EXPERIMENTALNEUROLOGY 102,102-108 (1988) Anatomical and Behavioral Correlates of a Xenograft-Mediated Pupillary Reflex HENRY KLASSEN’ AND RAYMOND D. LUND Department of Neurobiology, Anatomy and Cell Science, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15261 Retinae from mouse embryos were transplanted to the midbrain of neonatal rats which were unilaterally enucleated at the time of transplantation. At maturity the host’s remaining optic nerve was cut and the skull over the transplant was removed. Illumination of the transplant caused pupilloconstriction of the host eye, a response abolished by removal of the transplant or damage to the olivary pretectal nucleus which is the pupilloconstriction center of the brain stem. The degree of constriction correlated with the intensity of trans- plant illumination. Using mouse-specific monoclonal autibodies, we demonstrated that the grafts projected only to areas of the brain stem normally innervated by the eye. The density of label in the olivary pretectal nu- cleus did not, however, correlate with the briskness of the graft-mediated pupillary reflex. In summary, this study shows that ectopic neural xenografts are capable of making specific connections which can drive an ap- propriate response to a natural stimulus in the host ani- mal. While important in showing the ability of trans- plants to make specific connections, this study also pro- vides a simple assay system for examining conditions which may influence the efficacy of host innervation by the transplant. 0 1988 Academic Press, Inc. INTRODUCTION Neural tissue transplantation into the mammalian nervous system has attracted considerable attention be- cause of the demonstrated ability of certain grafts to en- hance recovery of function following lesions. Functional recovery depends, in most of these situations, on local production by the graft of an appropriate chemical in the host brain (2, 6) and not necessarily on the establish- ment of precise neural connections between transplant and host. We have defined a system here in which it is possible to examine the direct relationship between a simple reflex behavior and the precise neuroanatomical connections that underlie it. ’ To whom reprint requests should be addressed at University of Pittsburgh, Department of Neurobiology, Anatomy and Cell Science, School of Medicine, 3550 Terrace St., Pittsburgh, PA 15261. Previous work has shown that fetal retinae, both allo- typic and xenotypic, can survive when placed in the rat brain and project only to retinorecipient areas of the brain stem (7, 15, 16). Furthermore, allotypic retinal grafts can mediate a pupillary reflex in the host (10). The pupillary reflex in normal animals requires an optic pro- jection to the pupilloconstriction neurons in the olivary pretectal nucleus (OPN) (5, 21). These neurons project in turn to preganglionic parasympathetic neurons of the Edinger-Westphal nucleus (1,3,9,19) which innervate the iris muscles by way of the oculomotor nerve and re- lay neurons in the ciliary ganglion. In this study we wished to examine whether a graft- mediated reflex could also be driven by a retina taken from a different species of animal, in this case mouse. There were three principal reasons for this approach: first, we have shown previously that mouse-specific anti- bodies can be used to trace mouse graft projections in rat brains at both light and electron microscopic levels (7, 11). Combined with measurement of graft-mediated pu- pillary reflex activity this permits the examination of re- lationships between structure and function. Second, we were interested in whether the molecular mechanisms that underlie the formation of the specific connections involved in this function are common across speciesbar- riers. Third, specific connectivity exhibited by xeno- grafts may ultimately be of relevance to humans, since such an application would be worthy of consideration under circumstances where autografts or allografts are not feasible. Therefore, the degree of interaction possi- ble between xenograft and host brain needs to be ex- plored in carefully controlled situations. METHODS Retinae were dissected from the donor CD-l mice at Embryonic Day 13 and injected into the brains of neona- tal Sprague-Dawley rats. Recipients were unilaterally enucleated at this time in order to enhance innervation of the host by the transplant (16). Retinae were placed either on the surface of the midbrain or into the cerebral aqueduct. No immunosuppressive agents were adminis- tered since previous studies have shown that mouse reti- nae can survive for prolonged periods in recipient rat brains, after being implanted at birth (13, 14). At 3 or 0014~4a86/&3 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved. 102