97

Behavioural Brain Research, 38 (1990) 97-108 Elsevier BBR01044

Research Reports

Classical nictitating membrane conditioning in rabbits with varying interstimulus intervals and direct activation of cerebellar mossy fibers as the CS J o s e p h E. S t e i n m e t z Department of Psychology, Program in Neural Science, Indiana University, Bloomington, IN47405 (U.S.A.) (Received 25 April 1989) (Revised version received 23 November 1989) (Accepted 24 November 1989)

Key words: Classical conditioning; Nictitating membrane; Pontine stimulation; Conditioned stimulus; Cerebellum; Interstimulus interval; Conditioned stimulus trace

The rate and level of classical nictitating membrane (NM)/eyelid conditioning in rabbits established by pairing a pontine nucleus stimulation conditioned stimulus (CS) with an air puff unconditioned stimulus (US) were studied at 6 interstimulus intervals (ISis). Similar to earlier studies which used peripheral CSs, an inverted U-shaped function relating ISI and conditioning was generated. Interstimulus intervals of 250 and 500 ms produced the highest levels of conditioning, 100, 1000 and 2000 ms ISis resulted in lower levels of conditioning, and no conditioning was established with a 50 ms ISI. These results demonstrate that a normal ISI function can be established when direct activation of cerebellar mossy fibers is used as a CS instead of conventional peripheral CSs.

INTRODUCTION

An intriguing aspect of classical conditioning procedures is that for a number of response systems, an interval of time must be allowed between the onset of the conditioned stimulus (CS) and the onset of the unconditioned stimulus (US) for conditioned responses (CRs) to develop 9. Furthermore, a number of studies demonstrated that systematic manipulations of the CS-US interval (i.e. the interstimulus interval, ISI) produced characteristic inverted U-shaped functions between frequency of CRs and 18I 8'35-37. In general, studies

involving classical conditioning of the rabbit nictitating membrane (NM)/eyelid response have shown that when US onset precedes CS onset, when CS and US onsets are simultaneous, or when the CS-US interval is less than 80-100 ms, no CR acquisition occurs. However, as the CS-US interval is increased CR acquisition becomes possible with ISis of 200-500 ms generally producing the most rapid and robust levels of conditioning. CS-US intervals greater than 500 ms generally produce lower rates and levels of acquisition with no conditioning viewed with ISis greater than about 3000 ms.

Correspondence." J.E. Steinmetz, Department of Psychology, Program in Neural Science, Indiana University, Bloomington, IN 47405, U.S.A. 0166-4328/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

98 Explanations of the observed ISI function have generally centered around stimulus trace notions first proposed by Hull 1°,11. These accounts hypothesize that the onset of the CS initiates a stimulus trace within the nervous system which increases in intensity for a period of time until a maximum is reached after which the trace decreases back to baseline levels. Maximum rates of conditioning are thought to occur when the peak of the stimulus trace is at the point of contiguity with the US and/or UR. The characteristic ISI function is thought to reflect the changes in CS trace intensity over time (i.e. the rate and levels of conditioning are greatest with ISis of 200-500 ms because activity in the stimulus trace is at a maximum during that range of times). Support for the stimulus trace notion has been obtained in behavioral studies such as a study that examined alterations in CR topography with ISI manipulation 6 and a study that monitored changes in unconditioned response (UR) amplitude after manipulation of the IS112. In addition, the timing of the stimulus trace was manipulated when microstimulation of the inferior colliculus was used as a CS 31. Whereas a number of studies had demonstrated that CR acquisition was not seen with an acoustic CS and a 50-ms ISI (e.g. ref. 37), a significant level of conditioned responding was observed with an inferior colliculus stimulation CS and a 50-ms ISI. Because the stimulation CS potentially bypassed a portion of the normal CS pathway, it was argued that the initial recruitment time of the CS trace was reduced and the minimal ISI necessary for conditioning was also decreased. The basic idea of the CS trace has also been included successfully in a variety of computational models of NM conditioning (e.g. refs. 29, 33,34). For example, in the attentional-associative model of Schmajuk and Moore 33'34, the CS is assumed to produce a neural trace after a delay which increases over time to a maximum, stays at that level for a period of time independent of CS duration, and then gradually decays back to zero. The model assumes that the shape of the CS trace reflects the pattern of afferent neural activity at the locus of learning. Moreover, the pattern of the CS trace is thought to be dependent on the time

