University of Southern California Dissertations and Theses

A context for timing, conditioning and modification of cerebellar function

(USC Thesis Other)

A context for timing, conditioning and modification of cerebellar function

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content A CONTEXT FOR TIMING, CONDITIONING AND MODIFICATION OF CEREBELLAR FUNCTION Copyright 2004 by Andrew Makoto Poulos A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirement for the Degree DOCTOR OF PHILOSPHY (PSYCHOLOGY) December 2004 Andrew Makoto P o u lo s Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 3155462 INFORMATION TO USERS 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 bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send 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. ® UMI UMI Microform 3155462 Copyright 2005 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Dedication To my mother, who has worked tirelessly to provide for my brother, sister and I giving us the freedom, encouragement and love to follow our dreams. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Acknowledgements I owe a deep debt of gratitude to my advisor Dr. Richard F. Thompson for opening his laboratory to me, for imparting his knowledge, expertise and guidance in pursuit of the most important question in modern neuroscience, the neural basis of learning and memory. Indeed, I am grateful for his tremendous patience and generosity over the last 5 years. I would also like to thank Dr. David Lavond for his technical expertise and helpful suggestions. In addition, I also want thank Drs. Larry Swanson, Stephen Madigan and Ernest Greene for taking the time and effort to be part of my thesis committee. I would especially like to thank Judith Thompson for being a constant source of encouragement and a good friend. She is a great example of courage, strength and commitment. I would like to thank the members of the Thompson Lab both past and present, David King, Richard Hinchliffe, Kimberly Christian, Andrea Scicli, Benjamin Tran, Shawn Mojtahedian, Karla Robleto, Ka Hung Lee, Ingrid Liu, Yu Zheng and Narawut Pakaprot from whom I have learned and garnered so much support. During my initial training David King and Richard Hinchliffe were of tremendous importance in both learning techniques and experimental design. I would like to iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. thank Karla Robleto and Kim Christian for their friendship and many long discussions that made coming to the lab each day more enjoyable. In addition, I want to thank all the undergraduate students that have worked with me, Jackie Lopez, Hiroko Nobuta, Benjamin Mahdi and Connor Schaye, which have made life a little easier and taught me a lot about the process of teaching. Finally, I want thank Kimberly Rapp my very significant other who has provided unwavering support over the past year and half and has been a constant source of encouragement and love. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table of Contents Dedication..............................................................................................................................ii Acknowledgements............................................................................................................. iii List of Tables....................................................................................................................... vii List of Figures.................................................................................................................... viii Abstract................................................................................................................................. xii Chapter 1 General Introduction..................................................................................................1 Neural Basis of Eyeblink Conditioning.................................................................4 Sites of Critical Plasticity........................................................................................9 Intracellular Mediators of Plasticity.................................................................... 14 Generalization to Humans.....................................................................................18 Overview of experiments....................................................................................... 19 Chapter 2 Timing of Conditioned Responses Utilizing Electrical Stimulation in the Region of the Interpositus Nucleus as a CS Introduction............................................................................................................. 21 Materials and Methods..........................................................................................26 Results...................................................................................................................... 30 Discussion................................................................................................................36 Chapter 3 Evidence of Learning-Related Interpositus Nucleus Plasticity Introduction............................................................................................................. 42 Materials and Methods..........................................................................................46 Results...................................................................................................................... 51 Discussion................................................................................................................55 Chapter 4 Effects of Sensory Stimulation on Acute Measures of Interpositus Nucleus Excitability Introduction............................................................................................................. 62 Material and Methods............................................................................................67 Results...................................................................................................................... 71 Discussion..............................................................................................................76 v Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 5 Cerebellar GABAa Receptor Mediated Eyeblink Responses Introduction..............................................................................................................81 Material and Methods............................................................................................ 84 Results...................................................................................................................... 89 Discussion..............................................................................................................102 Chapter 6 Effects of Post-Training Exposure to the Training Apparatus Following Delay Eyeblink Conditioning Introduction............................................................................................................109 Material and Methods.......................................................................................... 112 Results.................................................................................................................... 115 Discussion..............................................................................................................121 Chapter 7 General Discussion..................................................................................................126 References...........................................................................................................................131 vi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. List of Tables Table 4-1. Lists the sequence of trials for a given experimental session. Time of stimulation (TOS) in the following order at specific threshold levels........... 70 Table 5-1. Depicts the modality and intensity of a stimulus and the total volume of picrotoxin infused for a given experimental session as well as indicating the number of animals run in each condition...................................................... 86 Table 5-2. List of eyeblink conditioning experiments that have infused picrotoxin in the IPN (interpositus nucleus). Concentrations, volumes and effect upon eyeblink conditioning are described..............................................106 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. List of Figures Figure 1-1. A simplified schematic of the putative neural circuitry essential for delay eyeblink conditioning. Arrows and fork terminals denote excitatory synapses; bar terminals denote inhibitory synapses. Solid ovals denote fibers (axons) and dotted ovals denote proposed sites of plasticity underlying eyeblink conditioning. A/V, auditory and visual pathways; CC, cerebellar cortex; cf, climbing fiber; CS, conditioned stimulus; CR/UR, conditioned response/unconditioned response; GC, granule cell; 10, inferior olive; IPN, interpositus nucleus; mf, mossy fibers; MN, motor nuclei; PC, Purkinje cell; pf, parallel fiber; PN, pontine nucleus; RN, red nucleus; RF, reticular formation; TN, trigeminal nucleus; US, unconditioned stimulus. (Modified from Christian & Thompson, 2003).................................................................... 5 Figure 2-1. Procedure used to produce eyeblink conditioning. Stimulation of left IPN through a pair of electrodes was used as a conditioned stimulus (CS) and corneal airpuff delivered to the left eye was used as an unconditioned stimulus (US)........................................................................................................ 25 Figure 2-2. Conditioned response (CR) percentages (%) plotted for each of half of a training session. Rabbits in the Pre-CS group received stimulation alone (session 1,2) followed by paired stimulation and airpuffs (sessions 3,4) under a 250 ms interstimulus interval (ISI). Rabbits in group I-ISI were initially trained to a 250 ms ISI during sessions 1 and 2 and shifted to a 500 ms ISI for sessions 3 and 4. Rabbits in group D-ISI were initially trained to a 500 ms ISI for sessions 1,2 and shifted to a 250 ms ISI during sessions 3 and 4........... 31 Figure 2-3. Mean number of trials to learning criterion (8 CRs within 8 consecutive 9 consecutive paired trials). All rabbits reached learning criterion (8 CRs with 9 consecutive trials) by the end 2n d paired (CS-US) training session. Training utilizing an initially shorter interstimulus interval (I-ISI) showed a faster rate of learning then the group initially trained with a longer interval (D-ISI) or receiving stimulation alone (Pre-CS) prior to paired training..................................................................................................................... 32 Figure 2-4. Mean onset and peak conditioned response (CR) latencies as measured in milliseconds (ms) over the course of training blocks, consisting of 1 CS alone and 8 paired trials. A. group I-ISI; B. group D-ISI. Arrow indicates ISI shift....................................................................................................33 vm Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2-5. Illustration of coronal sections through the rabbit cerebellum. Filled circles depict electrode tips used for electrical stimulation as a CS. IPN, interpositus nucleus. A. Plate of a cerebellar section 0.5 mm anterior to lambda. B. Plate of a cerebellar section 1.0 mm anterior lambda...................35 Figure 3-1. Experimental procedures used to investigate any conditioning-related changes in interpositus nucleus stimulation to eyeblink thresholds. Following surgery and recovery (days 1-8) rabbits were adapted to the training apparatus (days 9-10). Thereafter, baseline threshold measurements were made and eyeblink conditioning began (day 11). Following a 2n d session of conditioning, post-acquisition thresholds were measured (day 12). The next day, extinction (CS alone) training started (day 13), followed the next day by a 2n d session of extinction and post-extinction threshold measurements (day 14). Finally rabbits were presented with a last conditioning session followed by post-reacquisition threshold measurements...................................................49 Figure 3-2. Mean conditioned response (CR) percentages (%) plotted for each of half of a training session. Each acquisition (Acq) session consisted of 108 trials divided into 12 blocks of 9 trials (1 CS alone trial, 8 paired trials). Extinction (Ext) sessions consisted of 100 CS alone trials, while a session of reacquisition (Reacq) was identical to an acquisition session........................52 Figure 3-3. Electrical stimulation to eyeblink thresholds measured as current (uA). Baseline thresholds (Baseline Thres.) were measured just prior to acquisition. Following acquisition training, thresholds were measured again (Post-acq. Thres.)....................................................................................................53 Figure 3-4. Illustration of coronal sections through the rabbit cerebellum. Filled circles depict electrode tips used for electrical stimulation as a CS. IPN, interpositus nucleus. A. Plate of a cerebellar section 0.5 mm anterior to lambda. B. Plate of a cerebellar section 1.0 mm anterior lambda................... 54 Figure 4-1. Depicts asymptote level of percentage CRs as a function of delay eyeblink conditioning as a function of interstimulus interval. Adapted from Gormezano (1976)..................................................................................................63 Figure 4-2. Procedure used to measure stimulation to eyeblink thresholds during the presentation of auditory stimuli..................................................................... 66 Figure 4-3. Depicts the time pints of stimulation during the onset of an 85 or 95 decibel (DB) tone....................................................................................................69 ix Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 4-4. Mean percent eyeblink amplitude as measured in millimeters (mm) elicited by electrical stimulation, measured at multiple time points prior to (baseline) and after the onset of an 85 decibel (dB) tone................................. 73 Figure 4-5. Mean percent eyeblink amplitude as measured in millimeters (mm) elicited by electrical stimulation, measured at multiple time points prior to (baseline) and after the onset of an 95 decibel (dB) tone.......................,.........74 Figure 4-6. Illustration of coronal section through the rabbit cerebellum estimated at 1 mm anterior to lambda. Filled circles depict marking lesions used for identifying the final position of stylet................................................................. 75 Figure 5-1. Mean percentage of eyeblink responses produced during an 85 or 95 decibel (dB) tone over a 2 block pre-infusion baseline period and following an 8 block 1.5 /d picrotoxin (PTX) test period.................................................. 90 Figure 5-2. Percentage distribution of eyeblink onset latencies produced during a 95 decibel (dB) tone following infusion 1.5 /d of picrotoxin.......................... 92 Figure 5-3. Mean amplitude of eyeblinks as measured in millimeters (mm) during a 95 decibel (dB) tone over a two-block pre-infusion period and following infusion of 1.5/d of picrotoxin (PTX).................................................................93 Figure 5-4. Mean percentage of eyeblink responses produced during an 95 dB tone over a 2 block pre-infusion baseline period and following an 8 block 1.0 /d picrotoxin (PTX) infusion test period.............................................................94 Figure 5-5. Mean percentage of eyeblink responses produced during a 5 and 24 lumen light over a 2 block pre-infusion baseline period and following an 8 block 1.5 ]A picrotoxin (PTX) infusion test period........................................... 95 Figure 5-6. Percentage distribution of eyeblink onset latencies during a 24-lumen light following infusion of 1.5 /d of picrotoxin..................................................96 Figure 5-7. Mean percentage change in airpuff evoked eyeblink amplitudes as measured in millimeters (mm) prior to and following infusion 1.5 /d of picrotoxin................................................................................................................99 Figure 5-8. Conditioned response (CR) percentages (%) plotted for sessions of acquisition (acql-acq7), with (MUS) and without infusion of muscimol (RET) and sessions of extinction (extl-ext7) and a session with infusion of picrotoxin (PTX)................................................................................................... 100 x Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5-9. Illustration of coronal sections through the rabbit cerebellum. Filled circles depict electrode tips used to deliver marking lesions. IPN, interpositus nucleus. A. Plate of a cerebellar section 0.5 mm anterior to lambda. B. Plate of a cerebellar section 1.0 mm anterior lambda................................................101 Figure 6-1. Mean conditioned response (CR) percentages (%) plotted for each of sessions of acquisition (acql-acq6), exposure solely to the conditioning apparatus (cxtl-cxt6), sessions of conditioned stimulus alone extinction training (extl-ext6) and a session of reacquisition (reacq) training.............117 Figure 6-2. Mean percent conditioned responses (CRs) during the first session of extinction training following either conditioning apparatus alone exposure (CXT) or homecage maintained (HMC) animals (* denotes significant difference < .05).................................................................................................... 118 Figure 6-3. Mean percent point difference of conditioned response (CRs) between the final session of acquisition and the initial session of extinction..............119 Figure 6-4. Mean percentage conditioned responses (CRs) during reacquisition training................................................................................................................... 120 Figure 6-5. Illustrates Kehoe’s (2004) model that accounts for the present results by predicting associations between the context (CXT) and conditioned stimulus (CS) and unconditioned stimulus (US)..............................................125 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Abstract There is substantial evidence that essential memory traces for classically conditioned discrete motor responses are established and maintained in the interpositus nucleus (IPN) of the cerebellum. The studies presented in this thesis, utilizing intracerebellar brain stimulation, pharmacological and behavioral methods attempt to further characterize the mechanisms of cerebellar plasticity underlying the acquisition, storage and expression of conditioned eyeblink responses. The first experiment, utilized electrical stimulation of the IPN as conditioned stimulus (CS) and a corneal airpuff unconditioned stimulus (US) to measure timing of conditioned responses (CR) following temporal changes in contingency. Conditioning, which was very rapid and robust, adapted to changes in CS-US intervals suggesting CR timing maybe regulated by climber fibers input. The second experiment, utilized stimulation thresholds required to elicit eyeblinks directly stimulating the IPN before and following acquisition and extinction of eyeblink CRs. Acquisition resulted in a decrease in stimulation thresholds, whereas extinction resulted in a modest increase in thresholds. Interestingly, prior to training stimulation in some animals elicited behaviors other than eyeblinks, however with conditioning, stimulation exclusively produced eyeblink responses even following extinction of CRs. Results suggest potential learning related changes in intracerebellar somatotopic representations. xii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The third experiment measured changes in IPN stimulation to eyeblink amplitudes following the presentation of a tone in the untrained animal. The auditory stimulus produced some increases in eyeblink amplitudes that persisted even following the offset of the tone. The fourth experiment, utilized intracerebellar infusion of GABAa receptor antagonist, picrotoxin in the untrained under different levels and modalities of sensory stimulation. Higher levels of visual or acoustic stimuli following infusions of large volumes of picrotoxin were sufficient to evoke eyeblink responses. These results suggest sufficient activation of mossy fiber-IPN inputs combined with release of cerebellar cortical inhibition are capable of expressing eyeblink responses. The final experiment, examined the effects of repeated conditioning apparatus exposure on the expression of eyeblink CRs. Measures of CRs during initial extinction training session revealed a significant decline as compared to homecage control animals. These results and those of previous studies demonstrate that the conditioning context can modulate the expression of CRs during extinction. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 1 Neural Basis of Classically Conditioned Eyeblink Responses Introduction Fundamental to the survival of an organism is the ability to utilize cues in one’s immediate environment to anticipate the occurrence of biologically significant events. Inherent in such a process is the establishment of associations between stimuli that predict aversive or appetitive events in close temporal or spatial proximity. For instance, in response to cues that predict potential danger, an organism must be able to produce defensive or avoidance behaviors to minimize potential harm. Alternatively, if a particular stimulus predicts the occurrence of events that promote appetitive behaviors, including feeding and sexual reproduction, it is critical that an association be established. Central to associative learning is that sensory information must be encoded then established in a learned contingency and retained over a period of time and thus expressed under appropriate conditions. If conditions change, such that the occurrence of environmental cues no longer predicts biologically significant events, the organism must be able to decrease the expression of conditional behaviors. In Pavlov’s initial description of classical conditioning in the dog, the beating of a metronome was paired with the application of meat powder in the mouth. Prior to 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and during the initial pairings the application of meat powder elicited a reflexive salivary response. However, with repeated pairings the metronome could conditionally express the production of saliva, in the absence of meat powder. Hence, an association between two contiguous and contingent stimuli resulted in the establishment of a conditioned reflex. In addition, with repeated presentations of the conditional stimulus (CS) alone, the expression of the conditioned reflex gradually extinguished. However, if the CS was once again paired with the unconditional stimulus (US) performance of the conditioned behavior returned at a rate faster than the initial conditioning. Such results suggested that the extinction of the conditioned reflex did not abolish the original memory trace. To date, one of the most widely used classical conditioning paradigms to investigate the neurobiological basis of associative memory is eyeblink conditioning. This paradigm, originally developed in the rabbit by Isidore Gormezano (1962) typically involves the presentation of an initially neutral tone conditioned stimulus (CS) and a mildly aversive corneal airpuff. Presentation of the unconditioned stimulus (US) airpuff results in the reflexive eyelid closure and extension of the nictitating membrane (NM). Following repeated CS-US pairings, the tone comes to elicit conditioned eyeblink and NM responses (CR) prior to, and maximal at, the onset of the airpuff. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Over the past thirty years Richard Thompson and colleagues have utilized the rabbit eyeblink conditioning paradigm combining lesion/inactivation, pharmacological and electrophysiological methods to successfully identify neural correlates underlying the acquisition and expression of conditioned eyeblink memory traces. The collective results obtained and corroborated by other laboratories strongly argue that cerebellum and its associated brainstem regions are critical for the establishment, maintenance and expression of Pavlovian eyeblink responses. 3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Neural Circuits The core neural component for eyeblink conditioning is the cerebellum, a brain structure located just caudal to the cerebral hemispheres and overlying the dorsal surface of the brainstem. The neuronal cell bodies of the cerebellum form a thick cortical layer that covers the underlying white matter (axons) and deep cerebellar nuclei. These nuclei, organized in medial-lateral orientation, consist of the fastigial, interpositus and dentate nuclei. The relays of cerebellum are organized in highly regular manner with well-defined sensory input and motor output pathways. Sensory information corresponding to potential tone or light CSs is relayed to the cerebellum by auditory and visual pathways primarily via pontine mossy fibers (see figure 1). Mossy fibers form monosynaptic connections to neurons in the interpositus nucleus (IPN) and disynaptic connections to the Purkinje cells of the cerebellar cortex by way of granule cell parallel fibers. Damage to appropriate portions of the pontine nuclei can abolish CRs established to a tone CS, while leaving conditioning to a visual CS, such as light, intact (Steinmetz et al 1987). At the level of the IPN single neurons receive CS information from multiple sensory modalities. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. p f Cerebellum PN mf RN RF TN A/V MN CS CR/UR US Figure 1.1 A simplified schematic of the putative neural circuitry essential for delay eyeblink conditioning. Arrows and fork terminals denote excitatory synapses; bar terminals denote inhibitory synapses. Solid ovals denote fibers (axons) and dotted ovals denote proposed sites of plasticity underlying eyeblink conditioning. A/V, auditory and visual pathways; CC, cerebellar cortex; cf, climbing fiber; CS, conditioned stimulus; CR/UR, conditioned response/unconditioned response; GC, granule cell; IO, inferior olive; IPN, interpositus nucleus; mf, mossy fibers; MN, motor nuclei; PC, Purkinje cell; pf, parallel fiber; PN, pontine nucleus; RN, red nucleus; RF, reticular formation; TN, trigeminal nucleus; US, unconditioned stimulus. (Modified from Christian & Thompson, 2003). 5 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. For example, single cell recordings reveal IPN neurons that respond singly and in combination to both tone and light CSs and airpuffs (Tracy et al, 2001) and appropriately directed lesions of the IPN prevent conditioning to all CS modalities tested (Yeo et al., 1985, Lavond et al., 1985; Steinmetz et al., 1992; Ivkovich et al., 1993; Krupa & Thompson, 1994). Substitution of peripheral CSs with direct electrical stimulation of either the pontine nucleus or IPN serves as a very powerful CS, producing conditioning that is more rapidly acquired than either to light or tone (Steinmetz, 1986; Tracy et al., 1998). Somatosensory information corresponding to an airpuff or mild eye-shock US arrives at the cerebellum primarily by climbing fibers from the inferior olive (see figure 2). This essential US pathway includes the trigeminal nucleus, which in turn innervates the inferior olive, which sends axonal processes to the cerebellum via climbing fibers. The climbing fibers and their collaterals synapse on Purkinje cells and IPN neurons of the cerebellum and utilize glutamate and CRH as primary neurotransmitters (Ito, 2002). Lesions of the inferior olive prior to training prevent eyeblink conditioning, whereas similar lesions following conditioning results in extinction with continued paired training (McCormick et al, 1985). Conversely, electrical stimulation of the inferior olive that elicits behavioral responses when used as a US produces conditioning at a rate, magnitude and topography similar to peripheral USs (Mauk et al., 1986). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The IPN is not only a primary site of CS-US convergence, but also a primary cerebellar motor output responsible for the generation of the conditional eyeblink response. The IPN, like the motor cortex, contains a somatotopic representation of the entire body (Schultz et al, 1979). For example, in the rabbit, direct electrical stimulation of the medial aspects of IPN elicit movements in the lower trunk and hindlimb areas, whereas stimulation of lateral portions of IPN evoke movements in head area that include eyelid closure (McCormick et al, 1983). This eyelid region of the IPN sends heavy projections to contralateral magnocellular red nucleus. From the red nucleus, projections to a set of motor nuclei trigger the expression of the conditional eyeblink response. The same motor nuclei also receive direct and indirect projections from the trigeminal nucleus, responsible for producing the unconditional eyeblink reflex. Under simple delay conditioning procedures the cerebellum and its associated brainstem regions are necessary and sufficient for the acquisition and expression conditional eyeblink responses (figure 1.1). However, damage to the medial septum, a primary source of cholinergic projections to the hippocampus, retards the rate of delay conditioning (Berry & Thompson, 1979). Conversely, intra-septal injections of scopolamine, an acetylcholine receptor antagonist, which suppress hippocampal functioning, also slow eyeblink conditioning (Solomon & Gottfried, 1981; Salvatierra & Berry, 1989). However, if the hippocampus is lesioned first, scopolamine no longer impairs learning (Solomon et al, 1983). Thus, for delay 7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. eyeblink conditioning, a functionally compromised hippocampus is more detrimental to learning than the absence of the hippocampus. However, the introduction of a stimulus free period between the CS and US requires the hippocampus. Lesions of the hippocampus, which produce no discernable effects on delay conditioning, markedly impair trace eyeblink conditioning (Solomon et al., 1986; Beylin et al., 2001). In trace conditioning, lesions made immediately after training abolish the trace CR but lesions made 1 month following training do not impair performance of trace CRs (Kim et al., 1995), in a manner that parallels hippocampus-dependent consolidation of context fear (Kim & Fanselow, 1992). Anatomical evidence suggests that interactions between the cerebellum and hippocampus occur indirectly, with the hippocampus modulating CS and US input by interacting with the pontine nucleus and inferior olive (Lee & Kim, 2004). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Sites of Critical Plasticity In eyeblink conditioning it is quite clear that both neurons of IPN and Purkinje cells of the cerebellar cortex receive CS and US inputs. Therefore, it is conceivable that plasticity at one or both sites mediate the formation and storage of eyeblink memory traces. Furthermore, anatomical evidence reveals that both cerebellar regions send and receive reciprocal connections among each other (see figure 1.1). For this reason, determining the relative contributions of IPN and cerebellar cortex has been difficult. Initial findings by McCormick et al., (1982) demonstrated that electrolytic lesions of dentate-IPN region and large aspirations of cerebellar cortex and nuclei completely abolished all expression of conditional eyeblink responses without affecting the UR. Later studies revealed that lesions of the anterior lateral IPN as small as 1 mm3, are sufficient to completely abolish CRs (Lavond et al., 1984). Further, the use of temporary inactivation methods demonstrate that inactivation of the IPN during training completely prevents learning, as evidenced by the lack of CRs following inactivation (Krupa & Thompson, 1995; Clark et al, 1992; Krupa & Thompson, 1997). In contrast inactivation of an efferent pathway or its target the red nucleus, which completely abolishes CR expression, does not prevent learning at all (Krupa et al., 1993; Clark & Lavond, 1993; Krupa & Thompson, 1995). These results suggests that the essential plasticity can not occur efferent to the IPN and that essential memory trace(s) are established and maintained in the cerebellum. Single and multiple unit recordings in the IPN show learning-related unit activity, also 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. found in the cerebellar cortex that precede and predict occurrence of the behavioral CR (McCormick & Thompson, 1984; Berthier & Moore, 1986; Berthier & Moore, 1990; Gould & Steinmetz, 1994; King et al., 2001; Rogers et al., 2001). Moreover, neuronal recording of conditioning-related unit activity in many regions of the essential eyeblink circuitry (see figure 1.1) are abolished by lesions or temporary inactivation of the IPN (Thompson & Krupa, 1994). Removal of cerebellar cortical tissue has been reported to result in varying effects in eyeblink conditioning. While it has been reported that lesions of cerebellar cortex prevent the acquisition of conditional eyeblink responses (Garcia, et al., 1999), all others studies to date report either no effect or impairments in acquisition, retention and/or CR timing (Logan et. al, 1994; Perrett et. al, 1993; McCormick and Thompson, 1983; Lavond and Steinmetz, 1989). Perhaps the clearest interpretation of these lesion studies is provided by Purkinje cell degeneration (PCD) mice, in which Purkinje cells, the sole output of the cerebellar cortex, completely degenerates several weeks after birth. PCD mice acquire eyeblink conditioning at a slower rate and lower level than wild type mice, but they do learn (Chen et al., 1996). Further, CRs expressed tended to have shorter peak latencies. More recent work by Bao et al, (2002) suggests that CR expression and timing may be completely dissociable and that memory traces for eyeblink CRs may be encoded in a functionally distinct manner in the both cerebellar cortex and IPN. In their study, rabbits displaying well-timed CRs were given sequential IPN application of the 10 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. GABAa agonist muscimol and the GABAa antagonist picrotoxin that resulted in the expression of reduced onset and peak CR latencies poorly timed to the US. Such results are consistent with studies by Mauk and associates using high concentrations of picrotoxin (Garcia and Mauk, 1998; Medina et al., 2001). This disruption of CR timing following blockade of cortical outputs suggests that the memory traces for learned timing and for basic associative eyeblink memory maybe expressed within two distinct sites of plasticity, the cerebellar cortex and IPN, respectively. Early cerebellar learning theories proposed that plasticity developed within the cerebellar cortex, specifically at the parallel fiber-Purkinje cell (PF-PC) synapses, as a general mechanism of motor learning (Marr, 1969; Albus, 1971). Albus (1971) further hypothesized that the high tonic-firing rate of Purkinje cells resulting in sustained GABAergic inhibition of the IPN could be released by a decrease in PF-PC synaptic strength. Empirical evidence for this theory came with the discovery of long-term depression (LTD), resulting from simultaneous low frequency stimulation of parallel fibers and climbing fibers, could result in persistent decreases in PF-PC synaptic efficacy (Ito, 1982). However, under such parameters of simultaneous CS and US presentation, eyeblink conditioning does not develop. Further work by Chen and Thompson (1995) demonstrated that under specific in vitro conditions that did not block GABA receptors, LTD could be induced by repeated parallel fiber and climbing fiber stimulation separated by 250 msec, matching the optimal eyeblink conditioning interstimulus intervals (ISI). In contrast, simultaneous stimulation of 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. parallel and climbing fibers can elicit LTD in the presence of the GABA antagonist Biccuculline. Indeed, in the intact animal, direct electrical stimulation of parallel fibers as a CS and climbing fibers as a US produces appropriately timed eyeblink conditioning (Shinkman et al., 1996). More recent investigations both in vivo and in vitro have begun to examine plasticity specific to the IPN and its synapses. One attractive mechanism of memory formation in eyeblink conditioning is long-term plasticity of the mossy fiber-IPN synapse. Racine (1986) previously reported that tetanic electrical stimulation of mossy fibers induces LTP of the mossy fiber-IPN synapse. Perhaps the most compelling evidence of conditioning-specific synaptic plasticity comes from recent findings by Kleim and colleagues (2002) using unbiased stereological synapse quantification methods. In this study rats trained in delay eyeblink conditioning exhibited an increased number of excitatory IPN synapses compared to unpaired and untrained controls. Moreover, since expression of CRs is driven solely by the CS, increases in excitatory IPN synapses are likely to occur along the CS pathway, specifically at mossy fiber-IPN synapses. There was no increase in inhibitory synapses in the IPN (presumably from Purkinje axons). A form of non-synaptic plasticity has also been implicated in cerebellar learning. Linden and associates (2000) showed that direct high frequency stimulation of deep nuclear neurons of the cerebellum under reduced levels of inhibition result in persistent increases in maximum firing rate. Interestingly, IPN stimulation thresholds yielding eyeblinks are markedly reduced following eyeblink 12 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. conditioning, suggesting possible conditioning related increases in IPN excitability (Poulos et al, 2002). Conversely, infusion of NMD A antagonist APV in the IPN, which in vitro blocks the induction of intrinsic excitability (Aizenman & Linden, 2000), selectively impairs conditioning but not expression of eyeblink CRs (Chen and Steinmetz, 2000). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Intracellular Mediators of Plasticity In eyeblink conditioning the cumulative evidence from several levels of analysis strongly suggests that the development and maintenance of the adaptive (well timed) associative memory (CRs) are meditated by plasticity occurring at least two cerebellar regions, cortex and IPN. Moreover, determination of which forms of plasticity contribute to learning has yet to be directly identified in vivo, therefore employing molecular and genetic methods to isolate key components of plasticity and learning is imperative. At the level of the IPN, if an increase in synaptic strength is a mechanism of the primary CS-US memory, then alterations of intracellular mediators of LTP or synaptogensis should compromise conditioning. Further, if depression of synaptic function in the cortex mediates the appropriate timing of the associative memory, utilizing methods to manipulate expression of key molecular components of cerebellar cortical LTD should affect conditional responding and timing. The demonstration that eyeblink conditioning is associated with an increase in the number of IPN excitatory synapses prompts important questions as to possible mechanisms of synapse formation in the adult central nervous system that may mediate memory formation. Almost certainly any physical or chemical changes in neurons involve alterations in gene expression and/ or protein structure. Gomi et al, (1999) demonstrated that inhibition of RNA synthesis in the IPN profoundly impairs 14 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. learning but not the expression of eyeblink CRs. Inhibition of protein synthesis similarly reduces the rate of conditioning (Bracha et al., 1998). Gomi et al, (1999) further identified a kinase whose expression was increased in the IPN with eyeblink conditioning. Isolation of cDNA and sequence analysis revealed that the expressed gene was KKIAMRE kinase, a member of CDC2-related and mitogen activated protein (MAP) family. Both inhibitors of protein kinases and specific MAP kinase p38 markedly impair conditioning independent of learning, but not CR expression (Chen & Steinmetz, 2000; Zhen et al., 2001). Evidence of synaptogensis or gene transcription and translation, does not preclude a role for of mossy fiber- IPN LTP as a mechanism of learning, such that LTP may be an antecedent or act in concert with synaptogensis to promote memory formation. Like amygdala dependent fear conditioning and hippocampal dependent maze learning, application of an NMDA receptor antagonist, which prevents learning and amygdala and hippocampal LTP, markedly impairs eyeblink conditioning (Chen & Steinmetz, 2000). A reduction in PF-PC synaptic strength at multiple and spatially distinct synapses is an attractive model which could promote the appropriate temporal release of IPN inhibition and hence expression of appropriately timed conditional eyeblink responses. Indeed, single unit recordings of Purkinje cells reveal decreases and increases in firing rates that immediately precede the expression of well-timed CRs (King et al., 2001). The requirements for eyeblink conditioning, like those of PF-PC LTD, are typically associative. Both can be induced by parallel fiber and Purkinje 15 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cell stimulation. To date, molecular mechanisms of cerebellar LTD are becoming more clearly recognized. The induction of cerebellar LTD is initially triggered by activation of mGluRl metabotropic receptor and AMPA receptors. Mutant mice deficient in mGluRl and impaired in the induction of LTD, are also impaired in eyeblink conditioning (Aiba et al, 1994). Further, blockade of cerebellar cortical AMPA receptors, which block the induction of LTD (Wang & Linden, 2000), also has been reported to impair the acquisition and expression of CRs (Attwell et al, 1999; Attwell et al, 2001). Following activation of glutamate receptors, transient activation of protein kinase (PKC) is essential. Transgenic mice with Purkinje cell specific inhibition of PKC, which prevent the induction of LTD, show impairments in eyeblink conditioning (Koekkoek et al, 2003). Conversely, eyeblink conditioning results in an increase in membrane bound PKC specific to cerebellar cortical tissue (Freeman et al, 1998). Further, inhibition of nitric oxide, which blocks the induction of LTD (Shibuki and Okada, 1991) results in attenuated eyeblink acquisition and learning-related neural activity in the IPN (Allen & Steinmetz, 1996). Expression of PF-PC LTD is expressed postsynaptically, possibly as a reduction in the number of functional AMPA receptors produced by endocyotosis. Interestingly, quantitative autoradiography reveals eyeblink conditioning-related decreases in (3 H) AMPA binding to synaptic subpopulations of cerebellar cortical AMPA receptors (Hauge et al, 1998). Additional support for the LTD hypothesis comes from mice deficient in glial fibrillary acidic protein (GFAP) that show normal excitatory synaptic 16 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. transmission and no observable motor deficits, but are markedly deficient in PF-PC LTD and impaired eyeblink conditioning (Shibuki et al, 1996). It is likely that eyeblink conditioning results in plasticity at a number of sites. Here we have chosen to focus primarily at two critical sites of plasticity, the IPN and cerebellar cortex. It should be noted that within each of these regions, synaptic plasticity at sites other then PF-PC and mossy fiber-IPN synapses have been identified such possible sites and mediators of plasticity and their potential contributions to eyeblink conditioning have been reviewed by Hansel et al. (2001). However, these potential mechanisms have yet to be tested in eyeblink conditioning. As pointed out earlier, much of the research in cerebellar synaptic plasticity has focused primarily on PF-PC LTD. However since most of the evidence suggest that eyeblink conditioning can occur independent of the cerebellar cortex, it seems likely that elucidating the molecular mechanisms of IPN plasticity (LTP and/or synaptogensis) may yield for the first time a cellular substrate of cerebellar memory. 17 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Generalization to Humans The results obtained in animal studies of eyeblink conditioning correspond closely with the evidence available in human eyeblink conditioning. The combination of lesion, neuro-imaging and observation in amnesic patients confirm that the cerebellum plays a critical role in eyeblink conditioning in humans as well. Positron emission tomography (PET) in humans reveal changes in glucose metabolism in the cerebellum correlated with eyeblink conditioning (Molchan et al., 1994; Logan & Grafton, 1995; Blaxton et al., 1996; Schreurs et al., 1997). Moreover, functional magnetic resonance imaging (fMRI) show increases in cerebellar cortex and deep nuclear activity over the course of eyeblink conditioning (Lemieux & Woodruff-Pak, 2000). In addition, both PET and fMRI also reveal activation of the medial temporal lobe, which include the hippocampus (Lemieux & Woodruff-Pak, 2000; McIntosh & Schreurs, 2000). In amnesic patients suffering from bi-lateral medial temporal lobe damage, trace conditioning under a long trace interval is markedly impaired, whereas delay eyeblink conditioning is normal (McGlinchey-Berroth et al., 1997; Clark & Squire, 1998; McGlinchey-Berroth, 2000). However, patients with bilateral as well as with unilateral cerebellar lesions ipsilateral to the trained eye are severely impaired in delay and trace eyeblink conditioning (Lye et al., 1988; Solomon et al., 1989; Daum et al., 1993; Topka et al., 1993). In general, across a wide spectrum of mammals including humans the cerebellum is critical for the acquisition, retention and expression of conditional eyeblink responses. 18 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Brief overview of experiments As discussed above there is overwhelming evidence that the cerebellum is the neural hub for the conditioning of discrete motor responses. In addition it is clear that the primary association between the CS and US is established and maintained in the interpositus nucleus. Further, within the cerebellum there is a distribution of function for the specific attributes of eyeblink conditioning. The combination of analytic methods reveals that elements for timing, conditioning and extinction of conditioned eyeblink responses may be distributed between the cerebellar cortex and IPN. Hence, there is no unitary region within the cerebellum that accounts for all aspects of the conditioned response. In spite of this, what is apparent is that lesions or inactivation of the IPN are devastating to the timing, conditioning and extinction of eyeblink CRs. In the following chapters, we present evidence from a series of experiments intended to better characterize the neural components and mechanisms underlying timing, conditioning, expression and extinction of conditioned eyeblink responses. In the initial experiments, we examine timing and conditioning in which electrical stimulation of the IPN is utilized as a CS and as an index of learning-related cerebellar plasticity. Next, we examine the effects of sensory stimulation in the naive animal during acute measures of excitability under control conditions and with IPN 19 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. infusions of GABAa receptor antagonist picrotoxin. Finally, we examine the effects of post-training exposure to the conditioning apparatus following delay eyeblink conditioning. 20 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 2 Timing of Conditioned Responses Utilizing Electrical Stimulation in the Region of the Interpositus Nucleus as a Conditioned Stimulus Introduction A wide array of neurobiological techniques has contributed to delineation of the essential neuroanatomical substrates underlying eyeblink conditioning. Indeed, more than half a century ago, Brogden and Gantt (1942) provided the initial evidence demonstrating a cerebellar role in learning and memory. In these pioneering studies, electrical stimulation of the dog cerebellar cortex produced a number of behavioral responses, including leg flexion, head turns, and more notably, eyeblinks. After repeated pairings in which these responses followed the presentation of a bell, such responses came to be elicited by the presentation of the bell. Today it is well established that the cerebellum is essential for the acquisition and retention of discrete motor responses (McCormick et al., 1983; McCormick & Thompson, 1984a; Lavond et al., 1985; Yeo et al., 1985a; Berthier & Moore, 1986; Mauk et al., 1986; Steinmetz et al., 1986; Thompson & Krupa, 1994, Christian & Thompson, 2003). However, in debate is the functional role of the cerebellar cortex and interpositus nucleus (IPN) in the establishment, storage and expression of adaptively timed conditioned eyeblink responses. Although some have reported that lesions or disruption of cerebellar cortical output completely prevent the acquisition of 21 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. conditioned eyeblink responses (Yeo et al., 1984, 1985b; Garcia, et al., 1999), others only report impairments in acquisition, retention and/or CR timing (McCormick and Thompson, 1983; Lavond and Steinmetz, 1989; Perrett et. al, 1993; Logan et. al, 1994; Chen et al., 1996). It is clear that lesions or inactivations of the anterior lateral IPN completely prevent the formation of eyeblink memory traces in the naive animal (rabbits, rats and mice), and that similar lesions in the trained animal completely prevent the expression of the CR and its distributed neural signature (McCormick et al., 1983 Lavond et al., 1985; Lavond et al., 1990; Clark et al., 1992; Krupa et al., 1993; Chen et al., 1996; Clark & Lavond, 1996). The standard delay paradigm, as originally developed by Gormezano (1962) involves pairing of an initially neutral tone conditioned stimulus (CS) with a coterminating corneal airpuff unconditioned stimulus (US). Following repeated pairing of these stimuli, the tone comes to elicit a conditioned eyeblink response timed to be maximal near the onset of the airpuff. Remarkably, following the establishment of well timed conditioned motor responses, such learning retains an extraordinary degree of temporal flexibility. That is, timing of well established conditioned responses at a specific CS-US interval will with little delay adapt to a novel interval (Coleman & Gormezano, 1971). The results from electrophysiological, pharmacological, tract tracing and lesioning studies reveal that CS and US related neural information converge at the cerebellum. 22 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Both the cerebellar cortex and IPN receive mossy fiber and climbing fiber projections from peripheral CS and US, respectively. Conditioned response expression is conveyed by projections of the IPN to the motor nuclei via the red nucleus. Microstimulation of the IPN and its efferents results in the expression of a number of stereotyped movements, including eyeblinks, indicating that the IPN contains a somatotopic representation of the body (Courville, 1966; Black-Cleworth et al., 1975; Cegavske et al., 1976; Schultz et al., 1979; Rispal-Padel et al., 1982; McCormick & Thompson, 1984; Chapman et, al., 1988; Thach et al., 1993). Interestingly, such stimulation of the IPN in the rabbit at subthreshold levels as a CS when paired with a corneal airpuff results in rapid and robust acquisition of conditioned eyeblink responses. Moreover, such learning shows substantial savings of training when transferred to a tone CS (Tracy, et, al., 1999). Further, previous studies in the cat demonstrate forelimb conditioning to electrical stimulation of the IPN as a CS (Rispal-Padel & Meftah, 1992; Pananceau & Rispal-Padel, 2000) as well. Here we investigate eyeblink conditioning by directly engaging CR pathways via microstimulation of the IPN as a CS with a coterminating corneal airpuff US (see Figure 2.1). 23 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. We examine timing characteristics of the CR in the well trained animal and changes in timing that reflect shifts in the interstimulus interval (ISI) independent of direct peripheral activation of the lateral pontine nucleus or cerebellar cortex. Our results reveal that learning established under these conditions not only results in rapid and robust CRs, but also in changes of peak latencies that are timed appropriately to changes in CS-US interval, just as in conditioning utilizing a tone CS. 24 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Airpuff (US) Cerebellar cortex Inferior Olive Pontine Nucleus IPN CR Figure 2.1 Procedure used to produce eyeblink conditioning. Stimulation of left IPN through a pair of electrodes was used as a conditioned stimulus (CS) and corneal airpuff delivered to the left eye was used as an unconditioned stimulus (US). 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Materials and Methods Subjects Twenty-one adult male naive New Zealand White rabbits (Oryctolagus cuniculus) weighing between 2 and 3 kg were individually housed in a vivarium (Hedco Neurosciences, University of Southern California) on a 12 hr light-dark cycle with ad-lib access to food and water. All procedures and animal care were in accordance with NIH guidelines. Surgery All rabbits were initially anesthetized by subcutaneous injections of xylazine (.08 ml/kg of 100 mg/ml solution) and ketamine (.6 ml/kg of 100 mg/ml solution), and maintained on halothane (2-3%) for the duration of surgery. Bipolar electrodes were created from two stainless steel insect pins, dipped in epoxylite insulation with tips exposed 250 to 350 pm. Insect pins were bound in parallel via dental acrylic with tips set 1 mm apart. Electrodes were stereotaxically positioned in the vicinity of the left anterior interpositus nucleus 5 mm lateral, 0.5-1.0 mm anterior and 13.5-14.5 mm ventral to lambda. Electrode placements in some rabbits were contingent upon the elicitation of eye-blink responses to current application (15-250 pA). Dental acrylic applied over the skull created a head mount where both the final position of the bipolar electrode and a plastic connector piece were fixed. 26 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Apparatus Nictitating membrane movements were measured by a minitorque potentiometer attached via a thread lead hooked through a nylon suture stitched into the left nictitating membrane. Voltage changes were measured by an IBM/PC, where data were analyzed offline. Stimulations during training and threshold tests were delivered to the cerebellum using a Grass Instruments (Quincy, MA) Model S48 stimulator and a constant current isolation unit. Behavioral Training After 7 days of recovery, all animals were habituated to the training apparatus for 3 sessions. Rabbits were then divided into one of three groups. The D-ISI group (N=10) consisted of rabbits that were initially trained to a 500 ms delay (500 ms ISI) then shifted to a decreased 250 ms delay (250 ms ISI). The I-ISI group (N=6) was trained initially with a 250 msec delay and then shifted to an increased 500 ms delay. The Pre-CS group (N=5) was initially presented with CS alone trials and then trained with a 250 ms delay (250 ms ISI) to control for any non-associative effects. 