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The cerebellum is necessary for rabbit classical eyeblink conditioning with a non-somatosensory (photic) unconditioned stimulus
Behavioural Brain Research 104 (1999) 105 – 112 www.elsevier.com/locate/bbr
Research report
The cerebellum is necessary for rabbit classical eyeblink conditioning with a non-somatosensory (photic) unconditioned stimulus Ronald F. Rogers *, Abbey F. Fender, Joseph E. Steinmetz Program in Neural Science, Department of Psychology, Indiana Uni6ersity, 1101 East 10th Street, Bloomington, IN 47405 -7007, USA Received 1 June 1998; received in revised form 29 March 1999; accepted 2 April 1999
Abstract The present research investigated the acquisition of classically conditioned eyeblinks in rabbits using a light flash unconditioned stimulus (US), as well as the contribution of deep cerebellar nuclei to such an association. Two independent groups of animals experienced three phases of training: (1) pre-lesion delay conditioning using either a light- (Group 1) or an air puff-US (Group 2), (2) post-lesion testing of response performance, and (3) post-lesion acquisition to the opposite US. During the initial acquisition (720 trials), the groups did not differ with regard to their rate of learning or their overall level of responding. To assess the contribution of the cerebellum to the maintenance of responding, the interpositus nucleus was electrolytically lesioned and animals were given 8 days of additional training. Both groups exhibited a profound reduction in conditioned responding (CR) and showed no signs of recovery over the remainder of this phase (480 trials). Animals were then shifted to the opposite US (same eye) and given 12 days of training to assess the effect of interpositus lesions on the acquisition of CRs to a novel US. No learning was observed during this phase, regardless of whether animals experienced the light- or air puff-US. These results demonstrate: (1) the ability of a non-somatosensory stimulus to serve as a US during classical eyeblink conditioning; and (2) a common reliance on deep cerebellar nuclei for both somatosensory- and non-somatosensory-based reflexive motor learning. The findings are discussed in reference to the processing of conditioning stimuli within the brainstem-cerebellar circuitry that underlies eyeblink conditioning. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Classical conditioning; Cerebellum; Eyelid; Interpositus nucleus; Electrolytic lesions; Light
1. Introduction Neurobiologically speaking, what are the defining features of the unconditioned stimulus (US) used in the classically conditioned nictitating membrane/eyeblink response? One definition that appears to suffice is that the US constitutes an activation of the trigeminal nuclear system by somatosensory stimuli (e.g. mild periorbital shock or corneal air puff). This information is then conveyed to Larsell’s lobule HVI and the interpositus nucleus of the cerebellum via climbing fibers * Corresponding author. Tel.: +1-812-855-9592; fax: + 1-812-8554691. E-mail address: [email protected] (R.F. Rogers)
projecting from the inferior olive [3]. A more parsimonious statement might be that any stimulus that engages the olivary-climbing fiber system may serve as a reasonable US during eyeblink conditioning, e.g. [16,22]. In light of its representation within this system, one might predict that visual stimuli should function well in a US capacity. Visual information is relayed to the cerebellum through climbing fibers by three major structures: the superior colliculus, pretectal nuclei, and nuclei of the accessory optic tract [12]. These nuclei relay visual information to localized areas of the inferior olive which exhibits evoked cellular activity following electrical or peripheral stimulation of visual afferents [2,4]. Furthermore, visual stimulation induces widely spread climbing fiber responses in regions of the
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cerebellar cortex, including lobule HVI [1,9,17]. Finally, both the interpositus and dentate nuclei of the cerebellum exhibit cellular activity that is highly correlated with light flash-evoked blinks [11]. Despite the responsiveness of the olivo-cerebellar system to visual stimulation, initial attempts failed to demonstrate learning when a light US was used during conditioning of the nictitating membrane response [5]. Bruner (1965) was the first to examine the behavioral properties of a light US during conditioning of the nictitating membrane response. He found that despite the ability of a 200-W chamber light to reliably elicit blink responses, animals failed to condition when a tone was paired with this photic US. Interestingly, when tone-light trials were interspersed with tone-air puff trials (50%/50%), a facilitation in learning was observed relative to animals receiving only 50% tone-air puff trials. This latter finding implied that while the light US could not support conditioning on its own, properties of the photic stimulus were such that it could modulate ongoing learning. Regardless of this modulatory effect, Bruner’s data showed that a stimulus that is now known to engage US-specific neural circuitry, did not support eyeblink conditioning. One factor that may have contributed to this negative result is the well-documented effect of stimulus intensity on conditioning. That is to say that as the US intensity increases, so does the strength of conditioning [20]. With this in mind, one might hypothesize that Bruner’s negative finding resulted from the use of a US intensity that, while sufficient to activate the reflex circuitry mediating the unconditioned eyeblink, was not sufficient to engage the neural circuitry needed for conditioned response (CR) acquisition (i.e. the climbing fiber system). The possibility then remains that a stronger photic US may in fact support classical conditioning of the eyeblink response. It is reported here that animals successfully conditioned using a light flash US positioned directly in front of the eye and that their performance did not differ from animals trained with corneal air puff. Furthermore, as with somatosensory-based eyeblink conditioning [6,8], the photically-based conditioning was dependent on an intact cerebellar complex.
2. Materials and methods
2.1. Subjects Thirty male, New Zealand White, rabbits (1.75– 2.0 kg) obtained from Myrtle’s Rabbitry (Thompson Station, TN) served as subjects throughout this experiment. The animals were individually housed and given free access to food ( 150 g/day) and water. All experimental procedures were conducted during light phases
of 12/12 h light/dark cycles. The rabbits were acclimated to the colony procedures for 2 weeks prior to the onset of the study and were maintained on antibiotics (Sulmet) for the first week of this period to minimize risk of infection.
2.2. Surgery All surgeries were performed 7 days prior to behavioral training and under aseptic conditions. Rabbits were anesthetized with a combination of ketamine (60 mg/kg; im) and xylazine (6 mg/kg; sc), and maintained throughout surgery with 1.0 cc injections (im) of a ketamine/xylazine cocktail (2:1 ratio) administered every 45 min. Once anesthetized, animals were placed into a stereotaxic headholder with bregma 1.5 mm above lambda and two ground screws were placed in the skull over the posterior regions of the cerebral cortex. Standard stereotaxic procedures were utilized to position a unilateral pair of insulated, stainless steel, lesion electrodes (0.5 mm tip exposure) in either the left or right interpositus nucleus (counterbalanced across animals) of the cerebellum (AP= − 0.7; ML= 9 5.5; DV= − 14.5 mm relative to lambda). Electrode placement was further facilitated by monitoring characteristic multiunit activity throughout positioning. Placement of the electrodes was accomplished by removing a small portion of skull above the cerebellum, replacing the area with bone wax, introducing the electrodes, and securing the structure to the skull with dental acrylic. Finally, a head bolt, used to attach the light US/blink detector assembly, was embedded within the acrylic, and the scalp was sutured about the headstage. Provodine ointment was liberally applied to the wound as a prophylactic treatment for infection.
