Brain Research, 532 (1990) 211-221 Elsevier
211
BRES 16010
Neuronal activity in the mediodorsal and intralaminar nuclei of the dorsal thalamus during classical heart rate conditioning D . A . Powell 1"2'3, Karen L. Watson I and Shirley L. B u c h a n a n 1'2 lNeuroscience Laboratory, VA Medical Center, Columbia, SC 29201, 2Department of Psychology, University of South Carolina, Columbia, SC 29208, and 3Department of Neuropsychiatry and Behavioral Science, University of South Carolina School of Medicine, Columbia, SC 29208 (U.S.A.) (Accepted 15 May 1990) Key words: Learning; Neuronal plasticity; Mediodorsal nucleus; Intralaminar nuclei of the thalamus; Rabbit; Heart rate
Multiple unit activity (MUA) was recorded from chronically implanted electrodes in either the mediodorsal (MD) or the intralaminar (IL) nuclei of the dorsal thalamus in separate groups of rabbits during (a) habituation of the cardiac orienting reflex, (b) Pavlovian heart rate (HR) conditioning, and (c) extinction of the HR conditioned response (CR). Other animals with similar recording electrodes received explicitly unpaired presentations of the conditioned stimulus (CS) and unconditioned stimulus (US). The cardiac orienting reflex and the HR CR consisted of bradycardia. However, tone-evoked tachycardia was obtained in animals that received CS/US unpaired presentations. MUA evoked by the CS consisted of a short latency (20-40 ms) increase under all conditions, which reached its maximum 200-300 ms after CS onset. This response habituated greatly during tone-alone pretraining, but was considerably greater in the paired than unpaired group during the later trials of conditioning in animals with MD, but not IL, placements. Instead, a longer latency increase (>500 ms) in MUA occurred in the paired but not in the unpaired animals in the IL group. The MUA increases in both instances, including the early, short latency increase in the MD group, and the longer latency increase in the IL group, were trial-related, and declined to pretraining levels during extinction, indicating that these neuronal changes had an associative basis. These findings suggest that neuronal activity in both MD and IL is related to the early events involved in Pavlovian conditioning, but that the relative roles of these two closely related thalamic nuclei in associative learning must be somewhat different. INTRODUCTION We have previously d e m o n s t r a t e d that the midline prefrontal cortex (PFC) is a m a j o r focus for the heart rate ( H R ) decelerations and blood pressure (BP) depressor responses observed during classical conditioning in rabbits. These cardiovascular adjustments occur in response to a previously neutral stimulus (conditioned stimulus, CS) that precedes an aversive unconditioned stimulus (US), and can be contrasted with instrumental conditioning in which the presentation of the US (or reinforcer) is d e p e n d e n t upon the occurrence (or nonoccurrence) of an o p e r a n t response. The present research focused solely on the bradycardia elicited by classical conditioning contingencies. Lesions of the midline P F C dramatically attenuate the magnitude of the conditioned bradycardia normally o b t a i n e d during classical conditioning 3'4. Electrical stimulation of the midline P F C elicits inhibitory cardiovascular changes which are similar to those observed during classical conditioning 7 and neuronal activity in the midline prefrontal area e v o k e d by classical conditioning contingencies is correlated with the magnitude of conditioned b r a d y c a r d i a 13.
It has been suggested that the prefrontal cortex in subprimate species be defined as that part of the prefrontal area that receives the projections of the m e d i o d o r s a l ( M D ) nucleus of the thalamus 26. This includes the midline P F C , as described above, as well as a m o r e lateral region of the prefrontal cortex along the dorsal b o r d e r of the rhinal fissure. H o w e v e r , lesions of the latter, so-called agranular insular cortex, have little effect on conditioned b r a d y c a r d i a , although stimulation of this more lateral prefrontal a r e a also produces inhibitory cardiovascular changes 25. F u r t h e r , M U A r e c o r d e d from the agranular insular cortex is not strongly associated with conditioned b r a d y c a r d i a , unlike that described above for the midline P F C 14. H o w e v e r , M D has also long been implicated in learning and m e m o r y processes 17" 20~21,22,33. Cells in the lateral p o r t i o n of M D project to the midline P F C 8'11'15, which has, as noted, been demonstrated to be intimately involved in the e l a b o r a t i o n of conditioned bradycardia. A n i m p o r t a n t question thus concerns whether neuronal activity r e c o r d e d from M D during classical conditioning is also r e l a t e d to conditioned bradycardia. In addition, the i n t r a l a m i n a r (IL) nuclei of the thalamus, including the c e n t r o m e d i a n , paracentral
Correspondence: D.A. Powell, Neuroscience Laboratory (151A), VA Medical Center, Columbia, SC 29201, U.S.A. 0006-8993/90/$03.50 ~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
212 a n d t h e c e n t r o l a t e r a l n u c l e i , h a v e a s t r o n g p r o j e c t i o n to t h e p r e f r o n t a l c o r t e x 8"12'~6"28. T h e s e nuclei also p r o j e c t t o the neostriatum, including the caudate nucleus, p u t a m e n , g l o b u s p a l l i d u s , a n d n u c l e u s a c c u m b e n s ~'12"16"28. H o w ever, whether MD
p r o j e c t s to t h e s e nuclei is at t h e
p r e s e n t t i m e c o n t r o v e r s i a l t'~5. D u e to t h e similar p r o j e c t i o n s o f t h e s e t w o nuclei to t h e P F C ,
unit discharges were generated during specific time epochs of interest during pretraining, classical conditioning, and extinction by the online computer. The window discriminator was set for each animal so that the pre-tone baseline activity recorded was approximately 10-15 Hz. MUA and HR was displayed on an oscilloscope and acoustically monitored to ensure that any subtle movement artifacts or baseline shifts could be documented and thus be excluded from the data analysis.
