The Effects of Reversible Inactivation of the Red …learnmem.cshlp.org/content/3/6/519.full.pdf · The Effects of Reversible Inactivation of the Red Nucleus on Learning-Related and

The Effects of Reversible Inactivation of the Red Nucleus on Learning-Related and Auditory-Evoked Unit Activity in the Pontine Nuclei of Classically Conditioned Rabbits M. Claire Cartford, 1 Elizabeth B. Gohl, Maria Singson, and David G. Lavond Departments of Psychology and Biological Sciences

University of Southern California

Los Angeles, California 90089-2520

Abstract

The p o n t i n e nucle i c a r ry aud i to ry cond i t i oned s t imulus i n f o r m a t i o n to the ce r ebe l l um dur ing classical cond i t ion ing of the nict i ta t ing m e m b r a n e r e s p o n s e in rabbits . In wel l - t ra ined an ima l s l ea rn ing- re la ted as wel l as s t imulus -evoked un i t activity can be r eco rded t h r o u g h o u t the p o n t i n e nucle i bu t pa r t i cu la r ly in the la teral and dorso la te ra l pons . Recent w o r k in ou r l abo ra to ry has p rov ided ev idence tha t the l ea rn ing- re la ted un i t activity in the pons is d e p e n d e n t o n the in te rpos i tus nuc leus and tha t the p o n s is no t a site o f essent ia l plast ici ty for the l ea rned re sponse . In the p r e s e n t s tudy we cons ide red the ques t ion of w h e t h e r l ea rn ing- re la ted un i t activity m i g h t be p ro jec ted f r o m the in te rpos i tus nuc leus to the p o n s t h r o u g h the r ed nucleus , a p r i m a r y ou tpu t target o f the in te rpos i tus and a s t ruc ture k n o w n to be essent ia l for exp re s s ion of the l ea rned re sponse . Mult iple un i t r ecord ings w e r e t aken f r o m la tera l and dorso la te ra l p o n t i n e locat ions in wel l - t ra ined rabbi ts before and du r ing cool ing of the red nucleus . Analysis o f poo led data for all r eco rd ing locat ions w i th in the la teral and dorso la te ra l p o n s indica ted tha t revers ib le inact iva t ion o f r ed nuc leus abo l i shed b o t h s t imulus -evoked a n d lea rn ing- re la ted un i t activity. However , we also f o u n d discrete r e co rd ing locat ions w h e r e s t imulus -evoked and lea rn ing- re la ted

1Corresponding author.

uni t activity w e r e a t t enua ted bu t no t abo l i shed by r ed nucleus cool ing.

Introduction

In simple delay classical conditioning of the rabbit nictitating membrane response, neural unit activity that models the learned behavioral eye- blink motor response occurs in brainstem and cerebellum (Clark and Lavond 1993, 1996; Krupa et al. 1993; Krupa et al. 1996; Clark et al. 1992, this issue). Permanent and reversible lesion studies as well as anatomical pathway tracing studies have produced a growing body of evidence describing the circuitry of the eye-blink response and the flow of learning-related unit activity within that circuitry. Much of the data presented to date supports the hypothesis that the interpositus (IP) nucleus of the cerebellum is the locus of the essential association and the source of learning-related unit activity recorded in other structures within the circuit (Chapman et al. 1990; Clark et al. 1984, 1992, 1996; Clark and Lavond 1993; Clark et al., this issue). Less is known about the functional roles that may be played by each of the other structures that participate in the acquisition and expression of the learned behavior.

Within the conditioned nictitating membrane response circuitry the lateral and dorsolateral pontine nuclei carry conditioned stimulus information to the cerebeUum and receive projections from cerebellar deep nuclei (Eller and Chan-Pelay 1976; Watt and Mihailoff 1983; Gerrits and Voogd 1987; Steinmetz and Sengelaub 1992; Gould et al. 1993; J.K. Thompson, W.J. Spangler, and R.F. Thompson, unpubl.). There are known primary auditory projections to the lateral pontine area (Aitkin and Boyd

LEARNING & MEMORY 3:519-531 �9 1997 by Cold Spring Harbor Laboratory Press ISSN1072-0502/97 $5.00

& 519

L E A R N / N G M E M O R Y

Cold Spring Harbor Laboratory Press on August 2, 2018 - Published by learnmem.cshlp.orgDownloaded from

http://learnmem.cshlp.org/

http://www.cshlpress.com

Cartford et al.

1978; Steinmetz et al. 1987). Electrophysiological data indicate that stimulus-evoked responses occur in the pons to both light- and tone-conditioned stimuli, as well as learning-related "models" that are increases in unit activity correlated to the rabbit eye-blink behavior or conditioned response (Steinmetz et al. 1987; McCormick et al. 1983). There is also some evidence that the pontine nuclei receive unconditioned stimulus somatosensory information (Lavond et al. 1981; McCormick et al. 1983). These anatomical and electrophysiological findings suggest that the pons could be a site of convergence of conditioned stimulus and unconditioned stimulus information, and therefore, could possibly be a locus of essential association for the learned response.

Recently, Clark et al. (this issue) undertook investigation of the question of whether the pontine nuclei might be a site of essential plasticity for the learned response. Their results indicate that learning-related unit activity in the pons is dependent on the activity of the IP nucleus. This result lends further support to the hypothesis that the IP nucleus is the site of essential association and that learning-related unit activity that develops in the IP is projected subsequently to the pontine nuclei. On the basis of this result and the known anatomical and physiological connections between the IP and the lateral pons, we explored the question of whether inactivation of red nucleus, the major output source of the conditioned response and the major efferent of the IP, would have an effect on the learning-related unit activity in the pons. We used the technique of reversibly inactivating red nucleus by cooling probe while simultaneously recording from multiple units in varying locations within the lateral and dorsolateral pontine nuclei.

Materials and Methods

All methods used in this study have been out- lined in detail in previous submissions (see Clark et al. 1992). Briefly, our procedures included the fol- lowing.

SUBJECTS

The subjects were 14 adult male New Zealand white rabbits initially weighing 2.0 (+0.2) kg. Ani- mals were housed individually with free access to food and water and were kept on a 12-hr light/dark

cycle. The rabbits were cared for by the experi- menters and by the staff and veterinarians of the University of Southern California. All care and treat- ment of the animals was approved and governed by the guidelines of the American Association for Accreditation of Laboratory Animal Care (AAALAC), the American Psychological Association (APA), and the National Institutes of Health.