2.3. Beha6ioral training and testing 2.3.1. UR examination Twelve rabbits were used to characterize the unconditioned response (UR) to both light and air puff unconditioned stimuli. These rabbits were first given subcutaneous injections of a sedative (6.0 mg/kg of xylazine). The eyelid area was then shaved and cleansed, after which two fine, uninsulated, stainless steel wires that served as electromyographic (EMG) recording electrodes were placed in the dorsal musculature of the left eyelid. The wires were formed into two small loops and gold pins were clamped to their ends. The procedure concluded with the application of provodine ointment to the eyelid and animals were returned to their home cages for 1–2 days. After the recovery period, the rabbits were placed in standard Plexglas restraint boxes and positioned in dark, soundand light-attenuating chambers. The light US consisted of a 150-ms light flash provided by a 7.5-V incandescent
R.F. Rogers et al. / Beha6ioural Brain Research 104 (1999) 105–112
bulb (CM51; 0.22 A) positioned 1.0 – 2.0 cm from the eye. A small ‘warm-up’ voltage was delivered to the bulb filament between trials in order to minimize the rise time necessary to reach full illumination during light presentations. The air puff US consisted of a 150-ms puff of air (3.0 psi) positioned 1.0 – 2.0 cm from the cornea of the eye. Eyeblinks were monitored by recording EMG activity from the dorsal musculature of the eye. The EMG activity was amplified 1000 × , filtered (300–3000 Hz) and routed to an AC-bridge integrator. The integrated signal was then scaled and fed to a microcomputer that also controlled the delivery and timing of the stimuli [7]. Two groups of animals (i.e. light- or air puff-US) received one session of 40 trials with each trial consisting of a single, unsignaled US presentation and separated by an average of 30-s. On each trial, the EMG signal was digitally sampled for 2000 ms with the light onset occurring 1000 ms into this interval. In this manner, the UR was characterized by calculating the average UR amplitude, onset latency and latency to the peak response for each animal across the 40 trials.
2.3.2. Eyeblink conditioning Throughout classical conditioning, animals were positioned in standard Plexiglas restraint boxes housed within dark, sound- and light-attenuating chambers. Two unconditioned stimuli were used at different phases throughout conditioning. The light and air puff USs were equivalent to the stimuli described above. All animals were exposed to the same 900-ms tone-conditioned stimulus (CS; 1000 Hz, 85 db SPL) that was presented through a speaker positioned above the animal’s head and co-terminated with the US resulting in an interstimulus interval (ISI) of 750-ms. Eye movements were monitored using an infrared detection system, the output of which was sent to a microcomputer that also controlled the timing and presentation of the conditioning stimuli. Daily paired training sessions consisted of 60 trials, with each session broken into six blocks of ten trials each. The animal experienced a CS-alone probe trial on the first trial of each block with paired CS – US presentations occurring on the remaining nine trials of the block. Intertrial intervals were pseudo-randomly determined and ranged between 40- and 60-s. Regardless of the trial type (i.e. paired or CS-alone), a 250-ms pre-CS interval was examined for eyelid movements greater than 0.7 mm. If this threshold was surpassed the trial was discarded and not used in the analysis. A conditioned response was defined as a 0.5 mm or more closure of the eyelid that occurred during the 750-ms ISI on paired CS – US trials or anywhere after CS onset on CS-alone probe trials (1750 ms total duration).
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All animals progressed through three phases of training: (1) pre-lesion acquisition; (2) post-lesion testing with the initial US; and (3) post-lesion acquisition to a novel US. Following two 60-min adaptation sessions, animals were randomly assigned to one of two independent groups, distinguished by the type of US used during the initial acquisition training, i.e. light US (Group 1) or air puff US (Group 2). Consequently, the pre-lesion phase consisted of 12 days of paired tone CS/light US training for half the animals and paired tone CS/air puff US training for the remaining animals. Conditioning stimuli during paired training were always presented to the ipsilateral eye relative to the lesion electrode placement which was counterbalanced across animals. Immediately following this initial acquisition phase, each animal received lesions of the interpositus nuclear region by passing 1-mA dc (referenced to the ground screw) for 30 s through the two previously implanted electrodes. The second phase of conditioning began 24 h post-lesion and consisted of 8 subsequent days of paired CS–US training. During this phase, all of the conditioning parameters remained unchanged relative to the pre-lesion phase for each animal, thus allowing us to evaluate the effects of cerebellar lesions on the established conditioned responding (CR). The third phase of training allowed us to evaluate acquisition to a novel US under a lesioned state. To accomplish this, each animal received 12 days of paired tone CS/US training to the lesioned side. Rabbits that were initially trained using the light US (Group 1) were now shifted to the air puff US, while air puff US animals (Group 2) were shifted to the light US. Consequently, these three phases of training enabled a within-subject analysis of: (1) normal acquisition with both a non-somatosensory- and somatosensory-US; (2) the effects of cerebellar lesions on the maintenance of CR; and (3) acquisition with both a non-somatosensory- and somatosensory-US following cerebellar lesion. Given the proximity of the incandescent light source to the eye (i.e. 1–2 cm), a subset of animals were used to test the contribution of bulb-induced heat (i.e. a potential somatosensory stimulus) to the light-US conditioning. These separate animals received twelve days of paired CS–US conditioning as described above with the exception that a heat absorbing glass filter (Oriel, Stratford, CT; Model c 51950; 25.4 mm diameter) was positioned between the light source and the eye. The filter selectively removed spectral bands greater than 750 nm with an external transmittance of less than 0.15 at 800 nm and effectively zero transmittance at 1000 nm. At the completion of training, the rabbits were overdosed with an intravenous injection of pentobarbital (4.0 cc) and transcardially perfused with 0.9% saline followed by 10% formalin. The brains were then removed and placed in a formalin/sucrose solution for at
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Table 2 Percentage of interpositus nucleus lesion area Animal
Fig. 1. Representative unconditioned eyeblink responses evoked by corneal air puff and light flash. Traces are rectified electromyographic (EMG) signals from separate animals recorded from the dorsal musculature of the eye during a single trial.
least 10 days. Following this period, the brains were blocked in albumin-gelatin and frozen coronal sections (80-mm) were taken through the interpositus nucleus. The sections were mounted on gelatinized slides, stained with Cresyl violet, and examined microscopically to determine the extent of the lesion. The sections were subsequently digitized and the extent of the lesions were determined by calculating the percentage of lesioned interpositus area relative to the total nuclear area across four standard stereotaxic planes (0.0, 0.5, 1.0, and 1.5-mm anterior of lambda).
