Genetic Substrates of Memory: Cerebellum

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Cerebellum

The cerebellum has been traditionally regarded as a part of the motor system, mainly because of clinical and experimental observations that humans and animals with cerebellar damage are impaired in coordinated movement. Early theoretical consideration indicated that the unique and stereotyped cellular organization of the cerebellar cortex could also function as a learning machine (Albus, 1971; Marr, 1969). The cerebellar-learning hypotheses inspired broad experimental investigations. In 1982, Masao Ito and colleagues discovered that conjoint activation of the mossy fiber and climbing fiber led to long-lasting depression of the Purkinje-cell responses to mossy fiber activation, known as cerebellar long-term depression (LTD). Behavioral studies also indicate that the cerebellum is involved in several forms of motor learning, including classical eyeblink conditioning and adaptation of vestibulo-ocular reflex. In the 1990s, considerable progress was made in understanding the cellular and molecular mechanisms underlying cerebellum-dependent learning and memory. A number of spontaneous mouse mutants with relatively specific cerebellar deficits proved useful in dissecting the cerebellar circuitry involved in learning. Studies of gene knockout mice provided much insight into the molecular and cellular basis of learning and memory.

Classical Eyeblink Conditioning

Classical eyeblink conditioning is a form of motor learning, in which animals learn to associate an innocuous stimulus to a noxious one and to respond adaptively. For example, a naive animal will blink to a mild eyelid electrical shock but not to a brief tone. If the tone (serving as a conditioned stimulus, CS) is repeatedly paired with the eyelid shock (serving as an unconditioned stimulus, US), the animal will gradually learn to respond by eye blink to the tone. Richard F. Thompson and colleagues, among others, have characterized the brain circuitry involved in this learning. In essence, conditioned stimulus information is conveyed through mossy fibers, and unconditioned stimulus information is conveyed through climbing fibers. These converge in the cerebellum, where learning occurs and memory is stored (Thompson et al., 1996).

Cerebellar Cortex

Both the cerebellar cortex and the deep nuclei receive mossy fiber and climbing fiber projections and therefore are potentially capable of supporting learning and memory of the conditioned eyeblink response. The relative importance of the cortex and the deep nuclei in classical eyeblink conditioning is unclear, partly because it is virtually impossible to lesion the entire cerebellar cortex without damaging the deep nuclei. Lu Chen and colleagues (1996) examined classical eyeblink conditioning in Purkinje-cell degeneration (pcd) mutant mice to dissect the components of the cerebellar circuitry that are crucial for learning. These pcd mutant mice are born with Purkinje cells, but gradually lose all of them during the first four weeks of postnatal development. Other neural degenerations occur much later, allowing a time window to examine behavioral consequences associated with the loss of Purkinje cells. Because the Purkinje cells are the only output neurons in the cerebellar cortex, pcd mice are functionally equivalent to animals with complete cortical lesions. Classical delay (i.e., the US follows and overlays with the CS) eyeblink conditioning was impaired in the pcd mice in three aspects: the learning was slower; the maximal level of learning was lower; and the learned responses occurred earlier and were hence maladaptive. The low level of residual learning in pcd mice was completely abolished by lesions of the cerebellar deep nuclei, indicating the learned eyeblink response is stored in both the cerebellar cortex and the deep nuclei (Chen, Bao, and Thomson, 1999).

The Conditioned-Stimulus Pathway

The stargazer is another mutant with deficient cerebellar cortical circuit. The gene coding for stargazin, a protein that targets a-amino-3-hydroxy-5-methylisoxazoleproprionic acid (AMPA) receptors to their synaptic locations, is truncated in this mutant. The mutation results in a loss of AMPA receptors at the cerebellar mossy fiber-granule cell synapses, thereby disrupting the conditioned-stimulus pathway to the cerebellar cortex (Chen et al., 2000). This AMPA receptor defect is observed only in the cerebellar granule cells, possibly because in other neurons the loss of stargazing is compensated for by expression of functionally similar proteins. These stargazer mutant mice were impaired in classical eyeblink conditioning in similar ways to the pcd mutant mice, showing slower and reduced learning and disrupted response timing (Qiao et al., 1998). Further, the mutant mice performed normally in classical fear conditioning with a tone CS and a foot-shock US, a task that does not involve the cerebellum.

The Unconditioned-Stimulus Pathway

In early stages of rodent postnatal development, each Purkinje cell is innervated by several climbing fibers. By the end of third postnatal week, all but one of the climbing fibers are eliminated. In some mutants, a large portion of their Purkinje cells remain innervated by more than one climbing fiber even in adulthood. These mutants provide useful tools to study roles of climbing fibers as the US pathway in eyeblink conditioning.

For example, in the adult mutant mice deficient in the γ isoform of protein kinase C (PKC γ), 30 percent of the Purkinje cells are innervated by multiple climbing fibers. Other cerebellar physiological properties examined are all normal in this mutant. Multiple climbing fiber innervation presumably enhances US input (i.e., the teaching signal) to the cortex. In agreement with this view, PKC γ mutant mice showed facilitated eyeblink learning (Chen et al., 1995). Whereas wild-type mice attained maximal level of learning in five days, the mutant mice reached the same level in only two days. In addition, the learned eyeblink responses in the PKC γ mutant mice were more resistant to extinction by presentations of CS alone.

