Elsevier

Brain Research

Volume 861, Issue 1, 7 April 2000, Pages 45-58
Brain Research

Research report
Motor neurons are rich in non-phosphorylated neurofilaments: cross-species comparison and alterations in ALS

https://doi.org/10.1016/S0006-8993(00)01954-5Get rights and content

Abstract

The localization and distribution of non-phosphorylated neurofilaments (NP-NF) in the upper and lower motor neurons was investigated in the rat, the common marmoset, the rhesus monkey and man using the SMI-32 antibody. Within the spinal cord of all species studied, the most intense NP-NF immunoreactivity was observed within the ventral horn α-motor neurons. Concurrent staining for the cholinergic marker choline acetyltransferase (ChAT) demonstrated that virtually all of the ChAT-positive α-motor neurons contain NP-NF immunoreactivity. Although NP-NF staining was also observed in other neurons within the ventral and intermediate horns, these neurons were loosely scattered and contained a considerably lower staining intensity. The only other prominent NP-NF staining in the spinal cord occurred within the neurons of the dorsal nucleus of Clark and the intermediolateral cell column. Phosphorylated neurofilament (P-NF) immunoreactivity was found primarily in neuronal processes. Occasionally, a solitary motor neuron contained weak P-NF immunoreactivity. Within the brainstem, neurons in all cranial nerve motor nuclei contained intense NP-NF immunoreactivity. The distribution and apparent density of NP-NF immunoreactive neurons in these nuclei was virtually identical to that observed for neurons immunoreactive for ChAT. NP-NF immunoreactive neurons of relatively lower intensity were found in many other regions of the brainstem. All of the giant Betz cells of layer (L) V in the motor cortex contained dark NP-NF immunoreactivity. Within the spinal cord of amyotrophic lateral sclerosis (ALS) patients, both Nissl and NP-NF staining demonstrated the dramatic loss of α-motor neurons characteristic of this disorder. Some of the remaining motor neurons contained intense P-NF immunoreactivity. These observations suggest that NP-NF immunoreactivity is a good marker for motor neurons in health and disease and may be a useful tool for studies of motor neuron degeneration (MND).

Introduction

Skeletal muscles are under the direct influence of lower motor neurons within the brainstem (cranial nerve motor nuclei) and the ventral horns of the spinal cord (α-motor neurons). The lower motor neurons receive substantial input from the upper motor neurons in layer (L) V of motor cortex (Brodmann's area 4) 4, 26. Damage to neurons in various parts of this system or the axons which emanate from them produces distinct motor deficits [1]. In addition, some components of this system are vulnerable to neurodegenerative processes. For example, α-motor neurons, some cranial nerve motor nuclei, and to a smaller extent the upper motor neurons are vulnerable to degeneration in motor neuron diseases such as amyotrophic lateral sclerosis (ALS) 1, 9.

Because of their vulnerability to damage and degeneration at multiple levels of the neuraxis, and their crucial role in the execution of behavioral responses, the upper and lower motor neurons have been subject to intense investigation. One problem encountered in these investigations has been the correct identification of motor neurons 6, 8. In some locations, such as the ventral horns of the spinal cord, motor neurons are not always confined to closed nuclear boundaries [4] and must be distinguished from neurons in adjacent regions.

In the past, several markers have been utilized for the identification of motor neurons. The large size of these neurons can be used in Nissl preparations as one such marker 4, 23. However, all neurons are enriched in Nissl substance and adjacent, large non-motor neurons may be mistakenly identified as motor neurons. Because of their cholinergic phenotype, the cholinergic markers choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) have been used to identify somatic motor neurons within the brainstem and spinal cord 6, 8, 23. The synthetic cholinergic enzyme ChAT is specifically localized in cholinergic neurons [12]. However, this enzyme is exquisitely sensitive to fixatives and therefore difficult to visualize, particularly in formalin-fixed pathologic specimens (Geula, unpublished observations) 12, 18. The hydrolytic enzyme AChE is also present in all cholinergic neurons, but it is not a specific cholinergic marker and is found in a substantial population of non-cholinergic neurons within the ventral horns of spinal cord and in the brainstem 8, 12, 13. Furthermore, the upper motor neurons are not cholinergic and are therefore devoid of ChAT immunoreactivity but contain a high concentration of AChE, particularly in the human 13, 20. Differentiating brainstem and ventral horn motor neurons in the chick have been shown to express the homeobox gene Islet-1 and to contain Islet-1 protein 10, 27, 35. However, the presence of Islet-1 in adult motor neurons, particularly in the rat and the human, has not been established.

A reliable marker of somatic motor neurons should have several characteristics. First, it should allow the identification of virtually all motor neurons. Second, it should be relatively resistant to variations in tissue parameters such as duration and mode of fixation, post-mortem interval, etc. Third, it should identify motor neurons in a range of species. Here we report that immunohistochemical staining for non-phosphorylated neurofilaments (NP-NF), identified by the specific antibody SMI-32 30, 31, can serve as a relatively reliable marker of motor neurons, satisfying all of the criteria listed above. The enrichment of α-motor neurons in NP-NF has important implications for ALS in which these neurons have been shown to contain inclusions of the phosphorylated form of neurofilaments (P-NF) 19, 22, 24.

Section snippets

Tissue preparation

Four Sprague–Dawley rats (3 months old), four common marmosets (Callithrix jacchus, 2–8 years old), two rhesus monkeys (Macaca mulata, 5 years old) and post-mortem tissue from six neurologically normal human cases and three patients with the clinical and pathological symptoms of ALS were used in this study. Spinal cord, brainstem and motor cortex was available in all animals. Of the normal human cases, spinal cord was available in two and cortex and brainstem in four cases. From the ALS cases,

Results

Immunohistochemistry, using the NP-NF antibody SMI-32, stained a substantial population of neurons throughout the brain. The majority of these were large projection neurons. NP-NF immunoreactivity was granular in appearance, was distributed throughout the cytoplasm and extended into dendrites. The intensity of staining in NP-NF-positive neurons displayed a systematic increase as the phylogenetic scale was ascended. Thus, NP-NF-positive neurons in the rat brain displayed the lightest staining

Discussion

The results of the present study demonstrate the presence of a relatively high density of NP-NF immunoreactivity within virtually all motor neurons of the spinal cord, brainstem and motor cortex. Although neurons in some adjacent structures also contained NP-NF immunoreactivity, NP-NF staining in motor neurons was considerably more intense. NP-NF immunoreactivity in motor neurons was observed in all of the species examined. In addition, perikaryal NP-NF staining was obtained in motor neurons

Acknowledgements

We thank Nicholas Nagykery for expert technical assistance. This work is supported in part by the Milton Fund of the Harvard Medical School.

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