Motor Cortex Lesions Do Not Affect Learning or Performance of the Eyeblink Response in Rabbits

Transcription

1 Behavioral Neuroscience Copyright 1997 by the American Psychological Association, Inc. 1997, Vol. 111, No. 4, /97/$3.00 Motor Cortex Lesions Do Not Affect Learning or Performance of the Eyeblink Response in Rabbits Dragana Ivkovich and Richard E Thompson University of Southern California The possible modulatory role of motor cortex in classical conditioning of the eyeblink response was examined by ablating anterior neocortex in rabbits and training them with an auditory conditioned stimulus (CS) and an airpuff unconditioned stimulus (US) in either a delay (Experiment 1) or a trace (Experiment 2) conditioning paradigm. Topographic measures such as amplitude and onset latency were assessed during conditioning sessions for conditioned responses (CRs) and on separate test days for unconditioned responses (URs) by using a range of US intensities. No lesion effects were observed for learning or performance measures in acquisition or retention of either delay or trace conditioning. During trace conditioning, lesioned rabbits did, however, exhibit a trend toward impairment and demonstrated significantly longer CR latencies. Damage to motor and frontal cortex does not significantly affect eyeblink response performance or learning in either a delay or a trace conditioning paradigm. Classical conditioning of the rabbit eyeblink response has become a model preparation for the study of learning and memory (see Gormezano, Kehoe, & Marshall, 1983; Thompson, 1990, for reviews of the field). In the standard delay paradigm, the conditioned stimulus (CS; typically sound or light) and the unconditioned stimulus (US; typically corneal airpuff or periorbital shock) overlap, and the conditioned response (CR) is "adaptive" in the sense that it develops such that the peak amplitude occurs at about the onset of the US over the effective range of CS-US onset intervals that support robust eyeblink conditioning (Gormezano et al., 1983). The essential elements of the neural circuit underlying conditioning of the eyeblink reflex have been identified at the simplest level to involve structures of the brainstem and cerebellum (see Lavond, Kim, & Thompson, 1993; Thompson, 1989; Thompson & Krupa, 1994; Yet, 1991, for reviews). Indeed, specific damage to a localized region of the anterior interpositus nucleus of the cerebellum has been shown to permanently abolish both the ability to acquire and the ability to retain the conditioned eyeblink response (Lavond, Hembree, & Thompson, 1985; Lavond, McCormick, & Thompson, 1984; Yet, Hardiman, & Glickstein, Dragana Ivkovich and Richard E Thompson, Department of Psychology and Neuroscience Program, University of Southern California. This work is part of Dragana Ivkovich's doctoral dissertation. Support for this research was provided by grants from the National Science Foundation (IBN ), National Institute on Aging (AF05142), National Institute of Mental Health (5P01-MH52194), Office of Naval Research (N ), and Sankyo. We thank C. G. Logan, N. Chatterjee, and K. Jones for their assistance and M. E. Stanton for helpful comments on drafts of this article. Correspondence concerning this article should be addressed to Dragana Ivkovich, who is now at the Department of Psychology: Experimental, Duke University, Box 90086, Durham, North Carolina Electronic mail may be sent via Internet to acpub.duke.edu. 1985). However, higher brain structures (e.g., hippocampus; Berger, Berry, & Thompson, 1986; see General Discussion section) have been implicated in complex conditioning paradigms and in modulation of conditioning. In this article we focus on the possible modulatory role of the motor areas of the cerebral cortex in classical conditioning of the rabbit eyeblink/nictitating membrane (NM) response. The neuroanatomical connections between cerebral motor cortex and the cerebellar eyeblink circuit are numerous. There are four major motor cortical efferent structures that have the potential to modulate conditioned eyeblink behavior (see Figure 1). First, motor cortex projects heavily to the red nucleus (Allen & Tsukahara, 1974; Brodal, 1981; Ito, 1984), which has been implicated in adaptive timing of the CR (Krupa, Weng, & Thompson, 1994). From the red nucleus, motor cortex may affect behavior either directly via the efferent motor nuclei or indirectly by feedback to the trigeminal (Clark & Lavond, 1996; Davis & Dostrovsky, 1986), inferior olive (Weiss, McCurdy, Houk, & Gibson, 1985), and cerebellum (Rosenfield & Moore, 1995). Second, motor cortex projects to pontine nuclei (Allen & Tsukahara, 1974; Wiesendanger, 1986), which carry sensory information about the tone CS to the cerebellum as mossy fibers (Steinmetz et al., 1987). Third, motor cortex projects to the lateral reticular nucleus (LRN), which also receives projections from red nucleus (Brodal, 1981; Ito, 1984; Wiesendanger, 1986). The LRN itself projects back heavily to deep nuclei of the cerebellum as well as to cerebellar cortex, sites of convergence for CS and US information. Fourth, in addition to the indirect projections through red nucleus already mentioned, direct projections from motor cortex to the trigeminal nucleus (airpuff sensory relay) have been identified in primates and may also exist in lower mammals (Wiesendanger, 1986). Alternatively, the primary ascending influence on neocortex from the cerebellum is from the dentate nucleus via red nucleus projections through the ventrolateral nucleus (VL) of the thalamus. However, re- 727