27 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Prior to training IPN stimulation was applied to determine the minimum amount of current required to elicit a just detectable eye-blink or movement in the facial or head area. Interpositus nucleus stimulation consisted of a 350 ms electrical train delivered at a rate of 200 Hz with a pulse width of 0 .1 ms, at between 15 and 150 pA. Stimulation current was initially applied at 50 pA and decreased or increased at 4 pA increments until responses were observed. Thresholds for eye-blink responses were determined by the potentiometer, where response amplitudes as small 0.2 mm were measured. Movements in facial or head areas were verified by observers. Following threshold measurements, all rabbits were trained to delay (250 or 500 ms ISI) eye blink conditioned procedures with exception of group Pre-CS. Each paired daily session consisted of 108 trials divided into 12 blocks, with each block consisting of 1 CS alone trial and 8 CS-US paired trials. Each CS alone daily session consisted of 100 CS alone trials divided into 10 blocks. Inter-trial intervals were randomly varied from 20 to 40 seconds. In all groups the CS consisted of either a 350 or 600 ms IP stimulation (200 Hz, pulse width 0.1 ms, 7.5 to 75pA), whereas the US consisted of a coterminating 100 ms corneal airpuff (3 psi) delivered to the left eye. Conditioned stimulus current for each animal was determined by taking 50% of threshold value required to elicit an eye-blink or movement in the head or facial area. During all training sessions CRs were defined as any extension of the left nictitating membrane equal to or greater than 0.5 mm occurring between 35 and 250 or 500 ms following CS onset. All animals were trained to criterion, 8 CRs within 9 28 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. consecutive trials within a given session, plus one additional session of acquisition training. A total of 6 rabbits, 5 from D-ISI and 1 from Pre-CS group did not meet learning criterion (8 CRs within 9 consecutive trials) within the first 2 sessions (216 trials) of acquisition training and were not included in the data analysis. Excluded animals were trained for an additional 2 sessions (total of 4 sessions), showing no more then 30 percent CRs within any given session. Further in two of the excluded animals, training under a tone CS for up to 3 sessions of delay conditioning were added during which no more than 15 percent CRs were observed within the final day of conditioning. Histology After the final training session, the location of the bipolar electrode was determined the by creation of marking lesions by passing 100 mA of dc for 10 s. Animals were overdosed with intravenous injections of euthasol solution (1 ml), then perfused with 0.9% saline and 10% formalin. Brains were extracted and embedded in a mixture of gelatin and albumin. Embedded brains were frozen and cut into 80 pm slices. Slices were mounted and stained using both Cresyl violet and Prussian blue. Electrode placements were assessed using a dissecting microscope. 29 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Results Figure 2.2 shows mean conditioned response percentages over the course of training sessions for groups I-ISI (250 ms ISI -> 500 ms ISI: N=6), D-ISI (500 ms ISI 250 ms ISI: N=5) and Pre-CS (CS alone-> 250 ms ISI: N=4). The Pre-CS group during IPN stimulation alone sessions produced CRs at no more than spontaneous levels. A significantly faster rate of acquisition was found in the I-ISI group initially trained to the 250 ms ISI, in comparison to the D-ISI group trained to the 500 ms ISI (Repeated Measures ANOVA, F (1,9) = 6.114. p < .05). Figure 3 indicates that all rabbits reached learning criterion (8 CRs within 9 consecutive trials) within the first two days of paired training. In addition, a significant difference between I-ISI (M trials to criterion = 36.67), D-ISI (M trials to criterion = 111.4) and Pre-CS (M trials to criterion = 63.6) groups was identified for mean number of trials to learning criterion (One way ANOVA, F(2,12)=3.926, p < .05). Further, Post Hoc (LSD) analysis revealed that Group I-ISI showed a significantly fewer trials to learning criterion than group D-ISI (g < .05). However, no significant differences were found between groups I-ISI and Pre-CS or groups D- ISI and Pre-CS. A closer inspection of data revealed a significant difference between the mean percent CRs during the initial half of the first conditioning session between group I-ISI and Pre-CS (One way ANOVA, F(l,8) = 5.503. p < .05). 30 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 0 0 90 80 70 60 50 40 30 20 10 0 3 4 1 2 Number of Sessions Figure 2.2 Conditioned response (CR) percentages (%) plotted for each of half of a training session. Rabbits in the Pre-CS group received stimulation alone (session 1,2) followed by paired stimulation and airpuffs (sessions 3,4) under a 250 ms interstimulus interval (ISI). Rabbits in group I-ISI were initially trained to a 250 ms ISI during sessions 1 and 2 and shifted to a 500 ms ISI for sessions 3 and 4. Rabbits in group D-ISI were initially trained to a 500 ms ISI for sessions 1,2 and shifted to a 250 ms ISI during sessions 3 and 4. 31 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ■ D -IS I ■ I-IS I □Pre-C S Figure 2.3 Mean number of trials to learning criterion (8 CRs within 8 consecutive 9 consecutive paired trials). All rabbits reached learning criterion (8 CRs with 9 consecutive trials) by the end 2n d paired (CS-US) training session. Training utilizing an initially shorter interstimulus interval (I-ISI) showed a faster rate of learning then the group initially trained with a longer interval (D-ISI) or receiving stimulation alone (Pre-CS) prior to paired training. 32 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. PEAK O N SET 800 i 700 - 600 - 500 - jS 300 200 - 100 - 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 B 800 700 - 600 - -5 T 500 - S '400- 2 300 200 100 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 T rain in g B lo ck s Figure 2.4 Mean onset and peak conditioned response (CR) latencies as measured in milliseconds (ms) over the course of training blocks, consisting of 1 CS alone and 8 paired trials. A. group I-ISI; B. group D-ISI. Arrow indicates ISI shift. 33 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2.4 shows the average eyeblink latencies over the course of training for groups I-ISI (figure 4a) and D-ISI (figure 4b). Group I-ISI following initial training blocks (12 trials) shows a substantial decrease in onset and peak eyeblink latencies final 6 blocks of 250 ms ISI training for group I-ISI yielded mean onset and peak latencies of 131.1 and 235.9 ms, respectively. Group D-ISI after initial training blocks also shows a considerable decrease in onset and peak eyeblink latencies with the final session 6 blocks of 500 ms ISI training exhibiting a mean onset latency of 222.2 ms and mean peak latency of 436.1 ms. Following shifts in the ISI the I-ISI group revealed a significant increase in peak eyeblink latencies between the final session of 250 ms and 500 ms ISI training (Matched Pairs T-test, t(9) = 4.709, g < .01). Conversely, the D-ISI groups following shifts in the ISI showed a significant decrease in mean peak eyeblink latencies between the final session of 500 ms and 250 ms ISI training (Matched Pairs T-test, t(9) = -6.801, g < .001). Figure 2.5 shows electrode placements based on histological reconstruction of lesions. Such evidence reveals that a majority of electrodes tips were positioned in the IPN (figure 5a: 9, figure 5b: 8) or its overlying white matter (figure 5a: 12, figure 5b: 11) with a few positioned in the cerebellar cortex (figure 5a: 1, figure 5b: 4) and dentate nucleus (figure 5a: 1, figure 5b: 3). 34 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IPN Figure 2.5 Illustration of coronal sections through the rabbit cerebellum. Filled circles depict electrode tips used for electrical stimulation as a CS. IPN, interpositus nucleus. A. Plate of a cerebellar section 0.5 mm anterior to lambda. B. Plate of a cerebellar section 1.0 mm anterior lambda. 35 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Discussion In the initial portion of this study it was demonstrated that subthreshold electrical stimulation of the IPN (including regions in close proximity) as a CS and a corneal airpuff US resulted in rapid and robust acquisition of conditioned eyeblink responses, in accordance with Tracy et al. (1999). Here, rabbits trained with a 250 ms CS - US interval met learning criterion on average in 36.67 trials. In contrast, rabbits trained with a tone CS, under similar conditions (250 ms ISI) typically reach learning criterion following approximately 140 trials (Thompson & Krupa, 1994). In addition, the rate of conditioning using IPN stimulation was faster under a shorter (250 ms) ISI then a longer (500 ms) ISI, as similarly found using peripheral CSs (Smith et al, 1969). Moreover, the CRs observed under these conditions at either a 250 or 500 ms CS-US interval peaked prior to and near the presentation of the US. Further, in a separate group (Pre-CS) of naive rabbits (N=4), half of which displayed stimulation evoked eyeblinks, subthreshold IPN stimulation (CS alone) over the course of 200 trials did not result in the expression of any measurable eyeblink responses. In addition, such repeated presentation of the CS prior to conditioning resulted in a subsequent retardation of learning a result consistent with theories of latent inhibition. 36 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Interestingly, in all rabbits regardless of the initial behavior elicited by electrical stimulation (head turn or whisker movement), conditioning resulted in the expression of conditioned eyeblink responses. Thus, eyeblink CRs established here following repeated pairings of the CS and US were not a result of a non-associative process (sensitization) but rather the acquisition of a well-timed discrete motor response. The primary results we report here indicate that conditioned eyeblink responses established with IPN stimulation as a CS adjust to abrupt shifts of the CS - US interval. Timing of CRs established under the initial long interval (500 ms) decreased following shortening of the ISI, so that CRs were once again timed prior to, and nearly maximal at the presentation of the US. Similarly, rabbits trained under the initial short (250 ms) ISI resulted in an increase of CR peak latency when shifted to a 500 ms ISI. These results are comparable with those of previous studies using tone (Coleman & Gormezano, 1971) or pontine nucleus stimulation CSs (Steinmetz, 1990; Perrett, 1998) where peak CR latencies in well trained animals contingently adjusted to widening and/or shortening of the ISI. Therefore an important implication of our results is that adaptive timing of CRs does not appear due to processes in the portion of the CS circuitry prior to the cerebellum. A number of studies suggest that timing of conditioned eyeblink responses depends on plasticity in the cerebellar cortex. Single and multiple unit recordings of cortical neurons exhibit firing patterns that model the expression of well-timed conditioned 37 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. eyeblink responses (McCormick & Thompson, 1984; Berthier & Moore, 1986; Foy et al, 1992; Katz & Steinmetz, 1997; Tracy & Steinmetz, 1998). Conversely, lesions of cerebellar cortex have been shown to disrupt timing of CRs as well as retard acquisition and retention (McCormick & Thompson, 1984b; but see Garcia et al., 1999). Mutant pcd mice completely void of Purkinje cells, the sole output of the cerebellar cortex, are impaired in eyeblink conditioning but do learn and CRs expressed tended to have shorter peak latencies then wild types (Chen et al., 1996). In addition, more recent work by Bao et al, (2002) suggests that CR expression and timing may be completely dissociable and that memories for eyeblink CRs may be encoded in a functionally distinct manner in both cerebellar cortex and IPN. In their study, rabbits displaying well-timed CRs were given sequential IPN application of the GABAa agonist muscimol and the GABAa antagonist picrotoxin which resulted in the expression of reduced onset and peak CR latencies poorly timed to the US. Such results are consistent with studies by Mauk and associates using high concentrations of picrotoxin (Garcia and Mauk, 1998; Medina et al., 2001). This disruption of CR timing in animals following blockade of cortical outputs suggests that the memory traces for learned response timing may be expressed by the cerebellar cortex. Moore and Choi (1997) have proposed that timing of responses occurs among an ensemble of cells that convey CS related information, including pontine nucleus mossy fibers, granules cells and cerebellar Purkinje cells. Mauk and colleagues (2003) argue that CR timing is established via temporal coding of granule 38 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cells resulting in long term changes in parallel fiber - Purkinje cell synapses. Bao et al, (2000) showed that when electrical stimulation of the lateral reticular nucleus was used as a CS, adaptive behavioral timing of the CR occurred even after the lateral pontine nucleus was inactivated, i.e. the pontine nuclei do not play a necessary role in adaptive timing of the CR, a result consistent with present findings. However, given the cerebellar circuitry (Figure 1.1) stimulation of the IPN was likely to activate reciprocal projections from the IPN to the pontine nucleus or the IPN to the inferior olive (inhibitory) or the IPN to the cerebellar cortex, therefore providing a number of means by which the cerebellar cortex maybe actively engaged during the acquisition or maintenance of well timed CRs. To a similar extent, stimulation of white matter overlying the IPN is likely to activate cerebellar cortical neurons. A number of mechanisms might be involved in the rapid and robust learning established here. An obvious interpretation is that stimulation of the IPN may have operated as a more salient CS resulting in faster learning. Indeed, eyeblink conditioning using a louder tone results in a faster rate of acquisition (Scavio & Gormezano, 1974). Such a strong auditory stimulus is likely to recruit more fibers along CS pathways resulting in more robust activation of cerebellar circuits. Similarly, direct electrical stimulation of a specific nucleus along the CS pathway is also more likely to activate pathways representing several sensory modalities. Such a region, the pontine nuclei, are indeed a major pre-cerebellar site of sensory convergence. Lesions of the middle cerebellar peduncle (MCP), a major cerebellar 39 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. projection of the pons, abolish CRs established by auditory, visual and somatosensory CSs (Lewis et al., 1987). In fact, direct electrical stimulation of the lateral and dorsal pons as a CS (airpuff US) results in rate of acquisition (M trials to criterion = 45) similar to the levels attained in this study (Tracy et al., 1998). Therefore, stimulation in the region of the IPN, a site of memory formation, is likely to depolarize a larger number of neurons then a standard 85 db tone. Such activation would thereby increase the number of potential neurons undergoing plasticity and collectively drive the earlier and robust expression of CRs. Interestingly, a closer inspection of the histology revealed that across all groups that animals that showed the fastest conditioning (M trials to criterion =43) all had electrode placements just above or lateral to the IPN, whereas animals with electrode placements that penetrated IPN showed slower learning (M trials to criterion =89.3). Moreover, animals which failed to reach learning criterion and produced no more then 30% CRs over the course of 4 sessions (432 trials) of training (excluded from the present results) all had IPN electrode placements more ventral then those showing conditioning. Therefore, in the present experiment learning was more efficient under conditions in which the IPN remained intact versus those in the IPN may have been damaged a result consistent with partial lesions of the IPN (Clark et al., 1984). The demonstration of latent inhibitory learning in the Pre-CS group suggest that neural systems involved in the retardation of eyeblink conditioning are not likely to occur afferent to the cerebellum. To date, the evidence from a limited number of 40 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. studies suggest that the hippocampus and/or entorhinal cortices my play important role in these latent learning effects. Both lesions of the hippocampus that include entorhinal cortex and those specific to the entorhinal cortex attenuate retardation to pre-conditioning exposure of a tone CS alone or uncorrelated presentations of CS and US (Shohamy et al., 2000; Allen et al., 2002). Thus, the present results could indicate that an interaction between the cerebellum and entorhinal cortex may exist efferent to the cerebellum. Overall these data suggest that learning and CR timing using IPN stimulation as a CS are consistent with conditioning using peripheral stimuli. Further, these results raise questions about possible mechanisms of CR timing, particularly involving sites afferent to the cerebellum, such as the pontine nuclei. 41 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 3 Evidence of Learning-Related Interpositus Nucleus Plasticity Introduction Considerable evidence suggests that essential memory traces for classically conditioned eyeblink responses are established and maintained in the interpositus nucleus (IPN) of the cerebellum. Information pertaining to both conditioned stimulus (CS) and unconditioned stimulus (US) converge at the IPN and cerebellar cortex. Neural unit recordings from IPN neurons reveal firing characteristics correlated with the development and expression of conditioned responses (CR) (McCormick et al. 1981; McCormick and Thompson 1984b; McCormick and Thompson 1984a; Berthier and Moore 1990; Sears & Steinmetz 1990b; Yang and Weisz 1992; Gould and Steinmetz 1996; Freeman and Nicholson 2000; Stanton, 2000; Steinmetz, 2000; Rogers et al. 2001; Tracy et al. 2001). Lesions of the IPN prevent learning in naive animals and permanently abolish CRs in well-trained animals but do not affect performance of URs (Lincoln et al, 1982; Lavond et al, 1984, 1987; Woodruff-Pak et al, 1985; Thompson, 1986; Sears and Steinmetz, 1990; Chen et al, 1996). 4 2 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Finally, reversible inactivation of the IPN or it efferents block expression of eyeblink CRs, but do not prevent learning at all (Clark et al, 1992; Krupa et al, 1993; Nordholm et al, 1993; Krupa and Thompson, 1995; Hardiman et al, 1996). Thus, such results suggest that IPN is essential for the acquisition and expression of eyeblink CRs and a prime candidate for learning-related plasticity. To date, evidence of learning-related IPN plasticity has been collected using an array of methods, including electrophysiology, electron microscopy, molecular biology and pharmacology. In spite of this, the relative accumulation of such evidence has been sparse. Infusions of transcription and translation inhibitors into IPN markedly impair acquisition but not performance of eyeblink CRs (Bracha et al, 1998; Gomi et al, 1999). Conversely, differential display PCR analysis reveals conditioning-specific gene expression of a cdc2 related mitogen activated protein KKIAMRE kinase specific to deep cerebellar nuclei (Gomi et al, 1999). In addition, unbiased stereological analysis of IPN tissue using electron microscopy reveals learning- specific increases in IPN excitatory synapses (Kleim et al, 2002). 43 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Further, it has been argued that pharmacological dissociation of the cerebellar cortex and IPN, which in the previously trained and extinguished animal results in the expression of short-latency eyeblink responses, is evidence of IPN plasticity (Garcia et al, 1999; Medina et al, 2000, 2001; Ohyama and Mauk, 2001; Bao et al, 2002; Ohyama et al, 2003). In addition, both in-vivo and in-vitro preparations suggest long term synaptic and cell specific candidate mechanisms that may underlie development and storage of eyeblink conditioning memory traces. The results described in chapter 2 suggest that the conditioning parameters using electrical stimulation of the IPN as a CS and a corneal airpuff US share similar learning characteristics to those using peripheral CSs. Moreover, this learning paradigm may provide an attractive method to evaluate potential learning related plasticity within a nucleus essential for the acquisition, retention and expression of discrete motor memories. Previously, Tracy et al. (1998) demonstrated that stimulation of the pontine nucleus as a CS results in learning-specific decreases in pontine-stimulation to eyeblink thresholds, whereas similar thresholds measured in ventral IPN remained relatively unchanged. Together these data suggest that conditioning-related plasticity maybe localized efferent to the pontine nucleus and afferent to ventral IPN. 4 4 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In the present study we intend to utilize similar methods directed towards measuring potential plasticity in one such candidate, the dorsal IPN, where lesions as small as 1 m m 3 can completely abolish the expression of eyeblink CRs (Lavond et al, 1984). Therefore, in the present experiment we intend to examine dorsal IPN electrical stimulation to eyeblink thresholds prior to and following the acquisition and extinction of classically conditioned eyeblink responses established by electrical stimulation of dorsal IPN as a CS. 45 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Materials and Methods Subjects Eleven adult male naive New Zealand White rabbits (Oryctolagus cuniculus) weighing between two and three kg were individually housed in a vivarium (Hedco Neurosciences, University of Southern California) on a 12 hr light-dark cycle with ad-lib access to food and water. All procedures and animal care were in accordance with NIH guidelines. Surgery All rabbits were initially anesthetized by subcutaneous injections of both rompun and ketamine, and maintained on halothane (2-3%) for the duration of surgery. Bipolar electrodes were created from two stainless steel insect pins, dipped in epoxylite insulation with tips exposed 250 to 350 um. Insect pins were bound in parallel via dental acrylic with tips set 1 mm apart. Electrodes were stereotaxically positioned in the vicinity of the left anterior interpositus nucleus 5 mm lateral, .5-1.0 mm anterior and 13.5-14.5 mm ventral to lambda. Electrode placements in some rabbits were contingent upon the elicitation of eye-blink responses to current application (15-250 uA). Dental acrylic applied over the skull created a head mount where both the final position of the bipolar electrode and a plastic connector piece were fixed. 46 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Apparatus Nictitating membrane movements were measured via a thread lead coming from a minitorque potentiometer, which was attached to a nylon suture stitched into the left nictitating membrane. Voltage changes were measured by an IBM/PC, where data were analyzed offline. Stimulations during training and threshold tests were delivered to the cerebellum using a Grass Instruments (Quincy, MA) Model S48 stimulator and a constant current isolation unit. Behavioral Training (see Figure 3.1) Following recovery from surgery all rabbits were adapted to the training apparatus as described in chapter 2. The next day rabbits were placed in the training apparatus during which electrical stimulation was applied to determine thresholds for eyeblink or movement in the facial or head area. Electrical stimulation consisted of a 350 ms electrical stimulation delivered at a rate of 200 Hz at a pulse width of .1 ms between 15 and 150 piA. Stimulation current was initially applied at 50 /M. and decreased or increased at 4 piA increments until responses were observed. 47 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Thresholds for eyeblinks were determined by potentiometer and defined as the minimum amount of current required to elicit a just measurable eyeblink ranging from .3 to .5 mm in amplitude on 2 of 3 trials. Thresholds for movements in facial or head areas were defined as the minimum amount of current required to elicit a just detectable movement as verified by second or third observers. Immediately following threshold measurements rabbits were trained to a 250 msec delay conditioning procedures as described in detail in chapter 2. The CS consisted of a 350 ms electrical stimulation (200 Hz, pulse width 0.1 ms, 7.5 to 75pA) delivered through stimulating electrode, whereas the US consisted of a coterminating 100 ms corneal airpuff (3 psi) delivered to the left eye. Conditioned stimulus current for each animal was determined by taking 50% of threshold value required to elicit an eye-blink or movement in the head or facial area. Animals were trained to an acquisition criterion defined as the production of 8 CRs within 9 consecutive trails within a given session, plus an additional session of overtraining the next day. Following the cessation of the final overtraining trial, thresholds measurement were again measured. 48 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Surgery / Baseline A daptation A daptation Threshold / recovery Acquisition Acquisition / Post- A cquisition Threshold Extinction Extinction / Reacquisition Post- / Post- Extinction Reacquisition Threshold Threshold Days 1-8 Day Day 9 10 Day 11 Day 12 Day 13 Day 14 Day 15 Figure 3.1 Experimental procedures used to investigate any conditioning-related changes in interpositus nucleus stimulation to eyeblink thresholds. Following surgery and recovery (days 1-8) rabbits were adapted to the training apparatus (days 9-10). Thereafter, baseline threshold measurements were made and eyeblink conditioning began (day 11). Following a 2n d session of conditioning, post-acquisition thresholds were measured (day 12). The next day, extinction (CS alone) training started (day 13), followed the next day by a 2n d session of extinction and post-extinction threshold measurements (day 14). Finally rabbits were presented with a last conditioning session followed by post-reacquisition threshold measurements. 49 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The next day extinction training sessions began, each session consisting of 100 CS alone trials. The criterion for extinction was defined as the expression < 25% CRs within a given session. Immediately following, thresholds were once more measured. The following day reacquisition training was initiated, which was identical to procedures used during acquisition. Then threshold measurements were determined once more. 50 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Results Figure 3.2 depicts mean percentage CRs over the course of two sessions of acquisition training, extinction training and one session of reacquisition training. All rabbit reached learning criterion within the first session of acquisition training. In addition, all rabbits reached extinction criterion within the first or second session of extinction training. Figure 3.3 shows mean electrical stimulation to eyeblink thresholds prior to acquisition, following acquisition, extinction and reacquisition. Wilcoxon matched pairs (dependent t tests), revealed significant differences between pre-acquisition and post acquisition mean thresholds (z = 9.316, p > .001). In addition, post-acquisition and post-extinction mean thresholds levels were significantly different (z = -3.506,p > .001). Significant differences were found between pre-acquisition and post extinction thresholds (z = 3.213, p > .05) and post-acquisition and post-reacquisition thresholds (z = 3.763, p > .01). Reconstruction of lesions based on histological analysis revealed bipolar electrode placements within dorsal anterior lateral portion IP as well as regions just dorsal and lateral to the IPN (figure 3.4). 