This study addresses several potential biological bases for selective cholinergic dysfunction in the aging rat brain. The project is designed to evaluate alterations in the level of expression of candidate plasticity genes in two major types of cholinergic neuronal systems, those in the basal forebrain and those in the upper brainstem reticular formation, in the behaviorally characterized Long-Evans rat. Our previous studies have implicated the septo-hippocampal cholinergic system in spatial memory impairment in this strain of rat. The upper brainstem contains cholinergic groups important in memory, but which do not degenerate in Alzheimer's disease. These latter cell groups differ from the rostral cholinergic groups by their expression of the neural type of nitric oxide synthase (b-NOS) and their independence of NGF. Since the free radical nitric oxide is implicated in some types of neurodegeneration and because some NOS-positive neurons appear to survive in certain neurodegenerative diseases, we hypothesize that the brainstem cholinergic system will exhibit a profile of gene expression responses in aging that will differ from that of the rostral, basal forebrain cholinergic system. The project will combine behavioral analyses of the aging Long-Evans rat with anatomical procedures for assessing gene expression in cholinergic neurons. The """"""""water maze will be used to train young and aged (27 months) rats and individual """"""""learning indices"""""""" will assigned. In situ hybridization histochemistry (ISHH) and immunohistochemistry will be combined to quantitatively assess the level of gene expression in cholinergic neurons and associated glia, permitting tests of hypotheses of selective cholinergic dysfunction. With some aged rats, deafferentation of the hippocampus of cholinergic or glutamatergic input, or chronic activation of hippocampal glia, will be used to gain insight into the causes of abnormal gene expression in the aging brain ISHH and immunocytochemical methods can be used quantitatively and powerfully to determine alterations in gene expression at the cellular level in phenotypically identified neurons. We have shown that, b-NOS mRNA is expresssed not only in the brainstem cholinergic neurons but also in a subset of basal forebrain cholinergic neurons, and that mRNAs for the plasticity protein GAP-43 and the oxidative stress indicator superoxide dismutase (Mn-SOD) are expressed at high levels in most or all cholinergic neurons. In studies of aged Long-Evans rat using these methods, we have found that the hippocampus of the impaired rat exhibits abnormal levels of mRNA for glial fibrillary acidic protein (GFAP), a marker of astrogliosis, in a way highly correlated (P less than O.0001) with the degree of spatial memory impairment. Additionally, the hippocampus of these animals expresses higher levels of mRNA for beta- amyloid precursor protein (BAPP-695), Mn-SOD, and brain-derived neurotrophic factor (BDNF). We have devised experiments to evaluate the degree to which glial activation, neurotrophin dysfunction, neuronal plasticity reactions, and oxidative stress are involved in differential effects of aging on the basal forebrain and brain stem cholinergic systems and on brain regions related by connectivity. BAPP and GAP-43 will be evaluated as neuronal plasticity markers, while GFAP (for astrocytes) and complement antigen, TNF-a, and IL-1b (for microglia) will be used as markers of glial involvement. The nitric oxide phenotype and oxidative stress indicators (Mn-SOD, Cu/Zn-SOD, and heat shock protein 70) will be quantitated in the two cholinergic cell types to test our hypothesis that only the rostral cholinergic system will be involved in age-related spatial memory dysfunction. The elements of the neurotrophin system to be studied include NGF, BDNF, the low-affinity NGF receptor, trk, and trkA. Thus, a combination of markers and techniques will be applied in a focused study of behaviorally-characterized rodents to test hypotheses of selective cholinergic vulnerability in aging.

National Institute of Health (NIH)
National Institute on Aging (NIA)
Research Project (R01)
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Neurological Sciences Subcommittee 1 (NLS)
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Mayo Clinic, Rochester
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