Role Of Cerebellum During The Acquisition And Expression Of Classically Conditioned Eyeblink Responses In Guinea Pigs: Comparison Of Delay And Trace Paradigms

Aims and methods:Numerous lines of evidence ranging from purely theoretical considerations to human functional imaging studies have consistently implicated the cerebellum as being critically involved in a number of learning related tasks. Among the tasks in which the cerebellum is critically involved, a number of studies from several laboratories have demonstrated that the cerebellum is essentially involved in acquisition and expression of classically conditioned discrete skeletal movements, in particular, the classically conditioned eyeblink response.Classical conditioning of eyeblink reflex is a widely-used model system for elucidating the mechanisms of cerebellar involvement in motor learning, which involves paired presentation of a behaviorally neutral conditioned stimulus (CS, e.g., a tone) and an aversive unconditioned stimulus (US, e.g., a corneal airpuff in the eye). Initially, the organism produces only a reflexive unconditioned response (UR) to the US. However, after repeated pairings of the CS and US, the alone CS begins to elicit anticipatory conditioned eyeblink response (CR), suggesting that an association between the CS and US has been learned. There are two paradigms of eyeblink conditioning that differ in the temporal relationship between the CS and US. In the delay paradigm, the CS precedes, overlaps, and coterminates with the US. On the other hand, in the trace paradigm, the CS and the US are separated by a stimulus-free time gap (called trace interval, TI).Cumulative evidence from the lesion, recording, stimulation, reversible inactivation and neuroimaging studies has proved the cerebellum to be the basic locus of delay eyeblink conditioning. However, the relative contributions of the cerebellar cortex and interpositus nucleus to the acquisition of delay eyeblink conditioning are far to be determined. On the other hand, significant activations of the cerebellar cortex and interpositus nucleus during trace eyeblink conditioning have recently been observed. At this time, it is unclear what role does the cerebellar cortex and interpositus nucleus play during trace eyeblink conditioning.Consequently, the present study was undertaken to (1) investigate the role of cerebellar interpositus nucleus during the acquisition of delay and trace eyeblink conditioning in guinea pigs; (2) investigate changes of the synaptic ultrastrure in the guinea pig interpositus nucleus after delay and trace eyeblink conditioning; (3) investigate the cerebellar cortical function duting the expression of acquired trace-conditioned eyeblink responses in guinea pigs.Results:(1) Role of the unilateral cerebellar interpositus nucleus during the acquisition of delay eyeblink conditioning.1) The CR acquisition increased sharply during the sessions 2 to 4;2) A dose of 2.5 ug/kg muscimol had a sufficient inactivation effect;3) Microinjections of muscimol, a GABAA receptor agonist, into the left intermediate cerebellum before training completely prevented acquisition of the left CRs in guinea pigs;4) Microinjections of muscimol into the right intermediate cerebellum before training retarded acquisition of the left CRs at the early stage of training, whereas the CR acquisition was not affected at the later stage of traning relative to that of the Control animals;5) Microinjections of either aCSF or muscimol into the intermediate cerebellum did not affect the performance of tone-airpuff evoked UR;(2) Role of the unilateral cerebellar interpositus nucleus during the acquisition of trace eyeblink conditioning.1) Inactivations of the left cerebellar interpositus nuelceus with the GABAA receptor agonist muscimol during training prevent the acquisition of left TEBC using a relatively short (50 ms) TI, instead of the acquisition of left TEBC using a relatively long (250 ms) TI;2) Inactivations of the left cerebellar interpositus nucleus abolished the established left trace CRs regardless of the time length of TI;(3) Changes of synaptic ultrastructure in the cerebellar interpositus nucleus after delay and trace eyeblink conditioning.1) The PSD thickness of the excitatory and inhibitory synapses in the guinea pig interpositus nucleus increased after 10 daily sessions of delay eyeblink conditioning;2) However, the PSD thickness of the excitatory and inhibitory synapses in the guinea pig interpositus nucleus did not significantly change after 10 daily sessions of trace eyeblink conditioning;(4) Role of the cerebellar cortex during the expression of acquired trace-conditioned eyeblink responses in guinea pigs.1) Topography of the acquired trace-conditioned eyeblink responses was disrupted after blockade of cerebellar cortical input to the interpositus nucleus with bicuculline methiodide;2) Expression of the acquired trace-conditioned eyeblink response was totally abolished after microinjections of the GABAA agonist muscimol into the intermediate cerebellum;Conclusions:1) In contrast to the essential role of the ipsilateral cerebellar interpositus nucleus, the contralateral cerebellar interpositus nucleus is potentially involved in the acquisition of unilaterally classical eyeblink conditioning during the early stage of training.2) While the ipsilateral cerebellar interpositus nucleus is essential for the expression of trace CRs, its contribution to the acquisition of trace CRs appears to mainly depend on the length of trace interval.3) Delay, but not trace eyeblink conditioning is associated with significant changes of synaptic ultrastructure in the guinea pig interpositus nucleus.4) Blockade of cerebellar cortical input to the cerebellar interpositus nucleus disrupts the topography of acquired trace-conditioned eyeblink responses in guinea pigs.