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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 55:B233-B241 (2000)
© 2000 The Gerontological Society of America

Age-Dependent Changes in the Midsized Neurofilament Subunit in Sensory-Motor Systems of the Cat Brainstem

An Immunocytochemical Study

Jian-Hua Zhanga, Sharon Sampognaa, Francisco R. Moralesa and Michael H. Chasea

a Department of Physiology and the Brain Research Institute, University of California—Los Angeles School of Medicine

Michael H. Chase, Department of Physiology, UCLA School of Medicine, University of California\|[mdash ]\|Los Angeles, Los Angeles, CA 90024 E-mail: mchase{at}ucla.edu.

Decision Editor: Jay Roberts, PhD


   Abstract
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 Abstract
Methods
 Results
 Discussion
 References
 
This study documents age-related changes in the immunoreactivity of the medium-molecular weight subunit of neurofilaments in sensory and motor neurons in the brainstem of the cat. In old age, there was a clear decrease in immunoreactivity in the following brainstem sensory and motor nuclei: sensory trigeminal, gracile, cuneate, and facial motor. Only a few neuronal perikarya and dendrites were labeled in these nuclei in old cats; moreover, when present, the labeling was weak. In contrast, in adult cats, these nuclei contained intensely stained neuronal perikarya and dendrites. In other sensory and motor nuclei of the brainstem, there was an obvious age-related increase in the immunoreactivity of the medium-molecular weight subunit of neurofilaments in the perikarya. Despite different patterns of age-related alterations in immunoreactivity within perikarya and dendrites in distinct brainstem regions, most sensory and motor axons in old cats were smaller than those in adult cats. A decrease in the medium-molecular weight neurofilament subunit in the dendrites may be the basis for the dendritic atrophy that has been shown to occur in sensory nuclei in old animals. The decrease in axonal size is likely to be one of the causes of the decrease in axonal conduction velocity, in these neurons, that was reported in our previous studies.

DURING the aging process, a great many physiological changes occur in neurons and their processes (1)(2)(3)(4)(5). For example, using intracellular recording and stimulating techniques, we have found that there are significant changes in the electrophysiological properties of motoneurons in the spinal cord and brainstem, such as a decrease in axonal conduction velocity, an increase in input resistance, and an increase in the membrane time constant (6)(7)(8)(9)(10)(11)(12). These changes indicate that during old age there are significant alterations in axons as well as in the somadendritic compartments of motoneurons. At an ultrastructural level of analysis, using electron microscopic techniques, we have found age-related morphological changes in the axons of motoneurons including a significant reduction in axonal diameter and demyelination (10)(13)(14)(15). Machado-Salas and colleagues (16) demonstrated, with Golgi methodology, that the somadendritic portions of neurons in certain sensory and motor nuclei of the mouse brainstem undergo atrophic changes during the aging process, such as a decrease in size, a change in shape of the soma, and a progressive distortion of the dendrites (swelling, constriction, and fragmentation). To date, to our knowledge, although many age-related morphological changes have been found in brainstem sensory and motor neurons, the reason for these changes are unknown. We have previously hypothesized that age-related alterations in neurofilament structure could account for the proceeding atrophic neuronal changes that occur in old age (17).

Neurofilaments (NFs) are the neuron-specific intermediate filaments that are believed to contribute to the structural integrity of neurons and neuronal processes (18)(19)(20)(21). In mature neurons, NFs are composed of three different polypeptide subunits, with molecular weights of approximately 68 kd (NF-L), 160 kd (NF-M), and 200 kd (NF-H). These three subunits, each of which is encoded by its own gene, are synthesized and assembled into the NF protein triplet in the perikarya. Subsequently, the NF protein triplet is transported into neuronal processes (dendrites and axons; (19), (22)(23)). Within axons, the NF protein triplet undergoes somatofugal translocation by means of slow axonal transport (24)(25). Finally, the transported NF protein triplet is degradated by a variety of proteases in the axon terminal (26)(27).