of propagation along afferent pathways and through synaptic delays as well as the graded activation of different neural populations. Progress has recently been made in delineating essential circuitry involved in classical eyelid conditioning, including elements of the circuit which may constitute the normal CS pathway for auditory stimuli (see ref. 46 for review). In brief, a number of recording, lesion, pharmacology, and stimulation studies have shown that regions of the cerebellar cortex and/or the cerebellar interpositus nucleus are involved critically in the acquisition and retention of classically conditioned eyelid responses 2,5,22,23,48. These studies have suggested that the critical plasticity that underlies CR formation occurs in regions of the cerebellum. In addition, hypotheses concerning possible pathways responsible for relaying an acoustic CS and corneal air puff US to the cerebellum have been formulated from data collected in a number of studies 2°'21'39-41 These studies have suggested that an auditory CS is relayed to the cerebellum via mossy fibers that originate from cells in the lateral pontine nuclear region while a corneal air puff US is relayed to the cerebellum via climbing fibers that originate in the dorsal accessory division of the inferior olive. Plasticity underlying formation of the eyelid CR is thought to occur in regions of the cerebellum (either cortex, deep nuclei, or both) that receive convergent CS and US input. Other hypotheses concerning the involvement of the cerebellum in classical NM conditioning maintain that critical plasticity associated with NM conditioning occurs outside the cerebellum and that the plasticity is projected through the cerebellum to motor nuclei responsible for executing the CR 47. Still other hypotheses suggest that essential learning-related plasticity could occur both inside and outside of the cerebellum27, 28. Evidence for the involvement of cells within lateral regions of the pontine nuclei in projecting acoustic CSs to the cerebellum during conditioning comes from a variety of studies. First, through anatomical and electrophysiological studies, a number of auditory-related structures (e.g. dorsal and ventral cochlear nuclei and inferior colliculus) have been shown to send projec-

99 tions to lateral regions of the pontine nuclei 1'3"13'30'40"44'45. Second, horseradish peroxidase (HRP) studies in the rabbit have demonstrated that cells within lateral regions of the pontine nuclei project to regions of the cerebellum that are known to be involved in classical NM conditioning. These include projections to Larsell's lobule HVI of cerebellar cortex and possibly direct projections to the interpositus nucleus. The existence of pontine projections to cerebellar cortex has been well-established (e.g. ref. 49), but the existence of direct pontine projections to the interpositus nucleus has recently been challenged (e.g. ref. 4). However, preliminary retrograde and anterograde HRP studies and electrophysiological studies suggest that some projections from lateral regions of the pontine nuclei may terminate in anterior regions of the interpositus nucleus 43 (also J.E. Steinmetz and D.R. Sengelaub, unpublished data). Third, lesions of the middle cerebellar peduncle (MCP) which consists of axons of pontine cells prevented acquisition and abolished retention of classically conditioned responses when tone, light, tactile and pontine stimulation CSs w e r e u s e d 18,38. Although these lesions also encompassed portions of cerebellar cortex dorsal to the MCP, lesions of these regions of cortex alone have not abolished CRs to a tone CS 15"17, thus suggesting that damage to the MCP might interrupt CS projections to the cerebellum. Fourth, lesions placed in the lateral pontine nuclear region abolished selectively CRs established with a tone CS while leaving intact CRs established with a light CS 4°. These lesions encompassed large regions of the dorsolateral and lateral pontine nuclei as well as small portions of the lateral lemniscus just dorsal to the pontine nuclei. The effective lesions included pontine regions known to exhibit auditory-related activity and also known to receive projections from auditory structures 1'13'3°'4°. A final line of evidence supporting the involvement ofpontine mossy fibers in projecting the CS to the cerebellum includes studies which demonstrated that microstimulation of the lateral pontine nuclear region or portions of the MCP served as an effective CS when forward-paired with an air puff US 41,42. The use of electrical brain stimu-