51 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 0 0 90 80 70 60 50 40 30 20 10 0 Acq 1 Acq 2 Ext 1 Ext 2 Reacq Figure 3.2 Mean conditioned response (CR) percentages (%) plotted for each of half of a training session. Each acquisition (Acq) session consisted of 108 trials divided into 12 blocks of 9 trials (1 CS alone trial, 8 paired trials). Extinction (Ext) sessions consisted of 100 CS alone trials, while a session of reacquisition (Reacq) was identical to an acquisition session. 52 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ■ Baseline Thres. BPost-acq. Thres □ Post-Ext. Thres. □ Post-Reacq. Figure 3.3 Electrical stimulation to eyeblink thresholds measured as current (uA). Baseline thresholds (Baseline Thres.) were measured just prior to acquisition. Following acquisition training, thresholds were measured again (Post-acq. Thres.). 53 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. interpositus nucleus Figure 3.4. Illustration of coronal sections through the rabbit cerebellum. Circles depict electrode tips used for electrical stimulation as a CS. IPN, interpositus nucleus. A. Plate of a cerebellar section 0.5 mm anterior to lambda. B. Plate of a cerebellar section 1.0 mm anterior lambda. 54 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Discussion The present findings provide strong evidence that classical eyeblink conditioning is associated with a general modification of cerebellar excitability. Under the present conditions, acquisition using electrical stimulation of the IPN as a CR was both rapid and robust, as in the previous chapter. In addition, extinction training yielded a relatively rapid reduction in CR expression compared to extinction using peripheral CSs. Results of the present study demonstrate that acquisition of eyeblink CRs yield a dramatic decrease in IPN stimulation to eyeblink thresholds, whereas extinction is associated with a relative increase in eyeblink thresholds. Moreover, reacquisition training, which resulted in the return of CRs at a level similar to the second session of paired training, was correlated with a return of post acquisition threshold levels. In a separate group of animals (n=4) described in the previous chapter, pre-training CS alone trials, which did not result in the expression of eyeblink responses, revealed no significant change in IPN stimulation to eyeblink thresholds. The stability of such thresholds following 200 CS alone trials indicates that the decrease in thresholds following acquisition is not likely due to desensitization, but suggests rather an associative mechanism. The present results have implications for the types of IPN plasticity that may underlie associative memory established in delay eyeblink conditioning. As described earlier, such candidate mechanisms of IPN plasticity include long-term 55 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. potentiation and development of new synapses as well as intrinsic increases in IPN excitability. Indeed, a cell-specific increase in IPN excitability is an attractive model for plasticity underlying eyeblink conditioning and consistent with the present results. An important characteristic of deep cerebellar neurons is the pronounced rebound depolarization that can trigger a series of action potentials, which are typically generated by the offset of inhibitory post-synaptic potentials (IPSPs). A primary source of IPSPs in deep cerebellar neurons are provided by Purkinje cell action potentials triggered by climbing fiber input (Aizenman et al, 2003). Aizenman and Linden (2000) demonstrated in vitro that stimulation of mossy fibers coupled with the blockade of GABAergic input resulted in increased deep cerebellar neuron synaptic spikes. In the present experimental conditions, airpuff related activation of climbing fibers, could trigger Purkinje cell action potentials, coupled with direct stimulation of the IPN as a CS could in a similar fashion promote intrinsic increases in IPN excitability. If such is the case, subsequent IPN stimulation could sufficiently activate IPN neurons to trigger the expression of eyeblink CRs. Further, such results could account for the learning related decrease in IPN stimulation to eyeblink thresholds in the present study. Long-term potentiation (LTP) of mossy-fiber synapses has also been proposed as a mechanism of IPN plasticity. However, as of yet there is only a single study demonstrating that tetanus stimulation of mossy fibers, coupled with depolarization of the IPN neurons could result in LTP (Racine et al, 1986). To date, very little is 56 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. known about the intracellular mechanisms that may govern mossy fiber-IPN LTP. Perhaps, the strongest direct evidence of a mechanisms that my underlie eyeblink memory traces comes from a study by Kleim et al (2002) suggesting synaptogensis of mossy fiber-IPN synapses. As described earlier, eyeblink conditioned rats showed more excitatory synapses per neuron within the IPN than both explicitly unpaired and untrained controls. Such an increase in synapses would be permissive to enhancing the responsiveness of IPN neurons to sensory input such as an auditory CS. In the present study, it is likely that the process of stimulating the IPN may have also resulted in the activation of mossy fiber terminals. This, coupled with the airpuff, could have established a similar increase in mossy fiber-IPN synapses. Therefore, the methods used to index IPN thresholds here could conceivably have detected conditioning-related increases in mossy-IPN synapses. One limiting factor in a mechanism such as synaptogenesis would be the time course of its development versus rate of learning in eyeblink conditioning. Eyeblink conditioning, as described earlier in chapter 2, typically develops slowly over the course of 140 trials separated over a two-day period. However, it is not unusual for learning to be established during an initial session of training over the course of < 1 hour. Indeed, in the present experiment conditioning was very rapid requiring on average of 40 trial or ~ 20 min, with one animal learning after only 7 trials or ~ 3.5 min. Examination of the time course of synaptogenesis in hippocampal neurons reveals development of synapses as fast as 1 hour. Therefore, if synaptogenesis at 57 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IPN synapses occurs at a similar rate it is unlikely that synapse formation represents initial learning, but might rather serve a mechanism for the long-term maintenance of memory traces. Hence, the initial development of learning may result from non- morphological changes such as LTP or cell specific increase in excitability that may set the context for growth of new synapses (dendritic spines). The present results do not rule out the possibility that the learning-related increase in IPN stimulation to eyeblink thresholds results from changes outside of the cerebellum. It is conceivable in the present experiment that threshold changes could represent excitability changes downstream from the IPN. Known IPN efferents include reciprocal projection to both pontine nucleus and inferior olive, and direct projections to thalamus and red nucleus. Studies have reported limb flexion conditioning-related changes in red nucleus and thalamo-cortical pathway excitability in the cat (Murakami et al, 1988; Meftah and Rispal-Padel, 1994). However, if such changes develop in eyeblink conditioning they are not critical, given that inactivation of the superior cerebellar peduncle, the sole output of the IPN, which prevents expression of eyeblink CRs but does not prevent learning at all (Krupa & Thompson, 1995). Further, the results by Tracy et al (1998) which demonstrate that ventral IPN stimulation to eyeblink remains unchanged following eyeblink conditioning suggests that in the present study conditioning related changes in excitability may not be due to changes efferent to the ventral IPN such as the red nucleus. 58 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Also, very interesting in the present results are the increases in eyeblink thresholds following extinction of eyeblink CRs. Close examination of thresholds revealed a significant increase in eyeblink thresholds, which however remained significantly less than the initial baseline (pre-training) levels. Such results may be evident of residual cerebellar plasticity retained following extinction of CRs. An abundance of behavioral data from this and prior studies demonstrate that post-extinction reacquisition of CRs occurs considerably faster than initial conditioning, suggesting two possibilities: that extinction results in the formation of a separate memory trace, or that extinction results in inhibition of the original memory trace or of course both. A recent study by Mauk and colleagues (2000) report that infusions of GABAa receptor antagonist picrotoxin following extensive extinction training for as long as 45 sessions resulted in the unmasking of residual IPN plasticity as indexed by production of short latency ‘CRs’. However to date, all electrophysiological studies of the IPN have yet to report any evidence of a sustained neural model of the CR following extinction. Interestingly, a study by Steinmetz and colleagues (1997), using single-cell recordings, reveal a continued pattern of neural discharge in the cerebellar cortex that persists even after CRs are no longer observed. Thus, in the present study the maintenance of cerebellar plasticity following extinction could represent cerebellar cortical inhibition overlying acquisition related IPN plasticity. 59 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Perhaps most interesting in the present study, and also evident in chapter 2, was that electrical stimulation of the IPN prior to training elicited a range of behaviors, including head turns, whisker movements, lip curls, forepaw flexion and even eyelid opening. However, with conditioning, in all animals observed, IPN stimulation evoked exclusively eyeblink responses. Further, in chapter 2 untrained control animals not producing eyeblinks to stimulation, receiving 200 stimulations of the IPN did not produce any observable changes in evoked behavior. In the present experiment, even following extinction of eyeblink CRs and subsequently increasing the level of stimulation, which returned expression of eyeblinks, did not produce an overt expression of pre-training behaviors. These results are strong evidence that somatotopic representations maintained in the cerebellum, in particular the IPN, maybe reorganized by conditioning of discrete motor responses. Indeed, learning-dependent alterations in motor representation have been previously demonstrated in human (Cohen et al, 1993; Pearce et al, 2000), monkey (Nudo et al, 1996), and rodent (Kleim et al, 1998; Remple et al, 2001; Conner et al, 2003) motor cortex. Interestingly, in a recent report by Kleim et al, (2004) training of skilled reaching tasks in rats was correlated with an increase synapse formation and reorganization of motor cortex representations. Altogether the present results are consistent with models of cerebellar learning that predict that the establishment of classically conditioned discrete motor responses 60 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. result from long lasting increases in IPN synaptic/neuronal excitability and/or morphology. Moreover, the effects of subsequent extinction training provide evidence that neuronal inhibition may play a prominent role in the extinction of conditioned eyeblink responses. Further, the present results reveal evidence of conditioning-dependent changes in cerebellar somatotopic representations. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 4 The Effects of Sensory Stimulation on Acute Measures of Interpositus Nucleus Excitability Introduction Central to cellular models of associative learning is that development of plasticity and its product, memory, result from the coincidental activation of stimulus inputs converging on a given population of neurons. Examination of this hypothesis requires defining in detail critical neural elements fundamental to a particular learning paradigm. Pavlovian eyeblink conditioning is one such paradigm in which the underlying neural circuits have been well delineated. However, to date there is little evidence in cerebellar dependent eyeblink conditioning that the conditioned stimulus (CS) and the unconditioned stimulus (US) simultaneous activate cerebellar neurons. Neural unit recording studies reveal single IPN neurons that respond singly and in combination to both CS and US input. Tone related activation of IPN neurons is typically characterized by a relatively short onset (42.4 ms) and transient pattern of discharge, offsetting well before the onset of the airpuff US. Therefore, under standard delay eyeblink conditioning procedures in which a 250 or 500 ms CS-US interval is utilized, some neural signature of the CS must be maintained over the course of the interstimulus interval (ISI). Seemingly contradictory is that eyeblink 62 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 0 0 90 - 80 ■ 70 ■ 6 0 - u +J c HI 5 0 - a. 4 0 - 3 0 - 2 0 - 10 - -50 0 50 100 200 800 400 ISI (ms) Figure 4.1. Depicts asymptote level of percentage CRs as a function of delay eyeblink conditioning at different interstimulus intervals (ISI). Adapted from Gormezano (1976). 63 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. conditioning which can be established to a maximum CS-US interval of 3000 ms does not occur at intervals less than 50 ms (Gormezano et al, 1983). Figure 4.1, depicts conditioning as a function of ISI revealing optimal levels of conditioning are established under ISIs roughly between 250 and 400 ms (Kehoe, 1976). Regardless of the ISI, timing of CRs in well trained animals consistently onset just prior to and maximal to the presentation of the US. To date, results from lesion, pharmacological and genetic analysis studies demonstrate timing of conditioned eyeblink response are markedly affected by disruption of cerebellar cortical function. In mice deficient of Purkinje cells the sole output of the cerebellar cortex, conditioning which is significantly slower than wild type mice show reduced amplitude and onset latencies of CRs (Chen et al, 1996). Aspirations of selective regions of the cerebellar cortex reveal deficits in conditioning, amplitude and timing of learned eyeblink responses (McCormick and Thompson, 1983; Lavond and Steinmetz, 1989; Perrett et al, 1993; Logan et al, 1994; Chen et al, 1996). Further, it has been argued that infusions of GABAa receptor antagonist in the IPN, functionally and selectively disconnect the cerebellar cortex from IPN resulting in the expression of poorly timed eyeblink responses. In contrast, permanent and temporary lesions of IPN prior to training prevent conditioning, while lesions in the well trained completely abolish the expression of eyeblink responses. Therefore, it has been hypothesized that timing functions established in the cerebellar cortex are transmitted to the IPN and combined with 64 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. representations of the associative memory. Alternatively, if timing of CRs can be established with lesions of the cerebellar cortex, as has also been demonstrated, do cells of the IPN contain a template of excitability or responsiveness that maybe capable of yielding CR timing functions integrated with associative memories? During the early adaptation of rabbit eyeblink conditioning paradigm for the neural analysis of learning and memory, Young et al, (1976) demonstrated that eyeblink responses evoked by direct electrical stimulation of the abducens motor nuclei the final common output of the eyeblink CR and UR prior to conditioning could be altered during the presentation of a auditory stimulus. In particular, stimulation following the initial 160 ms segment of a 350 ms tone resulted in potentiation of eyeblink amplitudes that persisted even following the offset of the auditory stimulus. Such results, as suggested by the authors resembled eyeblink conditioning ISI functions and were potential evidence of a molar stimulus trace (Young et al, 1976). In the present experiment, we intend to examine responsiveness of IPN neurons to electrical stimulation as indexed by the expression of eyeblink responses under different levels of auditory stimulation in the naive animal (see figure 4.2). Moreover, we intend to sample the responsiveness of these neurons at multiple time points throughout the duration of an acoustic stimulus (see figure 4.3). 65 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Cerebellar cortex~^> Pontine Nucleus Inferior Olive IPN TONE Red Nucleus 85 or 95 dB ABN Eyeblink Figure 4.2 Procedure used to measure stimulation to eyeblink thresholds during the presentation of auditory stimuli. 66 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Materials and Methods Subjects and Surgery Twelve New Zealand White rabbits weighing between 2 and 2.2 kg were used in the present experiment. All animals were given subcutaneous injections of xylazine, followed by ketamine while maintained on 1.5 to 2.5% halothane over the course of surgery. Bipolar stimulating electrodes were implanted in the left IPN as described in chapter 2. Following 7 days of recovery, animals were adapted to the training apparatus for 2 daily sessions. The next day animals were returned to training apparatus and electrical current was administered through the bipolar electrodes. Threshold to eyeblink responses were determined and measured by potentiometer for three levels of eyeblink amplitudes. The minimum current required to elicit a just measurable eyeblink responses was identified. Next, current levels were increased until threshold to eyeblink amplitudes reached maximum height, thus defining high amplitude thresholds. Thresholds to mid level eyeblink responses were next determined by the expression of median levels of eyeblink amplitudes. Each threshold value was determined by the expression of at least 2 of 3 eyeblink responses to each of the above criterion. 67 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Thereafter, mid and high amplitude eyeblink current levels were administered in a 3 trial series. Each series of stimulation was given at 8 different time points during the onset of an 85 or 95-db 350 ms tone (1 KHz). Specifically, each series of trials was administered at 50, 100, 150, 200, 250, 300 and 350 msec during the tone-on period, plus an additional series after the offset of the tone (400 ms). The order of each series for a given single session is indicated in table 4.1. The order of every other session was presented in reverse sequence. A total of 4 sessions for each animal was presented: 1)2 sessions under an 85 db tone 2) 2 sessions under an 95 db tone. For each level of tone the sequence of stimulation was counterbalanced. Histology After the final experimental session, the location of the bipolar electrode was determined by passing 100 pA of dc for 10 s. Animals were overdosed with intravenous injections of euthasol solution (1 ml), then perfused with 0.9% saline and 10% formalin. Brains were removed and embedded in a combination of gelatin and albumin. Embedded brains were frozen and cut into 80 pm slices. Brain slices were mounted and stained using both Cresyl violet and Prussian blue. Electrode placements were assessed using a dissecting microscope. 68 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 85 or 95 dB (1 kHz) Tone on 50 100 150 200 250 Tone of? 300 400 Figure 4.3 Depicts the time points of stimulation during the onset and offset of an 85 or 95 decibel (dB) tone. 69 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TOS-I Th-1 TOS-II Th-II TOS-III Th-III 1. 50 ms Low 9. 150 ms Mid 17. No tone High 2. 100 ms Mid 10. 250 ms Low 18.400 ms Mid 3. 150 ms High 11. No tone High 19. 300 ms Low 4. 200 ms Low 12. 50 ms Mid 20. 250 ms High 5. 250 ms Mid 13.300 ms Low 21. 200 ms Mid 6. 300 ms High 14. 400 ms High 22. 150 ms Low 7. 400 ms Low 15. 200 ms Mid 23. 100 ms High 8. No tone Mid 16. 100 ms Low 24. 50 ms Mid Table 4.1 Lists the sequence of trials for a given experimental session. Time of stimulation (TOS) in the above order at these specific threshold levels (Th). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Results Of the twelve animals used in the present experiment only three produced reliable stimulation evoked eyeblink responses. The amplitude of the eyeblink response to IPN stimulation averaged over six trials for all animals were collected for each of the following conditions in the present study. la) 85 db tone + Mid level stimulation lb) 85 db tone + High-level stimulation 2a) 95 db tone + Mid level stimulation 2b) 95 db tone + High-level stimulation For each session, amplitude of eyeblink responses with or without the presentation of a tone and to each level and time point of stimulation was averaged. 85 decibel Tone Sensory Stimulation Figure 4.4 shows the mean percentage of mid and high-level IPN stimulation evoked eyeblink response amplitudes following the onset of an 85-db tone versus IPN stimulation alone. Regardless of the level of IPN stimulation, stimulation over the duration of a 350 msec tone elicited a trend of increase eyeblink amplitudes relative to IPN stimulation alone eyeblink amplitudes. As expected, high-level IPN stimulation produced the largest overall tone-related increase in eyeblink amplitudes. 71 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. However, Matched Pairs T-test revealed that only stimulation time points at 250 (t(20)=-3.47, p< .001), 300 (t(17)=-2.352, p<05) and 400 (t(14)=-3.192, p<01) msec showed a significant increase in eyeblink amplitude in comparison to baseline eyeblink amplitudes. 95 decibel Tone Sensory Stimulation Figure 4.5 illustrates the mean percentage of mid and high-level IPN stimulation elicited eyeblink amplitudes after the onset of a 95-db tone and to IPN stimulation alone (baseline). Similar to the eyeblink amplitudes during the 85-db tone, a trend of increased in overall IPN stimulation evoked eyeblink amplitudes during and after the onset of a 95-db tone was observed. However, the level mid or high of IPN stimulation evoked eyeblink amplitudes did not reveal an overall increase in tone- related eyeblink amplitudes. The largest mid-level IPN stimulation produced eyeblink amplitude occurred at 150 ms following the onset of the tone, whereas the largest high-level IPN stimulation was evoked at 200 ms. Figure 4.6 shows electrode placements based on histological reconstruction of lesions. Such evidence reveals that a majority of electrodes tips were positioned in the IPN. 72 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. E n 4 - * Baseline 50 ms 100 ms 150 ms 200 ms 250 ms 300 ms 400 ms □ Mid Thres ■ H I Thres Tone on (85 dB) Tone off Figure 4.4 Mean percent eyeblink amplitude measured in millimeters (mm) as elicited by electrical stimulation, measured at multiple time points prior to (baseline) and after the onset of a 85 dB tone. 73 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. □ Mid T h res Hi T hres B aseline 50 m s 100 m s 150 m s 20 0 m s 250 m s 30 0 m s 4 0 0 m s Tone on (95 dB) Tone off Figure 4.5 Mean percent eyeblink amplitudes measured in millimeters (mm) as elicited by electrical stimulation, measured at multiple time points prior to and after the onset of a 95 dB tone. 7 4 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 4.6 Illustration of coronal sections through the rabbit cerebellum. Filled circles depict marking lesions used for identifying the final position of the electrodes. 75 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Discussion The present results indicate that in the naive animal presentations of an 85 dB tone increases the excitability of IPN to direct stimulation at specific time points during (250 and 300 msec) and after auditory stimulation (400 msec). However the inability to acquire statistical significance at other time points during and following the onset of auditory stimuli is likely due to a lack of power attained with the small sample size tested here. In spite of this, the present results remain consistent with the original hypothesis that auditory stimulation increases the responsiveness of IPN neurons to direct stimulation as indexed by changes eyeblink amplitudes. However, it remains untested whether such changes are mediated or a product of down stream targets of the IPN. Equally possible is that the present results and those by reported by Young et al, (1976) resulted from direct changes in abducens motor neuron excitability. Evidence for plasticity in motor neurons is sparse and has been limited to in vitro experiments. Bracha and colleagues contend that eyeblink conditioning results in plasticity both in the IPN and brainstem nuclei. Primary evidence for this comes from eyeblink conditioning experiments revealing conditioning specific increases in eyeblink reflexes (Schreurs, 2003). However, regardless of the induction of plasticity along any nuclei along the reflex pathways, inactivation of the IPN abolishes any 76 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. conditioning related enhancement of the unconditioned responses (Wikgren & Korhonen, 2001). As suggested by Young et al (1976) the sustained increase in IPN responsiveness under the following conditions may represent evidence for the maintenance of a stimulus trace in the IPN or its cerebellar circuits. As described earlier neural recordings reveal short duration stimulus evoked increases in IPN action potential discharge. However, such activity develops and disappears well before the onset of the unconditioned stimulus. In the present experiment, the tone related enhancement of IPN stimulation to eyeblink amplitudes, which persist over the duration of tone and even following its offset may provide a highly sensitive method to index small threshold increases in neuronal populations created by extracellular currents. Alternatively such sustained increases may reflect inherent properties of neural pathways being activated. Anatomical evidence reveals cerebellar feedback loops along pathways involved in the transmission of CS related information that include: pontine nucleus Purkinie cells IPN pontine nucleus, pontine nucleus IPN pontine nucleus and dorsal cochlear nucleus pontine nucleus IPN dorsal cochlear nucleus. Indeed, it has been hypothesized that sensory information initiates the activation of neural circuits that “reverberate” for a short period of time until they are consolidated in to long lasting memories resulting in permanent alterations in neural circuits (Hebb, 1949). 