Although the role of NFs in the nervous system is not yet completely understood, several recent observations support the hypothesis that NFs are a primary determinant of axon caliber in myelinated fibers. For example, it has been shown that the number of NFs is closely correlated with the cross-sectional area of axons in both the central and the peripheral nervous system in adult and aged animals (22)(28)(29). In addition, NF content increases in axons that exhibit radical growth during postnatal development (30)(31) and, conversely, it decreases in atrophic axons that exhibit a reduction in their cross-sectional area either in aged animals or in adult animals following axotomy (32)(33). An increase or decrease in NF content in an axon is always accompanied by an increase or decrease, respectively, in the level of NF gene expression in the neuron (32)(33)(34).

Changes in the level of NF gene expression have been correlated with changes in the size of the neuronal perikarya. For example, in the postnatal development of the dorsal root ganglion (DRG) neuron, the onset of perikaryal growth is associated with an increase in NF gene expression (35). Reductions in the size of the perikarya of atrophic DRG neurons that occur following axotomy or aging (32)(33) are correlated with a decrease in NF gene expression in these neurons. Recently, using transgenic techniques, Kong and colleagues (36) proposed that NFs also contribute to maintaining the normal dendritic arborization of motoneurons in adult animals. Taken together, these data suggest that NFs play an important role in regulating the size of neuronal perikarya and their processes (axons and dendrites). Therefore, it is reasonable to expect that age-related changes in the NF protein triplet might contribute significantly to the morphological changes and subsequent functional degradation that have been observed in sensory and motor neurons of the cat brainstem during the aging process.

Accordingly, in the present experiment, age-related alterations in the NF-M subunit in the sensory and motor systems of the cat brainstem were examined by using immunocytochemical techniques.


   Methods
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 Abstract
 Methods
Results
 Discussion
 References
 
Tissue Processing
Three adult cats (controls) and four aged cats (experimentals) were used in the present study (Table 1 ). All animals were obtained from and determined to be in good health by the University of California—Los Angeles Division of Laboratory Animal Medicine. The cats were deeply anesthetized with nembutal and perfused transcardially with 1 L of ice-cold saline followed by 2.5 L of a fixative containing 4% paraformaldehyde, 0.2% saturated picric acid, and 0.25% glutaraldehyde in 0.1 M of phosphate-buffered saline (PBS; pH 7.4). After perfusion, the brainstem was removed and postfixed overnight in a solution of 2% paraformaldehyde and 0.2% saturated picric acid in 0.1 M of PBS at 4°C. Next, the brainstem was rinsed in 20% sucrose (wt/vol) in 0.1 M of PBS at 4°C overnight. After it was frozen with dry ice, the brainstem was cut into 15-µm coronal sections with a Reichert–Jung cryostat. The sections were collected in 0.1 M of PBS and stored in a solution of 0.1 M of PBS containing 0.3% Triton X-100 and 0.1% sodium azide at 4°C for later use.


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  		Table 1. Number of Axons/Unit Area and Axonal Cross-Sectional Areas Observed in the ML and MLF of Adult and Aged Cats

 
Immunohistochemistry
All immunocytochemical procedures were carried out at room temperature, unless otherwise specified. Free-floating sections were rinsed several times in ice-cold PBST (0.1 M of PBS with 0.3% Triton X-100); they were then incubated with anti-NF-M serum (Chemicon International, CA; diluted 1:1200) in PBST solution. After overnight incubation, the sections were treated according to the protocol for the avidin-biotin peroxidase complex (ABC) method (Vectastain ABC Kit, Vector Laboratories, Burlingame, CA), which was previously described in the literature (37). Briefly, after they were rinsed four times in PBST for a total period of 30 minutes, the sections were incubated for 90 minutes in PBST containing biotinylated anti-rabbit IgG (Vector Laboratories; diluted at 1:300), followed by incubation in the ABC complex (diluted at 1:200) for 90 minutes. The color reaction was carried out by incubating the sections in 50 mM of Tris buffer (pH 7.5) containing 0.02% 3,3'-diaminobenzidine (DAB) and 0.015% H2O2 for 15–30 minutes. After the DAB reaction, sections were rinsed in PBST several times, mounted on gelatin-coated glass slides and dried in air. The sections were then dehydrated and coverslipped in Permount (Fisher Scientific, Fairlawn, NJ). An Olympus BX60 light microscope (Olympus Optical Co, Ltd, Tokyo, Japan) was used to examine and photograph the sections. In order to compare the intensity of NF-M immunoreactivity between adult and old cats, sets of sections from both groups were treated simultaneously under identical conditions.

