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Inactivation of the interpositus nucleus prevents transfer of the rabbit's classically conditioned eyeblink response from a light to a tone-conditioned stimulus

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Inactivation of the interpositus nucleus prevents transfer of the rabbit's classically conditioned eyeblink response from a light to a tone-conditioned stimulus

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Content INFORMATION TO USERS This manuscript has been reproduced from the microfiim master. UM! films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. 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INACTIVATION OF THE INTERPOSITUS NUCLEUS PREVENTS TRANSFER OF THE RABBIT’S CLASSICALLY CONDITIONED EYEBLINK RESPONSE FROM A LIGHT TO A TONE CONDITIONED STIMULUS by Benjamin David Cipriano A Thesis Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment o f the Requirements for the Degree MASTER OF ARTS (Behavioral Neuroscience) December 1998 Copyright 1998 Benjamin D. Cipriano Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 1394793 UMI Microform 1394793 Copyright 1999, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UNIVERSITY O F S O U T H E R N C A L IF O R N IA t h e o r a o u a t e s c h o o l UNIV ERSITY P A R R LOS A N S E L E S . C A L IF O R N IA * 0 0 0 7 T h is thesis, written by - j j k y . J f l ' T M . y 0 * —^ ___ _______ ___ under the direction of k jik Thesis C om m ittee, and approved by all its m embers, has been pre~ sented to and accented by the D ean o f T he G raduate School, in partial fu lfillm en t o f the requirem ent: fo r the degree of jjate September, 25 1998 THESIS COMMITTEE Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Inactivation of the Interpositus Nucleus Prevents Transfer o f the Rabbit's Classically Conditioned Eyeblink Response from a Light to a Tone Conditioned Stimulus Groups I and II received 7 sessions of light-airpuff training. Prior to session 8. group I was infused with 200ng muscimol. Groups II and III were infused with saline. Session 8: 120 tone-airpuff trials (20 interpolated light trials). Session 9: 30 tone alone trials. Session 10: identical to session 8 excluding infusion. Session 9’s results indicate that group I did not learn during session 8. Group II showed learning during session 8. The percentage of CRs performed by group II to tone during session 8 and 9 was significantly higher than the percentage performed by group III, demonstrating a strong transfer of learning. These results indicate that the cerebellum is essential for transfer training from one CS to a second, contradicting a report by Welsh and Harvey. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. T able of Contents • List of Figures page iii • Introduction--------------------------------- page 1 • Methods page 4 • Results page 8 • Discussion-------------------------------------page 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. List of Figures Figure 1.................................................... page Figure 2.................................................... page Figure 3.................................................... page Figure 4.................................................... page Figure 5.................................................... page Figure 6.................................................... page Figure 7.................................................... page Figure 8.................................................... page 21 22 23 24 25 26 27 28 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Introduction Extensive evidence based on lesions, neuronal recording, electrical stimulation, anatomical data, and behavioral procedures supports ihe hypothesis that the cerebellum and its associated circuitry is the essential neuronal system for classical conditioning of the eyeblink response, and, to the extent tested, other discrete skeletal muscle responses as well learned with an aversive unconditioned stimulus (US). The conditioned stimulus (CS) pathway includes the pontine- mossy fiber projections to the cerebellum, the US pathway inferior olive-climbing fiber projections to the cerebellum, and the conditioned response (CR) pathway includes the efferent projections from the cerebellar interpositus nucleus via the superior cerebellar peduncle to the magnocellular red nucleus and descending rubral pathways to premotor and motor nuclei (Hesslow & Ivarsson, 1996; Lavond et al„ 1985, 1992; McCormick & Thompson, 1984; McCormick et al„ 1985; Steinmetz et al., 1987; Thach et al„ 1992; Thompson & Krupa, 1994; Voneida et al., 1990; Yeo, 1991; Y eoet al., 1985). Recent evidence supports the further hypothesis that memory traces for this aspect of learning are formed in the cerebellum. Thus, there is unanimous agreement among studies using reversible inactivation during initial training that a localized region of the cerebellum is necessary for learning of the conditioned eyeblink response. Several methods of reversible inactivation (muscimol, baclofen, lidocaine, cooling) of portions of the anterior interpositus nucleus and overlying tissue of cortical lobule H VI during initial training result in prevention of learning of the conditioned response. As training continues after inactivation ceases, learning 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. develops with no savings, indicating that no learning at all occurred during training with activation (Clark el al., 1992; Hardiman « * t al., 1996; Krupa et al., 1993; Nordholm et al.. 1993: Ramirez et al., 1997). These same inactivations completely prevent expression of the CR in previously