent of any overt bias in responding. This suggests that striatal lesioned rats are exhibiting difficulties in switching between responding on the two sides, and the deficit may reflect a specific inability to select the correct response choice in the presence of a competing, previously-rewarded response opportunity. This inability to alternate may be over and above, or even instead of, a primary deficit in learning and executing the correct response according to the task demands.

5. Conclusion In summary, an operant delayed alternation task is introduced that allows collection and analysis not only of simple measures of choice accuracy, but also parameters of bias, speed and rates of responding, short-term memory and forgetting within a trial, and proactive interference and perseveration of incorrect responses between trials. Execution of the delayed alternation contingency is disrupted both by prefrontal and by striatal lesions, as predicted by the anatomical connectivity of these two structures within a discrete functional subsystem of the forebrain. Nevertheless, subtle differ-

ences in the profiles of impairments that result from the two lesions suggest different contributions of the prefrontal and striatal nuclei to integrated performance of the behaving animal. The results highlight the utility of operant strategies for dissecting frontostriatal function and the new task provides a powerful tool for the further analysis of strategies for repair following damage or disease within frontal and striatal systems of the forebrain. Acknowledgements This research was supported by grants from the Medical Research Council. Appendix A. Indices of perseveration Let P be the overall probability of a correct response (total correct/total responses) for any rat. Let P(L) be the probability of a correct (left) response when the previous response was right. Let P(R) be the probability of a correct (right) response when the previous response was left. In the absence of a side bias, P(L)= P(R). A left response bias will be represented by P(L)\ P(R), and vice versa. Within a block of trials, for each rat record the numbers of runs of repeated responses on the left side: L(0)=the number of occurrences of a single left response …RLR… (i.e. 0 repeated lefts). L(1)=the number of occurrences of two successive left responses …RLLR… (i.e. 1 repeated left). L(2)=the number of occurrences of three successive left response. …RLLLR… (i.e. 2 repeated lefts). And so on, for L(n) is the number of occurrences of n repeated left responses before a correct right, for all n] 0. Similarly, record the numbers of runs of repeated responses on the right side before a correct left response, R(n) for all n]0. And let C(n) be the combined numbers of runs of each length on either side, C(n)= L(n)+ R(n) for all n] 0. It is these values of C(n) for n= 0 … 10 that are shown in Fig. 9A. The conditional probability of a correct right response after n perseverative left responses in a row is: CpL(n)= L(n)/SL(n)+ L(n + 1)+ L(n + 2)+ …) CpL(n)= L(n)/SL(i ), for i = n … . Similarly, the conditional probability of a correct response after n perseverative right responses in a row is: CpR(n)= R(n)/SR(n)+ R(n + 1)+ R(n + 2)+ …) CpR(n)= R(n)/SR(i ), for i= n …

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And the combined conditional probability of a correct response after n perseverative responses on the same side (either left or right) is:

[6]

CpC(n)=(L(n)+ R(n)) /((L(n)+L(n +1) +L(n +2) + …)

[7]

+ (R(n)+ R(n +1) + R(n +2) + …)) CpC(n)=(L(n)+ R(n))/S(L(i ) + R(i )),

[8]

for i = n … It is these values of CpC(n) for n = 0 … 4 that are shown in Fig. 9B–D. If responding on each trial is independent of previous trials then CpC(i )= P for all i] 0. If performance is subject to proactive interference then CpC(0)B PB CpC(i ) for all i ]1. Indeed, if the alternation rule has been properly learned, then the longer the run of previous trials on the same side the greater the proactive facilitation of a correct choice on a given trial. In this case CpC(i ) ](CpC( j ), for all i\ j, Of course, if the overall probability of making a correct response, P, is high there will be fewer runs of repeated responses, than if P is low, so a higher incidence of runs per se cannot define the tendency to perseverate. Rather, we can define perseveration, independently of the overall chance to make a correct response, as an increased tendency to repeat a response if the response has already been repeated, i.e. CpC(i ) \ CpC(0) for all i ] 1.Since P is the overall probability correct, it must lie between the extremes. Thus in the case of perseveration, CpC(i) \P\ CpC(0) for all i] 1, and indeed, CpC(i ) ] CpC( j ) for all i\ j. Note that proactive interference and perseveration make directly complementary predictions of the conditional probability outcomes.

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