77 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Available evidence in delay eyeblink does not support that such a mechanism is critical, since conditioning can still be established with electrical stimulation of the lateral reticular nucleus as a CS during inactivation or lesions of the pontine nucleus (Bao et al, 2000). Another plausible mechanism could result from the intrinsic architecture of the cerebellar cortex, in this study electrical stimulation of the IPN is also likely to activate cortical neurons. Cerebellar cortical models of timing propose that CS information is conveyed by parallel fibers to Purkinje cell by progressively delayed synapses. Such staggered points of synaptic contact may individually convey temporally variable action potentials in Purkinje cells over the entire duration of CS, thus capable of maintaining a sufficient resolution of the CS to coincide with US related climbing fiber activation. Moreover, under conditions in which the CS and US are presented discontinuously as in trace conditioning it has been presumed that some signal of CS must be maintained to bridge the association across the trace period between the CS and US. Theories of trace conditioning have proposed that the training context itself may provide such a bridge or that the offset of the CS or the period of no CS may act as a “conditioning stimulus” to establish a relationship between the concrete CS and US. 78 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Here, we also demonstrate that even following the offset of the tone IPN responsiveness is sustained, such results may suggest a cerebellar mechanism at short trace intervals (< 400 ms) for which discontinuous stimuli become associated and thereby express conditioned responses. At trace intervals of 500 ms or greater lesions of the hippocampus markedly impair eyeblink conditioning, however at intervals of 400 ms or less hippocampal lesions do not significantly affect CRs. Also striking, in the present experiment is that the current procedures did not result in the expression of CRs a result consistent with Chapman et al (1988) in which a tone CS and eyeblink responses produced by IPN stimulation did not result in any expression of CRs. However, those results did reveal that subsequent conditioning to a tone CS and airpuff US were significantly enhanced suggesting that the preceding procedures could have resulted in latent excitatory learning effects. It has been demonstrated under higher levels of IPN stimulation as a US and a tone CS that expression of conditioned eyeblink responses can be established (Nowak et al, 1997). Indeed, one animal in this study, not part of the present results was given a single pairing of a tone and very high level of stimulation (1500 pi A) and resulted in the expression of CRs. Such large current levels and those used by Nowak et al (1997) are more likely to result in antidromic stimulation and thus activation of climbing 79 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. fibers. Experiments explicitly examining this, have demonstrated that stimulation of the inferior olive or it climbing fibers, which elicit eyeblink responses as a US, result in normal levels of conditioning (Mauk et al, 1986; Swain et al, 1992; Shinkman et al, 1996; Swain et al, 1999). Together, such evidence could suggest the present procedures resulted in the induction of cerebellar plasticity and that the expression of which is dependent upon climbing fiber activation of Purkinje cells. To summarize the present results demonstrate in the presumably naive animal tone related increases in IPN stimulation to eyeblink amplitudes. Perhaps, future experiments inactivating the IPN and stimulating accessory abducens nuclei during a auditory stimulus, as Young et al (1976) did, could better determine the locus of such actions. 80 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 5 Cerebellar GABAa Receptor Mediated Eyeblink Responses Introduction Cerebellar gamma amino butyric acid (GABAa) receptor function is critical for the acquisition and expression of classically conditioned eyeblink responses. Purkinje cells, the sole output of the cerebellar cortex, are a primary source for the synthesis and release of GABA within the cerebellum. The tonic discharge of action potentials generated intrinsically by Purkinje cells and its excitatory afferents maintain a robust inhibitory influence upon deep cerebellar nuclei including the interpositus nucleus (IPN). Previous studies have repeatedly demonstrated direct IPN infusions of GABAa receptor agonist muscimol, prevents the establishment and expression of eyeblink CRs (Krupa et al, 1993; Bracha et al, 1994; Hardiman et al, 1996; Krupa & Thompson, 1997; Bracha et al, 1998; Garcia & Mauk, 1998; Bracha et al, 2001). It is presumed that such actions are mediated by an overall increase in Cl' permeability maximally hyperpolarizing IPN neurons thereby decreasing effects of excitatory sensory input, thus depressing excitability (Cooper et al, 2003). Conversely, it has been demonstrated that cerebellar infusions of non-competitive GABAa receptor antagonist, picrotoxin blocks the acquisition and expression of eyeblink CRs (Mamounas et al, 1987; Bao et al, 2002). However, several reports by Mauk and colleagues reveal that IPN infusions of picrotoxin in the trained animal result in the expression of CS evoked short-latency eyeblink responses (Garcia & Mauk, 1998; 81 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Medina et al, 2001; Ohyama & Mauk, 2001; Ohyama et al, 2003). To date, such results have yet to be replicated by other laboratories (Bracha et al, 2000; Atwell et al, 2002; Bao et al, 2002). However, in a recent report Bao et al (2002) demonstrated only under conditions in which IPN infusion of muscimol preceded administration of picrotoxin did the expression of CS evoked short-latency eyeblink responses occur in the well-trained animal. Further, it was demonstrated in the cerebellar slice preparation that application of muscimol maximally hyperpolarizes deep cerebellar neurons, effectively abolishing all synaptic input. Subsequent application of picrotoxin returned resting potentials to near baseline levels (depolarized state), while occluding synaptic GABAa responses (Bao et al, 2002), thus, dissociating cerebellar cortical influence upon IPN neurons and limiting synaptic responding to glutamatergic excitatory inputs. Together, such results suggest that in the well- trained animal, excitatory mossy fiber input are sufficient to depolarize IPN neurons and drive the expression of eyeblink responses. In the present experiment, we examine the effects of IPN infusion of picrotoxin in the untrained animal during different levels and modalities of sensory input. We predicted in the naive animal that IPN infusions of picrotoxin under normal levels of sensory stimulation (i.e. 85 dB, 1 kHz tone) would produce no discernible expression of eyeblink responses, as found by Bao et al (2002). However, we predicted that under higher levels of sensory stimulation such as a 95 dB tone, combined with blockade of IPN Cl' channels resulting in occlusion of GABA mediated Purkinje cell 82 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. inhibition would thereby be sufficient to drive the expression of eyeblink response mediated through cerebellar conditioned response pathways. Hence, the expression of a behavioral correlate of memory via modification of cerebellar circuits independent of associative learning. 83 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Materials and Methods Subjects Subjects were 23 adult New Zealand White rabbits weighing between 2.0 and 3.0 kg housed in the Hedco Neuroscience vivarium under a 12-hour light/dark cycle with ad lib access to food and water. All experimental procedures were performed during the light phase of the cycle. Surgery All subjects underwent aseptic surgery prior to training procedures. All animals received subcutaneous injections of xylazine (.08 ml/kg of 100 mg/ml solution) followed by ketamine (.6 ml/kg of 100 mg/ml solution), and were maintained on 2 to 3% halothane (via respirator) for the duration of surgery. An incision was made across the midline of the scalp in which the tissue and skin were retracted exposing the skull. A hole approximately 3 to 4 mm in diameter was drilled 5 mm lateral to lambda. The orientation of the skull was adjusted so that lambda was located 1.5 mm ventral to bregma. All animals were implanted with a stainless steel outer cannula (24 gauge), 27 mm in length (stylet extending 1.5 mm from cannula tip), positioned 0.5 to 1.0 mm anterior, 5 mm lateral and 14 mm ventral to lambda. Dental acrylic applied over the skull and around the cannula created a head mount where both the final position of the cannula and a plastic connector piece were fixed. 84 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Apparatus Nictitating membrane movements were measured by potentiometer as described in chapter 2. We used a 2 mM / ]i\ solution of picrotoxin (Sigma Chemical Co., molecular weight = 602.6) dissolved in artificial cerebrospinal fluid (ACSF). All infusions were made via a 10-/d micro-syringe (Hamilton Instruments) connected to PVC tubing attached to a 30 mm injection cannula (31 gauge). Compression of the syringe was produced by a syringe pump (Harvard Apparatus) at rate of .54 /d/min. Quantity of injection was monitored by timing total duration of infusions and visually monitoring syringe movement. Behavioral Procedures After 7 days of recovery, animals were adapted to the experimental chambers for two daily sessions. The following day, animals were returned to the experimental chamber during which, an 85 or 95 tone or 5 (14 V/ 240 mA) or 24 lumen (2.33 V/ 270 mA) incandescent light (radioshack) was presented for 600 ms. Only one stimulus modality or intensity was presented in any given experimental session (see table 5.1). Each session consisted of a total of 100 stimulus trials with the first 20 85 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Stimulus Modality Intensity Picrotoxin volume ( j i l) Animals (N) Tone 95 dB 1.5 6 Tone 95 dB 1.0 4 Tone 85 dB 1.5 2 Light 5 lumen 1.5 4 Light 24 lumen 1.5 2 Airpuff 3 psi 1.5 4 Table 5.1 Depicts the modality and intensity of a stimulus and the total volume of picrotoxin infused for a given experimental session as well as indicating the number of animals run in each condition. 86 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. trials constituting the pre-infusion test period. Immediately following the 20th trial the training program was paused, the chamber door opened and the stylet was removed from the guide (outer) guide cannula and replaced with the injection (inner) cannula. At the termination of the infusion, the inner cannula remained in the guide cannula for an additional 3 minutes to allow for the diffusion of picrotoxin from the inner cannula. After which, the final 80 trials of the post-infusion period began. Hence, each session consisted of a 20 trial pre-infusion test period and an 80 trial post-infusion test period. Each of the conditions described in table 5.1 was presented for a total of 2 sessions. Three of the animals following the final stimulus-infusion test session received 7 daily sessions of standard delay eyeblink conditioning procedures. The CS consisted of a 600 ms 85 dB tone (1 kHz) followed by a 500 ms delay and a coterminating 100 ms airpuff (3 psi) US. The following day, animals were infused 1 /d of muscimol (.1 mg//d of .01 molar) at a rate of .54 /d/min (identical to the procedures described for infusion of picrotoxin). Following thirty minutes, animals were presented with a session of eyeblink conditioning. The next day animals were trained to eyeblink conditioning procedures once more without infusions of muscimol. The following day animals were trained for 7 daily sessions of CS-alone extinction training. An additional session of extinction following infusion of 1.5 /d of picrotoxin was presented in which the CS consisted of a 95 dB tone (1 kHz). 87 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Histology Following the final experimental session, a stimulating electrode, tip extending 1.5 mm from the end of the guide cannula, replaced the stylet and was used to determine the site of injection by passing 100 piA of dc for 10 s. Animals were overdosed with intravenous injections of euthasol solution (1 ml), then perfused with 0.9% saline and 10% formalin. Brains were removed and embedded in a combination of gelatin and albumin. Brains were frozen and cut into 80 pim slices. Brain slices were mounted and stained using both Cresyl violet and Prussian blue. Location of marking lesions was assessed using a dissecting microscope. 88 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Results Of the 23 animals used in the present experiment a total of 8 animals produced eyeblink responses ranging from 11 to 51.7% to light or tone stimulus following administration of picrotoxin and were included in present results. Figure 5.1 shows the mean percentage of eyeblink responses produced during the presentation of either an 85 or 95 dB tone over the course of the twenty trial pre-infusion test period and eighty trial post-infusion test period (n=6). The pre-infusion test period did not reveal a significant number of eyeblink responses, however following infusion 1.5 ]A of picrotoxin, both levels of tone produced a higher percentage of eyeblink responses. The 95 dB tone and 1.5 pi\ condition produced the largest overall percentage of eyeblink responses. Figure 5.2 reveals the total distribution of eyeblink onset latencies elicited by a 95 dB tone and 1.5 jd of picrotoxin. A majority of eyeblink responses developed between 60 and 140 ms (58.2 %) following onset of the tone. Figure 5.3 depicts the mean peak amplitude of 95 dB tone evoked eyeblink responses following 1.5 ji\ infusion of picrotoxin. Overall peak eyeblink response amplitudes averaged 4.64 mm. 89 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 45 40 35 - 30 - <o •X .£ 25 - I £ v « 20 - # 15 - 10 - 5 0 ■ 95 dB 85 dB B aseline Baseline Block 1 Block 2 Block 3 Block 4 Block 5 Block 6 Block 7 Block I 1 2 Figure 5.1 Mean percentage of eyeblink responses produced during an 85 or 95 decibel (dB) tone over a 2 block pre-infusion baseline period and following an 8 block 1.5 /d picrotoxin (PTX) infusion test period. 90 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5.4 illustrates the mean percentage of eyeblink responses produced over the duration of a 95 dB tone during pre-infusion and post-infusion of 1.0 fi 1 of picrotoxin test periods. No eyeblink responses were recorded prior to picrotoxin infusions, while after infusions only blocks 4 and 8 indicated greater than or equal to 10% eyeblink responses. Figure 5.5 shows the mean percent eyeblink responses elicited by an 5 and 24 lumen light during pre-infusion and post picrotoxin infusion test periods. During pre infusion test period no eyeblink responses were measured, however following infusion of 1.5 ]A a high percentage of eyeblink responses was elicited in animals presented an 24 lumen light, whereas an 5 lumen light did not evoke eyeblink responses greater than 10% for any block (10 trials) of training. The largest mean percentage of eyeblink responses were expressed by during a 24 lumen light (600 ms) during post-infusion blocks 4 and 7 (n=2). Figure 5.6 illustrates the total percentage distribution of eyeblink onset latencies elicited by a 24 lumen light. It is important to note that 140 ms was subtracted from all light evoked eyeblink onset latencies due to the fact that the maximal brightness of the light was delayed by 139 ms due to time required for light’s filament fully illuminate. Thus, a majority of eyeblink onset latencies occurred between 161 and 300 ms (55.9%). 91 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5.2 Percentage distributions of eyeblink onset latencies produced during a 95 dB tone following infusion of 1.5 ]A of picrotoxin. 92 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Baseline Baseline Block 1 Block 2 Block 3 Block 4 Block 5 Block 6 Block 7 Block 8 1 2 Figure 5.3 Mean amplitude of eyeblinks as measured in millimeters (mm) during a 95 dB tone over a two-block pre-infusion baseline period and following infusion of 1.5 ]A of picrotoxin (PTX). 93 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 60 50 40 J 2 _c 1 30 a P 20 10 PTX ■ - I B aseline B aseline Block 1 Block 2 Block 3 Block 4 Block 5 Block 6 Block 7 Block 8 1 2 Figure 5.4 Mean percentage of eyeblink responses produced during an 95 dB tone over a 2 block pre-infusion baseline period and following an 8 block 1.0 ]A picrotoxin (PTX) infusion test period. 9 4 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 90 - 1 80 - 70 - 60 - PTX •5 50 - < U 40 - 30 - 20 - 10 - B aseline Baseline Block 1 Block 2 Block 3 Block 4 Block 5 Block 6 Block 7 Block 8 124 Im 15 Im Figure 5.5 Mean percentage of eyeblink responses produced during a 5 and 24 lumen light over a 2 block pre-infusion baseline period and following an 8 block 1.5 ]A picrotoxin (PTX) infusion test period. 95 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. % ONSET LATENCY 1 4 1 12 - 10 - 8 - 6 - o o O o o o O o o O o o o o o o O o o o o o O O O o o o o o ( N V O 0 0 o P M V O C O o r M V O 0 0 o r M V O C O O r M V O 0 0 O P M V O C O o o i 1 t H rH rH rH rH ( N P M CM < N C M r o r o r o r o r o * T T T i n i n i n m i n V O < N V O 1—1 rH rH rH rH rH rH rH rH rH rH rH rH rH tH rH rH rH rH rH rH rH rH rH rH rH C O o ( N V O C O O r s i V O C O O r M V O 0 0 O r M V O C O O r M V O 0 0 t H rH rH rH rH pm P M rM CM r M r o r o r o r o r o Tf m i n i n i n i n Figure 5.6 Percentage distribution of eyeblink onset latencies during a 24-lumen light following infusion of 1.5 ji\ of picrotoxin. 96 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5.7 reveals the percent difference of corneal airpuff evoked eyeblink response amplitudes (mm) between pre-infusion and post-infusion test periods. Pre-infusion blocks 1 and 2 were averaged together as well as each pair of post-infusion blocks to detect any changes in eyeblink amplitudes. Post-infusion period 2 showed the largest percent increase in eyeblink amplitude (94.5%), followed by periods 1 (74.8%), 3 (73.7%) and 4 (65.4%). Figure 5.8 shows mean conditioned response percentages over the course of training sessions for a subset of 3 animals that expressed tone-evoked eyeblink responses following infusion of picrotoxin (PTX group) and a group of control animals (described in chapter 6) trained to the identical eyeblink conditioning procedures excluding stimulus and picrotoxin infusion testing. The group PTX revealed a notably slower rate of acquisition than the control group, but did acquire CRs to a level of 87.4%. Further, infusion of muscimol reduced CR percentages to 11.46, while training without infusion of muscimol returned CRs performance to 94.4%. 97 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Extinction training produced a gradual reduction of percentage CRs over the course of 7 sessions. Infusions of picrotoxin preceding a final session of extinction did not yield a significant number of eyeblink responses (M =2.5%). Figure 5.9 shows cannula placement based on histological reconstruction of marking lesions. Such evidence reveals that a majority of infusions were made in or above the IPN. 98 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. % difference 110 - i 90 - 70 - 50 - 30 - 10 - Drug Per.4 Baseline Drug Per.1 Drug Per.2 Drug Per.3 -10 J Figure 5.7 Mean percentage change in airpuff evoked eyeblink amplitudes as measured in (mm) prior to and following infusion 1.5 }A of picrotoxin. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 100 90 80 60 O 50 40 20 Control ptx group acql acq2 acq3 acq4 acq5 acq6 acq7 M U S R ET extl ext2 ext3 ext4 ext5 ext6 ext7 PTX C o n d itio n in g s e s s io n s Figure 5.8 Conditioned response (CR) percentages (%) plotted for sessions of acquisition (acql-acq7), with (MUS) and without infusion of muscimol (RET) and sessions of extinction (extl-ext7) and a session with infusion of picrotoxin (PTX). 100 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5.9 Illustration of coronal sections through the rabbit cerebellum. Filled circles depict electrode tips used to deliver marking lesions. IPN, interpositus nucleus. A. Plate of a cerebellar section 0.5 mm anterior to lambda. B. Plate of a cerebellar section 1.0 mm anterior lambda. 101 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Discussion The main results demonstrate that intracerebellar infusion of GABAa receptor antagonist, picrotoxin in the naive animal combined with strong sensory stimulation results in expression of eyeblink responses. Moreover, cessation of picrotoxin infusions and addition of an airpuff US resulted in a retarded but gradual acquisition of conditioned eyeblink responses. These results suggest that the development and expression of picrotoxin-mediated eyeblink responses in the untrained animal did not result in savings of any excitatory learning effects. Thereafter, infusion of muscimol in well-trained animals abolished nearly all expression of CRs, suggesting normal GABAa function. Finally, extinction of CRs proceeded normally with subsequent infusions of picrotoxin not yielding eyeblink responses greater than at spontaneous levels. In the present study, stimulus evoked eyeblink responses in untrained animals were most robust under conditions in which large volumes of picrotoxin were infused and a 95 dB tone or 24 lumen light was presented. Nevertheless, a small number of eyeblink responses were still produced by an 85 dB tone or 5 lumen light. Yet, infusions of smaller volumes of picrotoxin did not result in a significant number of eyeblink responses at the highest level of auditory stimulation. Examination of onset to eyeblink latencies in the high volume and high dB condition revealed a majority of responses developed between 61 and 120 ms following the presentation of the 102 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. tone, whereas onset to eyeblink latencies to a 24 lumen light occurred between 161 and 300 ms. Tone-evoked eyeblink response latencies are relatively consistent with previous reports in the well-trained animal that infusions of picrotoxin alone (Garcia & Mauk, 1998; Medina et al, 2001; Ohyama & Mauk, 2001; Ohyama et al, 2003) or muscimol followed by picrotoxin (Bao et al, 2002) result in the expression of short latency, 85 dB tone-evoked eyeblink responses. Aside from the 95 dB tone a fundamental difference in the present experiment was that none of the subjects ever received a single pairing of a tone/light and airpuff prior to and during picrotoxin infusions. Further, our results seemingly contradict previous reports that lower concentration of picrotoxin infused into the IPN alone attenuate or prevent the acquisition and expression of eyeblink CRs (Mamounas et al, 1987; Atwell et al, 2002; Bao et al, 2002). However, in the present experiment only high concentrations (2 mM) and volumes of picrotoxin (1.5 ]A) produced stimulus-evoked eyeblink responses. As described earlier, the absence of evidence for any learning-related savings suggests, the stimulus evoked eyeblink responses resulted from a temporary modification of cerebellar circuitry. Perhaps a reason for much of the variability between studies examining effects of picrotoxin is the apparent discrepancy in total volumes infused. Further complicating matters, in a recent report by Aksenov et al (2004) the quantities of picrotoxin used in previous studies were falsely reported. Conversely, Garcia and Mauk (1998) selectively reported that infusions of picrotoxin in well-trained animal by Mamounas 103 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. et al, (1987) only decreased CR amplitude using a .38 nM concentration, when in fact 3 different concentrations were used in the study with largest concentration .75 nM completely preventing the acquisition and expression of CRs. These initial studies examining the effects of intracerebellar infusions revealed a dose dependent impairment in the acquisition and expression of eyeblink CRs with the largest volume .75 nM (0.5 pi\ of 1.5 nM/ y\) completely prevented the acquisition and expression of CRs (Mamounas et al, 1987). Further, studies revealed infusions of 2 mM of total volume (1-1.5 nM / 2 mM //d), resulted in expression of tone-evoked short onset latency and reduced amplitude eyeblink responses, similar to those described here (Medina et al, 2001). Again, the total volume and concentration used in this study are nearly identical to those used by Mauk and associates. Table 5.2 summarizes the effects of different volumes of IPN infusion of picrotoxin in eyeblink conditioning experiments. At present the mechanisms of the picrotoxin related short latency eyeblink responses has been debated. Initial reports by Garcia and Mauk (1998) argued the picrotoxin, a chloride channel blocker, functionally disconnected the cerebellar cortex output to the IPN unmasking plasticity in the IPN independent of timing established in the cerebellar cortex. However, it has been recently argued that sustained increases in IPN output by picrotoxin produces excitability changes in brainstem pathways and shortens CR latency (Aksenov et al, 2004). The assumption in all of these accounts is that short-latency eyeblink responses are conditioned responses. Indeed, in the 104 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. present results all short-latency responses were produced in untrained animals receiving an robust tone or light stimuli. Further, in a group of animals tested, none showed any evidence that tone, light or airpuff alone trials combined with picrotoxin produced excitatory savings in subsequent eyeblink conditioning. A recent study by Aksenov et al (2004) reported that IPN neurons expressing CR related neural responses revealed an increase in tonic activity following infusions of picrotoxin (1.25 nM concentration), which saturated neural responses and eventually abolished expression of CRs. In the study, the initial infusion of picrotoxin reduced CR expression and slightly decreased CR latencies from 227 to 200 ms whereas a second infusion further reduced and eventually abolished CR expression. However, in the present study (figure 5.2) 58% of all tone elicited eyeblinks responses occurred between 61 and 140 ms, whereas only 8% were detected between 181 and 240 ms, a result consistent with previous findings (Bao et al, 2002; Medina et al, 2001). Therefore, the reported picrotoxin induced changes in single unit IPN activity may not represent neural responses underlying expression of short latency eyeblink responses. In contrast, the results by Bao et al (2002) that sequential infusion of muscimol and picrotoxin (10 nM) in the well-trained animal massively reduced average CR onset latencies from 143 to 92.4 msec at that similar application of muscimol and picrotoxin in whole cell recordings of deep nuclear 105 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Study Concentration Volume Effect Garcia & Mauk, 1998 2 mM//d saline 2-3 mM Short-latency eyeblink response in trained animals Medina & Mauk, 2001 2 mM//d saline 2 mM Short-latency eyeblink response in extinguished animals Atwell et al, 2002 0.5 mM//d 0.5 mM Attenuated expression Mamounas et al, 1987 0.28-0.56 nM/0.75 ]A Ringer’s solution 0.38-0.75 nM High dose: Prevented acquisition and abolished expression Bao et al, 2002 10 nM//d ACSF .052 mM Prevented acquisition and abolished expression Askenov et al, 2004 0.4-2.5 nM//d ACSF 2.5 nM Attenuated expression Table 5.2 List of eyeblink conditioning experiments that have infused picrotoxin in the IPN. Concentrations, volumes and effect upon eyeblink conditioning are described. 