trained animals. Inactivation of the superior cerebellar peduncle (with TTX), magnocellular red nucleus, and relevant motor nuclei (7th and accessory 6th) similarly prevent expression of the CR in trained animals; however, animals learn normally when trained during inactivation of these regions, as evidenced by asymptotic learned performance from the beginning of post-inactivation training (Clark & Lavond, 1993; Krupa & Thompson, 1995; Krupa et al., 1996). Most recently, training was done with very low doses of muscimol (1.0 nmol) limited in spread to the dorsal anterior interpositus nucleus, with identical results: no evidence of any learning at all having occurred, that is, on the first few post-inactivation trials or in terms of number of subsequent trials to learning criterion (Krupa & Thompson, 1997). Collectively, these results indicate that the memory trace must be formed at or beyond the anterior interpositus/ cortical H VI but before the efferent projection of the interpositus, the superior peduncle, thus strongly supporting the hypothesis that the memory traces for this aspect of learning are formed and stored in a localized region of the cerebellum (Thompson & Krupa, 1994; Kim & Thompson, 1997). The one apparent exception to this literature is a study by Welsh and Harvey (1991) where already-trained animals (light CS) were subsequently trained with lidocaine inactivation of the interpositus nucleus to a tone CS. They reported that the animals learned to the tone CS during inactivation training, as evidenced in post inactivation testing and training. Use of lidocaine for reversible inactivation during Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. training is very difficult; unlike muscimol or baclofen, it acts on both neurons and axons and it has a very short duration of action, thus requiring continuous infusion. Using naive animals, Nordholm et al. (1993) found that continuous lidocaine infusion in the dorsal portion of the anterior interpositus nucleus during training completely prevented learning (results cited above) but infusion in the ventral portion of the anterior interpositus nucleus, while preventing expression of the CR, did not prevent learning (Welsh & Harvey result). It seems that the effective locus of action of lidocaine can be extremely limited (see also Nordholm, 1993). In order to resolve this issue, it is necessary to use the same behavioral and testing procedures used by Welsh and Harvey, but utilizing a more reliable and robust method of reversible inactivation, namely muscimol, which we report here. We also incorporate a control group to test for possible transfer of training effect (see, e.g., Schreurs & Kehoe, 1987) which Welsh and Harvey did not do. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Material and Methods Subjects. Data from nineteen New Zealand White rabbits, weighing between 2.2 and 2.4 kg at the time of surgery, are presented in this paper. The rabbits were housed in individual cages and maintained on a 12-hour light/dark cycle. Food and water were made available ad-lib when the animals were not being trained. Surgery. All surgeries were performed using aseptic techniques. Animals were anesthetized with a ketamine (60 mg/kg) and xylazine (8 mg/kg) mixture and maintained throughout the surgical procedure with halothane in a 1-3.5% concentration in oxygen. The rabbits were positioned with a standard rabbit stereotaxic head-holding device so that the skull surface at lambda was 1.5mm below bregma. All animals were implanted with chronic stainless steel guide cannulae (23mm in length) fitted with stainless steel stylettes projecting 2.0mm beyond the tip of the guide cannulae. The cannulae were lowered stereotaxically to position the stylette tip in the region of the left IP (from lambda: 0.5mm anterior, 5.0mm lateral, and 14.5mm ventral). Acrylic dental cement was used to hold the cannula, as well as a headstage with connections for subsequent behavioral training, in place. Suture loops were placed in the left nictitating membrane (NM) using .7 metric Ethilon black microfilament nylon. Further details concerning the cannula implantation have been published previously (Chapman, et al 1990). All animals were allowed at least seven days of post-surgery recovery prior to any behavioral training. General Procedures. During all training sessions, rabbits were placed in standard Plexiglas restrainers inside sound-attenuating conditioning chambers. All 4 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. rabbiis received a one hour adaptation session in the restrainer and conditioning chamber one day prior to any conditioning. For conditioning sessions the airpuff was delivered through tygon tubing from a gate pressurized to 3 pounds per square inch (2.1N/cm2 ; 100ms duration). The tubing nozzle was positioned approximately 1cm from the cornea of the left eye. A minitorque potentiometer connected to the suture loop in the rabbit’s NM recorded the eyeblink response. The external eyelids of the left eye were held open with clips to restrict interference with NM recording. Both the airpuff tubing and the potentiometer were attached to the headstage cemented in place during surgery. Two different CSs were used: tone (1 KHz, 85 decibels; 300ms duration), delivered from a speaker positioned directly in front of the rabbit’s head, and two light sources (two 6-watt incandescent bulbs; 300ms flash duration) positioned in front of and slightly to the sides of the rabbit’s head. Session 1-7: Light CS conditioning. Rabbits were randomly assigned to one of three groups. The day following the adaptation session, group I (n=7) and group II (n=8) underwent their first