Involvement of the ventrolateral thalamic nucleus in rabbit classical eyeblink conditioning

BEHAVIOURAL BRAIN RESEARCH

ELSEVIER

Behavioural Brain Research 74 (1996) 105-117

Research report

Involvement of the ventrolateral thalamic nucleus in rabbit classical eyeblink conditioning Lonnie L. Sears, Sheree F. Logue and Joseph E. Steinmetz * Department of Psychology, Program in Neural Science, Indiana University, Bloomington, IN 47405, USA Received 8 March 1994; revised 18 July 1994; accepted 24 February 1995

Abstract

The involvement of the ventrolateral nucleus of the thalamus in relaying learning-related activity to higher brain structures during classical conditioning of the rabbit eyelid response was examined in two experiments. In the first study, multiple-unit ventrolateral thalamic nucleus activity was monitored before and after lesions of either the cerebellar interpositus nucleus or red nucleus were given. Before the lesions were given, conditioned response-related activity was observed in the ventrolateral thalamic nucleus. Lesions of the interpositus nucleus, but not the red nucleus, disrupted the conditioning-related activity in the ventrolateral thalamic nucleus, thus suggesting that an efferent copy of conditioned response-related activity is projected directly from the interpositus nucleus to higher brain areas by way of the ventrolateral thalamic nucleus. In the second study, multiple unit activity in the hippocampus was monitored before and after lesions were placed in the ventrolateral thalamic nucleus or red nucleus. Conditioning-related activity in the hippocampus was not affected by either lesion, thus suggesting that maintenance of trainingrelated activity in the hippocampus is not critically dependent on cerebellar information relayed through the ventrolateral thalamic nucleus or red nucleus. Keywords: Classical conditioning; Rabbit; Cerebellum; Thalamus; Hippocampus; Lesion; Multi-unit recording

1. Introduction

A variety of experiments have shown that the cerebellum is essentially involved in acquisition and performance of the rabbit classically conditioned eyeblink response. For example, single- and multiple-unit recordings from the cerebellum revealed cell activity related to the presentation of the conditioning stimuli as well as learning and performance of the eyeblink response [5,6,17]. Also, lesions of the interpositus nucleus prevented acquisition of conditioned responses [29] and abolished previously learned conditioned responses 1-13,16,31,35]. Evidence for critical plasticity in the cerebellum includes microstimulation studies of cerebellar afferents (e.g., [32]). Furthermore, lesions of afferent pathways relaying conditioned stimulus (CS) and unconditioned stimulus (US) information have produced abolition of the conditioned response (CR) [15,33,36], thus * Corresponding author. Fax: + 1 812-855-4691. e-mail: [email protected]. 0166-4328/96 $09.50 © 1996 Elsevier Science B.V. All rights reserved SSDI 0 1 6 6 - 4 3 2 8 ( 9 6 ) 0 0 1 7 1 - 9

supporting convergence of CS and US information in the cerebellum. Neural activity related to learning the conditioned response has been reported in other brain areas such as the hippocampus [4]. Even though lesions of the hippocampus do not abolish simple delay conditioned responses, hippocampal lesions do affect trace conditioned responses, thus suggesting that the hippocampus is involved in encoding important features of conditioning such as adaptive timing of the learned response (e.g., [ 19, 22, 30]). Furthermore, hippocampal plasticity during this type of motor learning appears to require an intact cerebellum. Lesions of the interpositus nucleus prevented training-related hippocampal plasticity [29] and abolished conditioning-related activity in the hippocampi of trained animals [ 11 ]. Even though it appears that interactions between neuronal activities in the cerebellum and hippocampus occur during conditioning, the neural pathways involved in these interactions have not been delineated. To date, we have investigated one system that could have been

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involved in projecting conditioning-related information from the cerebellum to the hippocampus; the septohippocampal system [ 18]. In that study, we found that lesions of the cerebellum that effectively abolished behavioral CRs did not affect the appearance of CS- and US-related activity characteristic of the medial septum but did abolish CR-related activity recorded from the lateral septum, a hippocampal efferent target. We thus concluded that essential conditioning-related activity from the cerebellum was not projected to the hippocampus by way of the medial septum. The present study evaluated the possible involvement of a second system that could play a role in projecting conditioning-related activity between the cerebellum and hippocampus, namely, projections from the interpositus nucleus to the red nucleus and the ventral lateral thalamic nuclei. This system is of interest because of its important involvement in motor control. The red nucleus contains cells that exhibited classical conditioningrelated activity. This activity, however, was abolished by reversible lesions of the interpositus nucleus [8]. The ventrolateral thalamic nucleus is of interest because it plays a critical role in relaying information between the cerebellum and motor cortex during voluntary movement (e.g., [1,27,28]). These cerebellar efferents, therefore, would seem to be potential indirect pathways of information flow for conditioning-related activity from the cerebellum to other higher structures like the hippocampus. Two experiments assessed the role of the red nucleus and ventrolateral thalamus in relaying conditioningrelated information from the cerebellum to higher brain structures. In the first experiment, anatomical and electrophysiological evidence is presented concerning the locations of training-related activity in the ventrolateral thalamus in rabbits during classical delay conditioning. Although both interpositus nucleus and red nucleus lesions abolished conditioned responses, only cerebellar lesions abolished training-related activity in the ventrolateral thalamic nucleus. The second experiment indicated that lesions of the same areas of the ventrolateral thalamus or of the red nucleus, where conditioned response-related activity could be found, had no effect on hippocampal training-related activity in contrast to the effects seen following cerebellar lesions.