we recorded
m u l t i p l e u n i t activity d u r i n g classical c o n d i t i o n i n g f r o m b o t h t h e I L a n d t h e M D nuclei in t h e p r e s e n t e x p e r i ments.
MATERIALS AND METHODS
Animals Experimentally naive New Zealand albino rabbits of both sexes were employed as experimental subjects. The animals were approximately 6 months old at the beginning of the study and weighed 2-3 kg on arrival from a local USDA-licensed supplier. The animals were housed individually in an animal facility accredited by the American Association for Accreditation of Laboratory Animal Care and were maintained on ad libitum food and water. A 07.00-19.00 light/dark cycle was employed; all animals were studied during the daylight portion of this cycle. Principles for the care and use of experimental animals, as outlined by the US Public Health Service, were strictly followed in these studies.
Surgery The animals were anesthetized with ketamine hydrochloride (55 mg/kg body weight, i.m.), supplemented with acepromazine malate (2.2 mg/kg, i.m.) and xylazine (3.0 mg/kg, i.m.). Electrolytically etched epoxalite insulated Tungsten microelectrodes (1-4/~m tip, impedance 1-2 M£2 at 1000 Hz) were implanted in either the MD or the IL of the dorsal thalamus under stereotaxic guidance. The coordinates for implantation of these electrodes varied from animal to animal with respect to the following coordinates: 2-4 mm posterior to bregma, 1-3 mm lateral to the midline suture, and 8.5-9.5 mm ventral to the dura 31. Leads from the recording electrode were connected to an amphenol socket, which was then secured to the skull with dental acrylic and stainless steel machine screws. After an injection of bicillin, each animal was allowed from 1-2 weeks post-surgical recovery, during which time it was observed daily and additional antibiotics and/or analgesics administered as needed.
Recording procedures During behavioral training the animals were restrained in a standard rabbit restrainer within a ventilated and sound-attenuating enclosure (Industrial Acoustics Co.). The conditioned stimulus was a 4-s, 1216-Hz, 75-db, re-20/t N/cm2, square wave tone generated by solid state audio-oscillator circuits. The tone was delivered through speakers located in the experimental chamber approximately 30 cm above the animal's head. The unconditioned stimulus was a paraorbital electric shock train consisting of a 250-ms, biphasic, 3-mA, 60-Hz stimulus, administered through 2 stainless steel wound clips positioned approximately 1 cm above and below the right eye. All experimental events were controlled by a Heath LSI 11 microcomputer. Electrocardiogram (ECG) and stimulus event markers were recorded on a Grass Model 7D polygraph. The ECG electrodes were stainless steel safety pins previously inserted over the right shoulder and left flank. As noted in our earlier studies 19"23, insertion of these pins does not produce any obvious discomfort or infection. MUA was conventionally amplified with a WPI Model DAM 80 amplifier. This signal was band-passed (0.5-10 kHz) and notch (60 Hz) filtered and subsequently fed into an amplitude-window discriminator (WPI Model 121) for signal analysis. Discriminable
Behavioral training The animals were first adapted to handling and restraint for 2 daily 30-min sessions. Subsequently, HR and neuronal activity were assessed during 2 consecutive daily training sessions. During the first day, animals received 10 presentations of the 4-s tone without the paraorbital electric shock US, to assess neuronal activity during elicitation and habituation of the cardiac orienting reflex (OR). This phase of training will be referred to as the 'pretraining' phase. This first phase was followed by four 10-trial blocks of classical conditioning, in which the tone CS was paired with the eyeshock US, with a CS/US interval of 4 s for separate groups of animals with either MD or IL placements (paired condition). To control for nonassociative changes in MUA and HR, a random sequence of 40 tones