Regulation of CGRP expression in rat spinal motoneurons

Abstract: The normal distribution and lesion-induced changes in the expression of neuropeptidesand growth-related proteins, with special reference to calcitonin gene-related peptide (CGRP),were studied in the motor system of the rat. In addition, factors that influence the expression ofCGRP in motoneurons were identified. The vast majority of adult motoneurons at the lumbar level of the spinal cord normallycontained detectable levels of CGRP-like immunoreactivity (-LI). The levels of CGRP-LI at thecell body level varied somewhat with the type and size of the motor units. CGRP-LI levels werehigher in motoneurons innervating the anterior tibial and lateral gastrocnemius muscles (whichcontain mainly fast, type II muscle fibers) than in those innervating the soleus muscle (wherethere is a predominance of slow, type I fibers) and flexor digiti minimi muscle (where motor unitscomprise relatively few muscle fibers). A large population of the small cells, presumably gammamotoneurons, lacked detectable levels of CGRP-LI. A sciatic nerve transection induced a robust increase in CGRP peptide and mRNA inlesioned motoneurons. Oligonucleotide probes that distinguish between the alpha and betaisoforms of CGRP showed that beta-CGRP is downregulated after axotomy, and thus that theincrease reflects an increased expression of alpha-CGRP. The change in CGRP expression on thelesioned side was similar after a ventral root transection, except that the low to moderate increasein CGRP expression on the unlesioned side was absent. Axotomy led to decreased expression ofcholine acetyltransferase (ChAT) and cholecystokinin (CCK) mRNAs, while the expression ofacidic fibroblast growth factor (aFGF) was unchanged. The protein expression pattern after ventral root avulsion differed from that seen afterperipheral nerve lesion. In both lesion models the expression of mRNAs encoding alpha-CGRPGAP-43, c-jun and the low affinity nerve growth factor receptor (p75) were initially upregulated.However, at one to three weeks after a sciatic nerve lesion, the expression pattern wascharacterized by elevated levels of alpha-CGRP, GAP-43, galanin message-associated peptide(GMAP), c-jun and p75 mRNAs, while, in contrast, GMAP, GAP-43 and nitric oxide synthase(NOS) mRNAs were upregulated after a ventral root avulsion. The expression pattern after aperipheral nerve lesion was different also immature rats, since alpha-CGRP was downregulatedin response to a sciatic nerve transection in newborn rats, but was upregulated in two week oldand in adult rats. A spinal cord transection led to lowered levels of CGRP below the lesion, while theopposite effect was seen after intraventricular administration of 5, 7-dihydroxytryptamine, aneurotoxic compound that lesions serotoninergic and catecholaminergic fiber tracts. However, inboth cases an increased CGRP expression could be elicited by a sciatic nerve transection. Nomajor effect on the CGRP expression in motoneurons was seen after a dorsal root transection. Basic FGF administered to the proximal cut end after a sciatic nerve transection abolishedthe axotomy-induced increase in alpha-CGRP mRNA, while ciliary neurotrophic factor (CNTF),brain derived neurotrophic factor (BDNF), and insulin-like growth factors-1 and -2 (IGF-1, IGF-2) were without effects. In early postnatal rats CNTF, bFGF, IGF-2 and the CGRP antagonistCGRP 8-37 increased the number of GAP-43 immunoreactive motor endplates. bFGF loweredthe number of CGRP immunoreactive motor endplates, while CNTF had the opposite effect.CNTF, bFGF, IGF-1 and IGF-2 increased the expression of GAP-43 mRNA in cultures ofembryonic rat motoneurons. In the same culture system, bFGF decreased the expression ofalpha-CGRP and increased neurite branching, whereas CNTF had the opposite effect, byincreasing alpha-CGRP expression and decreasing neurite branching. The present findings suggest that CGRP expression in spinal motoneurons is regulated, atleast in part, by target-derived factors, and that CGRP physiologically may be a nerve cell derivedsubstance that is involved in controlling sprouting during development and regeneration.Keywords; immunohistochemistry, in situ hybridization, motoneuron, axotomy, peptides,CGRP, trophic factor, FGF, IGF, BDNF, CNTF, GAP-43, cell culture, plasticity, mRNAregulation, regenerationISBN 91-628-2091-5