A rabbit polyclonal antibody (Chemicon International) was used to identify the NF-M subunit in the cat brainstem. According to a previous study (38) and the data sheet supplied by Chemicon International, this antibody specifically recognizes the C-terminal 168 amino acids of the NF-M polypeptide and has no cross-reactivity with NF-L or NF-H. In addition, the specific reaction of this NF-M antibody is independent of NF protein phosphorylation. For immunostaining controls, separate sections were incubated in the absence of the primary antibody. No immunoreactivity was found in these tissue sections.

Data Analysis
Two methods were used to quantify the difference between control and aged groups with respect to NF-M-ir (NF-M-immunoreactivity). The first was a five-point rating scale in which the presence of NF-M-ir throughout the brainstem was determined visually by using the following rating scale (Table 2 ): strong (+++), moderate (++), weak (+), very weak (±), or none (-). This rating scale was chosen because, in most cases, differences in NF-M-ir between adult and aged cats were immediately apparent upon visual inspection. For the second method of data analysis, the numbers and cross-sectional areas of axons in some fiber bundles were measured by using National Institutes of Health (Bethesda, MD) Image software (version 1.61) on a Macintosh computer. In this case, two coronal sections/animal were chosen from each control and aged animal at the same brainstem levels. The images were captured at the same magnification by using a Spot CCD camera (Diagnostic Instruments, Inc, Sterling Heights, MI). The mean number and mean cross-sectional area of axons in each individual animal were calculated before group means and standard deviation (SD) were computed. Differences between these group means were then analyzed by using the unpaired Student's t test. A difference between means was considered statistically significant when p < .05.


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  		Table 2. Comparison of NF-M-ir in Sensory-Motor Nuclei of the Brainstem in Adult and Aged Cats

 

   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
With the use of a light microscope, NF-M immunoreactivities were found in many regions of both the control and the aged cat brainstem. However, the intensity of staining was different between control and aged cats in these regions. In addition, there was no obvious variability in the immunostaining between individual control animals or between individual aged animals. Therefore, in the following descriptions, we only compare the difference of NF-M-ir between control and aged animal groups (Table 2 ).

General Somatosensory System
The general somatosensory system in the brainstem includes the sensory trigeminal nuclei and the dorsal column nuclei (39). The sensory trigeminal nuclei comprise the mesencephalic sensory nucleus (Me5), the principal sensory nucleus (Pr5), and the nucleus of the spinal tract (Sp5). The Sp5 is further subdivided into three parts: oral subnucleus (Sp5O), interpolar subnucleus (Sp5I) and caudal subnucleus (Sp5C) (39). In adult cats, NF-M-ir was found throughout the sensory trigeminal nuclei; however, the intensity of staining differed among these nuclei (Table 2 and Fig. 1). Moderate to strong NF-M-ir was found in the Me5, the Sp5O, and the Sp5C (Fig. 1). Although the Me5 contained a larger number of intensely stained perikarya (Fig. 1), the NF-M antibody heavily labeled many long dendrites in the Sp5C (Fig. 1) and Sp5O. Most of the labeled dendrites were primary and secondary dendrites, but some tertiary dendrites were also stained (Fig. 1). In contrast, only weak NF-M-ir was detected in the Pr5 (Fig. 1) and the Sp5I in which the perikarya and dendrites were faintly stained. All of these nuclei exhibited much weaker staining in the old cats (Table 1 and Fig. 1). The neuronal perikarya were faintly labeled (Fig. 1, Fig. 1, and Fig. 1), and only a few dendrites were stained in the Sp5 and the Pr5 (Fig. 1 and Fig. 1).