2 728 IVKOVICH AND THOMPSON J i i TONE III, Vl, avl, VII EYEBLINK Figure 1. Diagram of motor cortical efferents that provide links to the cerebellar learning circuit and have potential to modify behavioral output. Structures of the cerebellar circuit include the trigeminal (V) and ventral cochlear (VCN) sensory nuclei; inferior olive (IO); pontine nuclei (PN); cerebellar cortex, including Purkinje cells (shaded oval) and parallel fibers (curved line receiving PN projection); interpositus (IP); and the red nucleus (RN). Motor cortex forms a cerebral loop with basal ganglia (BG) and the ventrolateral thalamus (VL) but also projects down to RN, PN, lateral reticular nucleus (LRN), and V nucleus. All paths ultimately loop through either RN or V to influence motor nuclei III (oculomotor), VI (abducens), avi (accessory abducens), and VII (facial). Filled circles indicate known inhibitory connections. Numbers 1-4 apply to four major descending pathways described in text. cently Sears, Logue, and Steinmetz (1996) have also identified projections from the area of the cerebellar interpositus nucleus critically involved in classical conditioning to areas of VL demonstrating CR-related activity. Because of its interconnections with the cerebellar learning circuit, motor cortex is a reasonable candidate to exert modulatory actions on behavior during eyeblink conditioning (Houk, 1989; Woody, 1984). Bureg and Bracha (1990) suggested that motor cortex may be involved in "elaboration of discrete voluntary movements, the acquisition of new motor skills, and in the instrumentation of already existing unconditioned reflexes" (p. 213). Woody and colleagues (Woody & Engel, 1972; Woody, Yarowsky, Owens, Black- Cleworth, & Crow, 1974) used a 1-ms click CS and glabellar tap US to study the role of motor cortex in eyeblink conditioning in cats. Their measure was a very short-latency (<20 ms) electromyogram (EMG) component in muscles associated with eyelid closure. They reported that lesions of the motor cortex prevented learning of the short-latency eyeblink CR and that changes in neuronal unit activity occurred in motor cortex in learning, as did changes in neuronal excitability. Further, cortical spreading depression that disables motor cortex has been reported to prevent the performance of conditioned eyeblink responses without affecting unconditioned responses (URs; Papsdorf, Longman, & Gormezano, 1965; Megirian & Bureg, 1970). These profound effects of motor cortex inactivation on memory for the learned response are inconsistent with decerebration and

3 MOTOR CORTEX LESIONS AND EYEBLINK CONDITIONING 729 decortication studies in cats and rabbits (Mauk & Thompson, 1987; Norman, Buchwald, & Villablanca, 1977; Oakley & Russell, 1977), which suggest that motor cortex is not necessary for classical conditioning of the eyeblink response. In this article we address the functional role of motor cortex in classical conditioning of the eyeblink response in rabbits trained with either delay (Experiment 1) or trace (Experiment 2) conditioning paradigms. Motor cortex ablations were performed, eyeblink behavior was recorded, and measures of response topography were monitored for changes that might indicate modulation by motor cortex. Experiment 1 In the following study we focused on potential changes in CR and UR topography during acquisition and retention phases of delay conditioning in rabbits when motor cortex, including frontal cortex, was lesioned bilaterally. Acquisition and retention of delay conditioning were compared between lesioned and nonlesioned rabbits. Topographic measures of CR and UR responses, such as amplitude and onset latency, were also analyzed. Potential lesion effects on eyelid performance as well as nonspecific nonassociative effects were monitored on separate days over the course of training by measuring the eyeblink reflex to a range of airpuff intensities. The results clearly indicated that large bilateral, frontal-cortical lesions have no effect on either acquisition or retention of delay conditioning, nor do they affect any of the response characteristics measured. Me~od Subjects. Fourteen New Zealand white rabbits (Oryctolagus cuniculus; Irish Farms, Norco, CA) were used in this study. All procedures and rabbit care were in accordance with National Institutes of Health guidelines. Rabbits were housed individually in a room with a regulated 12-hr light-dark cycle. All rabbits were maintained on an ad lib food and water diet. Rabbit care was provided by the experimenters, staff, and veterinarians of the University of Southern California. Apparatus. For all training sessions and performance testing, rabbits were restrained and placed in a sound-attenuating chamber, and their external eyelids were retracted to prevent interference with NM response measurements. Eyelid movement was measured by placing a loop of 6-0 nylon suture into the temporal margin of the NM. A minitorque potentiometer was attached to this loop so that movement of the NM rotated the arm of the potentiometer and was transduced into a signal for data collection. Both stimulus presentation and data collection were achieved on-line with interface and software for the IBM-PC/XT (see Lavond & Steinmetz, 1989). Procedure. Seven rabbits received bilateral ablations of frontal cortex, including motor areas, before any training (acquisition group) and were then given 5 days of delay classical conditioning. Another 7 rabbits were trained for 5 days, lesioned, and then given 5 more days of training (retention group). All rabbits were adapted to the conditioning chamber for 2 days before any training and were allowed 7-10 days recovery following surgery. Each rabbit's reflexive responses to airpuff intensities of 1, 2, 3, and 4 psi (lbs/in. 2) were measured on separate test days before and after acquisition for both groups (I/O [input/output] Tests 1 and 2, respectively) and before and after postlesion training for the retention group (I/O Tests 3 and 4). Delay conditioning sessions consisted of 108 trials, 12 blocks of 9 trials, in which the first trial of each block was a tone-alone test trial followed by 8 tone-air paired trials. On paired trials the tone CS (1 khz, 85 db SPL-[sound pressure level], 348 ms) was presented 248 ms before the airpuff US (3 psi at the source), and both continued together for 100 ms to coterminate. The intertrial interval varied between 20 and 40 s (30-s average). Each conditioning session lasted about 50 min. Reflex responses were measured on separate days over the course of training as described previously (Steinmetz, Lavond, Ivkovich, Logan, & Thompson, 1992). These I/O tests consisted of 40 US-alone trials, 10 consecutive trials at each of four airpuff intensities (1, 2, 3, and 4 psi) presented in either ascending or descending series. Series order was determined at random for each subject but remained constant within subject. The standard training intensity was 3 psi. Each I/O trial consisted of a 100-ms corneal airpuff presentation with the intertrial interval varying from s (average 30 s). Between each block of 10 trials there was a pause while intensity was manually adjusted. The entire I/O session lasted about 20 min. Data analysis. On-line data collection sampled signals of NM movement every 4 ms during the 744-ms epoch making up each trial. The first 248 ms made up a baseline period during which no stimuli were presented. After the baseline period, the 348-ms tone CS was presented, if applicable. The last 100 ms coincided with the presentation of the airpuff US. The 248-ms period between CS onset and US onset is referred to as the CS period. Data collection continued for another 248 ms after US onset (referred to as the US period). Bad trials were identified by movements exceeding 0.7 mm in the baseline period or 0.5 mm within the first 25 ms of the CS period. These trials were excluded from analyses to prevent contamination of CR recordings by spontaneous movement artifact. The percentage of CRs was monitored as were measurements of CR and UR amplitude and CR onset latency, averaged for each block of training or I/O testing. CRs were defined as deflections exceeding 0.5 mm within the CS period, excluding bad trials. Onset latency was defined as the first time within a trial that NM movement exceeded 0.5 mm. Statistical analyses were performed, and a significance criterion of p <.05 was applied. Data were analyzed by using a betweengroups analysis of variance (ANOVA) with repeated measures (5 days of training) for each response measure collected during acquisition for both groups. Another within-subject repeated measures ANOVA, in which pre-post lesion (2 levels) and session (5 levels) were the variables, was run for the retention group. I/O amplitudes were analyzed separately for each group as two-factor repeated measures ANOVAs in which I/O test (2 levels for acquisition group and 4 levels for retention group) and intensity (4 air pressure levels) were variables. Surgery. Each rabbit was anesthetized with a mixture of ketamine (60 mg/kg) and xylazine (8 mg/kg) and was placed in a stereotaxic headholder. Anesthesia was maintained during surgery by using halothane (1-3% in oxygen). The surface of the brain was exposed by making an opening in the skull, bilaterally, which extended mm anterior to bregma and 3 mm posterior to bregma. In the medial-lateral (ML) direction, the opening began 3 mm lateral to the midline so as to leave a protective bridge over the saggital sinus, and extended to the orbital arch at the anterior end, following the arch posterolaterally to reach an extent of 7-8 mm lateral to the midline along the posterior edge. The opening, therefore, was about 15 mm anterior-posterior (AP) by 5 mm ML.

4 730 IVKOVICH AND THOMPSON Large lesions of motor cortex, including some somatosensory regions, were made by aspiration. Lesion dimensions were based on stimulation studies in rabbit (Brooks & Woolsey, 1940; Woolsey, 1958) with some reference to the motor cortex lesions of Hobbelen and van Hof (1986) and on detailed maps of rat cortex (Neafsey, 1990). The eye region of motor cortex in rabbit is believed to be located along the dorsal surface near the midline and the medial surface of the hemispheres. For this reason, aspirations extended further medially than the bone opening. Because the AP borders of motor cortex are rather vague, lesions also extended beyond the bone opening as far as possible anteriorly to include much of the anterior forebrain, and about 2 mm posteriorly. Because the amount of tissue removed was extensive, the cavity was loosely packed with sterile absorptive gelatinous sponge (Gelfoam, Upjohn, Kalamazoo, MI), and bonewax sealed the skull opening. Rabbits were closely monitored after surgery and were allowed 1 week recovery before behavioral training began (acquisition group) or resumed (retention group). Histology. On completion, rabbits were overdosed with 5 cc sodium pentobarbital, injected intravenously, and were perfused with physiological saline followed by 10% formalin. The brains were removed and stored in 10% formalin for at least a week. Drawings and measurements of the extents of the lesions were made on visual inspection of the fixed tissue. The brains were embedded in an albumin-gelatin mixture and sectioned on a freezing microtome 1 week later. Every fifth 80 ~tm coronal section was collected and stained with cresyl violet for further lesion reconstruction and identification of damage. Using the reconstructed sections, an independent qualified rater, blind to the behavioral data, ranked the lesions by size, and an analysis was performed to see if there were graded behavioral effects varying with extent of damage. Spearman's rank correlation (rs) coefficient was used to determine the interdependence of lesion size and rate of learning, as measured by the number of trials required to reach a criterion (trials to criterion) of eight out of nine consecutive CRs. Results Both lesioned (acquisition group) and unlesioned (retention group) rabbits acquired the conditioned eyeblink without difficulty. The average trials to criterion was SEM for the acquisition group and for the retention group. Percentage of CRs. There was no difference between groups in percentage of CRs over the course of 5 days of acquisition training, indicating that lesions of motor cortex had no effect on learning rate (see Figure 2). There was a significant effect of training session, F(4, 48) = 34.4, reflecting the increase in CRs observed with acquisition. The retention group showed no impairment after bilateral motor cortex lesion and responded at prelesion levels on the first day of retention training. CR amplitude. CR amplitudes on both CS-alone test trials and paired trials increased with acquisition in both groups of rabbits--cs alone, F(4, 48) = 14.0; paired, F(4, 48) = 19.7)--and were within the same range (see Figure 3A). CR amplitudes were unimpaired by subsequent motor cortical lesions for the retention group (Figure 3B). Postlesion amplitudes were significantly higher overall than prelesion amplitudes: CS alone, F(1, 6) = 15.4 and paired, F(1, 6) = t~ u e.