Regulation of CGRP expression in rat spinal motoneurons

Abstract

The normal distribution and lesion-induced changes in the expression of neuropeptides and 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 of CGRP in motoneurons were identified.

The vast majority of adult motoneurons at the lumbar level of the spinal cord normally contained detectable levels of CGRP-like immunoreactivity (-LI). The levels of CGRP-LI at the cell body level varied somewhat with the type and size of the motor units. CGRP-LI levels were higher in motoneurons innervating the anterior tibial and lateral gastrocnemius muscles (which contain mainly fast, type II muscle fibers) than in those innervating the soleus muscle (where there is a predominance of slow, type I fibers) and flexor digiti minimi muscle (where motor units comprise 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 in lesioned motoneurons. Oligonucleotide probes that distinguish between the alpha and beta isoforms of CGRP showed that beta-CGRP is downregulated after axotomy, and thus that the increase reflects an increased expression of alpha-CGRP. The change in CGRP expression on the lesioned side was similar after a ventral root transection, except that the low to moderate increase in CGRP expression on the unlesioned side was absent. Axotomy led to decreased expression of choline acetyltransferase (ChAT) and cholecystokinin (CCK) mRNAs, while the expression of acidic fibroblast growth factor (aFGF) was unchanged. The protein expression pattern after ventral root avulsion differed from that seen after peripheral 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 was characterized 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 a peripheral nerve lesion was different also immature rats, since alpha-CGRP was downregulated in response to a sciatic nerve transection in newborn rats, but was upregulated in two week old and in adult rats.

A spinal cord transection led to lowered levels of CGRP below the lesion, while the opposite effect was seen after intraventricular administration of 5, 7-dihydroxytryptamine, a neurotoxic compound that lesions serotoninergic and catecholaminergic fiber tracts. However, in both cases an increased CGRP expression could be elicited by a sciatic nerve transection. No major 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 abolished the 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 antagonist CGRP 8-37 increased the number of GAP-43 immunoreactive motor endplates. bFGF lowered the 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 of embryonic rat motoneurons. In the same culture system, bFGF decreased the expression of alpha-CGRP and increased neurite branching, whereas CNTF had the opposite effect, by increasing alpha-CGRP expression and decreasing neurite branching.

The present findings suggest that CGRP expression in spinal motoneurons is regulated, at least in part, by target-derived factors, and that CGRP physiologically may be a nerve cell derived substance that is involved in controlling sprouting during development and regeneration.