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  		Figure 1. Photomicrographs showing NF-M immunoreactivity in the sensory trigeminal nucleus complex of an adult cat (A, C, E) and an old cat (B, D, F). Strong NF-M immunoreactivity was present in axons in the mesencephalic tract of the trigeminal nerve (MT) and in perikarya in the mesencephalic sensory nucleus (Me5) in the adult cat (A), while such staining was weaker in the old cat (B). Note that there are fewer stained axons in the MT in the old cat compared with the adult control. Many stained dendrites and some perikarya were seen in the principal sensory nucleus (Pr5) in the adult cat (C), but such structures were rarely labeled in the old cat (D). In addition, the intensity of staining also decreased in the old cat compared with the adult cat. In the adult cat, the caudal subnucleus of the nucleus of the spinal tract (Sp5C) contained many perikarya and dendrites (arrows), which showed intense NF-M immunoreactivity. Fewer stained perikarya and dendrites and weaker staining were seen in this nucleus in the old cat (F). A–F: Normarski optics. .

 
The dorsal column nuclei include the gracile nucleus (Gr), the cuneate nucleus (Cu) and the external cuneate nucleus (ECu). These nuclei exhibited weak to moderate NF-M-ir (Fig. 2 and Fig. 2) in adult cats. Immunoreactivity was present in both perikarya and dendrites in the Gr and the Cu, but only in the perikarya in the ECu. In the old cats, the Gr and the Cu contained only a few weakly stained perikarya and no labeled dendrites (Fig. 2), whereas many strongly labeled perikarya as well as some labeled dendrites were found in the ECu (Fig. 2).



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  		Figure 2. NF-M immunoreactivity in the dorsal column nuclei (A–D) and the medial lemniscus (E, F) in an adult cat (A, C, E) and an old cat (B, D, F). Many perikarya and dendrites in the gracile nucleus (Gr) were moderately stained in the adult cat (A), but only a few perikarya were stained in the old cat and the staining was very weak (B). In the external cuneate nucleus (ECu), weak to moderate staining appeared in the neuronal perikarya in the adult cat (C), but moderate to strong immunoreactivity was present in these perikarya in the old cat. The medial lemniscus (ML) contained many labeled axons in both the adult cat (E) and the old cat (F). However, the labeled axons in the old cat were smaller in size than those in the adult cat (E). C–F: Normarski optics. .

 
The mesencephalic tract of the trigeminal nerve (MT) is composed of the axons of Me5 neurons, whereas the medial lemniscus (ML) contains axons of Gr and Cu neurons as well as neurons of the Pr5 and Sp5 (39). Consequently, we were able to examine age-related changes in NF-M-ir of axons in the MT and ML in addition to immunoreactivity in the perikarya and dendrites of neurons in the sensory trigeminal nuclei and the dorsal column nuclei. In both adult and aged cats, there were many stained axons in the MT and the ML (Fig. 1, Fig. 1; Fig. 2, Fig. 2). In the ML, there was no statistically significant change in the mean number of axons between adult and old cats (7.53 ± 0.31/1000 µm2 vs 7.10 ± 0.21/1000 µm2, p > .05; Table 1 ). However, there was a statistically significant reduction in the mean cross-sectional area of the axons in the aged cats compared with those in the control animals (18.07 ± 2.54 µm2 vs 37.38 ± 2.80 µm2, p < .0001; Table 1 , Fig. 3). This indicates that the axons in the ML became smaller in the old cats.



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  		Figure 3. Frequency distributions of the cross-sectional areas of axons in the medial lemniscus (ML; A) and the medial longitudinal fasciculus (MLF; B) in both adult cats and aged cats. Note that in both ML and MLF, the histograms of cross-sectional areas shift to the left, indicating that the mean size of axons is smaller in aged cats. Data are shown as mean ± SD.

 
Auditory System
The auditory system is one of the brainstem special somatosensory systems; the other is the vestibular nucleus complex. At the level of the brainstem, the auditory system includes the cochlear nucleus (Co; Fig. 4), the superior olivery complex (SOC; Fig. 5), the nucleus of the lateral lemniscus (NLL), and the inferior colliculus (IC; Fig. 6). Each of these complexes of nuclei has several subnuclei or subregions ((39); Table 2 ). The nuclei in the brainstem auditory system in adult cats exhibited only weak or very weak NF-M-ir (Fig. 4 and Fig. 4; Fig. 5 and Fig. 5; Fig. 6 and Fig. 6). In addition, although both perikarya and dendrites were labeled in most nuclei of the general somatosensory system, only perikarya were stained in the nuclei of the auditory system, with the single exception of the medial superior olive (MSO), which contained a few faintly labeled dendrites (Fig. 5).



