Expression of Subtypes NF-L, NF-M and NF-H of Neurofilament Triplet Proteins in the Developing Rat Inner Ear

Abstract

The expression of each subtype (NF-L, NF-M and NF-H) of neurofilament (NF) triplet proteins in the developing rat inner ear was analysed using the immunofiuorescence method (FITC). On the 15th gestational day (GD), immunostaining of NF subtypes did not occur in the otic region. On the 18th GD, positivity for NF-L and NF-M occurred in nerve fibres in both the sensory and the subepithelial layers of the inner ear but was lacking for NF-H. At 4 days after birth immunoreactivity was present for each NF subtype but with topographic differences in their distribution. The cell bodies of (different?) subpopulations of vestibular and spiral ganglion cells were immunostained with monoclonal antibodies for NF-L and NF-M, but a subpopulation of both vestibular and spiral ganglion cells showed immunoreactivity for all three NF subtypes. The vestibular nerve fibres and ganglion cells evidenced stronger positivity than cochlear nerve fibres and spiral ganglion cells. This study confirms that the cytoskeletal maturation of inner ear nerve fibres precedes the onset of at least the auditory function by several days.

Mapping of notch activation during cochlear development in mice: Implications for determination of prosensory domain and cell fate diversification

“…The polyclonal antibody against neurofilament-M (NF-M) that we used (Chemicon International) specifically labeled the nerve fibers from the spiral ganglion to the differentiating cochlear hair cells (Fig. 1), which is basically consistent with previous descriptions of results obtained using another monoclonal antibody against NF-M ( Nishizaki and Anniko, 1995). The anti-Notch1 antibody (M-20; Santa Cruz Biochemistry, Santa Cruz, CA) and anti-Jag1 antibody (C-20; Santa Cruz Biochemistry) stained a single band of 120 kD MW and 150 kD MW, respectively, on Western blot (manufacturer's technical information), and the staining pattern of these antibodies in the mouse inner ear (Notch1: Figs. 2A, 7C; Jag1: Figs.…”

Neurofilament localization and phosphorylation in the developing inner ear of the rat

“…In the cochlear nerve, the expression patterns of NF subunits are found to progress from base to apex, and from IHCs to OHCs. This spatiotemporal pattern in NF expression in the inner ear parallels the process of morphological and functional maturation in the vestibular and cochlear organ in general (Pirvola et al, 1991;Nishizaki and Anniko, 1995;Bruce et al, 2000;Rubel and Fritzsch, 2002).…”

“…As early as ED12 immunostaining of inner ear neurons can be observed when antibodies against NF-PI subunits are used. This is 4e6 days prior to the detection of NF-P subunits, and proves that NFs are much earlier present during development than was assessed before (Hafidi et al, 1990;Nishizaki and Anniko, 1995). Although these differences may result from variations in tissue preparation (Shi et al, 1991;Ezaki, 2000), they may be related as well to the fact that different, not further specified antibodies were used previously.…”

Technical report: Laser microdissection and pressure catapulting is superior to conventional manual dissection for isolating pure spiral ganglion fractions from the cochlea

“…NF-L is a neural tissue marker, present only in neural elements of the cochlea (i.e. auditory nerve with its cell bodies) (Berglund and Ryugo, 1991;Hafidi et al, 1990;Nishizaki and Anniko, 1995). Thus, it can be used as positive control for demonstrating that all examined samples contain neurofilamental mRNA from SG cells and its nerve fibres indicating constitutive neurofilament gene expression in all sample pools -manually dissected or laser microdissected -and, therefore intact tissue and cell samples.…”

“…Hence, both D2 and Cld-11 seem to be appropriate tissue specific markers of contamination to assess the purity of tissue/cell pools. Neurofilament light chain (NF-L) amplification was used as positive control for specific neuronal gene expression in all sample pools (Berglund and Ryugo, 1991;Hafidi et al, 1990;Nishizaki and Anniko, 1995). The results were used to demonstrate whether neighbouring tissues contaminated the manually and LMPC dissected SG cell samples.…”