and eyeshocks was presented that were specifically unpaired in a second group of animals with either MD or IL placements (unpaired condition). Day-2 training involved 2 additional 10-trial training blocks that were identical to the paired and unpaired training during the first day. These 20 training trials were followed by three 10-trial blocks of tone-alone presentations to assess neuronal and cardiac changes during experimental extinction. During pretraining, classical conditioning, and extinction trial-blocks, the tones were delivered at intervals of 75, 90 or 105 s on a random basis with a mean of 90 s. During training the interval separating the tones and eyeshocks in the unpaired groups was half that of the tone-tone interval for the paired groups, viz., 37.5, 45.0, or 52.5 s on a random basis with a mean of 45 s. Thus, the animals in the unpaired groups received the same number of tones and eyeshocks over the same period of time as the paired groups, but the tones and shocks were specifically never paired together in the unpaired condition. The 10th trial of each block of tone trials served as a tone-alone 'test' trial, during which the shock US was not presented. Heart rate was recorded on these trials during training and extinction, but was recorded during trials 1, 5, and 10 of the pretraining phase to 'track' the habituation of the OR.
Histology At the conclusion of the behavioral observations, the recording site was marked by passing an anodal current of 2.0-3.0 mA through the recording electrode for 20-25 s. Approximately 24 h later the animal was killed with sodium pentobarbital (75 mg/kg, i.v.) and perfused transcardially with physiological saline, followed by 10% formalin. After further immersion fixation at 4 °C in a solution containing 2% DSMO and 10% glycerol27, the brain was blocked in the transverse plane and frozen serial sections were taken at 40 ktm through the dorsal thalamus. These sections were counter-stained with .thionin and the electrode tract and electrolytic lesion subsequently plotted on plates adapted from the atlas of Urban and Richard 31 using a Zeiss drawing tube and microscope.
Data reduction and statistical analysis Heart period (interbeat interval duration) was measured in ms during a 4-s pre-tone baseline period and during the tone (CS) period on pretraining trials 1, 5, and 10, and on each of the test trials during training or extinction. CS-evoked changes in heart period on each of these trials was calculated by subtracting the mean 4-s baseline heart period from the heart period for each of the IBis during the tone. For purposes of data presentation heart period was converted to heart rate in all instances. As in our previous studies (i.e., ref. 13), MUA was evaluated by compiling peristimulus histograms for selected trial blocks in bins of 20 or 100 ms over 1-s
213 periods immediately preceding and following tone onset. Z-score equivalents for each of the tone bins was calculated by subtracting the mean baseline discharge from the discharge evoked by the tone, and dividing this difference by the standard deviation of the baseline discharge. Both the heart period and neuronal data of the MD and IL groups were submitted to repeated measures analysis of variance (ANOVAs), generally involving factors of (a) condition (viz. paired vs. unpaired), (b) interval during tone presentation (viz. ms) and (c) trial (for HR) or trial-block (for MUA). Significant effects were further analyzed by post-hoc application of Duncan's Multiple Range Test.
RESULTS
Histology Fig. 1 shows electrode placements in the M D (closed circles) and IL (closed squares) nuclei on line drawings through 4 A P levels of the thalamus. Fig. 1A shows placements of animals that received paired CS/US training, while Fig. 1B shows similar placements in animals that received unpaired, randomly presented CS/US presentations. As can be seen there were no systematic differences between placements in the paired and unpaired conditions of either the MD or IL groups.
