244 Developmental Brain Research, 56 (1990) 244--256 Elsevie~ BRESD 51162 Developmental changes in the heavy subunit of neurofilaments in the corpus callosum of the cat Ana Guadano-Ferraz*, Beat M. Riederer and Giorgio M. Innocenti Institute of Anatomy, University of Lausanne, Lausanne (Switzerland) (Accepted 19 June 1990) Key words: Neurofilament; Development; Corpus callosum; Cortex; Phosphorylation In the corpus callosum of the cat, the heavy subunit of neurofilaments (NFH) can be demonstrated with the monoelonal aatibody NE14, as early as Pll, not at P3, and only in a few axons. At P18-19 and more markedly at P29, many more callosai axons have become positive to NE14 and this is similar to what is found in the adult. In contrast, callosal axons become positive to the neurofilament antibody SMI-32 only between P29 and P39 and remain positive in the adult. Treatment with alkaline phosphatase prevents axonal staining with NE14, but results in SMI-32 staining of a few callosal axons as early as Pll, but not at P3. Between Pll and P19 the number of axons stained with SMI-32 after alkaline phosphatase treatment increases, in parallel with that of axons stained with NE14. Thus NE14 appears to recognize a phosphorylated form of NFH, while SMI-32 appears to recognize an epitope of NFH which is either masked by phosphate or inaccessible until between P29 and P39, unless the tissue is treated with alkaline phosphatase. These two forms of NFH appear towards the end of the period of massive developmental elimination of callosal axons ~. They are also synchronous with changes in the spacing of neurofilaments quantified in a separate ultrastructural studya. These cytoskeletal changes may terminate the juvenile-labile state of eallosal axons and allow further axial growth of the axon. INTRODUCTION A crucial step in the development of neural connec- tions is the 'decision' of whether a juvenile cortical axon will be maintained or eliminated. Many of the juvenile axons of cortical origin are eliminated in development 17' 19,26; at least about 70% of the callosal axons which are produced in cat 1 and monkey 2° and at least 50% of the axons in the pyramidal tract of the hamster 29. Presum- ably, the axons which remain differ from those which are eliminated in some crucial aspect of their maturation. Therefore we are in the process of analyzing the molecular and ultrastructural maturation of the cytoske- leton of callosal axons 2'3'9'3°. The earliest biochemical change in the axonal cytoskeleton detected thus far towards the end of the period of axonal elimination, and therefore presumably characteristic of the axons which are maintained is the appearance of the 200 kDa subunit of neurofilaments (NUn) 9. This protein can be heavily phosphorylated (for references and discussion see refs.4- 6,11,16,21,22,31,32, inter alios). In the adult nervous system, the phosphorylated form of NFH localizes in axons but not, or only little in cell bodies or dendrites, while the dephosphorylated NFH has an almost complementary distribution 21'33'34. The effects of phosphorylation on neurofilament (NF) subunits are debated and may in- clude susceptibility to degeneration by calpain 27, rate of axonal transport 22, activity in tubulin polymerization 25 and cross-linking of NFs (see Discussion). The state of phosphorylation of NFH was not inves- tigated in the developing corpus callosum (CC) of the cat. The present study indicates that the previously observed late appearing NFH 9 is in a phosphorylated form. Subsequently, however, CC axons become positive to an antibody which recognizes either a partially dephos- phorylated form of a NF protein, presumably NFH, or an epitope of it whose reactivity is developmentally regu- lated but can also be revealed by alkaline phosphatase treatment. MATERIALS AND METHODS The brains examined were from kittens sacrificed on the following postnatal (P) days: P3 (3 animals), Pll (3 animals), P18(1 animal), P19 (2 animals), P28 or 29 (2 animals), P39 (2 animals), adults of unknown age, but older than 1 year (2 animals). After heavy Nembutal anaesthesia (50 mg/kg) and perfusion with 200-300 mi of 0.1 M phosphate buffer (pH 7.4), brains were quickly removed, frozen with dry-ice and sectioned at 20 pm. Sections :through the occipital part of the brain and including the visual areas 17 and 18 and the caudal part of the CC were selected, fixed with methanol at -20 °C for 10 rain and air-dried. These procedures were similar to those used previously 9. Sections were washed with Tfis-buffered * Present address: Department of Histology, Faculty of Medicine, University of Alicante, 03690 Alicante, Spain. Correspondence. G.M. Innocenti, Institut d'Anatomie, Universit6 de Lausanne, Rue du Bugnon 9, 1005 Lausanne, Switzerland.