- o 60 o = n- O 20 o~ Acquisition 1" T T T-'~-T / i o&s -& lit Acquisition Group (N-7) Retention Group (N-7) Retention Figure 2. Percentage of conditioned responses on CS-alone trials during training. There was no difference in 5 days of acquisition between rabbits lesioned before any training (circle and solid line represent the acquisition group) and unlesioned controls (triangle and dotted line represent the retention group). Retention group rabbits subsequently received lesion of motor cortex bilaterally. There was no effect on retention, which was carried out for 5 days. Error bars represent standard error of the mean. CS = conditioned stimulus; CR = conditioned response. Unconditioned reflex response amplitudes. A withinsubject repeated measures ANOVA performed on the two I/O tests for the acquisition group (Figure 3A) indicated that the difference between pre- and postacquisition I/O tests approached significance, F(1, 6) = 5.6, p =.055. There was a significant effect for US intensity, F(3, 18) = 6.9, which was due to the much lower response amplitudes at 1 psi as compared with 2, 3, and 4 psi combined: contrast comparison, F(1, 6) = 9.8. For the retention group (Figure 3B), when analyzing the 2 (lesion: pre-post) X 2 (I/O tests) x 4 (US intensities) design, there was no difference across I/O tests overall; however, I/O Test 1 and I/O Test 4 were significantly different: contrast comparison, F(I, 6) = 6.7. There was no main effect for intensity, but there was a significant interaction between pre-post lesion and intensity, F(3, 18) = 4.9. Post hoc analyses revealed that this effect was due to increased responsiveness at 4 psi postlesion, F(1, 6) = 7.4, and that there was no difference between pre- and postlesion response trends for 1, 2, or 3 psi. Response onset latencies. Average onset latencies (in milliseconds relative to tone onset) decreased across acquisition sessions in both lesioned and unlesioned rabbits as the ratio of URs:CRs decreased, CS alone, F(4, 48) = 42.2, and paired, F(4, 48) = 58.0; the shorter latencies were maintained postlesion for the retention group (see Figure 4). On both CS-alone and paired trials there was a significant difference between pre- and postlesion latencies for the retention group----f(1, 6) = 61.3 and F(1, 6) = 25.5, respectively--reflecting the adaptive learning process taking place prelesion and maintained during postlesion overtraining. Learning was also reflected in the significant effect of

5 MOTOR CORTEX LESIONS AND EYEBLINK CONDITIONING 731 A A E 12 8 ~ 4.i x T o Acquisition psi -- ~ 3 psi psi psi --~-- CRs (CS-alone) surface, while the largest did not, and both the smallest and largest lesions extended laterally 8-9 mm. Cresyl stained coronal sections (see Figures 6B and 6C) indicate that the dorsal hippocampus, subiculum, and entorhinal cortex remained intact in all cases, although swelling in the cavity of some rabbits may have exerted pressure causing some displacement or distortion. Lesion size was not correlated with rate of learning for rabbits lesioned before acquisition of the delay conditioned eyeblink (rs =.39, where p =.05 was.79). Rabbits with the largest lesions performed in the middle range of trials to criterion (see Table 1, acquisition group). Rabbits lesioned after acquisition, given 1 week of recovery, and returned to conditioning training performed at prelesion levels within the first session, and there was no relationship between extent of lesion and learning or performance. Discussion psi E psi v "0 --&-- 2psi ~ psi E 4.. _.,,,,,, o Acquisition Retention --D-- CRs {CS-alone) Figure 3. Reflex and conditioned response amplitudes over the course of training. A: In the acquisition group, rabbits lesioned before training develop CRs across 5 days of training and exhibit reflex enhancement (US-alone I/O tests) with experience. All measures are comparable between the lesioned rabbits in A and nonlesioned rabbits, as seen in the acquisition phase for retention rabbits in B. B: In the retention group, no performance deficit was measured for either CRs or URs within subject after motor cortex (Ctx) lesions. Error bars represent standard error of the mean. Overlapping error bars have been removed from I/O tests for clarity. CR = conditioned response; CS = conditioned stimulus; I/O = input/output; psi = pounds per square inch. training session---cs alone, F(4, 24) = 13.6, and paired, F(4, 24) = and interaction between lesion and session---cs alone, F(4, 24) = 13.1, and paired, F(4, 24) = 9.8--in which latencies prelesion approached asymptote with acquisition training and then remained there through postlesion retention training. Histology. Surface measurements and sketches of the frontal cortical ablations were made and are reconstructed in Figure 5. Even the severest lesions spared 5-7 mm of the most anterior frontal pole. The smallest AP extent was 13 mm, and the largest was about 17.5 ram. The smallest ML aspect of the ablation spared some tissue along the midline As had been expected, motor cortex lesions did not affect the acquisition or retention of the conditioned eyeblink when rabbits were trained under standard delay conditions. Further, there were no effects on topography of CRs or URs, as represented by amplitude or