Cerebellar circuits and synaptic mechanisms involved in classical eyeblink conditioning

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1 S.A. Fisher et al. Short-term synaptic enhancement 106 Tang, Y-G. and Zucker, R.S. (1995) Soc. Neurosci. Abstr. 21, Werth, J.L., Usachev, Y.M. and Thayer, S.A. (1996) J. Neurosci. 16, Lee, W-L., Anwyl, R. and Rowan, M. (1987) Brain Res. 426, Benham, C.D., Evans, M.L. and McBain, C.J. (1992) J. Physiol. 455, Gunter, T.E. and Pfeiffer, D.R. (1990) Am. J. Physiol. 258, C755 C Fischer, T.M. and Carew, T.J. (1993) J. Neurosci. 13, Walters, E.T. and Byrne, J.H. (1984) Brain Res. 293, Landgren, S., Phillips, C.G. and Porter, R. (1962) J. Physiol. 161, Racine, R.J. and Milgram, N.W. (1983) Brain Res. 260, Fischer, T.M., Zucker, R.S. and Carew, T.J. (1996) Soc. Neurosci. Abstr. 22, Blazis, D.E.J. et al. (1994) Soc. Neurosci. Abstr. 20, Fisher, S.A., Fischer, T.M. and Carew, T.J. (1995) Soc. Neurosci. Abstr. 21, Buonomano, D., Hickmott, P. and Merzenich, M. (1995) Soc. Neurosci. Abstr. 21, Buonomano, D.V. and Merzenich, M.M. (1995) Science 267, Nelson, S.B. et al. in Computational and Neural Systems (in press) 121 Abbott, L.F. in Computational and Neural Systems (in press) 122 Atwood, H.I. and Bittner, G.D. (1971) J. Physiol. 34, Bittner, G.D. (1968) J. Gen. Physiol. 51, Wojtowicz, J.M., Marin, L. and Atwood, H.L. (1994) J. Neurosci. 14, Acknowledgements We thank T.J. Sejnowski and R.S. Zucker for very helpful comments on an earlier draft of this manuscript. This work was supported by NIMH grant MH48672 to TJC and NIMH grant MH10334 to TMF. Cerebellar circuits and synaptic mechanisms involved in classical eyeblink conditioning Jeansok J. Kim and Richard F. Thompson There is increasing evidence that, in addition to its major functional role in the regulation of fine motor control, the cerebellum is involved in other important functions, such as sensory motor learning and memory. Classical conditioning of the eyeblink or nictitating membrane response (and other discrete behavioral responses) is a form of sensory motor learning that depends crucially upon the cerebellum. Within the cerebellum, however, the relative importance of the cerebellar cortex and the deep cerebellar nuclei in eyeblink conditioning is unclear and disputed. Recent studies employing various mutant mice provide an effective approach to resolving this controversy. Eyeblink conditioning in spontaneous mutant mice deficient in Purkinje cells, the exclusive output neurons of the cerebellar cortex, indicate that both the cerebellar cortex and the interpositus nucleus are important. Furthermore, studies involving gene knockout mice suggest that long-term depression, a process of synaptic plasticity occurring in Purkinje cells, might be involved in eyeblink conditioning. Trends Neurosci. (1997) 20, APREREQUISITE to understanding the neural mechanisms by which an organism acquires and retains information is the identification of the site(s) of learning and memory storage. While substantial progress has been made in elucidating the memory mechanisms in simpler invertebrate systems 1 3, for many years, the task of localizing learning and memory has been the main obstacle in understanding memory mechanisms in mammalian systems. It now appears that in mammals there are different forms or aspects of memory that are subserved by different brain structures 4. For example, the hippocampus seems to be important for spatial, contextual and relational memories 4 6, whereas the amygdala appears to be crucial for emotional (fear) memories 7,8. The cerebellum, by contrast, is necessary for classical conditioning of the eyeblink, or nictitating membrane response, and other discrete behavioral responses 9,10. Classical or Pavlovian conditioning is the simplest form of associative learning by which animals, including humans, learn relations among events in the world so that their future behaviors are better adapted to their environments 11. For biological analysis, eyeblink conditioning (and other types of classical conditioning) provides an important advantage over complex forms of learning in that the stimuli involved are well defined and can be precisely controlled. Typically, a discrete conditioned stimulus (CS; usually a tone) is paired with a discrete unconditioned stimulus (US; usually an airpuff to the eye) with particular temporal relationships between the CS and US. The naive animal exhibits eyeblinks only to the airpuff US; this unlearned eyeblink behavior to the US is referred to as the unconditioned response (UR). Over the course of training, the animal gradually develops a conditioned response (CR) to the CS that mimics the UR, precedes the US in onset time, and peaks at about the time of US onset. As only two stimuli are involved, the learning or association of CS and US has to occur at the brain sites where the two forms of information converge. Jeansok J. Kim is at the Dept of Psychology, Yale University, New Haven, CT , USA. Richard F. Thompson is at the Neuroscience Program, University of Southern California, Los Angeles, CA , USA. Copyright 1997, Elsevier Science Ltd. All rights reserved /97/$17.00 PII: S (96) TINS Vol. 20, No. 4,

2 J.J. Kim and R.F. Thompson Mutant mice and eyeblink conditioning Cerebellum and eyeblink conditioning Converging lines of evidence from lesioning, recording, stimulation, reversible inactivation and brain-imaging studies indicate that the cerebellum is essential for eyeblink conditioning, a type of discrete sensory motor learning 9,10. In brief, selective lesions (electrolytic or chemical) of the cerebellum prevent the acquisition and retention of conditioned eyeblink responses. Correspondingly, electrophysiological studies indicate that cells in specific regions of the cerebellum undergo learning-induced changes during eyeblink conditioning. For example, many Purkinje cells in the cortex (particularly in lobule HVI) decrease their activity 9 while cells in the interpositus nucleus increase their activity 12, which is consistent with inhibitory projections from the cortex to the interpositus nucleus. The involvement of the cerebellum in eyeblink conditioning is also supported by studies that show that electrical stimulation of the two major afferents to the cerebellum the mossy fibers from the pontine nuclei and the climbing fibers from the inferior olive can substitute for the peripheral CS and US, respectively 9. Because limited lesions of the pontine nuclei (specifically the lateral region) abolish CRs to a tone CS but not to a light CS (Ref. 13), and localized lesions of the inferior olive in well-trained animals result in behavioral extinction (of conditioned eyeblink responses 14 and conditioned limbflexion responses 15 ; but see Yeo and associates 16 who found abolition of CRs following dorsal accessory olive lesions) with continued CS US paired training, these afferent structures are not likely to be the major sites of memory storage. (Sears and Steinmetz 17 reported that neuronal unit activity recorded in the dorsal accessory olive exhibited no responses to the tone CS and a clear elicited increase in unit activity to the onset of the corneal airpuff US before training, suggesting that the memory trace is not in the dorsal accessory olive. Moreover, this US-elicited neuronal activity decreases as animals learn and perform the CR but is still fully present on trials to the US alone, a finding consistent with the hypothesis that the inferior olive, via its climbing fiber projections, provides the US or reinforcing input to the cerebellum.) Most important, perhaps, is that reversible inactivation of the anterior interpositus nucleus and overlying cerebellar cortex by cooling, or application of muscimol (a GABAreceptor agonist) or lidocaine (an open Na + -channel blocker) during training completely prevents learning of the CR, whereas inactivation of the efferents (superior cerebellar peduncle, red nucleus) does not prevent learning at all Interestingly, Ramnani and Yeo 24 recently reported that reversible cerebellar inactivation also prevents extinction of the eyeblink CR. Finally, recent human brain-imaging studies reveal changes in activities related to eyeblink learning in specific regions of the brain, including the cerebellum 25,26. The essential role of the cerebellum in eyeblink conditioning has been challenged on the ground that cerebellar lesions (or reversible inactivations) simply produced a performance deficit rather than a learning