sisted of bradycardia that was greatest during the initial block of 10 trials, and declined somewhat over subsequent blocks of trials, but which was still considerably greater on these trial-blocks than during pretraining or extinction. The H R changes in the animals presented unpaired tones and paraorbital shocks, however, consisted of tachycardia, also as previously described 13'14. Fig. 3 illustrates these findings. This figure shows CS-evoked H R changes from pre-CS baseline as a function of postoCS IBis during test trials associated with the first 4 blocks of 10 trials of acquisition training. Also shown are the H R changes from pre-tone baseline for the last trial of pretraining and the last trial of extinction. These data are shown separately for the M D and IL placements and for the paired vs. unpaired conditions. A N O V A of these data revealed significant condition (viz., paired vs. unpaired) differences for both M D and
Heart rate: pretraining The initial cardiac response to the first tone during trial 1 of pretraining consisted of a dramatic decrease in H R , which reached its maximum magnitude by the 10th IBI, just prior to tone offset in most animals. However, this response had declined greatly in magnitude by trial 5, and had virtually disappeared by trial 10, representing virtually complete habituation of the cardiac OR. Figure 2 illustrates tone-evoked changes in H R in the paired and unpaired groups during the first, middle, and last trial of pretraining. A N O V A revealed no differences between the M D and IL groups. Thus, the H R data shown are pooled for the electrode placement dimension. These data are plotted in beats/min (BPM) change from pre-CS baseline as a function of 10 post-CS IBis. Separate A N O V A s of the H R data for the IL and MD groups revealed significant IBI and trial effects for both groups ( I B I : F9,17 l = 5.31; P < 0.0001, and F9,1o8 = 2.92, P < 0.003; and trial: F2.38 = 27.9, P < 0.0001 and F2,24 = 13.6, P < 0.0001 for, the M D and IL placement groups, respectively). However, the condition (viz. paired vs. unpaired) main effect was not significant nor were any condition interactions significant for either group. Thus, the H R response to the tone was comparable in the paired and unpaired conditions prior to the initiation of classical conditioning or unpaired CS/US training.
Heart rate., classical conditioning As has been found in previous studies 3'19"23, CSevoked H R changes during classical conditioning con-
'
Fig. 1. Electrode placements located in the mediodorsal nucleus (O) and in the intralaminar nuclei ( . ) of the thalamus. Drawings represent A-P levels that are approximately 2.0, 3.0, 4.0 and 5.0 mm posterior to bregma, based on plates adapted from the atlas of Urban and Richard 31. A shows placements in animals that received paired CS/US training and B shows similar placements in animals that received unpaired randomly presented CS/US presentations.
214 IL placements (MD: Fl,19 = 5.37, P < 0.03 and IL: Fl.12 = 8.3, P < 0.01), as well as a condition × IBI interaction (MD: F9,17~ = 8.6, P < 0.0001; IL: F9,ao8 = 7.8, P < 0.0001). Post-tests of the H R change in the paired conditions revealed that the greatest magnitude change, which occurred during the 10th IBI, at the end of the 4-s CS, was significantly larger on acquisition trial-block 1, compared to trial-blocks 2, 3, and 4. The latter were not significantly different from each other. All 4 acquisition trial-blocks were significantly different from both extinction and pretraining (largest P < 0.01). In the unpaired conditions, the tachycardia associated with the 10th IBI was significantly greater during the 4 training trial-blocks, compared to extinction and pretraining (largest P < 0.05), but the training trial-blocks did not differ significantly from each other.
Heart rate: day 2 training CS-evoked H R changes during training on day 2 were very similar to those during day 1. This response again consisted of conditioned H R decelerations, beginning at tone onset in both the IL and MD groups, which became greater over the tone interval to reach a maximum by the 10th interbeat interval. Also like session 1, both unpaired groups revealed H R increases from baseline during tone presentation. The paired vs. unpaired condition and IBI main effects, and the condition x IBI interaction, were significant for both the IL and MD analyses (largest P < 0.01). Heart rate: extinction Tone-evoked H R changes declined during extinction from those obtained during conditioning (see Fig. 3). However, these H R changes did not completely extin-
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Multiple unit activity: pretraining Tone-evoked increases in M U A occurred in both the IL and MD placement groups during pre-training, regardless of whether the animals later received paired or unpaired CS/US training. However, the magnitude of the M U A increase, as well as its change over bins, was different for the two placement groups. Fig. 4 illustrates these changes in M U A during pretraining, separately for the paired and unpaired conditions with MD and IL placements. These data are Z-scores, as described above, shown as a function of ten 100-ms post-tone bins. Average M U A is shown separately for the first 3, second 3, and last 4 trials of pretraining. Tone-evoked M U A increases in MD showed a rapid augmentation over the first 3-4 100-ms bins, reaching a maximum Z-score of 6-7, after which M U A declined. This was true of all 3 stages of pretraining, but was most pronounced during trial-block 1, early during pretraining. In fact, the middle and last trial-blocks were considerably smaller than the first in both the paired and unpaired animals. ANOVA of these data for the MD groups revealed significant trial-block (F2,57 = 7.68, P < 0.001) and bin (F9,279 = 6.46, P < 0.0001) effects. However, the condition (viz.,
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guish. A N O V A revealed a significant change from baseline for all animals during extinction (largest P < 0.01). An A N O V A comparing the paired and unpaired conditions also revealed significant differences between the conditions for both the IL and MD placements (largest P < 0.01), again reflecting the different directions of H R change in the two conditions. However, as described above, the H R changes during extinction were significantly smaller than those during acquisition for both paired groups.