lation as a CS is not new. Indeed, a number of previous studies have employed brain stimulation as a US, including direct stimulation of a number of auditory structures 25"26'31. The use of pontine stimulation as a CS instead of peripheral stimuli, however, was an attempt to directly activate potential CS pathways to cerebellar areas suspected to be involved in classical N M conditioning. The stimulation studies have demonstrated rather rapid and robust rates of CR acquisition with a pontine microstimulation CS, normal extinction of CRs with CS-alone presentations, a lack of conditioning with unpaired presentations of the pontine stimulation CS and air puff US, and extremely rapid transfer of training effects when the CS stimulation site was transferred to the opposite side of the p o n s 38'41'42. Evidence for the activation of the cerebellum during training with a pontine CS was obtained when evoked activity in cerebellar cortex was observed with single pulse stimulation of the pontine implant sites (i.e. during electrode implantation) and when a loss of CRs was observed subsequent to lesions of the interpositus nucleus or MCP delivered after training with a pontine CS 41. In short, previous studies showed that training with a pontine stimulation CS produced conditioned responding in rabbits in a manner that paralleled closely conditioning with peripheral CSs such as tones, lights or tactile stimuli. The studies described above suggest that a rather direct CS pathway from the periphery to the cerebellum exists with initial CS information reaching the cerebellum about 12-20 ms after presentation of the tone (i.e. using a CS pathway consisting of the cochlea, cochlear nuclei, pontine nuclei and cerebellum). This speculation has been further substantiated by the observation of interpositus nucleus neurons in anesthetized or awake rabbits which respond to acoustic stimuli with latencies of 12-20 m s 7'23 (also Steinmetz, unpublished data). The relatively short length of the CS pathway is noteworthy since some accounts of the CS trace have suggested a somewhat longer route from periphery to the brain region where convergence between CS and US occurs (e.g. refs. 9,11,29,31). Indeed, it has been suggested that the minimal ISI necessary for conditioning

100 may be the interval between CS onset and the beginning of the CS trace recruitment and that this time period is at least partially determined by delays in sensory transduction and neural transmission within the CS pathway 29'31. The major goal of the present study was to explore the relationship between ISI and rate of conditioning observed when a pontine nucleus stimulation CS was paired with an air puff US. This study was undertaken for at least two major reasons. First, because a number of previous pontine stimulation studies had demonstrated a close correspondence between conditioning with a tone CS and conditioning with a pontine stimulation CS (e.g ref. 41), it seemed useful to determine if manipulating the ISI and using a pontine CS would produce a similar ISI function to that seen with peripheral CSs or if the ISI function could be altered, in some fashion, when the brain stimulation CS was used. Second, it also seemed possible to begin studying the neural basis of the hypothesized CS trace by directly stimulating neural elements suspected to constitute a portion of the neural circuitry involved critically in classical conditioning. MATERIALS AND METHODS

Subjects A total of 35 male, New Zealand white rabbits were used in the present study. Five rabbits failed to condition when the brain stimulation CS was used and their data were therefore not included in the study (see below). All rabbits were maintained on 12/12 h light/dark cycles, received food and water ad libitum and weighed between 2.5 and 3.0 kg at the time of surgery.

