Pharmacological analysis of cerebellar contributions to the timing and expression of conditioned eyelid responses
Introduction
Evidence from lesion, recording, and stimulation studies indicates that Pavlovian eyelid conditioning is mediated by plasticity in the cerebellum (Thompson, 1986, Mauk and Donegan, 1997). Briefly, mossy fiber and climbing fiber afferents to the cerebellum are activated respectively by the conditioned stimuli (CS) and unconditioned stimuli (US) used for Pavlovian eyelid conditioning (Aitkin and Boyd, 1978, Sears and Steinmetz, 1991). Electrical stimulation of mossy fibers and climbing fibers will support learning when substituted for the CS and US respectively (Mauk et al., 1986, Steinmetz et al., 1986, Steinmetz et al., 1989, Steinmetz, 1990), and lesions of these afferent pathways affect conditioning in ways identical to omitting the CS or US (McCormick et al., 1985, Lewis et al., 1987, Steinmetz et al., 1987). Similar studies have demonstrated that output from the cerebellar (anterior) interpositus nucleus is necessary and sufficient for the expression of conditioned eyelid responses. Neurons in the interpositus nucleus show increased activity that precedes the conditioned response (McCormick and Thompson, 1984a, McCormick and Thompson, 1984b, Chapman et al., 1990), stimulation in the same region in untrained animals elicits eyelid responses (McCormick and Thompson, 1984a, McCormick and Thompson, 1984b), and lesions of this nucleus permanently abolish conditioned response expression (Clark et al., 1984, McCormick and Thompson, 1984a, McCormick and Thompson, 1984b, Lavond et al., 1985, Yeo et al., 1985b). Together these data suggest that converging mossy fiber and climbing fiber inputs induce plasticity in the cerebellum, and that this plasticity allows the CS to activate cerebellar output and drive the expression of conditioned responses (Mauk, 1997).
Although there is, based on this sort of evidence, general agreement that eyelid conditioning is mediated by the cerebellum (see Welsh and Harvey, 1991, Bloedel, 1992, Llinas et al., 1997for alternative views), there remain debates regarding the specific cerebellar processes that mediate response acquisition and expression. Two of the more contentious issues have been the relative contributions of the cerebellar cortex and cerebellar nuclei, and the exact regions of cerebellar cortex that are involved. Surgical ablation of the cerebellar cortex or cerebellar nuclei, and more recently reversible pharmacological inactivation of the cerebellar nucleus, have been employed to address these issues. There is general agreement that both surgical lesions and reversible inactivation of the cerebellar anterior interpositus nucleus abolish the expression of conditioned responses (Clark et al., 1984, McCormick and Thompson, 1984b, McCormick and Thompson, 1984a, Lavond et al., 1985, Yeo et al., 1985b, Welsh and Harvey, 1991, Krupa et al., 1993, Nordholm et al., 1993, Hardiman et al., 1996, Ivarsson et al., 1997, Welsh and Harvey, 1989).
Results from lesions of the cerebellar cortex have been far less clear. Following the original studies showing that lesions of the cerebellar cortex have no effects (McCormick and Thompson, 1984a), Yeo et al. reported that lesions of the HVI region of cerebellar cortex abolish conditioned responses (Yeo et al., 1984, Yeo et al., 1985a). Subsequent studies from this group and others report less robust and less persistent effects of these lesions (Lavond et al., 1987, Lavond and Steinmetz, 1989, Yeo and Hardiman, 1992, Clark and Lavond, 1994). Lesions or genetic defects of the cerebellar cortex have also been reported to produce only modest effects on the acquisition of conditioned responses, and no effect on the extinction of previously learned responses (Lavond et al., 1987, Lavond and Steinmetz, 1989, Chen et al., 1996).
In previous studies we have reported quite different effects of cerebellar cortex lesions. When made in previously trained animals, lesions that included the anterior lobe of the cerebellar cortex did not abolish conditioned responses, but diminished their amplitude and disrupted their learned timing. Whereas normally the learned responses are delayed to peak near the onset of the US, post-lesion responses displayed a fixed, short latency to onset (Perrett et al., 1993). We have subsequently shown that these same lesions prevent the extinction of previously learned responses (Perrett and Mauk, 1995), and prevent the acquisition of responses using a second CS (Garcia and Mauk, 1995). Lesions of HVI without damage to the anterior lobe had no effect on response timing or amplitude and had no measurable effects on acquisition or extinction. These studies raise important issues regarding both the regions of cerebellar cortex that are involved in eyelid conditioning, and regarding sites of cerebellar plasticity.
On the basis of these and other studies, we have proposed that plasticity in both the cerebellar cortex and cerebellar anterior interpositus nucleus are involved in the acquisition and expression of conditioned eyelid responses (Raymond et al., 1996, Mauk and Donegan, 1997, Mauk et al., 1997). Plasticity in the cerebellum and not extra-cerebellar sites is suggested by both the abolition of conditioned responses by lesions or inactivation of the interpositus nucleus as well as by the ability of stimulation of mossy fibers and climbing fibers to substitute for the CS and US respectively in producing normal response acquisition. Plasticity in the interpositus nucleus is suggested by the expression of (improperly timed) conditioned responses following lesions of the cerebellar cortex. Plasticity in the cerebellar cortex is suggested by the ability of cortex lesions to abolish learned response timing.