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215 UNPAIRED INTRALAMINAR NUCLEI
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211
BRES 16010
Neuronal activity in the mediodorsal and intralaminar nuclei of the dorsal thalamus during classical heart rate conditioning D . A . Powell 1"2'3, Karen L. Watson I and Shirley L. B u c h a n a n 1'2 lNeuroscience Laboratory, VA Medical Center, Columbia, SC 29201, 2Department of Psychology, University of South Carolina, Columbia, SC 29208, and 3Department of Neuropsychiatry and Behavioral Science, University of South Carolina School of Medicine, Columbia, SC 29208 (U.S.A.) (Accepted 15 May 1990) Key words: Learning; Neuronal plasticity; Mediodorsal nucleus; Intralaminar nuclei of the thalamus; Rabbit; Heart rate
Multiple unit activity (MUA) was recorded from chronically implanted electrodes in either the mediodorsal (MD) or the intralaminar (IL) nuclei of the dorsal thalamus in separate groups of rabbits during (a) habituation of the cardiac orienting reflex, (b) Pavlovian heart rate (HR) conditioning, and (c) extinction of the HR conditioned response (CR). Other animals with similar recording electrodes received explicitly unpaired presentations of the conditioned stimulus (CS) and unconditioned stimulus (US). The cardiac orienting reflex and the HR CR consisted of bradycardia. However, tone-evoked tachycardia was obtained in animals that received CS/US unpaired presentations. MUA evoked by the CS consisted of a short latency (20-40 ms) increase under all conditions, which reached its maximum 200-300 ms after CS onset. This response habituated greatly during tone-alone pretraining, but was considerably greater in the paired than unpaired group during the later trials of conditioning in animals with MD, but not IL, placements. Instead, a longer latency increase (>500 ms) in MUA occurred in the paired but not in the unpaired animals in the IL group. The MUA increases in both instances, including the early, short latency increase in the MD group, and the longer latency increase in the IL group, were trial-related, and declined to pretraining levels during extinction, indicating that these neuronal changes had an associative basis. These findings suggest that neuronal activity in both MD and IL is related to the early events involved in Pavlovian conditioning, but that the relative roles of these two closely related thalamic nuclei in associative learning must be somewhat different. INTRODUCTION We have previously d e m o n s t r a t e d that the midline prefrontal cortex (PFC) is a m a j o r focus for the heart rate ( H R ) decelerations and blood pressure (BP) depressor responses observed during classical conditioning in rabbits. These cardiovascular adjustments occur in response to a previously neutral stimulus (conditioned stimulus, CS) that precedes an aversive unconditioned stimulus (US), and can be contrasted with instrumental conditioning in which the presentation of the US (or reinforcer) is d e p e n d e n t upon the occurrence (or nonoccurrence) of an o p e r a n t response. The present research focused solely on the bradycardia elicited by classical conditioning contingencies. Lesions of the midline P F C dramatically attenuate the magnitude of the conditioned bradycardia normally o b t a i n e d during classical conditioning 3'4. Electrical stimulation of the midline P F C elicits inhibitory cardiovascular changes which are similar to those observed during classical conditioning 7 and neuronal activity in the midline prefrontal area e v o k e d by classical conditioning contingencies is correlated with the magnitude of conditioned b r a d y c a r d i a 13.
