Quantitative in situ hybridization and RNA blotting methods were used to define the time course and magnitude of changes in expression of mRNAs encoding peripherin and the neurofilament (NF) triplet proteins in rat dorsal root ganglion (DRG) neurons during axonal regeneration. mRNA levels in adult rat L4 and L5 DRGs were examined in autoradiograms after in situ hybridization with specific 35S‐labeled cDNA probes 1–56 days following unilateral crush lesions of the sciatic nerve. The results of quantitative analyses indicated that peripherin mRNA levels were significantly increased in large‐sized (>1000 μm2) DRG neurons at 7, 14, and 28 days after axotomy while the mRNA levels for each of the NF triplet proteins were significantly decreased at these same time points. The mRNA levels of the low (NF‐L) and middle (NF‐M) sized NF subunits were significantly decreased as early as 1 day postaxotomy but the mRNA level of the large NF subunit (NF‐H) did not change until 7 days after axotomy. The maximal reduction in NF mRNA levels was observed at 14 days postaxotomy when NF‐L mRNA levels were only 35% of those in large‐sized, normal control neurons. Recovery toward normal levels of both NF and peripherin mRNAs was observed at 8 weeks postaxotomy. RNA blot analyses with total RNA obtained from DRGs at different postaxotomy times confirmed that NF‐L mRNA levels were reduced in the DRG during the first 4 weeks after axotomy but, interestingly, failed to detect an increase in peripherin mRNA levels. This difference concerning peripherin mRNA levels in axotomized preparations obtained by RNA blotting vs. in situ hybridization was attributed to the fact that RNA blots utilized total DRG RNA which includes mRNAs from both small and large‐sized DRG neurons. A recent in situ hybridization study showed that the small‐sized DRG neurons which contain the majority of the peripherin mRNA in the DRG do not increase their peripherin mRNA levels 14 days after axotomy (Oblinger et al., 1989b). This may mask any change in the large neuron response when total RNA is examined. Overall, the results of this study demonstrate (1) that type III (peripherin) and type IV (NF) intermediate filament genes are regulated differently during axonal regeneration, and (2) that the three NF genes are down‐regulated in a fairly coordinate manner during regeneration. These data suggest that an important component of the regeneration program is the alteration of the composition of the IF component of the cytoskeleton. A cytoskeleton with fewer Type IV IFs and more Type III IFs may be more compatible with the specific structural and functional requirements of regenerating neurons. Copyright © 1990 Wiley‐Liss, Inc.
