Target recognition and synapse formation by ciliary-ganglion neurons in tissue culture.

Cell-to-cell recognition must play an important role in the nervous system during the formation and maintenance of specific connections between neurons and their target cells. This process of recognition in the nervous system might be based on ( I ) complementarity of structural components in the presynaptic and postsynaptic membranes of appropriate pairs of cells, ( 2 ) the release of a chemical compound by the target cell, which acts as chemotactic ‘lure’ for the correct exploring nerve ending, (3) a profuse aspecific formation of cell-to-cell contacts followed by selective survival and maintenance of those connections that appear to be meaningful for the organism (in this case electrophysiological activity of the target cell or a biochemical process associated with this activity is supposed to be the crucial factor in recognition) and (4) a time-position mechanism in which differences in timing of maturation is a major factor in the control of the formation of the pattern of connections. For references see Gaze & Hope (1976) and Holliday et al. (1977). The relative contribution of any of these mechanisms is unknown. The formation of junctions between neurons, and between neurons and muscle cells can be studied in tissue culture of embryonic neurons and in mixed cultures of embryonic neurons and skeletal-muscle cells respectively. In many studies the neuronal cells are obtained from embryonic spinal cord and the muscle cells from embryonic leg or breast muscle. Although such mixed cultures are likely to contain the genuine motor neurons they are bound to contain a host of other neurons, although no methods are presently available to identify a motor neuron in culture with any degree of certainty. The preparation is therefore not very attractive for the study of the recognition process involved in the formation of the neuromuscular junction. A less complicated source of neurons suitable for this type of studies is the parasympathetic ciliary ganglion. In the pigeon and in the chick this ganglion is known tocontain only two classes of neurons, which both are cholinoceptive and cholinergic and that innervate the muscle fibres of the choroid plexus of the eye, the sphincter iridis and the ciliary body (Marwitt et al., 1971). The iris and the ciliary muscle in birds are both striated (Ovio, 1927), whereas in the mammal they are smooth. Neurons of the ciliary ganglion of the chick embryo can be cultivated in tissue culture (Hooisma et al., 1975) either in explants of whole ganglia or after dissociation, as individual cells. Branching neurites grow radially from the ganglia. A corona of neurites with a diameter of 500-1000pm surrounding the ganglia is formed during 2 days in culture. Mixed cultures of ciliary neurons and skeletal-muscle cells have been used for the study of the process of neuron-to-target recognition. The present paper deals with the following questions: (1) do the autonomic neurons of the chick ciliary ganglion recognize chick skeletal-muscle cells as appropriate target cells for functional innervation? ( 2 ) do neurons, which exclusively form neuromuscular connections with smoothmuscle cells in uiuo, recognize striated muscle fibres as target cells in vitro? (3) does recognition of chick skeletal-muscle fibres by chick ciliary-ganglion neurons lead to neurotrophic support of these muscle fibres, analogous to the neurotrophic influence of the motor neuron on its target cells? Muscle cells obtained by trypsin dissociation of leg muscles of the 11-day chick embryo were cultivated by the method of Slaaf (1977), on collagen-coated object glasses in Eagle’s minimum essential medium and Earle’s balanced-salt solution supplemented with 15 % horse serum and 5 embryo extract at 35°C and with a final osmolarity of 320mos~, in a humid atmosphere of C02/air (1 : 19). D-Arabinofuranosyl-