Surgery All rabbits were anesthetized with a mixture of ketamine (60 mg/kg) and xylazine (6 mg/kg) and their heads mounted in a standard stereotaxic head-holding device such that the bregma skull landmark was located 1.5 mm above the lambda skull landmark. After drilling a hole through the skull, a stimulating electrode was lowered stereotaxically into the right pontine nuclei located about 10.5 mm posterior to bregma, 2.7 mm to

the right of the midline and 20.0-23.5 mm below bregma. The electrode was an insulated, 00 stainless steel insect pin with a 100-150 #m exposed tip. Final electrode position in the pontine nuclei was determined by maximizing a population potential (3-5 ms latency) evoked by presentation of a click to the right ear (see ref. 41 for details). The electrode was then cemented into place with dental acrylic. A headstage device consisting of a plug with connections to the implanted stimulating electrode and to a ground screw placed in the skull and a threaded plastic block designed to secure a minitorque potentiometer and air puff nozzle for subsequent training sessions were also anchored to the skull with dental acrylic. The rabbits were allowed a 2-week recovery period before training procedures were initiated.

Procedure At the end of the recovery period each rabbit was placed in a standard restraint box, a headstage consisting of a minitorque potentiometer and air puff nozzle was mounted onto the previously implanted plug assembly, and the rabbit was then placed in a sound-attenuated training chamber for a 1-h adaptation session. Training sessions were given on subsequent days. Each training session consisted of 108 trials divided into 12 blocks. Each block was composed of one C S-alone presentation followed by 8 paired CS-US presentations. The CS was a train of monopolar, cathodal, square-wave stimulation delivered to lateral regions of the pontine nuclei (120 #A, 200 Hz, 0.1 ms pulses). The previously implanted skull screw served as the anode for stimulation. The US was a 100-ms air puff directed to the left eye (2.1 N/cm 2 = 3 lbs/in 2 or a displacement of 153 mm of HG). Initially, the air puff US elicited a brisk movement of the left nictitating membrane (NM) that was accompanied by closure of the left outer eyelid. In no case, however, did the stimulation CS produce a discernable NM, eyelid, or other bodily movement before training. Movement of the N M was monitored by the minitorque potentiometer attached to the N M via a hook and a length of suture. Delivery of stimuli and recording of behav-

101 ioral data was accomplished by a microcomputer programmed in Forth and machine language 16. Thirty rabbits were pseudorandomly assigned to 6 groups (n's = 5) and all rabbits were given 8 sessions of paired training with a pontine stimulation CS and air puff US. However, the length of the train of stimulation delivered as a CS during the 8 sessions differed for each of the 6 groups. The 6 groups of rabbits were given either a 150-, 250-, 350-, 600-, 1100-, or 2100-ms stimulation CS over the 8 sessions. During sessions 1-4, the air puff US coterminated with the stimulation trains thus creating 6 groups of rabbits with varying CS-US onset intervals (i.e. ISis) of 50, 100, 250, 500, 1000, or 2000 ms. The 6 ISis used in the present study were chosen because they represent a fair range of ISis used in a number of previous ISI studies (e.g. refs. 31,35,37). During sessions 5-8, the length of the stimulation train was identical to sessions 1-4 except that the 100 ms air puff was moved in time so that 250 ms were allowed to elapse between onset of the stimulation CS and onset of the air puff US for all rabbits. In this manner, the length of the stimulation train was held constant for each rabbit during the 8 sessions of C S-U S training. However, the interval between CS onset and US onset varied on sessions 1-4 for each group of rabbits (i.e. either 50, 100, 250, 500, 1000 or 2000 ms) and was a constant 250 ms for each rabbit on sessions 5-8. In sum, all rabbits received two phases of training. The first phase (sessions 1-4) tested for the relationship between ISI and conditioned responding, while the second phase (sessions 5-8) tested for adaptation to a shift in the ISI. Conditioned responses were defined as 0.5 mm of N M movement that occurred after onset of the CS but before onset of the US. Responding on CS-alone (i.e. stimulation-alone) trials was carefully monitored, especially in rabbits given ISis of 50 or 100 ms, because it was possible that CRs could be masked by execution of the UR in rabbits given paired training with short ISis.