106 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. neurons revealed muscimol hyperpolarized cells, while subsequent picrotoxin reduced holding currents, returning cells to depolarized states at the same time totally blocking synaptic GABAa input (Bao et al, 2002). In addition, application of picrotoxin alone resulted in a similar reduction in holding currents thus, increasing neuronal membrane excitability. The behavioral results by Bao et al (2002) and Mauk and colleagues revealing conditioning specific expression of short latency eyeblink responses suggest plasticity at mossy fiber-IPN synapses is critical. Indeed, eyeblink conditioning related increases in mossy fiber-IPN synapses has been previously reported (Kleim et al, 2002). Collectively these results, suggest that increasing the excitability of IPN neurons, by reducing GABAergic inhibition and increasing level of activity of mossy-IPN synapses maybe sufficient for the expression of cerebellar mediated eyeblink responses. Indeed, in the present experiment IPN infusions of a GABAa receptor antagonist, and presentations of supernormal sensory stimulus (95 dB tone or 24 lumen light), which has been shown to produce greater stimulus evoked activity in the IPN (Tracy, 1998; dissertation), were sufficient to result in the expression of eyeblink responses. Another possibility as Aksenov et al (2004) contend, is that these eyeblink responses are product of extracerebellar brainstem circuitry. In the present study UR amplitudes were significantly increased by IPN infusions of picrotoxin. These results suggest that, changes upstream (afferent) in the cerebellar circuitry are modifying reflex pathways. Conversely, previous studies have demonstrated eyeblink conditioning related increases in UR amplitudes (Schreurs, 2003), which were eliminated by 107 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. inactivation of the IPN (Wikgren & Korhonen, 2001). However, to date there have been no published reports in rabbit that acoustic stimuli can evoke a reliable alpha or startle eyeblink response. Reports of alpha response to light in rabbit have been demonstrated for onsets in the 20 to 30 ms range. Again, the onset latencies of eyeblink responses, reported here primarily occur between 61 to 140 ms and 161 to 300 ms to light. These results are consistent with reports of transmission times estimated by stimulation and recording studies. In brief, tone and light related activation of IPN neurons roughly occurs at latencies of 42 and 44 ms, respectively (Tracy, 1998: dissertation). Interpositus nucleus electrical stimulation to eyeblink latencies measured in chapters in 2,3 and 4 averaged 48 ms. Together such results, suggest that tone or light stimuli have the capacity to elicit eyeblink response at minimum latencies near 90 ms. In summary the finding of the present study reveal in the naive animal that cerebellar infusions of picrotoxin and strong sensory stimulation is sufficient to evoke expression of eyeblink responses. The timing of these responses were consistent with previous studies in well-trained animals that IPN infusions of picrotoxin resulted tone-elicited short latency to onset eyeblink responses. In conclusion, the present results are consistent with models of eyeblink conditioning that predict the release of cerebellar cortical-IPN synaptic inhibition together with enhancement of mossy fiber-IPN synapses are pivotal for the expression of classically conditioned eyeblink responses. 108 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 6 The Effects of Post-Training Exposure to the Training Apparatus following Delay Eyeblink Conditioning Introduction There is growing evidence that the conditioning context plays a vital role in learning and memory. Perhaps the clearest evidence of this comes from animal studies of Pavlovian fear conditioning, in which re-exposure to the conditioning context alone can evoke the expression of conditioned responses (CR). Further, studies have revealed that extinction of aversive and appetitive conditioning are both highly sensitive to alterations of the conditioning context. In stark contrast, there is relatively little evidence for significance of the conditioning context in the conditioning and extinction of classically conditioned eyeblink responses. Penick and Solomon (1991) reported that changes in context, following eyeblink conditioning in the rabbit, markedly attenuated expression of conditioned nictitating membrane responses. In the experiment, rabbits were trained to criterion, 8 CRs in any block of 10 trials, under delay eyeblink conditioning procedures. The following day conditioning was resumed in either the previous context or switched to a new context. Rabbits switched to the new context showed a temporary, but significant reduction in CR expression compared to animals continuing training in the same context. Interestingly, this context effect was abolished by pre-training hippocampal lesions. 109 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Various theories have implicated the hippocampus in coding of contextual information. Indeed, evidence of “place” cells in the hippocampus, which fire specifically to spatial orientation in rats, can yield robust spatial maps of the environment (Barnes et al, 1990). Likewise, lesions of hippocampus, which disrupt spatial navigation tasks in rats, prevent the acquisition and retention of contextual fear (Kim & Fanselow, 1992; Phillips and LeDoux, 1992; Selden et al, 1991). Recently, Kehoe et al (2004) demonstrated, in an experiment initially intended to measure cross modal CS extinction effects, that control animals well-trained in eyeblink conditioning showed a pronounced decrement in CR expression following repeated exposure to the conditioning apparatus alone. Further, in a separate experiment, reintroduction of the US in the conditioning context did not attenuate this effect. In the present study, we wanted to explicitly test whether such effects could be replicated, given the slight variation in experimental procedures used by Kehoe et al, (2004) and our laboratory’s standard conditioning procedures. In Kehoe’s experiments, conditioning was established to two different CSs (tone and light) modalities, the offset each occurring simultaneously with the onset of the US, a 50 ms electric current (3 mA, 50 Hz) delivered to the eye. In the present experiment conditioning occurred to a single CS (tone), which coterminated with an airpuff US directed towards cornea. It is conceivable that such variations in conditioning procedures, acquisition to multiple CS modalities, timing of CS-US presentations or 110 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. strength of the US could affect the salience of the context. Indeed, in trace fear conditioning increasing the CS-US interval results increases the expression of fear responding to the conditioning context. 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Material and Methods Subjects Fourteen New Zealand White rabbits weighing between 2 and 2.2 kg were individually housed in the Hedco Neurosciences vivarium on a 12 hour light-dark cycle and provided ad lib access to food and water. Apparatus Nictitating membrane movements were measured by a minitorque potentiometer attached via a thread lead hooked through a nylon suture stitched into the left nictitating membrane. Voltage changes were measure by an IBM/PC, where data were analyzed offline. Behavioral training Four days following arrival, adaptation procedures began. Animals were removed from their homecage by the experimenter and transported by basket to the conditioning room. Next, each animal was restrained in a Plexiglas box during which a pair of Velcro straps was secured around the head and muzzle, so that a 3” x 1” plastic head stage could be secured along the midline of the animals head. Animals 112 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. were then placed in a conditioning chamber during which a potentiometer and air nozzle were attached to the head stage. The chamber door was closed and remained so for the duration of 50 minutes. Then, animals were removed from the conditioning chamber and a nylon loop (~1 mm diameter) was stitched in the left nictitating membrane under an optical anesthetic (tetracaine hydrochloride) and returned to their homecage. Thereafter, throughout the course of the experiment the same experimenter handled the same animal. Following a second day of adaptation all rabbits were trained to delay (500 ms ISI) eye-blink conditioned procedures. Each acquisition session consisted of 100 pairings of a 600 ms tone (1 Khz) CS and a coterminating 100 ms left corneal airpuff (3 psi) US. Inter-trial intervals were randomly varied from 20 to 40 seconds. Each animal was trained for a total of 6 acquisition sessions. Animals were then divided into one of two groups. The CXT group (N=5) consisted of animals returned to the conditioning context on 6 successive days for 50 minutes (identical to adaptation procedures). The HMC group (N=5) consisted of animals that remained in homecages for 6 days. The next day, all animals were returned to the conditioning context and given extinction training. Each extinction session consisted of 100 CS alone trials spaced at 20 to 40 s intervals. A total of 6 successive days of extinction training was presented. The following day, animals were returned to the conditioning context and presented with an additional session of acquisition training. 113 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Conditioned responses were defined as any extension of the left nictitating membrane equal to or greater than 0.5 mm occurring between 35 and 500 ms following CS onset during acquisition training and 35 and 600 ms following CS onset during extinction training. 114 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Results Figure 6.1 shows the mean conditioned response percentage over the course of acquisition, context exposure, extinction and reacquisition training for groups CXT and HMC. Both groups produced CRs at a similar rate during acquisition training (Repeated Measures ANOVA, F (1,8) = .052, g > .05). In addition, by the final acquisition session (acq 6) both groups expressed CRs to a similar level (CXT M= 91.36%, SEM=3.7), (HMC M=88.3%, SEM=2.2), (One Way ANOVA, F (1,8) = .46, g >.05). During context exposure group CXT, showed low levels of responding ranging between 1.02% (SEM 1.26%) to 2.46% (SEM 2.37%). Figure 6.2 illustrates mean percent CRs during the first day of extinction training. During this period, group CXT showed 11.2% CRs (SEM=4.26) and group HMC showed 38.7% (SEM=7.86). A significant difference between groups CXT and HMC for the mean percent CRs was identified for this initial session of extinction (One way ANOVA, F (1,8) = 7.867, g < .05). Such low levels of responding represent an 81.6 % and 49.6 % point loss from the final acquisition session in the CXT and HMC groups, respectively. 115 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 6.3 indicates percentage point difference of mean CR expression between the final acquisition session and the first session of extinction training. Overall, group HMC showed a faster rate of extinction than group CXT (Repeated Measure ANOVA, F (1,8) = 6.966, p > .05). However this is likely do a floor effect. Figure 6.4 indicates following extinction training both groups reacquired CRs to a similar level. Analysis of variance revealed no significant difference between in CR expression between groups CXT and HMC (One Way ANOVA, F (1,8) = 1.569, p < .05). 116 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 100 9 0 - 80 - 70 - 60 - 2 y so - # 30 - vO 4-1 f M 4-1 c r u r n ( N r o i n v O n 4-1 tH 4-) training sessions Figure 6.1 Mean conditioned response (CR) percentages (%) plotted for each of sessions of acquisition (acql-acq6), exposure solely to the conditioning apparatus (cxtl-cxt6), sessions of conditioned stimulus alone extinction training (extl-ext6) and a session of reacquisition (reacq) training. 117 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ■ CXT (extl) ■ HMC (extl) Figure 6.2 Mean percent conditioned responses (CRs) during the first session of extinction training following either conditioning apparatus alone exposure (CXT) or homecage maintained (HMC) animals (* denotes significant difference < .05). 118 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 0 0 1 90 - ■ CXT (acq-ext) ■ HMC (acq-ext) Figure 6.3 Mean percent point difference of conditioned response (CRs) between the final session of acquisition and the initial session of extinction. 119 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. % CRs 1 0 0 - 1 90 - 80 - ■ CXT (reacq) ■ HMC (reacq) Figure 6.4 Mean percentage conditioned responses (CRs) during reacquisition training. 120 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Discussion Exposure to conditioning context alone over a period of six daily sessions resulted in a significant decrease in the expression of eyeblink CRs as compared to control animals that remained in the homecage. These results replicate previous work by Kehoe et al (2004) that exposure to the conditioning apparatus alone results in profound decrements in classically conditioned eyeblink responses. Given, that the results by Kehoe et al (2004) were demonstrated using non-standard conditioning procedures to test for cross modal CS extinction effects. In the present study standard delay eyeblink conditioning procedures in this laboratory, involving a the presentation of a single CS coterminating with an airpuff US resulted in CRs that were similarly attenuated by context exposure. Further, the larger decrement in CR expression following context exposure relative to homecage confined animals indicate that the reduction in CXT group could not be attributed solely to a temporal decay of associative memory. Therefore, the present results suggests that exposure to conditioning context alone can attenuate expression of classically conditioned eyeblink responses. Conditioning in other paradigm has demonstrated variable effects of the exposure to the learning context on cued performance of conditioned behaviors. Gabriel (1970) reported that prolonged exposure to training context produced decrements in a cued avoidance task. Further Marlin et al, (1981) using a cued fear lick suppression 121 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. paradigm and reported apparatus exposure produced decrement in lick suppression, whereas Miller and colleagues have demonstrated facilitated lick suppression (Kasprow, Schachtman & Miller, 1987). The present result demonstrate in the eyeblink conditioning paradigm that exposure to the conditioning context is sufficient to produce large decrements in CR expression. Moreover, these results are puzzlingly in light of most of the present learning theories. Kehoe et al, (2004) provide an exhaustive overview of how each of these theories predicts the effects of context exposure in associative learning. Here we provide only a brief overview. Neither response based nor context-CS associative theories predict that exposure solely to the context can account for the present results. Rescorla’s theory argues that the greater the amount of responding occurring during extinction the more robust the extinction process. Hence, the few eyeblink responses measured during the context alone condition should not decrease conditioned responding. Bouton contends that extinction occurs as a result of increases in associative strength between the context and CS. According to this model, exposure to the context independent of the CS should not reduce the expression of the CR. Further, comparator models (Gibbon & Balsam, 1981; Stout et al, 2003) argue that amount of responding reflects the difference in excitatory strength of CS-US versus Context-US. Hence, these models predict that acquisition is a product of increasing the excitatory value between the CS 122 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and US at the same time keeping the excitatory value between context and US low. Therefore, exposure to the context alone should not alter neither of these excitatory values or expression of learned behaviors. Brandon and Wagner’s AESOP model contends that contextual cues like CSs can acquire excitatory strength with the US, that simultaneous increase the motivation to respond. According to this model, exposure to conditioning context alone should reduce the motivation to respond to the CS. However, it incorrectly predicts that reinforced exposure to the conditioning context (Context + US) should not reduce CRs. In contrast to present theories that rely on Context-CS or Context-US associations, which have difficulty in reconciling the present results, Kehoe (2004) proposed that eyeblink conditioning results in formation of excitatory Context-CS-US associations. According to this theory, initial training results in the formation of Context-CS associations, however as conditioning progresses the association between the CS-US results in the expression of CRs (see figure 6.5). Thus, exposure to the context alone retrieves a representation of the CS, resulting in the extinction of conditioned responses. Moreover, under reinforced context exposure the elicited representation of CS is too temporally diffuse to maintain an excitatory CS-US association. Therefore, under either condition, context alone or context and US, expression of eyeblink CRs is significantly reduced. Interestingly, the present results and those of Kehoe et al, 123 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (2004) indicate that exposure to the conditioning context alone, or in combination with US produce a profound decline in conditioned eyeblink responses strikingly similar to that produced by CS alone extinction. Future studies could address whether both behavioral and neural interventions that effect CS alone extinction similarly affect both non-reinforced and reinforced apparatus exposure. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CXT Figure 6.5 Illustrates Kehoe’s (2004) model that accounts for the present results by predicting associations between the context (CXT) and conditioned stimulus (CS) in addition to CS and unconditioned stimulus (US). 125 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 7 General Conclusion For every act of memory, every exercise of bodily aptitude, every habit, recollection, train of ideas, there is a specific neural grouping, or co ordination, of sensations and movement, by virtue of specific growths in cell junctions (Bain, 1873). The contributions in fields of anatomy, physiology, psychology and the development of modern neuroscience encompassing the disciplines of genetics, chemistry, molecular biology and computation has told us a great deal about where memory is and is not and what memory maybe. The development of an “ideal” preparation eyeblink conditioning by Gormezano and colleagues and the later adaptation by Thompson and colleagues for the neural analysis of mammalian learning and memory has become a powerful tool in elucidating the neural circuits underlying the development of an associative memory. The collective evidence suggest that essential memory traces for eyeblink conditioning are established and maintained in the interpositus nucleus (IPN). However, there is growing evidence that plasticity in the cerebellar cortex may play an important role in the induction and expression of IPN memory traces. The data presented here confirm this, and further suggest potential mechanisms and substrates of Pavlovian conditioning of discrete motor responses. 126 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The results in chapter 2 demonstrate electrical stimulation of a region critical for the formation and expression eyeblink conditioning memory traces, the IPN, can be utilized as a highly effective conditioned stimulus (CS) producing very rapid learning. Further, the results that timing of conditioned responses (CRs) can be established and modified following temporal changes in contingency, independent of direct activation of the pontine nucleus or cerebellar cortex, suggest a critical input for CR timing maybe regulated by climbing fibers. Interestingly, corticotropin- releasing hormone (CRH) a neuropeptide immunoreactive in climbing fiber terminals (Sawchenko & Swanson, 1989) plays a permissive role for the induction parallel fiber-Purkinje cell LTD (Miyata et al, 1999). Perhaps future studies could address the role of CRH in eyeblink conditioning by utilizing CRH antagonists or inducible CRH knockout mice. In chapter 3 the results indicate that classical conditioning using stimulation of the IPN as CS results in an overall decrease in IPN stimulation-to-eyeblink thresholds. Such results are consistent with theoretical and empirical evidence that either synaptic and/or cell wide increases in IPN excitability or morphology are associated with eyeblink conditioning (Kleim et al, 2002; Aizenman & Linden, 2000). The fact that electrical stimulation of the IPN prior to training evoked a wide range of behavioral responses (e.g. head turn, whisker, eyelid opening, lip curls and forelimb flexion) other than eyeblinks, and that ensuing conditioning resulted in the exclusively expression of eyeblink responses may suggest evidence of reorganization 127 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. of cerebellar somatotopic representations. Further, given that non-eyeblink behaviors did not redevelop after extinction, suggests that such changes were maintained even following behavioral extinction. Future studies using large arrays of electrodes could initially better define somatotopic representations in the IPN, then classically condition specific sites of stimulation and determine to what extent representations are altered. The experiment in chapter 4 indicated in the untrained animal that auditory stimulation results in an overall increase in IPN stimulation to eyeblink amplitude, a change that persisted even following the offset of the stimulus. The largest increases corresponded roughly around the onset of CRs produced at optimal CS-US intervals. Moreover, such results may be evidence of a cerebellar stimulus trace that maintains the capacity to intercept US related climbing fiber activation of IPN neurons. It is important to note that, procedures in chapter 4 (Tone + IPN stimulation), unlike chapters 2 and 3 (IPN stimulation + airpuff) did not result in any overt conditioning. However, a prior study demonstrated that such procedures could facilitate subsequent eyeblink conditioning to a tone CS and airpuff US (Chapman et al, 1988). Such evidence could indicate that expression of CRs under normal conditions may require climbing fiber activation of Purkinje cells. 128 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In chapter 5, it was demonstrated that cerebellar infusion of picrotoxin in the naive animal combined with strong auditory or visual stimuli can evoke expression of short onset latency eyeblink responses. A result only previously reported in well-trained animals, this indicates modulating cortical inhibition and increasing the level of sensory input received by IPN neurons are sufficient for the expression of cerebellar mediated eyeblink responses. Further, these changes seem to occur independent of learning as indicated by subsequent tone-airpuff pairing, which resulted in normal levels and rates of conditioning. Indeed, the present results do not adequately indicate whether the production of eyeblink responses is directly mediated by neural responses generated by IPN or extracerebellar sites. Therefore, neural unit recording studies could reveal whether IPN neural activity models the development of picrotoxin mediated eyeblink response as found with conditioned responses produced by classical conditioning. The results in chapter 6 indicate the exposure to the conditioning context alone is sufficient to profoundly diminish the expression of conditioned eyeblink responses in extinction as previously demonstrated by Kehoe et al, (2004). Further, such decrements in CR expression are strikingly similar to those produced by CS alone extinction or even following US alone training. The one commonality in all of mentioned methods for reduction of CRs is the conditioning context. 129 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This evidence suggest carrying out experiments manipulating variables that affect CS alone extinction, such as prolonged intervals between training, resulting in spontaneous recovery of CRs similarly affect context-mediated reduction of CRs. An important study would be to examine to what extent the hippocampus, a region presumed to encode contextual-spatial cues, mediates the present results. Collectively the present results are consistent with evidence that plasticity within the cerebellum is critical for the acquisition and expression of conditioned eyeblink responses. Moreover, the present results suggest that mechanisms of plasticity may include changes in IPN somatotopic representations and/or an overall increase in IPN excitability. Further, the present results are consistent with evidence that release of cerebellar cortical inhibition and the activation of mossy fiber-IPN pathways are essential for the expression of conditioned eyeblink responses. Finally, the evidence suggests that mere exposure to conditioning context is capable of inhibiting expression of cerebellar dependent eyeblink CRs in extinction. Overall the present results have shed some light on to potential mechanisms underlying the storage and expression of cerebellar memory traces. 130 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. References Aiba A, Kano M, Chen C, Stanton ME, Fox GD, et al. 1994. Deficient cerebellar long-term depression and impaired motor learning in mGluRl mutant mice. Cell 79: 377-88 Aizenman CD, Huang EJ, Linden DJ. 2003. Morphological correlates of intrinsic electrical excitability in neurons of the deep cerebellar nuclei. J Neurophysiol 89: 1738-47 Aizenman CD, Linden DJ. 2000. Rapid, synaptically driven increases in the intrinsic excitability of cerebellar deep nuclear neurons. Nat Neurosci 3: 109-11 Aksenov D, Serdyukova N, Irwin K, Bracha V. 2004. GABA neurotransmission in the cerebellar interposed nuclei: involvement in classically conditioned eyeblinks and neuronal activity. J Neurophysiol 91: 719-27 Albus JS. 1971. A theory of cerebellar function. Math Biosci 10: 25-61 Allen MT, Chelius L, Gluck MA. 2002. Selective entorhinal and nonselective cortical-hippocampal region lesions, but not selective hippocampal lesions, disrupt learned irrelevance in rabbit eyeblink conditioning. Cogn Affect Behav Neurosci 2: 214-26 Allen MT, Steinmetz JE. 1996. A nitric oxide synthase inhibitor delays the formation of learning-related neural activity in the cerebellar interpositus nucleus during rabbit eyelid conditioning. Pharmacol Biochem Behav 53: 147-53 Attwell PJ, Ivarsson M, Millar L, Yeo CH. 2002. Cerebellar mechanisms in eyeblink conditioning. Ann N YAcad Sci 978: 79-92 Attwell PJ, Rahman S, Ivarsson M, Yeo CH. 1999. Cerebellar cortical AMPA- kainate receptor blockade prevents performance of classically conditioned nictitating membrane responses. J Neurosci 19: RC45 Attwell PJ, Rahman S, Yeo CH. 2001. Acquisition of eyeblink conditioning is critically dependent on normal function in cerebellar cortical lobule HVI. J Neurosci 21: 5715-22 Bain A. 1873 Mind and Body: The Theories of Their Relation (Henry King, London) Bao S, Chen L, Kim JJ, Thompson RF. 2002. Cerebellar cortical inhibition and classical eyeblink conditioning. Proc Natl Acad Sci U S A 99: 1592-7 131 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Bao S, Chen L, Thompson RF. 2000. Learning- and cerebellum-dependent neuronal activity in the lateral pontine nucleus. Behav Neurosci 114: 254-61 Bames CA, McNaughton BL, Mizumori SJ, Leonard BW, Lin LH. 1990. Comparison of spatial and temporal characteristics of neuronal activity in sequential stages of hippocampal processing. Prog Brain Res 83: 287-300 Berry SD, Thompson RF. 1979. Medial septal lesions retard classical conditioning of the nicitating membrane response in rabbits. Science 205: 209-11 Berthier NE, Moore JW. 1986. Cerebellar Purkinje cell activity related to the classically conditioned nictitating membrane response. Exp Brain Res 63: 341-50 Berthier NE, Moore JW. 1990. Activity of deep cerebellar nuclear cells during classical conditioning of nictitating membrane extension in rabbits. Exp Brain Res 83: 44-54 Beylin AY, Gandhi CC, Wood GE, Talk AC, Matzel LD, Shors TJ. 2001. The role of the hippocampus in trace conditioning: temporal discontinuity or task difficulty? Neurobiol Learn Mem 76: 447-61 Black-Cleworth P, Woody CD, Niemann J. 1975. A conditioned eyeblink obtained by using electrical stimulation of the facial nerve as the unconditioned stimulus. Brain Res 90: 45-56 Blaxton TA, Zeffiro TA, Gabrieli JD, Bookheimer SY, Carrillo MC, et al. 1996. Functional mapping of human learning: a positron emission tomography activation study of eyeblink conditioning. J Neurosci 16: 4032-40 Bracha V, Irwin KB, Webster ML, Wunderlich DA, Stachowiak MK, Bloedel JR. 1998. Microinjections of anisomycin into the intermediate cerebellum during learning affect the acquisition of classically conditioned responses in the rabbit. Brain Res 788: 169-78 Bracha V, Webster ML, Winters NK, Irwin KB, Bloedel JR. 1994. Effects of muscimol inactivation of the cerebellar interposed-dentate nuclear complex on the performance of the nictitating membrane response in the rabbit. Exp Brain Res 100: 453-68 132 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Bracha V, Zhao L, Irwin K, Bloedel JR. 2001. Intermediate cerebellum and conditioned eyeblinks. Parallel involvement in eyeblinks and tonic eyelid closure. Exp Brain Res 136: 41-9 Bracha V, Zhao L, Irwin KB, Bloedel JR. 2000. The human cerebellum and associative learning: dissociation between the acquisition, retention and extinction of conditioned eyeblinks. Brain Res 860: 87-94 Brogden, W.J. & Gantt, W.H. (1942) Intraneural conditioning: cerebellar conditioned reflexes. Arch. Neurol. Psychol. 