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  		Figure 4. Photomicrographs illustrating NF-M immunoreactivity in the dorsal cochlear nucleus (DCo; A, B; Normarski optics) and the anterior ventral cochlear nucleus (AVCo; C, D) in an adult cat (A, C) and an old cat (B, D). There were a few faintly stained perikarya in these two nuclei in the adult cat, but many labeled perikarya with heavy staining were found in the old cat. Note the labeling of some primary dendrites in the dorsal cochlear nucleus of the old cat (arrows in B). 1, molecular layer; 2, pyramidal layer; 3, polymorph layer.

 


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  		Figure 5. Photomicrographs showing NF-M immunoreactivity in the medial superior olive (MSO) (A, B) and the nucleus of the trapezoid body (MTz) (C, D) in an adult cat (A, C) and an old cat (B, D). In the MSO, staining was weak in the adult cat (A), but moderate to strong in the old cat (B). This increase in immunoreactivity was found in both the perikarya and the dendrites. In the MTz, NF-M immunoreactivity was also increased in the perikarya of neurons in the old cat (D). In contrast to the perikarya, fewer labeled axons, which proceed transversely through the MTz, were observed in the old cat than were present in the adult cat. A–D: Normarski optics. .

 


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  		Figure 6. NF-M immunoreactivity in the nucleus of the lateral lemniscus (NLL; A, B; Normarski optics) and the central nucleus of the inferior colliculus (IC; C, D) of an adult cat (A, C) and an old cat (B, D). In the adult cat, a few very weakly stained perikarya were found in these two nuclei, but there were some stained axons in the lateral lemniscus (LL; A). In the old cat, both nuclei exhibited a marked increase in the number of labeled perikarya as well as an increase in the intensity of the labeling (B, D). However, there were very few labeled axons in the LL of the old cat (B). .

 
In contrast to adult cats, in the aged animals most of the perikarya were moderately or strongly stained in the nuclei of the brainstem auditory system (Fig. 4 and Fig. 4; Fig. 5 and Fig. 5; Fig. 6 and Fig. 6). In addition to staining in the perikarya, NF-M-ir also appeared in the basal portion of primary dendrites in the dorsal cochlear nucleus (DCo; Fig. 4) and the MSO (Fig. 5). Therefore, in the old cats, NF-M-ir in the perikarya and primary dendrites increased in the nuclei of the auditory system.

Vestibular Nucleus Complex
There are several brainstem nuclei in the vestibular nucleus complex (39). These nuclei are, from the rostral to the caudal levels of the brainstem, the superior vestibular nucleus (SuVe), the lateral vestibular nucleus (LVe), the medial vestibular nucleus (MVe), and the spinal vestibular nucleus (SpVe). In adult cats, moderate NF-M-ir was present in the dorsal division of the LVe (DLVe; Fig. 7). Immunoreactivity in this division was observed in the perikarya, the basal portion of a few dendrites, and in some axons of passage. In contrast to the DLVe, all other nuclei in the vestibular nucleus complex exhibited very weak or weak NF-M-ir (Table 2 and Fig. 7).



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  		Figure 7. Photomicrographs showing NF-M immunoreactivity in an adult cat (A, C, E) and an old cat (B, D, F). In the adult cat, the dorsal division of the lateral vestibular nucleus (DLVe) contained many moderately stained perikarya (A). The intensity of staining in the perikarya increased in the old cat (B). In the superior vestibular nucleus (SuVe), very few perikarya with faint NF-M immunoreactivity were present in the adult cats (C), but this nucleus contained many perikarya with moderate staining in the old cat (D). In the adult cat, the medial longitudinal fasciculi (MLF) contained many heavily stained axons (E). However, the staining of the axons in the old cat was weaker than that in the adult cat (F). In addition, in the old cat, most of the labeled axons were smaller than those in the adult cat. A–D: Normarski optics. .