2. Experiment 1 In the first study, multiple-unit neuronal recordings from the ventrolateral thalamic nucleus were observed before and after lesions of the red nucleus or interpositus nucleus. These recordings were done in hopes of determining the relative effects of disrupting two major projection systems to the thalamus known also to be involved in projecting eyeblink conditioned responses to

brain stem nuclei responsible for generating the learned behavior. These projection systems include interpositus projections that course directly to the ventrolateral thalamus and interpositus projections that are relayed via the red nucleus to the thalamus. Also included in this experiment were a series of HRP injections designed to confirm that regions of the interpositus nucleus known to be involved in generating classically conditioned responses projected directly to those regions of the ventrolateral thalamic nuclei from which we recorded.

2.1. Method 2.1.1. Subjects Twenty-six male, New Zealand, albino rabbits that weighed 2.0-3.0 kg were used in Experiment 1. Due to inaccurate placement of recording electrodes revealed in subsequent histological analysis, data from four rabbits were not used in the analyses reported below. All rabbits were individually housed, given ad lib access to food and water, and maintained on 12/12-h light/dark cycles. 2.1.2. Surgery Surgery was performed under aseptic conditions. Surgical anesthesia was initiated and maintained with im injections of xylazine (6mg/kg) and ketamine (60 mg/kg). Rabbits were placed in a stereotaxic headholder with the bregma landmark positioned 1.5 mm above the lambda skull landmark. Recording electrodes (insulated stainless steel 00 insect pins, approx. 1 M~2 impedance) were lowered into the right and left ventral lateral thalamus using stereotaxic coordinates (5.0 mm posterior, 4.5 mm lateral, and 12 mm ventral to the bregma skull landmark). Neural activity was recorded while the electrodes were lowered to aid in determining final electrode placement. Lesion electrodes (insulated stainless steel 00 insect pins, 0.5 mm exposed tips) were also implanted in either the left interpositus nucleus (n-12) or bilaterally in the red nucleus (n--14) also using stereotaxic coordinates and observations of neural activity. Recording and lesion electrodes were cemented to the skull. A plug assembly designed to hold a headstage device to be used in behavioral training was also cemented into place. Following surgery the rabbits were given a 1-week recovery period. 2.1.3. Behavioral training For behavioral training, rabbits were placed in standard, padded Plexiglas restraint boxes inside a soundattenuating chamber. Following two 45-min adaptation sessions, paired presentations of a 90 dB, 1 kHz tone CS and air puff US (2.1 N/cm 2) were used to produce classical conditioning. The duration of the tone CS was 348 ms co-terminating with the 99 ms air puff US, thus creating a 249 ms interstimulus interval (ISI). During training, movement of the nictitating membrane (NM)

Lonnie L. Sears et al./Behavioural Brain Research 74 [ 1996) 105-117

was measured with a minitorque potentiometer connected by a thread to a suture placed in the rabbit's NM during surgery. The NM movements were transduced to voltage signals allowing for measurement of several response parameters such as percent CRs (defined as 0.5 mm of NM movement during the CS-US interval), response amplitude, and response latency. Presentation of conditioning stimuli and recording of neural and behavioral data were controlled by a computer [-14]. Each training session consisted of 120 trials divided for the purpose of data analysis into 12 blocks of 10 trials. The first trial of each block was a tone-alone test trial and the remaining 9 trials were CS-US paired presentations. Inter-trials intervals ranged from 20 to 30 s ( M = 2 5 s). Each rabbit received 12 training sessions. During Sessions 1-4 the air puff was presented to the left eye, while Sessions 5-8 were right-eye training sessions. Following Session 8, 30 s of 1 mA dc was passed through the lesioning electrode located in either the right and left red nucleus or left interpositus nucleus. During Sessions 9-12 the air puff was presented to the left eye, since left-eye conditioning should be abolished by either bilateral red nucleus and left interpositus lesions.

2.1.4. Neural recording Neural activity was amplified and band-pass filtered (500 to 5000Hz) prior to being sent to a window discriminator. Multiple units were then counted by a computer based on spikes breaking a preset level in consecutive 3 ms bins across the 747 ms trial (249 ms prior to the CS, 249 ms interval between CS and US onset, and 249 ms following US onset). The window discriminator was set before each session began to discriminate action potentials with amplitudes two or more times greater than background activity (i.e., 4 to 6 neurons per session). Neural activity was analyzed by dividing each trial into 6 periods. Four sequential CS periods were created consisting of 62 ms of discriminated unit activity (CS-1, CS-2, CS-3 and CS-4). Two US periods were created reflecting discriminated unit activity in consecutive 62 ms bins (US-1 and US-2). Raw unit counts and the behavioral data were stored on a floppy disk for subsequent off-line analysis. Raw unit counts were converted to standard scores for statistical analysis. Raw unit counts during 62 ms of the preCS period (i.e., baseline activity) for a 10-trial block was subtracted from block unit counts for each CS and US period and than divided by the standard deviation of average preCS activity across the entire session. This procedure produced a 12 × 6 Blocks × Periods matrix of standard scores. Session averages of period scores were used for subsequent statistical analyses.

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2.1.5. Histology Following completion of the 12 training sessions, rabbits were overdosed with an i.v. injection of pentobarbital (4 cc) and perfused via the ascending aorta with saline followed by 10% formalin. Ten seconds of 100 ~tA dc was passed through the recording electrode to mark recording sites. The brains were removed and placed in a 10% formalin/30% sucrose solution. After 1 week the brains were blocked in albumin/gelatin, frozen, and sectioned in the coronal plane. Sections were taken from locations where recording and lesion electrodes were placed. Ten sections were mounted on gelatinized slides and stained with cresyl violet and potassium ferrocyanide. Locations of recording electrodes and lesion areas were determined by viewing the stained sections under a microscope.