Histology After the last training session, the position of the stimulating electrode was marked by passing 100 pA ofdc for 10 s. The rabbits were then over-

dosed by injecting 4 ml of pentobarbital i.v., perfused via the ascending aorta with 0.9~o saline followed by 10~ formalin, and the brain was removed from the skull. Electrode positions were then assessed under a microscope after serial frozen sections (80/~m) through the pontine nuclei were cut, mounted on slides and stained with Cresyl violet and potassium ferrocyanide. RESULTS

Analysis of the data revealed differences in the rate and level of conditioning during the first 4 sessions of training when different ISis were used for the 6 groups of rabbits. The function relating conditioned responding with ISI could be described as inverted-U with maximum rate of conditioning observed with ISis of 250 and 500 ms and lesser or no conditioning present at longer or shorter ISis. All groups showed comparable numbers of CRs, however, by the eighth session when all rabbits were being trained with a 250 ms ISI. Differences in onset latencies and peak latencies were also observed between ISI groups and changes in the latencies were noted over the course of training when ISis were changed. Details of the statistical analyses performed on the present data are as follows (all P's < 0.05). Analysis of variance involving percent CRs recorded during the first 4 sessions when ISis between groups varied revealed a significant acquisition effect over sessions, F3,72 = 45.26, and a significant Group × Session interaction, F I 5 , 7 2 = 3.1. Further analysis of the significant interaction (Tukey's HSD) showed relatively rapid and similar rates of acquisition for rabbits in the 250- and 500-ms ISI groups, slower but equal rates of acquisition in the 100-, 1000-, and 2000-ms ISI groups, and no CR acquisition in 50-ms ISI rabbits. In addition, no behavioral responses were observed on CS-alone trials in the 50-ms ISI group, indicating that the failure to see learned responses in this group was not due to execution of CRs that were masked by reflexive responding to the US. Analysis of percent CRs during sessions 5-8 when all rabbits were switched to a 250-ms ISI condition revealed a

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significant increase in percent CRs over training, F3.72 = 26.95, and a significant Group x Session interaction effect, F]5,72 = 2.65. Analysis of the interaction effect demonstrated that increases in percent CRs between sessions 5 and 8 were greatest in the 50-ms ISI group and less pronounced (but equal) in the 100-, 1000-, and 2000-ms ISI groups. Significant increases were not detected for the 250- or 500-ms ISI groups. The interaction analysis further revealed that all groups were responding at equal levels by session 8. Fig. 1 shows percent CRs for the 6groups of rabbits across the 8 sessions of training. Fig. 2 shows the average percent CRs for the 6 ISI groups collapsed over sessions 1-4 and also the average percent CRs for the 6 ISI groups recorded during session 4 of paired training. Analyses involving onset latencies and peak response latencies were also conducted. Only 100 90

trials during which CRs were recorded (either test trials or paired trials) were used in the analyses. Because no conditioning was seen in rabbits given a 50 ms ISI, this group was not included in the analysis. Onset latency was defined as the point in time after CS onset when the first 05. mm of N M movement was detected while the peak latency was defined as the point in time after CS onset when the N M response was at a maximum. The interval of time between CS onset and the US onset as well as a 250-ms period after US onset was analyzed for each paired trial. Analysis of variance conducted on onset latencies recorded during sessions 1-4 revealed significant differences between ISI groups, F4,2o = 54.78, and between training sessions, F3,6o = 19.30. Further Tukey's H S D analysis showed significant differences involving comparisons made between any two groups. A significant decrease in onset latencies between session 1 and session 4 was also detected when session means of all groups were compared. Analysis of variance conducted for onset latencies observed during sessions 5-8 showed only a significant difference involving training sessions, F3,6o = 21.99, with a significant decrease in onset latencies observed between session 5 and session 8. These data indicate that onset latencies were related to ISI duration and that the onset latencies changed over training at a given ISI as well as changed when all ISis were shifted to 250 ms. Fig. 3 depicts onset latencies recorded over training for the 5 ISI groups. Similar to analysis of onset latencies, an analy2O00