Cerebellar Long-Term Depression

Conjoint activation of the mossy parallel fiber and the climbing fiber induces long-term depression of the parallel fiber-Purkinje cell synaptic transmission, which is considered a cellular mechanism for some forms of motor learning such as classical eye-blink conditioning. Cerebellar LTD probably reduces Purkinje-cell responses, resulting in increased activity in the deep cerebellar nuclei, which may code for the learned responses. Testing the role of cerebellar LTD in motor learning had been hindered by the lack of pharmacological agents that specifically block cerebellar LTD and by the difficulty in applying the agents to the entire cerebellar cortex. The advent of modern molecular biology made it possible to completely remove specific proteins from a mouse by deleting the corresponding genes from the mouse genome. Using this powerful technique, a number of gene knockout mice with deficient cerebellar LTD were generated, including the mice lacking mGluR1 (type 1 metabotropic glutamate receptor), GFAP (glial fibrillary acidic protein), GluR δ 2 (glutamate receptor δ 2 subunit) or PLC β 4 (phospholipase C β 4 isoform). Behavioral examinations indicated that they were all impaired in classical eyeblink conditioning (Aiba et al., 1994; Kishimoto et al., 2001; Miyata et al., 2001; Shibuki et al., 1996).

Mutation of a gene may have widespread effects in the brain, making any results difficult to interpret. In that respect, results obtained in GluR δ 2 knockout mice are particularly informative, because GluR δ 2 is expressed only in the dendritic spines of the Purkinje cells. Impairments in both cerebellar LTD and eye-blink learning suggest that cerebellar LTD is involved in learning conditioned eyeblink responses.

Vestibulo-Ocular Reflex Adaptation

Vestibulo-ocular reflex (VOR) adaptation is another form of motor learning that depends on an intact cerebellum. An animal normally moves its eyes opposite in direction to its head turn to keep visual images steady on the retina. This reflex is guided by vestibular inputs (hence called vestibulo-ocular reflex) and is present even in complete darkness. If an erroneous motion of images on retina is artificially introduced, for example, by fitting spectacles on the eyes or by moving the visual scene, the animal learns to adjust the amplitude of VOR to compensate for the error. Cerebellar LTD has been hypothesized as a molecular mechanism for this VOR adaptation.

Chris I. De Zeeuw and colleagues (1998) examined cerebellar LTD and VOR adaptation in L7-PKCI transgenic mice in which the protein kinase C (PKC) inhibitor is artificially expressed in the cerebellar Purkinje cells under the control of L7 promoter. Protein kinase C, required for cerebellar LTD, has several iso-forms coded by separate genes. Knockout of the γ isoform did not impair cerebellar LTD (see discussion above on PKC γ knockout mice), possibly due to compensatory expression of other isoforms of the kinase. In L7-PKCI mice, expression of a selective inhibitor to a broad range of PKC isoforms virtually blocked LTD induction. These L7-PKCI mice showed normal VOR. When subjected to visuo-vestibular training, in which the visual scene was rotated in addition to the head turn, the wild-type mice learned to adapt their VOR. In contrast, L7-PKCI exhibited no VOR adaptation. These results suggest that cerebellar LTD is a mechanism for VOR adaptation.

Complex Motor-Skill Learning

In addition to classical eyeblink conditioning and VOR adaptation, the learning of more complex motor skills has also been examined in many mutant mice with what is know as the rotorod test. Typically, a mouse is placed on either a stationary rod or a rod that rotates at a certain speed, and the time it remains on the rod is measured to assess motor-skill learning. Impaired motor-skill learning has been observed in various mutant mice with cerebellar deficits including altered Purkinje-cell excitability, altered short-term synaptic plasticity, impaired cerebellar long-term potentiation, impaired cerebellar LTD, and multiple climbing fiber innervation.

Mutant mice with deficient cerebellar LTD are generally impaired in learning complex motor skills, with the exception of the GFAP knockout mice. As mentioned before, the GFAP knockout mice showed deficient cerebellar LTD and impaired eyeblink conditioning. However, they learned to stay on the rotating rod as well as normal mice, suggesting that there may be other forms of cerebellar plasticity supporting motor-skill learning.

Chong Chen and colleagues (1995) proposed that multiple climbing fiber innervation of the Purkinje cells disrupts learning of component movement and, hence, learning of complex motor skills. This notion is supported by numerous studies but has been challenged by De Zeeuw and colleagues (1998). These investigators reported normal rotorod performance and multiple climbing fiber innervations in the L7-PKCI transgenic mice.

Conclusion

Impaired motor learning in the cerebellum-specific mutants such as the GluR δ 2 knockout and the L7-PKCI transgenic mice provides convincing evi dence that the cerebellum is a learning machine. Deficient cerebellar LTD and motor learning have been observed in vastly different mutants, suggesting a causal link between cerebellar LTD and motor learning. In addition to cerebellar LTD, other forms of synaptic plasticity may also be involved in cerebellum-dependent learning. The cerebellum may also be involved in spatial learning (Goodlett, Hamre, and West, 1992). Further studies using cerebellum-specific mutant mice should help illuminate some other aspects of this question.

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Learning and Memory