 
Compared with the adult cats, the old cats exhibited increased NF-M-ir in the DLVe (Fig. 7). Most neuronal perikarya and the basal portion of primary dendrites in this nucleus were strongly stained. In a fashion similar to that of the DLVe, the rest of the vestibular nucleus complex also exhibited stronger NF-M-ir in the old cats (Fig. 7). This increase in NF-M-ir occurred in many perikarya and a few primary dendrites in these nuclei (Fig. 7).

Fibers from all vestibular nuclei enter the medial longitudinal fasciculus (MLF; Parent, 1996). As in the case of the ML, there was no statistically significant difference in the mean number of axons within the MLF of adult and old cats (0.55 ± 0.14/100 µm2 vs 0.51 ± 0.15/100 µm2, p > .05, Table 1 ). However, there was a statistically significant reduction in the mean cross-sectional areas of the axons adult versus old cats (28.92 ± 13.82 µm2 vs 15.42 ± 11.18 µm2, p < .0001; Table 1 , Fig. 3).

Somatomotor and Branchimotor Systems
The somatomotor system of the brainstem is composed of the abducens nucleus and the hypoglossal nucleus, whereas the nuclei in the branchimotor system are the motor trigeminal nucleus (Mo5), the facial nucleus, and the nucleus ambiguus (39). In adult cats, NF-M-ir was very weak in the perikarya of all of these motor nuclei, except for the facial motor nucleus (Table 2 and Fig. 8, Fig. 8, and Fig. 8). During aging, NF-M-ir increased in the perikarya of motoneurons. In aged cats, the staining in the perikarya ranged from weak to moderate, depending on the nucleus (Fig. 8, Fig. 8, and Fig. 8). In contrast, in adult cats, within the brainstem the 5th, 6th, and 12th (Fig. 8) cranial nerves contained many heavily stained axons. In contrast, only a few axons in these cranial nerves of old cat were labeled and the intensity of the labeling was reduced (Fig. 8).



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  		Figure 8. Photomicrographs showing NF-M immunoreactivity in the somatomotor and branchimotor nuclei (A, B: motor trigeminal nucleus, 5; C, D: abducens nucleus, 6; E, F: hypoglossal nucleus, 12) and the hypoglossal nerve (12N; G, H) in an adult cat (A, C, E, G) and an old cat (B, D, F, H). In these motor nuclei, neuronal perikarya exhibited very weak to weak NF-M immunoreactivity in the adult cat, but moderate to strong staining were seen in the old cat. In contrast, although many axons were intensively stained in the hypoglossal nerve of the adult cat (G), only a few of these axons were labeled in the old cat. In addition, the intensity of the labeling decreased dramatically in the old cat (H). A–D, G and H: Normarski optics. ).

 
The age-related changes in the NF-M-ir of the facial nucleus were different from those observed in other somatomotor and branchimotor nuclei in the brainstem. In adult cats, both the perikarya and the dendrites of motoneurons were moderately labeled in the facial motor nucleus (Fig. 9), whereas the axons of these motoneurons in the facial nerve were intensely stained (Fig. 9 and Fig. 9). However, in old cats, only weakly stained perikarya and no labeled dendrites were found in the facial nucleus (Fig. 9). In the facial nerve, although there were still many stained axons, most of these axons appeared to be smaller than those observed in adult cats (Fig. 9).



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  		Figure 9. Photomicrographs showing changes in NF-M immunoreactivity in the motoneurons of the facial nucleus (7) and their axons with aging. A, B (Normarski optics): motoneurons in the facial nucleus of an adult (A) and an old (B) cat. Both the perikarya and the dendrites were intensely stained in the adult cat, but they were only weakly stained in the old cat; C, D: the genus of the facial nerve (7G) of an adult cat (C) and an old cat (D); E, F: enlargement of the labeled fibers in C and D, respectively. In the adult cat, there were many intensely stained axons in the facial nerve; however, the staining of axons in this nerve was weaker in the old cat. In addition, the size of the labeled axons in the facial nerve was also reduced in the old cat. (A, B); 50 µm (C, D); 10 µm (E, F).