It has been suggested that the prefrontal cortex in subprimate species be defined as that part of the prefrontal area that receives the projections of the m e d i o d o r s a l ( M D ) nucleus of the thalamus 26. This includes the midline P F C , as described above, as well as a m o r e lateral region of the prefrontal cortex along the dorsal b o r d e r of the rhinal fissure. H o w e v e r , lesions of the latter, so-called agranular insular cortex, have little effect on conditioned b r a d y c a r d i a , although stimulation of this more lateral prefrontal a r e a also produces inhibitory cardiovascular changes 25. F u r t h e r , M U A r e c o r d e d from the agranular insular cortex is not strongly associated with conditioned b r a d y c a r d i a , unlike that described above for the midline P F C 14. H o w e v e r , M D has also long been implicated in learning and m e m o r y processes 17" 20~21,22,33. Cells in the lateral p o r t i o n of M D project to the midline P F C 8'11'15, which has, as noted, been demonstrated to be intimately involved in the e l a b o r a t i o n of conditioned bradycardia. A n i m p o r t a n t question thus concerns whether neuronal activity r e c o r d e d from M D during classical conditioning is also r e l a t e d to conditioned bradycardia. In addition, the i n t r a l a m i n a r (IL) nuclei of the thalamus, including the c e n t r o m e d i a n , paracentral
Correspondence: D.A. Powell, Neuroscience Laboratory (151A), VA Medical Center, Columbia, SC 29201, U.S.A. 0006-8993/90/$03.50 ~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
212 a n d t h e c e n t r o l a t e r a l n u c l e i , h a v e a s t r o n g p r o j e c t i o n to t h e p r e f r o n t a l c o r t e x 8"12'~6"28. T h e s e nuclei also p r o j e c t t o the neostriatum, including the caudate nucleus, p u t a m e n , g l o b u s p a l l i d u s , a n d n u c l e u s a c c u m b e n s ~'12"16"28. H o w ever, whether MD
p r o j e c t s to t h e s e nuclei is at t h e
p r e s e n t t i m e c o n t r o v e r s i a l t'~5. D u e to t h e similar p r o j e c t i o n s o f t h e s e t w o nuclei to t h e P F C ,
unit discharges were generated during specific time epochs of interest during pretraining, classical conditioning, and extinction by the online computer. The window discriminator was set for each animal so that the pre-tone baseline activity recorded was approximately 10-15 Hz. MUA and HR was displayed on an oscilloscope and acoustically monitored to ensure that any subtle movement artifacts or baseline shifts could be documented and thus be excluded from the data analysis.
we recorded
m u l t i p l e u n i t activity d u r i n g classical c o n d i t i o n i n g f r o m b o t h t h e I L a n d t h e M D nuclei in t h e p r e s e n t e x p e r i ments.
MATERIALS AND METHODS
Animals Experimentally naive New Zealand albino rabbits of both sexes were employed as experimental subjects. The animals were approximately 6 months old at the beginning of the study and weighed 2-3 kg on arrival from a local USDA-licensed supplier. The animals were housed individually in an animal facility accredited by the American Association for Accreditation of Laboratory Animal Care and were maintained on ad libitum food and water. A 07.00-19.00 light/dark cycle was employed; all animals were studied during the daylight portion of this cycle. Principles for the care and use of experimental animals, as outlined by the US Public Health Service, were strictly followed in these studies.
Surgery The animals were anesthetized with ketamine hydrochloride (55 mg/kg body weight, i.m.), supplemented with acepromazine malate (2.2 mg/kg, i.m.) and xylazine (3.0 mg/kg, i.m.). Electrolytically etched epoxalite insulated Tungsten microelectrodes (1-4/~m tip, impedance 1-2 M£2 at 1000 Hz) were implanted in either the MD or the IL of the dorsal thalamus under stereotaxic guidance. The coordinates for implantation of these electrodes varied from animal to animal with respect to the following coordinates: 2-4 mm posterior to bregma, 1-3 mm lateral to the midline suture, and 8.5-9.5 mm ventral to the dura 31. Leads from the recording electrode were connected to an amphenol socket, which was then secured to the skull with dental acrylic and stainless steel machine screws. After an injection of bicillin, each animal was allowed from 1-2 weeks post-surgical recovery, during which time it was observed daily and additional antibiotics and/or analgesics administered as needed.
Recording procedures During behavioral training the animals were restrained in a standard rabbit restrainer within a ventilated and sound-attenuating enclosure (Industrial Acoustics Co.). The conditioned stimulus was a 4-s, 1216-Hz, 75-db, re-20/t N/cm2, square wave tone generated by solid state audio-oscillator circuits. The tone was delivered through speakers located in the experimental chamber approximately 30 cm above the animal's head. The unconditioned stimulus was a paraorbital electric shock train consisting of a 250-ms, biphasic, 3-mA, 60-Hz stimulus, administered through 2 stainless steel wound clips positioned approximately 1 cm above and below the right eye. All experimental events were controlled by a Heath LSI 11 microcomputer. Electrocardiogram (ECG) and stimulus event markers were recorded on a Grass Model 7D polygraph. The ECG electrodes were stainless steel safety pins previously inserted over the right shoulder and left flank. As noted in our earlier studies 19"23, insertion of these pins does not produce any obvious discomfort or infection. MUA was conventionally amplified with a WPI Model DAM 80 amplifier. This signal was band-passed (0.5-10 kHz) and notch (60 Hz) filtered and subsequently fed into an amplitude-window discriminator (WPI Model 121) for signal analysis. Discriminable
