* To whom correspondence should be addressed. Journal of Neurocytology 27, 187–196 (1998) Regenerated retinal ganglion cell axons form normal numbers of boutons but fail to expand their arbors in the superior colliculus D. A. CARTER, G. M. BRAY* and A. J. AGUAYO Centre for Research in Neuroscience, McGill University and The Montreal General Hospital Research Institute, 1650 Cedar Avenue, Montre ´ al, Que ´ bec, Canada, H3G 1A4 Received 17 November 1997; revised 10 February 1998; accepted 13 February 1998 Summary Regenerated retinal ganglion cell (RGC) axons can re-form functional synapses with target neurons in the superior colliculus (SC). Because preterminal axon branching determines the size, shape and density of innervation fields, we investigated the branching patterns and bouton formation of individual RGC axons that had regrown along peripheral nerve (PN) grafts to the SC. Within the superficial layers of the SC, the regenerated axons formed terminal arbors with average numbers of terminal boutons that were similar to the controls. However, axonal branches were shorter than normal so that the mean area of the regenerated arbors was nearly one-tenth that of control arbors and the resulting fields of innervation contained greater than normal numbers of synapses concentrated in small areas of the target. Our results have delineated a critical defect in the reconstitution of retino-collicular circuitry in adult mammals: the failure of terminal RGC branches to expand appropriately. Because recent studies have documented that brain-derived neurotrophic factor (BDNF) can specifically lengthen RGC axonal branches not only during development in the SC but also within the adult retina after axotomy, the present quantitative studies should facilitate experimental attempts to correct this deficit of the regenerative response.  1998 Chapman and Hall Introduction In previous studies in adult rodents with peripheral nerve (PN) grafts connecting the eye with the mid- brain, we showed that regenerated retinal ganglion cell (RGC) axons penetrated the superior colliculus (SC) for short distances and reformed well-differen- tiated synapses in the appropriate retino–recipient laminae (Vidal-Sanz et al., 1987, 1991; Carter et al., 1989, 1994). When light was flashed into such grafted eyes, the regenerated RGC terminals activated post- synaptic SC neurons (Keirstead et al., 1989; Sauve ´ et al., 1995), elicited pupillary responses (Thanos et al., 1992), and appeared to influence behaviour (Sasaki et al., 1993, 1996). While these observations prove that axonal re- growth can lead to the re-establishment of functional synapses, little is known about the axonal branching patterns that define the distribution of synaptic inter- actions between the afferent fibers and their target neurons. Electrophysiological studies of neuronal re- sponses in the SCs of hamsters indicated that the terminal arborizations of regenerated RGC axons synapsed with multiple neurons in the SC (Sauve ´ et al., 1995). Here, we have used intra-axonal labelling tech- niques to delineate the main features of the regen- erated arbors made by RGC axons that have been guided to the SC through a PN graft in adult rodents (Carter et al., 1989, 1994; Vidal-Sanz et al., 1987, 1991). The results of these studies should facilitate the understanding of functional effects of axonal regrowth and connectivity in the injured mammalian CNS. Fur- thermore, a better characterization of the connections made by regenerating axons in the adult brain is es- sential for the appropriate use of molecules that may influence terminal arborizations. The ability of neuro- trophins to cause the elongation of terminal RGC axons during development (Cohen-Cory & Fraser, 1995) and regeneration (Sawai et al., 1996) underscore the need for this information. Materials and methods In 9 female hamsters (Mesocricetus auratus, 90–120 days old), the right optic nerve (ON) was transected close to the 0300–4864  1998 Chapman and Hall