session of standard delay conditioning with light as the CS. The session consisted of 10 blocks of twelve light-airpuff pairings for a total of 120 pairings per session. The intertrial interval was randomized between 20 and 40 seconds. Groups I and II continued with one session per day for a total of seven days. Group HI (n=4) was restrained in the conditioning chamber for seven days following the adaptation session but was not exposed to any CS or US presentation. Session 8: Infusion and Tone CS conditioning. On the eighth session day, 60 minutes prior to conditioning, subjects in group I were infused with 200ng 5 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. muscimoi (lng/nl) using 25mm infusion cannulae inserted into the guide cannulae. Group I subjects were exposed to 10 light alone trials to assess for effective IP inactivation. Groups II and III were infused with saline to control for infusion effects. All three groups then underwent one session of tone-airpuff paired conditioning. The session consisted of ten blocks each containing 12 tone-airpuff pairings and 2 light-airpuff pairings to test for retention of the CR to light in groups I and II. All subjects received a total of 120 tone-air pairings and 20 light-air pairings on session day eight. Session 9: Tone alone test. Forty eight hours after session eight (muscimol’s effect wears off after 3-4 hours), all groups were exposed to 30 tone- alone trials to test for evidence of tone CR acquisition. Session JO: Tone CS conditioning. Immediately following the 30 tone- alone trials, all subjects received a second session of tone-airpuff conditioning (120 tone-airpuff, 20 light-airpuff). Histology. Following the conclusion of behavioral procedures, five animals from group I were infused with 200ng ^ H- muscimol to determine the pattern of diffusion. The animals were lightly anesthetized with halothane and decapitated immediately following the infusion. The brains were removed and immediately frozen. Coronal sections forty microns thick were taken on a cryostat. The slices were exposed to photographic film which, following development, was compared to slices to determine the extent of diffusion. The rest of the animals were overdosed with sodium pentobarbital following marking lesions for determination of cannulae placement. The animals were then perfused transcardially with 10% saline followed by 10% formalin. The brains were removed and stored in 10% 6 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. formalin. Frozen coronal sections were sliced at a thickness of 40 microns. Slices were mounted onto slides and stained with Prussian blue and cresyl violet stains. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Results By the seventh day of paired light training, subjects in group I and II had ail shown significant signs of learning (p< .001), responding with a well-timed CR on an average of 78.7% +-8.6 and 74.1% +-9.0 of the trials respectively (see Fig. 1). A similar pattern of acquisition was shown by an increase in the amplitude of the conditioned eyeblink across sessions for both groups (see Fig. 2). Both groups showed the same level of acquisition of the light CR (p= .902). During the eighth session of training (see Fig. 3 and 4), the muscimol infusion administered to group I successfully blocked CRs to the paired tone and the interpolated light trials. Group II produced CRs to tone on 45.4% +-7.6 of the tone trials during session 8. In contrast, group III (no pre-exposure to paired light training) responded with 4.9% +-2.3 CRs. Clearly, group II is showing a transfer of learning from the earlier light-airpuff training, as evidenced by performance significantly higher than group Eli (p< .001; Fig. 3). Group II, on average, reached learning criteria (8 CRs out of nine consecutive trials) in 54 trials, whereas group m did not reach criteria. Further evidence of transfer is provided by comparing the performance of group II on session 1 (light CRs) to its performance on session 8 (tone CRs); the percentage of CRs shown on the first exposure to tone is significantly higher than the percentage to light (p< .001). When comparing group II on session 1 to group III on session eight (first paired training for both groups), no significant difference is found. During session 9,48 hours later (see Fig. 5), group II responded to the 30 tone alone trials with the highest percentage of CRs, 52.8%+- 7.6, followed by group I with 17.7%+-6.9, and group IE with 10.0%+- 5.0. Analysis of variance 8 permission of the copyright owner. Further reproduction prohibited without permission. revealed differences between the groups (p< .005), followed by Dunnet’s test for treatment mean minus control mean (family error rate = .05) which showed group II significantly higher than the statistically indistinguishable groups I and HI. The percentage of CRs performed by group I indicates a degree of transfer of learning from sessions 1-7, but conspicuously lacks the high level of response that effective transfer on session 8 would produce. The level of response from group I to the 30 tone alone trials suggests that the infusion of muscimol prior to session 8 successfully blocked transfer of learning during that session. The above base-line performance of group I on session 9 does not indicate any acquisition of a tone CR during session 8, rather it is the result of transfer of learning just as seen in the enhanced performance of group n. Immediately after session 9, the second paired tone session was administered. During session 10 (see Fig. 6), group II again performed the highest percentage of CRs to tone with 75.9%+-3.5. Group I performed 50.4%+-8.7, and group