Role Of Cerebellular Dentate-interpositus Nuclei During Establishment Of Trace Eyeblink Conditioning Inguinea Pigs

Background and goal:Learning and memory are two high-rank functions of the brain. The former is a process that the central nervous system adapts change of environment, acquires new knowledge, behaviors, skills, or understanding, and creates new behaviors and habits. The later refers to an ability to store retain and recall information after study. Cerebellum (CE), the classical subcortical center for motor control, is regarded as an organ which functions as controlling and adjusting movements, especially random and skillful movements, and as adjusting gesture and muscle tonus and maintaining body balance. Among all its functions, the most important one is to coordinate the random movements. But in recent years, more and more convincing evidence shows that the cerebellum has other important functions except for taking part in coordinating fine motors, for example, learning and memory of sensorimotor. In recent years, overseas and domestic researches showed that nictitating membrane/eyeblink classical conditioning (NM/EBCC) is an ideal model for the study of motor learning. A series of studies on the model improved the cerebellum has important function during the establishment of EBCC.The basic training process of EBCC reflex is: conditioned reflex (CS), such as tone or light signal, and unconditioned reflex (US), such as air puff to the cornea or electric stimulus to the orbicularis oculi which are paired and repeatedly give in order to train the animal to establish conditioned reflex (CR) according to the conditioned stimulus (CS). CR can be divided into trace eyeblink conditioning (TEBC) and delay eyeblink conditioning (DEBC), depending on whether there is the overlapping of time for CS and US. For the successful trained EBCC animal, its CR eyeblink behavior is observed prior to UR so that the animal can avoid nociceptive stimulus and get self protection. The establishment of eyeblink conditioning results from intensive coordinative movement of a lot of motor nucleus and muscle, and the whole behavior training is a process that the animal creates defense reaction against achromatic stimulus. Some evidence has verified that the cerebellum and hippocampus are involved in the process of DEBC and TEBC.Anatomically, the cerebellum consists of two parts, i.e, the cerebellar cortex and deep nuclei. The deep nuclei of the cerebellum act as the main centers of communication, and the four different nuclei of the cerebellum (dentate, interpositus, fastigial, and vestibular) receive and send information to specific parts of the brain. Among these deep nuclei, the dentate-interpositus nuclei (D-I nuclei) act as the most important output nuclei of the cerebellum. The D-I nuclei have special fiber connection with the hippocampus of limbic system, which is well known as a structure related to memory. Therefore, based on this connection, the cerebellum may be involved in cognition and motor.Previous studies found that the establishment of eyeblink conditioning is mainly related to the hippocampus, and some recent studies indicate that the hippocampus CA3 is of high necessity in the process of establishment of trace eyeblink conditioning, while the cerebellum is for the delay eyeblink conditioning. However, even when the cerebra cortex, hippocampus and thalamus were removed, damaged, or given microinjection of Muscimol, the animal still was able to learn TEBC partially, which suggests that the establishment of TEBC is far more complicated than the establishment of DEBC. Krupa et al adopted 3H-muscimol injection devitalization technology and found that TEBC mnemonic trace was formed and stored at deeper nucleus region of the cerebellum. Linden DJ used an in vivo electric activity recording system at cellular level to observe the eyeblink conditioning model, recorded animal's total behavioral response and simultaneously the electric movement of single cell, such as neuron. No matter the study on the animal as a whole or at cellular level, the results are similar. But whether the cerebellum and the deeper nuclei of the cerebellum are involved in TEBC, and what is the underlying mechanism on the synapse function? There is no exact conclusion until now.Synaptic plasticity is of significance in the change of central neuron activity, especially in high cognitive functions such as learning and memory. Syn is a specific protein on the membrane of synaptic vesicle which is closely related to the structure and function of synapse. In nervous tissue, Syn is located selectively on all the membrane of synaptic vesicle of axon terminal, so it can be regarded as one of the specific marker of synapse, reflecting the structural distribution of axon terminal. In recent years, many researches observed that the expression of Syn was changed in the learning and memory, especially