Robert H. Helfert

Regional induction of fos immunoreactivity in the brain by anticonvulsant stimulation of the vagus nerve

Electrical stimulation of the vagus nerve exerts an antiepileptic effect on human partial-onset epilepsy, but little is known about the brain structures that mediate this phenomenon. Fos is a nuclear protein that is expressed under conditions of high neuronal activity. We utilized fos immunolabeling techniques on Sprague-Dawley rat brains to identify regions that are activated by antiepileptic stimulation of the left vagus nerve. Vagus nerve stimulation (VNS) induced specific nuclear fos immunolabeling in several forebrain structures, including the posterior cortical amygdaloid nucleus, cingulate and retrosplenial cortex, ventromedial and arcuate hypothalamic nuclei. In the brainstem, there was specific immunolabeling in vagus nerve nuclei, in the A5 and locus ceruleus noradrenergic nuclei, and in the cochlear nucleus. No labeling of these structures occurred in sham-operated, unstimulated control animals. Intense labeling also occurred in habenular nucleus of thalamus after vagus nerve stimulation, whereas only mild staining occurred in unstimulated animals. Several of the brain structures activated by VNS are important for genesis or regulation of seizures in the forebrain. These structures may mediate the antiepileptic effect of VNS.

Central auditory aging: GABA changes in the inferior colliculus

Age-related hearing loss (presbycusis) is a complex state that reflects pathologic changes along the entire auditory neuraxis. Loss of speech understanding, decreased ability to localize sounds, and a decreased ability to detect and extract signals in noise are characteristic problems encountered by the elderly. Central (neural) presbycusis frequently results in a dramatic loss in speech understanding without a parallel change in pure-tone thresholds. In spite of evidence that suggests these deficits cannot be fully explained by peripheral changes alone, few studies have examined the neurochemical basis of central auditory dysfunction in aging. Age-related alterations in neural circuits involved in the processing of acoustic information could reflect changes in the synthesis, degradation, uptake, release, and receptor sensitivity of neurotransmitters, perhaps secondary to cell loss and/or progressive deafferentation. A series of studies designed to test this hypothesis has examined aging in the central auditory system of the F344 rate. Age-related changes associated with GABA neurotransmitter function in an important auditory midbrain structure, the inferior colliculus, have been investigated. These studies found: (1) decreased numbers of GABA immunoreactive neurons; (2) decreased basal levels (concentrations) of GABA; (3) decreased GABA release; (4) decreased glutamic acid decarboxylase activity; (5) decreased GABAB receptor binding; (6) decreased numbers of presynaptic terminals; and (7) subtle GABAA receptor binding changes. Collectively, these age-related changes suggest altered GABA neurotransmitter function in the IC. Identification of specific neurotransmitter changes in structures important in speech processing could eventually lead to the development of pharmacotherapy for selective types of age-related hearing loss.

Balance and aging

Objectives/Hypothesis: To provide a basic science and clinical review of normal balance changes with age, and to provide a current review for the evaluation and treatment of elderly patients with balance disorders. As we age, we lose balance function through loss of sensory elements, the ability to integrate information and issue motor commands, and because we lose musculoskeletal function. Diseases common in aging populations lead to further deterioration in balance function in some patients. Treatment of balance dysfunction in aging populations is based on the knowledge of normal aging processes and on an evaluation of the individuals balance loss and remaining balance elements. Prevention and rehabilitation play a major role in treatment; medical and surgical therapy also have a place.

Age-related synaptic changes in the central nucleus of the inferior colliculus of Fischer-344 rats