III performed 40.6+-12.4. ANOVA revealed group differences (p= .012) and Dunnet’s test (family error rate = .05) found group I significantly higher than groups I and III which did not differ. The average number of trials to reach learning criteria during session 10 was 12.75 for group II and 55.1 for group; group II reached criteria in statistically fewer trials (p< .005). The performance of group I during session 10 appears similar to the performance of group II during session 8 (see Fig. 7). While the average trial to criteria was 54 for group n during session 8, it was 55.1 during session 10 for group I. Similarly the average percentage of CRs performed by group II during session 8 was not statistically different than CRs performed by group I during session 10 (p= .618). These data 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. further support the conclusion that the muscimol infusion administered to group I just prior to session 8 prevented any build-up of associative strength between the tone CS and the airpuff. The performance of group I on subsequent sessions is most simply accounted for by transfer of learning from seven days of paired light training. The mounted brain slices and autoradiographs were examined for determination of cannula placement. The members of group I all had placements which allowed for diffusion of muscimol to cover the critical region of the anterior interpositus. Figure 8 shows a representative slice from group I with the labeled muscimol at its greatest level of diffusion. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Diacussion In sum, muscimol inactivation of the dorsal portion uf the cerebellar anterior interpositus nucleus and variable amounts of overlying cortex of lobule H VI during one day of training to a tone CS in animals previously trained to a light CS completely prevented learning to the tone, as evidenced in post-inactivation testing and training (see Figs. 5 and 6). Specifically, these animals (Group I) show only spontaneous levels of CRs on the initial tone-alone test trials the day after muscimol inactivation, levels comparable to the transfer control group (III) given no prior training at all ( see Fig. 5). In marked contrast, the group given prior light-airpuff training and the tone-airpuff training with saline on session 8 showed a very significant greater number of CRs on the tone-alone test trials on session 9. On the following session (10) of tone-airpuff training, Group I animals (muscimol inactivation in initial tone training) show rapid acquisition of the CR in a manner identical to the performance of the saline control group (D) on their first day of tone-airpuff training (see Fig. 7). The identical performance of the prior muscimol group (I) on the second session of tone-airpuff training and the saline group (II) on the first session of tone- airpuff training (see Fig. 7) demonstrates that no learning occurred during the tone- airpuff training with muscimol infusion. However, group II shows very much higher levels of conditioned responses than the transfer control group (ID), given no training prior to their saline infusion tone-airpuff training on session 8 (see Figs. 1, 3, 5, and 6); indeed, they show virtually no CRs. Consequently, group II, given seven days of training to a light CS, shows a very substantial transfer of 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. training to the tone CS (see below). The transfer effect in group I is present but due to training procedures (post inactivation exposure to tone alone trials) it is difficult to distinguish its performance from group III which received effective training on session 8 allowing it to perform as well as group I on subsequent tone tests and training sessions. Results reported here are in complete agreement with all previous studies where the anterior interpositus nucleus and varying degrees of the overlying cerebellar cortex of lobule H VI were inactivated (with cooling, muscimol, lidocaine, and baclofen) during initial tone-airpuff training. Because we replicated exactly the training and testing procedures of Welsh and Harvey (1991), the incompatibility of their results with all other reversible inactivation studies cannot be due to their training and testing procedures. There is a very substantial effect when animals are first trained to a light CS and then a tone CS, to be discussed below, but this cannot fully account for the results reported by Welsh and Harvey (1991). But we note here that because acquisition to a tone CS following training to a light CS occurs so rapidly, it is essential in studies such as Welsh and Harvey’ s that inactivation of the critical region of the interpositus must remain stable and complete throughout the entire conditioning session, as it was in the present study with muscimol inactivation. Efficacy of Lidocaine Infusion As noted earlier (Introduction), Nordholm et al. (1993) were able to replicate the basic Welsh and Harvey (1991) finding that infusions of lidocaine can prevent expression of CRs but not learning of CRs, but only if the infusions are ventral in the interpositus; dorsal infusions completely prevent learning. Further, 12 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. reversible inactivation (with TTX) of the superior cerebellar peduncle immediately efferent to the interpositus nucleus, which contains all the efferent projections from the interpositus nucleus to extracerebellar sites, completely prevents expression of the CR but does not prevent learning at all. This finding is completely consistent with the ventral infusions o f Nordholm et al. (1993). Nordholm et al. (1993, footnote, p.883), Nordholm (1993) and Welsh and Harvey (1991) all report that many lidocaine infusions were ineffective (i.e., in blocking CR performance). Indeed, use o f lidocaine infusion during acquisition is very problematic; because o f its short duration of action, it must be infused continuously and appears to have a very localized region o f action (see Nordholm, 1993). In an effort to estimate the region of interpositus which was inactivated by lidocaine in their study, Welsh and Harvey injected a single bolus 1 ul of [3H]-lidocaine into the interpositus of one rabbit and e<amined the spread o f the [3H]-!idocaine within the structure using autoradiographic techniques. They present a figure (figure 9B, p.474:) which shows the extent of diffusion o f [3H]-lidocaine in which the concentration exceeded a value of 0.8 mM. The authors state that this concentration of lidocaine (0 .8 mM) is sufficient to block both pre- and post- synaptic activity within the interpositus. However, the estimation o f 0.8 mM as the minimal concentration necessary to block all pre- and post-synaptic activity is based upon assumptions related to experiments using an entirely different preparation and does not account for uncontrolled variables such as extracellular pH. Welsh and Harvey base their estimation o f 0.8 mM upon a series of m vitro studies which examined the effects of lidocaine on nerve impulse conduction. Each of these studies used isolated, frog myelinated spinal fibers. However, as Hille 13 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (1966) points out, this preparation is different from 'nearly every other common nerve preparation' because the nodes of Ranvier are exceptionally well exposed to the external bathing medium. Thus, the concentrations of lidocaine sufficient to block nerve impulse conduction in this preparation would likely be much lower than concentrations necessary to block conduction in other neural tissues, such as interpositus neurons, where access to excitable membranes would be obstructed by dense extracellular attachments and ensheathments. Problems in comparing effective lidocaine doses in one preparation with another are further highlighted by Martin (1991) who points out that full recovery from lidocaine inactivation of cortical or subcortical structures occurs in 15-45 minutes (see Demer & Robinson, 1982; Martin & Ghez, 1988) whereas recovery from similar inactivation of spinal cord take up to 90 minutes (see Sandkuhler, Maisch, & Zimmermann, 1987) to recover. Further, the in vitro studies (above) employed very stable, well controlled external solution; in particular, pH was maintained between 1.2-1 A. The effectiveness of lidocaine blockage of voltage gated Na+ channels, however, has been shown to vary with differing pH. Hille (1977) demonstrated that decreasing pH causes a reduction in both potency and rate of action of lidocaine. The pH of the lidocaine solution used by Welsh and Harvey was 4.6. Their single autoradiograph was based upon rapid injection of 1 ul of lidocaine solution. This volume would replace much of the extracellular fluid throughout much of the interpositus nucleus with a lidocaine solution of pH 4.6, thereby reducing the effectiveness of the lidocaine throughout the structure. Thus, the conclusion by Welsh and Harvey that all neural function was abolished within the region of the 14 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. interpositus nucleus shown in their figure 9B must be regarded as highly suspect. Their assumptions regarding the effectiveness of 0.8 mM lidocaine are based upon results obtained with a very different preparation known to be unusually sensitive to lidocaine. Further, Welsh and Harvey did not control for variations in pH resulting C from injection of a highly acidic lidocaine solution which would reduce the effectiveness of the drug in the interpositus nucleus. Finally, it should be pointed out that figure 9B reveals that concentrations of lidocaine throughout a significant portion of the anterior interpositus nucleus were, in fact, well below the value of 0.8 mM. Even if the estimate of 0.8 mM were accurate and even if the effects of pH were non-significant, the assumption, by Welsh and Harvey, that the spread of [3H]-lidocaine (shown in figure 9B) was stable throughout the entire conditioning session remains highly questionable. Their autoradiograph was based upon a single, rapid injection of [3H]-lidocaine followed, almost immediately (5 min.). by sacrifice of the animal and removal of the brain. As such, the spread of lidocaine shown in figure 9 (Welsh & Harvey, 1991, p.474) represents the lidocaine distribution at the very beginning of training. An entire conditioning session would have continued for 73 minutes following the initial infusion of lidocaine. The authors provide no information at all about possible distributions of lidocaine at different time intervals throughout the session. Nordholm (1993) did in fact subsequently infuse [3H]-lidocaine in exactly the same manner as in training in animals where the dorsal infusions prevented learning (rate of 0.229 ul/min.). After the animals were subsequently trained (tone-airpuff trials) without infusion and were reliably performing CRs, lidocaine infusion was begun and continued 15 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. until the CRs were completely abolished, at which point the animals were rapidly sacrificed. Under these conditions, the distribution of [3H]-lidocaine extended throughout the anterior dorsal interpositus nucleus and some overlying cortex of lobule H VI but did not extend beyond the cerebellum. A recent study by Martin (1991) examined the time course of changes in