SURGERY

Animals were operated before initial training to control for any changes in learning that might be caused by the presence of the implanted cold probe or recording electrode. The rabbits were preanesthetized with xylazine (5 mg/kg) and ket- amine (50 mg/kg) and maintained on 1.5%-2.5% halothane for the duration of aseptic surgery.

A cooling probe consisting of 19-gauge stainless steel tubing (based on a design by Zhang et al. 1986) was implanted in one of two possible locations. We inserted the cold probe into the brain from a location anterior to the red nucleus so that the body of the cold probe that remained above the surface of the rabbit brain would not interfere with placement of the base stand for the pontine recording electrode micromanipulator. To achieve proper placement of the probe it was inserted at a 15 ~ angle from perpendicular to the brain surface with the distal cooling tip of the probe located either at coordinates AP +10, ML -2.5, DV -16, or AP +12, ML -1.5, DV -16 (coordinates were de- rived from previous studies in this laboratory and were based originally on the stereotaxic atlas of McBride and Klemm 1968). From these locations cooling spread to the red nucleus either from a location lateral to the nucleus or from a location anterior to the nucleus. All coordinates are in mil- limeters from lambda with lambda being placed 1.5 mm lower than bregma. The minus sign for ML coordinates indicates placement on the right side of the rabbit's head.

In addition to the cold probe, a recording electrode constructed from a 00 stainless steel insect pin, insulated with a minimum of six baked coats of Epoxylite, with 20-40 lam exposed tip was chronically implanted in the contralateral IP using coordinates AP +0.5, ML +5, DV -14.5.

To obtain recordings from pontine cells, a 5-mm diameter hole was made in the right cranium using coordinates AP +8.5, ML -2.5 (at the center of the hole). The hole was filled with sterile bone

& 520





PONTINE NUCLEI AND EYE-BLINK CONDITIONING

w a x and a stainless steel base stand was cemen ted in place over the hole using dental acrylic. The base stand would later be used for at taching a Na- rishige microelect rode manipula tor (MO-95) so that recording electrodes could be lowered into varying regions of the pons during testing days.

Finally, a socket for a t tachment of a mini- torque poten t iometer and air puff hose was f ixed to the anterior por t ion of the cranium. This socket, along wi th the cold probe, base stand, and Fixed recording electrode were secured to the rabbit skull using dental acrylic anchored by three stain-

less steel screws. After all surgical procedures were completed,

but before the animal was revived, a small loop of 6-0 Ethilon monof i lament surgical suture was secured in the distal margin of the left nictitating m e m b r a n e of the rabbit. This loop was used later to secure the mini torque po ten t iomete r for mea- surement of the eye-blink response.

Two of the 14 subjects unde rwen t surgery as descr ibed above wi th the excep t ion that they did not have a f ixed recording electrode placed in the IP nucleus. These subjects were naive controls that were tested for tone-evoked unit activity in the lateral pons before and during cooling of red nucleus. We were interested in the effects of cooling on tone-evoked activity for compar ison wi th the trained animals.

TRAINING

Rabbits were al lowed to recover f rom surgery

for 7 days. On the first day after the recovery per iod each

animal was habi tuated for 1 hr to a plexiglass rabbit restrainer p laced inside an Industrial Acoustics sound-attenuating chamber . Training sessions be- gan on the day after habituation. Each daily training session consisted of 12 blocks of 9 trials for a total of 108 trials. Each block of trials consisted of one tone alone trial, one air puff alone trial, and seven paired trials. The tone alone trial occurred at the beg inn ing of each block and was fol lowed by three paired trials, then the air alone trial, and finally, four more paired trials. The condi t ioned st imulus (CS) was a 1-kHz, 85-dB (SPL), 352-msec tone. The uncondi t ioned st imulus (US) was a 2 .1-N/cm 2 (source pressure), 98-msec corneal air puff.

After each animal reached cri terion in acquisi- t ion training it was overtrained 1 day. The animal was then ready for recording from pont ine cells.

Recording electrodes identical to those descr ibed above were lowered using the Narishige micromanipulator. Stereotaxic atlas coordinates were used to guide in sampl ing locations along the rostral/ caudal axis of the pont ine nucle i (from lambda AP +8.5 + 2.0; ML -2 .0 + 0.5).

Each test session consis ted of the fol lowing procedures. An electrode was lowered into the pons, trait activity was isolated, and then a block of trials was begun. Unit activity was moni tored for recordings that reflected stimulus-evoked or learning-related elevations in unit activity for at least one full block of trials. The cooling probe was then activated and a second block of trials was run at that recording location. The electrode was then moved to another location and the process was begun again. Marking lesions were made at the end of testing along each electrode pa thway by passing 100 ~ of current through the recording electrode for 5 sec. Lesions were made every 5 m m starting at the lowest recording location, then moving dor- sally through the brain tissue. In some instances additional marking lesions were made at locations w h e r e both recording and cooling blocks had been completed. The testing p rocedure was repeated, one session each day, for as long as we were able to cool the red nucleus or unti l three marking lesion tracks had been made. In our exper ience , clear dec ipher ing of recording locations became too difficult w h e n more than three tracks were marked.

The two control animals were not given eye- bl ink condit ioning. After surgery and 7 days of recovery these animals were given 1 hr of habituation to the test c h a m b e r fol lowed the nex t day by testing. Testing procedures were as descr ibed above for recording from the lateral pont ine nuclei. Rabbits were p laced in the test c h a m b e r and given tone alone training trials that fol lowed all normal parameters wi th the excep t ion that they had no uncondi t ioned st imulus delivery (no air puff). Recording locations in the pons were sampled until tone-evoked responses were found. W h e n locations wi th evoked responses were found, data were collected for several blocks of trials. Cooling was then initiated for one block of trials. Subsequent to cooling the electrode was moved to a n e w location as descr ibed above.