latency. The largely negative effects on eyeblink conditioning cannot be attributed to the absence of behavioral effects of the lesions. Although the overall behavior of these rabbits was quite normal for the most part, despite massive tissue damage, they did show an inability to cease running once initiated. When a rabbit was retrieved from its home cage, its initial response often, though not always, was an escape attempt characterized by hyperactivity. These symptoms are characteristic of frontal lobe damage in lower mammals (Warren & Akert, 1964). Experiment 2 The results of Experiment 1 indicated that motor cortex is not involved in simple delay conditioning of the eyeblink response in rabbits. The possibility remains that this higher cortical region and other forebrain areas may become involved when the conditioning task becomes more difficult. For example, damage to the hippocampus, a forebrain structure in which neural activity models the development of Table 1 Individual Rankings of Trials to Criterion and Lesion Size for Rabbits Lesioned Before Conditioning Experiment 1: Acquisition group Experiment 2: Trace group Rabbit TTC Lesion rank Rabbit TI'C Lesion rank , , ,160 a 6 Note. TTC = trials to criterion. athis subject never reached criterion and was assigned the maximum number of possible trials.

6 732 IVKOVICH AND THOMPSON 1 t= 500,- 400 o! m 300 O e- o >" o 200 r- -J 100 ~T Acquisition Group (N-7) Retention Group (N=7) i v-.i.- I.t 1 e,- o 0 I I I I I I I I I I Acquisition Retention Figure 4. Response onset latencies on CS-alone trials during training. There was no difference in 5 days of acquisition between rabbits lesioned before any training (circle and solid line represent the acquisition group) and unlesioned controls (triangle and dotted line represent the retention group). All subjects showed a decreasing latency to respond across acquisition days. Retention group rabbits maintained short response onsets postlesion. Error bars represent standard error of the mean. CS = conditioned stimulus. learning (Berger & Thompson, 1978; Berry & Oliver, 1982), alters and markedly impairs acquisition and performance of trace but not delay conditioning (Kim, Clark, & Thompson, 1995; Moyer, Deyo, & Disterhoft, 1990; Port, Romano, Steinmetz, Mikhail, & Patterson, 1986; Solomon, Vander Schaaf, Thompson, & Weisz, 1986). Several studies that have found motor cortex to be important in conditioningrelated phenomena (Megirian & Buret, 1970; Woody et al., 1974) have used a trace conditioning paradigm. Experiment 2 was designed to assess the effects of motor cortex lesions on acquisition of the conditioned eyeblink response by using a trace conditioning paradigm similar to that used by Woody et al. (1974). This involved using a I-ms auditory click, instead of a tone, for the CS and a 500-ms trace interval between presentations of the CS and airpuff US. Motor cortex lesions did not prevent acquisition of trace conditioning or affect performance measures. Method Subjects. Thirteen New Zealand white rabbits (Oryctolagus cuniculus; Irish Farms,.Norco, CA) were included in this study. Rabbit care and living conditions were the same as in Experiment 1. Apparatus and procedure. The apparatus was the same one used in Experiment 1. Seven rabbits received bilateral ablations of motor cortex, as described in Experiment 1, before any training (lesion group). After 1 week of recovery, each rabbit received training on the trace conditioning paradigm with a click CS and an airpuff US until they achieved two consecutive sessions at or above criterion performance (eight out of nine consecutive CRs) with a maximum of 25 days of training. Another 6 rabbits served as unlesioned controls receiving the same behavioral training (control group). Trace conditioning sessions involved presentations of a 1-ms click CS followed by a 500-ms trace interval, during which no stimuli were presented, and a 100-ms airpuff US. Each daily session consisted of 108 trials, (12 blocks of 9 trials), just as with delay training in the previous experiment. In addition to trace conditioning, for each rabbit in the lesion group, reflexive responses to airpuff intensities of l, 2, 3, and 4 psi were measured on separate test days (I/O tests described in Experiment 1) before and after acquisition (I/O Tests 1 and 2, respectively). Data analysis. In the case of trace conditioning, on-line data were sampled for a 1,250-ms trial length. The baseline period was 250 ms, CS period was 500 ms, and US period was 500 ms. CRs and bad trials were detected and analyzed as described previously. An independent t test was used to compare lesion and control rabbits on the basis of trials to criterion. Measures such as percentage of CRs, CR and UR amplitude, and CR onset latency were compared across first, middle, and criterion days of training and between groups by means of a between-groups repeated measures ANOVA. These points of training were chosen to standardize comparisons across rabbits with widely varying learning rates. Rabbits were trained to a criterion and not for a standard number of days because trace conditioning is more difficult than delay conditioning and yields more variable learning rates. The familiar method of Vincentizing data to obtain learning curves (distributing trials across the average number of training days, in this case 9 days) would have generated individual data points based on as few as 5 trials for rapid learners and as many as 3 days, or more than 300 trials, for the slowest learners. For this reason, comparison of subjects on the basis of each individual's first, middle, and criterion days of conditioning was chosen to enable comparison of rabbits at similar points of acquisition and allowing for individual differences in learning rates. All significant differences reported use the criterion p <.05. Histology. Histological procedures, lesion reconstruction, and analysis were the same as outlined for the motor cortex lesioned rabbits in Experiment 1.

