In these studies we propose to examine the ability of estrogen to stimulate the compensatory sprouting response of the cholinergic system in an area that is first and most profoundly affected in Alzheimer's disease (AD), the entorhinal cortex (EC). Several reports have described increased synaptic sprouting in response to estrogen, and have implicated this hormone in the upregulation of cholinergic function in the basal forebrain. Based on these studies and on our own results, we hypothesize that estrogen may induce cholinergic sprouting and reactive synaptogenesis in response to cell loss, and perhaps bring about some degree of functional recovery. The basic premise of the proposed experimentation is that estrogen has a neuroprotective effect that restores and prolongs the function of damaged basal forebrain cholinergic neurons. Male and female Fisher 344 rats, young (3 months old) and aged (24 months old), both gonadectomized and intact, will receive a lesion that mimics the cortical cholinergic denervation in AD. Unilateral lesions (allowing for the contralateral side to act as an internal control) will be produced in the nucleus of the horizontal limb of the diagonal band of Broca (HDB) using 192 IgG-saporin immunotoxin complex as the lesioning tool. This immunotoxin specifically targets cholinergic neurons. Treatment with estrogen or placebo will begin four weeks after lesioning, when maximal EC denervation has occurred.
For specific aim 1 we will use standard immunohistochemical techniques to measure the change in lesion-induced cholinergic sprouting in the EC that results from estrogen treatment.
In specific aim 2 we will employ a modification of the Fonnum method to assay choline acetyltransferase (ChAT) activity in the same EC area.
In specific aim 3 we will examine the regenerative effects of estrogen on surviving basal forebrain cholinergic neurons by semi-quantitative RT/PCR analysis of changes in the mRNA expression of two cholinergic markers, the synthetic enzyme ChAT and the vesicular acetylcholine transporter (VAChT). Finally, in specific aim 4 we will test the hypothesis that estrogen can ameliorate lesion- and age-dependent behavioral impairments. Experimental animals will be tested before and after estrogen treatment using an odor discrimination task that is sensitive to EC damage. The resulting measures of behavior will be correlated with cortical fiber sprouting and with levels of cholinergic activity in the cortex and basal forebrain.
