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Pharmacological analysis of the magnocellular red nucleus during classical conditioning of the rabbit nictitating membrane response
Brain Research, 454 (1988) 131-139
131
Elsevier BRE 13711
Pharmacological analysis of the magnocellular red nucleus during classical conditioning of the rabbit nictitating membrane response Deborah A. Haley 1, Richard F. Thompson 2 and John Madden IV 3 1Department of Psychology, Stanford University, Stanford, California 94305 (U.S.A.), 2Department of Psychology, University of Southern California, Los Angeles, California 90089 (U.S.A.) and 3Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, California 94305 (U.S.A.) (Accepted 19 January 1988)
Key words: Red nucleus; Nictitating membrane response; 7-Aminobutyric acid; Classical conditioning; Bicuculline methiodide; Picrotoxin
Previous experiments have suggested that the red nucleus is an essential structure in the neural pathways subserving the conditioned responses (CRs) elicited in several simple associative learning paradigms. The present investigation confirms the involvement of the magnocellular red nucleus in production of the classically conditioned nictitating membrane response in the rabbit and suggests that 7-aminobutyric acid (GABA) processes within this structure are involved in expression of the CR. Specifically, these studies demonstrate that microinfusion of a GABA antagonist (either picrotoxin or bicuculline methiodide) into the magnocellular red nucleus can selectively and reversibly reduce or abolish retention of the CR, without altering the unconditioned reflex response. Furthermore, these pharmacological manipulations that disrupt the CR are both anatomically and pharmacologically specific, and demonstrate a predictable dose-dependent function. These findings suggest that GABAergic processes within the magnocellular red nucleus are part of the critical circuitry subserving the CR.
INTRODUCTION
project to the contralateral, magnocellular r e d nucleus 19'25. In turn, a p o r t i o n of the efferent fibers
Classical conditioning is a form of simple associative learning. To elucidate the neurobiological basis of this form of learning, an accurate delineation of the neural pathways, which process information about either the conditioned (CS) or unconditioned stimulus (US), must be established. Such neural characterization has been initiated for classical conditioning of the nictitating m e m b r a n e (NM) response in the rabbit. These investigations indicate that cerebellar loci, including the anterior interpositus nucleus and perhaps restricted areas of the cerebellar cortex, are essential structures in the circuitry mediating the conditioned response (CR) 7-9,16,17,20,43-46.
from the red nucleus p r o j e c t to the contralateral accessory abducens nucleus 1°'22'23, an anatomical structure that controls reflexive retraction of the eyeball and passive closure of the N M through projections to the retractor bulbi muscles via the VIth nerve 2'3'1s'28.
A n a t o m i c a l tracing studies, c o n d u c t e d in a n u m b e r of species including the rabbit, d e m o n s t r a t e that the m a j o r efferent fibers from the interpositus nucleus
ings, obtained from the red nucleus during conditioning, d e m o n s t r a t e a p a t t e r n of unit discharge that precedes and models the C R 21.
Interestingly, the red nucleus has b e e n extensively described as a structure that displays b o t h substantial synaptic plasticity26'33'36'38 and essential involvement in the p e r f o r m a n c e of several l e a r n e d responses 11'29' 30,32,35. In classical conditioning of the rabbit N M response, lesions of the magnocellular red nucleus abolish the C R on the eye contralateral to the lesion site 11,29,3°. M o r e o v e r , electrophysiological record-
Correspondence: J. Madden IV, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305, U.S.A. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B .V. (Biomedical Division)
132 In the present investigation, pharmacological manipulations of the red nucleus are employed to substantiate that this structure is part of the essential circuitry mediating the CR and to initiate characterization of those neurotransmitter systems involved in expression of the CR. Previous experiments have confirmed the presence of 7-aminobutyric acid (GABA)-containing neurons within the red nucleus and have strongly implicated their role in synaptic transmission 1,13,15,24,26, 31,41. Furthermore, the GABAergic synapses in the magnocellular portion of the red nucleus have exhibited plasticity following experimental manipulation of the interpositus nucleus 12'27,42. Noting the potential significance of these GABAergic processes in associative learning, the present experimental manipulations were directed at determining whether micro-infusion of G A B A antagonists within the red nucleus could affect retention of the classically conditioned NM response. MATERIALS AND METHODS Preliminary studies, involving both unit recordings and electrical stimulation of the red nucleus, were carried out to confirm that the magnocellular region displayed characteristics that were consistent with involvement in the conditioned NM response. Following this assessment, pharmacological experiments were initiated to determine the involvement of GABAergic processes within the magnocellular red nucleus in retention of this form of simple associative learning.
Subjects Twelve male, New Zealand Albino Rabbits (Oryctolagus cuniculus), weighing approximately 2.5 kg at the beginning of the experiment, were used. During the investigation, all animals were individually housed in facilities set on a 12 h light/12 h dark cycle and were given ad libitum access to food and water. Rabbits were allowed to adapt to colony conditions for approximately 7 days before experimental procedures were initiated.
Surgicalprocedures During surgical procedures, a cannula/electrode assembly, which would permit the infusion of neuro-
transmitter antagonists during behavioral testing procedures, was implanted into the red nucleus. Each cannula/electrode assembly consisted of a 26gauge stainless-steel guide cannula, a 33-gauge internal stylet (Plastic Products, Roanoke, VA) that extended 1.25 mm beyond the tip of the guide cannula, and a recording electrode that was immediately adjacent to the guide cannula and extended 0.5 m m ventral to the tip of the internal stylet. To initiate surgical procedures, rabbits were anesthetized with a mixture of halothane gas ( 2 - 3 % ) and oxygen and then secured in a stereotaxic head holder (Josef Biela Instruments, Anaheim, CA). The skull was exposed and then positioned such that the lambda bone suture was 1.5 mm ventral to the bregma bone suture. The cannula/electrode assembly was stereotaxically implanted in the right red nucleus (coordinates: 0.8 _+ 0.3 mm lateral to the midline, 9.0 + 0.5 mm posterior, and 14.0 + 1.0 mm ventral to the skull bone at bregma), using electrophysiological monitoring from the recording electrode to guide the cannula placement. The cannula/electrode assembly and a small headstage, designed to accommodate the stimulus delivery/micropotentiometer system used during behavioral conditioning procedures, were secured to the skull using skull screws and dental acrylic. The incision area was cleaned; treated with an antibacterial agent; and sutured closed, leaving only the headstage and the internal stylet of the cannula/ electrode assembly accessible. A small loop of silk thread was then placed in the left NM for subsequent monitoring of NM movements during conditioning procedures. Animals were allowed a minimum of 5 days to recover from these procedures before behavioral training was initiated.