2.1.6. WGA-HRP analysis Eight additional adult albino rabbits were used to examine projection patterns from the anterior interpositus nucleus to areas of the thalamus to verify anatomical connections corresponding to the recorded neural activity. It should be noted that these anatomical tracings were not undertaken to provide a thorough and complete description of the projection patterns from the cerebellum to the thalamus in the rabbit, but rather to provide independent, anatomical confirmation that the area we lesioned in the interpositus nucleus sent axons directly the area of the thalamus where we obtained the present recordings. The rabbits were anesthetized with a mixture of xylazine (6mg/kg) and ketamine (60 mg/kg). Wheat germ agglutinin conjugated to HRP (WGA-HRP; 2.0%) was pressured injected with a stereotaxically placed Hamilton microliter syringe into the anterior interpositus nucleus. Injection values ranged from 0.05 to 0.3 ~tl. Five min after positioning the syringe needle, the WGA-HRP conjugate was injected in approx. 0.02 ~tl increments over several minutes. After completing the injection, the needle was left in place for an additional 5 min and then slowly withdrawn. After 5 days were allowed for uptake and transport, the rabbits were overdosed with pentobarbital and perfused through the aorta with saline followed by cold 1% paraformaldehyde/1.25% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2-7.4). The brains were removed and placed in cold fixative for 5 h and then stored in cold sucrose/phosphate buffer overnight. The following day, brains were embedded in albumin gelatin, the blocks rapidly hardened with phosphate buffered glutaraldehyde, and frozen sectioned coronally at 40 ~tm. Sections were collected in cold phosphate buffer and reacted immediately with tetramethyl benzidine. After processing, the sections were mounted on gelatin-subbed slides and counterstained with thionin. Injection sites were examined using a projection microscope and the maximal extent of the injection was plotted on a standard

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set of reference sections. Brain sections were examined microscopically under both brightfield and darkfield illumination (final magnification = 312.5 ×). The location of HRP-labeled cell bodies or terminal fields was noted on reference sections.

2.1.7. Results Neural activity and behavioral responses were analyzed by comparing data from 3 groups of rabbits. The non-effective lesion group included rabbits with missed or partial interpositus nucleus or red nucleus lesions (n=8), i.e., all had greater than 50% CRs after the lesions. The interpositus lesion group had less than 10% CRs following the lesion (n= 7). The red nucleus lesion group also had less than 10% CRs on any session after the lesion (n = 7). All rabbits had significant CR-related activity prior to lesioning (see below for criteria). 2.1.8. Behavioral training Fig. 1A shows percent CRs for the three groups across the 12 sessions. Percent CRs were analyzed with a Session × Group within factor analysis of variance. There was a significant interaction effect of Session and Group on percent CRs [F(22,176)= 7.16, P < 0.001 ] with a post hoc Tukey tests (P values <0.05) yielding the following results: Sessions 1-4. No differences in the three groups were found in percent CRs across initial left-eye training. There was a significant increase in percent CRs between Session 1 and Session 4 in all three groups. Sessions 5-8. During right-eye training there were no differences between the groups on percent CRs indicated by non-significant Group main or interaction effects. There was a significant Session effect with an increase in percent CRs from Session 5 to Session 8. Session 9-12. During sessions following the lesion there was a significant difference between groups. The non-effective lesioned group increased from 59% CRs on Session 9 to 67% CRs on Session 12. The interpositus nucleus and red nucleus lesion groups had significantly fewer CRs than the non-effective lesioned group on postlesion training. Both the interpositus nucleus and red nucleus lesion groups had fewer than 5% CRs on each of the final sessions. Summary. Rabbits in all three groups achieved similar levels of conditioned responding during initial right- and left-eye sessions. Lesions encompassing either the interpositus or red nucleus abolished conditioned responding (see Histological Analysis). Partial lesions or lesions outside of these structures produced either reductions in CRs or no effect on percent CRs, respectively. 2.1.9. Neural recording Laterality differences. An analysis of variance was used to assess differences in right and left ventral lateral thalamus activity prior to lesioning. Of the 22 rabbits in

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Fig. 1. (A) Average percent conditioned responses for interpositus nucleus (O), red nucleus (A), and noneffective (HI) lesion groups across training sessions. (B) Average standard scores of ventral lateral thalamus multiple-unit activity for the CS-4 period across all training sessions for interpositus nucleus (circles), red nucleus (triangles) and noneffective (squares) lesion groups. Sessions 1 to 4, left eye training; Sessions 5 to 8 right eye training; Sessions 9 to 12, post-lesion left eye training.

the 3 groups, 12 had recording electrodes located in posterior regions of the right ventral lateral thalamus and 10 had electrodes in the left ventral lateral thalamus (see Histological Analysis). Results of the Training Side x Recording Side analysis of variance indicated that there were no significant differences between right and left ventrolateral thalamic nucleus unit activity overall, and no differences in ventrolateral thalamic nucleus unit activity related to the eye receiving air puff presentation. Therefore for the following analyses data from both right and left ventrolateral thalamic nucleus were combined and analyzed together for all three groups. A Sessions x Group within factor analysis of variance of the standard scores of neural activity indicated a significant interaction effect of Session and Group for neural activity in all CS and US periods (all P values