7 MOTOR CORTEX LESIONS AND EYEBL1NK CONDITIONING 733 A "nbil~-t) Figure 5. A: Surface reconstruction of the smallest (filled) and largest (hatched) extent of lesion for acquisition and retention groups combined. B: Photos of representative lesions in 2 rabbits: top and side views (black bar is 5 ram). Results Both lesioned (lesion group) and unlesioned (control group) rabbits acquired the trace conditioned eyeblink with some difficulty. One rabbit in the lesion group never reached the criterion of eight out of nine consecutive CRs, and training was discontinued after 25 days (Rabbit ; see Table 1). One rabbit in each group reached criterion once, but not twice, and training was discontinued after 20 days. All others reached criterion at least twice, and training was terminated at that point. The average trials to first criterion was 1, for the lesion group and 900 -_+ 252 for the control group. Though the learning rates were not significantly different for the two groups, the lesioned rabbits tended to be slower learners. This nonsignificant trend toward poorer performance for the lesion group was observed consistently on all of the following measures. Percentage of CRs. Control rabbits achieved 59 ± 6% CRs on the 1st criterion day, whereas rabbits in the lesion group reached 40 10% CRs (see Figure 7). The groups were not, however, significantly different. There was a significant effect for training day, F(2, 20) = 41.6, p <.05, indicating a considerable increase in CRs, but there was no interaction between group and training day. CR amplitude. CR amplitudes on both CS-alone and paired trials increased significantly during the course of training, F(2, 20) = 9.9 and 9.8, respectively. There was no

8 734 IVKOVICH AND THOMPSON A B C D -13 Fr Ctx.,~ 0 ~0 (3O -8 Corona._L_~l I I~ ~A--SS Radiata ~ F 3 acing cau,,e >Tss Lat Septat N ~ F h F +2 ~ ss ~rr~t~tw, Figure 6. Serial reconstructions of the common and largest extent of damage presented in the coronal plane from cresyl stained sections at 5-mm increments (labels relative to bregma) for acquisition group (B), retention group (C), and trace group (D), with reference sections (A). Fr Ctx = frontal cortex; Mtr = motor cortex; SS = somatosensory cortex; Lat Septal N = lateral septal nucleus; Rh F = rhinal fissure; LOT = lateral olfactory tract; CC = corpus callosum; acing = anterior cingulate cortex; pcing = posterior cingulate cortex; d Hip and v Hip = dorsal and ventral hippocampus, respectively; lat vent = lateral ventricle; sub = subiculum; hip = hippocampus. significant difference between groups, though the lesioned rabbits performed slightly more poorly than controls (see Figure 8). There was also no interaction effect. Unconditioned reflex response amplitude. 110 tests were given before and after training for the lesion group, and the c O 6 O O o~ T, // T ACQ1 MID CRIT Training Day i CONTROL (N=6) -.-B-- LESION (N-7) Figure 7. Percentage of conditioned responses on CS-alone trials on the first day (ACQI), middle day (MID), and criterion day (CRIT) of trace conditioning. There was no significant difference between lesion (square and dotted line) and control (circle and solid line) groups. Error bars represent standard error of the mean. CS = conditioned stimulus; CR = conditioned response. characteristic increase in amplitude with experience was observed. There was a significant increase for all US intensities posttraining, F(1, 6) = 9.2. There was no difference across subjects in response amplitude to any particular airpuff intensity, and there was no interaction effect (see Figure 8). A group comparison of UR amplitude can be made from data on training days, although later response amplitudes were confounded with learning. Lesioned rabbits were not significantly impaired. There was a significant effect of training day, F(2, 20) = 10.7, as response amplitudes increased with training. Response onset latencies. Average onset latency on CS-alone trials during trace conditioning was the only measure significantly affected by motor cortex lesions, F(1, 10) = 6.5, with lesioned rabbits consistently responding slower than controls. There was no significant difference between lesion and control groups on paired trials, F(1, 10) = 4.2, p =.06. Average onset latencies on CS-alone trials got significantly shorter, F(2, 20) = 36.1, over the course of training for both groups as CRs developed (see Figure 9A). The interaction of group and training day was not significant, F(2, 20) = 1.7. A post hoc analysis was conducted taking into account onset latency only on those CS-alone trials on which a CR occurred. Because of the slow learning rates, only the criterion day had sufficient data in both groups for a comparison analysis (see Figure 9B for

9 MOTOR CORTEX LESIONS AND EYEBLINK CONDITIONING A E E v 03 "O E,< i O T ACQ1 MID CRIT O4 o 4 psi _~. 800 s 3 psi ~ 700.t 2 psi c O a 600 I 1 psi n C~ 500 " O 400 CONTROL >,, CRs c o LESION -~" = 200 CRs.