Behavioral trainingprocedures Prior to behavioral training, rabbits were allowed to adapt to the experimental apparatus in the absence of stimuli presentation. During this adaptation period, the animal was positioned in a Plexiglas restrainer formed to restrict head and body movements and then situated within a ventilated, sound-attenuated test chamber. The headstage was connected to a headmount that held an airpuff delivery nozzle and minitorque potentiometer. The airpuff delivery nozzle was positioned 1 cm in front of the animal's
133 cornea, but was not activated during the adaptation period. The wire wiper arm of the potentiometer was attached to the silk suture loop in the left NM of the animal and was used to monitor NM movements. The adaptation session was carried out over a 1 h period. On the next day, conditioning procedures were initiated. Animals were placed in the Plexiglas restrainer and fitted with the headmount, airpuff delivery nozzle, and the wiper arm of the potentiometer as described for adaptation; however, animals now received paired presentations of an auditory CS (a 350 ms, i kHz tone at 85 dB SPL) and a co-terminant, unconditioned airpuff to the cornea on the left eye (US: 100 ms, 2.1 N/cm 2 airpuff). Each training session consisted of 117 trials arranged into 13 blocks. Each block included 1 CS-alone test trial and 8 CS-US paired trials. The trials were delivered every 20-40 s (mean = 30 s). Conditioned and unconditioned responses were determined by calculating the peakNM-response amplitude (expressed in ram) during these CS and US periods respectively. A C.R was defined as a NM extension of at least 0.5 mm, occurring within 250 ms of the CS onset and before the US onset. Training sessions were conducted on consecutive days until the animal exhibited at least a 95% CR proficiency in a given training session.
sion and all testing sessions were conducted 4 days apart.
Experimental design A series of 3 experiments were used to assess the effects of GABAergic and glycinergic antagonists, micro-infused into the red nucleus, during retention of the classically conditioned NM response. In Experimentl, three doses (0.19, 0.37, and 0.75 nmol) of the G A B A antagonist picrotoxin and the vehicle (0.75/A of artificial CSF) were administered to each of 6 rabbits. The micro-infusion of each dose or the vehicle occurred on separate behavioral testing days and the order of the 'dose administrations was randomized. In Experiment 2, each of 6 rabbits received 3 doses of a second G A B A antagonist, bicuculline methiodide (0.75, 1.5, and 3.0 nmol) or vehicle (0.75 ~1 of artificial CSF) on separate behavioral testing days and in randomized dose sequence. In Experiment 3, the 4 animals that were sensitive to bicuculline methiodide in the second study were administered the glycine antagonist strychnine (3.0 nmol), bicuculline methiodide (3.0 nmol), or vehicle (0.75/~1 of artificial CSF) on separate behavioral testing days and in randomized dose sequence.
Tissuepreparation Behavioral testingprocedures During behavioral testing, animals first received 4 blocks of paired CS-US presentations as described under behavioral training procedures. Immediately following this baseline conditioning, the internal stylet of the chronic, guide cannula was removed and an approximately matched internal cannula, which was connected to a microsyringe via polyethylene tubing, was inserted. Designated pharmacological compounds in appropriate amounts were dissolved in a 0.75/~1 volume of artificial cerebrospinal fluid (CSF, pH 7.4) and were then continuously infused over a 1 min period. To allow for adequate diffusion of an infused compound, a 5 min period intervened between the micro-infusion and resumption of behavioral testing. To determine the behavioral effects of an infused substance, animals were then monitored for an additional 15 blocks of paired conditioning procedures. To minimize possible tachyphylaxis, only one micro-infusion was administered in a given testing ses-
Following behavioral testing, each animal was deeply anesthetized with sodium pentobarbital and then perfused through the heart with a 10% formalin solution. The brain was then removed from the skull; embedded in albumin, which was allowed to gel; and then sectioned at 80/~m through areas containing the cannula/electrode track. Once mounted on prepared slides, all brain sections were stained with Cresyl violet. RESULTS
Preliminary electrophysiologicalanalysis A representative multiple unit peristimulus histogram (Fig. 1), obtained from the magnocellular red nucleus during one training session, demonstrated a pattern of unit discharge that modeled and preceded the NM response in time. Furthermore, electrical stimulation of this region elicited a selective eyeball retraction, eyelid closure, and NM extension of the
134
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Fig. 1. A representative histogram obtained from the magnocellular red nucleus during classical conditioning of the NM response over one training period (117 trials). The upper trace of this histogram indicates the average NM extension during the training period. The lower trace depicts the corresponding magnocellular red nucleus multiple unit peristimulus histogram. The histogram bar spans a 9 ms time bin. The first vertical line in the histogram indicates tone onset; the second vertical line indicates air puff onset. Note, the pattern of unit discharge from the red nucleus appears to model the NM response and to precede the response in time.
contralateral eye (Fig. 2). Collectively, these observations suggested that the magnocellular red nucleus was involved in expression of the NM response and thus, provided a rationale for pharmacological analysis.
Histological reconstruction of micro-infusion sites To determine the precise site of antagonist infusion for each animal, a histological reconstruction of each cannula tip placement was prepared (Fig. 3), using the atlas of Urban and Richard 4°. Such reconstructions demonstrated that those animals, which had shown either a significant diminution or a complete abolition of the CR following microinfusion of either picrotoxin or bicuculline methiodide, in fact, had cannulae placements within 0.5 mm of the right magnocellular red nucleus (Expt. 1 : 4 of 6 animals had such placements; Expt. 2:4 of 6 animals). In contrast, the remaining rabbits, which had shown no reduction of the CR peak amplitude following such infusions, had cannulae placements more distal to the magnocellular red nucleus. The behavioral testing data from those animals with cannulae tip placements within 0.5 mm of the magnocellular red nucleus were further analyzed to determine the specific dose-dependent function of the various pharmacological agents, which could either markedly reduce or completely abolish conditioned responding.
