Cell suspension grafts of noradrenergic locus coeruleus neurons in rat hippocampus and spinal cord: reinnervation and transmitter turnover

Abstract

Fetal noradrenergic neurons from the brain stem locus coeruleus region can be successfully grafted as a dissociated cell suspension provided that the dissociation is done in the absence of any trypsin digestion step. The survival, fiber outgrowth and biochemical function of locus coeruleus neurons, taken from 13- to 15-day-old rat embryos, have been studied after injection into the dorsal hippocampal formation and the thoracolumbar spinal cord in adult rats. All rats were treated with an i.v. injection of 6-hydroxydopamine prior to grafting to remove the intrinsic locus coeruleus projections to these areas, and they were taken for fluorescence histochemical or biochemical analyses 2-7 months after transplantation. Up to 330 surviving noradrenaline neurons were found at each implantation site (injected with 2-3 microliters of cell suspension) which represents an estimated survival rate of about 40%. In the most successful cases the entire dorsal hippocampal formation, and an approximately 4 cm long segment of the thoracolumbar spinal cord, was supplied with a new noradrenaline-containing terminal network, which reached normal densities in the regions closest to the grafts. In the hippocampal formation, in particular, the ingrowing axons re-established a laminar innervation pattern which resembled that of the normal locus coeruleus afferents. In the hippocampus, two 2-microliters injections of locus coeruleus cell suspension restored the total hippocampal noradrenaline content to an average of 55%, and the noradrenaline synthesis rate (as assessed by the rate of DOPA accumulation after synthesis inhibition) was found to be close to normal in the graft-reinnervated specimens. In the spinal cord, two 3-microliters injections restored the noradrenaline level in the thoracolumbar cord (a 4.5 cm long segment) to an average of 22% of normal, with the highest individual levels being close to normal. Determinations of the noradrenaline metabolite 3,4-dihydroxy-phenylethyleneglycol indicated that the rate of noradrenaline metabolism in the graft-reinnervated spinal cord was close to that of the normal intact spinal cord. The results demonstrate the potential of the suspension grafting technique for extensive noradrenergic reinnervation of the hippocampal formation or large portions of the spinal cord. Fetal locus coeruleus neurons implanted in this way can re-establish fairly normal terminal innervation patterns and reinstate noradrenaline turnover and metabolism in a previously denervated central target.