An electrophysiological study of synaptic connections in tissue cultures of the mouse cerebellum.

The aim of this study was to explore the feasibility of the use of tissue cultures for studying some of the mechanisms of synaptic transmission in the mammalian CNS. The cerebellar cultures are particularly suitable because their morphological features have been studied in detail, offering a possibility of correlating electrophysiological and morphological data. Another advantage of the cerebellum is that its structure and function are relatively well known in vivo so the validity of the tissue culture model can be well tested in this case. It is well known from previous studies that functional synapses develop in the cerebellar cultures. However, the patterns of synaptic connections in cultures, in particular the interactions between cortex (Cx), deep cerebellar nuclei (DN) and brain stem (BS) have not been investigated. Electrophysiological experiments were performed on fully developed cultures 3-4 weeks old. The cultures were placed in a perfusion chamber mounted on the stage of an inverted microscope so the electrodes could be placed near the neurones under visual control. The temperature of the perfusing solution (Earle's balanced salt solution) was maintained at 35-37°C and the pH at 7.3-7.4. One region of the cultures was usually activated by brief electrical pulses and synaptically evoked responses were sought in a different region. Stimulation of the DN area resulted in a synaptic activation of about 50% of the Cx neurones whereas a stimulation of the Cx area evoked 30% of the DN neurones antidromically. On the basis of known conduction velocities and latencies of the synaptically evoked responses it has been concluded that in culture DN neurones form a synaptic projection to the Cx area, which resembles a mossy fiber pathway found in the intact cerebellum (Tolbert et al. , 1976). Electron microscopic observations in culture give supporting evidence for the mossy type projection from the DN to the Cx (Hendelman et al., 1978). Another synaptic pathway which has been well documented in culture is the monosynaptic inhibitory projection from the Cx region to the DN. Satisfactory electrophysiological and morphological evidence has been obtained to suggest that the projection is monosynaptic and that it can be routinely reproduced in culture. Electrophysiological characteristics of the pathway resemble those described in vivo by Ito et al. (1970a). Using pharmacological blockers, such as Bicuculline, Picrotoxin and Strychnine, it has been shown that GABA is probably the neurotransmitter responsible for the inhibition. This observation is similar to that of Obata et al. (1970a) who studied an analogous synaptic connection in the lateral vestibular nucleus of cats. In addition to the two major pathways demonstrated in cultures, evidence for recurrent axon collaterals of DN neurones and possibly Purkinje neurones has been obtained which again is in agreement with previous morphological observations in culture as well as in vivo. The presence of inhibitory interneurones in the Cx area was also evident. Neurones from the BS area, which can be included with the cerebellar cultures, have also been studied in spite of their uncertain identity. No firm evidence could be obtained as to the terminations of these neurones in other areas of the cultures but the results suggest that these neurones are excitatory and that they are interconnected by excitatory synapses which are possibly formed by the terminals of their own recurrent axon collaterals. A sparse inhibitory projection from the Cx to the BS area has been shown. Such a detailed demonstration of the synaptic interconnections in tissue cultures which resemble those in vivo provides a background for further experimentation. The tissue culture model of the Cb can be used for investigation of the mechanisms of synaptic transmission and the action of various pharmacological agents on the neurones. The advantage of such a model system lies primarily in its accessibility to pharmacological manipulations which are often difficult in the intact brain