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103 sis of variance involving peak latencies recorded during sessions 1-4 demonstrated a significant difference between ISI groups, F4,2o = 28.71, and between training sessions, F3,6o = 19.23. Tukey's H S D analysis revealed significant differences between all groups and between all sessions with peak latencies decreasing with paired training. Analysis of variance conducted on peak latency data collected during sessions 5 - 8 revealed a significant ISI group effect, F4,2o = 5.31, and a significant sessions effect, F3,6o = 10.41. Significant differences were observed when comparisons of any two groups were made. A significant decrease in peak latency was observed between session 5 and session 8. These results show that similar to onset latencies, the timing o f the peak latencies were also related to the ISI and that the peak latencies changed over training and when the ISis were shifted to 250 ms. See Fig. 4 for a summary of peak latency data collected for the 5 ISI groups. Histological procedures showed that all stimulating electrodes used in the present study had exposed tips located in the lateral pontine nuclear region Five rabbits failed to condition over the 8 days o f stimulation training and were therefore not used in the present study. Their electrodes were found to be located in regions o f the reticular formation above the level of the pontine nuclei (n = 3) or in regions of the brainstem just rostral and caudal to the pontine nuclei (n = 2). The 5 rabbits that failed to condition with a stimulation CS did, however, show acquisition of CRs when a tone CS was employed, thus suggesting 25OO -~ -o-ii-~-

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Fig. 5. Schematic representation ofcoronal sections through the pontine nuclear region depicting locations of all stimulating electrodes used in the present study. The coronal section located 10.5 mm caudal to the bregma skull landmark is identified and shows divisions of the pontine nuclei. Filled squares show stimulation sites for the 50-ms ISI group, filled circles show 100-ms ISI group sites, filled triangles show 250-ms ISI group sites, open squares show 500-ms ISI group sites, open circles show 1000-ms ISI group sites, open triangles show 2000-ms ISI group sites, and stars depict non-effective stimulation sites, cp, cerebral peduncle; DL, dorsolateral nucleus; L, lateral nucleus; NRTP, nucleus reticularis tegmenti pontis; P, peduncular nucleus; PM, paramedian nucleus; V, ventral nucleus. that their failure to demonstrate conditioned responding with a stimulation CS was due to either activation of brain sites that were ineffective as CS sites or possibly to malfunctioning electrodes. Fig. 5 provides a summary of stimulation sites used in the present study. DISCUSSION

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Fig. 4. Peak latencies recorded during the 8 training sessions for rabbits given a 100-, 250-, 500-, 1000-, or 2000-ms ISI.

The results o f the this study can be summarized as follows: (1) the characteristic inverted U-shaped function relating ISI with conditioned responding was generated when mossy fiber after-

104 ents to the cerebellum (i.e. cells in the pontine nuclei) were activated as a CS and paired with an air puff US. Onset latencies recorded during training with a pontine CS and air puff US were related to the ISI with response onsets occurring later in rabbits given longer ISis. (2) Once established, the onset latency recorded during training with a pontine CS and air puff US was changed when the ISI was altered. This was demonstrated in the present study when rabbits trained with a 2000, 1000, 500, or 100 ms ISI were shifted to a 250-ms ISI. Peak latencies recorded during training with a pontine CS and air puff US were also related to the ISI (i.e. the longer the ISI, the later the recorded peak latency). Similar to the onset latency, the peak latency was also altered when the ISI was changed. The first goal of the present experiment was to demonstrate further parallels between eyelid conditioning established with pontine nucleus stimulation as a CS and eyelid conditioning established with more conventional, peripheral CSs. These parallels are evident when one considers both the relationship between the ISI and the rate or level of conditioning and the relationship between the ISI and timing of the CR. Identical to previous ISI studies involving conventional CSs (e.g. refs. 14,36), presentation of pontine CSs produced maximal rates and levels of conditioning with ISis of 250 or 500 ms, less robust conditioning with slightly longer or shorter I S Is, and no conditioning when 50 ms ISis were employed. Together, these data produce the characteristic, inverted-U function relating ISI with level of conditioning. Analyses of onset and peak latencies observed at the various ISis in the present study also indicated that the relationship between timing of the CR and the ISI was similar to previous studies which used peripheral CSs. In previous studies, onset latencies generally decreased systematically with training, while the peak latencies shifted until they were recorded consistently around the time of the U S / U R (see ref. 9 for review). Also, a number of previous studies demonstrated changes in the onset and peak latencies when the ISI was shifted (e.g. ref. 24). Identical ISI-related changes involving the onset and peak latencies were observed in the present