deficit For example, Welsh and Harvey 29 reported that with extensive pre- and postoperative training, cerebellar-lesioned (specifically the interpositus nucleus) animals exhibited a small residual CR with low amplitude, low frequency of occurrence, and long-onset latency on CS-alone trials. Furthermore, they postulate that effective cerebellar lesions (that is, lesions that abolished CRs) produce a significant decrement on UR performance at low US intensities. However, Welsh and Harvey did not actually show this because they did not report the amplitude of the URs before making the lesions. Moreover, Steinmetz et al. 31 failed to find systematic or persisting decrements in the UR amplitude following fully effective lesions of the interpositus nucleus (that is, no residual CRs). Furthermore, when a low-intensity US was used for training that was at the threshold level to yield learning (the URs and CRs were of low and equal amplitude before the lesion), interpositus nucleus lesion completely abolished the CR and had no effect at all on the threshold level UR (Ref. 32). But perhaps more important than the performance issue is the fact, noted above, that several laboratories have now shown that reversible inactivation of the cerebellum during training prevents learning of the conditioned eyeblink response. Recently, Bracha, Bloedel and associates 33 (see also Ref. 34) infused a protein-synthesis inhibitor anisomycin into the rabbit cerebellum and examined eyeblink conditioning. During the CS US paired training, in the presence of anisomycin, the animals exhibited some CRs (perhaps analogous to short-term CR memory?). However, when retention was assessed in the absence of anisomycin, almost no CRs were observed, that is, anisomycin-injected animals showed less than 10% CRs, whereas vehicle-injected animals showed greater than 80% CRs, during the CS-alone test trials 34. According to Bracha et al. 33, animals showing anisomycin-affected retention of CRs acquired the CRs at a normal rate after the drug injections were discontinued (p. 1222). As intracerebellar infusions of anisomycin did not affect CR expression in welltrained animals, this suggests that anisomycin selectively blocked learning, but not performance, of CRs. Based on their anisomycin work, Bracha and Bloedel 34 stated that the formation of plastic changes underlying the classical conditioning of the nictitating membrane response in the rabbit is dependent on protein synthesis at the cerebellar nuclear region (p. 191). In our view, the anisomycin data provide strong support for the cerebellar hypothesis of eyeblink conditioning. A hypothetical eyeblink-conditioning model is presented in Fig. 1 (similar models have been presented before 9,35 ). According to the model, both the cerebellar cortex and the interpositus nucleus receive information about the CS, conveyed by the mossy fiber emanating from the pontine nucleus, and information about the US, relayed by the climbing fiber (CF) originating from the inferior olive. In the cortex, the mossy fibers form synapses with the granule cells, which in turn send parallel fibers (PF) that make synapses on the Purkinje cells. As both the interpositus nucleus and the cerebellar cortex receive information about the CS and the US, both structures are potentially capable of supporting CS US associations. Consistent with this view, long-term depression (LTD) and long-term potentiation (LTP), two forms of synaptic plasticity implicated in associative learning, have been demonstrated in the cerebellar cortex 36 and the interpositus nucleus 37, respectively. De Schutter 38 recently pointed out that the required relative timing of the CF and PF inputs necessary to 178 TINS Vol. 20, No. 4, 1997