The central nucleus of the inferior colliculus (ICc) is a major processing center for the ascending auditory pathways. Gamma‐aminobutyric acid (GABA) and excitant amino acids (EAAs) are essential for coding many auditory tasks in the IC. Recently, a number of neurochemical and immunocytochemical studies have suggested an age‐related decline in GABAergic inhibition in the ICc, and possibly excitant‐amino‐acid‐mediated excitation as well. The objective of this study was to compare quantitatively changes in the synaptic organization of the ICc among three age groups (3, 19, and 28 months) of Fischer‐344 rats. Immunogold electron microscopic methods were used to determine if there were age‐related changes in the density, distribution, or morphology of GABA‐immunoreactive (+) and GABA‐immunonegative (–) synapses in the ICc. The data suggest similar losses of excitatory and inhibitory synapses in the ICc. There were significant reductions in the densities of GABA+ and GABA– synaptic terminals (∼30% and ∼24%, respectively) and synapses (∼33% and ∼26%, respectively) in the ICc of 28‐month‐old rats relative to 3‐month‐olds. The numeric values, which were adjusted to consider changes in volume of the IC with age, depict similar effects, although the effect magnitude for the adjusted values was reduced by approximately 9%. For both types of synapses, the decreases did not differ significantly from each other. The reductions in synaptic numbers appeared to be related to a similar numeric decline in dendrites, in particular those with calibers of between 0.5 and 1.5 μm. The number and distribution of synaptic terminals on the remaining dendrites of GABA– neurons appeared not to undergo major age‐related changes. GABA+ neurons, on the other hand, may have evolved patterns of synaptic and dendritic change during aging in which the distribution of synaptic terminals shifts to dendrites of larger caliber. In the 19‐month group, the synaptic areas were elevated in terminals apposed to dendrites with calibers of 1.5 μm or less. However, this increase in synaptic size did not persist in the aged animals. No neuronal losses were detectable among the three age groups. Thus, the decrease in GABA and EAAs identified in the IC by previous studies may be attributable to synaptic and dendritic declines, rather than cell loss. J. Comp. Neurol. 406:285–298, 1999.

Three classes of inhibitory amino acid terminals in the cochlear nucleus of the guinea pig

Electron microscopic postembedding immunocytochemistry was used to analyze and assess the synaptic distribution of glycine (GLY) and γ‐amino butyric acid (GABA) immunoreactivities in the guinea pig cochlear nucleus (CN). Three classes of endings were identified containing immunolabeling for glycine, GABA, or both glycine and GABA (GLY/GABA). All classes were similar in that the terminals contained pleomorphic vesicles and formed symmetric synapses with their postsynaptic targets. A fourth class, which labeled with neither antibody, contained round vesicles and formed asymmetric synapses. Glycine endings predominated in the ventral CN, while GLY/GABA endings were prevalent in the dorsal CN. GABA endings were the least common and smallest in size. Glycine, GLY/GABA, and GABA endings differed in their proportions and patterns of distribution on the different classes of projection neurons in the CN, including spherical bushy, type I stellate/multipolar, and octopus cells in the ventral CN and fusiform cells in the dorsal CN. The vast majority of anatomically‐defined, putative inhibitory endings contain GLY, GABA, or both, suggesting that most of the inhibition in the cochlear nucleus is mediated by these three cytochemically and, probably, functionally distinct classes of endings. The results of this study also suggest that a large proportion of the GABA available for inhibition in the CN coexists in terminals with glycine.

Age‐Related Glycine Receptor Subunit Changes in the Cochlear Nucleus of Fischer‐344 Rats

Previous studies have shown that levels of binding for the strychnine‐sensitive glycine receptor in the cochlear nucleus (CN) of Fischer(F344) rats decrease with age. Given the major role glycine plays in normal CN function, changes in glycine‐receptor activity may contribute to central presbycusis. To further evaluate the impact of age on glycine receptors, in situ hybridization was used to assess, in three age groups of F344 rats, changes in levels of gene expression for four of its subunits. When compared with the 3‐month‐old rats, expression of mRNAs for α1 and β subunits in the anteroventral CN decreased significantly in the 18‐ and 27‐month‐old age groups, while mRNA expression for the α2 subunit increased. If protein expressions are similar, these subunit changes may alter the function of glycine receptors, thereby affecting binding to its ligands.

Deafness induced cell size changes in rostral AVCN of the guinea pig

The right cochleae of 250-350 g guinea pigs were lesioned by topical administration of neomycin in the middle ear cavity. Eight weeks after the lesion, the cochleae and cochlear nuclei were analyzed. Cochlear hair cell loss was assessed, and cell areas of spherical bushy cells in the rostral anteroventral cochlear nucleus (AVCN) were compared between the lesioned and normal hearing sides for each animal. In five animals with both inner and outer hair cell loss in the lesioned cochlea, the average area of neuronal somata in the rostral AVCN in the lesioned side was 22% smaller than the average area of these cells in the normal hearing side. In two animals with outer hair cell loss but inner hair cells remaining, there was no difference in cell size between the lesioned and non-lesioned AVCN. These results provide evidence that there is significant shrinkage in AVCN cell size in the mature mammal after hearing loss associated with inner hair cell loss.