Experiment h effects of picrotoxin on the N M response Microinfusion of picrotoxin into the magnocellular red nucleus markedly reduced or abolished the CR peak amplitude in the well-trained animal (Fig. 4). Furthermore, this effect was dose-dependent. A twoway analysis of variance (drug treatment x 2 grouped blocks of trials) with repeated measures over both factors revealed a significant dose x block interaction (F24,72 = 2.61; P < 0.001) effect on CR amplitude. Posthoc analysis showed that the postinfusion CR amplitudes for animals receiving the two highest drug dose levels (0.37 and 0.75 nmol) were significantly different from the amplitudes of animals receiving vehicle. The CR amplitude for the 0.37 nmol dose of picrotoxin remained significantly different from vehicle during the first 2 blocks postinfusion (blocks 5-6), while the 0.75 nmol dose of picrotoxin remained significantly different for 6 blocks postinfusion (blocks 5-10). In contrast to this effect on the CR, picrotoxin produced no significant effect on the amplitude of the unconditioned response (UR). Experiment 2: effects of bicuculline methiodide on the N M response Microinfusion of bicuculline methiodide into the magnocellular red nucleus also produced a significant dose-dependent diminution or abolition of conditioned responding (Fig. 5). A two-way analysis of variance (drug treatment x 2 grouped blocks of trials) with repeated measures revealed a significant dose x block interaction (F24,72 = 7.99; P < 0.001) effect on the CR peak amplitude. Posthoc analysis showed that the postinfusion CR amplitudes for animals receiving each of the 3 drug dose levels (0.75, 1.5 and 3.0 nmol) were significantly different from the amplitudes of animals receiving vehicle. The CR amplitude for the 0.75 and 1.5 nmol doses of bicuculline methiodide remained significantly different from vehicle during the first 2 blocks postinfusion (blocks 5-6), while the 3.0 nmol dose remained significantly different for 4 blocks postinfusion (blocks 5-8). However, bicuculline methiodide produced no significant effect on the U R peak amplitude. Experiment 3: effects of strychnine on the N M response In contrast to the effects of the G A B A antagonists,
135 microinfusion of the glycine antagonist, strychnine, into the magnoceUular red nucleus had no apparent effect on the amplitude of either the C R or U R . These observations were confirmed statistically. DISCUSSION Evidence from previous lesion 11'29'3° studies suggested that the magnocellular red nucleus is a critical structure within the essential neuronal circuitry mediating both acquisition and retention of the classically conditioned N M response. The present investigation substantiates the later finding and suggests that the functional integrity of G A B A e r g i c processes
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within this structure are critical for retention of the CR. Analysis of unit recordings, obtained from the magnocellular red nucleus during conditioning, demonstrated a pattern of unit discharge that preceded and modeled the C R (Fig. 1). Furthermore, direct electrical stimulation of this area produced selective eyeball retraction, NM extension, and eyelid closure on the contralateral eye (Fig. 2). These observations were consistent with previous findings 631'21. Micro-infusion of a G A B A antagonist, either picrotoxin or bicuculline methiodide, into this region produced a selective, marked diminution or abolition of the CR without altering the UR. This effect ap-
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Fig. 3. Histological reconstructions of infusion sites. Effective infusion sites for GABA antagonists, picrotoxin and bicuculline methiodide, are denoted by solid circles (n = 8). Collectively, these sites lie within 0.5 mm of the magnocellular red nucleus. Ineffective infusion sites for these antagonists are denoted by open circles (n = 4). BCI, nucleus brachii colliculi inferioris; CM, centrum medianum; CP, nuclei commissurae posterioris; CSU, nucleus centralis superior; D, nucleus Darkschewitsch; EW, nucleus Edinger-Westphal; FHI, fissura hippocampi; FR, formatio reticularis; GLD, corpus geniculatum laterale dorsale; GM, corpus geniculatum mediale; GYD, gyrus dentatus hippocampi; HID, hippocampus dorsalis; HIV, hippocampus ventralis; HL, nucleus habenulae lateralis; HM, nucleus habenulae medialis; IFLM, nucleus interstitialis fasciculi longitudinalis medialis; IMD, nucleus intermediodorsalis; IP, nucleus interpeduncularis; N III, nucleus nervi oculomotorii; PF, nucleus parafascicularis; PL, nucleus pontis lateralis; PM, nucleus pontis medialis; PO + PLV, posterior thalamic complex + pulvinar; PT, nucleus paratenialis; PTA, nucleus pretectalis anterior; PTP, nucleus pretectalis posterior; PVP, nucleus paraventricularis thalami posterior; R, nucleus tuber; RT, nucleus reticularis tegmenti Bechterew; SG, nucleus suprageniculatum; SGC, substantia grisea centralis; SGS, stratum griseum superficiale colliculi superioris; SN, substantia nigra; TO, nucleus tracti optici; VP, nucleus ventralis posterior thalami; bci, brachium colliculi inferioris; bp, brachium pontis; cc, corpus callosum; ccs, commissura colliculi superioris; chd, commissura hippocampi dorsalis; cin, cingulum; cp, commissura posterior; dpcs, decussatio pedunculorum cerebellaris superior; drs, decussatio tracti rubrospinalis; dts, decussatio tracti tectospinalis; tim, fasciculus longitudinalis medialis; fr, fasciculus retroflexus; fpd, fasciculus predorsalis; 11, lemniscus lateralis; lm, lemniscus medialis; pcm, pedunculus mammillaris; pcsd, pedunculus cerebellaris superior pars descendens; ped, pedunculus cerebri; py, pyramis; r III, radix nervi oculomotorii; tc, tractus tegmentalis centralis; to, tractus opticus; tp, tractus tectoponticus; ts, tractus tectospinalis. (Adapted from I. Urban and P. Rickard, A Stereotaxic Atlas of the New Zealand Rabbit's Brain, 197840.)
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Fig. 4. The effect of microinfusion of picrotoxin into the magnocellular red nucleus on the NM response. Mean UR (upper) and CR (lower) peak amplitudes are displayed (in mm) during 4 blocks of preinfusion baseline conditioning (blocks 1-4) and 15 blocks of postinfusion testing (blocks 5-19).
peared anatomically specific. Microinfusion of G A B A antagonists into several areas distal to the red nucleus did not affect the CR peak amplitude. Furthermore, the effect appeared pharmacologically specific. The glycine antagonist strychnine, infused at dosage levels equimolar to those of the G A B A antagonists, did not alter either conditioned or unconditioned responding. The fact that glycine also shares seizure-inducing properties with G A B A antagonists supports the premise that non-specific effects, such as possible seizures, are an unlikely explanation for the selective effects of G A B A antagonists on retention of the CR. While previous lesion studies of the red nucleus 11'29'30 could not distinguish whether abolition of the conditioned NM response was due to the disruption of fibers of passage (cerebello-thalamic) or to the disruption of the intrinsic circuitry of the red nucleus; the current pharmacological manipulations suggest that GABAergic processes, localized within the magnocellular red nucleus, are critically involved in expression of the CR. Although the present analysis does not precisely distinguish the relevant neurons, current evidence suggests that the most likely sources of G A B A within the red nucleus are inhibito-
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Fig. 5. The effect of microinfusion of bicuculline methiodide into the magnocellular red nucleus on the NM response. Mean UR (upper) and CR (lower) peak amplitudes are displayed (in mm) during 4 blocks of preinfusion baseline conditioning (blocks 1-4) and 15 blocks of postinfusion testing (blocks 5-19). ry interneurons ]5'26'31'41.These interneurons seem to form somatic and dendritic connections with both magnocellular and parvocellular red nucleus neurons and to demonstrate plasticity following experimental manipulations of the cerebellar deep nuclei 12'42. Further experimentation is necessary to precisely determine which GABAergic processes within the magnocellular red nucleus are essential and whether the adjacent parvocellular red nucleus also has a role in retention of the conditioned NM response. In sum, the red nucleus appears to be part of the essential circuitry for classical conditioning of motoric responsesll'29'3°'32'35; however, the precise nature of its involvement remains unclear. Tsukahara et al. 38'39 propose that the red nucleus is a probable site for critical plastic changes during classical conditioning of the forelimb flexion response in the cat. These investigators suggest that the red nucleus is subject to prolonged depolarization and accompanying synaptic changes as the result of impulse reverberation along a cerebelloreticular excitatory loop, which includes the nucleus interpositus, the nucleus reticularis tegmenti pontis, and the nucleus reticularis paramedianis 34,37. Kennedy TM, however, proposes that the red nucleus is more likely a 'phasic control structure' that maintains the efficiency of motor learning, particularly in primates. This model sug-
138 gests that learned movements, which are relayed
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from the deep cerebellar nuclei to the red nucleus, could also activate the rubro-olivary tract, which
ACKNOWLEDGEMENTS
would influence the inferior olive. As a result of such feedback, the inferior olive could modify its neural transmission to the cerebellar cortex. Specifically, the inferior olive could eliminate r e d u n d a n t instructions to the cortex once the motor task had been learned, and in this m a n n e r promote the efficiency of this learning circuit. In contrast, several investigators 4-6'30 suggest that the red nucleus is an essential relay for the expression of the CR, but that this structure is not the site of learning-induced modifications per se. Each of these hypotheses suggests quite a different role for the red nucleus in m o t o r learning and emphasizes the need for extended examination of
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The authors are grateful to Michael D. Mauk for his guidance in the stimulation experiment and Kimberly K. Powlishta for her valuable contributions to the statistical analysis. We are indebted to Joseph E. Steinmetz for his valuable criticism of this manuscript and Jack D. Barchas for his e n c o u r a g e m e n t during the investigation. This research was supported by funds from the National Institute of Mental Health Grant MH-23861 to J . D . B . ; National Science F o u n dation G r a n t BNS-81-17115, Office of Naval Research G r a n t N00014-83-K-0238, and a grant from The McKnight F o u n d a t i o n to R . F . T .
brane response, Brain Research Bull., 10 (1983) 765-773. 10 Desmond, J.E., Rosenfield, M.E. and Moore, J.W., An HRP study of the brainstem afferents to the accessory abducens region and the dorsolateral pons in the rabbit: implications for the conditioned nictitating membrane response, Brain Research Bull., 10 (1983) 747-763. 11 Haley, D.A., Lavond, D.G. and Thompson, R.F., Effects of contralateral red nuclear lesions on retention of the classically conditioned nictitating membrane/eyelid response, Soc. Neurosci. Abstr., 9 (1983) 643. 12 Katsumaro, H., Murakami, F., Wu, J.Y. and Tsukahara, N., Sprouting of GABAergic synapses in the red nucleus after lesions of the nucleus interpositus in the cat, J. Neurosci., 6 (1986) 2864-2874. 13 Katsumaro, H., Murakami, F., Wu, J.Y. and Tsukahara, N., GABAergic intrinsic interneurons in the red nucleus of the cat demonstrated with combined immunocytochemistry and anterograde degeneration methods, Neurosci. Res., 1 (1984) 35-44. 14 Kennedy, P.R•, The rubro-olivo-cerebellar teaching circuit, Med. Hypoth., 5 (1979) 799-807. 15 Kubota, M., Sakaguchi, H. and Tsukahara, N., Release of endogenous GABA from the cat red nucleus slice, Brain Research, 270 (1983) 190-192. 16 Lincoln, J.S., McCormick, D.A. and Thompson, R.F., Ipsilateral cerebellar lesions prevent learning of the classically conditioned nictitating membrane/eyelid response, Brain Research, 242 (1982) 190-193. • 17 Mamounas, L.A., Thompson, R.F. and Madden, IV., J., 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 (1987) 2101-2105. 18 Marek, G.J., McMaster, S.E., Gormezano, I. and Harvey, J., The role of the accessory abducens nucleus in the rabbit nictitating membrane response, Brain Research, 299 (1984) 215-229. 19 Massion, J., The mammalian red nucleus, Physiol. Rev., 47
139 (1967) 383-436. 20 McCormick, D.A., Clark, G.A., Lavond, D.G. and Thompson, R.F., Initial localization of the memory trace for a basic form of learning, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 2731-2742. 21 McCormick, D.A., Lavond, D.G. and Thompson, R.F., Neuronal responses of the rabbit brainstem during performance of the classically conditioned nictitating membrane/eyeblink response in the rabbit, Brain Research, 271 (1983) 73-88. 22 Mizuno, N., Mochizuki, K., Akimoto, C., Matsushima, R. and Nakamura, Y., Rubrobulbar projections in the rabbit. A light and electron microscopic study, J. Comp. Neurol., 147 (1973) 267-279. 23 Mizuno, N. and Nakamura, Y., Rubral fibers to the facial nucleus in the rabbit, Brain Research, 28 (1971) 545-549. 24 Murakami, F., Katsumara, H., Wu, J.Y., Matsuda, T. and Tsukahara, N., Immunocytochemical demonstration of GABAergic synapses on identified rubrospinal neurons, Brain Research, 267 (1983) 357-360. 25 Nakamura, Y. and Mizuno, N., An electron microscopic study of the interposito-rubral connections in the cat and rabbit, Brain Research, 35 (1971) 283-286. 26 Nieoullon, A., Neuronal plasticity in the red nucleus and the ventrolateral thalamus of the adult cat: a biochemical approach, Adv. Neurol., 40 (1984) 107-116. 27 Nieoullon, A. and Dusticier, N., Increased glutamate decarboxylase activity in the red nucleus of the adult cat after cerebellar lesions, Brain Research, 224 (1981) 129-139. 28 Rosenfield, M.E., Dovydaitus, A. and Moore, J.W., Brachium conjunctivum and rubrobulbar tract: brain stem projections of the red nucleus essential for the conditioned nictitating membrane response, Physiol. Behav., 34 (1985) 751-759. 29 Rosenfield, M.E. and Moore, J.W., Red nucleus lesions disrupt the classically conditioned nictitating membrane response in rabbits, Behav. Brain Res., 10 (1983) 393-398. 30 Rosenfield, M.E. and Moore, J.W., Red nucleus lesions impair acquisition and of the classically conditioned nictitating membrane response but not eye-to eye savings or unconditioned response amplitude, Behav. Brain Res., 17 (1983) 77-81. 31 Sakaguchi, H., Kubota, M., Nakamura, M. and Tsukahara, N., Effects of amino acids on cat red nucleus neurons in vitro, Exp. Brain Res., 54 (1984) 150-156. 32 Smith, A.M., The effects of rubral lesions and stimulation on conditioned forelimb flexion responses in the cat, Physiol. Behav., 5 (1970) 1121-1126. 33 Tsukahara, N., Synaptic plasticity in the red nucleus. In
C.W. Cotman (Ed.), Neuronal Plasticity, Raven, New York, 1978, pp. 113-131. 34 Tsukahara, N., Plastic and dynamic properties of the red nucleus neurons: a physiological study. In M.A.B. Brazier (Ed.), Brain Mechanisms in Memory and Learning: From Single Neuron to Man, Raven, New York, 1979, pp. 65-70. 35 Tsukahara, N., Neuronal plasticity: sprouting and the neuronal basis of learning, Trends Neurosci., 4 (1981) 234-237. 36 Tsukahara, N., Plasticity and specificity in the red nucleus. In P.A. Buser, W.A. Cobb and T. Okuma (Eds.), Kyoto Symposia, Elsevier, Amsterdam, 1982, pp. 153-157. 37 Tsukahara, N., Bando, T., Murakami, F. and Oda, Y., Properties of cerebello-precerebellar reverberating circuits, Brain Research, 274 (1983) 249-259. 38 Tsukahara, N. and Fujito, Y., Neuronal plasticity in the newborn and adult feline red nucleus. In H. Flohr and W. Precht (Eds.), Lesion-Induced Plasticity in Sensorimotor Systems, Springer, Berlin, 1981, pp. 64-74. 39 Tsukahara, N., Oda, Y. and Notsu, T., Classical conditioning mediated by the red nucleus in the cat, J. Neurosci., 1 (1981) 72-79. 40 Urban, I. and Richard, P., A Stereotaxic Atlas of the New Zealand Rabbit's Brain, Thomas, Springfield, 1978, 86 pp. 41 Vuillon-Cacciuttolo, G., Bosler, O. and Nieoullon, A., GABA neurons in the cat red nucleus: a biochemical and immunohistochemical demonstration, Neurosci. Lett., 52 (1984) 129-134. 42 Vuillon-Cacciuttolo, G., Bosler, O. and Nieoullon, A., Immunohistochemical evidence of plasticity of 7-aminobutyric acid neurons in the red nucleus and adjacent reticular formation after contralateral cerebellectomy in the adult cat, Neurosci. Lett., 70 (1986) 308-313. 43 Yeo, C.H., Hardiman, N.J. and Glickstein, M., Discrete lesions of the cerebellar cortex abolish the classically conditioned nictitating membrane response in the rabbit, Behav. Brain Res., 13 (1984) 261-266. 44 Yeo, C.H., Hardiman, N.J. and Glickstein, M., Classical conditioning of the nictitating membrane response of the rabbit. I. Lesions of the cerebellar nuclei, Exp. Brain Res., 60 (1985) 87-98. 45 Yeo, C.H., Hardiman, N.J. and Glickstein, M., Classical conditioning of the nictitating membrane response of the rabbit. II. Lesions of the cerebellar cortex, Exp. Brain Res., 60 (1985) 93-113. 46 Yeo, C.H., Hardiman, N.J. and Glickstein, M., Classical conditioning of the nictitating membrane response of the rabbit. III. Connections of the cerebellar lobule HVI, Exp. Brain Res., 60 (1985) 114-126.
131
Elsevier BRE 13711
Pharmacological analysis of the magnocellular red nucleus during classical conditioning of the rabbit nictitating membrane response Deborah A. Haley 1, Richard F. Thompson 2 and John Madden IV 3 1Department of Psychology, Stanford University, Stanford, California 94305 (U.S.A.), 2Department of Psychology, University of Southern California, Los Angeles, California 90089 (U.S.A.) and 3Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, California 94305 (U.S.A.) (Accepted 19 January 1988)
Key words: Red nucleus; Nictitating membrane response; 7-Aminobutyric acid; Classical conditioning; Bicuculline methiodide; Picrotoxin
Previous experiments have suggested that the red nucleus is an essential structure in the neural pathways subserving the conditioned responses (CRs) elicited in several simple associative learning paradigms. The present investigation confirms the involvement of the magnocellular red nucleus in production of the classically conditioned nictitating membrane response in the rabbit and suggests that 7-aminobutyric acid (GABA) processes within this structure are involved in expression of the CR. Specifically, these studies demonstrate that microinfusion of a GABA antagonist (either picrotoxin or bicuculline methiodide) into the magnocellular red nucleus can selectively and reversibly reduce or abolish retention of the CR, without altering the unconditioned reflex response. Furthermore, these pharmacological manipulations that disrupt the CR are both anatomically and pharmacologically specific, and demonstrate a predictable dose-dependent function. These findings suggest that GABAergic processes within the magnocellular red nucleus are part of the critical circuitry subserving the CR.
INTRODUCTION
project to the contralateral, magnocellular r e d nucleus 19'25. In turn, a p o r t i o n of the efferent fibers
Classical conditioning is a form of simple associative learning. To elucidate the neurobiological basis of this form of learning, an accurate delineation of the neural pathways, which process information about either the conditioned (CS) or unconditioned stimulus (US), must be established. Such neural characterization has been initiated for classical conditioning of the nictitating m e m b r a n e (NM) response in the rabbit. These investigations indicate that cerebellar loci, including the anterior interpositus nucleus and perhaps restricted areas of the cerebellar cortex, are essential structures in the circuitry mediating the conditioned response (CR) 7-9,16,17,20,43-46.
from the red nucleus p r o j e c t to the contralateral accessory abducens nucleus 1°'22'23, an anatomical structure that controls reflexive retraction of the eyeball and passive closure of the N M through projections to the retractor bulbi muscles via the VIth nerve 2'3'1s'28.
A n a t o m i c a l tracing studies, c o n d u c t e d in a n u m b e r of species including the rabbit, d e m o n s t r a t e that the m a j o r efferent fibers from the interpositus nucleus
ings, obtained from the red nucleus during conditioning, d e m o n s t r a t e a p a t t e r n of unit discharge that precedes and models the C R 21.
Interestingly, the red nucleus has b e e n extensively described as a structure that displays b o t h substantial synaptic plasticity26'33'36'38 and essential involvement in the p e r f o r m a n c e of several l e a r n e d responses 11'29' 30,32,35. In classical conditioning of the rabbit N M response, lesions of the magnocellular red nucleus abolish the C R on the eye contralateral to the lesion site 11,29,3°. M o r e o v e r , electrophysiological record-
Correspondence: J. Madden IV, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305, U.S.A. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B .V. (Biomedical Division)
132 In the present investigation, pharmacological manipulations of the red nucleus are employed to substantiate that this structure is part of the essential circuitry mediating the CR and to initiate characterization of those neurotransmitter systems involved in expression of the CR. Previous experiments have confirmed the presence of 7-aminobutyric acid (GABA)-containing neurons within the red nucleus and have strongly implicated their role in synaptic transmission 1,13,15,24,26, 31,41. Furthermore, the GABAergic synapses in the magnocellular portion of the red nucleus have exhibited plasticity following experimental manipulation of the interpositus nucleus 12'27,42. Noting the potential significance of these GABAergic processes in associative learning, the present experimental manipulations were directed at determining whether micro-infusion of G A B A antagonists within the red nucleus could affect retention of the classically conditioned NM response. MATERIALS AND METHODS Preliminary studies, involving both unit recordings and electrical stimulation of the red nucleus, were carried out to confirm that the magnocellular region displayed characteristics that were consistent with involvement in the conditioned NM response. Following this assessment, pharmacological experiments were initiated to determine the involvement of GABAergic processes within the magnocellular red nucleus in retention of this form of simple associative learning.
Subjects Twelve male, New Zealand Albino Rabbits (Oryctolagus cuniculus), weighing approximately 2.5 kg at the beginning of the experiment, were used. During the investigation, all animals were individually housed in facilities set on a 12 h light/12 h dark cycle and were given ad libitum access to food and water. Rabbits were allowed to adapt to colony conditions for approximately 7 days before experimental procedures were initiated.
Surgicalprocedures During surgical procedures, a cannula/electrode assembly, which would permit the infusion of neuro-
transmitter antagonists during behavioral testing procedures, was implanted into the red nucleus. Each cannula/electrode assembly consisted of a 26gauge stainless-steel guide cannula, a 33-gauge internal stylet (Plastic Products, Roanoke, VA) that extended 1.25 mm beyond the tip of the guide cannula, and a recording electrode that was immediately adjacent to the guide cannula and extended 0.5 m m ventral to the tip of the internal stylet. To initiate surgical procedures, rabbits were anesthetized with a mixture of halothane gas ( 2 - 3 % ) and oxygen and then secured in a stereotaxic head holder (Josef Biela Instruments, Anaheim, CA). The skull was exposed and then positioned such that the lambda bone suture was 1.5 mm ventral to the bregma bone suture. The cannula/electrode assembly was stereotaxically implanted in the right red nucleus (coordinates: 0.8 _+ 0.3 mm lateral to the midline, 9.0 + 0.5 mm posterior, and 14.0 + 1.0 mm ventral to the skull bone at bregma), using electrophysiological monitoring from the recording electrode to guide the cannula placement. The cannula/electrode assembly and a small headstage, designed to accommodate the stimulus delivery/micropotentiometer system used during behavioral conditioning procedures, were secured to the skull using skull screws and dental acrylic. The incision area was cleaned; treated with an antibacterial agent; and sutured closed, leaving only the headstage and the internal stylet of the cannula/ electrode assembly accessible. A small loop of silk thread was then placed in the left NM for subsequent monitoring of NM movements during conditioning procedures. Animals were allowed a minimum of 5 days to recover from these procedures before behavioral training was initiated.
Behavioral trainingprocedures Prior to behavioral training, rabbits were allowed to adapt to the experimental apparatus in the absence of stimuli presentation. During this adaptation period, the animal was positioned in a Plexiglas restrainer formed to restrict head and body movements and then situated within a ventilated, sound-attenuated test chamber. The headstage was connected to a headmount that held an airpuff delivery nozzle and minitorque potentiometer. The airpuff delivery nozzle was positioned 1 cm in front of the animal's
133 cornea, but was not activated during the adaptation period. The wire wiper arm of the potentiometer was attached to the silk suture loop in the left NM of the animal and was used to monitor NM movements. The adaptation session was carried out over a 1 h period. On the next day, conditioning procedures were initiated. Animals were placed in the Plexiglas restrainer and fitted with the headmount, airpuff delivery nozzle, and the wiper arm of the potentiometer as described for adaptation; however, animals now received paired presentations of an auditory CS (a 350 ms, i kHz tone at 85 dB SPL) and a co-terminant, unconditioned airpuff to the cornea on the left eye (US: 100 ms, 2.1 N/cm 2 airpuff). Each training session consisted of 117 trials arranged into 13 blocks. Each block included 1 CS-alone test trial and 8 CS-US paired trials. The trials were delivered every 20-40 s (mean = 30 s). Conditioned and unconditioned responses were determined by calculating the peakNM-response amplitude (expressed in ram) during these CS and US periods respectively. A C.R was defined as a NM extension of at least 0.5 mm, occurring within 250 ms of the CS onset and before the US onset. Training sessions were conducted on consecutive days until the animal exhibited at least a 95% CR proficiency in a given training session.
sion and all testing sessions were conducted 4 days apart.
Experimental design A series of 3 experiments were used to assess the effects of GABAergic and glycinergic antagonists, micro-infused into the red nucleus, during retention of the classically conditioned NM response. In Experimentl, three doses (0.19, 0.37, and 0.75 nmol) of the G A B A antagonist picrotoxin and the vehicle (0.75/A of artificial CSF) were administered to each of 6 rabbits. The micro-infusion of each dose or the vehicle occurred on separate behavioral testing days and the order of the 'dose administrations was randomized. In Experiment 2, each of 6 rabbits received 3 doses of a second G A B A antagonist, bicuculline methiodide (0.75, 1.5, and 3.0 nmol) or vehicle (0.75 ~1 of artificial CSF) on separate behavioral testing days and in randomized dose sequence. In Experiment 3, the 4 animals that were sensitive to bicuculline methiodide in the second study were administered the glycine antagonist strychnine (3.0 nmol), bicuculline methiodide (3.0 nmol), or vehicle (0.75/~1 of artificial CSF) on separate behavioral testing days and in randomized dose sequence.
Tissuepreparation Behavioral testingprocedures During behavioral testing, animals first received 4 blocks of paired CS-US presentations as described under behavioral training procedures. Immediately following this baseline conditioning, the internal stylet of the chronic, guide cannula was removed and an approximately matched internal cannula, which was connected to a microsyringe via polyethylene tubing, was inserted. Designated pharmacological compounds in appropriate amounts were dissolved in a 0.75/~1 volume of artificial cerebrospinal fluid (CSF, pH 7.4) and were then continuously infused over a 1 min period. To allow for adequate diffusion of an infused compound, a 5 min period intervened between the micro-infusion and resumption of behavioral testing. To determine the behavioral effects of an infused substance, animals were then monitored for an additional 15 blocks of paired conditioning procedures. To minimize possible tachyphylaxis, only one micro-infusion was administered in a given testing ses-
Following behavioral testing, each animal was deeply anesthetized with sodium pentobarbital and then perfused through the heart with a 10% formalin solution. The brain was then removed from the skull; embedded in albumin, which was allowed to gel; and then sectioned at 80/~m through areas containing the cannula/electrode track. Once mounted on prepared slides, all brain sections were stained with Cresyl violet. RESULTS
Preliminary electrophysiologicalanalysis A representative multiple unit peristimulus histogram (Fig. 1), obtained from the magnocellular red nucleus during one training session, demonstrated a pattern of unit discharge that modeled and preceded the NM response in time. Furthermore, electrical stimulation of this region elicited a selective eyeball retraction, eyelid closure, and NM extension of the
134
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Fig. 1. A representative histogram obtained from the magnocellular red nucleus during classical conditioning of the NM response over one training period (117 trials). The upper trace of this histogram indicates the average NM extension during the training period. The lower trace depicts the corresponding magnocellular red nucleus multiple unit peristimulus histogram. The histogram bar spans a 9 ms time bin. The first vertical line in the histogram indicates tone onset; the second vertical line indicates air puff onset. Note, the pattern of unit discharge from the red nucleus appears to model the NM response and to precede the response in time.
contralateral eye (Fig. 2). Collectively, these observations suggested that the magnocellular red nucleus was involved in expression of the NM response and thus, provided a rationale for pharmacological analysis.
Histological reconstruction of micro-infusion sites To determine the precise site of antagonist infusion for each animal, a histological reconstruction of each cannula tip placement was prepared (Fig. 3), using the atlas of Urban and Richard 4°. Such reconstructions demonstrated that those animals, which had shown either a significant diminution or a complete abolition of the CR following microinfusion of either picrotoxin or bicuculline methiodide, in fact, had cannulae placements within 0.5 mm of the right magnocellular red nucleus (Expt. 1 : 4 of 6 animals had such placements; Expt. 2:4 of 6 animals). In contrast, the remaining rabbits, which had shown no reduction of the CR peak amplitude following such infusions, had cannulae placements more distal to the magnocellular red nucleus. The behavioral testing data from those animals with cannulae tip placements within 0.5 mm of the magnocellular red nucleus were further analyzed to determine the specific dose-dependent function of the various pharmacological agents, which could either markedly reduce or completely abolish conditioned responding.
Experiment h effects of picrotoxin on the N M response Microinfusion of picrotoxin into the magnocellular red nucleus markedly reduced or abolished the CR peak amplitude in the well-trained animal (Fig. 4). Furthermore, this effect was dose-dependent. A twoway analysis of variance (drug treatment x 2 grouped blocks of trials) with repeated measures over both factors revealed a significant dose x block interaction (F24,72 = 2.61; P < 0.001) effect on CR amplitude. Posthoc analysis showed that the postinfusion CR amplitudes for animals receiving the two highest drug dose levels (0.37 and 0.75 nmol) were significantly different from the amplitudes of animals receiving vehicle. The CR amplitude for the 0.37 nmol dose of picrotoxin remained significantly different from vehicle during the first 2 blocks postinfusion (blocks 5-6), while the 0.75 nmol dose of picrotoxin remained significantly different for 6 blocks postinfusion (blocks 5-10). In contrast to this effect on the CR, picrotoxin produced no significant effect on the amplitude of the unconditioned response (UR). Experiment 2: effects of bicuculline methiodide on the N M response Microinfusion of bicuculline methiodide into the magnocellular red nucleus also produced a significant dose-dependent diminution or abolition of conditioned responding (Fig. 5). A two-way analysis of variance (drug treatment x 2 grouped blocks of trials) with repeated measures revealed a significant dose x block interaction (F24,72 = 7.99; P < 0.001) effect on the CR peak amplitude. Posthoc analysis showed that the postinfusion CR amplitudes for animals receiving each of the 3 drug dose levels (0.75, 1.5 and 3.0 nmol) were significantly different from the amplitudes of animals receiving vehicle. The CR amplitude for the 0.75 and 1.5 nmol doses of bicuculline methiodide remained significantly different from vehicle during the first 2 blocks postinfusion (blocks 5-6), while the 3.0 nmol dose remained significantly different for 4 blocks postinfusion (blocks 5-8). However, bicuculline methiodide produced no significant effect on the U R peak amplitude. Experiment 3: effects of strychnine on the N M response In contrast to the effects of the G A B A antagonists,
135 microinfusion of the glycine antagonist, strychnine, into the magnoceUular red nucleus had no apparent effect on the amplitude of either the C R or U R . These observations were confirmed statistically. DISCUSSION Evidence from previous lesion 11'29'3° studies suggested that the magnocellular red nucleus is a critical structure within the essential neuronal circuitry mediating both acquisition and retention of the classically conditioned N M response. The present investigation substantiates the later finding and suggests that the functional integrity of G A B A e r g i c processes
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Fig. 3. Histological reconstructions of infusion sites. Effective infusion sites for GABA antagonists, picrotoxin and bicuculline methiodide, are denoted by solid circles (n = 8). Collectively, these sites lie within 0.5 mm of the magnocellular red nucleus. Ineffective infusion sites for these antagonists are denoted by open circles (n = 4). BCI, nucleus brachii colliculi inferioris; CM, centrum medianum; CP, nuclei commissurae posterioris; CSU, nucleus centralis superior; D, nucleus Darkschewitsch; EW, nucleus Edinger-Westphal; FHI, fissura hippocampi; FR, formatio reticularis; GLD, corpus geniculatum laterale dorsale; GM, corpus geniculatum mediale; GYD, gyrus dentatus hippocampi; HID, hippocampus dorsalis; HIV, hippocampus ventralis; HL, nucleus habenulae lateralis; HM, nucleus habenulae medialis; IFLM, nucleus interstitialis fasciculi longitudinalis medialis; IMD, nucleus intermediodorsalis; IP, nucleus interpeduncularis; N III, nucleus nervi oculomotorii; PF, nucleus parafascicularis; PL, nucleus pontis lateralis; PM, nucleus pontis medialis; PO + PLV, posterior thalamic complex + pulvinar; PT, nucleus paratenialis; PTA, nucleus pretectalis anterior; PTP, nucleus pretectalis posterior; PVP, nucleus paraventricularis thalami posterior; R, nucleus tuber; RT, nucleus reticularis tegmenti Bechterew; SG, nucleus suprageniculatum; SGC, substantia grisea centralis; SGS, stratum griseum superficiale colliculi superioris; SN, substantia nigra; TO, nucleus tracti optici; VP, nucleus ventralis posterior thalami; bci, brachium colliculi inferioris; bp, brachium pontis; cc, corpus callosum; ccs, commissura colliculi superioris; chd, commissura hippocampi dorsalis; cin, cingulum; cp, commissura posterior; dpcs, decussatio pedunculorum cerebellaris superior; drs, decussatio tracti rubrospinalis; dts, decussatio tracti tectospinalis; tim, fasciculus longitudinalis medialis; fr, fasciculus retroflexus; fpd, fasciculus predorsalis; 11, lemniscus lateralis; lm, lemniscus medialis; pcm, pedunculus mammillaris; pcsd, pedunculus cerebellaris superior pars descendens; ped, pedunculus cerebri; py, pyramis; r III, radix nervi oculomotorii; tc, tractus tegmentalis centralis; to, tractus opticus; tp, tractus tectoponticus; ts, tractus tectospinalis. (Adapted from I. Urban and P. Rickard, A Stereotaxic Atlas of the New Zealand Rabbit's Brain, 197840.)
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peared anatomically specific. Microinfusion of G A B A antagonists into several areas distal to the red nucleus did not affect the CR peak amplitude. Furthermore, the effect appeared pharmacologically specific. The glycine antagonist strychnine, infused at dosage levels equimolar to those of the G A B A antagonists, did not alter either conditioned or unconditioned responding. The fact that glycine also shares seizure-inducing properties with G A B A antagonists supports the premise that non-specific effects, such as possible seizures, are an unlikely explanation for the selective effects of G A B A antagonists on retention of the CR. While previous lesion studies of the red nucleus 11'29'30 could not distinguish whether abolition of the conditioned NM response was due to the disruption of fibers of passage (cerebello-thalamic) or to the disruption of the intrinsic circuitry of the red nucleus; the current pharmacological manipulations suggest that GABAergic processes, localized within the magnocellular red nucleus, are critically involved in expression of the CR. Although the present analysis does not precisely distinguish the relevant neurons, current evidence suggests that the most likely sources of G A B A within the red nucleus are inhibito-
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138 gests that learned movements, which are relayed
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from the deep cerebellar nuclei to the red nucleus, could also activate the rubro-olivary tract, which
ACKNOWLEDGEMENTS
would influence the inferior olive. As a result of such feedback, the inferior olive could modify its neural transmission to the cerebellar cortex. Specifically, the inferior olive could eliminate r e d u n d a n t instructions to the cortex once the motor task had been learned, and in this m a n n e r promote the efficiency of this learning circuit. In contrast, several investigators 4-6'30 suggest that the red nucleus is an essential relay for the expression of the CR, but that this structure is not the site of learning-induced modifications per se. Each of these hypotheses suggests quite a different role for the red nucleus in m o t o r learning and emphasizes the need for extended examination of
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