Bruce H. Wainer, MD, PhD - US grants

Central cholinergic neurons of the basal forebrain and possibly cerebral cortex contribute the cognitive processes. Dysfunctions of this system, as in Alzheimer's Disease, result in cognitive deficits. The long term goals of this project are to better understand the anatomical and functional organization of this system, and the abnormalities that can result in disease. In the present proposal, monoclonal antibodies previously developed against the cholinergic marker choline acetyltransferase (ChAT) will be employed to study the synaptic organization and development of cholinergic pathways in the basal forebrain and cerebral cortex. The topography and neurochemical identity of basal forebrain cortical projections will be studied by combination horseradish peroxidase retrograde transport and ChAT immunohistochemistry. The neuronal targets and synaptic organization of cholinergic cortical innervation will be studied at the light/electron microscopic levels by combinations of ChAT immunohistochemistry, Golgi impregnation, lesioning, and retrograde transport. The development of the cholinergic basal forebrain system and its projections to cortex will be studied by [3H]-thymidine autoradiography to determine neuronal birth dates and possible migration patterns; ChAT-immunohistochemistry for determination of neuronal phenotype expression; and retrograde tracing to determine the developmental time course of cortical projections. In order to gain greater resolution of cholinergic synapses and targets, monoclonal antidodies will be developed against the high affinity choline transport system and against muscarinic receptors, which are preferentially localized in cholinergic terminal fields. In addition, reagents and techniques will be developed to allow for immunocytochemical studies of cholinergic systems in ordinary post-mortem human tissues. The information provided from these studies will offer a better understanding of cholinergic function in health and in disease.

tissue /cell culture; disease /disorder model; Alzheimer's disease;

Central neurons which utilize acetylcholine as a neurotransmitter affect a number of important brain functions such as processing of sensory information, arousal, and learning and memory. Loss of cortical cholinergic innervation can result in severe behavioral deficits. A cholinergic deficit is also partly responsible for the dementia of Alzheimer's Disease. The studies proposed here will extend previous work of this laboratory on the synaptic organization of central cholinergic pathways that affect transmission in the cerebral cortex, either directly or via subcortical circuitries. These studies will utilize somatosensory pathways in the rat brain as a model system to identify anatomically the synaptic sites at which cholinergic transmission might modulate the processing of sensory information. The specific pathway to be studied is the mystacial vibrissal system which conveys tactile information that is essential for exploratory behavior in this species. Preliminary evidence from this laboratory suggests that both forebrain and brainstem cholinergic cell groups influence sensory pathways at several levels of the neuraxis including the midbrain, thalamus, and cortex. The proposed studies will utilize correlated light and electron microscopic analyses to identify such connections. First, putative sensory afferents to cholinergic neurons of the brainstem pedunculopontine tegmental nucleus (PPTn) will be studied by injecting the retrograde tracer wheatgerm agglutinin-horseradish peroxidase (WGA-HRP) into the PPTn. Identified retrogradely- labeled sensory relay nuclei will then be injected in a second set of experiments with anterograde tracers including WGA-HRP and phaseolus vulgarus leukoagglutinin. The resulting anterogradely- labeled sensory axons and terminals will be co-visualized with choline acetyltransferase immunoreactive neurons of the PPTn. Correlated light and electron microscopic analyses will verify synaptic contacts between anterogradely-labeled sensory axons and PPTn neurons. Second, cholinergic PPTn projections to the ventrobasal thalamus will be studied by placing simultaneous injections of anterograde tracers in the PPTn and retrograde tracers in the barrel field of somatosensory cortex. Light and ultrastructural visualization of anterogradely-labeled fibers and retrogradely-labeled cells will permit identification of cholinergic-PPTn inputs onto thalamic relay cells responsible for transmission of vibrissal tactile information. Third, tissue from the same animals, which contains retrogradely-labeled cholinergic and noncholinergic basal forebrain neurons will be processed in a similar manner to identify putative PPTn-basal forebrain connections. Finally, cortical targets of basal forebrain projections to the barrel field of somatosensory cortex will be identified by anterograde tracing of these projections combined with either Golgi impregnation of cortical neurons, GABA immunocytochemistry, or retrograde tracing from known cortical- cortical and cortico-fugal pathways. The results of these studies will provide a detailed understanding of a specific information- processing system in which the modulatory role of cholinergic transmission can be studied in detail at both an anatomical and physiological level.