DATA COLLECTION AND ANALYSIS

Each trial had a 252-msec basel ine per iod of no stimuli, a 252-msec CS period, and a 252-msec US

& 521





Cartford et aL

period. During the triM, data were sampled and extracted at 4-msec intervals. The behavioral-de- penden t measure was an extens ion of the nictitating m e m b r a n e as measured by a mini torque poten- tiometer. The neural unit act ivi ty-dependent measure was amplif ied wi th an A-M Systems four- channe l amplif ier (model 1700, 10k gain, 300-5000 Hz bandpass filtering). These neural spikes were passed to an ampl i tude discriminator that digitized the activity. This information was then collected by the compu te r to construct a peri- st imulus t ime histogram. All st imulus presenta- tions, sessions, data collection, and data analyses were control led by IBM-PC/XT clones that were equ ipped wi th an interface (Lavond and Steinmetz 1989) and programs wr i t ten in 8088 mach ine code and Forth.

Behavioral-condit ioned responses (CR) were counted if nictitating m e m b r a n e closure exceeded 0.5 m m during the CS per iod on paired trials, and during the CS and US periods on tone alone trials. The learning cri terion was the first t ime that eight CRs occurred on n ine consecut ive trials. We de- f ined bad trials as 0.7 m m of eyelid movemen t in the 160-msec before CR onset or 0.5 m m of eyelid closure in the first 25 msec of the CS period. Con- di t ioned responses, trials to criterion, amplitude, latency, and area under the curve for both CS and US per iods were calculated and pr in ted at the end of each training and testing session.

We used a m i n i m u m cri terion of 2:1 signal-to- noise ratio based on visualization of unit data on the oscilloscope. Most recordings were 3"1 or bet- ter. We est imate the n u m b e r of units we typically discr iminated (based on height) to be 3 to 5 units.

Unit activity f rom IP nucleus and from the pons were analyzed wi th peris t imulus t ime histograms and statistical analysis wi th z scores to quan- tify activity associated wi th learning (Thompson et al. 1976). Each trial had three 252-msec periods: (1) a p recondi t ioned st imulus (baseline) period, (2) a CS period, and (3) a US period. These three st imulus per iods were each subdivided into three 84-msec epochs (epochs 1, 2, and 3). Significant activity in CS epoch 1 was def ined as tone-evoked unit activity. Significant activity in CS epochs 2 and 3 was def ined as learning-related unit activity.

Significant unit activity was de te rmined by z score analysis. For each daily training session a grand mean and standard error for behavioral responses were calculated across all trials using the 84-msec epoch immedia te ly before the onset of the CS. The grand mean and standard error were used

to calculate z scores for the three 84-msec CS period epochs and the three 84-msec US per iod epochs for each individual trial. Any increase in unit activity that resulted in a z score of 1.65 or greater (a one-tailed significance level of 0.05) in CS epochs 2 and 3 was scored as a learned unit response or model. Any individual animal that showed these learned unit responses on at least 80% of the trials for a given day was considered to show activity that mode led the learned behavior.

For each testing session, baseline means and standard errors were calculated separately for behavioral and unit responses for each block of trials. For statistical analysis, z scores were calculated by blocks to take into account differences in normal versus cooling blocks and differences in recording locations.

HISTOLOGY

Each rabbit was sacrificed wi th an overdose of intravenous sodium pentobarbi ta l and perfused in- tra-aortically wi th saline, fol lowed by 10% formalin. Marking lesions were made of the chronical ly im- p lanted IP recording electrode locations by passing 100 pA through the electrode for 10 sec. The brain was removed and postf ixed in 10% sucrose formalin solution. Each brain was blocked and embed- ded in a ge la t in-a lbumin matrix, frozen and cut on a micro tome at a thickness of 80 jxm, and moun ted onto c h r o m e - a l u m subbed slides. The tissues were stained wi th Prussian Blue for marking lesions and cresyl violet for cells, and were analyzed using a light microscope to reconstruct electrode and cold probe placements .

Results

BEHAVIORAL RESULTS

Eleven subjects trained to a 1-kHz tone reached cri terion wi th in 5 days of training in 256 _+ 102 (mean _+ S.D.) trials to criterion. Clark et al. (1992) found comparable trims to cri terion data for 12 animals wi th implanted cold probes wi th the mean trials to cri terion equal to 271 +_ 43.0. In the present study, one additional rabbit required 7 days of training to reach cri terion using a 5-kHz tone as the CS. The 5-kHz tone was used to achieve greater filtering of recorded neural units f rom possible tone artifacts in the recording. Those data are

& 522

L E A R N I N G M E M O R Y





not included in our report on acquisition, but are included in all o ther aspects of the study.

Figure 1 illustrates behavioral results of our exper iment . Inactivating red nucleus abol ished the learned nictitating m e m b r a n e behavior. This is consistent wi th prior inactivation of red nucleus studies (Clark and Lavond 1993, 1996; Krupa et al. 1993). Condi t ioned response ampl i tude data for all subjects was pooled for paired trials during each block preceding cooling and for each block during cooling. Pooled condi t ioned response ampli tude during normal training was 5.1 _+ 1.2 m m and during cooling was 0.2 _+ 0.1 mm, t(15) = 4.063, P < 0.001. Similar to previous studies (Clark and

Lavond 1993, 1996), cooling red nucleus also attenuated but did not abolish uncondi t ioned response (UR) amplitude. Uncondi t ioned response data were pooled for all subjects using air puff alone trials f rom each block preced ing cooling and from each block during cooling. Pooled UR amplitude during normal training was 5.9 -+ 1.5 m m and during cooling was 2.8 _+ 1.0 ram, t(15) = 2.4717, P < 0.05.

Figure 1: Results of pooled behavioral measure of nictitating membrane extension for conditioned responses (CRs) and unconditioned responses (URs) during normal and cooling sessions.

RECORDING RESULTS FROM NORMAL ANIMALS

LATERAL PONS

For the 12 normal subjects, 16 recording locations wi th in the pons had comple te recording and cooling data. z Scores based on the block of trials preceding cooling and during cooling were analyzed for significant levels of unit activity recorded in the pons during each of the three epochs wi th in the CS period. For pooled data f rom all recording locations a two-factor analysis of variance (ANOVA) (normal versus cooling) wi th six repeated measures ( three CS epochs and three US epochs) showed a main effect of cooling

(F1,32 = 7.33, P < 0.01). z Scores for basel ine activity in the pons over

blocks preceding and those during cooling were analyzed using a related measures t-test and showed that cooling did not significantly change basel ine neural activity, t(15) = -0 .8885, P > 0.05. Of the 16 recording locations 13 showed an increase in unit activity during the basel ine per iod wi th cooling and 3 showed a decrease. Based on compar i son of the group mean z score precool ing and during cooling there was an overall increase of basel ine unit activity by 27% during cooling.