Behavioral training The animals were first adapted to handling and restraint for 2 daily 30-min sessions. Subsequently, HR and neuronal activity were assessed during 2 consecutive daily training sessions. During the first day, animals received 10 presentations of the 4-s tone without the paraorbital electric shock US, to assess neuronal activity during elicitation and habituation of the cardiac orienting reflex (OR). This phase of training will be referred to as the 'pretraining' phase. This first phase was followed by four 10-trial blocks of classical conditioning, in which the tone CS was paired with the eyeshock US, with a CS/US interval of 4 s for separate groups of animals with either MD or IL placements (paired condition). To control for nonassociative changes in MUA and HR, a random sequence of 40 tones and eyeshocks was presented that were specifically unpaired in a second group of animals with either MD or IL placements (unpaired condition). Day-2 training involved 2 additional 10-trial training blocks that were identical to the paired and unpaired training during the first day. These 20 training trials were followed by three 10-trial blocks of tone-alone presentations to assess neuronal and cardiac changes during experimental extinction. During pretraining, classical conditioning, and extinction trial-blocks, the tones were delivered at intervals of 75, 90 or 105 s on a random basis with a mean of 90 s. During training the interval separating the tones and eyeshocks in the unpaired groups was half that of the tone-tone interval for the paired groups, viz., 37.5, 45.0, or 52.5 s on a random basis with a mean of 45 s. Thus, the animals in the unpaired groups received the same number of tones and eyeshocks over the same period of time as the paired groups, but the tones and shocks were specifically never paired together in the unpaired condition. The 10th trial of each block of tone trials served as a tone-alone 'test' trial, during which the shock US was not presented. Heart rate was recorded on these trials during training and extinction, but was recorded during trials 1, 5, and 10 of the pretraining phase to 'track' the habituation of the OR.
Histology At the conclusion of the behavioral observations, the recording site was marked by passing an anodal current of 2.0-3.0 mA through the recording electrode for 20-25 s. Approximately 24 h later the animal was killed with sodium pentobarbital (75 mg/kg, i.v.) and perfused transcardially with physiological saline, followed by 10% formalin. After further immersion fixation at 4 °C in a solution containing 2% DSMO and 10% glycerol27, the brain was blocked in the transverse plane and frozen serial sections were taken at 40 ktm through the dorsal thalamus. These sections were counter-stained with .thionin and the electrode tract and electrolytic lesion subsequently plotted on plates adapted from the atlas of Urban and Richard 31 using a Zeiss drawing tube and microscope.
Data reduction and statistical analysis Heart period (interbeat interval duration) was measured in ms during a 4-s pre-tone baseline period and during the tone (CS) period on pretraining trials 1, 5, and 10, and on each of the test trials during training or extinction. CS-evoked changes in heart period on each of these trials was calculated by subtracting the mean 4-s baseline heart period from the heart period for each of the IBis during the tone. For purposes of data presentation heart period was converted to heart rate in all instances. As in our previous studies (i.e., ref. 13), MUA was evaluated by compiling peristimulus histograms for selected trial blocks in bins of 20 or 100 ms over 1-s
213 periods immediately preceding and following tone onset. Z-score equivalents for each of the tone bins was calculated by subtracting the mean baseline discharge from the discharge evoked by the tone, and dividing this difference by the standard deviation of the baseline discharge. Both the heart period and neuronal data of the MD and IL groups were submitted to repeated measures analysis of variance (ANOVAs), generally involving factors of (a) condition (viz. paired vs. unpaired), (b) interval during tone presentation (viz. ms) and (c) trial (for HR) or trial-block (for MUA). Significant effects were further analyzed by post-hoc application of Duncan's Multiple Range Test.
RESULTS
Histology Fig. 1 shows electrode placements in the M D (closed circles) and IL (closed squares) nuclei on line drawings through 4 A P levels of the thalamus. Fig. 1A shows placements of animals that received paired CS/US training, while Fig. 1B shows similar placements in animals that received unpaired, randomly presented CS/US presentations. As can be seen there were no systematic differences between placements in the paired and unpaired conditions of either the MD or IL groups.