 
Viscerosensory and Visceromotor Systems
In the brainstem, the nucleus of the solitary tract (So) receives both general and special visceral afferents, whereas the dorsal motor nucleus of the vagus nerve (DMV) gives rise to general visceral efferent fibers (39). In both adult and old cats, the So showed very weak NF-M-ir (Table 1 ). However, the DMV showed a slight increase in NF-M-ir in the perikarya, which ranged from very weak to weak in old animals (Table 2 ).


   Discussion
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 Abstract
 Methods
 Results
 Discussion
References
 
Nucleus-Specific Alterations of NF-M-ir in the Sensory and Motor Nuclei of the Cat Brainstem with Aging
In the present study, NF-M-ir in the brainstem sensory and motor systems of adult cats was compared with that in the same systems of old cats. A marked decrease was observed in NF-M-ir in most nuclei of the general somatosensory system (the sensory trigeminal nuclei, the Gr, and the Cu) and the facial motor nucleus with aging. The perikarya and dendrites of neurons in these nuclei were intensively stained in adult cats, but the staining was weak or very weak in aged cats.

These results suggest that the amount of NF-M subunit declines in the perikarya and dendrites of these neurons with aging. Recent studies have also indicated that there is an age-related decrease in NF-M-ir in the cerebellum (40) and in NF-M content in the perikarya and axons of DRG neurons in the rat (33). In DRG neurons, the level of NF-M gene expression declines with aging (33). Similarly, NF-M gene expression may also be reduced in the neurons that show an age-related decrease in NF-M-ir in the present experiment.

Using the Golgi method, Machado-Salas and colleagues (16) found many age-related degenerative morphological changes in the sensory trigeminal nuclei and the reticular formation in the brainstem of mice. These atrophic changes occurred in all portions of the neuron, including the perikarya (changes in size and contour), dendrites (swelling, constriction, and fragmentation), and axons (shriveling of the initial portion). It is noteworthy that in both the sensory trigeminal nuclei and the reticular formation of the cat, marked decreases were observed in NF-M-ir in all neuronal compartments in the old cats (17).

In contrast to the nuclei described above, the ECu of the general somatosensory system and nuclei of the auditory system, the vestibular nucleus complex, the visceromotor system, and somatomotor nuclei (which include the Mo5, the abducens nucleus, the hypoglossal nucleus and the nucleus ambiguus) showed a marked increase in the NF-M-ir in neuronal perikarya and the basal parts of primary dendrites with aging. This increase may indicate the accumulation of the NF-M subunit in both the perikarya and the basal parts of primary dendrites.

A similar age-dependent perikaryal accumulation of NF-M was observed by Vickers and colleagues (41) in the pyramidal neurons of the CA1 regions of the human hippocampus. In addition, they reported that both NF-L-ir and NF-H-ir show the same levels of accumulation that is exhibited by NF-M-ir in the hippocampus during aging (41). Furthermore, it has been shown that the three NF subunits colocalize in most neurons of the central and the peripheral nervous systems (42)(43)(44); they always exhibit the same age-related changes in the regions that have been examined thus far (e.g., an increase in the hippocampus and a decrease in the DRG and the cerebellum; 33, 40, 41). Therefore, it is likely that the NF-L and NF-H subunits also accumulate in the perikarya of neurons that we found to exhibit elevated NF-M-ir in the sensory and motor nuclei of old cats.

At the present time, the reasons for the accumulation of NF-M in these perikarya remain unknown. In the mature neuron, the three NF subunits, each encoded by its own gene, are synthesized and then assembled into NF polymers in the perikarya (20)(34). NFs are subsequently transported into the axon and dendrites. Within the axon, NFs travel to the terminals by means of slow axonal transport (24)(25). Little degradation of NFs occurs until they reach the axon terminals, where NFs are rapidly catalyzed by a variety of proteases, particularly those activated by calcium (26)(27). Therefore, several factors can contribute to the accumulation of NF-M in the perikarya of the sensory and motor neurons that have been observed in the present study, such as (1) a diminished ability of the neuronal axon to transport the NF protein, (2) an increase in the synthesis of the NF protein, and, less likely, (3) a lack of degradation of NF protein in the terminals.