the lidocaine concentration and distribution in cerebral cortex following injection of a single dose of lidocaine (the same dose as that used by Welsh & Harvey). Ten minutes following the injection, the distribution of lidocaine was similar to that shown in Welsh and Harvey figure 9. However, the concentration of lidocaine at just 20 minutes after the injection was substantially lower; the peak concentration had diminished to just one tenth of the peak concentration at 10 minutes post infusion. By 40 minutes, the concentration of lidocaine at the injection site had reduced to near background levels. Note that at 40 minutes, the training session of Welsh and Harvey would only be slightly more than halfway complete. These data, therefore, clearly demonstrate that the distribution of lidocaine shown by Welsh and Harvey (figure 9) cannot be considered to be representative of lidocaine distribution throughout the session. Following the initial injection of 1 ul of lidocaine at the very start of the tone conditioning session, Welsh and Harvey continuously infused lidocaine into the interpositus nucleus. However, the infusion rate was reduced to 0.1 ul/min., just one tenth the rate used to create their autoradiograph. The authors provide no information at all regarding the distribution of lidocaine using this much lower rate of infusion (as Nordholm has done, see above). However, considering factors such as pH effects on lidocaine effectiveness and its lipid solubility, as well as the 16 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. fact that lidocaine concentration diminishes rapidly following infusion, this very low rate would likely result in a much smaller radius of effective inactivation than the value cf 1.4 mn. reported. This possibility appears to be confirmed by the histology (figure 7. p.471) presented by Welsh and Harvey. Although the authors claim that all activity was abolished within a radius of at least 1.4 mm from the tip of the cannula, their histology does not support this claim. Examination of cannula placements reveals that the effectiveness of lidocaine infused through cannulae placed less than 1 mm apart varied dramatically. For instance, cannula placements in the dorsal aspects of the interpositus shown in plates P0.5-Hit and A0.5-Hit resulted in complete abolition of the light evoked CR. Yet, cannula placements less than 1 mm away (in some instances in nearly the exact same location) resulted in only a partial reduction (or no reduction at all) in performance of light evoked CRs (see plates P0.5-Part, P0.5-Miss, APO-Part, and A0.5-Part). These results argue that the effective spread of lidocaine was significantly smaller than the 1.4 mm estimate of Welsh and Harvey. In conclusion, all the available data are consistent with the view that the lidocaine inactivations of the interpositus nucleus in the Welsh and Harvey (1991) study were not complete, particularly for the cortical dorsal anterior region of the interpositus nucleus, which must be completely inactivated to prevent learning of the conditioned eyeblink response. In contrast, inactivation of the ventral anterior interpositus (Nordholm et al., 1993; many Welsh and Harvey cannulae) and its immediate efferent, the superior cerebellar peduncle (Krupa et al., 1996), which abolishes response of the CR, do not prevent learning. 17 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Long term depression in the interpositus nucleus? Recently, Perret and Mauk (1995) speculated that inactivation of the interpositus nucleus with muscimol during eyeblink conditioning did not block learning per se, but instead resulted in a depression of mossy fiber synaptic efficacy within this nucleus which apparently masked plasticity that had occurred in cerebellar cortex during training. However, these authors offer not a shred of evidence to support this idea. Numerous lines of evidence rule out this possibility. First, in a previous muscimol study (Krupa et al., 1993), both the interpositus nucleus as well as cerebellar cortex were inactivated with muscimol during conditioning. In Krupa and Thompson (1997), using a much lower dose of muscimol, only the interpositus nucleus was inactivated. However, percent CRs as well as the rate of CR acquisition on the first session without muscimol inactivation were identical for both groups. These measures were also identical to controls infused with saline. If muscimol infusions were somehow causing a synaptic depression within the interpositus, it would seem likely that there would be differences in CR performance by these different groups of rabbits, but there were none. Second, other studies have used local cooling (Clark et al., 1992) and lidocaine (Nordholm et al., 1993) (not cited by Perret and Mauk, 1995) to inactivate the interpositus nucleus during the interpositus nucleus during eyeblink conditioning. These methods of inactivation would abolish both pre- as well as post-synaptic activity within the interpositus, which would prevent any form of synaptic plasticity from occurring. Use of these methods of inactivation also 18 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. completely prevented eyeblink conditioning from occurring. The performance of animals on the first session without inactivation in these studies, however, was the same as performance of rabbits infused with muscimol. If muscimol infusions into the interpositus had resulted in a long term depression, the rates of acquisition in each of these studies should be different, but they are not. Third, Yeo and colleagues (Hardiman et al, 1996; Ramnani and Yeo, 1996) recently tested the possibility of muscimol-induced depression