Figure 2 is a map of the 16 pont ine recording locations, z Scores for each location are listed on Table 1 for precool ing and cooling blocks of CS epoch 1 and CS epoch 3 unit activity. In addition, we have represented changes in unit activity that occurred attributable to cooling by listing in the boxed co lumns of Table 1 the ari thmetic difference of the precool ing and cooling z scores.

We fotmd three types of unit activity response pat terns wi th in our pont ine recording sites: (1) stimulus-only unit activity, (2) learning-related unit activity, and (3) both st imulus and learning-related unit activity. Wi th in the 16 recording locations we found nine sites that reflected stimulus-evoked unit responses, that is, responses wi th z scores above the critical value in CS epoch 1. We found 10 sites wi th learning-related unit activity or activity wi th z scores above the critical value in CS epoch 3, and we found six sites wi th both types of activity.

Table 2 provides a list of those locations whe re st imulus-evoked responses were recorded and indicates those locations w h e r e these responses were abolished (z scores fell be low the critical level for unit activity during cooling). Table 3 provides a list of those locations w h e r e learning-related unit activity was recorded. Locations are in-

& 523





Cartford et aL

Figure 2: Summary of recording locations. Each location is noted (0) and identified by a corresponding number represented to the side of the location. Location numbers correspond to those listed in Tables 1,2, and 3 and in Figs. 3 and 4.

dicated w h e r e these models were abolished (z scores fell be low the critical level for unit activity during cooling).

The data for individual recording sites indicate that cooling did not cause stimulus-evoked or learning-related unit activity to fall be low the significant level in all locations. Figure 3 shows that there are four recording locations whe re cooling red nucleus at tenuated but did not abolish learning-related unit activity (locations 1, 2, 8, and 16). z Scores for unit data from locations whe re models were at tenuated but not abol ished by red nucleus cooling were analyzed using a related measures t- test. Cooling did not cause a statistically significant reduct ion in learning-related unit activity z scores, t(4) - 2.91, P > 0.05. z Score changes for recording location 2 before and during cooling suggest that cooling may have unmasked a normally inhibi tory effect at this location.

Figure 4 shows that there are four sites w h e r e st imulus-evoked unit activity was not abol ished by red nucleus cooling (locations 1, 3, 10, and 16). z Scores for unit data f rom locations whe re stimulus- evoked activity was not abol ished by red nucleus cooling were also analyzed using a related mea-

sures t-test. Cooling did not cause a statistically significant reduct ion in tone-evoked unit activity z scores in these locations, t(4) = 1.15, P > 0.05. Re- cording location 12 showed an increase in stimulus-evoked unit activity during cooling. We believe the cooling may have in terrupted an inhibi tory effect in this location.

Attenuation of unit activity was observed for both stimulus-evoked as wel l as learning-related unit activity in the pont ine nuclei in locations w h e r e cooling did not abolish unit activity. In the recording locations whe re st imulus-evoked unit activity was not abol ished by cooling (Table 2 z scores n o t marked wi th asterisk), there was an overall a t tenuation of activity of 24% in compar i son to group mean z scores for CS epoch 1 before and during cooling. However, no z score for an individual recording location falls be low the 1.65 cri- tef lon for significant activity as compared to baseline. Likewise, w h e n mean z scores for CS epoch 3 are compared before and during cooling for locations w h e r e cooling did not abolish unit activity (Table 3 z scores n o t marked wi th an asterisk), a 44% reduct ion in overall activity is noted. How- ever, once again, no individual z scores fell be low

& 524






Table 1 : Pontine recording locations stimulus-evoked (CS1) and learning-related (CS3) unit activity and the effects of red nucleus cooling on that unit activity (as reflected in the arithmetic difference between normal and cooling z-scores from CS epoch 1 and CS epoch 3 blocks of data)

Recording Normal Cooling Norm-Cool Normal Cooling Norm-Cool location CS epoch 1 CS epoch 1 CS epoch 1 CS epoch 3 CS epoch 3 CS epoch 3

1. 3.610 2.820 .790 4.990 3.270 1.720 2. 2.120 4.600 -2.480 1.460 4.180 -2.720 3. 1.770 .520 1.250 2.590 1.700 .890 4. 4.290 1.080 3.210 5.140 1.41 0 3.730 5. 3.490 .800 2.690 5.870 .530 5.340 6. .740 .150 .590 3.340 1.61 0 1.730 7. .1 70 .320 - .015 1.360 .640 .720 8. 3.340 2.260 1.080 1.450 1.930 -0.480 9. 1.020 .010 1.01 0 4.430 .290 4.140

10. .800 - .160 .960 6.380 2.570 3.810 11. .480 .360 .120 1.550 .400 1.1 50 12. .120 -1.240 1.360 2.230 -1 .150 3.380 13. 2.830 .570 2.260 8.690 .850 7.840 14. 1.1 60 -1 .020 2.180 0.000 - .830 -0.830 15. 5.450 - .430 5.880 .370 - .050 0.420 16. 6.440 2.290 4.150 3.510 2.140 1.370

the 1.65 cri terion for significant levels of activity w h e n compared to baseline.

INTERPOSITUS

Table 4 includes z scores for CS epoch 3 unit activity recorded in IP nucleus (there were no

Table 2- Effects of red nucleus cooling on stimulus-evoked unit activity in pontine recording locations (reflected in z scores from CS epoch 1)

Stimulus-evoked unit activity

location a normal cool

1. 3.610 2.820 2. 2.120 4.600 3.* 1.770 .520 4.* 4.290 1.080 5.* 3.490 .800 8. 3.340 2.260

13.* 2.830 .570 15.* 5.450 - .430 16. 6.440 2.290

aft) Locations where cooling had a significant effect on unit activity.

Figure 3: z Scores calculated for tone-evoked unit activity for individual recording locations represent unit activity for one block (nine trials) before cooling and one block (nine trials) of cooling. Four of the nine recording locations continue to show significant levels of unit activity during cooling of the red nucleus. These data are rank-ordered from highest z score to lowest (before cooling). The numbers below the bars identify the locations from which these recordings were made as plotted on Fig. 2.