study indicating that the fundamental temporal characteristics of the CR execution were intact when direct activation of the cerebellum via pontine stimulation was used as a CS. These data further demonstrate a close correspondence between conditioning with peripheral CSs and conditioning with a mossy fiber stimulation CS. This correspondence was expected since pontine stimulation has been hypothesized to activate directly the CS pathway normally used for classical eyelid conditioning4~. A second goal of the present study was to use the pontine nucleus stimulation preparation to begin studying the neural bases for the hypothetical CS trace and its involvement in the generation of the characteristic ISI function. Under traditional formulations of the CS trace (e.g. refs. 9-11), the CS was thought to create a neural trace in brain regions that received convergent information about US occurrence. The trace was postulated to have a inverted-U time course with a 100-ms latency, a 200-500 ms peak latency, and a slowly decaying inactivation phase. The notion of the CS trace easily accounts for the relationship observed between ISI and CR acquisition. Since CS trace activity would be greatest 200-500 ms after CS onset, CR acquisition rates would be highest when the U S / U R occurred at that time (i.e. when a 200-500 ms ISI was used). Longer ISis (i.e. up to about 3000 ms) and slightly shorter ISis (i.e. as low as 100 ms) would produce lower CR acquisition rates because CS activity would not be maximally contiguous with U S / U R activity. Conditioning with ISis less than 80-100ms would be impossible because the onset latency for the trace was thought to be about 80-100 ms. Evidence for the CS trace has come mostly from the behavioral literature from studies designed to manipulate the CS trace by altering properties of the CS or studies designed to show that the CS trace was capable of controlling precisely CR topography. In general, the latter studies examined changes in CR topography produced by manipulations of the ISI (e.g. ref. 24) and have concentrated on changes involving the onset latency or peak latency (see above). Attempts to directly manipulate neural activity

105 underlying an auditory CS trace by manipulating properties of the CS have also been made. Presentations of a pulsed-tone CS resulted in more rapid rates of CR acquisition than did presentations of a constant-tone CS 8. For at least CSs presented in the range of 65-85 dB, acquisition of eyelid CRs was found to be a direct function of the intensity of the CS 32. Also, more rapid rates of CR acquistion were generally noted during delay conditioning which typically used longer duration CS s as compared to trace conditioning when relatively brief CSs were used 35. Effect of manipulating CS parameters were noted informally when stimulation parameters were being established for the original study involving a mossy fiber stimulation CS 4~. First, stimulation frequencies below 50-60 Hz were not effective for producing conditioned responding. Second, CR acquisition was more rapid when a 100-200/~A train of stimulation was delivered than when 10-50 #A train was used. Third, conditioned responding was established more rapidly when long trains of pontine stimulation were used than when short trains were used 19. It appears that similar to manipulating features of peripheral C Ss, manipulating parameters of a pontine stimulation CS can affect the rate and level of conditioning. Although not supported to date by physiological studies, it seems possible that changes in the rate of conditioning through CS manipulations might occur through manipulations of the hypothetical CS neural trace. Additional evidence for the CS trace has been obtained in a study which used brain stimulation as a CS. Patterson 3~ paired an inferior colliculus stimulation CS with an air puff US and observed the rate and level of classical N M conditioning at a variety of ISis. Unlike other ISI studies, robust conditioned responding was observed at a 50-ms ISI. This reduction of the minimal ISI was interpreted as evidence for an alteration in the CS trace caused by the use of a brain stimulation CS instead of peripheral CSs. Specifically, it was suggested that the timing of the initial CS trace recruitment in critical brain regions was partially determined by delays caused by sensory transduction and afferent neural transmission. Stimulating the inferior colliculus as a CS was thought

