Role Of Cerebellar D-I Nuclei During Acquisition Of The Classically Conditioned Eyeblink Responses In Guinea Pigs

Aims and methods:Numerous lines of evidence ranging in diversity 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 many 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 eye-blink response.The classically eyelid conditioning involves paired presentations of a neutral conditioned stimulus (CS) such as a tone, and a reinforcing unconditioned stimulus (US) such as a puff of air in the eye. Whereas initially the tone does not elicit an eyelid response, after a few hundred tone+puff training trials the eyelid will close in response to the tone. A variety of techniques that include lesion, recording, stimulation, reversible inactivation, brain-imaging and computer simulations indicate that the cerebellum is essential for the acquisition and expression of these conditioned eyeblink responses.It is well known that, The cerebellum is comprised of two anatomical components , the cerebellar cortex and nuclei. Despite much progress, their relative contributions to motor learning remain a fundamentally important issue that is largely unresolved and hotly debated. Some experts said cerebella cortex play the key role in the classically eyelid conditoning, whereas others have suggested that the cerebellar cortex is involved mainly in the proper performance of learned eyelid responses or in their adequate timing but not in their acquisition and storage, in recent years, cerebellar nucleus is suggested as the putative sites where this type of associative learning takes place.It is meaningful to explore the role of the cerebellar nucleus during the acquisition of classically eyeblink conditioning, which will be helpful to reveal the neural mechanisms of learning and memory. The present study were divided into three parts: Firstly, we establish the classically eyeblink conditioning, then we observe the activity of single D-I neuron during the classically eyeblink conditioning, which allowed us to determine whether the D-I involved in the neural circuit important for the acquisition of classically eyeblink conditioning; Secondly, we use low dose muscimol to inactivate the Dentate-Interpositus nuclues of cerebellum and test its involvement in acquisition of classically eyelid conditioning in some subjects;Thirdly, we explore the relationship of the changes of classically eyeblink conditioning behavior and the changes of the parameter of synapse structure in the Dentate-Interpositus nuclei of cerebellum.Results:1) Activities of N-I nuclei in the cerebellum of guinea pigs during the acquisition of classically eyeblink conditioning.In present study, the paired group animals acquired the classically eyeblink conditioned responses across the 3 daily training sessions, and the N-I nuclei neurons exhibited tow different activity patterns during the acquisition of classically eyeblink conditioning. 40% (12/30) N-I nuclei neurons were observed with increases in activity during the appearance of CR and increased their firing in parallel with the CR rate and amplitude in acquisition of classically eyeblink conditioning; 60% (18/30) N-I nuclei did not modified their firing several trails in advance of the appearance of eyelid conditioned responses.2) The effect of microinjections of muscimol into cerebellar D-I nuclei during the acquisition of classically eyeblink conditioning.In the present study, very low doses of muscimol were used to selectively inactivate the N-I nuclei of guinea pigs during three conditioning sessions. Animals performed no significant levels of CRs during those sessions. Training was continued six more sessions without any inactivations to test whether any learning had occurred during the previous three sessions. Detailed analysis of responses during session five revealed that learning was completely blocked by the low doses of muscimol infused into the interpositus during the previous three sessions. Animals subsequently acquired the CR normally.3)The ultrastructure changes of N-I nuclei neurons synapse after the acquisition of classically eyeblink conditioning. The paired group animals acquired the classically eyeblink conditioned responses across the 6 daily training sessions,the behavioral learning and memory following training was not accompanied by alteration in the number density of synapses in cerebellar D-I nuclei,but the length of synaptic active zone and post-synaptic density thickness were significantly increased in the guinea pigs of paired conditioned group in comparing with those of the unpaired group(P<0.05).Conclusions:1)The N-I nuclei contain neurons significantly related to classically conditioned eyeblink responses,suggest that N-I nuclei take part in the acquisition of classically eyeblink conditioning;2) Reversible inactivation of the cerebellar N-I nuclei with muscimol prevent the acquisition and performance of conditioned eyeblink responses,demonstrate that cerebellar N-I nuclei is important for both acquisition and performance of conditioned eyeblink responses.3) There are some changes of the parameter of synapse ultrastructure in the Dentate-Interpositus nuclei of cerebellum after acquisition of conditioned eyeblink responses.The cerebellar N-I nuclei participate in the neural circuit important for the aquisition of classically eyeblink conditioning.