Lonnie L. Sears et al./Behavioural Brain Research 74 (1996) 105 117

<0.05): CS-1 period, [F(22,176)=2.24]; CS-2 period, [F(22,176)=2.79]; CS-3 period, [F(22,176)=2.18]; CS-4 period, [/7(22,176)=2.94]; US-1 period, [F(22,176) = 3.67]; and US-2 period, [F(22,176) = 2.27)]. Tukey tests revealed the following results. Sessions 1-4. There were no significant differences in standard scores of neural activity between the three groups during Sessions 1 to 4. There was a significant increase in neural activity from Session 1 to 4 in all groups for CS-3, and CS-4 periods (i.e., the latter half of the CS period). Fig. 1B shows neural activity across sessions for the CS-4 period (i.e., when substantial CR-related activity is expected within each trial). The CS-4 standard scores increased from 1.3 during Session 1 to 2.5 for Session 4. The CS-3 activity followed a similar pattern across sessions. Standard scores for CS-3 increased from 0.8 during Session 1 to 2.1 in Session 4. The CS-1 and CS-2 period neural activities were low and did not change over training. The increased neural activity during the CS-3 and CS-4 period corresponded well with the increase in percent CRs and reflected an increase in training-related neural activity in the ventrolateral thalamic nucleus. In fact, the ventrolateral thalamic nucleus activity could be described as CR-related since significant standard scores (i.e., scores greater than 1.96) were seen in the CS period, the onset of this increased neural activity was at the time of behavioral CR onset, and the unit activity correlated with CR topography at r values greater than 0.70 (using crosscorrelational procedures). Sessions 5-8. No significant differences between groups existed for Sessions 5 to 8 (see Fig. 1B). Activity during Session 5, the first session of right-eye training, was not different than neural activity seen during Session 1, the first day of left-eye training. Average standard scores for the three groups increased significantly during CS-3 and CS-4 periods with training on the right eye. The CS-3 average standard score on Session 5 was 0.9 while the CS-3 standard score for Session 8 was 1.8. Average CS-4 neural activity increased from 1.3 on Session 5 to 2.3 during Session 8. The increased neural activity during the CS-3 and CS-4 period paralleled the development of conditioned responding. Sessions 9-12. Following electrolytic lesions there was a significant difference between the interpositus nucleus group and the other two groups in CS-2, CS-3, CS-4, US-1 and US-2 period neural activity (see Fig. 1B). The interpositus nucleus group had no significant neural activity in any of these periods evidenced by no standard scores above 0.4. In contrast the non-effective lesion and red nucleus lesion groups showed significant activity during the CS and US periods with standard scores during CS-3 and CS-4 above 2.0. Complete lesions of the interpositus nucleus, but not lesions of the red nucleus or partial lesions of the red nucleus or interpositus nucleus, abolished training related activity.

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Summary. Of particular interest was the abolition of training-related neural activity in the animals with interpositus lesions but not in the other groups. This effect was apparent in CS-4 period standard scores illustrated in Fig. lB. There was, however, a slight decrease in CS-4 activity from Session 8 to Session 9 in the red nucleus group, but this group was at pre-lesion levels of trainingrelated neural activity by Sessions 10-12. The red nucleus lesions, therefore, appeared to cause only a temporary disruption of neural activity in the ventral lateral thalamus. Other thalamic areas. Several animals had recording electrodes located in other areas of the thalamus. Neural activities in these animals across training sessions were analyzed. Two rabbits with electrodes in medial and posterior areas of the ventral posterior thalamus (see Histological Analysis) had significantly increased standard scores for the US-1 period indicating air puffevoked activity. There was no significant difference across sessions in US-1 activity and this activity was not affected by lesions of either the red nucleus or interpositus nucleus. Average standard scores for these 2 animals were 2.5 during Sessions 1-4, 2.3 for Sessions 5-8, and 2.4 for Sessions 9-12. One rabbit evidenced both tone CS and air puff US evoked activity during initial left-eye training. The recording electrode was located in medial and anterior areas of the right medial geniculate nucleus (MGN). As percent CRs increased the air puff evoked activity decreased while activity following the tone was similar across sessions. Thus, there appeared to be an across training suppression of air puff evoked auditory activity in this area of the medial geniculate. Other researchers have reported conditioning-related activity in the MGN. During trace conditioning, O'Connor el al. [20] observed training related MGN activity during the CS and trace intervals that seemed to be time-locked to CS-offset, possibly suggesting a role for the MGN in CR timing. 2.1.10. Histological analysis Locations of recording electrodes are shown in Fig. 2. Recording sites where training-related activity was seen are indicated. According to the rabbit brain atlas of Urban and Richard [34], all of these locations were located bilaterally in medial and posterior regions of the ventrolateral thalamic nucleus. It should be noted that in other species, such as the monkey, finer anatomical subdivisions of this thalamic region have been described. This region of the rabbit thalamus seems to correspond best with the posterior ventrolateral nucleus (VLp) described by Jones [ 12]. In a stereotaxic monkey atlas authored by Olszewski [21], the ventral portion of the VLp that lies anterior to the ventral posterior nucleus was labeled the pars oralis of the ventral posterolateral nucleus (VPLo), the dorsal part of the VLp was called

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the pars caudalis of the ventral lateral nucleus (VLc), the posterior tail of the VLp was called the pars postrema of the ventral lateral nucleus (VLps), and its large anterior extension was referred to as the nucleus X. If these subdivisions are present in the rabbit, it is likely that the present recording sites included locations within each of these regions since they all receive input from the cerebellar nuclei [2,3]. The sites in the ventral posterior thalamus that evidenced air puff-evoked activity and the site in the medial geniculate with CS- and US-evoked neural activity are also indicated. Areas where no stimulus or response related activities were recorded are also shown. These areas were located in fiber tracts or in other thalamic nuclei. Fig. 3 shows both the smallest and largest areas encompassed by lesions in rabbits in the interpositus nucleus group. Lesions that abolished conditioned responding included large areas of the anterior interpositus nucleus. Rabbits in the non-effective lesion group had lesions lateral and posterior to this area of the interpositus. Fig. 4 indicates the smallest and largest

lesion areas for rabbits in the red nucleus lesion group. Lesions involving caudal regions of the red nucleus abolished conditioned responding. Rabbits in the noneffective lesion group had lesions in areas posterior and lateral to caudal regions of the red nucleus.