& 5 2 5





Cartford et al.

Figure 4: z Scores calculated for learning-related unit activity for individual recording locations represent unit activity for one block (nine trials) before cooling and one block (nine trials) of cooling. Four of the 10 recording locations continue to show significant levels of unit activity during cooling of the red nucleus. These data are rank ordered from highest z score to the lowest (before cooling). The numbers below the bars identify the locations from which these recordings were made as plotted on Fig. 2.

cases w h e r e significant increases in unit activity were observed in CS epoch 2). Four animals had accurate implanted IP electrode placements . The data f rom these animals were analyzed for effects of red nucleus cooling on learning-related unit activity. A related measures t-test showed no significant reduct ion in learning-related unit activity z scores during cooling of red nucleus, t(4) = 0.9261, P > 0.05. Interpositus data from these subjects closely matches that reported by Clark and Lavond (1993) whe re cooling the red nucleus had no statistically significant effect on learning-related unit activity in IP during acquisi- t ion and re tent ion of the eye-blink response.

Like recordings from pont ine areas whe re cooling did not abolish unit activity, the z scores for IP indicate an at tenuation of activity attributable to red nucleus cooling. This at tenuation rep- resents a 10% drop in unit activity based on group m e a n z scores for CS epoch 3. With one except ion, this a t tenuat ion of activity did not cause unit activity to decrease be low the critical level of 1.65 as compared to basel ine and, in fact, in two locations

Table 3: Effects of red nucleus cooling on learning-related unit activity in pontine recording locations (reflected in z scores from CS epoch 3)

Learning-related unit activity

location a normal cool

1. 4.990 3.27O 3. 2.590 1.700 4.* 5.140 1.410 5.* 5.870 .530 6.* 3.340 1.610 9.* 4.430 .290 O. 6.380 2.570 2.* 2.230 -1 .150 3.* 8.690 .850 6. 3.510 2.140

a(,) Locations where cooling had a significant effect on unit activity.

the unit activity increased during cooling of red nucleus.

Baseline unit activity for the IP nucleus recording locations showed an overall increase in activity by 14% during cooling. However, three of the recording locations showed a modes t decrease in activity and one showed a large increase. The difference in z scores before and during cooling was not statistically significant, t(4) = 0.40618, P > 0.05.

Two different cold p robe p lacements near the red nucleus were used in this study to control for interference that might be caused by cooling w h e n recordings were taken in the nearby rostral lateral pons. Using t-tests for i ndependen t means w e verified that no differences in unit data activity, e i ther tone-evoked or learning-related activity, could be accounted for by the different location of the cold

Table 4: Effects of red nucleus cooling on learning-related unit activity in interpositus nucleus (reflected in z scores from CS epoch 3)

& 526

I nterpositus learning-related unit activity

normal cool

7.49 5.75 2.0 3.15 2.71 4.65 4.03 1.01






probes. For tone-evoked unit activity z scores, t(14) = 0.2773, P > 0.05, and for learning-related unit activity, t(14) = 0.1817, P > 0.05.

Figure 5 is a peristimulus time histogram of multiple unit recordings from IP nucleus and pontine nuclei during a block of trials preceding cooling and a block of trials during cooling. Learning- related model activity can be seen in both recordings before and during cooling. These data replicate earlier findings of Chapman et al. (1990) and Clark and Lavond (1993) for IP recording with red nucleus inactivation. The data illustrate atten- tuation but not loss of learning-related unit models in a dorsolateral pontine recording location before and during red nucleus cooling.

The variable results that we report both in pontine as well as IP recording locations may be an indication of a widespread inhibitory influence ex- erted on the eye-blink system perhaps controlled by or routed through the red nucleus. This could explain, for instance, locations where unit activity increased during cooling of red nucleus. However, with our small number of observations it is difficult to find significant effects. What the data indicate consistently is a variability of response patterns during cooling, including increases in unit activity, decreases in unit activity, as well as a few locations where no changes occur whatsoever.

RECORDING RESULTS FROM CONTROL ANIMALS

Figure 6 includes histology (A), z scores 03), and peristimulus time histograms (C) for each naive subject tested for stimulus-evoked responses in the lateral pons before and during cooling of red nucleus. We obtained one excellent recording

with successful cooling in each subject. Subject 96-279 showed a clear short latency stimulus- evoked response that was significantly reduced by red nucleus cooling. Subject 96-280 showed a long onset latency and long duration response to the tone stimulus before cooling. During red nucleus cooling this response changed to a short onset latency.

The initial recording from rabbit 96-280 looks very much like learning-related unit activity recorded in well-trained animals (see Fig. 5), yet ex- amination of our data from normal subjects shows no such shift in unit activity onset latency during cooling. It is possible that changes in unit activity associated with learning had already masked or caused changes in unit activity associated with the conditioning stimulus. The fact that we have seen evidence in normal subjects for inhibitory feedback processes being unmasked by red nucleus cooling (recording location 15 in particular) leads us to believe that red nucleus cooling is somehow releasing or interfering with an inhibitory process that normally controls the onset of the stimulus- evoked response.

D i s c u s s i o n

Our results show that temporary inactivation of the red nucleus has a significant effect on learning-related and stimulus-evoked unit activity found in the pontine nuclei. The results also show that sites within central regions of the rostral/caudal extent of the dorsolateral and lateral pons maintain significant, although attenuated learning-related unit activity and stimulus-evoked activity during red nucleus inactivation.

& 527

Figure 5: Peristimulus time histograms taken from one animal showing behavioral nictitating membrane responses (nmr) and unit activity recorded from lateral pons (LPN) and interpositus nucleus (IP). These data represent the average activity over one block (nine trials) before cooling of red nucleus (Normal) and one block (nine trials) during cooling of red nucleus (Cooling). The behavioral response is abolished with cooling. Tone- evoked unit activity is abolished and learning-related unit activity is reduced in both interpositus and lateral pons recordings.

L E A R N / N G M E M 0 R Y




Cartford et aL

Figure 6: Data and histology for control subjects 96-279 and 96-280. (A) Peristimulus time histograms for neural recording during one block of normal and one block of cooling trials. (B) z Scores for CS epoch 1 and CS epoch 3 for the block of normal and the block of cooling trials represented in the peristimulus time histograms. (C) Recording locations where multiple unit data was obtained.