GABA and Glycine Inputs Control Discharge Rate within the Excitatory Response Area of Primary-Like and Phase-Locked AVCN Neurons

The studies presented in this chapter were designed to identify potential functions for noncochlear inputs onto spherical bushy cells in the anteroventral cochlear nucleus (AVCN). Compounds related to glycine and gamma aminobutyric acid (GABA) were iontophoretically applied in a number of different acoustic paradigms.

Age-related reductions in the activities of antioxidant enzymes in the rat inferior colliculus.

The inferior colliculus (IC) is a major relay and processing center of auditory signals in the midbrain and receives inputs from most other auditory nuclei. A number of studies have indicated age-related declines in the GABAergic and excitatory amino acid systems in the IC, including losses in both GABA immunoreactive (+) and GABA immunonegative (-) synapses. The goal of this project was to identify potential biochemical and morphological changes in the IC that may contribute to deficits in the functions of these neurotransmitters, using three age groups of Fischer-344 rats. Homogenates obtained from the IC showed age-dependent reductions in activities of the antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), with a concomitant increase in lipid peroxidation. Dephosphorylation of IC homogenates with alkaline phosphatase reduced the activities of SOD and CAT in all age groups, which could be restored by protein kinase C (PKC)-dependent phosphorylation. Restoration of enzyme activity was specific to the PKC-alpha isozyme, but not to the beta1, beta2, delta or gamma forms. No age-dependent change in the levels of PKC isoforms (alpha, beta1, beta2 and gamma) was detectable in IC homogenates. Morphological analyses indicate decreases in mitochondrial density in the somata of both GABA+ and GABA- IC neurons in 19- and 28-month-old rats when compared to 3-month-olds, along with significantly higher matricial abnormalities. These data indicate age-related increases in oxidative stress in the IC, which could be partially restored by PKC. The progressive increase in oxidative stress with age may underlie changes in neuronal morphology and function of the IC.

Age-related changes in levels of tyrosine kinase B receptor and fibroblast growth factor receptor 2 in the rat inferior colliculus : Implications for neural senescence

Brain-derived neurotrophic factor and fibroblast growth factor 2, and their respective binding sites, tyrosine kinase B receptor and fibroblast growth factor receptor 2, are known to regulate neurite outgrowth and antioxidant enzyme activity. Several studies suggest that brain-derived neurotrophic factor and fibroblast growth factor are contained in the inferior colliculus. Previous work in our laboratories revealed dendritic and synaptic losses in the inferior colliculus of aged Fischer-344 rats, along with coincident increases in lipid peroxidation possibly linked to a decrease in activity of antioxidant enzymes. In an effort to identify potential causal mechanisms underlying age-related synaptic and dendritic losses that occur in the inferior colliculus, the present study attempted to determine if inferior colliculus levels of tyrosine kinase B receptor and fibroblast growth factor receptor 2 expression are altered with age. Immunocytochemistry was performed in the inferior colliculus, hippocampus and cerebellum of 3-month-old F344 rats to study distributions of the full-length and truncated isoforms of tyrosine kinase B receptor, and fibroblast growth factor receptor 2. The latter two brain regions served as positive controls. For all three antigens, immunolabeling was localized primarily in somata and proximal dendrites in all subdivisions of the inferior colliculus, and in the dentate gyrus and Ammons horn of the hippocampus. In the cerebellum, the somata and dendrites of the Purkinje cells were also immunolabeled.A significant reduction in levels of the full-length form of tyrosine kinase B receptor in 18- and 25-month-old rats (respectively, approximately 20% and 30% relative to 3-month-olds) was revealed using western blot analyses. Inferior colliculus and hippocampal levels of the truncated form were modestly decreased ( approximately 7%) as well in the two older age groups. In contrast, levels of fibroblast growth factor receptor 2 in the inferior colliculus and hippocampus were elevated by approximately 35% in the two older age groups when compared to 3-month-olds. Changes in cerebellar levels of tyrosine kinase B receptor and fibroblast growth factor receptor 2, while similar to those in the inferior colliculus and hippocampus among the age groups, did not achieve statistical significance in this study. These findings give rise to the possibility that age-related reductions in tyrosine kinase B receptor levels could be a causal factor in the degenerative changes observed in the inferior colliculus of aged animals, including mitochondrial damage and dendritic regression. The observed increases in fibroblast growth factor receptor 2 levels may be compensatory to the increased oxidative stress. The effectiveness of the fibroblast growth factor receptor 2 response is questionable given the damage that occurs in the inferior colliculus and hippocampus of aged animals. However, the deficits could worsen in the absence of an increase in fibroblast growth factor receptor 2.