Dennis Higgins - US grants

Electrical coupling and/or gap junctions (the structures generally responsible for coupling) have been observed in every metazoan embryo that has been examined; although such structures were first described 20 years ago, their role in embryonic development remains unknown. The experiments in this proposal will use an in vitro model to identify and characterize agents that regulate the formation of gap junctions in peripheral neural tissue. This laboratory has found that fetal rat sympathetic neurons can be maintained in vitro under conditions in which virtually all or virtually none of the neurons are electrically coupled; this is the first neural population in which it has been possible to chronically manipulate electrical coupling. Initially experiments will be done to identify agents capable of inducing electrical coupling among neurons. Subsequent experiments will extensively characterize the mechanism of action and the chemical nature of a crude material obtained from fetal calf serum that acts chronically to decrease coupling among sympathetic neurons in vitro; if this material acts in vivo as it does in vitro, it will provide a powerful tool for analyzing the developmental role of gap junctions. After agents capable of chronically altering junctional communication among sympathetic neurons have been characterized, a comparison will be made of the regulation of coupling in vitro among cells that are coupled in situ (ciliary neurons) and cells that are not coupled in situ (sympathetic neurons). Such a comparison will determine whether agents that alter coupling among sympathetic neurons have a general action upon other peripheral neural tissues; it may also be helpful in determining why only a subpopulation of neural tissue is coupled in adult animals. The identification of agents that chronically alter coupling among fetal neurons is necessary to allow future investigations that examine the types of developmental information that can be conveyed across gap junctions. Thus, this study of the regulation of electrical coupling in fetal neural tissue represents initial progress towards defining the role of gap junctions in neural development.

Extracellular matrix (ECM) molecules such as laminin and fibronectin stimulate the growth of neuronal processes. The biochemical characterization of these molecules has yielded substantial information about the amino acid sequences which promote neuritic growth and the receptors which mediate this response. However, a fundamental question remains unanswered: what is the nature of the processes which form in the presence of laminin or fibronectin; are they axons or dendrites? To address this issue, Dr. Higgins is currently determining how chronic (1-4 week) exposure to ECM components affects the morphological development of embryonic rat sympathetic neurons in tissue culture. A combination of techniques (intracellular dye injection, immunocytochemistry, electron microscopy) is being used to distinguish axons from dendrites. His preliminary experiments indicate that: 1) laminin promotes only axonal growth in sympathetic neurons; and 2) a basement membrane extract causes the extension of dendrites. These data suggest that axons and dendrites have different growth requirements and that ECM molecules may play a role in determining the shape of sympathetic neurons. This research project will use the same bioassay to determine: 1) are there other neurite-promoting factors which act like laminin in selectively stimulating axonal growth; and 2) what is the identity of the dendrite-program molecule? They will also test the hypothesis that process-specific interactions occur because axons and dendrites have different types of ECM receptors on their surfaces. Such data will help to more clearly define the role of ECM in the morphogenesis of the nervous system.

The processes extending from nerve cells are called axons and dendrites. Dendrites receive messages from other cells, while the axon conducts messages often over long distances. Compared to axons, we know far less about dendrites, and little about how the distinctions between the two arise during development. This project extends studies on the molecular identity of a compound believed to be a protein that appears to act specifically as a dendrite-promoting factor. A novel neuronal culture system has been established to test the effects of this compound on isolated growing cells. Microscopic analysis of cells along with protein chemistry will be used to assay and isolate the purified molecule. The purification of such a unique factor will have a great impact on developmental neuroscience, and the potential opportunity for further biotechnological development of such a product is very high.

9808565 HIGGINS Dendrites are the primary site of synapse formation in the nervous system, and so it is important to learn how the growth of these processes are regulated. During the previous period of support, Dr. Higgins found that bone morphogenetic proteins induce dendritic growth in several classes of neurons, including embryonic rat sympathetic, hippocampal and spinal motor neurons. The current project explores the possibility that leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), and related neuropoietic cytokines have an opposing activity, and that they function as negative regulators of dendritic growth. The study of such inhibitory factors is important because dendritic regression is widespread in the nervous system, having been seen in many regions of the developing brain and also in neurodegenerative disorders and after acute neural injury. CNTF promotes the survival of postnatal sympathetic neurons; yet, in the same population, it causes dendritic regression. If CNTF exerts a similar dendrite-retracting influence on motor and striatal neurons in vivo, this would be expected to interfere with synaptic function and compete with the influence of dendrite-promoting molecules such as bone morphogenetic proteins. CNTF appears to have a dominant negative influence on dendritic growth; i.e., it induces dendritic regression even in the presence of optimal concentrations of other dendrite-promoting factors. We need to know more about how a survival-promoting molecule can simultaneously adversely affect neural function. To further characterize the effects of neuropoietic cytokines, the planned studies will: a) characterize the receptors and signal transduction system mediating dendritic retraction; and b) determine whether CNTF and other neuropoietic cytokines also promote dendritic retraction in neurons from the central nervous system.

Bone morphogenetic proteins (BMPs) regulate many aspects of neural development and they are also neuroprotective in mature animals. It is, therefore, important to understand the mechanism of action of these neurotrophic factors. Previous studies of BMP signaling pathways have focused on the transcriptional activities of these proteins and have emphasized their ability to alter neural cell fate by inducing or repressing gene expression. Preliminary studies from this laboratory, however, indicate that BMPs can also induce rapid (<1 second) changes in the conductance state of ionotropic glutamate receptors and that this occurs in both mature (adult human retinal) and developing (rat hippocampal) neural cell populations. Thus, there may be a second, parallel pathway for BMP signaling that acts independently of nuclear events and that alters neural function by acutely regulating existing ion channels in cultured neurons. The experiments in this project will characterize this pathway by comparing the effects of BMPs on the three types of ionotropic glutamate receptors and by determining whether BMPs also affect voltage-sensitive ion channels. In addition, the project will characterize the types of receptors mediating the effects of BMPs on glutamate receptors and will determine whether BMPs modulate glutamatergic synaptic transmission in tissue culture. It is anticipated that these experiments will substantially expand our knowledge of BMP signaling mechanisms and that they will also provide an initial assessment of the ability of BMPs to function as synaptic modulators.