These data support earlier findings that the pontine nuclei are an integral part of the conditioned stimulus pathway in rabbit eye-blink classical conditioning. Extensive mapping of recording information taken from rabbits trained using a tone-conditioned stimulus has verified that models of learning-related unit activity as well as tone- evoked unit activity occur extensively throughout the pontine midbrain (McCormick et al. 1983). Steinmetz et al. (1985 and 1986) have shown that direct stimulation of either the middle cerebellar peduncle or the dorsolateral, lateral, and medial pontine nuclei may serve as the conditioned stimulus in this paradigm. Lesions of the dorsolateral pons, the caudal lateral pons, and the lateral lem- niscus abolish conditioned responses to a tone- conditioned stimulus (Steinmetz et al. 1987). Ana- tomical and physiological data substantiate a direct and reciprocal connection between the IP nucleus and the dorsolateral and lateral pontine nuclei (Eller and Chan-Palay 1976; Watt and Mihailoff 1983; Gerrits and Voogd 1987; Steinmetz et al. 1987; Steinmetz and Segelaub 1992; Gould et al. 1993; J.K. Thompson, w.J. Spangler, and R.F. Thompson, unpubl.).

The data from our study, considered with that of Clark et al. (this issue), confirm the integral re- lationship of the pontine nuclei within the eye- blink circuitry and support the hypothesis that the learning-related unit activity we and others have seen in the pons is dependent on the function of the IP. We now also see an indication that feedback to the pontine nuclei from the red nucleus may play a functional role in the eye-blink circuit, although we have found no evidence in the litera- ture for a direct anatomical pathway from red nucleus to the pons.

Involvement of the red nucleus in the eye- blink circuitry has been established from both anatomical and physiological data. This nucleus has been shown to be an essential part of the output of the cerebellum for the conditioned eye-blink response (Tsukahara et al. 1981; Des- mond and Moore 1982; Tarnecki 1988; Chap- man et al. 1990; Clark and Lavond 1993; D.A. Haley, D.G. Lavond, and R.F. Thompson, unpubl.) and is now known to have reciprocal connections to the IP (Courville and Brodal 1966; J. Lock- ard and D.G. Lavond, unpubl.). However, there is no evidence of direct anatomical connections

& 528






between red nucleus and dorsolateral or lateral

pons. Our recording data from the IP do not provide

conclusive evidence for a theory that changes in unit activity in the pons could be caused by anti- dromic effects from red nucleus cooling passing back to IP and from there to the pons. Attenuation of unit activity in the IP during red nucleus cooling is not significant either in our findings or those of Clark and Lavond (1993) and is far less than attenuation that occurs in the pons. It is possible that the effect in the pons is somehow amplified over that seen in the IP. Given the evidence for both anatomical and physiological connections between dorsolateral and lateral pons and the IP, further studies are warranted to explore possible interac- tions occurring among these structures.

In 1993 Clark and Lavond found that cooling red nucleus during acquisition prevented expression of the learned response that was formed in the IP during training. Very recently our laboratory has shown that the trigeminal complex, a major locus for uncondit ioned stimulus information traveling to the cerebellum, receives learning-related information from IP through red nucleus excitatory projections (Clark and Lavond 1996). This study also showed evidence of inhibitory connections between red nucleus and pars oralis of the trigeminal complex. Our current data show attenuation or abolition of neuronal unit activity related to the conditioned stimulus and to learned behavior in all parts of the lateral and dorsolateral pontine nuclei as a consequence of red nucleus inactivation. Taken together, these data suggest that the red nucleus is serving as more than output for the conditioned response. It also seems to function as an important feedback source of information to midbrain structures that are active in sending stimulus information to the cerebellum.

The effects of red nucleus cooling on stimulus- evoked unit responses in the pons surprised us and led us to question whether at least some of our results may be explained by the location of the cold probe. For instance, we wondered whether there could be a generalized effect from cooling in the lateral reticular formation adjacent to the red nucleus. Irvine and Jackson (1983) found stimulus- sensitive cells in the central tegmental field with complex and broad response properties that would suggest both a convergence of sensory mo- ralities as well as multiple origins (i.e., inputs through the auditory pathways as well as cerebellar components). Anatomical analysis of these re-

Ocular formation cells indicated that the majority of projections come from the lateral lemniscal auditory system. If our lateral placement of the cold probe effectively inactivated this system as well as the red nucleus, we might expect to see a loss or reduction of tone-evoked unit activity within the projection area of these cells.

We do not believe cooling had a generalized effect on the lemniscal system in our subjects. If there were such a generalized effect from cooling it would be difficult to explain the retention of tone-evoked information in the middle regions of the dorsolateral and lateral pons. Descending as well as ascending auditory fibers travel through the lemniscal projection and both have direct connections within the lateral pons (Matano et al. 1966; Aitkin and Boyd 1978; Kandler and Herbert 1991). Also, if the effects were caused by the cold probe placement we would expect that there be no effect from cooling in those subjects with more anterior and medial cold probe placements. We saw no significant differences in unit activity in the pons that corresponded to different cold probe placements. Finally, there were no significant effects on baseline neural unit activity between normal and cooling blocks of recordings for any cold probe placement. Thus, it does not seem that the location of the cold probe was having any generalized effect on the regions from which we were taking recordings.

The role of the pontine nuclei within the eye- blink circuit remains to be elucidated. The importance of the pontine projection for carrying CS information to the cerebellum has been well established (Steinmetz and Sengelaub 1992; Steinmetz et al. 1986, 1987, 1989). More recently, Tracy (1995) found that stimulation thresholds in the dorsolateral pons were lowered during training with a tone-conditioned stimulus, whereas IP thresholds were not. In addition, J. Steinmetz (pers. comm.) has found evidence that tuning curves of pontine cells become sharper as a consequence of training. This suggests that modulation in cells sensitive to auditory stimuli may be occurring at the level of the pons, although it is also possible that tuning of cells responsive to auditory stimuli occurs afferent to the pons and shows in the response properties of pontine neurons (Woody et al. 1992, 1994; Weinberger 1993). The tuning of pontine neurons may also be a result of feedback from efferent connections such as IP, or red nucleus by IP. We plan to explore the pontine area further for evidence of adaptations to the conditioned stimulus that might

L E A R N I N G I M E M 0 R Y




Cartford et al.

suggest a funct ional role for the pons in a mecha- n ism such as selective attention.