in the spatial learning, suggesting that it plays a role in the process. As the important form of synaptic plasticity related to associated learning, long-term depression and long-term potentiation are always accompanied with the EBCC establishment. It is wildly accepted that the hippocampus LTD play an important role in the establishment of TEBC. In the other hand, some evidence shows that the cerebellum LTD may be mainly involved in the motor learning process of the cerebellum. Both cerebellum and cerebellar deep nucli LTD is very necessary in the establishment of DEBC. But researchers have different ideas on the function of the cerebellum during the establishment and maintenance of TEBC, especially the function of deep nuclei. Some other researches found that the D-I nuclei and cerebellar cortex in the cerebellar circuit loop, receive CS and US information simultaneously, and both of them may be involved in the integral process between CS and US in view of the structural connection. Therefore, the change of synaptic plasticity of the D-I nuclei in the establishment of TEBC is worthy for our close attention.To investigate the role of the D-I nuclei in the establishment of TEBC, is helpful not only to confirm the function of cerebellum in motor and learning and the possible mechanism, but also to fully understand the nerve mechanism of learning and memory.Methods:Our experiments were mainly as follows.1) Through eyeblink electromyography recording system and"follow-up"air puff stimulus device, we implemented the experimental requirement of fully"follow-up"eyeblink recording and penalty stimulus device. We established TEBC on the conscious and unfixed guinea pigs and found a technical basis for further research on the mechanism of cerebellum in movement and learning.2) An in vivo extracellular recording system was used to record and observe the discharge activity of the neurons in the D-I nuclei of guinea pigs. Peri-stimulus time histogramme (PSTH) before and after stimulus reflected the neuron activity. The activity changes of the neurons in the cerebellum D-I nuclei in the establishment of TEBC was explored.3) Immunohistochemical staining was used to detect the expression of Syn in the D-I nuclei of cerebellum after establishment of TEBC for the morphological mechanism of the cerebellum in the process.Results:1. With the aid of metal electrode embedding technique, eyeblink electromyography motor recording system and simple"follow-up"air puff stimulus device, we took tone as CS, and air puff as US, and used eyeblink electromyography to record the eyeblind behavior. With the elapse of training time, the CR acquisition rate was increasing. The rate was at a relatively stable state at 10 days, and still high in 15 and 20 days after training. The CR acquisition rate was gradually rising with the training time went by, and was maintained for a long time. The model of TEBC was successfully established on conscious and unfixed guinea pigs.2. An in vivo extracellular recording system was used to record and observe the discharge of the D-I nucleus neuron of cerebellum in TEBC guinea pigs. There were three types of neurons in the cerebellar D-I nuclei, that is, the stimulus-related neurons, the CR acquisition-related neurons and the discharge-irrelevant neurons. 1) Stimulus-related neurons consisted of CS stimulus-related neurons and US stimulus-related neurons, whose discharging activity was related to CS and US, but had no obviously relevance to the training of behavior or the CS-US paired or not. 2) The CR acquisition-related neurons, also as the trace-related neurons, had a close relationship of its discharging activity with CR, and were only recorded in CS-US paired group, This type of neuron may be involved in the establishment of TEBC. 3) The discharge activity of discharge-irrelevant neurons had no relevance with the CS, US and the establishment of TEBC.3. Immunohistochemical staining displayed that the expression of synaptophysin in the D-I nucleus of cerebellum had changed before and after the establishment of TEBC. In CS-US paired group, the positive cells and intensity of Syn staining were increased with the elapse of TEBC behavior training. The fiber bundles positive to Syn were formed in 6 day, and well arranged and increased more in 10 and 15 days. Our results showed that the expression and changes of the Syn were related with the training of behavior at the same time period.Conclusion:To sum up, the D-I nucleus of cerebellum is a critical part in the nerve circuit loops for the establishment of TEBC. It functions as the important encephalic region of acquisition and memory storage in the process of TEBC establishment. It actively participates in the establishment through changing the activity mode of neuron. The change of synaptic quantity and distribution may be the morphological base for the cerebellum functions in motor learning and memory.