It has been shown that NF accumulation in the perikarya and proximal axons is characteristic of many motoneuron diseases such as human amyotrophic lateral sclerosis and accelerated hereditary canine spinal muscular atrophy. In both of these diseases, there is no evidence of an overproduction of NF protein; instead, NF protein synthesis in the neurons remains unchanged in amyotrophic lateral sclerosis (45) but decreases in hereditary canine spinal muscular atrophy (46)(47). However, slow axonal transport in the sciatic nerve is severely retarded in these diseases (21)(48). Therefore, impairment of NF protein transport appears to be one of the major causes of NF accumulation in the perikarya. Similar reasons may also account for the age-related accumulation of NF-M in the perikarya observed in the present study because (1) an age-related retardation of slow axonal transport has been observed in many fiber systems (49), including motor fibers (sciatic nerve; 50,51) and sensory fibers (dorsal root; 52); (2) only a decrease in NF protein synthesis has so far been detected in the nervous system during aging (33)(40)(41); and (3) a decrease in NF degradation would most likely cause NFs to accumulate in the axons (53), which is contrary to the observation of the present study.

Age-related Decrease in NF-M-ir in the Axons of Brainstem Sensory and Motor Neurons
As discussed above, during aging, the NF-M-ir in neuronal perikarya decreased in the sensory trigeminal nuclei, the Gr, and the Cu, while it increased in the vestibular nuclei. It has been well established that axons from neurons in the sensory trigeminal nuclei, the Gr, and the Cu form the ML, whereas neurons in all vestibular nuclei send their axons to the MLF (39). In the present experiment, we have found that although there were no statistically significant changes in the mean number of axons per unit area in either the ML or the MLF between the control and the aged cats, there were statistically significant reductions in the mean cross-sectional areas of the axons within these fiber bundles. This indicates that the size of the axons in these fiber bundles decreases with aging. The reason for the reduction in axonal size is unclear at present. However, two possibilities should be considered. First, because NFs are the major determinant of axonal caliber (32)(35), the observed decrease in axon size may be due to a decline in the number of NFs within the axons caused by either an age-related decrease in NF protein synthesis (32)(33)(40)(41) or by the retardation of slow axonal transport in aged animals (18)(49)(50)(51)(52). A decline in NF number may also explain why axonal size in both the ML and MLF decreases in aged cats, whereas the neurons contributing axons to these fiber bundles show different changes in NF-M-ir in their soma. Second, recent studies have shown that NF-M may contribute to the spacing between the NFs (19)(20)(21). Therefore, a reduction of NF-M only within the axon suggests that there may be a more compact filamentous network within the axon that could be either a predisposing factor for, or the result of, the decreased diameter of the axons. A similar decrease in the NF number may also be present in the axons of neurons in the cranial motor nucleus, because most axons in the cranial nerve (5th, 6th, 7th, and 12th) were obviously smaller in aged cats than in control cats. In addition, in a previous study we observed a statistically significant decrease in mean axon diameter in masseter nerves in old cats compared with the mean diameter observed in the masseter nerves of adult cats (7).

In previous experiments, we have found that the conduction velocity of motor nerves from both the brainstem and the spinal cord decreases significantly during aging (6)(7)(8)(9)(10)(11)(12). In addition, a decrease in motor axon conduction velocity has been consistently reported in aged humans (54)(55). One of the major causes for this reduction in conduction velocity is likely to be a decrease in axon diameter, because it is well known that axon diameter is one of the key factors that determine the velocity of conduction in axons (6)(7)(8)(9)(10)(11)(12). Therefore, it is reasonable to postulate that a similar reduction in conduction velocity may occur in axons of brainstem sensory and motor neurons. In summary, a common pattern appears to emerge throughout the sensory and motor systems that we have examined, which consists of a reduction both in axonal caliber and conduction velocity in old age.


   Acknowledgments
 
We thank Dr. J. K. Engelhardt for critically reading an earlier version of this manuscript. We also thank Ms. N. Vokshori for helping us with computer imaging. This study was funded by USPHS Grant AG 04307.

Received January 5, 1999

Accepted September 21, 1999


   References
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 Abstract
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 References
 

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