directly. They first trained rabbits to perform the conditioned response. They then infused muscimol into the cerebellum and presented the animals with tone alone extinction training. They then tested the animals without muscimol to determine whether any extinction had occurred. They found that the rabbits immediately performed CRs at preinfusion rates: no decrement in responding had occurred while the interpositus nucleus was inactivated with muscimol. This result alone would appear to rule out the possibility of a synaptic depression in the interpositus during these studies. In the present study, in the muscimol inactivation session (8), muscimol completely prevented CRs to either the tone or light CS. Subsequently we tested the animals in the absence of muscimol and found that learning to the tone CS had been blocked by the muscimol inactivation but performance of CRs to the light CS was completely unaffected. If a depression in the interpositus nucleus had prevented acquisition of CRs to the tone CS, then performance to the previously learned light CS should also have been reduced or eliminated but it was not. Finally, if muscimol infusions cause a synaptic depression, this process would apparently be unique to the interpositus nucleus, since inactivation of the red nucleus or brainstem motor nuclei with muscimol does not cause this effect. 19 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. However, we are aware of no evidence of a synaptic mechanism of long term depression in the cerebellum which would fit the requirements of the one proposed by Perret and Mauk; namely, depression occurring when the postsynaptic neuron is hyperpolarized and presynaptic input occurs in repetitive bursts of high frequency pulses as would be expected by mossy fiber responses to the tone CSs presented throughout a conditioning session. In summary, all of the available evidence rules out the possibility that muscimol inactivation of the interpositus nucleus might result in some form of long term depression within this nucleus which prevents performance of CRs on test sessions following removal of muscimol inactivation. 20 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Iff X u Figure 1 Tone o o Light 80 60 40 20 0 0 1 2 3 4 5 6 7 8 9 10 Session Percentage of CRs to light(l*7) or tone (8-10) for all groups on all days trained. Group I Group II GroupIII 21 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2 a a 4-1 3- 7 8 9 10 4 Group t Group II GroupIII Session CR amplitude for light (session 1-7) or tone (sessions 8-10) for all groups on all days trained. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3 100 80 60 40 20 0 0 1 2 3 4 5 6 7 8 9 10 Block # Percentage of tone CRs by block on session 8 (muscimol infusion in group I; saline infusion for groups II and III). Group I Group II GroupIII Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 4 100 r - □ c o n tro l session 8 session 10 Percentage of Light CRs on session 8 (group I- muscimol infusion; group II- saline infusion) and session 10. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5 00 r- m 60 O S u ^ 40 ■ Group I □ Group 1 1 IDD Group III Session 9- Tone alone test Percentage of tone CRs during session 9 (30 tone alone trials). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 6 100 0*60 40 0 1 2 3 4 5 6 7 8 9 10 Block # Percentage of tone CRs by block on session 10. Group I Group II GroupIII Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 7 Group II Session 8 Group I Session 10 100 80 60 40 20 0 0 1 2 3 4 5 6 7 8 9 10 Block Comparison of tone CR performance of group I on session 10 with performance of group II on session 8. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 28 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. References Bowery, N.G., Doble, A., Hill, D.R., Hudson, A.L., Shaw, J . S . , Turnbull, M.J., & Warrington, R. (1981). Bicuculline insensitive GAB A receptors on peripheral autonomic nerve terminals. European Journal of Pharmacology. 71. 53- 70. Bormann, J. (1988). Electrophysiology of GABA^ and GABAg receptor subtypes. Trends in Neuroscience 11(3). 112-116. Chapman, P.F., Steinmetz, J.E., Sears, L.L., & Thompson, R.F. (1990). Effects of lidocaine injection in the interpositus nucleus and red nucleus on conditioned behavioral and neural responses. Brain Research. 537. 149-156. Chapman, P.F., Steinmetz, J.E., & Thompson, R.F. (1988). Classical conditioning does not occur when direct stimulation of the red nucleus or cerebellar nuclei is the unconditioned stimulus. Brain Research. 537. 149-156. Clark, R.E., Zhang, A.A., & Lavond, D.G., (1992). Reversible lesions of the cerebellar interpositus nucleus during acquisition and retention of a classically conditioned behavior. Behavioral Neuroscience. 106. 879-888. Clark, R.E., & Lavond, D.G. (1993). Reversible lesions of the red nucleus during acquisition and retention of a classically conditioned behavior in rabbits. Behavioral Neuroscience. 107. 264-270. Demer, J.L., and Robinson, D.A. (1982). Effects of reversible lesion and stimulation of olivocerebellar system on vestibular reflex plasticity. J. Neurophvsiol.. 47. 1084-1107. Hardiman, M.J., Ramnani, N., and Yeo, C. (1996). Reversible inactivations of the cerebellum with muscimol prevent the acquisition and extinction of conditioned nictitating membrane responses in the rabbit. Exp. Brain Res.. 110. 235-247. Hesslow, G. and Ivarsson, M. (1994). Suppression of cerebellar Purkinje cells during conditioned response in ferrets. Neuroreport. 5. 649-652. Hille, B. (1966). Common mode of action of three agents that decrease the transient change in sodium permeability in nerves. Nature (London). 210. 