The results of this study as wel l as those of Clark et al. (this issue) provide support for our hypothesis regarding m e m o r y formation wi th in the putative eye-blink circuitry. Our cooperative memory hypothesis states that the fundamenta l association b e t w e e n tone and air puff and the learned behavior occurs in the IP nucleus, that different memor ies about different aspects of the condit ioning are represen ted in a distr ibuted m a n n e r in other brain structures, and w h e n combined, result in a coordinated memor ia l representation.

The exact nature of the role of each of the structures involved in the eye-blink circuit remains to be discovered. For instance, the roles of the cerebel lar cor tex and deep nuclei have been of part icular interest in this learning paradigm. In con- trast to our results (Clark et al. 1997a) that have s h o w n that cooling IP nucleus abolished learning- related unit activity in cerebel lar cortex, and results f rom the Thompson laboratory (Chen 1996) showing eye-blink learning in mutant mice lacking Purkinje cells, Katz and Steinmetz (1997) report that learning-related unit activity remains in cerebellar cor tex after chemica l lesions of the IP.

The results of our present study suggest that the pon t ine nucle i are subject to modifying influ- ences f rom the output pa thway of the learned behavior in addit ion to the inf luence of the IP nucleus shown by Clark et al. (this issue). In combina t ion wi th studies suggesting that there may be plasticity associated wi th auditory stimuli in the pont ine nuclei, our results support the not ion of a unique, integral role for the pont ine nuclei wi th in the circuitry of the learned eye-blink.

In conclusion, this study has shown that, like the IP, the red nucleus exerts an inf luence on learning-related unit activity wi th in the lateral and dorsolateral pont ine nuclei. Unlike the IP, the red nucleus also exerts an inf luence on stimulus- evoked unit activity wi th in the lateral and dorsolateral pon t ine nuclei. Given these results it seems likely that the pont ine nuclei part icipate in the neural circuitry of the learned nictitating m e m b r a n e response in a role that is more complex than the s imple relay of condi t ioned st imulus information to the cerebel lum. What exactly this role might be is a subject for future investigation.

Acknowledgments This research was supported by National Institute of

Mental Health 1 R01 MH51197. Special thanks to Bob Clark

for his generous and invaluable assistance throughout this project�9

References Aitkin, L.M. and J. Boyd. 1978. Acoustic input to the lateral pontine nuclei. Hearing Res. 1" 67-77�9

Chapman, P.F., J.E. Steinmetz, L.L. Sears, and R.F. Thompson. 1990. Effects of lidocaine injection in the interpositus nucleus and red nucleus on conditioned behavioral and neuronal responses. Brain Res. 537" 140-156.

Chen, L., S. Bao, J.M. Lockard, J.J. Kim, and R.F. Thompson. 1996. Impaired classical eyeblink conditioning in cerebellar-lesioned and purkinje cell degeneration (pcd) mutant mice. J. Neurosci. 16: 2829-2838.

Clark, R.E. and D.G. Lavond. 1993. Reversible lesions of the red nucleus during acquisition and retention of a classically conditioned behavior in rabbits. Behav. Neurosci. 107: 264-270.

�9 1996. Neural unit activity in the trigeminal complex with interpositus or red nucleus inactivation during classical eyeblink conditioning�9 Behav. Neurosci. 110" 13-21.

Clark, G.A., D.A. McCormick, D.G. Lavond, and R.F. Thompson�9 1984. Effects of lesions of cerebellar nuclei on conditioned behavioral and hippocampal neuronal responses. Brain Res. 291 : 125-136.

Clark, R.E., A.A. Zhang, and D.G. Lavond. 1992. Reversible lesions of the cerebellar interpositus nucleus during acquisition and retention of a classically conditioned behavior. Behav. Neurosci. 106: 879-888�9

�9 1997a. The importance of cerebellar cortex and facial nucleus in acquisition and retention of eyeblink/NM conditioning: Evidence for critical unilateral regulation of the conditioned response. Neurobiol. Learn�9 Mem. 67:96-111.

Clark, R.E., E.B. Gohl, and D.G. Lavond. 1997b. The learning-related activity that develops in the pontine nuclei during classically eye-blink conditioning is dependent on the interpositus nucleus�9 Learn. & Mem. (this issue)�9

Courville, J. and A. Brodal. 1966. Rubrocerebellar connections in the cat: An experimental study with silver impregnation methods�9 J. Comp. Neurol. 126" 471-485.

Desmond, J.E. and J.W. Moore. 1982. A brain stem region essential for classically conditioned but not unconditioned nictitating membrane response�9 Physiol. Behav. 28:1029-1033.

Eller, T. and V. Chan-Palay. 1976. Afferents to the cerebellar lateral nucleus. Evidence from retrograde transport of horseradish peroxidase after pressure injections through micropipettes. J. Comp. Neurol. 166: 285-302.

Gerrits, N.M. and J. Voogd. 1987. The projection of the nucleus reticularis tegmenti pontis and adjacent regions of

& 530





PONTINE NUCLEI A N D EYE-BLINK CONDIT IONING

the pontine nuclei to the central cerebellar nuclei in the cat. J. Comp. Neurol. 258: 52-69.

Gould, T.J., L.L. Sears, and J.E. Steinmetz. 1993. Possible CS and US pathways for rabbit classical eyelid conditioning: Electrophysiological evidence for projections from the pontine nuclei and inferior olive to cerebellar cortex and nuclei. Behav. Neural Biol. 60" 172-185.

lrvine, D.R.F. and G.D. Jackson. 1983. Auditory input to neurons in mesencephalic and rostral pontine reticular formation: An electrophysiological and horseradish peroxidase study in the cat. J. Neurophysiol. 49:1319-1333.

Kandler, K. and H. Herbert. 1991. Auditory projections from the cochlear nucleus to pontine and mesencephalic reticular nuclei in the rat. Brain Res. 562: 230-242.

Katz, D.B. and J.E. Steinmetz. 1997. Single-unit evidence for eye-blink conditioning in cerebellar cortex is altered, but not eliminated, by interpositions nucleus lesions. Learn. & Mem. (in press).