2.1.11. WGA-HRP analysis Histoanatomical analysis of the 8 rabbits with WGAH R P injections revealed 6 rabbits with injections sites in the anterior interpositus nucleus and areas of white matter dorsal to the nucleus. Microscopic examination of all 6 rabbits revealed H R P reaction product characteristic of labeled axon terminals in the contralateral ventral lateral thalamus (as described in the Urban and Richard [34] rabbit brain atlas). These areas corresponded well with the thalamic locations where training-related neural activity had been observed in rabbits used for the recording portion of this study. A small amount of terminal labeling was also observed in the dorsal-most aspects of the ventral posterior thalamic nuclei. No labeling in the ipsilateral thalamus was observed. Fig. 5

Lonnie L. Sears et aL/Behavioural Brain Research 74 (1996) 105-117

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2.1.12. Experiment 2

In the second experiment, the involvement of the ventrolateral thalamic nucleus and red nucleus pathways in potentially relaying training-related information from the cerebellum to the h i p p o c a m p u s was assessed. In this experiment, training-related h i p p o c a m p a l activity and

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after the lesion, rabbits in both groups were given 3 more sessions (Sessions 4 to 6) of left-eye training. 2.1.14. Results

Fig. 5. Schematic drawings of coronal sections through the cerebellum (upper right) and thalamic nuclei (lower left) from a representative rabbit that received an WGA-HRP injection into the region of the deep cerebellar nuclei. The cross-hatched areas of the cerebellar sections show the extent of the HRP injection as revealed in brightfield microscopy. The individual dots in the thalamic sections depict the location of HRP reaction product characteristic of terminal labeling. (Abbreviations: MD, nucleus medialis dorsalis thalami; VL, nucleus ventralis lateralis thalami; VM, nucleus ventralis medialis thalami; VP, nucleus ventralis posterior thalami.)

2.1.15. Behavioral training Red nucleus lesion group. Rabbits in the red nucleus group showed a significant increase in the percentage of conditioned responses from Session 1 (15% CRs) to Session 3 (94% CRs) (see Fig. 6A). Following bilateral red nucleus lesions there was a significant decrease in percent CRs IF(l,5) =1349.55, P<0.001]. The red nucleus group had 2% CRs on Session 4, 2% CRs on Session 5 and 4% CRs during Session 6. No rabbits had over 10% conditioned responses for Session 4 to 6. Ventral lateral thalamus lesion group. In ventrolateral thalamic nucleus-lesioned rabbits, similar to the red nucleus lesion group, there was a significant increase in conditioned responding from Session 1 (5% CRs) to Session 3 (85% CRs). In contrast to the red nucleus lesion group, however, rabbits with ventrolateral thala-

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2.1.13. Method Eighteen rabbits were used in Experiment 2. Data from 13 rabbits with accurate electrode placements were used for the data analyses described below. Surgical procedures were similar to those described above except recording electrodes were placed bilaterally in the hippocampus and lesion electrodes were placed bilaterally in either the ventral lateral thalamus or red nucleus. Rabbits with both recording electrodes located in the hippocampus and lesion electrodes in the red nucleus comprised the red nucleus lesion group (n =6). Rabbits with hippocampal recording electrode placements and with lesion electrodes located bilaterally in posterior regions of the ventral lateral thalamus were included in the ventrolateral thalamic nucleus lesion group (n= 7). Behavioral training and neural recording procedures were as described in Experiment 1. Rabbits in the red nucleus and ventrolateral thalamic nucleus lesion groups were given 3 daily sessions of left-eye training (Sessions 1 to 3) and then given a 30 s, 1 mA dc lesion. One day

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Fig. 6. (A) Average percent CRs for the red nucleus lesion group ( 0 ) and ventrolateral thalamic nucleus lesion group (11). Sessions 1-3 were given before the lesions while sessions 4-6 were given after the lesions. (B) Hippocampal activity during the CS-4 period across Sessions 1-6 for the red nucleus lesion group ( 0 ) and the ventrolateral thalamic nucleus lesion group (11).

Lonnie L. Sears et al./Behavioural Brain Research 74 (1996) 105-117

mic nucleus lesions had only a small, non-significant decrease in percent CRs from Session 3 to Session 4 following the lesion (see Fig. 6A) based on a within analysis of variance. Summary. Lesions of the red nucleus abolished conditioned responses in trained rabbits, in agreement with previous studies of red nucleus lesion effects [7-9,26]. Ventrolateral thalamic nucleus lesions, in contrast, had no effect on the percentage of conditioned responses observed following the lesion.

2.1.16. Hippocampal unit activity Red nucleus lesion group. Fig. 6B shows standard scores of unit activity recorded during the CS-4 period over the 6 sessions of training. A Session x Period analysis of variance followed by a Tukey test was done on standard scores from Sessions 3 and 4, the sessions before and after the lesion, to assess changes in hippocampal activity due to the lesion. Results of the analysis indicated that there was no significant difference in neural activity between Session 3 and 4 [F(1,7)=0.16, NS]. There was, however, a significant increase in the CS-2, CS-3, CS-4, and US-1 periods [F(5,30=15.85, P <0.001 ] for both sessions. The increase in hippocampal activity during these training has been previously reported [4] and was correlated highly with the increase in percent CRs. The CS-4 standard scores increased from 1.2 on Session 1 to 3.1 on Session 3 while CS-4 standard scores averaged 3.0 on Session 4 followed by 2.4 for Session 5 and 2.2 for Session 6. The increase in hippocampal unit activity followed by a decrease in activity across training sessions, producing an inverted "U"-shaped function, has been previously reported [29]. Ventrolateral thalamic nucleus lesion group. Fig. 6B indicates standard scores of hippocampal multiple unit activity during the CS-4 period across the 6 training sessions. Rabbits in this group showed training-related increases in unit activity during the CS-2, CS-3, CS-4, and US-1 periods based on an analysis of variance of Sessions 3 and 4 [F(5,30)= 13.03, P<0.001] and Tukey test. The CS-4 standard scores increased from 1.5 on Session 1 to 2.2 during Session 3. Post lesion standard scores were 2.9 on Session 4, 2.8 on Session 5 and 2.8 on Session 6. There were no significant differences preand post-lesion in hippocampal activity based on an analysis of variance of Sessions 3 and 4. Also, this group failed to show the decrease in hippocampal activity after 3-4 days of training noted in the interpositus lesion group as well as in previous experiments [29]. Summary. Recordings of hippocampal unit activity across 6 training sessions indicated that lesions of either the red nucleus or ventral lateral thalamus had no immediate effect on training-related neural activity. Hippocampal activity in both groups increased with acquisition of conditioned responding during periods

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approx. 70 ms following CS presentation with a maximal increase occurring during the CS-4 and US-1 periods.