sisted of bradycardia that was greatest during the initial block of 10 trials, and declined somewhat over subsequent blocks of trials, but which was still considerably greater on these trial-blocks than during pretraining or extinction. The H R changes in the animals presented unpaired tones and paraorbital shocks, however, consisted of tachycardia, also as previously described 13'14. Fig. 3 illustrates these findings. This figure shows CS-evoked H R changes from pre-CS baseline as a function of postoCS IBis during test trials associated with the first 4 blocks of 10 trials of acquisition training. Also shown are the H R changes from pre-tone baseline for the last trial of pretraining and the last trial of extinction. These data are shown separately for the M D and IL placements and for the paired vs. unpaired conditions. A N O V A of these data revealed significant condition (viz., paired vs. unpaired) differences for both M D and
Heart rate: pretraining The initial cardiac response to the first tone during trial 1 of pretraining consisted of a dramatic decrease in H R , which reached its maximum magnitude by the 10th IBI, just prior to tone offset in most animals. However, this response had declined greatly in magnitude by trial 5, and had virtually disappeared by trial 10, representing virtually complete habituation of the cardiac OR. Figure 2 illustrates tone-evoked changes in H R in the paired and unpaired groups during the first, middle, and last trial of pretraining. A N O V A revealed no differences between the M D and IL groups. Thus, the H R data shown are pooled for the electrode placement dimension. These data are plotted in beats/min (BPM) change from pre-CS baseline as a function of 10 post-CS IBis. Separate A N O V A s of the H R data for the IL and MD groups revealed significant IBI and trial effects for both groups ( I B I : F9,17 l = 5.31; P < 0.0001, and F9,1o8 = 2.92, P < 0.003; and trial: F2.38 = 27.9, P < 0.0001 and F2,24 = 13.6, P < 0.0001 for, the M D and IL placement groups, respectively). However, the condition (viz. paired vs. unpaired) main effect was not significant nor were any condition interactions significant for either group. Thus, the H R response to the tone was comparable in the paired and unpaired conditions prior to the initiation of classical conditioning or unpaired CS/US training.
Heart rate., classical conditioning As has been found in previous studies 3'19"23, CSevoked H R changes during classical conditioning con-
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Fig. 1. Electrode placements located in the mediodorsal nucleus (O) and in the intralaminar nuclei ( . ) of the thalamus. Drawings represent A-P levels that are approximately 2.0, 3.0, 4.0 and 5.0 mm posterior to bregma, based on plates adapted from the atlas of Urban and Richard 31. A shows placements in animals that received paired CS/US training and B shows similar placements in animals that received unpaired randomly presented CS/US presentations.
214 IL placements (MD: Fl,19 = 5.37, P < 0.03 and IL: Fl.12 = 8.3, P < 0.01), as well as a condition × IBI interaction (MD: F9,17~ = 8.6, P < 0.0001; IL: F9,ao8 = 7.8, P < 0.0001). Post-tests of the H R change in the paired conditions revealed that the greatest magnitude change, which occurred during the 10th IBI, at the end of the 4-s CS, was significantly larger on acquisition trial-block 1, compared to trial-blocks 2, 3, and 4. The latter were not significantly different from each other. All 4 acquisition trial-blocks were significantly different from both extinction and pretraining (largest P < 0.01). In the unpaired conditions, the tachycardia associated with the 10th IBI was significantly greater during the 4 training trial-blocks, compared to extinction and pretraining (largest P < 0.05), but the training trial-blocks did not differ significantly from each other.
Heart rate: day 2 training CS-evoked H R changes during training on day 2 were very similar to those during day 1. This response again consisted of conditioned H R decelerations, beginning at tone onset in both the IL and MD groups, which became greater over the tone interval to reach a maximum by the 10th interbeat interval. Also like session 1, both unpaired groups revealed H R increases from baseline during tone presentation. The paired vs. unpaired condition and IBI main effects, and the condition x IBI interaction, were significant for both the IL and MD analyses (largest P < 0.01). Heart rate: extinction Tone-evoked H R changes declined during extinction from those obtained during conditioning (see Fig. 3). However, these H R changes did not completely extin-
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Multiple unit activity: pretraining Tone-evoked increases in M U A occurred in both the IL and MD placement groups during pre-training, regardless of whether the animals later received paired or unpaired CS/US training. However, the magnitude of the M U A increase, as well as its change over bins, was different for the two placement groups. Fig. 4 illustrates these changes in M U A during pretraining, separately for the paired and unpaired conditions with MD and IL placements. These data are Z-scores, as described above, shown as a function of ten 100-ms post-tone bins. Average M U A is shown separately for the first 3, second 3, and last 4 trials of pretraining. Tone-evoked M U A increases in MD showed a rapid augmentation over the first 3-4 100-ms bins, reaching a maximum Z-score of 6-7, after which M U A declined. This was true of all 3 stages of pretraining, but was most pronounced during trial-block 1, early during pretraining. In fact, the middle and last trial-blocks were considerably smaller than the first in both the paired and unpaired animals. ANOVA of these data for the MD groups revealed significant trial-block (F2,57 = 7.68, P < 0.001) and bin (F9,279 = 6.46, P < 0.0001) effects. However, the condition (viz.,
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