to bypass a portion of the normal CS pathway and therefore allow CRs to form with a 50-ms ISI because of a reduction in the recruitment time of the trace. Like the behavioral studies described above, the inferior colliculus stimulation study provided strong support for the idea that a pattern of neural activity, represented as a CS trace, partially determined CR topography and the range of C S - U S intervals that would produce conditioning. Although the present study demonstrated the same relationship between CR topography and ISI that was seen in a number of other ISI studies, CRs were not observed when a 50-ms ISI was used in conjunction with a pontine stimulation CS. This result is not in agreement with observations of conditioning with a 50-ms ISI when inferior colliculus stimulation was used as a CS. However, consideration of recent data concerning neural circuitry involved in classical eyelid conditioning suggest reasons why a discrepancy between studies may have been observed. A number of studies have suggested that lateral regions of the pontine nuclei are an essential part of the acoustic CS pathway 4°-42. This region of the pontine nuclei is known to receive afferents from a variety of primary auditory areas, including the inferior colliculus 1,13,3°,4° and send projections to the cerebellar c o r t e x 49 a s well as possibly to the interpositus nucleus 43. In short, the lateral pontine nuclear region could be thought of as a point of convergence for a variety of auditory inputs capable of activating regions of the cerebellum that also receive US input. A major feature of this proposed CS pathway is that it is relatively short with auditory information reaching the cerebellum in less than 20 ms after CS onset. Because the CS pathway to the cerebellum is relatively short, it seems doubtful that stimulating either the inferior colliculus or pontine nuclei as a CS would reduce substantially the recruitment time of the CS trace and therefore account for the ability to produce conditioned responding with a 50-ms ISI. However, several possibilities could account for the differences in conditioned responding noted with relatively short ISis in the two studies. First, stimulation of the inferior colliculus could produce conditioned responding via circuitry that

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does not include the cerebellum. Second, inferior colliculus stimulation might activate cerebellar areas either directly or through pathways that do not include the pontine nucleus (e.g. through pathways with shorter synaptic delays). Third, pontine stimulation could activate cerebellar or non-cerebellar circuitry that in some fashion prevents or inhibits CR acquisition with relatively short ISI while inferior colliculus stimulation does not. Fourth, because ISis between 50 and 100 ms were not tested in the present study, it is possible that the difference in the minimal ISI for conditioning is not very great (e.g. maybe conditioning would occur with pontine stimulation as a CS when a 65- or 80-ms ISI is used). Finally, if one assumes that the cerebellum is not the putative site for plasticity associated NM conditioning, it is possible that the inferior colliculus is closer to the putative site than the pontine nuclei (or, for that matter, inputs from the periphery). It appears that future studies of differences between conditioning with an inferior colliculus CS and conditioning with a pontine CS while using relatively short ISis could aid our understanding of the neural bases of the CS trace and ISI function. In summary, the observations of similar ISI functions established with peripheral and pontine CSs suggest further parallels between classical eyelid conditioning with auditory stimuli and classical eyelid conditioning with direct activation of mossy fibers as a CS. Given recent progress made in delineating circuitry involved in this simple form of mammalian learning, it seems likely that the neural basis for the ISI function and CS trace will be detailed eventually. Furthermore, this delineation is likely to be facilitated by studying the various interactions between structures within this circuitry that relate to manipulating temporal aspects of the conditioning paradigm.

ACKNOWLEDGEMENTS

This research was supported by a National Institute of Mental Health Grant (MH44052) and an Indiana University Summer Faculty Research Fellowship. I thank K. Eby and S. Steinmetz for technical assistance during this experiment.

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