4 8 .437-455 Cegavske CF, Thompson RF. 1976. Mechanisms of efferent neuronal control of the reflex nicitating membrane response in rabbit (Oryctolagus cuniculus). J Comp Physiol Psychol 90: 411-23 Chapman PF, Steinmetz JE, Thompson RF. 1988. Classical conditioning does not occur when direct stimulation of the red nucleus or cerebellar nuclei is the unconditioned stimulus. Brain Res 442: 97-104 Chen C, Thompson RF. 1995. Temporal specificity of long-term depression in parallel fiber— Purkinje synapses in rat cerebellar slice. Learn Mem 2: 185-98 Chen G, Steinmetz JE. 2000a. Intra-cerebellar infusion of NMDA receptor antagonist AP5 disrupts classical eyeblink conditioning in rabbits. Brain Res 887:144-56 Chen G, Steinmetz JE. 2000b. Microinfusion of protein kinase inhibitor H7 into the cerebellum impairs the acquisition but not the retention of classical eyeblink conditioning in rabbits. Brain Res 856: 193-201 Chen L, Bao S, Lockard JM, Kim JK, Thompson RF. 1996. Impaired classical eyeblink conditioning in cerebellar-lesioned and Purkinje cell degeneration (pcd) mutant mice. J Neurosci 16: 2829-38 Christian KM, Thompson RF. 2003. Neural substrates of eyeblink conditioning: acquisition and retention. Learn Mem 10: 427-55 Clark GA, McCormick DA, Lavond DG, Thompson RF. 1984. Effects of lesions of cerebellar nuclei on conditioned behavioral and hippocampal neuronal responses. Brain Res 291: 125-36 133 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Clark RE, Lavond DG. 1993. Reversible lesions of the red nucleus during acquisition and retention of a classically conditioned behavior in rabbits. Behav Neurosci 107: 264-70 Clark RE, Lavond DG. 1996. Neural unit activity in the trigeminal complex with interpositus or red nucleus inactivation during classical eyeblink conditioning. Behav Neurosci 110: 13-21 Clark RE, Squire LR. 1998. Classical conditioning and brain systems: the role of awareness. Science 280: 77-81 Clark RE, Zhang AA, Lavond DG. 1992. Reversible lesions of the cerebellar interpositus nucleus during acquisition and retention of a classically conditioned behavior. Behav Neurosci 106: 879-88 Cohen LG, Brasil-Neto JP, Pascual-Leone A, Hallett M. 1993. Plasticity of cortical motor output organization following deafferentation, cerebral lesions, and skill acquisition. Adv Neurol 63: 187-200 Coleman SR, Gormezano I. 1971. Classical conditioning of the rabbit's (Oryctolagus cuniculus) nictitating membrane response under symmetrical CS-US interval shifts. J Comp Physiol Psychol 77: 447-55 Cooper JR, Bloom FE, Roth RH. 2003. The biochemical basis of neuropharmacology. Oxford University Press Conner JM, Culberson A, Packowski C, Chiba AA, Tuszynski MH. 2003. Lesions of the Basal forebrain cholinergic system impair task acquisition and abolish cortical plasticity associated with motor skill learning. Neuron 38: 819-29 Courville J. 1966. Somatotopical organization of the projection from the nucleus interpositus anterior of the cerebellum to the red nucleus. An experimental study in the cat with silver impregnation methods. Exp Brain Res 2: 191-215 Daum I, Ackermann H, Schugens MM, Reimold C, Dichgans J, Birbaumer N. 1993. The cerebellum and cognitive functions in humans. Behav Neurosci 107: 411-9 Freeman JH, Jr., Nicholson DA. 2000. Developmental changes in eye-blink conditioning and neuronal activity in the cerebellar interpositus nucleus. J Neurosci 20: 813-9 Freeman JH, Jr., Shi T, Schreurs BG. 1998. Pairing-specific long-term depression prevented by blockade of PKC or intracellular Ca2+. Neuroreport 9: 2237-41 134 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Foy MR, Thompson RF. 1986. Single unit analysis of Purkinje cell discharge in classically conditioned and untrained rabbits._Soc Neurosci Abs 12, 518. Gabriel M. 1970. Intersession exposure of rabbits to conditioning apparatus, avoidance extinction, and intertrial behavior. J Comp Physiol Psychol 72: 244-249 Garcia KS, Mauk MD. 1998. Pharmacological analysis of cerebellar contributions to the timing and expression of conditioned eyelid responses. Neuropharmacology 37: 471-80 Gibbon J, Balsam P. 1981. Spreading association in time. In C. M. Locurto & H. S. Terrace & J. Gibbon (Eds.), Autoshaping and conditioning theory (pp. 219- 253). New York: Academic Press. Garcia KS, Steele PM, Mauk MD. 1999. Cerebellar cortex lesions prevent acquisition of conditioned eyelid responses. J Neurosci 19: 10940-7 Gomi H, Sun W, Finch CE, Itohara S, Yoshimi K, Thompson RF. 1999. Learning induces a CDC2-related protein kinase, KKIAMRE. J Neurosci 19: 9530-7 Gormezano I, Schneiderman N, Deaux E, Fuentes I. 1962. Nictitating membrane: classical conditioning and extinction in the albino rabbit. Science 138: 33-4 Gould TJ, Sears LL, Steinmetz JE. 1993. Possible CS and US pathways for rabbit classical eyelid conditioning: electrophysiological evidence for projections from the pontine nuclei and inferior olive to cerebellar cortex and nuclei. Behav Neural Biol 60: 172-85 Gould TJ, Steinmetz JE. 1994. Multiple-unit activity from rabbit cerebellar cortex and interpositus nucleus during classical discrimination/reversal eyelid conditioning. Brain Res 652: 98-106 Gould TJ, Steinmetz JE. 1996. Changes in rabbit cerebellar cortical and interpositus nucleus activity during acquisition, extinction, and backward classical eyelid conditioning. Neurobiol Learn Mem 65: 17-34 Gormezano I, Kehoe EJ, Marshall BS. 1983. Twenty years of classical conditioning with the rabbit. Progress Psychobiol Psych 10: 197-274 Hansel C, Linden DJ, D'Angelo E. 2001. Beyond parallel fiber LTD: the diversity of synaptic and non-synaptic plasticity in the cerebellum. Nat Neurosci 4: 467- 75 135 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Hardiman MJ, Ramnani N, Yeo CH. 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: 235-47 Hauge SA, Tracy JA, Baudry M, Thompson RF. 1998. Selective changes in AMPA receptors in rabbit cerebellum following classical conditioning of the eyelid- nictitating membrane response. Brain Res 803: 9-18 Hebb DO. 1949. The Organization of Behavior: A neuropsychological theory. New York: Wiley Ito M. 2002. Historical review of the significance of the cerebellum and the role of Purkinje cells in motor learning. Ann N Y Acad Sci 978: 273-88 Ito M, Kano M. 1982. Long-lasting depression of parallel fiber-Purkinje cell transmission induced by conjunctive stimulation of parallel fibers and climbing fibers in the cerebellar cortex. Neurosci Lett 33: 253-8 Ivkovich D, Lockard JM, Thompson RF. 1993. Interpositus lesion abolition of the eyeblink conditioned response is not due to effects on performance. Behav Neurosci 107: 530-2 Kasprow WJ, Schachtman TR, Miller RR. 1987. The comparator hypothesis of conditioned response generation: Manifest conditioned excitation and inhibition as a function of relative excitatory associative strengths of CS and conditioning context at the time of test. J Exp Psychol: Anim Behav Process 13: 395-406 Katz DB, Steinmetz JE. 1997. Single-unit evidence for eye-blink conditioning in cerebellar cortex is altered, but not eliminated, by interpositus nucleus lesions. Learn Mem 4: 88-104 Kehoe EJ. 1976. Effects of serial compound stimuli on stimulus selection in classical conditioning of the rabbit nictitating membrane response. Doctoral Dissertation, University o f Iowa Kehoe EJ, Weidemann G, Dartnall S. 2004. Apparatus exposure produces profound declines in the rabbit's nictitating membrane response to discrete stimuli. J Exp Psychol: Anim Behav Process 3: Kim JJ, Fanselow MS. 1992. Modality-specific retrograde amnesia of fear. Science 256: 675-7 136 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. King DA, Krupa DJ, Foy MR, Thompson RF. 2001. Mechanisms of neuronal conditioning. Int Rev Neurobiol 45: 313-37 Kleim JA, Freeman JH, Jr., Bruneau R, Nolan BC, Cooper NR, et al. 2002. Synapse formation is associated with memory storage in the cerebellum. Proc Natl AcadSci U S A 99: 13228-31 Kleim JA, Hogg TM, VandenBerg PM, Cooper NR, Bruneau R, Remple M. 2004. Cortical synaptogenesis and motor map reorganization occur during late, but not early, phase of motor skill learning. J Neurosci 24: 628-33 Kleim JA, Swain RA, Armstrong KA, Napper RM, Jones TA, Greenough WT. 1998. Selective synaptic plasticity within the cerebellar cortex following complex motor skill learning. Neurobiol Learn Mem 69: 274-89 Koekkoek SK, Hulscher HC, Dortland BR, Hensbroek RA, Elgersma Y, et al. 2003. Cerebellar LTD and learning-dependent timing of conditioned eyelid responses. Science 301: 1736-9 Krupa DJ, Thompson JK, Thompson RF. 1993. Localization of a memory trace in the mammalian brain. Science 260: 989-91 Krupa DJ, Thompson RF. 1995. 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: 5097-101 Krupa DJ, Thompson RF. 1997. Reversible inactivation of the cerebellar interpositus nucleus completely prevents acquisition of the classically conditioned eye- blink response. Learn Mem 3: 545-56 Lavond DG, Hembree TL, Thompson RF. 1985. Effect of kainic acid lesions of the cerebellar interpositus nucleus on eyelid conditioning in the rabbit. Brain Res 326: 179-82 Lavond DG, Knowlton BJ, Steinmetz JE, Thompson RF. 1987a. Classical conditioning of the rabbit eyelid response with a mossy-fiber stimulation CS: II. Lateral reticular nucleus stimulation. Behav Neurosci 101: 676-82 Lavond DG, Lincoln JS, McCormick DA, Thompson RF. 1984. Effect of bilateral lesions of the dentate and interpositus cerebellar nuclei on conditioning of heart-rate and nictitating membrane/eyelid responses in the rabbit. Brain Res 305: 323-30 137 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Lavond DG, Logan CG, Sohn JH, Garner WD, Kanzawa SA. 1990. Lesions of the cerebellar interpositus nucleus abolish both nictitating membrane and eyelid EMG conditioned responses. Brain Res 514: 238-48 Lavond DG, Steinmetz JE. 1989. Acquisition of classical conditioning without cerebellar cortex. Behav Brain Res 33: 113-64 Lavond DG, Steinmetz JE, Yokaitis MH, Thompson RF. 1987b. Reacquisition of classical conditioning after removal of cerebellar cortex. Exp Brain Res 67: 569-93 Lee T, Kim JJ. 2004. Differential effects of cerebellar, amygdalar, and hippocampal lesions on classical eyeblink conditioning in rats. J Neurosci 24: 3242-50 Lemieux SK, Woodruff-Pak DF. 2000. Functional MRI studies of eyeblink classical conditioning. See Woodruff-Pak & Steinmetz 2000, Vol. 1, pp. 71— 93 Lewis JL, Lo Turco JJ, Solomon PR. 1987. Lesions of the middle cerebellar peduncle disrupt acquisition and retention of the rabbit's classically conditioned nictitating membrane response. Behav Neurosci 101: 151-7 Lincoln JS, McCormick DA, Thompson RF. 1982. Ipsilateral cerebellar lesions prevent learning of the classically conditioned nictitating membrane/eyelid response. Brain Res 242: 190-3 Logan CG, Grafton ST. 1995. Functional anatomy of human eyeblink conditioning determined with regional cerebral glucose metabolism and positron-emission tomography. Proc Natl Acad Sci U S A 92: 7500-4 Logan CG, Lavond DG, Wong JT, Thompson RF. 1994. Acquisition of classically conditioned eyeblink response following bilateral lesions of flocculus and paraflocculus. Behav Neural Biol 61: 102-6 Lye RH, O ’Boyle DJ, Ramsden RT, Schady W. 1988. Effects of unilateral cerebellar lesions on the acquisition of eye-blink conditioning in man. 7. Physiol. 403:58P Mamounas LA, Thompson RF, Madden Jt. 1987. Cerebellar GABAergic processes: evidence for critical involvement in a form of simple associative learning in the rabbit. Proc Natl Acad Sci U S A 84: 2101-5 Marlin NA. 1981. Within-compound associations between the context and the conditioned stimulus. Learn Motiv 13: 526-531 138 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Marr D. 1969. A theory of cerebellar cortex. J Physiol 202: 437-70 Mauk MD, Garcia KS, Medina JF, Steele PM. 1998. Does cerebellar LTD mediate motor learning? Toward a resolution without a smoking gun. Neuron 20: 359-62 Mauk MD, Steinmetz JE, Thompson RF. 1986. Classical conditioning using stimulation of the inferior olive as the unconditioned stimulus. Proc Natl Acad Sci U S A 83: 5349-53 McCormick DA, Lavond DG, Clark GA, Kettner RE, Rising CE, Thompson RF. 1981. The engram found?. Role of the cerebellum in classical conditioning of nictitating membrane and eyelid responses. Bull Psychon Soc 18: 103-105 McCormick DA, Clark GA, Lavond DG, Thompson RF. 1982. Initial localization of the memory trace for a basic form of learning. Proc Natl Acad Sci U S A 79: 2731-5 McCormick DA, Lavond DG, Thompson RF. 1983. Neuronal responses of the rabbit brainstem during performance of the classically conditioned nictitating membrane (NM)/eyelid response. Brain Res 271: 73-88 McCormick DA, Steinmetz JE, Thompson RF. 1985. Lesions of the inferior olivary complex cause extinction of the classically conditioned eyeblink response. Brain Res 359: 120-30 McCormick DA, Thompson RF. 1984a. Cerebellum: essential involvement in the classically conditioned eyelid response. Science 223: 296-9 McCormick DA, Thompson RF. 1984b. Neuronal responses of the rabbit cerebellum during acquisition and performance of a classically conditioned nictitating membrane-eyelid response. J Neurosci 4: 2811-22 McGlinchey-Berroth R. 2000. Eyeblink classical conditioning in amnesia. See Woodruff-Pak & Steinmetz 2000, Vol. 1, pp. 205— 27 McGlinchey-Berroth R, Carrillo MC, Gabrieli JD, Brawn CM, Disterhoft JF. 1997. Impaired trace eyeblink conditioning in bilateral, medial-temporal lobe amnesia. Behav Neurosci 111: 873-82 McIntosh AR, Schreurs BG. 2000. Functional networks underlying human eyeblink conditioning. See Woodruff-Pak & Steinmetz 2000, Vol. 1, pp. 51— 77 139 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Medina JF, Garcia KS, Mauk MD. 2001. A mechanism for savings in the cerebellum. J Neurosci 21: 4081-9 Meftah EM, Rispal-Padel L. 1994. Synaptic plasticity in the thalamo-cortical pathway as one of the neurobiological correlates of forelimb flexion conditioning: electrophysiological investigation in the cat. J Neurophysiol 72: 2631-47 Miyata M, Okada D, Hashimoto K, Kano M, Ito M. 1999. Corticotropin-releasing factor plays a permissive role in cerebellar long-term depression. Neuron 22: 763-75 Molchan SE, Sunderland T, McIntosh AR, Herscovitch P, Schreurs BG. 1994. A functional anatomical study of associative learning in humans. Proc Natl Acad Sci U S A 91: 8122-6 Moore JW, Choi JS. 1997. Conditioned response timing and integration in the cerebellum. Learn Mem 4: 116-29 Moore JW, Desmond JE. 1982. Latency of the nictitating membrane response to periocular electrostimulation in unanesthetized rabbits. Physiol Behav 28: 1041-6 Murakami F, Oda Y, Tsukahara N. 1988. Synaptic plasticity in the red nucleus and learning. Behav Brain Res 28: 175-9 Nowak AJ, Marshall-Goodell B, Kehoe EJ, Gormezano I. 1997. Elicitation, modification, and conditioning of the rabbit nictitating membrane response by electrical stimulation in the spinal trigeminal nucleus, inferior olive, interpositus nucleus, and red nucleus. Behav Neurosci 111: 1041-55 Nudo RJ, Milliken GW, Jenkins WM, Merzenich MM. 1996. Use-dependent alterations of movement representations in primary motor cortex of adult squirrel monkeys. J Neurosci 16: 785-807 Ohyama T, Mauk M. 2001. Latent acquisition of timed responses in cerebellar cortex. J Neurosci 21: 682-90 Ohyama T, Nores WL, Mauk MD. 2003a. Stimulus generalization of conditioned eyelid responses produced without cerebellar cortex: implications for plasticity in the cerebellar nuclei. Learn Mem 10: 346-54 Ohyama T, Nores WL, Murphy M, Mauk MD. 2003b. What the cerebellum computes. Trends Neurosci 26: 222-7 140 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Pananceau M, Rispal-Padel L. 2000. Functional plasticity in the interposito-thalamo- cortical pathway during conditioning. Role of the interstimulus interval. Exp Brain Res 132: 314-27 Pearce AJ, Thickbroom GW, Byrnes ML, Mastaglia FL. 2000. Functional reorganisation of the corticomotor projection to the hand in skilled racquet players. Exp Brain Res 130: 238-43 Penick S, Solomon PR. 1991. Hippocampus, context, and conditioning. Behav Neurosci 105: 611-7 Perrett SP. 1998. Temporal discrimination in the cerebellar cortex during conditioned eyelid responses. Exp Brain Res 121: 115-24 Perrett SP, Ruiz BP, Mauk MD. 1993. Cerebellar cortex lesions disrupt learning- dependent timing of conditioned eyelid responses. J Neurosci 13: 1708-18 Phillips RG, LeDoux JE. 1992. Differential contribution of amygdala and hippocampus to cued and contextual fear conditioning. Behav Neurosci 106: 274-85 Poulos AM, Robleto K, Thompson RF. 2002. Effects of sensory stimulation and learning on excitability of the interpositus nucleus. Abstr Soc Neurosci 28: 79.10 Racine RJ, Wilson DA, Gingell R, Sunderland D. 1986. Long-term potentiation in the interpositus and vestibular nuclei in the rat. Exp Brain Res 63: 158-62 Remple MS, Bruneau RM, VandenBerg PM, Goertzen C, Kleim JA. 2001. Sensitivity of cortical movement representations to motor experience: evidence that skill learning but not strength training induces cortical reorganization. Behav Brain Res 123: 133-41 Rispal-Padel L, Cicirata F, Pons C. 1982. Cerebellar nuclear topography of simple and synergistic movements in the alert baboon (Papio papio). Exp Brain Res 47: 365-80 Rispal-Padel L, Meftah EM. 1992. Changes in motor responses induced by cerebellar stimulation during classical forelimb flexion conditioning in cat. J Neurophysiol 68: 908-26 141 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Rogers RF, Britton GB, Steinmetz JE. 2001. Learning-related interpositus activity is conserved across species as studied during eyeblink conditioning in the rat. Brain Res 905: 171-7 Salvatierra AT, Berry SD. 1989. Scopolamine disruption of septo-hippocampal activity and classical conditioning. Behav Neurosci 103: 715-21 Scavio MJ, Jr., Gormezano I. 1974. CS intensity effects on rabbit nictitating membrane conditioning, extinction and generalization. Pavlov J Biol Sci 9: 25-34 Schreurs BG. 2003. Classical conditioning and modification of the rabbit's (Oryctolagus cuniculus) unconditioned nictitating membrane response. Behav Cogn Neurosci Rev 2: 83-96 Schreurs BG, McIntosh AR, Bahro M, Herscovitch P, Sunderland T, Molchan SE. 1997. Lateralization and behavioral correlation of changes in regional cerebral blood flow with classical conditioning of the human eyeblink response. J Neurophysiol 77: 2153-63 Schultz W, Montgomery EB, Jr., Marini R. 1979. Proximal limb movements in response to microstimulation of primate dentate and interpositus nuclei mediated by brain-stem structures. Brain 102: 127-46 Sears LL, Steinmetz JE. 1990a. Acquisition of classically conditioned-related activity in the hippocampus is affected by lesions of the cerebellar interpositus nucleus. Behav Neurosci 104: 681-92 Sears LL, Steinmetz JE. 1990b. Haloperidol impairs classically conditioned nictitating membrane responses and conditioning-related cerebellar interpositus nucleus activity in rabbits. Pharmacol Biochem Behav 36: 821- 30 Selden NR, Everitt BJ, Jarrard LE, Robbins TW. 1991. Complementary roles for the amygdala and hippocampus in aversive conditioning to explicit and contextual cues. Neuroscience 42: 335-50 Shibuki K, Gomi H, Chen L, Bao S, Kim JJ, et al. 1996. Deficient cerebellar long term depression, impaired eyeblink conditioning, and normal motor coordination in GFAP mutant mice. Neuron 16: 587-99 Shibuki K, Okada D. 1991. Endogenous nitric oxide release required for long-term synaptic depression in the cerebellum. Nature 349: 326-8 142 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Shinkman PG, Swain RA, Thompson RF. 1996. Classical conditioning with electrical stimulation of cerebellum as both conditioned and unconditioned stimulus. Behav Neurosci 110: 914-21 Shohamy D, Allen MT, Gluck MA. 2000. Dissociating entorhinal and hippocampal involvement in latent inhibition. Behav Neurosci 114: 867-74 Smith MC, Coleman SR, Gormezano I. 1969. Classical conditioning of the rabbit's nictitating membrane response at backward, simultaneous, and forward CS- US intervals. J Comp Physiol Psychol 69: 226-31 Solomon PR, Gottfried KE. 1981. The septohippocampal cholinergic system and classical conditioning of the rabbit's nictitating membrane response. J Comp Physiol Psychol 95: 322-30 Solomon PR, Pomerleau D, Bennett L, James J, Morse DL. 1989. Acquisition of the classically conditioned eyeblink response in humans over the life span. Psychol Aging 4: 34-41 Solomon PR, Solomon SD, Schaaf EV, Perry HE. 1983. Altered activity in the hippocampus is more detrimental to classical conditioning than removing the structure. Science 220: 329-31 Solomon PR, Vander Schaaf ER, Thompson RF, Weisz DJ. 1986. Hippocampus and trace conditioning of the rabbit's classically conditioned nictitating membrane response. Behav Neurosci 100: 729-44 Stanton ME. 2000. Multiple memory systems, development and conditioning. Behav Brain Res 110: 25-37 Steinmetz JE. 1990. Classical nictitating membrane conditioning in rabbits with varying interstimulus intervals and direct activation of cerebellar mossy fibers as the CS. Behav Brain Res 38: 97-108 Steinmetz JE. 2000. Brain substrates of classical eyeblink conditioning: a highly localized but also distributed system. Behav Brain Res 110: 13-24 Steinmetz JE, Logan CG, Rosen DJ, Thompson JK, Lavond DG, Thompson RF. 1987. Initial localization of the acoustic conditioned stimulus projection system to the cerebellum essential for classical eyelid conditioning. Proc Natl Acad Sci U S A 84: 3531-5 143 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Steinmetz JE, Logue SF, Steinmetz SS. 1992. Rabbit classically conditioned eyelid responses do not reappear after interpositus nucleus lesion and extensive post-lesion training. Behav Brain Res 51: 103-14 Steinmetz JE, Rosen DJ, Chapman PF, Lavond DG, Thompson RF. 1986. Classical conditioning of the rabbit eyelid response with a mossy-fiber stimulation CS: I. Pontine nuclei and middle cerebellar peduncle stimulation. Behav Neurosci 100: 878-87 Steinmetz JE, Sengelaub DR. 1992. Possible conditioned stimulus pathway for classical eyelid conditioning in rabbits. I. Anatomical evidence for direct projections from the pontine nuclei to the cerebellar interpositus nucleus. Behav Neural Biol 57: 103-15 Stout SC, Chang R, Miller RR. 2003. Trial spacing is a determinant of cue interaction. Journal of Experimental Psychology: Animal Behavior Processes, 29, 23-38. Swain RA, Shinkman PG, Nordholm AF, Thompson RF. 1992. Cerebellar stimulation as an unconditioned stimulus in classical conditioning. Behav Neurosci 106: 739-50 Swain RA, Shinkman PG, Thompson JK, Grethe JS, Thompson RF. 1999. Essential neuronal pathways for reflex and conditioned response initiation in an intracerebellar stimulation paradigm and the impact of unconditioned stimulus preexposure on learning rate. Neurobiol Learn Mem 7 1: 167-93 Thach WT, Perry JG, Kane SA, Goodkin HP. 1993. Cerebellar nuclei: rapid alternating movement, motor somatotopy, and a mechanism for the control of muscle synergy. Rev Neurol (Paris) 149: 607-28 Thompson RF. 1986. The neurobiology of learning and memory. Science 233: 941-7 Thompson RF, Krupa DJ. 1994. Organization of memory traces in the mammalian brain. Annu Rev Neurosci 17: 519-49 Topka H, Valls-Sole J, Massaquoi SG, Hallett M. 1993. Deficit in classical conditioning in patients with cerebellar degenerations. Brain 116:961— 69 Tracy, JA. 1995. Brain and behavior correlates in classical conditioning of the rabbit eyeblink response. Doctoral Dissertation, University o f Southern California Tracy JA, Green JT, Steinmetz JE. 1999 Extracellular interpositus stimulation as a conditioned stimulus during eyeblink conditioning. Soc Neurosci Abs, 25, 95. 144 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Tracy JA, Britton GB, Steinmetz JE. 2001. Comparison of single unit responses to tone, light, and compound conditioned stimuli during rabbit classical eyeblink conditioning. Neurobiol Learn Mem 76: 253-67 Tracy JA, Steinmetz JE. 1998. Purkinje cell responses to pontine stimulation CS during rabbit eyeblink conditioning. Physiol Behav 65: 381-6 Tracy JA, Thompson JK, Krupa DJ, Thompson RF. 1998. Evidence of plasticity in the pontocerebellar conditioned stimulus pathway during classical conditioning of the eyeblink response in the rabbit. Behav Neurosci 112: 267- 85 Wang YT, Linden DJ. 2000. Expression of cerebellar long-term depression requires postsynaptic clathrin-mediated endocytosis. Neuron 25: 635-47 Wikgren J, Korhonen T. 2001. Interpositus nucleus inactivation reduces unconditioned response amplitude after paired but not explicitly unpaired treatment in rabbit eyeblink conditioning. Neurosci Lett 308: 181-4 Woodruff-Pak DS, Lavond DG, Thompson RF. 1985. Trace conditioning: abolished by cerebellar nuclear lesions but not lateral cerebellar cortex aspirations. Brain Res 348: 249-60 Yang BY, Weisz DJ. 1992. An auditory conditioned stimulus modulates unconditioned stimulus-elicited neuronal activity in the cerebellar anterior interpositus and dentate nuclei during nictitating membrane response conditioning in rabbits. Behav Neurosci 106: 889-99 Yeo CH, Hardiman MJ, Glickstein M. 1984. Discrete lesions of the cerebellar cortex abolish the classically conditioned nictitating membrane response of the rabbit. Behav Brain Res 13: 261-6 Yeo CH, Hardiman MJ, Glickstein M. 1985a. Classical conditioning of the nictitating membrane response of the rabbit. I. Lesions of the cerebellar nuclei. Exp Brain Res 60: 87-98 Yeo CH, Hardiman MJ, Glickstein M. 1985b. Classical conditioning of the nictitating membrane response of the rabbit. II. Lesions of the cerebellar cortex. Exp Brain Res 60: 99-113 145 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Young RA, Cegavske CF, Thompson RF. 1976. Tone-induced changes in excitability of abducens motoneurons and of the reflex path of nictitating membrane response in rabbit (Oryctolagus cuniculus). J Comp Physiol Psychol 90: 424-34 Zhen X, Du W, Romano AG, Friedman E, Harvey JA. 2001. The p38 mitogen- activated protein kinase is involved in associative learning in rabbits. J Neurosci 21: 5513-9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Cerebellar cortical-nuclear projections and classical eyeblink conditioning