1220-1222. Hille, B. (1977). The pH-dependent rate of action of local anesthetics on the node of Ranvier. J. General Physiol.. 69, 475-496. Kehoe, E.J., & Holt, P.E. (1984). Transfer across CS-US intervals and sensory modalities in classical conditioning of the rabbit. Animal Learning and Behavior. I2» 122-128. 29 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Kim, J.J., and Thompson, R.F. (1997). Cerebellar circuits and synaptic mechanisms involved in classical eyeblink conditioning. Trends Neurosci., 20, 177-181. Krupa, D.G., and Thompson, R.F. (1995). Inactivation of the superior cerebellar peduncle blocks expression but not acquisition of the rabbit's classically conditioned eyeblink response. Proc. Natl. Acad. Sci.. 92, 5097-5101. Krupa, D.G., and Thompson, R.F. (1997). Reversible inactivation of the cerebellar interpositus nucleus completely prevents acquisition of the classically conditioned eyeblink response. Learning and Memory. 3, 545-556. Krupa, D.J., Thompson, J.K., & Thompson, R.F. (1993). Localization of a memory trace in the mammalian brain. Science. 260. 989-991. Krupa, D.G., Weng, J., Thompson, R.F. (1996). Inactivation of brainstem motor nuclei blocks expression but not acquisition of the rabbit’ s classically conditioned eyeblink response. Beha. Neurosci.. 110,219-227. Lavond, D.G., Hambree, T.L., and Thompson, R.F. (1985) Effects of kainic acid lesions of the cerebellar interpositus nucleus on eyelid conditioning in the rabbit. Brain Res.. 326, 179-182. Martin, J.H. (1991). Autoradiographic estimation of the extent of reversible inactivation produced by microinjection of lidocaine and muscimol in the rat. Neuroscience Lett.. 127, 160-164. Martin, J.H., and Ghez, C. (1988). Red nucleus and motor cortex: Parallel motor systems for the initiation and control of skilled movement. Behav. Brain Res.. 28. 217-223. McCormick, D.A., & Thompson, R.F. (1984). Cerebellum: essential involvement in the classically conditioned eyelid response. Science. 223. 296-299. McCormick, D.A., Steinmetz, J. E., and Thompson, R.F. (1985). Lesions of the inferior olivary complex cause extinction of the classically conditioned eyeblink response. Brain Res.. 359, 120-130. Nordholm, A.F., Thompson, J.K., Dersarkissian, C. and Thompson, R.F. (1993). Lidocaine infusion in a critical region of cerebellum completely prevents learning of the conditioned eyeblink response. Behav. Neurosci.. 107, 882-886. Perret, S.P., and Mauk, M.D. (1995). Extinction of conditioned eyelid responses requires the anterior lobe of cerebellar cortex. J. Neurosci.. 15, 2074-2080. Ramirez, O.A., Nordholm, A.F., Gellerman, D., Thompson, J.K.., and Thompson, R.F. (1997). The conditioned eyeblink response: A role for the GABA- B receptor. Pharmacol. Biochem. Behav. .(in press). 30 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ramnani, N., and Yeo, C.H. (1996). Reversible inactivations of the cerebellum prevent the extinction of conditioned nictitating membrane responses in rabbits. L Phvsiol.. 495.1, 159-168. Sandkuhler, J., Maisch, B., and Zimmermann, M. (1987). The use of local anesthetic microinjections to identify central Pathways: A qualitative evaluation of the time course of the neuronal block. Exp. Brain Res., 68, 168-178. Scheurs, B.G., & Kehoe, E.J. (1987). Cross-modal transfer as a function of initial training level in classical conditioning with the rabbit. Animal Learning and Behavior. 15. 47-54. Steinmetz, J.E., Logan, C.G., Rosen, D.J., Thompson, J.K., Lavond, D.G., and Thompson, R.F. (1987). Initial localization of the acoustic conditioned stimulus projection system to the cerebellum essential for classical eyelid conditioning. Proc. Natl. Acad. Sci. USA. 84, 3531-3535. Thach, W.T., Goodkin, H.P., and Keating, J.G. (1992). The cerebellum and adaptive coordination of movement. Annu. Rev. Neurosci.. 15, 403-442. Thompson, R.F., and Krupa, D.J. (1994). Organization of memory traces in the mammalian brain. Annu. Rev. Neurosci.. 17, 519-549. Voneida, T., Christie, D., Boganski, R., and Chopkp, B. (1990). Changes in instrumentally and classically conditioned limb-flexion responses following inferior olivary lesions and olivocerebellar tractotomy in the cat. J. Neurosci.. 10, 3583- 3593 Welsh, J.P., & Harvey, J.A. (1991). Pavlovian conditioning in the rabbit during inactivation of the interpositus nucleus. Journal of Physiology. 444, 459-480. Welsh, J.P., Harvey, J.A. (1989). Cerebellar lesions and the nictitating membrane reflex: Performance deficits of the conditioned and unconditioned response. Journal of Neuroscience. 9.299-311. Yeo, C.H. (1991). Cerebellum and classical conditioning of motor response. Ann. NY Acad. Sci.. 627, 292-304. Yeo, C.H., Hardiman, M.J., Glickstein, M. (1985). Classical conditioning of the nictitating membrane response of the rabbit I. Lesions of the cerebellar nuclei. Exp. Brain Res.. 60, 87-98. 31 permission of the copyright owner. Further reproduction prohibited without permission.

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Asset Metadata

Creator Cipriano, Benjamin David (author)

Core Title Inactivation of the interpositus nucleus prevents transfer of the rabbit's classically conditioned eyeblink response from a light to a tone-conditioned stimulus

School Graduate School

Degree Master of Arts

Degree Program Behavioral Neuroscience

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, neuroscience,OAI-PMH Harvest,psychology, experimental,psychology, physiological

Language English

Contributor Digitized by ProQuest (provenance)

Advisor [illegible] (committee chair), [illegible] (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-30367

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