2.1.17. Histological analysis Histological analysis for Experiment 2 followed procedures described above. The location of recording electrodes in the hippocampus are represented in Fig. 7. Recording electrodes were located in sites that have been previously shown to exhibit training-related neural activity [4,29]. Rabbits in the red nucleus lesion group had electrodes located in either the right or left CA1 region of the hippocampus. The ventrolateral thalamic nucleus lesion group had electrodes in either the right of left areas of CA1 and CA4 areas of the hippocampus. Fig. 8 shows representative bilateral lesions from rabbits with both the smallest and largest areas of lesions encompassing caudal regions of the red nucleus. Locations of both the smallest and largest bilateral lesions of the posterior ventrolateral thalamic nucleus are shown in Fig. 9. These lesions were in the same areas where training-related activity was recorded in Experiment 1. 2.1.18. General discussion Results from these experiments can be summarized as follows: (1) Cerebellar interpositus efferents projected to areas of the ventral lateral thalamus that evidenced learning-related activity during acquisition of the classically conditioned eyeblink response. (2) Lesions of either the red nucleus or interpositus nucleus abolished conditioned responding, in agreement with previous studies, while bilateral ventral lateral thalamic lesions did not disrupt conditioned responding. (3) Effective interpositus nucleus lesions, but not red nucleus lesions, abolished training-related activity in the ventral lateral thalamus. (4) Bilateral lesions of the ventral lateral thalamus or red nucleus did not abolish learning-related hippocampal activity in contrast to previous studies involving interpositus nucleus lesions. Monosynaptic projections from the interpositus contact cells in the ventral lateral thalamus as do bisynaptic connections through the red nucleus [-23,24,25]. In agreement with these observations, the recording and anatomical tract-tracing data from the present study demonstrated that areas of the interpositus essentially involved in classical eyeblink conditioning project to the ventral lateral thalamus. Injections of WGA-HRP into the region of the interpositus nucleus consistently labeled axon terminals in the contralateral ventral lateral thalamic nucleus. No labeling on the ipsilateral side was seen. The unilateral labeling observed is interesting given the fact that no significant differences in right- versus leftside thalamic activity was observed when training involved either the left or the right eye (e.g., training of the left eye produced training-related thalamic activity in both the fight and left ventral lateral thalamus and

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-6.0

-5.5

-5.0 Fig. 7. Locations of hippocampal recording electrodes for rabbits in the the red nucleus (0) and ventrolateral thalamic nucleus (11) lesion groups. Numbers indicate distance posterior to the bregma skull landmark. (Abbreviations: HP, hippocampus; DG, dentate gyrus.) The drawings were adapted from the Urban and Richard [34] rabbit brain atlas.

-8.5

-8.0

-7.5

-7.0

Fig. 8. Smallest (top) and largest (bottom) red nucleus lesions in rabbits from which hippocampal recordings were taken. Numbers refer to distance posterior to bregma. The drawings were adapted from the Urban and Richard 1-34] rabbit brain atlas. u n i l a t e r a l lesions of the c e r e b e l l u m a b o l i s h e d trainingrelated activity in b o t h the right a n d left thalamus). M o r e o v e r , when t r a i n i n g was switched to the o p p o s i t e eye, the n e u r o n a l m o d e l s d i d n o t a p p e a r until C R s were

established o n t h a t side (see Fig. 1). These r e c o r d i n g d a t a indicate t h a t p o p u l a t i o n s of n e u r o n s within b o t h the right a n d left t h a l a m u s receive i n f o r m a t i o n a b o u t C R s even t h o u g h training involves only one eye. Several

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- ~x~V L

-6.5

-6.0

-5.5

-5.0

Fig. 9. (Top) Smallest and (Bottom) largest ventrolateral thalamic nucleus lesions in rabbits from which hippocampal recording were taken. Numbers refer to distance posterior to bregma. [Abbreviations: VL, ventral lateral thalamus; VP, ventral posterior thalamus.] The drawings were adapted from the Urban and Richard[34] rabbit brain atlas.

explanations for this finding could be advanced including the possibility that (1) neuronal models are bilaterally established in the cerebellum, at least weakly, during unilateral training, (2) neuronal activity is projected bilaterally to the thalamus via the red nucleus, or (3) intra-thalamic projections transmit learning-related neuronal activity to the opposite side of the thalamus. The ventrolateral thalamic nucleus neuronal activity, recorded during classical eyeblink conditioning, models the amplitude - time course of the behavioral response. This amplitude - time course model is also observed in the interpositus nucleus [ 17]. The ventrolateral thalamic nucleus activity observed in the present study, however, was abolished by lesions of the interpositus nucleus, suggesting that an independent, essential site of plasticity sufficient to support the conditioned response, was not located in the ventrolateral thalamic nucleus. Learningrelated changes in the ventrolateral thalamic nucleus may have been produced by activation of monosynaptic cerebellar afferents from the interpositus since lesions of the red nucleus produced only a small, temporary decrement in ventrolateral thalamic nucleus activity with neuronal activity reaching pre-lesion levels by the second postlesion session. Thus, ventrolateral thalamic nucleus training-related activity during classical eyeblink conditioning appears to represent an efferent copy of the conditioned response projected from the cerebellar nuclei. A comparison of the effects of red nucleus versus interpositus nucleus lesions on ventrolateral thalamic