3 J.J. Kim and R.F. Thompson Mutant mice and eyeblink conditioning induce LTD is a serious problem for the cerebellar hypothesis of motor learning. Typically, LTD is optimally induced when the CF stimulation is concurrent with (or just precedes) the PF stimulation 39. In eyeblink conditioning, CRs develop when the CS is presented slightly before (for example, 250 ms) the US, but not when the US is presented slightly before or simultaneously with the CS. Several caveats must be considered when interpreting the timing property of LTD (Ref. 40). For example, GABA-receptor antagonists are often used in LTD experiments. Because inhibitory interneurons (Golgi cells, basket cells and stellate cells) in the cerebellar cortex outnumber Purkinje cells by far (for example, there are about 16 stellate cells for each Purkinje cell) 41, GABA-receptor antagonists might significantly alter the timing property of LTD induction (see Fig. 1 for inhibitory connections in the cerebellar cortex). In the absence of GABA-receptor antagonists, the temporal properties of LTD are consistent with those of eyeblink conditioning; LTD was induced (recording field-potentials) with PF activation preceding CF activation, with an interstimulus interval of 250 ms (Ref. 40). Regardless of the precise temporal condition for inducing LTD, the most important feature about LTD is the associative property, that is, the PF and CF activity must be paired to produce LTD (Ref. 42). [Interestingly, relatively intense stimulation of the PF, resulting in accumulations of Ca 2+ similar in peak levels to those following CF stimulation, appears to be capable of inducing LTD (Ref. 43).] Purkinje cell degeneration (pcd) mutant mice The relative importance of the cerebellar cortex and the deep cerebellar nuclei (specifically the interpositus nucleus) in terms of supporting eyeblink conditioning is not clear. Cerebellar lesions that include the interpositus nucleus abolish CRs completely and permanently; however, the effects of lesions of the cerebellar cortex are less clear. Some have consistently found CRs after large cerebellar cortical lesions 44, but others found substantial and persisting impairments of CRs (Ref. 45). Given the fractured somatotopy of cerebellar cortex 46, the conflicting reports of lesion effects might result from variability in the lesions. It is virtually impossible to lesion the entire cerebellar cortex without damaging the interpositus nucleus (and other cerebellar nuclei). In order to dissect the components of the cerebellar circuitry that are crucial for learning, we recently examined eyeblink conditioning in mutant mice deficient in Purkinje cells, the sole output neurons of the cerebellar cortex 47. These Purkinje cell degeneration (pcd) mutant mice are born initially with Purkinje cells, but during the course of development they are all lost by the third and fourth postnatal weeks. Other neural degenerations secondary to Purkinje cell degeneration occur in pcd mice, but they tend to occur with a much slower timecourse, allowing a window of opportunity to examine behavioral consequences associated primarily with the loss of Purkinje cells. Because pcd mice are completely devoid of Purkinje cells (there is no neural output from the cerebellar cortex), they are functionally equivalent to animals with complete cerebellar cortical lesions. The pcd mutants exhibited a significant reduction in the acquisition of conditioned eyeblink responses Cerebellum Eyeblink UR, CR US CS BA ST PC Int Ns VI and VII Reflex path V Coch N GR in comparison to the wild-type mice (Fig. 2A). [Eyeblink responses to the CS occurred only when the CS and US were paired, indicating that eyeblink conditioning is associative and feasible in mice (Fig. 2B).] However, following ten days of extensive training, the pcd mice developed a modest level of eyeblink CRs (and showed behavioral extinction); the CRs tended to have shorter peak latencies than the CRs displayed by the wild types. Alterations in CR timing have also been reported in rabbits with cerebellar cortical lesions 44,48. In accordance with pcd findings, the number of Purkinje cells correlates highly with the amount of eyeblink conditioning in rabbits and humans; that is, organisms with more Purkinje cells show stronger eyeblink conditioning than those with fewer Purkinje cells 49. As has been shown in other mammals, cerebellar lesions that include the interpositus nucleus completely blocked eyeblink conditioning in mice. These results from pcd mice suggest that the Purkinje cells and thus the cerebellar cortex is important for normal eyeblink conditioning. Because the pcd mice manifest classic cerebellar symptoms such as impaired motor co-ordination and mild ataxia, it is possible that the