THE HYALURONAN RECEPTOR RHAMM IN NORADRENERGIC FIBERS CONTRIBUTES TO AXON GROWTH CAPACITY OF LOCUS COERULEUS NEURONS IN AN INTRAOCULAR TRANSPLANT MODEL J. I. NAGY,*§ M. L. PRICE,† W. A. STAINES,‡ B. D. LYNN* and A.-CH. GRANHOLM† *Department of Physiology, Faculty of Medicine, University of Manitoba, 770 Bannatyne Avenue, Winnipeg, Manitoba, Canada, R3E 0W3 †Department of Basic Science, University of Colorado HSC, Denver, Colorado, U.S.A. ‡Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada Abstract––The hyaluronan receptor for hyaluronic acid-mediated motility (RHAMM) plays a role in cell migration and motility in many systems. Recent observations on the involvement of RHAMM in neurite motility in vitro suggest that it might also be important in axon outgrowth in situ. This was addressed directly by investigating both RHAMM expression in the rat CNS and the ability of anti-RHAMM reagents to interfere with tissue growth and axon outgrowth in intraocular brainstem transplants. By western blotting, anti-RHAMM antibody detected a RHAMM isoform of 75,000 mol. wt in both whole brain homogenate and synaptosome preparations, and a 65,000 mol. wt isoform in synaptosomes. Immunofluorescence of adult brain sections revealed RHAMM-like immunoreactivity in varicose fibers that were also positive for the noradrenergic marker dopamine--hydroxylase. Not all noradrenergic fibers contained RHAMM, nor was RHAMM detected in other monoaminergic fiber types. Lesions of noradrenergic fiber systems with -halobenzylamine-N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) eliminated RHAMM-positive fibers, but noradrenergic axons that sprouted extensively after this treatment were strongly RHAMM-positive. To assess RHAMM’s role in fiber outgrowth, fetal brainstem tissue containing noradrenergic neurons was grafted into the anterior chamber of the eye. Treatment of grafts with anti-RHAMM antibody caused significant inhibition of tissue growth and axon outgrowth, as did a peptide corresponding to a hyaluronan binding domain of RHAMM. These agents had no such effects on transplants containing serotonergic and dopaminergic neurons. These results suggest that RHAMM, an extracellular matrix receptor previously shown to contribute to migratory and contact behavior of cells, may also be important in the growth and/or regenerative capacity of central noradrenergic fibers originating from the locus coeruleus.  1998 IBRO. Published by Elsevier Science Ltd. Key words: axon growth, locus coeruleus, noradrenergic fibers, extracellular matrix, RHAMM. The projections of central noradrenergic (NA) neurons located in the locus coeruleus (LC) exhibit remarkable sprouting and growth capacity compared with most other neuronal populations in the adult CNS. 5,9,12,34,52,54 Cholinergic deafferentation of the hippocampus or partial damage to NA projections to the cerebellum, for example, leads to sprouting of NA fibers in denervated as well as non-denervated regions of the brain. 10,44,45 Following neurotoxic lesions which result in the extensive depletion of NA fibers in the brain and the death of over 60% of the NA neurons in the LC, fiber sprouting from the surviving neurons ultimately results in the complete reinnervation of forebrain structures. 21 This reinner- vation appears to be under the control of target tissue or target-specific factors. 2,21 While various members of the neurotrophin family increase survival of cul- tured LC NA neurons or provide neuroprotection of these neurons after LC lesions in adult rat brain, 3,4,18 to our knowledge no factors have been reported that are directly linked to sprouting of LC NA neurons. Identification of such factors is pertinent to the hypothesized role of these neurons in memory and learning, 65 as well as their loss of function and fiber sprouting capacity in senile dementia of the Alzheimer type, 30,35,46 and in animal models of age-related dementia. 32,38,50 Quite apart from neurotrophic mechanisms, factors that govern axonal outgrowth may include those involved in analogous processes, notably cell §To whom correspondence should be addressed. Abbreviations: BSA, bovine serum albumin; DA, dopamin- ergic; DH, dopamine--hydroxylase; DSP-4, -halo- benzylamine-N-(2-chloroethyl)-N-ethyl-2-bromobenzyl- amine; ECM, extracellular matrix ; HBD, hyaluronan binding domain; 5-HT, serotonergic; IR, immunoreactiv- ity; LC, locus coeruleus; NA, noradrenergic; PB, phos- phate buffer; PBS, phosphate-buffered saline; RHAMM, receptor for hyaluronic acid-mediated motility; TBS, Tris-buffered saline; TH, tyrosine hydroxylase. Pergamon Neuroscience Vol. 86, No. 1, pp. 241–255, 1998 Copyright  1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 PII: S0306-4522(98)00017-7 241