nucleus neural activity further supports the essential involvement of the interpositus in NM conditioning and argues against an independent critical site of conditioning-related plasticity in the red nucleus. This finding is in agreement with observations of previous studies comparing the effects of temporary chemical or cold-probe lesions of either the red nucleus [7,9] or interpositus nucleus [8,10]. The essential involvement of the interpositus is also demonstrated in the present study by the abolition of CRs following interpositus lesions. Lesions of the ventral lateral nucleus, in contrast, had no effect on CR performance. This finding argues against the essential involvement of the thalamus in plasticity underlying classical eyeblink conditioning although involvement of the ventrolateral thalamic nucleus in the acquisition of learned responses, as suggested by RispalPadel and Meftah [23], cannot be ruled out. Previous studies indicated that an intact cerebellum is essential for the establishment of learning-related hippocampal activity during classical eyeblink conditioning [29]. Lesions of areas of the ventrolateral thalamic nucleus receiving projections from the interpositus did not affect hippocampal training-related activity. Hippocampal activity following ventrolateral thalamic nucleus lesions follows the pattern observed in nonlesioned rabbits [29]. Furthermore, lesions of the red nucleus had no effect on hippocampal activity but abolished conditioned responding as expected given the role of the red nucleus in the CR motor output pathway

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[-8]. These findings suggest that information a b o u t the CR, that is essential for h i p p o c a m p a l plasticity, does not involve cerebellar efferents to the ventral lateral thalamus or red nucleus and must involve another, as of yet undefined system that links cerebellar o u t p u t with limbic system function. O u r recent data, however, suggest that the cerebellar - h i p p o c a m p a l interactions are not mediated by way of the medial septum [ 1 8 ] . Cerebellar lesions do not affect the sensory-evoked medial septal responses normally observed during classical conditioning. Thus, critical conditioning-related activity, sufficient to generate conditioning-related activity in the hippocampus, does not appear to be relayed to the hippocampus from the cerebellum via the medial septum. Results of the present study suggest that ventrolateral thalamic nucleus neural activity observed during classical eyeblink conditioning m a y reflect previously described interactive cerebellar and m o t o r cortical loops involved in coordination of voluntary m o v e m e n t (e.g., refs E27,28 ]). A cerebellar efferent copy of the learned response m a y project to m o t o r cortex as part of this coordinated system for programming, coordinating and fine-tuning movements. In the context of the present study this cerebellocortical system is not essential for classical eyeblink conditioning as m o t o r cortex is not required for this type of m o t o r learning. This type of motor-related information, however, m a y be essential for integrating this very simple learned response with more complex ongoing movements. Moreover, the observation that lesions of the interconnecting loops in this m o t o r system do not effect hippocampal activity suggests that cerebellar and hippocampal interaction m a y be processing other n o n - m o t o r features of the learned response. Further studies using the classical eyeblink conditioning paradigm should help to describe the interactions that occur between m o t o r and n o n - m o t o r systems involved in learning and m e m o r y processes.

Acknowledgment This research was supported by a grant from the N I M H (MH44052) and by funds from the Indiana University Center for the Integrative Study of Animal Behavior. Lonnie L. Sears is currently at the D e p a r t m e n t of Pediatrics, University of Louisville School of Medicine, Louisville, Kentucky. Correspondence concerning this paper should be addressed to Joseph E. Steinmetz, D e p a r t m e n t of Psychology, P r o g r a m in Neural Science, Indiana University, Bloomington, IN, 47405.

References [ 1] Angaut, P., The ascending projections of the nucleus interpositus posterior of the cat cerebellum: an experimental anatomical study using silver impregnation methods, Brain Res., 24 (1970) 377-394.

[2] Asanuma, C., Thach, W.T. and Jones, E.G., Cytoarchitectonic delineation of the ventral lateral thalamic region in monkeys, Brain Res. Rev., 5 (1983) 219-235. [3] Asanuma, C., Thach, W.T. and Jones, E.G., Distribution of cerebellar terminations and their relation to other afferent terminations in the thalamic ventral lateral region of the monkey, Brain Res. Rev, 5 (1983) 237-265. [4] Berger, T.W. and Thompson, R.F., Neuronal plasticity in the limbic system during classical conditioning of the rabbit nictitating membrane response. I. The hippocampus, Brain Res., 145 (1978) 323-346. [5] Berthier, N.E. and Moore, J.W., Cerebellar purkinje cell activity related to the classically conditioned nictitating membrane response, Exp. Brain Res., 63 (1986) 341-350. [-6] Berthier, N.E. and Moore, J.W., Activity of deep cerebellar nuclear cells during classical conditioning of nictitating membrane extension in rabbits, Exp. Brain Res., 83 (1990) 44-54. [-7] Bracha, V., Stewart, S.L. and Bloedel, J.R., The temporary inactivation of the red nucleus affects performance of both conditioned and unconditioned nictitating membrane responses in the rabbit, Exp. Brain Res., 94 (1993) 225-236. [8] Chapman, P.F., Steinmetz, J.E., Sears, L.L., and Thompson, R.F., Effects of lidocaine injection in the interpositus nucleus and red nucleus on conditioned behavioral and neuronal responses, Brain Res., 537 (1990) 149 156. [9] Clark, R.E. and Lavond, D.G., Reversible lesions of the red nucleus during acquisition and retention of a classically conditioned behavior in rabbits, Behav. Neurosci., 107 (1993) 264-270. [10] Clark, R.E., Zhang, A.A. and Lavond, D.G., Reversible lesions of the cerebellar interpositus nucleus during acquisition and retention of a classically conditioned behavior, Behav. Neurosci., 106 (1992) 879-888. [ 11] Clark, G.A., McCormick, D.A., Lavond, D.G. and Thompson, R.F., Effects of lesions of cerebellar nuclei on conditioned behavioral and hippocampal neuronal responses, Brain Res., 291 (1984) 125-136. E12] Jones, E.G. The Thalamus (1985) New York: Plenum Press. [13] Lavond, D.G., Hembree, T.L. and Thompson, R.F., Effect of kainic acid lesions of the cerebellar interpositus nucleus on eyehd conditioning in the rabbit, Brain Res., 326 (1985) 179-182. E14] Lavond, D.G. and Steinmetz, J.E., An interface for the IBM-XT/AT and compatibles, Behav. Res. Methods Instr. Comp., 21 (1989) 435-440. [15] McCormick, D.A., Steinmetz, J.E. and Thompson, R.F., Lesions of the inferior olivary complex cause extinction of the classically conditioned eyelid response. Brain Res., 359 (1985) 120-130. [16] McCormick, D.A. and Thompson, R.F., Cerebellum: Essential involvement in the classically conditioned eyelid response, Science, 223 (1984) 296-299. [17] McCormick, D.A., and Thompson, R.F., Neuronal responses of the rabbit cerebellum during acquisition and performance of a classically conditioned nictitating membrane-eyelid response, J. Neurosci., 4 (1984) 2811-2822. [18] Miller, D.P. and Steinmetz, J.E., Lesions of the rabbit cerebellar interpositus nucleus affect classical NM conditioned-related activity in the lateral but not the medial septum, Physiol. Behav., 52 (1992) 83-89. [193 Moyer, J.R., Deyo, R.A. and Disterhoft, J.F., Hippocampectomy disrupts trace eye-blink conditioning in rabbits, Behav. Neurosci., 104 (1990) 243 252. [203 O'Connor, K.N., Allison, T.L., Rosenfield, M.E. and Moore, J.W., Modulation of neuronal activity in the medial geniculate during auditory trace conditioning, Soe. Neurosci. Abstr., 19 (1993) 1006. [213 Olszewski, J. The Thalamus of the Macaca mulatta: An Atlas for Use with the Stereotaxic Instrument (1952) Basel: Karger. [223 Port, R.L., Romano, A.G., Steinmetz, J.E., Mikhail, A.A. and Patterson, M.M., Retention and acquisition of classical trace condi-

