Robert W. Baughman, Ph.D. - US grants

The overall aim of this study is to characterize the neurotransmitters of the central visual pathways. Four approaches will be pursued: 1) Release of endogenous compounds from tissue slices; including determination of the effect of cortical lesions on release of amino acids, in particular aspartate and glutamate, from tissue slices of lateral geniculate nucleus and superior colliculus; analysis of the release of low level constituents from cortical tissue slices with electron capture and nitrogen specific gas chromatography; use of electrochemical HPLC to monitor release of norepinephrine and serotonin from cortical tissue slices; investigation of neurotransmitter release mechanisms; and effects of putative neurotransmitters on release. 2) Pharmacological and bioassay studies with in vitro recording from tissue slices from visual cortex; including determination of pharmacological responses of different types of identified neurons in tissue slices by puffer micropipette application followed by intracellular marking with fluorescent dyes or horseradish peroxidase; and use of intracellular recording and puffer micropipette application of extracts from cortex onto tissue slices as a bioassay system for possible neurotransmitters. 3) Immunohistochemical and autoradiographic localization of neurotransmitter systems; including preparation of monoclonal or serum antibodies to rat brain choline acetyltransferase; use of new or existing antibodies to choline acetyltransferase to more precisely localize cholinergic neurons in retina and in visual cortex; preparation of monoclonal or serum antibodies to glutaminase from calf brain to attempt to achieve immunohistochemical localization of glutaminergic pathways; and use of neurotransmitter-specific autoradiographic tracing with radioactively labelled transmitters or precursors. 4) Fluorescent neuronal labelling, enzymatic dissociation and fluorescence-activated cell sorting; including optimizing dye labelling and cell sorting conditions; measurement of levels of endogenous compounds in sorted cells; and analysis of proteins present in sorted cells. Identification of the neurotransmitters used in the central visual pathway is important for understanding and treating clinical disorders of this and possibly other sensory systems.

Neurotransmitter function of identified cortical neurons will be studied in tissue culture. Cortical neurons, e.g. layer 5 corticocollicular cells, will be labeled by retrograde transport of fluorescent latex microspheres (beads) and put into monolayer tissue culture. The synaptic physiology and pharmacology of these identified neurons will be studied by simultaneously recording intracellularly from a labelled cell and a nearby cell to create a "driver/follower" pair in which driven postsynaptic potentials can be recorded. The effects of various excitatory amino acid agonists and antagonists on these psp's will be determined. Immunocytochemistry and autoradiography will be used to provide neurotransmitter-specific labeling of the cells in culture. Identified cortical neurons labeled with fluorescent beads will be sorted on an EPICS-V cell sorter. The endogenous pattern of amino acids and related compounds and of proteins will be determined in the sorted neurons. This may identify transmitter or other molecules related to specialized neuronal function. Homogeneous sorted cells will be placed in culture to study intrinsic cellular properties of identified cells and the effects of interactions between different cell types on neuronal function. The nature of geniculate afferent and layer 6 recurrent input to cells in layer 4 will be investigated in an in vitro tissue slice preparation. Neurotransmitter systems will be characterized with histochemical staining. Combined retrograde HRP and ChAT immunocytochemical staining will be used to attempt to localize the source of cholinergic input to the LGN, superior colliculus, and vestibulo-ocular cerebellum (mossy fiber inputs). In the long term, our goal is to determine what the connections and roles are for the various neurochemically or anatomically identifiable cell types in the visual pathways. In addition to being important for understanding normal function, these studies are important for understanding abnormal function. Many neurological deficits appear to be related to failures in specific types of molecular signaling, and many neurological diseases appear to be associated with the degeneration of specific neurotransmitter pathways. Our results may have importance not only for learning more about normal visual processing, but also for learning more about neuropathological mechanisms.

Cholinergic input to cerebral cortex, which largely originates in the basal forebrain, appears to be involved in arousal and memory and is greatly reduced in Alzheimer's disease. Although the cholinergic innervation is distributed throughout cortex, it is concentrated particularly in layer 5, the major source of cortical efferents. We have developed techniques for a) labelling specific classes of cortical neurons by retrograde filling, in particular layer 5 corticocollicular neurons, and b) dissociating postnatal cortical or basal forebrain neurons and maintaining the neurons in tissue culture under conditions where the labelled cells can be identified over several weeks. We have shown that the labelled neurons can be penetrated with micropipettes for recording pharmacological responses and the "driver/follower" recordings, including a labelled cell and a nearby cell with which it is synaptically connected, can be obtained. With this approach we have also succeeded in establishing criteria for identifying inhibitory Gabaergic neurons in the cultures. The aim of this proposal is to use our tissue culture preparation to characterize interactions of cholinergic basal forebrain neurons with identified cortical cells. In cultures prepared from postnatal rats, layer 5 corticocollicular neurons, previously labelled in vivo by retrograde transport, Gabaergic interneurons, identified by physiological criteria, and cholinergic basal forebrain neurons, labelled by retrograde transport, will be penetrated for intracellular recording with micropipettes. The pharmacology and ionic basis of the responses of these cells will be investigated with bath applied agonists and with synaptically driven postsynaptic responses. To study the pharmacology of synaptic interactions between cholinergic basal forebrain neurons and cortical neurons, co-cultures of basal forebrain and cerebral cortex will be prepared. Both cholinergic input from basal forebrain neurons onto cortical cells and excitatory amino acid input from cortical pyramidal cells onto basal forebrain neurons will be investigated. In a parallel set of experiments, the possibility that cortical cells provide NGF-like trophic support for cholinergic basal forebrain neuron will be tested in tissue culture. These experiments will increase our understanding of how cholinergic basal forebrain cells interact with cerebral cortex and thus are important for gaining a better understanding of the etiology of Alzheimer's disease.