Although these hypotheses are consistent with existing lesion data and provide concrete ideas regarding conditioned response acquisition and expression, testing these ideas has been hampered by two key limitations. The first of these is the continued debate regarding the exact regions of cerebellar cortex that may be involved. This issue is exacerbated by the inherent difficulty in determining the exact functional, and not just anatomical, extent of surgical ablations or electrolytic lesions. For example, it is difficult on the basis of histology alone to exclude the possibility that cerebellar cortex lesions abolish conditioned response expression due to inadvertent damage to input or output pathways that are necessary for response expression. Secondly, and perhaps more seriously, studies of the cerebellar cortex are potentially confounded by the degeneration of the climbing fibers that such lesions produce (Yeo et al., 1984, Yeo et al., 1985a). Indeed, this is a specific example of a more general concern regarding the potential for permanent lesions to produce their effects through unintended compensatory mechanisms rather than the strict absence of the cerebellar cortex. Thus, it is always possible with permanent lesions that post-lesion responding is mediated by neuronal processes that differ from normal (see Mauk and Thompson, 1987).
Here, we attempt to address these issues by using microinfusion of GABA antagonists into the interpositus nucleus to produce a rapid and reversible block of the GABA-mediated inhibitory input. Since this inhibitory projection represents the sole output from the cerebellar cortex, infusion of picrotoxin produces a reversible disconnection of the cerebellar cortex from the output cells of the interpositus nucleus. Moreover, infusion of the GABA agonist muscimol through the same cannula can be used to produce reversible inactivation of the interpositus nucleus neurons. Since these pharmacological manipulations are rapid and reversible, this approach obviates previous confounds from damage to climbing fibers, or from lesion-induced compensatory mechanisms. Moreover, these infusions obviate debates regarding which sites of cerebellar cortex are crucial since the abolition of responses with muscimol indicates the critical nucleus follower cells are affected, regardless of the anatomical source of their inhibitory Purkinje cell inputs.
We report that infusion of picrotoxin into the anterior interpositus nucleus diminishes the amplitude and disrupts the learned timing of conditioned eyelid responses. Infusion of the GABA agonist muscimol through the same cannula completely abolishes the expression of responses, and infusion of saline has no effect.
Section snippets
Animals
Data were obtained from four male New Zealand albino rabbits (Oryctolagus cuniculus), weighing 2.5–3.0 kg each. The animals were individually housed and had free access to food and water. Treatment of the animals and surgical procedures were in accordance with an approved animal welfare protocol.
Surgical preparation
Animals were first prepared with a cannula implanted in the anterior interpositus nucleus and with a head bolt cemented to the skull. Animals were pre-anesthetized with 5 mg/kg acepromazine, and their
Results
As shown in Fig. 1, infusion of picrotoxin into the nucleus significantly decreased the amplitude and onset latency of conditioned responses without affecting the rate of responding. In contrast, muscimol infusion abolished conditioned responses altogether. A two-way, repeated measures ANOVA demonstrated significant effects on the percentage of conditioned responses for trial blocks [F(3,1)=178.51, P<0.001], drug treatment [F(2,6)=89.13, P<0.001], and a significant blocks by treatment
Discussion
We have demonstrated that pharmacological block of cerebellar cortex output reversibly disrupts the timing and decreases the amplitude of conditioned eyelid responses. In contrast, pharmacological block of cells in the cerebellar interpositus nucleus abolishes responses completely, whereas infusion of equal volumes of vehicle has no measurable effects. These results replicate previous studies that demonstrated reversible inactivation of the interpositus nucleus abolishes the expression of
Acknowledgements
We thank Dodie Baccigalopi for assistance with surgery and post-surgical care of the animals. This work was supported by NIMH 46904-06.
References (47)
- et al.
Acoustic input to the lateral pontine nuclei
Hearing Res.
(1978) - et al.
Effects of lidocaine injection in the interpositus nucleus and red nucleus on conditioned behavioral and neuronal responses
Brain Res.
(1990) - et al.
Effects of lesions of the cerebellar nuclei on conditioned behavioral and hippocampal neuronal responses
Brain Res.
(1984) - et al.
Reacquisition of eyeblink classical conditioning following large cerebellar cortical lesions in dutch belted rabbits
Behav. Brain Res.
(1994) - et al.
Effect of kainic acid lesions of the cerebellar interpositus nucleus on eyelid conditioning in the rabbit
Brain Res.
(1985) - et al.
Acquisition of classical conditioning without cerebellar cortex
Behav. Brain Res.
(1989) - et al.
Retention of classically conditioned eyelid responses following acute decerebration
Brain Res.
(1987) - et al.
Lesions of the inferior olivary complex cause extinction of the classically conditioned eyeblink response
Brain Res.
(1985) - et al.
Classical conditioning of the rabbit’s nictitating membrane response under fixed and mixed CS-US intervals
Learn. Motiv.
(1977) - et al.
Dorsal accessory inferior olive activity diminishes during acquisition of the rabbit classically conditioned eyelid response
Brain Res.
(1991)