Krupa, D.J., J.K. Thompson, and R.F. Thompson. 1993. Localization of a memory trace in the mammalian brain. Science 260: 989-991.

Krupa, D.J., J. Weng, and R.F. Thompson. 1996. Inactivation of brainstem motor nuclei blocks expression but not acquisition of the rabbit's classically conditioned eyeblink response. Behav. Neurosci. 110:219-227.

Lavond, D.G. and J.E. Steinmetz. 1989. An inexpensive interface for IBM-PC/XT and compatibles. Behav. Res. Methods Instrum. Computers 21: 435-440.

Lavond, D.G., D.A. McCormick, G.A. Clark, D.T. Holmes, and R.F. Thompson. 1981. Effects of ipsilateral rostral pontine reticular lesions on retention of classically conditioned nictitating membrane and eyelid responses. Physiol. Psychol. 9" 335-339.

Lavond, D.G., J.J. Kim, and R.F. Thompson. 1993. Mammalian brain substrates of aversive classical conditioning. Ann. Rev. Psychol. 44:317-342.

Matano, S., T. Ariviki, T. Kozeki, and T. Ban. 1966. Some observations on the efferent fibers of the inferior colliculus in the rabbit. Med. J. Osaka Univ. 16." 259-269.

McBride, R.L. and W.R. Klemm. 1968. Stereotaxis atlas of rabbit brain, based on the rapid method of photography of frozen unstained sections. Comm. Behav. Biol. Part A 2: 179-215.

McCormick, D.A., D.G. Lavond, and R.F. Thompson. 1983. Neuronal responses of the rabbit brainstem during performance of the classically conditioned nictitating membrane (NM)/eyelid response. Brain Res. 271: 73-88.

Steinmetz, J.E. and D.R. Sengelaub. 1992. Possible conditioned stimulus pathway for classical eyelid conditioning in rabbits. I. Anatomical evidence for direct

projections from the pontine nuclei to the cerebellar interpositus nucleus. Behav. Neural Biol. 57:103-115.

Steinmetz, J.E., D.G. Lavond, and R.F. Thompson. 1985. Classical conditioning of the rabbit eyelid response with mossy fiber stimulation as the conditioned stimulus. Bull. Psychonomic Soc. 23: 245-248.

Steinmetz, J.E., D.J. Rosen, P.F. Chapman, D.G. Lavond, and R.F. Thompson. 1986. Classical conditioning of the rabbit eyelid response with a mossy fiber stimulation CS. I. Pontine nuclei and middle cerebellar peduncle stimulation. Behav. Neurosci. 100: 871-880.

Steinmetz, J.E., C.G. Logan, D.J. Rosen, J.K. Thompson, D.G. Lavond, and R.F. Thompson. 1987. Initial localization of the acoustic conditioned stimulus projection system to the cerebellum essential for classical eyelid conditioning. Proc. Natl. Acad. Sci. 84" 3531-3535.

Steinmetz, J.E., D.G. Lavond, and R.F. Thompson. 1989. Classical conditioning in rabbits using pontine nucleus stimulation as a conditioned stimulus and inferior olive stimulation as an unconditioned stimulus. Synapse 3" 225-233.

Tarnecki, R. 1988. Functional connections between neurons of interpositus nucleus of the cerebellum and the red nucleus. Behav. Brain Res. 28" 117-125.

Thompson, R.F., T.W. Berger, D.F. Cegavski, M.M. Pattersonn, R.A. Roemer, T.J. Teyler, and R.A. Young. 1976. The search for the engram. Am. Psychol. 31" 209-227.

Tracy, J. 1995. "Brain and behavior correlates in classical conditioning of the rabbit eyeblink response." Ph.D. dissertation. University of Southern California, Los Angeles, CA.

Tsukahara, N., Y. Oda, and T. Notsu. 1981. Classical conditioning mediated by the red nucleus in the cat. J. Neurosci. 1" 72-77.

Watt, C.B. and G.A. Mihailoff. 1983. The cerebellopontine system in the rat. I. Autoradiographic studies. J. Comp. Neurol. 215:312-330.

Weinberger, N.M. 1993. Learning-induced changes of auditory receptive fields. Curr. Opin. Neurobiol. 3: 570-577.

Woody, C.D., X.F. Wang, E. Gruen, and J. Landeira-Fernandez. 1992. Unit activity to click CS changes in dorsal cochlear nucleus after conditioning. NeuroReport 3" 385-388.

Woody, C.D., X.F. Wang, and E. Gruen. 1994. Response to acoustic stimuli increases in the ventral cochlear nucleus after stimulus pairing. NeuroReport 5:513-515.

Zhang, A.A., H. Ni, and R.M. Harper. 1986. A miniaturized cryoprobe for functional neuronal blockade in freely moving animals. J. Neurosci. 16" 79-87.

Received January 28, 1997; accepted in revised form March 25, 1997.

L E A R N I N G I M E M 0 R Y




10.1101/lm.3.6.519Access the most recent version at doi: 3:1997, Learn. Mem.

M C Cartford, E B Gohl, M Singson, et al. nuclei of classically conditioned rabbits.learning-related and auditory-evoked unit activity in the pontine The effects of reversible inactivation of the red nucleus on

References

http://learnmem.cshlp.org/content/3/6/519.full.html#ref-list-1

This article cites 36 articles, 4 of which can be accessed free at:

License

ServiceEmail Alerting

click here.top right corner of the article or

Receive free email alerts when new articles cite this article - sign up in the box at the

Copyright © Cold Spring Harbor Laboratory Press


http://learnmem.cshlp.org/lookup/doi/10.1101/lm.3.6.519

http://learnmem.cshlp.org/content/3/6/519.full.html#ref-list-1

http://learnmem.cshlp.org/cgi/alerts/ctalert?alertType=citedby&addAlert=cited_by&saveAlert=no&cited_by_criteria_resid=protocols;10.1101/lm.3.6.519&return_type=article&return_url=http://learnmem.cshlp.org/content/10.1101/lm.3.6.519.full.pdf



Documents

The Effects of Reversible Inactivation of the Red …learnmem.cshlp.org/content/3/6/519.full.pdf · The Effects of Reversible Inactivation of the Red Nucleus on Learning-Related and