3. Results
3.1. Properties of the UR to light Fig. 1 illustrates characteristic unconditioned eyeblink responses from separate animals given either a 150-ms presentation of light (n =6) or an air puff (n =6) on a single trial. Eyeblinks evoked by the two stimuli differed with regard to onset latency and rise times (i.e. velocity), but did not differ with regard to their overall magnitudes (see Table 1). Specifically, the UR to light exhibited a delayed onset, slower rise time, Table 1 Unconditioned response (UR) properties Measure
Amplitude (mm) Peak latency (ms) Onset latency (ms) Rise time (ms)
Light US
Air puff US
Mean 9S.E.M.
Mean9 S.E.M.
5.5 91.0 262.6 918.4
5.39 1.0 72.49 11.5
0.18 8.77*
119.2 95.7
45.69 10.5
6.14*
143.4 9 21.1
26.79 6.53
5.27*
t-score
* PB0.05; S.E.M., standard error of the mean; t test based on ten degrees of freedom.
1 2* 3** 4 5 6 7 8 9 10 11
Stereotaxic coordinates (mm from lambda)
Total area
0.0
0.5
1.0
1.5
0.00 0.00 28.36 0.00 0.00 34.15 0.00 0.00 0.00 0.00 0.00
15.18 0.00 55.74 0.00 19.24 23.58 8.40 0.00 20.42 0.00 8.58
2.80 7.86 43.81 27.55 0.00 19.06 0.00 17.52 0.00 3.43 21.14
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
4.56 2.00 32.48 7.00 4.89 19.50 2.13 4.45 5.18 0.18 7.55
* Lesion profile is illustrated in Fig. 2 as black area. ** Lesion profile is illustrated in Fig. 2 as gray area.
and longer duration relative to the air puff-elicited blink. Onset latencies ranged between 104.0 and 135.7 ms for light compared to 23.75–94.25 ms for air puff. The longer latencies for visually-evoked blinks have been observed elsewhere [5,11,15] and appear to be, at least in part, a consequence of retinal processing. For instance, electrical stimulation of the optic tract induces evoked potentials at latencies of 8–11 ms in the inferior olive [2] and 11–14 ms in the cerebellar cortex [13,14]. In contrast, visual stimulation of the retina (light flash) results in cellular response latencies of 25–28 ms for the inferior olive [4] and 30–40 ms at the cerebellar cortex [14]. In fact, delays of 13–40 ms are observed when single units are recorded from the optic nerve following visual stimulation using a light flash [21].
3.2. Histology and lesion extent Histological examination revealed that lesions in two animals from the light US group failed to disrupt any portion of the interpositus nucleus, but were instead located in the overlying white matter and cortical tissues. Additionally, one other animal in the air puff US group had no measurable lesion. Consequently, data from these animals were not included in the subsequent analyses, but will be discussed briefly below. In the remaining animals (n= 11), effective lesions encompassed portions of the anterior interpositus, medial border of the dentate nucleus, and/or white matter dorsal to the interpositus nucleus. Table 2 presents interpositus lesion area data expressed as the percentage of total area across four stereotaxic planes. As is evident from Table 2, anterior interpositus nucleus lesions varied considerably in size and location, ranging between 2.0 and 32.5% of the total area. Despite this variability, all animals had in common some degree of
R.F. Rogers et al. / Beha6ioural Brain Research 104 (1999) 105–112
damage to the dorsolateral portion of the anterior interpositus nucleus. Fig. 2 illustrates the largest (gray) and smallest (black) interpositus nucleus lesions across three stereotaxic planes. These lesions are also representative with regard to the disruption of the overlying white matter which was observed in ten of 11 animals.
3.3. Pre-lesion beha6ioral acquisition and post-lesion maintenance of responding The percentage CR during initial acquisition are plotted in Fig. 3 (Acquisition) for animals receiving a tone CS paired with either a light flash- (n = 5) or an air puff-US (n= 6). Both groups exhibited a slow, but incremental acquisition of CR, culminating in 69.3% (Light US) and 67.2% (Air puff US) CRs by day 12. These data were analyzed using a two-factor analysis of variance (ANOVA) with repeated measure which found a main effect of training sessions, F(11, 99) = 17.88, PB 0.001, consistent with the observed behavioral ac-
Fig. 2. Representative interpositus nucleus lesions depicting the smallest (black area) and largest (gray area) areas that were lesioned. (The numbers indicate the distance anterior to lambda. Abbreviations: ANS, ansiform lobule; DE, dentate nucleus; FA, fastigial nucleus; icp, inferior cerebellar peduncle; IO, inferior olive; IN, interpositus nucleus; PF, paraflocculus lobule; VN, vestibular nucleus.)
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quisition across training sessions. Although there appears to be some trend toward slower acquisition rates for light US animals, no significant differences were found with regard to the type of US used (light vs air puff) or in the rates of acquisition as indicated by the lack of a significant interaction between US type and session number (P\ 0.05). Furthermore, the number of trials necessary to reach a criterion of seven CRs within any ten consecutive trials did not differ between light US (381 9 91.5) and air puff US (294 979.1) groups, t(9)= 0.723, ns. The variability in CR observed in both groups is most likely due to the protracted ISI (750 ms) which is known to have a detrimental effect on conditioning relative to shorter ISIs (e.g. 250 ms) [20]. Conditioned response topographies were evaluated on CS-alone trials for both the light- and air puff-US conditions and are illustrated in Fig. 4. The topographies of the responses were very similar between the two conditions. Both groups exhibited responses that were comparable in duration, shape (mono- and biphasic), and were well timed to the US onset. Quantitatively, no differences were observed between groups with regard to their peak CR amplitudes, latency to peak response, or response onset latency on the final day of training (see Table 3). In order to assess the contribution of bulb-induced heat (i.e. somatosensory stimulus) to the light-US conditioning, a third group of animals (n= 4) underwent paired tone-light training equivalent to the other light US group with the exception that a heat absorbing glass filter was positioned between the light source and the eye. The results indicate little or no contribution of heat to the development of conditioning responding using a light US. As with the previous groups, these animals exhibited an incremental acquisition of CR across the 12 days of training, culminating in a mean percent of 64.3 9 3.45 by day 12. These data were compared using a single-factor ANOVA in which a main effect of training session was found, F(11, 47)= 2.06, PB 0.01. CR was significantly diminished by electrolytic lesions of the deep nuclei (Fig. 3; Same US). Twenty four hours following the lesion light US animals exhibited a 65% decrease in CRs compared to a 81% decrease in the response rates of air puff US animals. This post-lesion decrement in CR was found to be significant when compared to pre-lesion (day 12 of acquisition) rates, t(4)= 7.24, PB 0.01. Furthermore, no recovery in CR was observed for either group over 8 subsequent days of post-lesion training as determined by two-factor ANOVA with repeated measure (P \ 0.05).
3.4. Post-lesion acquisition to a no6el US Disruption of the deep cerebellar nuclei blocked any subsequent CR acquisition using a novel US. Animals
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Fig. 3. The mean percentage of conditioned responses are plotted for groups that differed with regard to their unconditioned stimulus (US), i.e. light or air puff, during the initial acquisition phase. The groups were trained further using the same US following lesions of the interpositus nucleus. Under this lesioned state, conditioned response acquisition was examined by shifting animals to the other US. Error bars indicate standard error of the mean.
were conditioned using the same tone CS, but during this phase the tone was paired with the other US. For instance, animals receiving tone – light pairings during the initial acquisition were now trained with tone–air puff pairings. The percentage of conditioned responses are presented in Fig. 3 (new US) for this phase of training. The interpositus lesions were sufficient to block any savings effect that may have occurred given the conditioning history with the tone CS. Response rates on the first day of this training, although more variable, did not differ from the initial day of acquisition (i.e. day 1 of the initial acquisition phase) for either group (P \0.05). Moreover, despite 12 days of training, neither group (i.e., light- or air puff-US) exhibit any subsequent acquisition to the tone when using the novel USs. These observations were confirmed using a two-factor ANOVA with repeated measure in which no main effects were found (P \0.05).
4. Discussion Using the classically conditioned eyeblink response, we report here that rabbits acquired CR to a tone CS that was paired with a light flash US. Furthermore, this learning did not differ from more traditional somatosensory-based conditioning (i.e. air puff US) with regard to the rate and overall level of acquisition. Two results suggest that both the non-somatosensory and somatosensory forms of this learning rely on common neural circuitry, namely the cerebellar complex, for their formation. First, the conditioned responses resulting from light and air puff training did not differ with
regard to the timing of the response, the overall magnitude, or the general response topography. Second, disruption of the deep cerebellar nuclei through electrolytic lesions profoundly disrupted CR regardless of the US used in training. Moreover, subsequent conditioning using either a light or air puff US was blocked by these lesions. While this research indicates a clear involvement of the cerebellum in non-somatosensorybased eyeblink conditioning, it is not possible to distinguish between the relative contributions of the deep nuclei to this learning. Although the majority of lesions compromised the interpositus nucleus, the lesions were not specific enough to rule out the possible disruption of information reaching the dentate nucleus. For instance, ten of 11 animals had damage to the overlying white matter which may have disrupted axons projecting to the dentate. Another three animals also received some damage to the medial dentate border. As mentioned previously, two animals were not included in the analyses because they lacked any damage to the interpositus nucleus, but instead had lesions exclusively involving the overlying white matter and cortical tissue. Interestingly, both of these animals developed CRs using the light US and exhibited a severe disruption of responding following the lesions, but unlike animals with interpositus damage, these rabbits subsequently developed CRs using the novel air puff US. Although only suggestive, the disruption of non-somatosensorybased conditioning in these animals, while leaving the somatosensory-based conditioning intact, may indicate a dissociation with regard to the specific cerebellar structures mediating these two forms of learning. Further research employing more discrete lesions will be
R.F. Rogers et al. / Beha6ioural Brain Research 104 (1999) 105–112
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Fig. 4. Representative conditioned responses from animals receiving paired training using a light US (top traces) or a corneal air puff US (bottom traces). The data were acquired on tone-only trials, although the vertical dashed lines indicate the time at which US presentations would have occurred.
necessary to clarify if in fact there is a distinction between the relative contributions of the deep nuclei to somatosensory- versus non-somatosensory-based eyeblink conditioning. This research stands in contrast to previous attempt to condition the nictitating membrane response using a Table 3 Conditioned response (CR) propertiesa Measure
Amplitude (mm) Peak latency (ms) Onset latency (ms)
Light US
Air puff US
Mean9 S.E.M.
Mean 9 S.E.M.
6.5 9 1.24 750.6 9 82.3
5.1 91.44 759.99 52.4
0.729 −0.098
458.6 9 63.8
482.3 9 56.6
−0.279
t-score
a S.E.M., standard error of the mean; t tests based on nine degrees of freedom.
more diffuse light US [5]. The primary distinction between the present research and this earlier report appears to be the intensity of the light serving as the US. In Bruner’s experiment the US consisted of indirect chamber illumination provided by a 200-W incandescent lamp that was superimposed upon a continuous illumination of the chamber by an adjacent 6-W bulb. In the present report, the light flash US was positioned directly in front of the cornea (1–2 cm) and darkness was maintained within the chamber between trials. It is reasonable to assume that this increase in intensity carried with it an increase in the ability of the US to promote learning. In fact, it is well known that the rate of learning is positively correlated with US intensity [20]. Two interesting and perhaps related questions emerge from the above statement: (1) what does it mean to increase a stimulus’ ability to promote learning from a neurobiological standpoint?; and (2) what processes allow light to serve as a CS under one set of
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conditions and a US under another set of conditions? Pavlov himself addressed the latter of these questions by proposing that the role of a stimulus in conditioning (i.e. CS vs US) is determined, at least in part, by the biological response elicited by the stimulus [18]. In other words, a US is define so not because of its modality, but rather, because it elicits a larger response relative to the CS. One implication of this criteria is that a single stimulus could serve both as a CS and a US depending on its intensity, i.e. a CS at low intensities and a US at high intensities. In fact, both corneal air puffs [10] and peri-orbital electrical stimulation [19] have been shown to support classical eyeblink conditioning using such a configuration. Taking this one step further, it can be speculated that intensity might ultimately determine what neural structures and systems are activated during conditioning. The present data argues that if sufficiently intense light is used as a US, the inferior olive (climbing fiber system) may be engaged and thus conditioning produced. The laboratory is currently investigating the hypothesis that the efficacy of a stimulus as an US is determined by the extent to which it engages the olivocerebellar system and that this ‘engagement’ is directly related to the intensity of the stimulus in question, regardless of its sensory modality.
Acknowledgements Our appreciation is extended to Jeremy Novak for his assistance in the collection of the data. This work was supported by a grant from the National Institute of Mental Health to R.F. Rogers (cMH11550) and J.E. Steinmetz (c MH51178). Correspondence should be addressed to Ronald F. Rogers, Program in Neural Science, Department of Psychology, Indiana University, Bloomington, IN 47405, USA.
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Research report
The cerebellum is necessary for rabbit classical eyeblink conditioning with a non-somatosensory (photic) unconditioned stimulus Ronald F. Rogers *, Abbey F. Fender, Joseph E. Steinmetz Program in Neural Science, Department of Psychology, Indiana Uni6ersity, 1101 East 10th Street, Bloomington, IN 47405 -7007, USA Received 1 June 1998; received in revised form 29 March 1999; accepted 2 April 1999
Abstract The present research investigated the acquisition of classically conditioned eyeblinks in rabbits using a light flash unconditioned stimulus (US), as well as the contribution of deep cerebellar nuclei to such an association. Two independent groups of animals experienced three phases of training: (1) pre-lesion delay conditioning using either a light- (Group 1) or an air puff-US (Group 2), (2) post-lesion testing of response performance, and (3) post-lesion acquisition to the opposite US. During the initial acquisition (720 trials), the groups did not differ with regard to their rate of learning or their overall level of responding. To assess the contribution of the cerebellum to the maintenance of responding, the interpositus nucleus was electrolytically lesioned and animals were given 8 days of additional training. Both groups exhibited a profound reduction in conditioned responding (CR) and showed no signs of recovery over the remainder of this phase (480 trials). Animals were then shifted to the opposite US (same eye) and given 12 days of training to assess the effect of interpositus lesions on the acquisition of CRs to a novel US. No learning was observed during this phase, regardless of whether animals experienced the light- or air puff-US. These results demonstrate: (1) the ability of a non-somatosensory stimulus to serve as a US during classical eyeblink conditioning; and (2) a common reliance on deep cerebellar nuclei for both somatosensory- and non-somatosensory-based reflexive motor learning. The findings are discussed in reference to the processing of conditioning stimuli within the brainstem-cerebellar circuitry that underlies eyeblink conditioning. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Classical conditioning; Cerebellum; Eyelid; Interpositus nucleus; Electrolytic lesions; Light
1. Introduction Neurobiologically speaking, what are the defining features of the unconditioned stimulus (US) used in the classically conditioned nictitating membrane/eyeblink response? One definition that appears to suffice is that the US constitutes an activation of the trigeminal nuclear system by somatosensory stimuli (e.g. mild periorbital shock or corneal air puff). This information is then conveyed to Larsell’s lobule HVI and the interpositus nucleus of the cerebellum via climbing fibers * Corresponding author. Tel.: +1-812-855-9592; fax: + 1-812-8554691. E-mail address: [email protected] (R.F. Rogers)
projecting from the inferior olive [3]. A more parsimonious statement might be that any stimulus that engages the olivary-climbing fiber system may serve as a reasonable US during eyeblink conditioning, e.g. [16,22]. In light of its representation within this system, one might predict that visual stimuli should function well in a US capacity. Visual information is relayed to the cerebellum through climbing fibers by three major structures: the superior colliculus, pretectal nuclei, and nuclei of the accessory optic tract [12]. These nuclei relay visual information to localized areas of the inferior olive which exhibits evoked cellular activity following electrical or peripheral stimulation of visual afferents [2,4]. Furthermore, visual stimulation induces widely spread climbing fiber responses in regions of the
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cerebellar cortex, including lobule HVI [1,9,17]. Finally, both the interpositus and dentate nuclei of the cerebellum exhibit cellular activity that is highly correlated with light flash-evoked blinks [11]. Despite the responsiveness of the olivo-cerebellar system to visual stimulation, initial attempts failed to demonstrate learning when a light US was used during conditioning of the nictitating membrane response [5]. Bruner (1965) was the first to examine the behavioral properties of a light US during conditioning of the nictitating membrane response. He found that despite the ability of a 200-W chamber light to reliably elicit blink responses, animals failed to condition when a tone was paired with this photic US. Interestingly, when tone-light trials were interspersed with tone-air puff trials (50%/50%), a facilitation in learning was observed relative to animals receiving only 50% tone-air puff trials. This latter finding implied that while the light US could not support conditioning on its own, properties of the photic stimulus were such that it could modulate ongoing learning. Regardless of this modulatory effect, Bruner’s data showed that a stimulus that is now known to engage US-specific neural circuitry, did not support eyeblink conditioning. One factor that may have contributed to this negative result is the well-documented effect of stimulus intensity on conditioning. That is to say that as the US intensity increases, so does the strength of conditioning [20]. With this in mind, one might hypothesize that Bruner’s negative finding resulted from the use of a US intensity that, while sufficient to activate the reflex circuitry mediating the unconditioned eyeblink, was not sufficient to engage the neural circuitry needed for conditioned response (CR) acquisition (i.e. the climbing fiber system). The possibility then remains that a stronger photic US may in fact support classical conditioning of the eyeblink response. It is reported here that animals successfully conditioned using a light flash US positioned directly in front of the eye and that their performance did not differ from animals trained with corneal air puff. Furthermore, as with somatosensory-based eyeblink conditioning [6,8], the photically-based conditioning was dependent on an intact cerebellar complex.
2. Materials and methods
2.1. Subjects Thirty male, New Zealand White, rabbits (1.75– 2.0 kg) obtained from Myrtle’s Rabbitry (Thompson Station, TN) served as subjects throughout this experiment. The animals were individually housed and given free access to food ( 150 g/day) and water. All experimental procedures were conducted during light phases
of 12/12 h light/dark cycles. The rabbits were acclimated to the colony procedures for 2 weeks prior to the onset of the study and were maintained on antibiotics (Sulmet) for the first week of this period to minimize risk of infection.
2.2. Surgery All surgeries were performed 7 days prior to behavioral training and under aseptic conditions. Rabbits were anesthetized with a combination of ketamine (60 mg/kg; im) and xylazine (6 mg/kg; sc), and maintained throughout surgery with 1.0 cc injections (im) of a ketamine/xylazine cocktail (2:1 ratio) administered every 45 min. Once anesthetized, animals were placed into a stereotaxic headholder with bregma 1.5 mm above lambda and two ground screws were placed in the skull over the posterior regions of the cerebral cortex. Standard stereotaxic procedures were utilized to position a unilateral pair of insulated, stainless steel, lesion electrodes (0.5 mm tip exposure) in either the left or right interpositus nucleus (counterbalanced across animals) of the cerebellum (AP= − 0.7; ML= 9 5.5; DV= − 14.5 mm relative to lambda). Electrode placement was further facilitated by monitoring characteristic multiunit activity throughout positioning. Placement of the electrodes was accomplished by removing a small portion of skull above the cerebellum, replacing the area with bone wax, introducing the electrodes, and securing the structure to the skull with dental acrylic. Finally, a head bolt, used to attach the light US/blink detector assembly, was embedded within the acrylic, and the scalp was sutured about the headstage. Provodine ointment was liberally applied to the wound as a prophylactic treatment for infection.