Lonnie L. Sears et al./Behavioural Brain Research 74 (1996) 105 117

[23]

[24]

[25]

[26]

[27]

[28]

[29]

tioned responses by rabbits with hippocampal lesions, Behav. Neurosei., 100 (1986) 745-752. Rispal-Padel, L., and Meftah, E.-M., Changes in motor responses induced by cerebellar stimulation during classical forelimb flexion conditioning in cat, J. Neurophysiol., 68 (1992) 908-926. Rispal-Padel, L., Harnois, C., and Troiani, D., Converging cerebellofugal inputs to the thalamus. I. Mapping of monosynaptic field potentials in the ventrolateral nucleus of the thalamus, Exp. Brain Res., 68 (1987) 47-58. Rispal-Padel, L., Troiani, D. and Harnois, C., Converging cerebellofugal inputs to the thalamus. II. Analysis and topography of thalamic EPSPs induced by convergent monosynaptic interpositus and dentate inputs, Exp. Brain Res., 68 (1987) 59-72. Rosenfield, M.E. and Moore, J.W.,, Red nucleus lesions impair acquisition of the classically conditioned nictitating membrane response but not eye-to-eye savings or unconditioned response amplitude, Behav. Brain Res., 17 (1985) 77 81. Sasaki, K., Gemba, H., Hashimoto, S. and Mizuno, N., Influences of cerebellar hemispherectomy on slow potentials in the motor cortex preceding self-paced hand movements in the monkey, Neurosci. Lett., 15 (1979) 23-28. Sasaki, K., Kawaguchi, S., Matsuda, Y. and Mizuno, N., Electrophysiological studies on cerebello-cerebral projections, Exp. Brain Res., 16 (1972) 75 89. Sears, L.L. and Steinmetz, J.E., Acquisition of classically conditioned-related activity in the hippocampus is affected by lesions

[30]

[31]

[32]

[33]

[34] [35]

[36]

117

of the cerebellar interpositus nucleus, Behav. Neurosci., 104 (1990) 681-692. Solomon, P.R., Vander Schaaf, E.R., Norbe, A.C., Weisz, D.J. and Thompson, R.F., Hippocampus and trace conditioning of the rabbit"s nictitating membrane response, Behav. Neurosci., 100 (1986) 729-744. Steinmetz, J.E., Lavond, D.G., Ivkovich, D., Logan, C.G. and Thompson, R.F., Disruption of classical eyelid conditioning after cerebellar lesions: Damage to a memory trace system or a simple performance defcit? J. Neurosci., 12 (1992) 4403-4426. Steinmetz, J.E., Lavond, D.G. and Thompson, R.F., Classical conditioning in rabbits using pontine nucleus stimulation as a conditioned stimulus and inferior olive stimulation as an unconditioned stimulus, Synapse, 3 (1989) 225-233. Steinmetz, J.E., Logan, C.G., Rosen, D.J., Thompson, J.K., Lavond, D.G. and Thompson, R.F., Initial localization of the acoustic conditioned stimulus projection system to the cerebellum during classical eyelid conditioning, Proc. Nat. Acad. Sci. (USA), 84 (1987) 3531 3535. Urban, I. and Richard, P., A stereotaxic atlas of the New Zealand rabbit's brain (1972) Springfield, IL: Charles C. Thomas. Yeo, C.H., Hardiman, M.J. and Glickstein, M., Classical conditioning of the nictitating membrane response of the rabbit I. Lesions of the cerebellar nuclei, Exp. Brain Res., 60 (1985) 87-90. Yeo, C.H., Hardiman, M.J. and Glickstein, M., Classical conditioning of the nictitating membrane response of the rabbit IV. Lesions of the inferior olive, Exp. Brain Res., 63 (1986~ 81-92.