reduction in eyeblink conditioning is due to impairments in motor PF N V (sp) MF CF GO Midline Fig. 1. A putative eyeblink conditioning circuit based on experimental findings and gross anatomy of the cerebellum and the brain stem. The conditioned stimulus (CS) pathway consists of excitatory (+) mossy fiber (MF) projections from the pontine nuclei (PN) to the interpositus nucleus (Int) and to the cerebellar cortex. In the cortex, the mossy fibers form synapses with granule cells (GR) that in turn send excitatory parallel fibers to the Purkinje cells (PC). The Purkinje cells are the exclusive output neurons from the cortex and they send inhibitory ( ) fibers to deep nuclei such as the interpositus. The unconditioned stimulus (US) pathway consists of excitatory climbing fiber (CF) projections from the inferior olive (IO) to the interpositus nucleus and to the Purkinje cells in the cerebellar cortex. Within the cerebellar cortex, Golgi (GO), stellate (ST) and basket (BA) cells exert inhibitory actions on their respective target neurons. The efferent conditioned response (CR) pathway projects from the interpositus nucleus to the red nucleus (RN) and via the descending rubral pathway to act ultimately on the eyeblink reflex path. Additional abbreviations: V Coch N, ventral cochlear nucleus; N V (sp), spinal fifth cranial nucleus; N VI, sixth cranial nucleus; N VII, seventh cranial nucleus; UR, unconditioned response. Adapted from Ref. 9. IO RN PN TINS Vol. 20, No. 4,

4 J.J. Kim and R.F. Thompson Mutant mice and eyeblink conditioning Fig. 2. Acquisition of conditioned eyeblink responses in mice. (A) Conditioned response (CR) percentage measures from wild-type and Purkinje cell degeneration (pcd) mice. Animals underwent 10 d of CS US paired trials followed by 4 d of CS-alone extinction trials. The daily paired training consisted of 100 trials, and the CS and US were a 352 ms tone and a co-terminating 100 ms periorbital shock, respectively, with a 252 ms inter-stimulus interval and a randomized intertrial interval between 20 and 40 s. The daily extinction training consisted of 100 CS-alone trials. The pcd mice are significantly impaired in eyeblink conditioning compared to wild types even after ten days of extensive training. (B) CR percentage measures from paired (paired extinction; 5 d of CS US paired trials followed by 4 d of CSalone trials), pseudo-random (unpaired paired; 5 d of CS US pseudorandom trials followed by 5 d of CS US paired trials), and cerebellarlesioned (interpositus lesioned; 10 d of CS US paired trials) wild-type mice. Associative eyeblink learning occurred when the CS and US were presented in a paired manner, but not when the CS and US were randomly presented. Moreover, the acquired CRs extinguished with repeated CS-alone presentations. Lesions of the cerebellum (specifically the interpositus nucleus) completely abolished eyeblink conditioning in mice. From Ref. 47. control. However, we found that pcd mice did not differ from wild-type mice in terms of sensitivity to the shock US nor UR performance to the US. In addition, other studies with mutant mice indicate that impairments in motor co-ordination can be dissociated from reductions in eyeblink conditioning (Table 1). Interestingly, the pcd mice show deficits in other forms of learning, such as the Morris water-maze task 56. They are severely impaired in hidden platform training, a spatial memory task widely thought to be hippocampal-dependent, but are normal in visible platform training, a non-spatial memory task. The fact that pcd mice also show deficits in the spatial memory task is consistent with the current view that, in addition to motor control and sensory motor learning, the cerebellum might also be involved in higher cognitive functions Gene knockout mice Reductions in eyeblink conditioning have also been observed in recently generated gene knockout mice that lack mglu 1 (metabotropic glutamate receptor type 1) and GFAP (glial fibrillary acidic protein) (Table 1). Unlike random mutations generated by chemical or X-ray mutagenesis (as is the case with pcd mice), with homologous recombination technology, specific genes can be targeted ( knocked out ) to assess their roles in learning and memory 60. Both mglu 1 and