Localization of the cerebellar cortical zone mediating acquisition of eyeblink conditioning in rats
Introduction
The cerebellum is necessary for acquisition and retention of associative eyeblink conditioning (McCormick, Clark, Lavond, & Thompson, 1982). Delay eyeblink conditioning consists of a conditioned stimulus (CS; e.g., a tone) paired with an unconditioned stimulus (US) that elicits an eyelid closure unconditioned response (UR) before training. Repeated CS–US pairings result in the development of an eyelid closure conditioned response (CR) which precedes the onset of the US. Numerous studies demonstrated the essential role of the cerebellum in acquisition and retention of the CR using lesions, reversible inactivation, electrical stimulation, unit recording, genetic manipulations, neuropharmacology, and quantitative neuroanatomy (for review see Freeman & Steinmetz, 2011).
Acquisition and retention of the eyeblink CR are completely abolished by lesions or inactivation of the anterior interpositus nucleus (Clark et al., 1992, Freeman et al., 2005, Krupa et al., 1993, Lavond et al., 1985, Lincoln et al., 1982, McCormick et al., 1982, Steinmetz et al., 1992, Steinmetz et al., 1992, Yeo et al., 1985a). Lesions or inactivation of the cerebellar cortex also produce deficits in acquisition; however, the severity of the deficit has differed between studies (Attwell et al., 2001, Garcia et al., 1999, Hardiman et al., 1996, Lavond and Steinmetz, 1989). Mice with the Purkinje cell degeneration mutation (pcd) which lose most of their Purkinje cells during development are impaired during acquisition, but show an increase in CRs across training that is significantly greater than unpaired controls (Chen et al., 1996, Chen et al., 1999). A similar set of results was found in rats that had Purkinje cells destroyed by OX7-saporin, an immunotoxin, as adults (Nolan & Freeman, 2006). The findings from the pcd studies in mice and the OX7-saporin study in rats suggest that the cerebellar cortex plays a substantial role in acquisition of eyeblink conditioning but the cerebellar interpositus nucleus can support modest learning without the cortex (Chen et al., 1999, Nolan and Freeman, 2006). Indeed, lesions of the interpositus nucleus abolish CRs in pcd mice (Chen et al., 1999). The rodent studies were informative regarding the role of the cerebellar cortex in eyeblink conditioning but they did not identify the area within the cortex that plays a critical role in acquisition.
A recent study that used optogenetic inhibition of Purkinje cells in mice that were not given eyeblink conditioning identified an “eyeblink microzone” at the base of the primary fissure (Heiney et al., 2014). Electrophysiology, electrical stimulation, and optogenetics were used to demonstrate that inhibition of Purkinje cells in this area produced eyelid closure. This mouse eyeblink microzone is therefore a very good candidate for the cortical area that is critical for acquisition of eyeblink conditioning in rodents. The current study used lesions (Experiment 1) and reversible inactivation (Experiment 2) to localize the area of the cerebellar cortex that is critical for acquisition of eyeblink conditioning in rats. Electrolytic lesions were made in the base of the primary fissure (PFb) or in the lobules on either side of the primary fissure: hemispheric lobule VI (HVI) or lateral anterior lobules IV/V (AIV/V). Reversible inactivation was used to determine whether the base of the primary fissure plays a role in acquisition and retention of eyeblink conditioning in a within subjects design.
Section snippets
Subjects
Subjects were 78 male Long-Evans rats, 250–350 g at the beginning of the experiment. The rats were housed in Spence Laboratories of Psychology at the University of Iowa with a 12 h light–dark cycle and were given ad libitum access to food and water.
Surgery
One week prior to the onset of training, rats were removed from their home cage and anesthetized with isoflurane. For Experiment 1, unilateral lesions of the cerebellar cortex were produced by passing 1.0 mA of DC current for 10 s through an insect pin
Experiment 1: cerebellar cortical lesions before acquisition
Unilateral electrolytic lesions were made in the cerebellar cortex ipsilateral to the conditioned eye prior to acquisition of delay EBC. Following histological examination, rats were placed into one of five groups depending on the location of the lesion: control (n = 10), anterior lobe (AIV/V; n = 5), lobule HVI (HVI; n = 4), base of the primary fissure (PFb; n = 13), or cerebellar cortex and anterior interpositus nucleus (CCTX/AIN; n = 8). Two rats within the AIV/V group and 1 within the HVI group
Discussion
The goal of the current study was to identify the area of the cerebellar cortex that is critical for acquisition of eyeblink conditioning in rats. Lesions or inactivation of the cerebellar cortex ipsilateral to the trained eye resulted in impaired acquisition of eyeblink conditioning. Lesions that included the base of the primary fissure produced the most severe impairment. The small amount of conditioning observed in rats with lesions of the base of the primary fissure was abolished by
Acknowledgment
This work was supported by NIMH Grant MH080005 to J.H.F.
References (31)
- et al.
Imaging the spread of reversible brain inactivations using fluorescent muscimol
Journal of Neuroscience Methods
(2008) - et al.
Pharmacological analysis of cerebellar contributions to the timing and expression of conditioned eyelid responses
Neuropharmacology
(1998) - et al.
Effect of kainic acid lesions of the cerebellar interpositus nucleus on eyelid conditioning in the rabbit
Brain Research
(1985) - et al.
Acquisition of classical conditioning without cerebellar cortex
Behavioural Brain Research
(1989) - et al.
Ipsilateral cerebellar lesions prevent learning of the classically conditioned nictitating membrane/eyelid response
Brain Research
(1982) - et al.
Rabbit classically conditioned eyelid responses do not reappear after interpositus nucleus lesion and extensive post-lesion training
Behavioural Brain Research
(1992) - et al.
Cerebellar cortical lesions and reacquisition in classical conditioning of the nictitating membrane response in rabbits
Brain Research
(1993) - et al.
Cerebellar cortical AMPA-kainate receptor blockade prevents performance of classically conditioned nictitating membrane responses
The Journal of Neuroscience
(1999) - et al.
Acquisition of eyeblink conditioning is critically dependent on normal function in cerebellar cortical lobule HVI
The Journal of Neuroscience
(2001) - et al.
Cerebellar inactivation impairs cross modal savings of eyeblink conditioning
Behavioral Neuroscience
(2009)
Impaired classical eyeblink conditioning in cerebellar-lesioned and purkinje cell degeneration (pcd) mutant mice
The Journal of Neuroscience
Bilateral lesions of the interpositus nucleus completely prevent eyeblink conditioning in purkinje cell-degeneration mutant mice
Behavioral Neuroscience
Reversible lesions of the cerebellar interpositus nucleus during acquisition and retention of a classically conditioned behavior
Behavioral Neuroscience
Differential effects of cerebellar inactivation on eyeblink conditioned excitation and inhibition
The Journal of Neuroscience
Neural circuitry and plasticity mechanisms underlying delay eyeblink conditioning
Learning & Memory
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