Cerebellar circuits and synaptic mechanisms involved in classical eyeblink conditioning
Authors: Jeansok J. Kim, Richard E. Thompson
Journal: Trends in Neurosciences (1997)

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. 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. 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 systems1Alkon D.L. Sci. Am. 1989; 26: 42-50Crossref Scopus (98) Google Scholar, 2Carew T.J. Sahley C.L. Annu. Rev. Neurosci. 1986; 9: 435-487Crossref PubMed Scopus (321) Google Scholar, 3Kandel E.R. Schwartz J.H. Science. 1982; 218: 433-443Crossref PubMed Scopus (1039) Google Scholar, 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 structures4Squire L.R. et al.Annu. Rev. Psychol. 1993; 44: 453-495Crossref PubMed Scopus (818) Google Scholar. For example, the hippocampus seems to be important for spatial, contextual and relational memories4Squire L.R. et al.Annu. Rev. Psychol. 1993; 44: 453-495Crossref PubMed Scopus (818) Google Scholar, 5Kim J.J. Fanselow M.S. Science. 1992; 256: 675-677Crossref PubMed Scopus (1952) Google Scholar, 6O'Keefe J. Nadel L. The Hippocampus as a Cognitive Map. Oxford University Press, 1978Google Scholar, whereas the amygdala appears to be crucial for emotional (fear) memories7Davis M. Annu. Rev. Neurosci. 1992; 15: 353-375Crossref PubMed Scopus (1819) Google Scholar,8LeDoux J.E. Sci. Am. 1994; 270: 50-57Crossref PubMed Scopus (520) Google Scholar. The cerebellum, by contrast, is necessary for classical conditioning of the eyeblink, or nictitating membrane response, and other discrete behavioral responses9Thompson R.F. Philos. Trans. R. Soc. London Ser. B. 1990; 329: 161-170Crossref PubMed Scopus (175) Google Scholar,10Yeo C.H. Ann. New York Acad. Sci. 1991; 627: 292-304Crossref PubMed Scopus (69) Google Scholar. 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 environments11Rescorla R.A. Annu. Rev. Neurosci. 1988; 11: 329-352Crossref PubMed Scopus (481) Google Scholar. 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. 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' learning9Thompson R.F. Philos. Trans. R. Soc. London Ser. B. 1990; 329: 161-170Crossref PubMed Scopus (175) Google Scholar,10Yeo C.H. Ann. New York Acad. Sci. 1991; 627: 292-304Crossref PubMed Scopus (69) Google Scholar. 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 activity9Thompson R.F. Philos. Trans. R. Soc. London Ser. B. 1990; 329: 161-170Crossref PubMed Scopus (175) Google Scholar while cells in the interpositus nucleus increase their activity12Berthier N.E. Moore J.W. Exp. Brain Res. 1990; 83: 44-54Crossref PubMed Scopus (133) Google Scholar, 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, respectively9Thompson R.F. Philos. Trans. R. Soc. London Ser. B. 1990; 329: 161-170Crossref PubMed Scopus (175) Google Scholar. Because limited lesions of the pontine nuclei (specifically the lateral region) abolish CRs to a tone CS but not to a light CS (Ref. 13Steinmetz J.E. et al.Synapse. 1989; 3: 225-233Crossref PubMed Scopus (184) Google Scholar), and localized lesions of the inferior olive in well-trained animals result in behavioral extinction (of conditioned eyeblink responses14McCormick D.A. et al.Brain Res. 1985; 359: 120-130Crossref PubMed Scopus (208) Google Scholar and conditioned limb-flexion responses15Voneida T. et al.J. Neurosci. 1990; 10: 3583-3593PubMed Google Scholar; but see Yeo and associates16Yeo C.H. et al.Exp. Brain Res. 1986; 63: 81-92Crossref PubMed Scopus (127) Google Scholar 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 Steinmetz17Sears L.L. Steinmetz J.E. Brain Res. 1991; 545: 114-122Crossref PubMed Scopus (131) Google Scholar 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 GABA-receptor 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 all18Clark R.E. et al.Behav. Neurosci. 1992; 106: 879-888Crossref PubMed Scopus (126) Google Scholar, 19Clark R.E. Lavond D.G. Behav. Neurosci. 1993; 107: 264-270Crossref PubMed Scopus (60) Google Scholar, 20Hardiman M.J. et al.Exp. Brain Res. 1996; 110: 235-247Crossref PubMed Scopus (70) Google Scholar, 21Krupa D.J. et al.Science. 1993; 260: 989-991Crossref PubMed Scopus (342) Google Scholar, 22Krupa D.J. Thompson R.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5097-5101Crossref PubMed Scopus (63) Google Scholar, 23Nordholm A.F. et al.Behav. Neurosci. 1993; 107: 882-886Crossref PubMed Scopus (47) Google Scholar. Interestingly, Ramnani and Yeo24Ramnani N. Yeo C.H. J. Physiol. 1996; 495: 159-168PubMed Google Scholar 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 cerebellum25Logan C.G. Grafton S.T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7500-7504Crossref PubMed Scopus (177) Google Scholar,26Molchan S.E. et al.Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8122-8126Crossref PubMed Scopus (196) Google Scholar. 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 deficit27Bloedel J.R. Bracha V. Behav. Brain Res. 1995; 68: 1-44Crossref PubMed Scopus (125) Google Scholar, 28Welsh J.P. Neuroscience. 1992; 47: 1-19Crossref PubMed Scopus (47) Google Scholar, 29Welsh J.P. Harvey J.A. J. Neurosci. 1989; 9: 299-311PubMed Google Scholar, 30Welsh J.P. Harvey J.A. J. Physiol. 1991; 444: 459-480PubMed Google Scholar. For example, Welsh and Harvey29Welsh J.P. Harvey J.A. J. Neurosci. 1989; 9: 299-311PubMed Google Scholar 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.31Steinmetz J.E. et al.J. Neurosci. 1992; 12: 4403-4426PubMed Google Scholar 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. 32Ivkovich D. Lockard J.M. Thompson R.F. Behav. Neurosci. 1993; 107: 530-532Crossref PubMed Scopus (24) Google Scholar). 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 associates33Bracha V. et al.Soc. Neurosci. Abstr. 1995; 21: 1222Google Scholar (see also Ref. 34Bracha V. Bloedel J.R. Bloedel J.R. Ebner T.J. Wise S.P. The Acquisition of Motor Behavior in Vertebrates. MIT Press, 1996: 175-204Google Scholar) infused a protein-synthesis inhibitor aniso-mycin 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 trials34Bracha V. Bloedel J.R. Bloedel J.R. Ebner T.J. Wise S.P. The Acquisition of Motor Behavior in Vertebrates. MIT Press, 1996: 175-204Google Scholar. According to Bracha et al.33Bracha V. et al.Soc. Neurosci. Abstr. 1995; 21: 1222Google Scholar, '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 well-trained animals, this suggests that anisomycin selectively blocked learning, but not performance, of CRs. Based on their anisomycin work, Bracha and Bloedel34Bracha V. Bloedel J.R. Bloedel J.R. Ebner T.J. Wise S.P. The Acquisition of Motor Behavior in Vertebrates. MIT Press, 1996: 175-204Google Scholar 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 before9Thompson R.F. Philos. Trans. R. Soc. London Ser. B. 1990; 329: 161-170Crossref PubMed Scopus (175) Google Scholar,35Donegan N.H. Gluck M.A. Thompson R.F. Hawkins R.D. Bower G.H. Computational Models of Learning in Simple Neural Systems. Academic Press Inc, 1989: 108-156Google Scholar). 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 synap-tic plasticity implicated in associative learning, have been demonstrated in the cerebellar cortex36Ito M. et al.J. Physiol. 1982; 324: 113-134PubMed Google Scholar and the interpositus nucleus37Racine R.J. et al.Exp. Brain. Res. 1986; 63: 158-162Crossref PubMed Scopus (96) Google Scholar, respectively. De Schutter38De Schutter E. Trends Neurosci. 1995; 18: 291-295Abstract Full Text PDF PubMed Scopus (83) Google Scholar recently pointed out that the required relative timing of the CF and PF inputs necessary to 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 stimulation39Karachot L. Kado R.T. Ito M. Neurosci Res. 1994; 21: 161-168Crossref PubMed Scopus (70) Google Scholar. In eye-blink 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. 40Chen C. Thompson R.F. Learn. Mem. 1995; 2: 185-198Crossref PubMed Scopus (121) Google Scholar). 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)41Llinás R. Walton K. Shepherd G.M The Synaptic Organization of the Brain. 3rd edn. Oxford University Press, 1990: 214-245Google Scholar, 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 inter-stimulus interval of 250 ms (Ref. 40Chen C. Thompson R.F. Learn. Mem. 1995; 2: 185-198Crossref PubMed Scopus (121) Google Scholar). 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. 42Linden D.J. Neuron. 1994; 12: 457-472Abstract Full Text PDF PubMed Scopus (355) Google Scholar). [Interestingly, relatively intense stimulation of the PF, resulting in accumulations of Ca2+ similar in peak levels to those following CF stimulation, appears to be capable of inducing LTD (Ref. 43Hartell N.A. Neuron. 1996; 16: 601-610Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar).] 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 lesions44Lavond D.G. Steinmetz J.E. Behav. Brain Res. 1989; 33: 113-164Crossref PubMed Scopus (191) Google Scholar, but others found substantial and persisting impairments of CRs (Ref. 45Yeo C.H. et al.Exp. Brain Res. 1985; 60: 99-113Crossref PubMed Scopus (280) Google Scholar). Given the 'fractured somatotopy' of cerebellar cortex46Ito M. The Cerebellum and Neural Control. Raven Press, 1984Google Scholar, 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 cortex47Chen L. et al.J. Neurosci. 1996; 16: 2829-2838PubMed Google Scholar. 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 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 lesions44Lavond D.G. Steinmetz J.E. Behav. Brain Res. 1989; 33: 113-164Crossref PubMed Scopus (191) Google Scholar,48Gruart A. Yeo C.H. Exp. Brain Res. 1995; 104: 431-448Crossref PubMed Scopus (101) Google Scholar. 