Introduction ponent in large myelinated axons of the peripheral nervous

Brain Research 885 2000 32–44 www.elsevier.com locate bres Research report Electrophysiological properties of axons in mice lacking neurofilament subunit genes: disparity between conduction velocity and axon diameter in absence of NF-H a ,1 b b a , ˇ Jasna Kriz , Qinzang Zhu , Jean-Pierre Julien , Ante L. Padjen a Department of Pharmacology and Therapeutics , the Montreal General Hospital Research Institute, McGill University, ´ 3655 Sir William Osler Promenade ex-Drummond Street, Montreal, QC H3G 1Y6, Canada b Centre for Research in Neuroscience , the Montreal General Hospital Research Institute, McGill University, ´ 3655 Sir William Osler Promenade ex-Drummond Street, Montreal, QC H3G 1Y6, Canada Accepted 22 August 2000 Abstract Neurofilament proteins NFs are made by co-polymerization of three intermediate filament proteins, NF-L, NF-M and NF-H and constitute the most abundant cytoskeletal element in large myelinated axons. NFs have a well-established role as intrinsic determinants of axon caliber with all the functional implications, but the role of each individual NF subunit is much less clear. The aim of our study was to examine functional properties of large myelinated axons with altered morphology from mice bearing a targeted disruption of each NF genes NF-L 2 2, NF-M2 2 and NF-H 2 2 mice. Membrane properties, action potentials and single axon refractory period were measured in isolated sciatic nerves in vitro, using intra-axonal microelectrode recording in conjunction with current–clamp technique. Some results were obtained from whole nerves by sucrose-gap recording. The NF-knockout mice showed several deficits in physiological properties of low-threshold fibers. In keeping with smaller axon diameter, the conduction velocity was significantly decreased in NF-L 2 2 and NF-M 2 2 transgenic animals control, 39.961.8 m s, NF-M 2 2; 23.561.4 m s, and NF-L2 2; 12.060.7 m s, mean6S.E.M.; intra-axonal recording; similar ratios obtained by sucrose-gap recording; 22–268C. However, in spite of their preserved caliber, large myelinated axons in NF-H 2 2 mice also showed a significant decrease in conduction velocity 22.861.0 m s, mean6S.E.M.. Although action potential amplitudes, duration and shape did not differ between control axons and transgenic animals, the refractory period was prolonged in NF-H 2 2 and NF-M 2 2 animals. Intracellular injections of 200 ms depolarizing and hyperpolarizing currents revealed outward and inward rectification in all animal groups. In comparison to control animals, NF-H 2 2 mice expressed a significant decrease in outward rectification. Potassium channel blockers 4AP and TEA and cesium ions were able to block outward and inward rectification in all myelinated axons in qualitatively the same manner. These results suggest that NF-H may have a specific role in modulating ion channel functions in large myelinated fibers.  2000 Elsevier Science B.V. All rights reserved. Theme : Disorders of the nervous system Topic : Genetic models Keywords : Neurofilament; Transgenic mouse; Myelinated axon; Intra-axonal recording; Conduction velocity

1. Introduction ponent in large myelinated axons of the peripheral nervous

system PNS. The three neurofilaments subunits NF-L, Neurofilaments represent the major cytoskeletal com- 68 kDa; NF-M, 134 kDa and NF-H, 200 kDa [33,50] share a conserved domain, common with other inter- mediate filaments IF, involved in the formation of Corresponding author. Tel.: 11-514-398-3603; fax: 11-514-398- coiled–coil structure [23,24,74]. Neurofilaments form het- 4449. eropolymers, which in mice require NF-L with either E-mail address : alppharma.mcgill.ca A.L. Padjen. 1 NF-M or NF-H proteins [48,49]. Present address: Department of Pharmacology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia. The three neurofilaments are synthesized in the 0006-8993 00 – see front matter  2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 8 9 9 - 7 J . Kriz et al. Brain Research 885 2000 32 –44 33 perikaryon and transported down the axon with the slow structure–function relationship for three isoforms of NF component of axoplasmic transport [33]. Neurofilaments we studied the electrophysiological properties of large seem not to move along the axon as an organized network, myelinated axons in peripheral nerves of mice with as originally considered [44,45] but rather as a dynamic targeted disruption of NF genes. We report here that structure [8,52,53]. There is evidence that subunit ex- changes in expression of NF genes lead to functional change can occur along the axon [57] and that neurofila- changes in peripheral nerves of transgenic mice. Similar ment proteins can be transported in an unpolymerized form conclusion stems from our recent studies of mice express- along axonal microtubules [76]. ing human NF-H [41,42], as well mice expressing neuro- It is generally considered that one of the functions of filament-beta-galactosidase fusion protein [40]. Prelimin- neurofilaments is related to the control of radial growth of ary results of the present study have been published in an large myelinated axons. This notion is largely based on the abstract [43]. correlation between the number of neurofilaments and axonal diameter [22,26,30–32]. Direct support for this hypothesis has been obtained by studies of three animal

2. Material and methods