We hypothesized that there would be a decrease in the number of hypoglossal motoneurons and a reduction in neuronal size and the number of primary dendrites with increasing age. However, we found that while neuronal number and size remained stable throughout life in F344/BN rats, the number of primary dendrites was significantly reduced in the old animals. These data suggest that, unlike spinal motoneurons which show age-associated changes in neuronal number and soma size, hypoglossal motoneurons appear to be less vulnerable to these morphological changes. Nonetheless, fewer primary dendrites reduces the surface area available for synaptic input, and could lead to changes in function.
Our data are consistent with those of Sturrock [42
] who found no age-associated change in the number of hypoglossal motoneurons or motoneuron size in mice. Similarly, in a study of human patients with Parkinson disease, the control population (aged 61–88 years) did not show any loss of hypoglossal motoneurons [50
]. Sturrock [42
] hypothesized that the fine movements in which the tongue is involved on a daily basis during respiration, speech, and swallowing could account for the stable number of hypoglossal motoneurons. Sturrock also noted that the number of motoneurons in nuclei innervating the extra-ocular muscles, which are also activated continuously, do not decline with age [39
]. Sustained neuromuscular activity may upregulate neurotrophins that are involved in motoneuron survival. In the spinal cord, for example, neuromuscular activity can upregulate neurotrophin expression in motoneurons as well as in the muscles they innervate, and it has been suggested that the exercise-induced neurotrophins synthesized in muscle are transported in retrograde to spinal motoneurons [51
]. Consequently, a reduction in spontaneous locomotor activity that is frequently associated with aging may deprive spinal motoneurons of critical trophic support for survival, whereas tonic activity in select cranial motoneurons may enhance their survival.
The tongue is critically involved in breathing, and genioglossal motoneurons discharge rhythmically during each inspiratory phase of respiration [10
]. Thus, hypoglossal motoneurons may have unique properties that enable them to function optimally throughout life and resist the affects of aging and disease. In the SOD1 G93A transgenic rat model of amyotrophic lateral sclerosis (ALS), at a disease stage where there was considerable loss of spinal and phrenic motoneurons, hypoglossal motoneuron cell counts were relatively normal [52
]. In human patients with ALS, loss of phrenic motoneurons innervating the diaphragm with muscle atrophy and subsequent respiratory failure is the usual cause of death [54
Previous studies of lumbar motoneuron size in F344 rats showed a size increase with age, and the authors suggested that this effect may be an adaptive mechanism to accommodate the increased amount of lipofuscin that accumulates in neurons as they age [33
]. Our data suggest that larger motoneurons (>22.5 μm diameter) in the hypoglossal nucleus tend to increase slightly in size with age, although this was not statistically significant (). Our data also show that more large neurons are located in the rostrally than in the middle or caudal parts of the hypoglossal nucleus, which may reflect the topographic organization of neurons that innervate specific muscle of the tongue [6
It is possible that additional age-related changes occur in the hypoglossal nucleus, but that the experimental design we used was unable to detect them. Tongue injections of CTβ selectively labeled motoneurons innervating the primary protrudor muscle of the tongue, which is critical to respiration, but not retractor muscle motoneurons, which are involved in swallowing [55
]. Both protrudor and retractor muscles are involved in speech, mastication, and licking [55
]. As protrudor and retractor neuronal populations are approximately equal, any loss of neurons innervating retractor tongue muscles would have been detected. However, changes in neuronal cell size associated with retractor neurons would not have been detected in this study.
Although no age-associated changes in the number of genioglossal motoneurons were detected, it is possible that interneurons in the hypoglossal nucleus are selectively affected by aging [31
]. Interneurons were included in our neuronal counts, but they were excluded in measurements of neuronal size and dendrite number. Approximately 13% of neurons in the hypoglossal nucleus are interneurons [42
], thus it is unlikely that we would have detected any morphologic changes in this small population.
The number of primary dendrites associated with retrogradely-labeled genioglossal motoneurons increased significantly from young to middle-age and decreased from middle-age to old in the present study. Interestingly, the number of dendrites associated with genioglossal motoneurons in cats was reported to remain constant during development [58
]. Loss of primary dendrites in hypoglossal motoneurons with increasing age may contribute to the changes in tongue function that have been reported in older human and rats [48
]. Hypoglossal motoneurons receive synaptic inputs from premotor neurons in several brainstem nuclei [55
]. Premotor neurons help to orchestrate the complex interactions between concurrent orofacial behaviors, and it is likely that a reduction in their synaptic input to hypoglossal motoneurons, particularly if there are fewer primary dendrites, contributes to alterations in speech, swallowing, and upper-airway control seen in the elderly. Age-related synaptic loss has been described in a number of brain regions [62
] as well as in spinal motoneurons in rats [63
]. Neuromodulatory systems, including acetylcholine, norepinephrine, and serotonin, are also affected by aging, and previous studies in our laboratory have shown an age-associated decrease in serotonergic synaptic input to the hypoglossal nucleus in the F344 rat strain [45
]. In addition to the reduction in the number of primary dendrites from middle-aged to old in rats, it is possible that the morphology of more distal dendrites also changes with age. Studies in primates and rodents have described pruning of dendritic arbors with age in pyramidal neurons in the cerebral cortex (for a review, see [59
]), although proliferation of dendritic branches has been described in motoneurons supplying the intrinsic muscles of the foot in old cats [60
Despite no changes in the number or diameter of hypoglossal motoneurons, there remains the possibility that axons of hypoglossal nerves degenerate or demyelinate with increasing age, which could result in decreased tongue strength and impaired function [24
]. However, in a recent study of human hypoglossal nerve, the total number and diameter of myelinated fibers did not change when adult (<60 yrs) and elderly (>60 yrs) individuals were compared [61
]. Receptors for select neurotransmitters and neuromodulators on hypoglossal motoneurons may also show age-associated changes that could contribute to altered function [47
]. Age-associated changes in neurotrophic factors could impact the efficacy of synaptic transmission in hypoglossal neurons, as has been seen in the hippocampus [64
]. Johnson et al. [43
] described a decreased expression of neurotrophin receptors in spinal motoneurons of aged rats. Several lines of evidence suggest that oxidative stress and mitochondrial dysfunction play a major role in sarcopenia [65
], and in a recent study of human skeletal muscle, the transcriptome profile for many mitochondrial genes was shown to be significantly altered with aging [66
]. In summary, cranial neuromuscular dysfunction may result from a combination of factors that involves both the central and peripheral nervous system, the neuromuscular junction, and muscle cells.
Recently, progressive resistance training of the tongue for 8 weeks has been shown to increase maximal isometric pressure as well as swallowing pressures in the elderly [67
], suggesting that a decline in cranial neuromuscular function can be partially reversed. Similar positive effects of exercise on skeletal neuromuscular function in the elderly have been reported, although the underlying mechanism(s) are not clear [68
]. Muscle activity has been shown to enhance the expression of muscle-derived neurotrophins at the neuromuscular junction [71
], alter the regulation of central serotonergic activity [73
], and increase nerve terminal branching at the neuromuscular junction [74
]. Exercise can enhance the expression of anti-apoptotic markers in aging muscle (for a review, see [75
]). Additionally, the signature mitochondrial transcriptome profile of aged vs. young humans is substantially reversed following 6 months of resistance-exercise training in skeletal muscle [66
], suggesting that some of the beneficial effects of exercise are associated with energy balance within the muscle fiber itself. Thus, interventions aimed at reversing the effects of aging on orofacial behaviors, including speech, swallowing, and breathing, may need to be targeted to multiple sites in the cranial neuromuscular system.