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 cells49Woodruff-Pak D.S. et al.NeuroReport. 1990; 1: 165-168Crossref PubMed Scopus (51) Google Scholar. 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 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 task56Goodlett C.R. et al.Behav. Brain Res. 1992; 47: 129-141Crossref PubMed Scopus (100) Google Scholar. 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 functions57Lalonde R. Botez M.I. Brain Res. Rev. 1990; 15: 325-332Crossref PubMed Scopus (137) Google Scholar, 58Leiner H.C. et al.Trends Neurosci. 1993; 16: 444-447Abstract Full Text PDF PubMed Scopus (514) Google Scholar, 59Schmahmann J.D. Arch. Neurol. 1991; 48: 1178-1187Crossref PubMed Scopus (768) Google Scholar.Table 1Motor learning, motor symptoms and cerebellar synaptic abnormalities in various mutantsMutant miceEyeblink conditioningLTDMotor co-ordinationCF innervationRefspcdImpairedNAImpairedNA47Chen L. et al.J. Neurosci. 1996; 16: 2829-2838PubMed Google ScholarGFAPImpairedImpairedNormalNormal50Shibuki K. et al.Neuron. 1996; 16: 587-599Abstract Full Text Full Text PDF PubMed Scopus (297) Google ScholarmGlu1 receptorImpairedImpairedImpairedMultiple51Aiba A. et al.Cell. 1994; 79: 377-388Abstract Full Text PDF PubMed Scopus (376) Google Scholar,52Conquet F. et al.Nature. 1994; 372: 237-243Crossref PubMed Scopus (638) Google ScholarPKCγFacilitatedNormalImpairedMultiple53Chen C. et al.Cell. 1995; 83: 1233-1242Abstract Full Text PDF PubMed Scopus (277) Google Scholar,54Kano M. et al.Cell. 1995; 83: 1223-1231Abstract Full Text PDF PubMed Scopus (291) Google ScholarGluδ2Not testedImpairedImpairedMultiple55Kashiwabuchi N. et al.Cell. 1995; 81: 245-252Abstract Full Text PDF PubMed Scopus (498) Google ScholarAbbreviations: CF, climbing fiber; GFAP, glial fibrillary acidic protein; Gluδ2, the glutamate receptor δ subunit; LTD, long-term depression; mGlu1, metabotropic glutamate receptor type 1; NA, not applicable; pcd, Purkinje cell degeneration; PKCγ, γ isoform of protein kinase C. Adapted from Ref. 53Chen C. et al.Cell. 1995; 83: 1233-1242Abstract Full Text PDF PubMed Scopus (277) Google Scholar. Open table in a new tab Abbreviations: CF, climbing fiber; GFAP, glial fibrillary acidic protein; Gluδ2, the glutamate receptor δ subunit; LTD, long-term depression; mGlu1, metabotropic glutamate receptor type 1; NA, not applicable; pcd, Purkinje cell degeneration; PKCγ, γ isoform of protein kinase C. Adapted from Ref. 53Chen C. et al.Cell. 1995; 83: 1233-1242Abstract Full Text PDF PubMed Scopus (277) Google Scholar. Reductions in eyeblink conditioning have also been observed in recently generated gene knockout mice that lack mGlu1 (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 memory60Grant S.G.N. Sil va A.J. Trends Neurosci. 1994; 17: 71-75Abstract Full Text PDF PubMed Scopus (90) Google Scholar. Both mGlu1 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 PKCγ mutants (deficient in the 7 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 LTD-deficient knockout (for example, mGlu1 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 proposed53Chen C. et al.Cell. 1995; 83: 1233-1242Abstract Full Text PDF PubMed Scopus (277) Google Scholar.) 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 developmen-tally compensated by some other plasticity. 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 conditioning61Raymond J.L. et al.Science. 1996; 272: 1126-1131Crossref PubMed Scopus (475) Google Scholar, 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)46Ito M. The Cerebellum and Neural Control. Raven Press, 1984Google Scholar,61Raymond J.L. et al.Science. 1996; 272: 1126-1131Crossref PubMed Scopus (475) Google Scholar,62du Lac S. et al.Annu. Rev. Neurosci. 1995; 18: 409-441Crossref PubMed Scopus (186) Google Scholar, and our results indicating that the Purkinje cells are crucial for eyeblink conditioning are consistent with 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. The availability of various other cerebellar mutants (such as weavers, staggerers, stargazers and nervous), as well as gene knockout and transgenic mice, offer exciting means to dissect the cellular and molecular substrates of the cerebellar circuitry involved in eyeblink conditioning. We thank Larry W. Swanson for encouragement, John K. Lee for preparation of the figures, and the referees for helpful comments and suggestions. The work from our laboratory was supported by grants from National Institutes of Health 1F32MN10521-01 BNR (to JJK), National Science Foundation IBN9215069, National Institute on Aging AF05142, Office of Naval Research N00014-95-1-1152 and Sankyo Co., Ltd (to RFT).