GFAP mutants also show LTD deficits in parallel fiber Purkinje cell synapses after conjoint stimulation of the parallel fibers and the climbing fibers. In PKC mutants (deficient in the isoform of protein kinase C), where cerebellar LTD is normal and CF innervation of a Purkinje neuron is multiple, eyeblink conditioning is facilitated. It is significant to note that eyeblink conditioning is reduced in both pcd mutants, where there is no output from the cortex, and LTDdeficient knockout (for example, mglu 1 and GFAP) mutants, where there are Purkinje-cell outputs. This suggests that synaptic plasticity, such as the LTD that occurs on Purkinje cells, plays an important role in eyeblink conditioning, but other forms of synaptic plasticity in the cerebellum must be involved also as these mutants still showed significant, albeit reduced, conditioned eyeblink responses. According to Table 1, which summarizes eyeblink-conditioning, LTD and motor co-ordination results from various studies of mutant mice, it is clear that impairment in eyeblink conditioning correlates with deficits in LTD, while impairment in motor co-ordination correlates with multiple innervation by climbing fibers. (A model for the role of climbing fibers in motor learning and motor co-ordination has recently been proposed 53.) Interestingly, in GFAP mutants, where there is no multiple climbing fiber innervation of the Purkinje cell, motor co-ordination (as measured by the rotating rod test) is normal although LTD is impaired. This suggests that the LTD of parallel fiber Purkinje cell synapses might not be the only synaptic plasticity used for the refinement of motor co-ordination; moreover, the lack of LTD might have been developmentally compensated by some other plasticity. Concluding remarks Cerebellar mutants such as pcd mice provide further evidence that the cerebellum plays a crucial role in eyeblink conditioning. In the absence of Purkinje cells, eyeblink conditioning is reduced significantly. Lesions of the interpositus nucleus block eyeblink conditioning completely in mice, as previously documented in other animals. Thus, it appears that within the cerebellum there might be a parallel or distributed learning system that can support eyeblink conditioning 61, that is, memory traces for eyeblink conditioning might occur in both the cerebellar cortex and the interpositus nucleus. The cerebellar cortex is also important for other forms of motor learning, such as plasticity of the vestibulo ocular reflex (VOR) 46,61,62, and our results indicating that the Purkinje cells are crucial for eyeblink conditioning are consistent with 180 TINS Vol. 20, No. 4, 1997

5 J.J. Kim and R.F. Thompson Mutant mice and eyeblink conditioning TABLE 1. Motor learning, motor symptoms and cerebellar synaptic abnormalities in various mutants Mutant mice Eyeblink conditioning LTD Motor co-ordination CF innervation Refs pcd Impaired NA Impaired NA 47 GFAP Impaired Impaired Normal Normal 50 mglu 1 receptor Impaired Impaired Impaired Multiple 51,52 PKC Facilitated Normal Impaired Multiple 53,54 Glu 2 Not tested Impaired Impaired Multiple 55 Abbreviations: CF, climbing fiber; GFAP, glial fibrillary acidic protein; Glu 2, the glutamate receptor subunit; LTD, long-term depression; mglu 1, metabotropic glutamate receptor type 1; NA, not applicable; pcd, Purkinje cell degeneration; PKC, isoform of protein kinase C. Adapted from Ref. 53. VOR results. Although the genetic approach to studying learning and memory is powerful, appropriate applications of this approach require identification of the neural circuitry involved in the form of learning and memory being analysed. 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The work from our laboratory was supported by grants from National Institutes of Health 1F32MN BNR (to JJK), National Science Foundation IBN , National Institute on Aging AF05142, Office of Naval Research N and Sankyo Co., Ltd (to RFT). Coming next month! Sniffer-patch technique for detecting neurotransmitter release Layer-specific sprouting in the hippocampus Presynaptic inhibition of neurotransmitter release The functional neuroanatomy of episodic memory The DnaJ-like cysteine string protein and neurotransmitter release TINS Vol. 20, No. 4,