- Email: [email protected]
Inactivation of the brachium conjunctivum prevents extinction of classically conditioned eyeblinks
Brain Research 1045 (2005) 175 – 184 www.elsevier.com/locate/brainres
Research report
Inactivation of the brachium conjunctivum prevents extinction of classically conditioned eyeblinks Wijitha U. Nilaweera, Gary D. Zenitsky, Vlastislav Bracha* Department of Biomedical Sciences, Iowa State University, 2032 Vet Med, Ames, IA 50011, USA Accepted 15 March 2005 Available online 22 April 2005
Abstract It is well established that the intermediate cerebellum is involved in the acquisition of classically conditioned eyeblink responses (CRs). Recent studies that inactivated the interposed nuclei (IN) demonstrated that blocking the intermediate cerebellum also interrupts CR extinction. Is this extinction deficit related to interrupting the information flow to efferent targets of the IN? To address this question, we inactivated axons of IN neurons in the brachium conjunctivum (BC). This treatment blocked the output of the intermediate cerebellum without directly affecting neurons in the deep cerebellar nuclei. Rabbits were trained in a delay classical conditioning paradigm, using a tone as the conditioned stimulus (CS) and a corneal air puff as the unconditioned stimulus (US). Then, the BC was microinjected with a sodium channel blocker, tetrodotoxin, during 4 extinction sessions in which rabbits were presented only with the CS. Tests performed after the 4-day injection period revealed that CRs did not extinguish in BC inactivation sessions but extinguished at a normal rate in the absence of the drug. CRs were then re-acquired. These data show that the normal flow of information along axons of cerebellar nuclear cells is required for CR extinction. D 2005 Elsevier B.V. All rights reserved. Theme: Neural basis of behavior Topic: Learning and memory: systems and functions—animals Keywords: Eyeblink; Classical conditioning; Cerebellum; Extinction; Superior cerebellar peduncle; Associative learning
1. Introduction Neuronal circuits that control the acquisition of classically conditioned eyeblinks in rabbits are among the best described neural substrates of associative learning in mammals. The expression and acquisition of classically conditioned eyeblinks are controlled by brainstem eyeblink circuits and by the intermediate cerebellum. Significant progress is being made in delineating the possible sites of plasticity that underlie information storage in this paradigm [3,4,7]. In contrast to advances made in understanding the expression and acquisition of conditioned eyeblink * Corresponding author. Fax: +1 515 294 2315. E-mail address: [email protected] (V. Bracha). URL: http://www.vetmed.iastate.edu/faculty_staff/users/vbracha/ (V. Bracha). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.03.015
responses, relatively little is known about the process of extinction. In the classical conditioning paradigm, animals are repeatedly exposed to an irrelevant stimulus (conditioned stimulus—CS) that is predictably followed by a biologically significant stimulus (unconditioned stimulus—US). In these conditions, the animal learns to produce a new response (conditioned response—CR) to the CS. Interestingly, the animal can extinguish CRs if the CS ceases to signal the US. This process is studied in the extinction paradigm whereby the subject is exposed to the CS without US reinforcement. In this condition, the frequency of CRs gradually decreases until they disappear. The CR is said to be extinguished. Are the same neural networks and the same, perhaps reversible, neural processes involved in both CR acquisition and extinction? A pivotal part of CR acquisition circuits is the intermediate cerebellum. Does the intermediate cerebellum
176
W.U. Nilaweera et al. / Brain Research 1045 (2005) 175 – 184
play the same role in CR extinction? Perrett and Mauk [25] reported that lesions of the cerebellar anterior lobe prevent extinction of short-latency eyeblink CRs in the rabbit. Since the disinhibition of the contralateral inferior olive following picrotoxin microinjections also prevented CR extinction [21] and because picrotoxin applied to the interposed cerebellar nuclei produced short-latency eyeblink CRs in previously extinguished animals [20], Medina et al. [21] proposed that a reciprocal plastic process at the same set of cerebellar synapses is involved in bi-directional learning during the acquisition and extinction of eyeblink CRs. A similar proposal was advanced by Ramnani and Yeo [26], who found that inactivating the cerebellar interposed nuclei prevents extinction. Since this result parallels reports of acquisition deficits induced by the same treatment [13,15,17], these authors concluded that both the acquisition and extinction of eyeblink CRs are dependent on cerebellar mechanisms. Inactivating the IN with GABA-A agonist muscimol produces local and remote physiological effects. The local effect is hyperpolarization and an associated block of IN neuronal firing [2]. The remote effect is on targets of IN projections that are prevented from receiving normal afferent information from the cerebellum. Both of these effects could potentially explain the disruption of CR extinction in animals with inactivations of the IN. To examine the role of remote effects, we temporarily inactivated the brachium conjunctivum (BC) in rabbits during extinction sessions. This procedure did not directly affect IN neurons, but it blocked the axons of IN cells. Here, we report that blocking the BC disrupts the normal extinction of CRs. This finding demonstrates that interrupting the information flow from the IN is sufficient for preventing CR extinction.
2. Materials and methods 2.1. Subjects Fourteen male New Zealand White rabbits (Harlan, Indianapolis, Indiana), weighing 2.5 –3.5 kg at the time of surgery, were housed individually on a 12/12 h light/dark cycle. All experiments were performed in accordance with the NIH ‘‘Principles of Laboratory Animal Care’’ (NIH publication No. 86-23, revised 1985) and the protocol approved by the Institutional Animal Care and Use Committee of Iowa State University. 2.2. Surgery Before any training, all rabbits were surgically implanted with a guide cannula (28-gauge stainless steel) aimed 1 mm dorsal to the left BC using sterile surgical techniques. Each rabbit was anesthetized with a mixture of Ketamine (50 mg/ kg), Xylazine (6 mg/kg), and Acepromazine (1.5 mg/kg).
During surgery, lambda was positioned 1.5 mm ventral to bregma, and the following stereotaxic coordinates were used: 12 –13 mm posterior, 2.8 mm lateral, and 14.2 mm ventral from bregma. A small Delrin head stage was mounted to the skull to accommodate the US delivery system and the eyeblink recording sensor. The implant and head stage were secured with anchoring screws and dental acrylic. The patency of the injection cannula was protected between experiments with a stainless steel stylet. The cannula was also protected with a removable lightweight Delrin cap. All animals were treated with antibiotics for 5 days after surgery and had a 1 week recovery period before experiments started. 2.3. Injection techniques Trained rabbits were tested in experiments in which the area of the BC was microinjected with solutions of tetrodotoxin (TTX, a potent and long-lasting sodium channel blocker, Calbiochem, 1 ng/Al = 3.1 pmol/Al), muscimol (GABA-A agonist, MP Biochemicals, 400 ng/Al = 3.5 nmol/Al), and 4% lidocaine (short-lasting sodium channel blocker). TTX and muscimol were dissolved in phosphate-buffered saline (PBS, pH 7.4). Injections of PBS were used in control experiments. All injections were delivered through a 33-gauge stainless steel needle inserted in the implanted guide tube. The injection needle was connected to a 10 Al Hamilton syringe (Hamilton Company, Reno, Nevada) using transparent Tygon tubing. The injected volume was measured by observing the movement of a small air bubble relative to gradation marks on the tubing connecting the needle to the syringe. Only one drug was injected on any given experimental day. Lidocaine was used for the functional localization of optimal injection sites. Other drug injections were performed at depths where lidocaine abolished CR expression. Muscimol was used for the axon-sparing local inactivation of neurons. TTX was used for inactivating the BC during extinction. All drugs were applied at the rate of 0.75 Al/min. For additional details of the drug injection protocol, see the corresponding sections of the experiment below. 2.4. Experiment outline The experiment consisted of 5 parts (Fig. 1). During the preparation phase, implanted rabbits were conditioned. Trained animals then received lidocaine injections targeting the BC at variable depths to determine an optimal injection site (CR expression abolished). Once the injection site was determined, Extinction Phase I began. Rabbits were injected with either TTX or with PBS before each experiment and trained for 4 days in the extinction paradigm. In Extinction Phase II, drug injections were discontinued and animals were trained in the extinction paradigm for 4 more days. Extinction Phase II was followed by re-acquisition and performance tests (2 days). In the final stage of the
W.U. Nilaweera et al. / Brain Research 1045 (2005) 175 – 184
177
injection site was determined, it was followed by a TTX test designed to establish the duration of TTX inactivation of the BC. Each rabbit was injected with 1.5 Al of TTX and presented with CS + US trials until CRs recovered. The general effect of this test TTX injection varied based on the specific location of the injection site. In 3 of the 7 animals in the experimental group, the initial dose of TTX not only abolished CRs, but also produced ophthalmic and postural side effects. To reduce these side effects while preserving CR abolition, the TTX dose was reduced to 0.75 Al in 2 rabbits and to 1.1 Al in one animal. In all animals, the abolition of CRs lasted 105 – 156 min. This duration of BC inactivation reliably exceeded the duration of the individual extinction sessions in the next stage of the experiment. Since the duration of the TTX effect averaged 120 min, the test sessions utilized groups of 5 trials alternating with 5 min pauses in stimulation. The purpose of this arrangement was to reduce stress to animals imposed by the large number of trials presented during a prolonged period of CR abolition. Fig. 1. Summary of the experiment and a brief description of procedures performed at each phase of the experiment. BC, brachium conjunctivum; CS, conditioned stimulus; PBS, phosphate-buffered saline; TTX, tetrodotoxin.
experiment, re-trained rabbits were injected with muscimol to test for effects of axon-sparing inactivation on CR expression. 2.4.1. Training Prior to any training, all rabbits were adapted to a standard rabbit restraining box (Plas Labs, Inc, Lansing, Michigan) and the experimental environment for 3 days, 30 min/day. After adaptation, they were conditioned in the standard delay classical conditioning paradigm. An 80 dB, 1 kHz, 450 ms tone was used as the CS and was superimposed on continuous 70 dB white noise. The US was a 100 ms, 210 kPa (at the source) air puff aimed to the caudal portion of the left cornea. The US co-terminated with the CS. The inter-stimulus interval was 350 ms. The inter-trial interval varied pseudorandomly between 15 and 25 s. Each experiment consisted of 100 paired-trial (CS + US) presentations, and the rabbits were trained until they had 90% CRs in 3 consecutive sessions. 2.4.2. Localizing the BC with lidocaine and TTX tests Before extinction experiments, the BC region was mapped with lidocaine injections to determine the optimal injection site. Rabbits were injected with 4 Al of lidocaine, starting 1 mm above the expected depth of the BC. In these experiments, the injection needle was inserted in the guide tube and 40 CS + US trials were delivered. Next, lidocaine was injected and the training was immediately resumed and continued until CRs recovered (60 to 80 trials.) If CRs were not abolished, the injection was made 0.5 mm deeper the next day. This continued until CRs were abolished for at least 6 min in any given experiment. After the optimal
2.4.3. Extinction Phase I After the optimal depth and TTX effect period were determined, 7 rabbits were injected with TTX (0.75 – 1.5 Al, see previous section) for 4 days at the beginning of each experiment. To maximize BC inactivation, extinction training began 20 min following the injection. Rabbits were then presented with 100 CS-alone trials per day. The control group (6 rabbits) underwent the same procedure as the TTX group but received PBS injections (1.5 Al). 2.4.4. Extinction test and Extinction Phase II After Extinction Phase I, rabbits had a 2-day rest during which no training was conducted and no injections were administered. The purpose of this pause was to prevent contamination of further tests by any possible lingering effects of TTX on CR performance. Following the rest period, all rabbits were trained for 4 additional days in extinction sessions consisting of 100 CS-alone presentations. No injections were administered on these days. The purpose of Extinction Phase II was to measure the degree of extinction achieved by the experimental group in the previous TTX sessions and to test the ability of the rabbits to extinguish CRs in normal conditions without injections. 2.4.5. Re-training and muscimol injections After extinction training, rabbits were re-trained with paired CS + US trials for 2 days to examine whether previous treatments affected the ability to re-acquire and express eyeblink CRs. Following CR re-acquisition, all animals were injected with 1.5 Al of muscimol to examine whether the effects of previous TTX injections on CR performance could be attributed to the inactivation of cell bodies located near injection sites. The muscimol test consisted of 40 trials before injection, a 20 min waiting period following the injection (comparable to that during the TTX injections), and then 60 additional trials.
178
W.U. Nilaweera et al. / Brain Research 1045 (2005) 175 – 184
2.5. Data acquisition and analysis Eyeblink recording was performed using techniques described previously [5]. Before each experiment, a small loop was attached to the upper eyelid and was coupled to a low friction electromechanical lever sensor to track the movements of the upper eyelid. The output of the sensor was amplified, digitized at 1 kHz with 12 bit resolution, and displayed and stored on a custom data acquisition system. Data were acquired for 1400 ms in each trial, starting 250 ms before CS onset. The data acquisition program measured alpha responses, CRs, and URs. Baseline eyelid movements exceeding 0.25 mm were recognized as spontaneous blinks and any blink exceeding CR threshold within 80 ms after CS onset was recognized as an alpha response. Trials containing spontaneous blinks and/or rare alpha responses were stored but were not included in further data analyses. Closure of the upper eyelid more than 0.1 mm after the alpha period and before the onset of the US was considered a CR in paired trials, and any blink that exceeded this threshold after the alpha response window was considered a CR in extinction trials. This relatively low threshold was selected to capture the small responses that are frequently expressed at later stages of extinction. In addition to the visualization of eyeblink traces on the computer monitor, the animal’s behavior was monitored using 2 digital video cameras. One camera provided a front view of the rabbit’s head and the other one was positioned on the side of the trained eye. This setup was used to monitor the general behavior of animals and was valuable in detecting drug injection side effects. Measurements of eyeblink responses were used for calculations of the CR incidence per session and also per block of 20 trials in each session. Data from individual animals were pooled together and statistically analyzed. Extinction days and 20-trial blocks were treated as repeated measures factors. Planned contrasts from repeated measures ANOVA were used to address hypotheses regarding patterns of CR incidence emerging from the interaction between treatments and extinction days and/or 20-trial blocks. Specifically, we contrasted days 1 and 4 from control Extinction Phase I with days 1 and 4 from TTX group Extinction Phase II. Furthermore, within Extinction Phase II, we contrasted day 1 of the control and TTX groups. A separate repeated measures ANOVA generated similar output for the analysis of effects of muscimol. All group data in the Results section represent group means T standard error of the mean. All calculations were performed using StatSoft Statistica software. 2.6. Histology After completing experiments, animals were deeply anesthetized, and the injection sites were marked by injecting 1 Al of tissue marking dye. The animals were
transcardially perfused with 1 l of PBS followed by 1 l of fixative (10% buffered formalin). Each perfused brain was removed from the skull and, following a post-fixation in 30% sucrose formalin, sectioned coronally at 50 Am on a freezing microtome. Sections were mounted onto gelatincoated slides and then stained with neutral red and Luxol fast blue.
3. Results 3.1. Location of injection sites The histological reconstruction of tracks produced by injection needles and deposits of tissue-marking dye revealed that, in all TTX and control animals, the injections were performed in the close vicinity of the BC (Fig. 2).
Fig. 2. Location of injection sites for TTX (black dots) and control (black stars) rabbits. All injection sites were found close to the BC, and the rostro-caudal spread was 1.5 mm. The depicted sections represent coronal sections through the region of interest and are spaced 0.5 mm apart with panel (A) being the most caudal and panel (D) the most rostral. The 2 white stars indicate the injection placement in rabbits that did not have sufficient CR abolition during the lidocaine test, and the dotted circle in panel (C) indicates the extent of BC damage in the rabbit whose CRs did not recover from the lidocaine injection. These 3 animals were excluded from the data analysis. IV, fourth ventricle; BC, brachium conjunctivum; IC, inferior colliculus; ll, lateral leminiscus; Mo5, motor trigeminal nucleus; MCP, middle cerebellar peduncle; Me5, mesencephalic trigeminal nucleus; NRTP, nucleus reticularis tegmenti pontis; PN, pontine nuclei; py, pyramidal tract; S5, sensory trigeminal nucleus; s5, root of the sensory trigeminal nerve; SC, superior colliculus; TN, trochlear nucleus; tz, trapezoid body; Tz, nucleus of the trapezoid body; VTg, ventral tegmental nucleus.
W.U. Nilaweera et al. / Brain Research 1045 (2005) 175 – 184
In most animals, injection sites were positioned just dorsal to the BC at the anterior –posterior level of 0 to 1 mm caudal to the caudal pole of the ventral tegmental nucleus.
179
93.0 T 3.1, 18.5 T 6.7, and 92.5 T 5.2% in controls (n = 4) (see Fig. 3). The histological analysis (Fig. 2) in conjunction with lidocaine mapping tests revealed that lidocaine failed to abolish CRs when injected at distances larger then 1 mm from the BC.
3.2. General observations 3.3. Effects of TTX on CR expression and TTX side effects Eleven of the 14 implanted rabbits were included in the results analysis. One animal was excluded because the BC was lesioned during the lidocaine test and lost the capacity to express CRs. Two additional rabbits designated as controls were also excluded since lidocaine test injections did not completely abolish the expression of CRs. Thus, 7 animals were included in the experimental TTX group and 4 animals in the control group. During the initial training, all rabbits reached the performance criterion (90% CRs for 3 consecutive days) in 5 training sessions on average. All trained animals were then injected with 4 Al of lidocaine in a functional mapping test to find an optimal location for TTX and PBS injections. A particular location was considered optimal when lidocaine abolished CRs for at least 6 min. A relatively short abolition criterion of 6 min was chosen to prevent advancing the injection needle too deep, thereby penetrating the BC with the injection needle, producing a permanent lesion to this structure resulting in a permanent CR deficit. The effect of lidocaine on CRs lasted between 6 and 23 min (mean 13.7 T 1.54 min, n = 11). Mean CR incidences before injection, after injection, and during recovery were 92.3 T 3.6, 4.4 T 1.7, and 80.0 T 6.4% respectively in the TTX group (n = 7) and
Once effective injection sites were determined in all rabbits, the TTX group received a TTX injection to examine the time period of CR abolition. Rabbits were injected with TTX at the beginning of the experiment and then presented with paired stimuli to observe the onset and duration of effect. Confirming previous studies [14], TTX abolished CR expression. This effect was immediate in all rabbits, and the duration ranged between 105 and 156 min (mean of 127.4 T 7.82 min, n = 7), confirming that the BC would be sufficiently inactivated for the 60 min duration of the planned extinction session. In addition to CR abolition, TTX injections in the BC elicited several notable side effects. The most frequent was unusual calmness in the restraining box. This was associated with a wide opening of the ipsilateral eyelids, and frequently with mild ipsilateral exophthalmus. The effects of BC inactivation were not restricted to the ipsilateral eye. Relatively frequently, animals lowered their heads and rotated it in the horizontal plane to the left side in addition to lowering their ipsilateral (left) ear. This is consistent with a generalized decrease of muscle tone in the ipsilateral face, head, and neck. Much less frequent and notably delayed was the induction of
Fig. 3. Summary of conditioned response incidence (means T SEM) in the experimental (TTX) and control (PBS) groups during all 5 phases of the experiment. All rabbits exhibited asymptotic CR incidence during the last 3 days of training. Lidocaine abolished CR incidence in both groups in the post-injection period (Post) and then these responses recovered (Recovery). During Extinction Phase I, the experimental group did not express CRs due to BC inactivation by TTX. At the same time, conditioned responses gradually extinguished in control animals. During Extinction Phase II, CR incidence in the control group was lower than in the TTX group. Most notably, the TTX group exhibited an extinction curve that resembled the extinction of CRs in the control group during Phase I, indicating that TTX during Phase I prevented normal extinction in these rabbits. Following Phase II, both groups were successfully re-trained. Finally, injections of muscimol failed to abolish CR incidence in either of the groups.
180
W.U. Nilaweera et al. / Brain Research 1045 (2005) 175 – 184
bilateral ocular nystagmus that was sometimes accompanied by synchronized oscillatory movements of the head. 3.4. Effects of BC inactivation on CR extinction Inactivating the BC with TTX completely abolished the expression of CRs for the duration of each Extinction Phase I session (Fig. 3). At the same time, control animals exhibited a typical decrease of CR frequency both in the individual extinction sessions (Fig. 4) as well as in the sequence of the 4 extinction sessions (Figs. 3 and 4). On the first day of Extinction Phase II (extinction with no injections), animals from the TTX group exhibited a large number of CRs that became less frequent toward the end of the session (Fig. 5). This behavior contrasted with the control group that expressed only a few scattered small-amplitude CRs (Fig. 5). At the group level, on the first day of Extinction Phase II, the TTX group exhibited 37.6 T 4.5% CRs. This level of responding was significantly different from the control group that demonstrated only 6.8 T 1.3% CRs ( F 1,9 = 26.631, P = 0.00060), and it was indistinguishable from the frequency of CRs the control group exhibited on the first day of Extinction Phase I (37.7 T 8.0%) ( F 1,9 = 0.00244, P = 0.96165). The high frequency of CRs exhibited by the TTX group on day 1 of Extinction Phase II clearly demonstrated that BC inactivation during Extinction Phase I disrupted normal CR extinction. During continued extinction training in the absence of any injections, CR frequency in the TTX group gradually decreased at a rate that was similar to the extinction rate of the control group in Extinction Phase I (Figs. 3 and 4; Fig 4 shows overlaid extinction curves of TTX Phase II
and control Phase I). On the fourth day of Extinction Phase II, the TTX group exhibited 13.1 T 3.5% CRs. This level of performance was nearly indistinguishable from controls on day 4 of Extinction Phase I (15.2 T 2.8% CRs) ( F 1,9 = 0.15050, P = 0.70710). Besides the similar aggregate daily frequency of CRs in the TTX group during Extinction Phase II and in the control group during Extinction Phase I, both groups exhibited a remarkably similar and statistically indistinguishable decline of CR frequency within individual sessions in corresponding phases of the experiment (Fig. 4, F 28,252 = 0.65457, P = 0.91028). These results demonstrated that BC inactivation prevented normal CR extinction and also that the BC inactivation using local injections did not produce a long-lasting dysfunction of the extinction circuits, since once the inactivation was discontinued, the extinction process occurred at a normal rate. Following Extinction Phase II, both groups were retrained in the paired CS + US paradigm for 2 sessions. Since both groups quickly re-acquired high levels of CRs comparable to pre-extinction performance (Fig. 3), it can be concluded that the injections of TTX did not alter the functionality of the circuits that are involved in normal re-acquisition and expression of eyeblink CRs. In the final experiment, all rabbits were injected with muscimol at the injection sites used for the TTX and PBS injections. The muscimol injections, which should have inactivated GABA-A receptor-containing neurons nearest the infusion sites, slightly decreased CR incidence but did not abolish CR expression (Fig. 3). These results suggest that the abolition of CRs following TTX injections was likely due to inactivating axons rather than cell bodies. Notably, there were no
Fig. 4. Detailed comparison of CR incidence (means T SEM) in Extinction Phase II of the TTX group and Extinction Phase I of the PBS group. Each session is represented by five 20-trial blocks. Note the gradual decline of CR incidence within each session and across sessions. This behavior is typical for normal extinction of eyeblink CRs. Also note that the extinction curves of both groups largely overlap. This indicates that the TTX group failed to extinguish during the prior 4 sessions that were conducted while the BC was inactivated with TTX.
W.U. Nilaweera et al. / Brain Research 1045 (2005) 175 – 184
181
Fig. 5. Examples of eyeblink mechanograms in a TTX and a control (PBS) rabbit during the last day of training (A,C) and during the extinction test (the first day of Extinction Phase II; B,D). Each stack plot represents a complete printout from a single training session and each trace represents the upper eyelid mechanogram for one trial. Vertical lines in each plot denote onsets of the CS and US. Upward deflections in mechanograms indicate eyelid closure. Eyelid closure between the CS and US markers in panels (A) and (C) are CRs. All eyelid closures following the CS marker in panels (B) and (D) are considered CRs. Trials are stacked with the first trial on the top and the last trial at the bottom of the plot. On the last day of pre-injection training (A,C), both rabbits expressed comparable and robust levels of CR performance. The animal that was injected with TTX during extinction training showed no signs of extinction, since it expressed a number of wellformed CRs at the beginning of the extinction test (B). In contrast to the TTX animal, CRs in the control rabbit extinguished during Phase I, as evidenced by the low number of CRs exhibited during the extinction test (D).
prominent side effects in the muscimol post-injection period, other than increased calmness, which was also observed following both lidocaine and TTX experiments. In several cases, though, rabbits tended to locomote in clockwise circles when taken out of the restraining box and put on the floor following the experiment.
4. Discussion The present experiments demonstrated that unilateral inactivation of the BC during extinction sessions blocked eyeblink CRs. BC inactivation, besides abolishing CR expression, also prevented normal extinction of CRs. These findings are the first to show that normal activity
182
W.U. Nilaweera et al. / Brain Research 1045 (2005) 175 – 184
of the BC is required for the extinction of eyeblink CRs in the rabbit.
both the CR performance and extinction effects observed in the present study are attributable to BC inactivation.
4.1. Specificity of BC inactivation
4.2. The role of the BC in circuits controlling the extinction of conditioned eyeblinks
The present study inactivated cerebellar output projections located in the BC using the sodium channel blocker, TTX. The BC is a fiber tract that contains axons originating in the deep cerebellar nuclei, including axons sent by the interposed nuclei. TTX blocks the generation of action potentials in cell bodies and also prevents the propagation of action potentials in neuronal axons. Microinjections of TTX in the present study blocked the expression of CRs. This confirms previous reports of the involvement of the BC in the execution of previously learned CRs [14]. More importantly, we found that TTX microinjections prevented CR extinction. Do these effects of TTX relate specifically to BC inactivation? Attributing the observed effects to the function of the BC requires eliminating the possibilities that the drug (a) did not spread to some other remote part or parts of the network that controls CRs; (b) did not inactivate CR-related cell bodies at the site of injection; and (c) did not inactivate some other CR-related fiber tracts at the injection site. To discuss these possibilities, the distance of the effective spread of TTX when injected in brain tissue should be considered. Zhuravin and Bures [27] conducted a functional test of effective TTX diffusion in the rat brain. They reported that 10 ng of TTX (6 to 13 times more than was injected in the present study) inactivated cells located up to 1.5 mm from the injection site. In our recent TTX diffusion study performed in the rabbit [24], we found that injections of 2 Al (2 ng total) of TTX abolished CRs only when injected 1 mm or closer to the BC. These data suggest that the amounts of TTX used in this study inactivate neural tissue in an area of less than 1.5 mm around the injection site. Considering this spread and the location of injections in the present study (Fig. 2), one can conclude that TTX either completely or near completely inactivated the BC. On the other hand, it is unlikely that TTX inactivated some of the known neighboring components of the eyeblink network, such as the cerebellar interposed nuclei, the red nucleus, pontine nuclei, the middle cerebellar peduncle, or the lateral lemniscus. All of these structures are located beyond the estimated spread of TTX. It is possible, however, that in addition to the BC, TTX inactivated some not yet known structures (either nuclear or axonal) located close to the injection sites. Since muscimol injection tests failed to abolish CR expression, it seems that the observed effects on CR expression were not due to inactivating cell bodies containing GABA-A receptors. This finding suggests that the effects of TTX on CR expression were most likely related to inactivating axons close to the injection site. Although the present experiment cannot exclude the possibility of TTX inactivating some as yet unknown CR-related fiber tracts proximate to the BC, we base our discussion on the hypothesis that
Previous experiments established that inactivating the cerebellar IN prevents the extinction of eyeblink CRs [26]. There are two interpretations of this finding. Specifically, one could speculate that inactivating the IN blocks an extinction-related and hyperpolarization-sensitive plastic process directly in the IN. An alternative explanation would be that inactivating the IN interrupts the supply of essential information to targets of IN projections. Results of the present experiment shed new light on this issue. Since we found that inactivating the BC without directly interfering with IN neuronal activity prevents CR extinction, it is clear that extinction depends on information supplied by deep cerebellar nuclei to efferent targets involved in CR extinction. A possible region for putative plastic components of eyeblink extinction neural circuits is the brainstem in the vicinity of the facial and accessory abducens nuclei. In a recent study, Krupa and Thompson [16] reported that inactivating this brainstem region prevents the extinction of CRs. It is possible that inactivating the BC upsets the extinction-related process at this site. Incidentally, changes in the excitability of eyeblink motoneurons were previously reported to be associated with the acquisition of eyeblink CRs in the cat [19]. Eyeblink motoneurons and premotoneurons could be affected by the IN either directly or indirectly through interposito-rubral projections [11,22]. Other candidates for the BC-efferent, extinction-related site of plasticity could be cerebellar afferent structures or, paradoxically, the cerebellum itself. It is known that the intermediate cerebellum is part of a recurrent, feedback-rich, neural network [4]. Some of the cerebellar output projections are directed to sites that are major suppliers of afferent information to the cerebellum. Thus, cerebellar output can regulate cerebellar input sources. The possible involvement of cerebellar nuclei and/or cerebellar afferents in extinction is supported by the results of Gould and Steinmetz [12], who showed that extinction of eyeblink responses is accompanied by a decrease of task-related neuronal activity in the interposed nuclei. More specifically, Medina et al. [21] suggested that the cerebello-inferior olivary-cerebellar feedback loop could be involved both in CR acquisition and extinction. The results of the present study are in agreement with this notion since inactivating the BC blocks cerebellar inhibitory projections to the inferior olive, and this is likely to produce an aggregate increase of inferior olivary firing. This in turn might prevent extinction-related learning in the cerebellar cortex and/or nuclei. Besides the cerebelloinferior olivary-cerebellar feedback system, other feedback loops, such as the cerebello-pontine nuclear cerebellar system, could be involved in regulating eyeblink-related
W.U. Nilaweera et al. / Brain Research 1045 (2005) 175 – 184
circuits in the intermediate cerebellum. Cerebellar control of cerebellar input sources in the rabbit was documented in previous studies demonstrating that inactivating the IN or the red nucleus affects neuronal activity in the pontine nuclei [6,9] and in the trigeminal nuclei [8]. Furthermore, involvement of cerebellar feedback loops in regulating cerebellar activity is supported by the finding that inactivating the BC with lidocaine affects the activity of IN neurons [23]. 4.3. CR acquisition vs. CR extinction Behavioral experiments suggest that the extinction of CRs is not a simple reversal of CR acquisition. It is believed that, during CR extinction, memory traces of CRs are not erased, but suppressed. If so, one could assume that either different neural networks or at least different physiological processes within the same neural network control CR acquisition and extinction. Several studies have documented that, indeed, some of the components of extinction circuits differ from those that govern CR acquisition. For example, inactivating the region of the ipsilateral facial/accessory abducens nuclei does not affect CR acquisition [10,18] but disrupts CR extinction [16]. Similar to medullar motoneuronal areas, lesions of the hippocampus do not prevent CR acquisition, but they seem either to prevent or at least significantly slow down CR extinction in well-trained rabbits [1]. The present study revealed an additional difference between substrates that control CR acquisition and extinction. Krupa and Thompson [14] reported that inactivating the BC with small injections of TTX did not prevent CR acquisition. Their finding contrasts with the results of the present study showing that BC inactivations block CR extinction. This finding is interesting when one considers that both acquisition and extinction are affected by inactivating the IN [15,26]. It appears that, while both of these processes depend on the intermediate cerebellum, only CR extinction requires the normal function of axons conveying information to cerebellar targets. The present results do not provide definite conclusions regarding the sites of plasticity for CR extinction since inactivating the BC could potentially block three qualitatively different types of processes: inactivating axons of cerebellar nuclear neurons could block the transmission of learning-specific information to cerebellar efferent targets, it could produce a nonspecific malfunction of cerebellar efferent targets due to the loss of tonic cerebellar input, and it could also interrupt cerebellar plastic changes due to blocking cerebellar feedback pathways. Dissociating these possibilities presents a challenge to future studies of classically conditioned eyeblink extinction.
Acknowledgments The authors would like to thank Mike Hord for assistance with the experimental set up, Kari Teeter for assistance with
183
histology, and Kristina Irwin for assistance with manuscript preparation. This research was supported by NIH grants R01 NS36210 and R01 NS21958.
References [1] E. Akase, D.L. Alkon, J.F. Disterhoft, Hippocampal lesions impair memory of short-delay conditioned eye blink in rabbits, Behav. Neurosci. 103 (1989) 935 – 943. [2] D.P. Aksenov, N.A. Serdyukova, J.R. Bloedel, V. Bracha, Glutamate neurotransmission in the cerebellar interposed nuclei: involvement in classically conditioned eyeblinks and neuronal activity, J. Neurophysiol. 93 (2005) 44 – 52. [3] P.J. Attwell, M. Ivarsson, L. Millar, C.H. Yeo, Cerebellar mechanisms in eyeblink conditioning, Ann. N. Y. Acad. Sci. 978 (2002) 79 – 92. [4] V. Bracha, J.R. Bloedel, The multiple pathway model of circuits subserving the classical conditioning of withdrawal reflexes, in: J.R. Bloedel, T.J. Ebner, S.P. Wise (Eds.), Acquisition of Motor Behavior in Vertebrates. MIT Press, Cambridge, MA, 1996, pp. 175 – 204. [5] V. Bracha, L. Zhao, K. Irwin, J.R. Bloedel, Intermediate cerebellum and conditioned eyeblinks: parallel involvement in eyeblinks and tonic eyelid closure, Exp. Brain Res. 136 (2001) 41 – 49. [6] M.C. Cartford, E.B. Gohl, M. Singson, D.G. Lavond, The effects of reversible inactivation of the red nucleus on learning-related and auditory-evoked unit activity in the pontine nuclei of classically conditioned rabbits, Learn. Mem. 3 (1997) 519 – 531. [7] K.M. Christian, R.F. Thompson, Neural substrates of eyeblink conditioning: acquisition and retention, Learn. Mem. 10 (2003) 427 – 455. [8] R.E. Clark, D.G. Lavond, Neural unit activity in the trigeminal complex with interpositus or red nucleus inactivation during classical eyeblink conditioning, Behav. Neurosci. 110 (1996) 13 – 21. [9] R.E. Clark, E.B. Gohl, D.G. Lavond, The learning-related activity that develops in the pontine nuclei during classical eye-blink conditioning is dependent on the interpositus nucleus, Learn. Mem. 3 (1997) 532 – 544. [10] R.E. Clark, A.A. Zhang, D.G. Lavond, The importance of cerebellar cortex and facial nucleus in acquisition and retention of eyeblink/NM conditioning: evidence for critical unilateral regulation of the conditioned response, Neurobiol. Learn. Mem. 67 (1997) 96 – 111. [11] K.D. Davis, J.O. Dostrovsky, Modulatory influences of red nucleus stimulation on the somatosensory responses of cat trigeminal subnucleus oralis neurons, Exp. Neurol. 91 (1986) 80 – 101. [12] T.J. Gould, J.E. Steinmetz, Changes in rabbit cerebellar cortical and interpositus nucleus activity during acquisition, extinction, and backward classical eyelid conditioning, Neurobiol. Learn. Mem. 65 (1996) 17 – 34. [13] M.J. Hardiman, N. Ramnani, C.H. Yeo, Reversible inactivations of the cerebellum with muscimol prevent the acquisition and extinction of conditioned nictitating membrane responses in the rabbit, Exp. Brain Res. 110 (1996) 235 – 247. [14] D.J. Krupa, R.F. Thompson, Inactivation of the superior cerebellar peduncle blocks expression but not acquisition of the rabbit’s classically conditioned eye-blink response, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 5097 – 5101. [15] D.J. Krupa, R.F. Thompson, Reversible inactivation of the cerebellar interpositus nucleus completely prevents acquisition of the classically conditioned eye-blink response, Learn. Mem. 3 (1997) 545 – 556. [16] D.J. Krupa, R.F. Thompson, Inhibiting the expression of a classically conditioned behavior prevents its extinction, J. Neurosci. 23 (2003) 10577 – 10584. [17] D.J. Krupa, J.K. Thompson, R.F. Thompson, Localization of a memory trace in the mammalian brain, Science 260 (1993) 989 – 991. [18] D.J. Krupa, J. Weng, R.F. Thompson, Inactivation of brainstem motor nuclei blocks expression but not acquisition of the rabbit’s classically conditioned eyeblink response, Behav. Neurosci. 110 (1996) 219 – 227.
184
W.U. Nilaweera et al. / Brain Research 1045 (2005) 175 – 184
[19] M. Matsumura, C.D. Woody, Long-term increases in excitability of facial motoneurons and other neurons in and near the facial nuclei after presentations of stimuli leading to acquisition of a Pavlovian conditioned facial movement, Neurosci. Res. 3 (1986) 568 – 589. [20] J.F. Medina, K.S. Garcia, M.D. Mauk, A mechanism for savings in the cerebellum, J. Neurosci. 21 (2001) 4081 – 4089. [21] J.F. Medina, W.L. Nores, M.D. Mauk, Inhibition of climbing fibres is a signal for the extinction of conditioned eyelid responses, Nature 416 (2002) 330 – 333. [22] S. Morcuende, J.M. Delgado-Garcia, G. Ugolini, Neuronal premotor networks involved in eyelid responses: retrograde transneuronal tracing with rabies virus from the orbicularis oculi muscle in the rat, J. Neurosci. 22 (2002) 8808 – 8818. [23] W.U. Nilaweera, K. Irwin, G.D. Zenitsky, D.P. Aksenov, V. Bracha, Are feedback loops involved in the cerebellar neuronal correlates of
[24]
[25]
[26]
[27]
conditioned eyeblinks? Abstract Viewer/Itinerary Planner, Society for Neuroscience, Washington, DC, 2002 (Program No. 79.6). W.U. Nilaweera, G.D. Zenitsky, K.B. Irwin, V. Bracha, Assessing diffusion of tetrodotoxin in the rabbit brain suppressing conditioned eyeblinks by brachium conjunctivum inactivation, Abstract Viewer/ Itinerary Planner, Society for Neuroscience, Washington, DC, 2004 (Program No. 136.4). S.P. Perrett, M.D. Mauk, Extinction of conditioned eyelid responses requires the anterior lobe of cerebellar cortex, J. Neurosci. 15 (1995) 2074 – 2080. N. Ramnani, C.H. Yeo, Reversible inactivations of the cerebellum prevent the extinction of conditioned nictitating membrane responses in rabbits, J. Physiol. 495 (Pt. 1) (1996) 159 – 168. I.A. Zhuravin, J. Bures, Extent of the tetrodotoxin induced blockade examined by pupillary paralysis elicited by intracerebral injection of the drug, Exp. Brain Res. 83 (1991) 687 – 690.
Research report
Inactivation of the brachium conjunctivum prevents extinction of classically conditioned eyeblinks Wijitha U. Nilaweera, Gary D. Zenitsky, Vlastislav Bracha* Department of Biomedical Sciences, Iowa State University, 2032 Vet Med, Ames, IA 50011, USA Accepted 15 March 2005 Available online 22 April 2005
Abstract It is well established that the intermediate cerebellum is involved in the acquisition of classically conditioned eyeblink responses (CRs). Recent studies that inactivated the interposed nuclei (IN) demonstrated that blocking the intermediate cerebellum also interrupts CR extinction. Is this extinction deficit related to interrupting the information flow to efferent targets of the IN? To address this question, we inactivated axons of IN neurons in the brachium conjunctivum (BC). This treatment blocked the output of the intermediate cerebellum without directly affecting neurons in the deep cerebellar nuclei. Rabbits were trained in a delay classical conditioning paradigm, using a tone as the conditioned stimulus (CS) and a corneal air puff as the unconditioned stimulus (US). Then, the BC was microinjected with a sodium channel blocker, tetrodotoxin, during 4 extinction sessions in which rabbits were presented only with the CS. Tests performed after the 4-day injection period revealed that CRs did not extinguish in BC inactivation sessions but extinguished at a normal rate in the absence of the drug. CRs were then re-acquired. These data show that the normal flow of information along axons of cerebellar nuclear cells is required for CR extinction. D 2005 Elsevier B.V. All rights reserved. Theme: Neural basis of behavior Topic: Learning and memory: systems and functions—animals Keywords: Eyeblink; Classical conditioning; Cerebellum; Extinction; Superior cerebellar peduncle; Associative learning
1. Introduction Neuronal circuits that control the acquisition of classically conditioned eyeblinks in rabbits are among the best described neural substrates of associative learning in mammals. The expression and acquisition of classically conditioned eyeblinks are controlled by brainstem eyeblink circuits and by the intermediate cerebellum. Significant progress is being made in delineating the possible sites of plasticity that underlie information storage in this paradigm [3,4,7]. In contrast to advances made in understanding the expression and acquisition of conditioned eyeblink * Corresponding author. Fax: +1 515 294 2315. E-mail address: [email protected] (V. Bracha). URL: http://www.vetmed.iastate.edu/faculty_staff/users/vbracha/ (V. Bracha). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.03.015
responses, relatively little is known about the process of extinction. In the classical conditioning paradigm, animals are repeatedly exposed to an irrelevant stimulus (conditioned stimulus—CS) that is predictably followed by a biologically significant stimulus (unconditioned stimulus—US). In these conditions, the animal learns to produce a new response (conditioned response—CR) to the CS. Interestingly, the animal can extinguish CRs if the CS ceases to signal the US. This process is studied in the extinction paradigm whereby the subject is exposed to the CS without US reinforcement. In this condition, the frequency of CRs gradually decreases until they disappear. The CR is said to be extinguished. Are the same neural networks and the same, perhaps reversible, neural processes involved in both CR acquisition and extinction? A pivotal part of CR acquisition circuits is the intermediate cerebellum. Does the intermediate cerebellum
176
W.U. Nilaweera et al. / Brain Research 1045 (2005) 175 – 184
play the same role in CR extinction? Perrett and Mauk [25] reported that lesions of the cerebellar anterior lobe prevent extinction of short-latency eyeblink CRs in the rabbit. Since the disinhibition of the contralateral inferior olive following picrotoxin microinjections also prevented CR extinction [21] and because picrotoxin applied to the interposed cerebellar nuclei produced short-latency eyeblink CRs in previously extinguished animals [20], Medina et al. [21] proposed that a reciprocal plastic process at the same set of cerebellar synapses is involved in bi-directional learning during the acquisition and extinction of eyeblink CRs. A similar proposal was advanced by Ramnani and Yeo [26], who found that inactivating the cerebellar interposed nuclei prevents extinction. Since this result parallels reports of acquisition deficits induced by the same treatment [13,15,17], these authors concluded that both the acquisition and extinction of eyeblink CRs are dependent on cerebellar mechanisms. Inactivating the IN with GABA-A agonist muscimol produces local and remote physiological effects. The local effect is hyperpolarization and an associated block of IN neuronal firing [2]. The remote effect is on targets of IN projections that are prevented from receiving normal afferent information from the cerebellum. Both of these effects could potentially explain the disruption of CR extinction in animals with inactivations of the IN. To examine the role of remote effects, we temporarily inactivated the brachium conjunctivum (BC) in rabbits during extinction sessions. This procedure did not directly affect IN neurons, but it blocked the axons of IN cells. Here, we report that blocking the BC disrupts the normal extinction of CRs. This finding demonstrates that interrupting the information flow from the IN is sufficient for preventing CR extinction.
2. Materials and methods 2.1. Subjects Fourteen male New Zealand White rabbits (Harlan, Indianapolis, Indiana), weighing 2.5 –3.5 kg at the time of surgery, were housed individually on a 12/12 h light/dark cycle. All experiments were performed in accordance with the NIH ‘‘Principles of Laboratory Animal Care’’ (NIH publication No. 86-23, revised 1985) and the protocol approved by the Institutional Animal Care and Use Committee of Iowa State University. 2.2. Surgery Before any training, all rabbits were surgically implanted with a guide cannula (28-gauge stainless steel) aimed 1 mm dorsal to the left BC using sterile surgical techniques. Each rabbit was anesthetized with a mixture of Ketamine (50 mg/ kg), Xylazine (6 mg/kg), and Acepromazine (1.5 mg/kg).
During surgery, lambda was positioned 1.5 mm ventral to bregma, and the following stereotaxic coordinates were used: 12 –13 mm posterior, 2.8 mm lateral, and 14.2 mm ventral from bregma. A small Delrin head stage was mounted to the skull to accommodate the US delivery system and the eyeblink recording sensor. The implant and head stage were secured with anchoring screws and dental acrylic. The patency of the injection cannula was protected between experiments with a stainless steel stylet. The cannula was also protected with a removable lightweight Delrin cap. All animals were treated with antibiotics for 5 days after surgery and had a 1 week recovery period before experiments started. 2.3. Injection techniques Trained rabbits were tested in experiments in which the area of the BC was microinjected with solutions of tetrodotoxin (TTX, a potent and long-lasting sodium channel blocker, Calbiochem, 1 ng/Al = 3.1 pmol/Al), muscimol (GABA-A agonist, MP Biochemicals, 400 ng/Al = 3.5 nmol/Al), and 4% lidocaine (short-lasting sodium channel blocker). TTX and muscimol were dissolved in phosphate-buffered saline (PBS, pH 7.4). Injections of PBS were used in control experiments. All injections were delivered through a 33-gauge stainless steel needle inserted in the implanted guide tube. The injection needle was connected to a 10 Al Hamilton syringe (Hamilton Company, Reno, Nevada) using transparent Tygon tubing. The injected volume was measured by observing the movement of a small air bubble relative to gradation marks on the tubing connecting the needle to the syringe. Only one drug was injected on any given experimental day. Lidocaine was used for the functional localization of optimal injection sites. Other drug injections were performed at depths where lidocaine abolished CR expression. Muscimol was used for the axon-sparing local inactivation of neurons. TTX was used for inactivating the BC during extinction. All drugs were applied at the rate of 0.75 Al/min. For additional details of the drug injection protocol, see the corresponding sections of the experiment below. 2.4. Experiment outline The experiment consisted of 5 parts (Fig. 1). During the preparation phase, implanted rabbits were conditioned. Trained animals then received lidocaine injections targeting the BC at variable depths to determine an optimal injection site (CR expression abolished). Once the injection site was determined, Extinction Phase I began. Rabbits were injected with either TTX or with PBS before each experiment and trained for 4 days in the extinction paradigm. In Extinction Phase II, drug injections were discontinued and animals were trained in the extinction paradigm for 4 more days. Extinction Phase II was followed by re-acquisition and performance tests (2 days). In the final stage of the
W.U. Nilaweera et al. / Brain Research 1045 (2005) 175 – 184
177
injection site was determined, it was followed by a TTX test designed to establish the duration of TTX inactivation of the BC. Each rabbit was injected with 1.5 Al of TTX and presented with CS + US trials until CRs recovered. The general effect of this test TTX injection varied based on the specific location of the injection site. In 3 of the 7 animals in the experimental group, the initial dose of TTX not only abolished CRs, but also produced ophthalmic and postural side effects. To reduce these side effects while preserving CR abolition, the TTX dose was reduced to 0.75 Al in 2 rabbits and to 1.1 Al in one animal. In all animals, the abolition of CRs lasted 105 – 156 min. This duration of BC inactivation reliably exceeded the duration of the individual extinction sessions in the next stage of the experiment. Since the duration of the TTX effect averaged 120 min, the test sessions utilized groups of 5 trials alternating with 5 min pauses in stimulation. The purpose of this arrangement was to reduce stress to animals imposed by the large number of trials presented during a prolonged period of CR abolition. Fig. 1. Summary of the experiment and a brief description of procedures performed at each phase of the experiment. BC, brachium conjunctivum; CS, conditioned stimulus; PBS, phosphate-buffered saline; TTX, tetrodotoxin.