2.3. Beha6ioral training and testing 2.3.1. UR examination Twelve rabbits were used to characterize the unconditioned response (UR) to both light and air puff unconditioned stimuli. These rabbits were first given subcutaneous injections of a sedative (6.0 mg/kg of xylazine). The eyelid area was then shaved and cleansed, after which two fine, uninsulated, stainless steel wires that served as electromyographic (EMG) recording electrodes were placed in the dorsal musculature of the left eyelid. The wires were formed into two small loops and gold pins were clamped to their ends. The procedure concluded with the application of provodine ointment to the eyelid and animals were returned to their home cages for 1–2 days. After the recovery period, the rabbits were placed in standard Plexglas restraint boxes and positioned in dark, soundand light-attenuating chambers. The light US consisted of a 150-ms light flash provided by a 7.5-V incandescent
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bulb (CM51; 0.22 A) positioned 1.0 – 2.0 cm from the eye. A small ‘warm-up’ voltage was delivered to the bulb filament between trials in order to minimize the rise time necessary to reach full illumination during light presentations. The air puff US consisted of a 150-ms puff of air (3.0 psi) positioned 1.0 – 2.0 cm from the cornea of the eye. Eyeblinks were monitored by recording EMG activity from the dorsal musculature of the eye. The EMG activity was amplified 1000 × , filtered (300–3000 Hz) and routed to an AC-bridge integrator. The integrated signal was then scaled and fed to a microcomputer that also controlled the delivery and timing of the stimuli [7]. Two groups of animals (i.e. light- or air puff-US) received one session of 40 trials with each trial consisting of a single, unsignaled US presentation and separated by an average of 30-s. On each trial, the EMG signal was digitally sampled for 2000 ms with the light onset occurring 1000 ms into this interval. In this manner, the UR was characterized by calculating the average UR amplitude, onset latency and latency to the peak response for each animal across the 40 trials.
2.3.2. Eyeblink conditioning Throughout classical conditioning, animals were positioned in standard Plexiglas restraint boxes housed within dark, sound- and light-attenuating chambers. Two unconditioned stimuli were used at different phases throughout conditioning. The light and air puff USs were equivalent to the stimuli described above. All animals were exposed to the same 900-ms tone-conditioned stimulus (CS; 1000 Hz, 85 db SPL) that was presented through a speaker positioned above the animal’s head and co-terminated with the US resulting in an interstimulus interval (ISI) of 750-ms. Eye movements were monitored using an infrared detection system, the output of which was sent to a microcomputer that also controlled the timing and presentation of the conditioning stimuli. Daily paired training sessions consisted of 60 trials, with each session broken into six blocks of ten trials each. The animal experienced a CS-alone probe trial on the first trial of each block with paired CS – US presentations occurring on the remaining nine trials of the block. Intertrial intervals were pseudo-randomly determined and ranged between 40- and 60-s. Regardless of the trial type (i.e. paired or CS-alone), a 250-ms pre-CS interval was examined for eyelid movements greater than 0.7 mm. If this threshold was surpassed the trial was discarded and not used in the analysis. A conditioned response was defined as a 0.5 mm or more closure of the eyelid that occurred during the 750-ms ISI on paired CS – US trials or anywhere after CS onset on CS-alone probe trials (1750 ms total duration).
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All animals progressed through three phases of training: (1) pre-lesion acquisition; (2) post-lesion testing with the initial US; and (3) post-lesion acquisition to a novel US. Following two 60-min adaptation sessions, animals were randomly assigned to one of two independent groups, distinguished by the type of US used during the initial acquisition training, i.e. light US (Group 1) or air puff US (Group 2). Consequently, the pre-lesion phase consisted of 12 days of paired tone CS/light US training for half the animals and paired tone CS/air puff US training for the remaining animals. Conditioning stimuli during paired training were always presented to the ipsilateral eye relative to the lesion electrode placement which was counterbalanced across animals. Immediately following this initial acquisition phase, each animal received lesions of the interpositus nuclear region by passing 1-mA dc (referenced to the ground screw) for 30 s through the two previously implanted electrodes. The second phase of conditioning began 24 h post-lesion and consisted of 8 subsequent days of paired CS–US training. During this phase, all of the conditioning parameters remained unchanged relative to the pre-lesion phase for each animal, thus allowing us to evaluate the effects of cerebellar lesions on the established conditioned responding (CR). The third phase of training allowed us to evaluate acquisition to a novel US under a lesioned state. To accomplish this, each animal received 12 days of paired tone CS/US training to the lesioned side. Rabbits that were initially trained using the light US (Group 1) were now shifted to the air puff US, while air puff US animals (Group 2) were shifted to the light US. Consequently, these three phases of training enabled a within-subject analysis of: (1) normal acquisition with both a non-somatosensory- and somatosensory-US; (2) the effects of cerebellar lesions on the maintenance of CR; and (3) acquisition with both a non-somatosensory- and somatosensory-US following cerebellar lesion. Given the proximity of the incandescent light source to the eye (i.e. 1–2 cm), a subset of animals were used to test the contribution of bulb-induced heat (i.e. a potential somatosensory stimulus) to the light-US conditioning. These separate animals received twelve days of paired CS–US conditioning as described above with the exception that a heat absorbing glass filter (Oriel, Stratford, CT; Model c 51950; 25.4 mm diameter) was positioned between the light source and the eye. The filter selectively removed spectral bands greater than 750 nm with an external transmittance of less than 0.15 at 800 nm and effectively zero transmittance at 1000 nm. At the completion of training, the rabbits were overdosed with an intravenous injection of pentobarbital (4.0 cc) and transcardially perfused with 0.9% saline followed by 10% formalin. The brains were then removed and placed in a formalin/sucrose solution for at
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Table 2 Percentage of interpositus nucleus lesion area Animal
Fig. 1. Representative unconditioned eyeblink responses evoked by corneal air puff and light flash. Traces are rectified electromyographic (EMG) signals from separate animals recorded from the dorsal musculature of the eye during a single trial.
least 10 days. Following this period, the brains were blocked in albumin-gelatin and frozen coronal sections (80-mm) were taken through the interpositus nucleus. The sections were mounted on gelatinized slides, stained with Cresyl violet, and examined microscopically to determine the extent of the lesion. The sections were subsequently digitized and the extent of the lesions were determined by calculating the percentage of lesioned interpositus area relative to the total nuclear area across four standard stereotaxic planes (0.0, 0.5, 1.0, and 1.5-mm anterior of lambda).