-I 100 o 0 Control Group ~! (N-S) Lesion Group 1 (N=7) i i i ACQ1 MID CRIT Figure 8. Reflex and conditioned response amplitudes over the course of training for lesion group (I/O tests and square and dotted line). Only CR amplitudes are presented for control rabbits (circle and solid line). Errors bars represent standard error of the mean. CR = conditioned response; CS = conditioned stimulus; psi = pounds per square inch; I/O = input/output; ACQ1 = first day of training; MID = middle day of training; CRIT = criterion day of training. individual data points). A t test performed on the group data on the first criterion day (CRIT) shows that lesioned rabbits produced significantly later CRs than nonlesioned rabbits, t(10) = Histology. Lesions were comparable to those made in Experiment 1 on delay conditioning. Cresyl stained coronal sections indicate that the dorsal hippocampus, subiculum, and entorhinal cortex remained intact in all cases, although swelling in the cavity of some rabbits may have exerted pressure, causing some displacement or distortion. Reconstructions of minimal and maximal damage are presented in Figure 6D. Lesion size was not correlated with rate of learning (see Table 1, trace group) for rabbits lesioned before acquisition of the trace conditioned eyeblink, although the significance criterion was closer than for delay conditioning (r 2 =.71, where p =.05 was.79). A second ranking of all the lesions from Experiment 1 and Experiment 2 taken together indicated there was no clear difference across experiments in extent of damage between delay and trace conditioned rabbits with motor cortex lesions. Discussion Bilateral frontal lesions did slightly, but not significantly, impair overall performance on the trace conditioning task. During trace conditioning, onset latencies were the only measure for which statistical significance was observed and, then, only on CS-alone trials. The delay in CR onset for lesioned rabbits may have been a secondary consequence of lower CR rate and amplitude trends and the interdependence between these measures and latency. However, the effects were not like the dramatic impairments in the timing of trace CRs observed following hippocampal damage or disruption B 600 :~ 500 t- O 400 o f- O 100 n- 0 Individual s = CONTROL LESION! Group Means i i i i i i ACQ1 MID CRIT ACQ1 MID CRIT Figure 9. A: Response onset latencies on CS-alone trials during training. All subjects showed a decreasing latency to respond across acquisition days. Rabbits lesioned before any training (square and dotted line represent lesion group) were consistently slower to respond than unlesioned controls (circle and solid line represent control group). B: Onset latencies for CRs only are presented as individual data (left) and group means (right). Lesioned rabbits produced significantly later CRs than controls when compared on their first criterion day (CRIT). Error bars represent standard error of the mean. CS = conditioned stimulus; CR = conditioned response; ACQ1 = acquisition day 1;MID = middle. of hippocampal efferents (Port et al., 1986; Solomon et al., 1986). Of the 7 rabbits in the present trace conditioning lesion group, 4 rabbits sustained minimal damage to anterior cingulate cortex, 2 sustained minimal damage to unilateral cortex (Rabbits and ), and 2 sustained minimal damage to bilateral cortex (Rabbits and ). One of these rabbits also received slight damage to dorsal anterior retrosplenial cortex bilaterally (Rabbit ), whereas 1 other rabbit had unilateral damage to anterior retrosplenium sparing a significant portion of cingulate cortex (Rabbit ). When lesions were ranked according to extent of damage, rabbits with lesions including these additional areas were ranked as most severe, but degree of damage did not correlate with rate of learning. 1

10 736 IVKOVICH AND THOMPSON UR performance on I/O tests before and after trace conditioning followed the same pattern of continued enhancement over the course of training as observed in Experiment 1 and reported elsewhere (Steinmetz et al., 1992). I/O data were not collected for unlesioned subjects in this experiment, but comparison to unlesioned subjects in Experiment 1 (see acquisition phase for the retention group) suggests that there is a possibility that motor cortex lesions facilitated UR enhancement from I/O Test 1 to I/O Test 2 in the trace condition. This may be an artifact of longer acquisition training in the trace conditioning paradigm and continued growth of UR amplitude. Still, rabbits with motor cortex lesions in Experiment 1 exhibited a general increase in response amplitude after lesioning. If this UR enhancement effect were to be significant for lesioned rabbits relative to controls, it would provide further evidence for cortical modulation of behavior. The only significant difference in Experiment 2, however, was in the onset latency of CRs on CS-alone trials, which may be attributable to trends toward fewer and smaller CRs in the lesion group. All other measures showed a nonsignificant trend toward impairment for the lesion group. General Discussion As our experiments demonstrated, motor cortex is not essential for conditioning of the eyeblink response in rabbits trained either with a delay paradigm or with a 500-ms trace procedure. Bilateral lesions of motor cortex, including frontal cortex, had no effect on learning or performance during delay conditioning. Similar lesions produced slight, though nonsignificant, impairment across all measures during trace conditioning. A significant difference in onset latency for CRs was observed in lesioned rabbits during trace conditioning, but may be attributable to fewer and smaller CRs. Although decerebration and decortication studies (Mauk & Thompson, 1987; Norman et al., 1977; Oakley & Russell, 1977) have demonstrated that structures above the level of the cerebellum-red nucleus are not essential for the acquisition of classical delay conditioning, other studies have indicated a modulatory role for these structures. In some cases, the involvement of higher brain structures is necessary for optimal learning and performance. Neurons in auditory regions of thalamus shift their optimal response to match the frequency of an auditory conditioning stimulus (e.g., Bakin & Weinberger, 1990; Diamond & Weinberger, 1986). Neural models of conditioned behaviors (eyeblink, jaw movement, and hindlimb flexion) have been recorded from hippocampus (Berger & Thompson, 1978; Berry & Oliver, 1982; Thompson et al., 1980, respectively), and stimulation of hippocampus can facilitate eyeblink conditioning (Prokasy, Kesner, & Calder, 1983). Further, the hippocampus has specifically been implicated in complex discrimination reversal tasks. Rabbits with damage to dorsal hippocampus bilaterally were impaired in reversal learning (Berger & Orr, 1983). In addition, dorsal hippocampal lesions altered performance for trace classical conditioning. Rabbits were massively impaired in acquisition of condi- tioned responding following tone and airpuff pairings separated by a 500-ms silent period. When the hippocampectomized rabbits did respond to the CS, the distinctive feature of these responses was a short onset occurring 600 ms before presentation of the US (Moyer et al., 1990; Port et al., 1986; Solomon et al., 1986). Another more recent study (Kim et al., 1995) has shown that hippocampectomy abolishes the memory of recently, but not remotely, acquired trace eyeblink CRs. Rabbits trained with a 500-ms trace interval and lesioned 1 day after reaching criterion did not retain conditioned responding when tested postlesion. However, rabbits lesioned 1 month following criterion showed perfect retention. Berger and Orr (1983) reported a massive deficit in reversal learning that required a change in conditioned behavior from one CS to another, previously unreinforced stimulus, as a result of hippocampal lesions. In fact, damage to retrosplenial cortex, which links the hippocampus to both cerebellum and motor cortex (Bassett, 1987), is sufficient to cause similar impairment (Berger, Weikert, Bassett, & Orr, 1986). Hippocampal information is transmitted to the retrosplenial cortex via the subiculum and is linked to cerebellum by way of projections to the ventral pontine nuclei. Other projections from retrosplenial cortex synapse in the ventrolateral thalamus, which feeds into motor cortex and can also influence cerebellar circuits (see Figure 1). Although retrosplenium was generally spared in the present study, the 2 rabbits in the trace conditioned group, which did sustain some damage to that area, were among the slowest learners. However, the 1 rabbit that did not learn in the allotted 25 days of training was spared damage to retrosplenial cortex. Other studies have specifically addressed the involvement of motor cortex in classical eyeblink conditioning. Woody and colleagues have used electrophysiology and lesion techniques to study the motor cortex in cats during trace eyeblink conditioning with a click CS and glabellar tap US (Woody & Engel, 1972; Woody et al., 1974). They have reported that motor cortex is essential for the development and production of short-latency (<20 ms) CRs, as measured by EMG, which represents the initiation of a behavioral response. In contrast, the present studies are of the longlatency adaptive conditioned eyeblink/nictitating membrane response that tends to peak at about the onset of the US over the effective range of CS-US onset intervals. Ablation does not significantly affect learning or performance of this standard conditioned response. We suggest that the very short-latency EMG response studied by Woody and associates may involve different neuronal circuits and may in fact be a component of the startle response to a sudden acoustic stimulus (Davis, 1984; Woody et al., 1974). The fact that spreading cortical depression induced in the neocortex abolishes the standard delay long-latency conditioned eyeblink response when motor cortex is depressed (Papsdorf et al., 1965) is of considerable interest in view of the fact that ablation of the motor cortex has no such effect. Solomon and associates found a similar result in the hippocampal system: Disruption of activity in the hippocampus was more detrimental than removing the structure in standard delay eyeblink conditioning (Solomon, Solomon,

11 MOTOR CORTEX LESIONS AND EYEBLINK CONDITIONING 737 Vander Schaff, & Perry, 1983). Cortical spreading depression spreads to subcortical systems (Marshall, 1959). In the present context, perhaps the process interferes with the critical cerebellar circuitry by way of motor cortex projections to the pontine nuclei (Wiesendanger & Wiesendanger, 1982). In a study of neural discharge patterns in motor cortex and red nucleus associated with skilled motor movements in monkeys, Martin and Ghez (1988) found both cell types to be related to initiation of movement, but motor cortical cells coded for direction of movement toward a target (see also Georgopoulos, Ashe, Smyrnis, & Taira, 1992), whereas red nucleus cells did not. The authors speculated that the red nucleus may be involved in the adaptive modification of motor patterns with changes in load or task requirements. The spatial nature of this type of motor task, as well as its operant characteristics such as presence of a cue and longer time course of behavior response, may be a determining factor for motor cortical modulation of movement and may explain why broader effects were not observed in the current experiments. Indeed, the observed small effects on CR timing in the present study may be a component of a larger effect on a system for integrating the simple conditioned eyeblink with other complex movements (see also Sears et al., 1996). In summary, the present study does not support the hypothesis that motor cortex is a structure critical for the conditioned eyeblink response in rabbits trained with tonealrpuff pairings. There are essentially no effects at all of extensive bilateral lesions of the motor and frontal neocortex on acquisition or retention of standard delay or trace classical conditioning of the eyeblink response in rabbits. References Allen, G. I., & Tsukahara, N. (1974). 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