experiment, re-trained rabbits were injected with muscimol to test for effects of axon-sparing inactivation on CR expression. 2.4.1. Training Prior to any training, all rabbits were adapted to a standard rabbit restraining box (Plas Labs, Inc, Lansing, Michigan) and the experimental environment for 3 days, 30 min/day. After adaptation, they were conditioned in the standard delay classical conditioning paradigm. An 80 dB, 1 kHz, 450 ms tone was used as the CS and was superimposed on continuous 70 dB white noise. The US was a 100 ms, 210 kPa (at the source) air puff aimed to the caudal portion of the left cornea. The US co-terminated with the CS. The inter-stimulus interval was 350 ms. The inter-trial interval varied pseudorandomly between 15 and 25 s. Each experiment consisted of 100 paired-trial (CS + US) presentations, and the rabbits were trained until they had 90% CRs in 3 consecutive sessions. 2.4.2. Localizing the BC with lidocaine and TTX tests Before extinction experiments, the BC region was mapped with lidocaine injections to determine the optimal injection site. Rabbits were injected with 4 Al of lidocaine, starting 1 mm above the expected depth of the BC. In these experiments, the injection needle was inserted in the guide tube and 40 CS + US trials were delivered. Next, lidocaine was injected and the training was immediately resumed and continued until CRs recovered (60 to 80 trials.) If CRs were not abolished, the injection was made 0.5 mm deeper the next day. This continued until CRs were abolished for at least 6 min in any given experiment. After the optimal
2.4.3. Extinction Phase I After the optimal depth and TTX effect period were determined, 7 rabbits were injected with TTX (0.75 – 1.5 Al, see previous section) for 4 days at the beginning of each experiment. To maximize BC inactivation, extinction training began 20 min following the injection. Rabbits were then presented with 100 CS-alone trials per day. The control group (6 rabbits) underwent the same procedure as the TTX group but received PBS injections (1.5 Al). 2.4.4. Extinction test and Extinction Phase II After Extinction Phase I, rabbits had a 2-day rest during which no training was conducted and no injections were administered. The purpose of this pause was to prevent contamination of further tests by any possible lingering effects of TTX on CR performance. Following the rest period, all rabbits were trained for 4 additional days in extinction sessions consisting of 100 CS-alone presentations. No injections were administered on these days. The purpose of Extinction Phase II was to measure the degree of extinction achieved by the experimental group in the previous TTX sessions and to test the ability of the rabbits to extinguish CRs in normal conditions without injections. 2.4.5. Re-training and muscimol injections After extinction training, rabbits were re-trained with paired CS + US trials for 2 days to examine whether previous treatments affected the ability to re-acquire and express eyeblink CRs. Following CR re-acquisition, all animals were injected with 1.5 Al of muscimol to examine whether the effects of previous TTX injections on CR performance could be attributed to the inactivation of cell bodies located near injection sites. The muscimol test consisted of 40 trials before injection, a 20 min waiting period following the injection (comparable to that during the TTX injections), and then 60 additional trials.
178
W.U. Nilaweera et al. / Brain Research 1045 (2005) 175 – 184
2.5. Data acquisition and analysis Eyeblink recording was performed using techniques described previously [5]. Before each experiment, a small loop was attached to the upper eyelid and was coupled to a low friction electromechanical lever sensor to track the movements of the upper eyelid. The output of the sensor was amplified, digitized at 1 kHz with 12 bit resolution, and displayed and stored on a custom data acquisition system. Data were acquired for 1400 ms in each trial, starting 250 ms before CS onset. The data acquisition program measured alpha responses, CRs, and URs. Baseline eyelid movements exceeding 0.25 mm were recognized as spontaneous blinks and any blink exceeding CR threshold within 80 ms after CS onset was recognized as an alpha response. Trials containing spontaneous blinks and/or rare alpha responses were stored but were not included in further data analyses. Closure of the upper eyelid more than 0.1 mm after the alpha period and before the onset of the US was considered a CR in paired trials, and any blink that exceeded this threshold after the alpha response window was considered a CR in extinction trials. This relatively low threshold was selected to capture the small responses that are frequently expressed at later stages of extinction. In addition to the visualization of eyeblink traces on the computer monitor, the animal’s behavior was monitored using 2 digital video cameras. One camera provided a front view of the rabbit’s head and the other one was positioned on the side of the trained eye. This setup was used to monitor the general behavior of animals and was valuable in detecting drug injection side effects. Measurements of eyeblink responses were used for calculations of the CR incidence per session and also per block of 20 trials in each session. Data from individual animals were pooled together and statistically analyzed. Extinction days and 20-trial blocks were treated as repeated measures factors. Planned contrasts from repeated measures ANOVA were used to address hypotheses regarding patterns of CR incidence emerging from the interaction between treatments and extinction days and/or 20-trial blocks. Specifically, we contrasted days 1 and 4 from control Extinction Phase I with days 1 and 4 from TTX group Extinction Phase II. Furthermore, within Extinction Phase II, we contrasted day 1 of the control and TTX groups. A separate repeated measures ANOVA generated similar output for the analysis of effects of muscimol. All group data in the Results section represent group means T standard error of the mean. All calculations were performed using StatSoft Statistica software. 2.6. Histology After completing experiments, animals were deeply anesthetized, and the injection sites were marked by injecting 1 Al of tissue marking dye. The animals were
transcardially perfused with 1 l of PBS followed by 1 l of fixative (10% buffered formalin). Each perfused brain was removed from the skull and, following a post-fixation in 30% sucrose formalin, sectioned coronally at 50 Am on a freezing microtome. Sections were mounted onto gelatincoated slides and then stained with neutral red and Luxol fast blue.
3. Results 3.1. Location of injection sites The histological reconstruction of tracks produced by injection needles and deposits of tissue-marking dye revealed that, in all TTX and control animals, the injections were performed in the close vicinity of the BC (Fig. 2).
Fig. 2. Location of injection sites for TTX (black dots) and control (black stars) rabbits. All injection sites were found close to the BC, and the rostro-caudal spread was 1.5 mm. The depicted sections represent coronal sections through the region of interest and are spaced 0.5 mm apart with panel (A) being the most caudal and panel (D) the most rostral. The 2 white stars indicate the injection placement in rabbits that did not have sufficient CR abolition during the lidocaine test, and the dotted circle in panel (C) indicates the extent of BC damage in the rabbit whose CRs did not recover from the lidocaine injection. These 3 animals were excluded from the data analysis. IV, fourth ventricle; BC, brachium conjunctivum; IC, inferior colliculus; ll, lateral leminiscus; Mo5, motor trigeminal nucleus; MCP, middle cerebellar peduncle; Me5, mesencephalic trigeminal nucleus; NRTP, nucleus reticularis tegmenti pontis; PN, pontine nuclei; py, pyramidal tract; S5, sensory trigeminal nucleus; s5, root of the sensory trigeminal nerve; SC, superior colliculus; TN, trochlear nucleus; tz, trapezoid body; Tz, nucleus of the trapezoid body; VTg, ventral tegmental nucleus.
W.U. Nilaweera et al. / Brain Research 1045 (2005) 175 – 184
In most animals, injection sites were positioned just dorsal to the BC at the anterior –posterior level of 0 to 1 mm caudal to the caudal pole of the ventral tegmental nucleus.
179
93.0 T 3.1, 18.5 T 6.7, and 92.5 T 5.2% in controls (n = 4) (see Fig. 3). The histological analysis (Fig. 2) in conjunction with lidocaine mapping tests revealed that lidocaine failed to abolish CRs when injected at distances larger then 1 mm from the BC.
3.2. General observations 3.3. Effects of TTX on CR expression and TTX side effects Eleven of the 14 implanted rabbits were included in the results analysis. One animal was excluded because the BC was lesioned during the lidocaine test and lost the capacity to express CRs. Two additional rabbits designated as controls were also excluded since lidocaine test injections did not completely abolish the expression of CRs. Thus, 7 animals were included in the experimental TTX group and 4 animals in the control group. During the initial training, all rabbits reached the performance criterion (90% CRs for 3 consecutive days) in 5 training sessions on average. All trained animals were then injected with 4 Al of lidocaine in a functional mapping test to find an optimal location for TTX and PBS injections. A particular location was considered optimal when lidocaine abolished CRs for at least 6 min. A relatively short abolition criterion of 6 min was chosen to prevent advancing the injection needle too deep, thereby penetrating the BC with the injection needle, producing a permanent lesion to this structure resulting in a permanent CR deficit. The effect of lidocaine on CRs lasted between 6 and 23 min (mean 13.7 T 1.54 min, n = 11). Mean CR incidences before injection, after injection, and during recovery were 92.3 T 3.6, 4.4 T 1.7, and 80.0 T 6.4% respectively in the TTX group (n = 7) and
Once effective injection sites were determined in all rabbits, the TTX group received a TTX injection to examine the time period of CR abolition. Rabbits were injected with TTX at the beginning of the experiment and then presented with paired stimuli to observe the onset and duration of effect. Confirming previous studies [14], TTX abolished CR expression. This effect was immediate in all rabbits, and the duration ranged between 105 and 156 min (mean of 127.4 T 7.82 min, n = 7), confirming that the BC would be sufficiently inactivated for the 60 min duration of the planned extinction session. In addition to CR abolition, TTX injections in the BC elicited several notable side effects. The most frequent was unusual calmness in the restraining box. This was associated with a wide opening of the ipsilateral eyelids, and frequently with mild ipsilateral exophthalmus. The effects of BC inactivation were not restricted to the ipsilateral eye. Relatively frequently, animals lowered their heads and rotated it in the horizontal plane to the left side in addition to lowering their ipsilateral (left) ear. This is consistent with a generalized decrease of muscle tone in the ipsilateral face, head, and neck. Much less frequent and notably delayed was the induction of
Fig. 3. Summary of conditioned response incidence (means T SEM) in the experimental (TTX) and control (PBS) groups during all 5 phases of the experiment. All rabbits exhibited asymptotic CR incidence during the last 3 days of training. Lidocaine abolished CR incidence in both groups in the post-injection period (Post) and then these responses recovered (Recovery). During Extinction Phase I, the experimental group did not express CRs due to BC inactivation by TTX. At the same time, conditioned responses gradually extinguished in control animals. During Extinction Phase II, CR incidence in the control group was lower than in the TTX group. Most notably, the TTX group exhibited an extinction curve that resembled the extinction of CRs in the control group during Phase I, indicating that TTX during Phase I prevented normal extinction in these rabbits. Following Phase II, both groups were successfully re-trained. Finally, injections of muscimol failed to abolish CR incidence in either of the groups.
180
W.U. Nilaweera et al. / Brain Research 1045 (2005) 175 – 184
bilateral ocular nystagmus that was sometimes accompanied by synchronized oscillatory movements of the head. 3.4. Effects of BC inactivation on CR extinction Inactivating the BC with TTX completely abolished the expression of CRs for the duration of each Extinction Phase I session (Fig. 3). At the same time, control animals exhibited a typical decrease of CR frequency both in the individual extinction sessions (Fig. 4) as well as in the sequence of the 4 extinction sessions (Figs. 3 and 4). On the first day of Extinction Phase II (extinction with no injections), animals from the TTX group exhibited a large number of CRs that became less frequent toward the end of the session (Fig. 5). This behavior contrasted with the control group that expressed only a few scattered small-amplitude CRs (Fig. 5). At the group level, on the first day of Extinction Phase II, the TTX group exhibited 37.6 T 4.5% CRs. This level of responding was significantly different from the control group that demonstrated only 6.8 T 1.3% CRs ( F 1,9 = 26.631, P = 0.00060), and it was indistinguishable from the frequency of CRs the control group exhibited on the first day of Extinction Phase I (37.7 T 8.0%) ( F 1,9 = 0.00244, P = 0.96165). The high frequency of CRs exhibited by the TTX group on day 1 of Extinction Phase II clearly demonstrated that BC inactivation during Extinction Phase I disrupted normal CR extinction. During continued extinction training in the absence of any injections, CR frequency in the TTX group gradually decreased at a rate that was similar to the extinction rate of the control group in Extinction Phase I (Figs. 3 and 4; Fig 4 shows overlaid extinction curves of TTX Phase II
and control Phase I). On the fourth day of Extinction Phase II, the TTX group exhibited 13.1 T 3.5% CRs. This level of performance was nearly indistinguishable from controls on day 4 of Extinction Phase I (15.2 T 2.8% CRs) ( F 1,9 = 0.15050, P = 0.70710). Besides the similar aggregate daily frequency of CRs in the TTX group during Extinction Phase II and in the control group during Extinction Phase I, both groups exhibited a remarkably similar and statistically indistinguishable decline of CR frequency within individual sessions in corresponding phases of the experiment (Fig. 4, F 28,252 = 0.65457, P = 0.91028). These results demonstrated that BC inactivation prevented normal CR extinction and also that the BC inactivation using local injections did not produce a long-lasting dysfunction of the extinction circuits, since once the inactivation was discontinued, the extinction process occurred at a normal rate. Following Extinction Phase II, both groups were retrained in the paired CS + US paradigm for 2 sessions. Since both groups quickly re-acquired high levels of CRs comparable to pre-extinction performance (Fig. 3), it can be concluded that the injections of TTX did not alter the functionality of the circuits that are involved in normal re-acquisition and expression of eyeblink CRs. In the final experiment, all rabbits were injected with muscimol at the injection sites used for the TTX and PBS injections. The muscimol injections, which should have inactivated GABA-A receptor-containing neurons nearest the infusion sites, slightly decreased CR incidence but did not abolish CR expression (Fig. 3). These results suggest that the abolition of CRs following TTX injections was likely due to inactivating axons rather than cell bodies. Notably, there were no
Fig. 4. Detailed comparison of CR incidence (means T SEM) in Extinction Phase II of the TTX group and Extinction Phase I of the PBS group. Each session is represented by five 20-trial blocks. Note the gradual decline of CR incidence within each session and across sessions. This behavior is typical for normal extinction of eyeblink CRs. Also note that the extinction curves of both groups largely overlap. This indicates that the TTX group failed to extinguish during the prior 4 sessions that were conducted while the BC was inactivated with TTX.
W.U. Nilaweera et al. / Brain Research 1045 (2005) 175 – 184
181
Fig. 5. Examples of eyeblink mechanograms in a TTX and a control (PBS) rabbit during the last day of training (A,C) and during the extinction test (the first day of Extinction Phase II; B,D). Each stack plot represents a complete printout from a single training session and each trace represents the upper eyelid mechanogram for one trial. Vertical lines in each plot denote onsets of the CS and US. Upward deflections in mechanograms indicate eyelid closure. Eyelid closure between the CS and US markers in panels (A) and (C) are CRs. All eyelid closures following the CS marker in panels (B) and (D) are considered CRs. Trials are stacked with the first trial on the top and the last trial at the bottom of the plot. On the last day of pre-injection training (A,C), both rabbits expressed comparable and robust levels of CR performance. The animal that was injected with TTX during extinction training showed no signs of extinction, since it expressed a number of wellformed CRs at the beginning of the extinction test (B). In contrast to the TTX animal, CRs in the control rabbit extinguished during Phase I, as evidenced by the low number of CRs exhibited during the extinction test (D).
prominent side effects in the muscimol post-injection period, other than increased calmness, which was also observed following both lidocaine and TTX experiments. In several cases, though, rabbits tended to locomote in clockwise circles when taken out of the restraining box and put on the floor following the experiment.
4. Discussion The present experiments demonstrated that unilateral inactivation of the BC during extinction sessions blocked eyeblink CRs. BC inactivation, besides abolishing CR expression, also prevented normal extinction of CRs. These findings are the first to show that normal activity
182
W.U. Nilaweera et al. / Brain Research 1045 (2005) 175 – 184
of the BC is required for the extinction of eyeblink CRs in the rabbit.
both the CR performance and extinction effects observed in the present study are attributable to BC inactivation.
4.1. Specificity of BC inactivation
4.2. The role of the BC in circuits controlling the extinction of conditioned eyeblinks
The present study inactivated cerebellar output projections located in the BC using the sodium channel blocker, TTX. The BC is a fiber tract that contains axons originating in the deep cerebellar nuclei, including axons sent by the interposed nuclei. TTX blocks the generation of action potentials in cell bodies and also prevents the propagation of action potentials in neuronal axons. Microinjections of TTX in the present study blocked the expression of CRs. This confirms previous reports of the involvement of the BC in the execution of previously learned CRs [14]. More importantly, we found that TTX microinjections prevented CR extinction. Do these effects of TTX relate specifically to BC inactivation? Attributing the observed effects to the function of the BC requires eliminating the possibilities that the drug (a) did not spread to some other remote part or parts of the network that controls CRs; (b) did not inactivate CR-related cell bodies at the site of injection; and (c) did not inactivate some other CR-related fiber tracts at the injection site. To discuss these possibilities, the distance of the effective spread of TTX when injected in brain tissue should be considered. Zhuravin and Bures [27] conducted a functional test of effective TTX diffusion in the rat brain. They reported that 10 ng of TTX (6 to 13 times more than was injected in the present study) inactivated cells located up to 1.5 mm from the injection site. In our recent TTX diffusion study performed in the rabbit [24], we found that injections of 2 Al (2 ng total) of TTX abolished CRs only when injected 1 mm or closer to the BC. These data suggest that the amounts of TTX used in this study inactivate neural tissue in an area of less than 1.5 mm around the injection site. Considering this spread and the location of injections in the present study (Fig. 2), one can conclude that TTX either completely or near completely inactivated the BC. On the other hand, it is unlikely that TTX inactivated some of the known neighboring components of the eyeblink network, such as the cerebellar interposed nuclei, the red nucleus, pontine nuclei, the middle cerebellar peduncle, or the lateral lemniscus. All of these structures are located beyond the estimated spread of TTX. It is possible, however, that in addition to the BC, TTX inactivated some not yet known structures (either nuclear or axonal) located close to the injection sites. Since muscimol injection tests failed to abolish CR expression, it seems that the observed effects on CR expression were not due to inactivating cell bodies containing GABA-A receptors. This finding suggests that the effects of TTX on CR expression were most likely related to inactivating axons close to the injection site. Although the present experiment cannot exclude the possibility of TTX inactivating some as yet unknown CR-related fiber tracts proximate to the BC, we base our discussion on the hypothesis that
Previous experiments established that inactivating the cerebellar IN prevents the extinction of eyeblink CRs [26]. There are two interpretations of this finding. Specifically, one could speculate that inactivating the IN blocks an extinction-related and hyperpolarization-sensitive plastic process directly in the IN. An alternative explanation would be that inactivating the IN interrupts the supply of essential information to targets of IN projections. Results of the present experiment shed new light on this issue. Since we found that inactivating the BC without directly interfering with IN neuronal activity prevents CR extinction, it is clear that extinction depends on information supplied by deep cerebellar nuclei to efferent targets involved in CR extinction. A possible region for putative plastic components of eyeblink extinction neural circuits is the brainstem in the vicinity of the facial and accessory abducens nuclei. In a recent study, Krupa and Thompson [16] reported that inactivating this brainstem region prevents the extinction of CRs. It is possible that inactivating the BC upsets the extinction-related process at this site. Incidentally, changes in the excitability of eyeblink motoneurons were previously reported to be associated with the acquisition of eyeblink CRs in the cat [19]. Eyeblink motoneurons and premotoneurons could be affected by the IN either directly or indirectly through interposito-rubral projections [11,22]. Other candidates for the BC-efferent, extinction-related site of plasticity could be cerebellar afferent structures or, paradoxically, the cerebellum itself. It is known that the intermediate cerebellum is part of a recurrent, feedback-rich, neural network [4]. Some of the cerebellar output projections are directed to sites that are major suppliers of afferent information to the cerebellum. Thus, cerebellar output can regulate cerebellar input sources. The possible involvement of cerebellar nuclei and/or cerebellar afferents in extinction is supported by the results of Gould and Steinmetz [12], who showed that extinction of eyeblink responses is accompanied by a decrease of task-related neuronal activity in the interposed nuclei. More specifically, Medina et al. [21] suggested that the cerebello-inferior olivary-cerebellar feedback loop could be involved both in CR acquisition and extinction. The results of the present study are in agreement with this notion since inactivating the BC blocks cerebellar inhibitory projections to the inferior olive, and this is likely to produce an aggregate increase of inferior olivary firing. This in turn might prevent extinction-related learning in the cerebellar cortex and/or nuclei. Besides the cerebelloinferior olivary-cerebellar feedback system, other feedback loops, such as the cerebello-pontine nuclear cerebellar system, could be involved in regulating eyeblink-related
W.U. Nilaweera et al. / Brain Research 1045 (2005) 175 – 184
circuits in the intermediate cerebellum. Cerebellar control of cerebellar input sources in the rabbit was documented in previous studies demonstrating that inactivating the IN or the red nucleus affects neuronal activity in the pontine nuclei [6,9] and in the trigeminal nuclei [8]. Furthermore, involvement of cerebellar feedback loops in regulating cerebellar activity is supported by the finding that inactivating the BC with lidocaine affects the activity of IN neurons [23]. 4.3. CR acquisition vs. CR extinction Behavioral experiments suggest that the extinction of CRs is not a simple reversal of CR acquisition. It is believed that, during CR extinction, memory traces of CRs are not erased, but suppressed. If so, one could assume that either different neural networks or at least different physiological processes within the same neural network control CR acquisition and extinction. Several studies have documented that, indeed, some of the components of extinction circuits differ from those that govern CR acquisition. For example, inactivating the region of the ipsilateral facial/accessory abducens nuclei does not affect CR acquisition [10,18] but disrupts CR extinction [16]. Similar to medullar motoneuronal areas, lesions of the hippocampus do not prevent CR acquisition, but they seem either to prevent or at least significantly slow down CR extinction in well-trained rabbits [1]. The present study revealed an additional difference between substrates that control CR acquisition and extinction. Krupa and Thompson [14] reported that inactivating the BC with small injections of TTX did not prevent CR acquisition. Their finding contrasts with the results of the present study showing that BC inactivations block CR extinction. This finding is interesting when one considers that both acquisition and extinction are affected by inactivating the IN [15,26]. It appears that, while both of these processes depend on the intermediate cerebellum, only CR extinction requires the normal function of axons conveying information to cerebellar targets. The present results do not provide definite conclusions regarding the sites of plasticity for CR extinction since inactivating the BC could potentially block three qualitatively different types of processes: inactivating axons of cerebellar nuclear neurons could block the transmission of learning-specific information to cerebellar efferent targets, it could produce a nonspecific malfunction of cerebellar efferent targets due to the loss of tonic cerebellar input, and it could also interrupt cerebellar plastic changes due to blocking cerebellar feedback pathways. Dissociating these possibilities presents a challenge to future studies of classically conditioned eyeblink extinction.
Acknowledgments The authors would like to thank Mike Hord for assistance with the experimental set up, Kari Teeter for assistance with
183
histology, and Kristina Irwin for assistance with manuscript preparation. This research was supported by NIH grants R01 NS36210 and R01 NS21958.
References [1] E. Akase, D.L. Alkon, J.F. Disterhoft, Hippocampal lesions impair memory of short-delay conditioned eye blink in rabbits, Behav. Neurosci. 103 (1989) 935 – 943. [2] D.P. Aksenov, N.A. Serdyukova, J.R. Bloedel, V. Bracha, Glutamate neurotransmission in the cerebellar interposed nuclei: involvement in classically conditioned eyeblinks and neuronal activity, J. Neurophysiol. 93 (2005) 44 – 52. [3] P.J. Attwell, M. Ivarsson, L. Millar, C.H. Yeo, Cerebellar mechanisms in eyeblink conditioning, Ann. N. Y. Acad. Sci. 978 (2002) 79 – 92. [4] V. Bracha, J.R. Bloedel, The multiple pathway model of circuits subserving the classical conditioning of withdrawal reflexes, in: J.R. Bloedel, T.J. Ebner, S.P. Wise (Eds.), Acquisition of Motor Behavior in Vertebrates. MIT Press, Cambridge, MA, 1996, pp. 175 – 204. [5] V. Bracha, L. Zhao, K. Irwin, J.R. Bloedel, Intermediate cerebellum and conditioned eyeblinks: parallel involvement in eyeblinks and tonic eyelid closure, Exp. Brain Res. 136 (2001) 41 – 49. [6] M.C. Cartford, E.B. Gohl, M. Singson, D.G. Lavond, The effects of reversible inactivation of the red nucleus on learning-related and auditory-evoked unit activity in the pontine nuclei of classically conditioned rabbits, Learn. Mem. 3 (1997) 519 – 531. [7] K.M. Christian, R.F. Thompson, Neural substrates of eyeblink conditioning: acquisition and retention, Learn. Mem. 10 (2003) 427 – 455. [8] R.E. Clark, D.G. Lavond, Neural unit activity in the trigeminal complex with interpositus or red nucleus inactivation during classical eyeblink conditioning, Behav. Neurosci. 110 (1996) 13 – 21. [9] R.E. Clark, E.B. Gohl, D.G. Lavond, The learning-related activity that develops in the pontine nuclei during classical eye-blink conditioning is dependent on the interpositus nucleus, Learn. Mem. 3 (1997) 532 – 544. [10] R.E. Clark, A.A. Zhang, D.G. Lavond, The importance of cerebellar cortex and facial nucleus in acquisition and retention of eyeblink/NM conditioning: evidence for critical unilateral regulation of the conditioned response, Neurobiol. Learn. Mem. 67 (1997) 96 – 111. [11] K.D. Davis, J.O. Dostrovsky, Modulatory influences of red nucleus stimulation on the somatosensory responses of cat trigeminal subnucleus oralis neurons, Exp. Neurol. 91 (1986) 80 – 101. [12] T.J. Gould, J.E. Steinmetz, Changes in rabbit cerebellar cortical and interpositus nucleus activity during acquisition, extinction, and backward classical eyelid conditioning, Neurobiol. Learn. Mem. 65 (1996) 17 – 34. [13] M.J. Hardiman, N. Ramnani, C.H. Yeo, Reversible inactivations of the cerebellum with muscimol prevent the acquisition and extinction of conditioned nictitating membrane responses in the rabbit, Exp. Brain Res. 110 (1996) 235 – 247. [14] D.J. Krupa, R.F. Thompson, Inactivation of the superior cerebellar peduncle blocks expression but not acquisition of the rabbit’s classically conditioned eye-blink response, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 5097 – 5101. [15] D.J. Krupa, R.F. Thompson, Reversible inactivation of the cerebellar interpositus nucleus completely prevents acquisition of the classically conditioned eye-blink response, Learn. Mem. 3 (1997) 545 – 556. [16] D.J. Krupa, R.F. Thompson, Inhibiting the expression of a classically conditioned behavior prevents its extinction, J. Neurosci. 23 (2003) 10577 – 10584. [17] D.J. Krupa, J.K. Thompson, R.F. Thompson, Localization of a memory trace in the mammalian brain, Science 260 (1993) 989 – 991. [18] D.J. Krupa, J. Weng, R.F. Thompson, Inactivation of brainstem motor nuclei blocks expression but not acquisition of the rabbit’s classically conditioned eyeblink response, Behav. Neurosci. 110 (1996) 219 – 227.
184
W.U. Nilaweera et al. / Brain Research 1045 (2005) 175 – 184
[19] M. Matsumura, C.D. Woody, Long-term increases in excitability of facial motoneurons and other neurons in and near the facial nuclei after presentations of stimuli leading to acquisition of a Pavlovian conditioned facial movement, Neurosci. Res. 3 (1986) 568 – 589. [20] J.F. Medina, K.S. Garcia, M.D. Mauk, A mechanism for savings in the cerebellum, J. Neurosci. 21 (2001) 4081 – 4089. [21] J.F. Medina, W.L. Nores, M.D. Mauk, Inhibition of climbing fibres is a signal for the extinction of conditioned eyelid responses, Nature 416 (2002) 330 – 333. [22] S. Morcuende, J.M. Delgado-Garcia, G. Ugolini, Neuronal premotor networks involved in eyelid responses: retrograde transneuronal tracing with rabies virus from the orbicularis oculi muscle in the rat, J. Neurosci. 22 (2002) 8808 – 8818. [23] W.U. Nilaweera, K. Irwin, G.D. Zenitsky, D.P. Aksenov, V. Bracha, Are feedback loops involved in the cerebellar neuronal correlates of
[24]
[25]
[26]
[27]
conditioned eyeblinks? Abstract Viewer/Itinerary Planner, Society for Neuroscience, Washington, DC, 2002 (Program No. 79.6). W.U. Nilaweera, G.D. Zenitsky, K.B. Irwin, V. Bracha, Assessing diffusion of tetrodotoxin in the rabbit brain suppressing conditioned eyeblinks by brachium conjunctivum inactivation, Abstract Viewer/ Itinerary Planner, Society for Neuroscience, Washington, DC, 2004 (Program No. 136.4). S.P. Perrett, M.D. Mauk, Extinction of conditioned eyelid responses requires the anterior lobe of cerebellar cortex, J. Neurosci. 15 (1995) 2074 – 2080. N. Ramnani, C.H. Yeo, Reversible inactivations of the cerebellum prevent the extinction of conditioned nictitating membrane responses in rabbits, J. Physiol. 495 (Pt. 1) (1996) 159 – 168. I.A. Zhuravin, J. Bures, Extent of the tetrodotoxin induced blockade examined by pupillary paralysis elicited by intracerebral injection of the drug, Exp. Brain Res. 83 (1991) 687 – 690.