3. Results
3.1. Properties of the UR to light Fig. 1 illustrates characteristic unconditioned eyeblink responses from separate animals given either a 150-ms presentation of light (n =6) or an air puff (n =6) on a single trial. Eyeblinks evoked by the two stimuli differed with regard to onset latency and rise times (i.e. velocity), but did not differ with regard to their overall magnitudes (see Table 1). Specifically, the UR to light exhibited a delayed onset, slower rise time, Table 1 Unconditioned response (UR) properties Measure
Amplitude (mm) Peak latency (ms) Onset latency (ms) Rise time (ms)
Light US
Air puff US
Mean 9S.E.M.
Mean9 S.E.M.
5.5 91.0 262.6 918.4
5.39 1.0 72.49 11.5
0.18 8.77*
119.2 95.7
45.69 10.5
6.14*
143.4 9 21.1
26.79 6.53
5.27*
t-score
* PB0.05; S.E.M., standard error of the mean; t test based on ten degrees of freedom.
1 2* 3** 4 5 6 7 8 9 10 11
Stereotaxic coordinates (mm from lambda)
Total area
0.0
0.5
1.0
1.5
0.00 0.00 28.36 0.00 0.00 34.15 0.00 0.00 0.00 0.00 0.00
15.18 0.00 55.74 0.00 19.24 23.58 8.40 0.00 20.42 0.00 8.58
2.80 7.86 43.81 27.55 0.00 19.06 0.00 17.52 0.00 3.43 21.14
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
4.56 2.00 32.48 7.00 4.89 19.50 2.13 4.45 5.18 0.18 7.55
* Lesion profile is illustrated in Fig. 2 as black area. ** Lesion profile is illustrated in Fig. 2 as gray area.
and longer duration relative to the air puff-elicited blink. Onset latencies ranged between 104.0 and 135.7 ms for light compared to 23.75–94.25 ms for air puff. The longer latencies for visually-evoked blinks have been observed elsewhere [5,11,15] and appear to be, at least in part, a consequence of retinal processing. For instance, electrical stimulation of the optic tract induces evoked potentials at latencies of 8–11 ms in the inferior olive [2] and 11–14 ms in the cerebellar cortex [13,14]. In contrast, visual stimulation of the retina (light flash) results in cellular response latencies of 25–28 ms for the inferior olive [4] and 30–40 ms at the cerebellar cortex [14]. In fact, delays of 13–40 ms are observed when single units are recorded from the optic nerve following visual stimulation using a light flash [21].
3.2. Histology and lesion extent Histological examination revealed that lesions in two animals from the light US group failed to disrupt any portion of the interpositus nucleus, but were instead located in the overlying white matter and cortical tissues. Additionally, one other animal in the air puff US group had no measurable lesion. Consequently, data from these animals were not included in the subsequent analyses, but will be discussed briefly below. In the remaining animals (n= 11), effective lesions encompassed portions of the anterior interpositus, medial border of the dentate nucleus, and/or white matter dorsal to the interpositus nucleus. Table 2 presents interpositus lesion area data expressed as the percentage of total area across four stereotaxic planes. As is evident from Table 2, anterior interpositus nucleus lesions varied considerably in size and location, ranging between 2.0 and 32.5% of the total area. Despite this variability, all animals had in common some degree of
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damage to the dorsolateral portion of the anterior interpositus nucleus. Fig. 2 illustrates the largest (gray) and smallest (black) interpositus nucleus lesions across three stereotaxic planes. These lesions are also representative with regard to the disruption of the overlying white matter which was observed in ten of 11 animals.
3.3. Pre-lesion beha6ioral acquisition and post-lesion maintenance of responding The percentage CR during initial acquisition are plotted in Fig. 3 (Acquisition) for animals receiving a tone CS paired with either a light flash- (n = 5) or an air puff-US (n= 6). Both groups exhibited a slow, but incremental acquisition of CR, culminating in 69.3% (Light US) and 67.2% (Air puff US) CRs by day 12. These data were analyzed using a two-factor analysis of variance (ANOVA) with repeated measure which found a main effect of training sessions, F(11, 99) = 17.88, PB 0.001, consistent with the observed behavioral ac-
Fig. 2. Representative interpositus nucleus lesions depicting the smallest (black area) and largest (gray area) areas that were lesioned. (The numbers indicate the distance anterior to lambda. Abbreviations: ANS, ansiform lobule; DE, dentate nucleus; FA, fastigial nucleus; icp, inferior cerebellar peduncle; IO, inferior olive; IN, interpositus nucleus; PF, paraflocculus lobule; VN, vestibular nucleus.)
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quisition across training sessions. Although there appears to be some trend toward slower acquisition rates for light US animals, no significant differences were found with regard to the type of US used (light vs air puff) or in the rates of acquisition as indicated by the lack of a significant interaction between US type and session number (P\ 0.05). Furthermore, the number of trials necessary to reach a criterion of seven CRs within any ten consecutive trials did not differ between light US (381 9 91.5) and air puff US (294 979.1) groups, t(9)= 0.723, ns. The variability in CR observed in both groups is most likely due to the protracted ISI (750 ms) which is known to have a detrimental effect on conditioning relative to shorter ISIs (e.g. 250 ms) [20]. Conditioned response topographies were evaluated on CS-alone trials for both the light- and air puff-US conditions and are illustrated in Fig. 4. The topographies of the responses were very similar between the two conditions. Both groups exhibited responses that were comparable in duration, shape (mono- and biphasic), and were well timed to the US onset. Quantitatively, no differences were observed between groups with regard to their peak CR amplitudes, latency to peak response, or response onset latency on the final day of training (see Table 3). In order to assess the contribution of bulb-induced heat (i.e. somatosensory stimulus) to the light-US conditioning, a third group of animals (n= 4) underwent paired tone-light training equivalent to the other light US group with the exception that a heat absorbing glass filter was positioned between the light source and the eye. The results indicate little or no contribution of heat to the development of conditioning responding using a light US. As with the previous groups, these animals exhibited an incremental acquisition of CR across the 12 days of training, culminating in a mean percent of 64.3 9 3.45 by day 12. These data were compared using a single-factor ANOVA in which a main effect of training session was found, F(11, 47)= 2.06, PB 0.01. CR was significantly diminished by electrolytic lesions of the deep nuclei (Fig. 3; Same US). Twenty four hours following the lesion light US animals exhibited a 65% decrease in CRs compared to a 81% decrease in the response rates of air puff US animals. This post-lesion decrement in CR was found to be significant when compared to pre-lesion (day 12 of acquisition) rates, t(4)= 7.24, PB 0.01. Furthermore, no recovery in CR was observed for either group over 8 subsequent days of post-lesion training as determined by two-factor ANOVA with repeated measure (P \ 0.05).
3.4. Post-lesion acquisition to a no6el US Disruption of the deep cerebellar nuclei blocked any subsequent CR acquisition using a novel US. Animals
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Fig. 3. The mean percentage of conditioned responses are plotted for groups that differed with regard to their unconditioned stimulus (US), i.e. light or air puff, during the initial acquisition phase. The groups were trained further using the same US following lesions of the interpositus nucleus. Under this lesioned state, conditioned response acquisition was examined by shifting animals to the other US. Error bars indicate standard error of the mean.
were conditioned using the same tone CS, but during this phase the tone was paired with the other US. For instance, animals receiving tone – light pairings during the initial acquisition were now trained with tone–air puff pairings. The percentage of conditioned responses are presented in Fig. 3 (new US) for this phase of training. The interpositus lesions were sufficient to block any savings effect that may have occurred given the conditioning history with the tone CS. Response rates on the first day of this training, although more variable, did not differ from the initial day of acquisition (i.e. day 1 of the initial acquisition phase) for either group (P \0.05). Moreover, despite 12 days of training, neither group (i.e., light- or air puff-US) exhibit any subsequent acquisition to the tone when using the novel USs. These observations were confirmed using a two-factor ANOVA with repeated measure in which no main effects were found (P \0.05).
4. Discussion Using the classically conditioned eyeblink response, we report here that rabbits acquired CR to a tone CS that was paired with a light flash US. Furthermore, this learning did not differ from more traditional somatosensory-based conditioning (i.e. air puff US) with regard to the rate and overall level of acquisition. Two results suggest that both the non-somatosensory and somatosensory forms of this learning rely on common neural circuitry, namely the cerebellar complex, for their formation. First, the conditioned responses resulting from light and air puff training did not differ with
regard to the timing of the response, the overall magnitude, or the general response topography. Second, disruption of the deep cerebellar nuclei through electrolytic lesions profoundly disrupted CR regardless of the US used in training. Moreover, subsequent conditioning using either a light or air puff US was blocked by these lesions. While this research indicates a clear involvement of the cerebellum in non-somatosensorybased eyeblink conditioning, it is not possible to distinguish between the relative contributions of the deep nuclei to this learning. Although the majority of lesions compromised the interpositus nucleus, the lesions were not specific enough to rule out the possible disruption of information reaching the dentate nucleus. For instance, ten of 11 animals had damage to the overlying white matter which may have disrupted axons projecting to the dentate. Another three animals also received some damage to the medial dentate border. As mentioned previously, two animals were not included in the analyses because they lacked any damage to the interpositus nucleus, but instead had lesions exclusively involving the overlying white matter and cortical tissue. Interestingly, both of these animals developed CRs using the light US and exhibited a severe disruption of responding following the lesions, but unlike animals with interpositus damage, these rabbits subsequently developed CRs using the novel air puff US. Although only suggestive, the disruption of non-somatosensorybased conditioning in these animals, while leaving the somatosensory-based conditioning intact, may indicate a dissociation with regard to the specific cerebellar structures mediating these two forms of learning. Further research employing more discrete lesions will be
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Fig. 4. Representative conditioned responses from animals receiving paired training using a light US (top traces) or a corneal air puff US (bottom traces). The data were acquired on tone-only trials, although the vertical dashed lines indicate the time at which US presentations would have occurred.
necessary to clarify if in fact there is a distinction between the relative contributions of the deep nuclei to somatosensory- versus non-somatosensory-based eyeblink conditioning. This research stands in contrast to previous attempt to condition the nictitating membrane response using a Table 3 Conditioned response (CR) propertiesa Measure
Amplitude (mm) Peak latency (ms) Onset latency (ms)
Light US
Air puff US
Mean9 S.E.M.
Mean 9 S.E.M.
6.5 9 1.24 750.6 9 82.3
5.1 91.44 759.99 52.4
0.729 −0.098
458.6 9 63.8
482.3 9 56.6
−0.279
t-score
a S.E.M., standard error of the mean; t tests based on nine degrees of freedom.
more diffuse light US [5]. The primary distinction between the present research and this earlier report appears to be the intensity of the light serving as the US. In Bruner’s experiment the US consisted of indirect chamber illumination provided by a 200-W incandescent lamp that was superimposed upon a continuous illumination of the chamber by an adjacent 6-W bulb. In the present report, the light flash US was positioned directly in front of the cornea (1–2 cm) and darkness was maintained within the chamber between trials. It is reasonable to assume that this increase in intensity carried with it an increase in the ability of the US to promote learning. In fact, it is well known that the rate of learning is positively correlated with US intensity [20]. Two interesting and perhaps related questions emerge from the above statement: (1) what does it mean to increase a stimulus’ ability to promote learning from a neurobiological standpoint?; and (2) what processes allow light to serve as a CS under one set of
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conditions and a US under another set of conditions? Pavlov himself addressed the latter of these questions by proposing that the role of a stimulus in conditioning (i.e. CS vs US) is determined, at least in part, by the biological response elicited by the stimulus [18]. In other words, a US is define so not because of its modality, but rather, because it elicits a larger response relative to the CS. One implication of this criteria is that a single stimulus could serve both as a CS and a US depending on its intensity, i.e. a CS at low intensities and a US at high intensities. In fact, both corneal air puffs [10] and peri-orbital electrical stimulation [19] have been shown to support classical eyeblink conditioning using such a configuration. Taking this one step further, it can be speculated that intensity might ultimately determine what neural structures and systems are activated during conditioning. The present data argues that if sufficiently intense light is used as a US, the inferior olive (climbing fiber system) may be engaged and thus conditioning produced. The laboratory is currently investigating the hypothesis that the efficacy of a stimulus as an US is determined by the extent to which it engages the olivocerebellar system and that this ‘engagement’ is directly related to the intensity of the stimulus in question, regardless of its sensory modality.
Acknowledgements Our appreciation is extended to Jeremy Novak for his assistance in the collection of the data. This work was supported by a grant from the National Institute of Mental Health to R.F. Rogers (cMH11550) and J.E. Steinmetz (c MH51178). Correspondence should be addressed to Ronald F. Rogers, Program in Neural Science, Department of Psychology, Indiana University, Bloomington, IN 47405, USA.
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