DESCRIPTION (Adapted from the applicant's abstract): The long term goals of this project are to understand the functional anatomy of central cholinergic pathways that contribute to behavioral state control mechanisms and higher cortical function. A substantial body of evidence suggests that the cholinergic brainstem nuclei, the pedunculopontine tegmental nucleus (PPT) and laterodorsal tegmental nucleus (LDT), modulate behavioral arousal through widespread innervation of the thalamus, and perhaps through innervation of the lateral hypothalamus and basal forebrain as well. Additional evidence suggests that cholinergic brainstem mechanisms may mediate the onset of rapid-eye-movement (REM) sleep including the generation of pontine-geniculate-occipital (PGO) waves that precede the onset of REM sleep, and the cortical desynchronization and postural atonia that accompany REM sleep. There is also evidence to suggest that brainstem cholinergic pathways may participate in the generation of patterned motor behavior. The major goals of the proposed project are to investigate the anatomical basis of the functional role of these nuclei in arousal and sleep/wakefulness mechanisms, as well as to better define the relationship of these nuclei to brainstem motor generating regions. The first studies will confirm putative PPT/LDT afferents from the periaqueductal gray, dorsal raphe and lateral hypothalamus using anterograde axonal tracing combined with choline acetyltransferase immunohistochemistry analyzed at the ultrastructural level. These putative afferents were identified previously by retrograde tracing and they have been implicated functionally in behavioral state control processes. A second set of experiments will determine if cholinergic PPT and LDT neurons receive synaptic input from catecholaminergic and/or serotonergic brainstem nuclei, using co-localization immunocytochemical techniques at the ultrastructural level. These monoaminergic systems have also been implicated in behavioral state control. The third set of experiments will examine PPT innervation of the midbrain extrapyramidal area, the medial pontine reticular formation, and the medullary gigantocellular field in order to identify anatomical connections which mediate the physiological correlates of REM sleep (ie. PGO waves, rapid eye movements, and postural atonia). The fourth series of experiments will investigate the hypothesis that the midbrain extrapyramidal area might participate in the atonia that accompanies REM sleep by further examination of its afferent and efferent connections. Finally, putative connections of the brainstem cholinergic system with the basal forebrain cholinergic system will be investigated using anterograde tracing from the PPT and LDT to the nucleus basalis and lateral septal areas. The results of these studies will provide anatomical evidence for participation of the PPT/LDT complex in specific physiological processes related to behavioral state.

A salient feature of Alzheimer's and other age-associated neurodegenerative diseases is the selective vulnerability of particular neural pathways. Since the development and maintenance of neural connections is supported by neural trophic factors, trophic dysfunction represents one possible pathogenetic mechanism for such disorders. The long range research interests of the Principal Investigator are to understand the nature of neural trophic interactions and how they may be perturbed in human disease. In previous years of support from this grant, primary reaggregating cell cultures have been utilized and immortalized central nervous system neurons have been generated to study the trophic interactions that establish and maintain the septohippocampal pathway, which plays an essential role in cognitive function and is prominently affected in Alzheimer's Disease. The goals of the present renewal application are to: I) To utilize reaggregating cell cultures and primary neuronal monolayer cultures to further study the cellular and molecular mechanisms that regulate the expression and actions of nerve growth factor and its receptor in this system; II) To further characterize the cellular and molecular properties of immortal central neural cell lines generated from the septal region as a model system to study the neuronal response to trophic signals from the hippocampal region; and III) To further characterize the cellular and molecular properties of central neural cell lines generated from the hippocampal region. In particular, immunochemical and biochemical techniques will be employed to characterize a putative novel "Neuronal Trophic Activity" expressed by one of the hippocampal-derived cell lines, HN10, which elevates the activity of choline acetyltransferase, the biosynthetic enzyme for acetylcholine, in primary septal monolayer cultures. The results of these studies will further exploit the utility of somatic cell fusion techniques for generation of clonal central nervous system cell lines, particularly from cell populations that are post- mitotic, as a strategy to investigate neural trophic mechanisms and for the identification of novel trophic substances. Such cell lines represent a potential source for isolation of such factors, and also a potential "delivery system" via neural grafting techniques. The technology and information that is generated from these investigations will serve as a strategy to study trophic interactions in other brain circuits in future years and to investigate possible changes or dysfunctions that occur both in the aging brain and in age-associated brain diseases. (Note: Bold-face type indicates sections where significant revisions or new information was added).