PDF

Apoptotic pathways involved in kainate excitotoxicity

PDF

Displaced aggression as a function of target power

PDF

Estrogen effects on excitability and plasticity in hippocampus

PDF

Interhemispheric interaction in bilateral redundancy gain: Effects of physical similarity

PDF

Involvement of the secretory pathway for AMPA receptors in the expression of LTP

PDF

Behavioral and neural investigations of perceptual affect

PDF

Effects Of Physostigmine On Avoidance Conditioning And Retention In The Isolated Cockroach Ganglion

PDF

Dynamic regulation of vasopressin, a mnemonic neuropeptide, induction of calcium signaling and V1a vasopressin receptors in the rat cerebral cortex

PDF

Development of orthographic knowledge in a consonantal script: Children's invented spellings in Farsi

PDF

Autonomic and cognitive indices of semantic conditioning and generalization

PDF

Acquisition, consolidation and storage of an associative memory in the cerebellum

PDF

Biochemical evidence for calpain's involvement in long term synaptic plasticity

PDF

Fluid balance and neuropeptides in the lateral hypothalamic area

PDF

The Role Of Cerebellar Cortex In Classical Conditioning Of Discrete Motor Movements: Intracranial Electrical Stimulation Studies

PDF

Functional impacts of morphology on synaptic transmission

PDF

Atypicality: Benefit and bane. Provocation, trigger, typicality, and the expression of aggression and generalization

PDF

A phenomenological inquiry into the essential meanings of the most intense experience of religiosity and spirituality

PDF

An Examination Of Positive And Negative Reinforcement In Classical And Operant Conditioning Paradigms In The Primary Psychopath

PDF

Functional regulation and trafficking mechanism of rat plasma membrane GABA transporter 1

Asset Metadata

Creator Poulos, Andrew Makoto (author)

Core Title A context for timing, conditioning and modification of cerebellar function

School Graduate School

Degree Doctor of Philosophy

Degree Program Psychology

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

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

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Thompson, Richard (committee chair), Greene, Ernest (committee member), Madigan, Stephen (committee member), Swanson, Larry (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-421922

Unique identifier UC11340396

Identifier 3155462.pdf (filename),usctheses-c16-421922 (legacy record id)

Legacy Identifier 3155462.pdf

Dmrecord 421922

Document Type Dissertation

Rights Poulos, Andrew Makoto

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA