Robin F. Krimm - US grants

The long term objective of these studies is to understand how NT3 influences sensory receptor organ development and innervation. NT3 affects proliferation and survival of both neuronal and nonneuronal cells during development and mediates communication between sensory end organs and neurons. This proposal will focus on a sensory mechanoreceptor, the touch dome-Merkel cell-neurite complex, because NT3 overexpression in skin has been shown to increase touch dome size, innervation and numbers of associated Merkel cells (Albers et al., 1996). The studies of this proposal will 1) determine the importance of NT3 derived only from skin on touch dome formation and innervation, 2) determine the role, if any, innervation has on mediating the receptor organ enlargement seen with NT3 overexpression, 3) determine the developmental time course for NT3 mediated enlargement of receptor end organs, and 4) determine what neurotrophin receptors are expressed in touch domes during that time. These studies are the first step to identify the mechanisms which underlie changes induced by NT3 in the skin-derived sensory targets of neurons. Future studies will attempt to identify (using representational difference analysis; RDA) genes expressed in both the target and neuron that temporally regulate development of touch dome end organs. By examining how NT3 acts on a sensory receptor organ, the mechanisms by which NT3 acts on both neuronal and nonneuronal cell types in the same system and how NT3 could be involved in the communication between these two cell types during development will begin to be uncovered. From a clinical perspective, NT3 may act via similar mechanisms to reestablish and maintain sensory receptor end organs after nerve damage or in skin grafts.

The long term objective of these studies is to understand how NT3 influences sensory receptor end organ development and innervation. NT3 affects proliferation and survival of both neural and nonneural cells during development and mediates communication between sensory end organs and neurons. This proposal will focus on a sensory mechanoreceptor, the touch dome Merkel cell neurite complex, because NT3 overexpression in skin increases touch dome size and number of associated Merkel cells between postnatal days 12 and 16. The studies of this proposal will focus on this developmental period to 1) determine if increased innervation to individual touch domes precedes increases in Merkel cell number 2) determine if the action of NT3 on the neural innervation of touch domes is the result of increased trkC expression by these neurons. 3) determine if there is a postnatal critical period for the action of NT3 on touch dome development. By examining how NT3 acts on a sensory receptor organ, the mechanisms by which NT3 acts on both neuronal and nonneuronal cells and how NT3 may mediate communication between these two cell types during development will begin to be uncovered. Elucidating how neurotrophins affect sensory end organs has important implications for use of these growth factors as therapeutic agents to enhance sensory regeneration following nerve injury or in disease states that cause sensory degeneration, e.g., diabetic neuropathies.

DESCRIPTION (provided by applicant): The neurotrophins - brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4) - are important survival factors for the developing taste system. Preliminary data shows that altering the pattern of BDNF or NT4 expression disrupts targeting of the chorda tympani. Specifically, when BDNF or NT4 is over-expressed in the basal epithelium of transgenic mice, taste axons of the chorda tympani nerve are misdirected to inappropriate non-taste bud targets. The mechanism underlying this misdirection of chorda tympani fibers is unclear. The long-term goal of the proposed research is to elucidate the mechanisms by which BDNF and NT4 over expression disrupts target selection in the fungiform taste system. The Specific Aims of this application will test the following hypotheses: 1) BDNF and/or NT4 over- expression in the basal layer of epithelia misdirects chorda tympani fibers during initial target innervation; and 2) BDNF over-expression can only disrupt chorda tympani targeting during a defined prenatal critical period, and not during postnatal development. The proposed studies will use anatomical, in situ hybridization, and immunohistochemical methodologies to: 1) determine the onset of BDNF and NT4 over-expression; and 2) determine how over-expression of BDNF or NT4 in tongue epithelium correlates temporally and spatially with the development of altered patterns of chorda tympani innervation in BDNF and NT4 over-expressing mice. The proposed studies will also use an inducible transgenic model of BDNF overexpression in which transgene expression is regulated by the Cre recombinase. This model system will allow us to test whether the sensitivity of chorda tympani fibers to BDNF overexpression is limited to a prenatal critical period or also occurs in adult systems. These experiments will provide requisite knowledge for future studies that will examine: 1) the receptors and internal signaling pathways mediating chorda tympani responses to BDNF and NT4; 2) interactions between neurotrophins and other axonal guidance cues; and 3) whether the location of BDNF and NT4 expression directs axonal regeneration. Uncovering these mechanisms will provide new insights into how taste neurons select their correct targets during development and following injury.

DESCRIPTION (provided by applicant): The neurotrophin (BDNF) is essential for the development and proper innervation of taste buds. Analysis of the taste system in wildtype and overexpresser transgenic mouse models has shown that BDNF acts, at least in part, as a target-derived factor that modulates axon targeting and innervation density. During early embryonic periods, BDNF is expressed in the epithelium of fungiform papillae. By later embryonic times BDNF expression in the tongue becomes focused and is only found in cells comprising developing taste buds. This refined expression pattern suggests that BDNF synthesis by taste bud cells is crucial for normal taste innervation. In support of this possibility, we found that transgenic mice that overexpress BDNF across the entire tongue epithelium had severely disrupted innervation to taste buds and fewer taste buds, even though these mice had more gustatory (geniculate) neurons. We now propose to advance this analysis and test how the level of BDNF in the taste bud itself affects neuron survival and innervation. This will be done using a new transgenic mouse model in which the keratin 19-gene promoter will be used to target BDNF gene expression to receptor cells of taste buds. Transgenic mice that overexpress K19 in taste cells (K19-BDNF mice) will be used to examine how target-derived BDNF modulates taste bud development and innervation. Specifically, we will use RT-PCR, in situ hybridization, retrograde labeling, and basic histological approaches to determine if enhanced expression of BDNF in taste cells, 1) increases innervation to fungiform papillae, 2) increases the size or number of taste buds in fungiform papillae, 3) disrupts the positive co-relation that exists between taste bud size and innervation, and 4) increases gelliculate ganglion cell survival. These mice will also provide a valuable model for studies on the receptor mechanisms and internal signaling pathways that underlie the action of BDNF in gustatory development. This approach provides a new and powerful means to manipulate gene expression in the taste end organs, allowing study of the intrinsic factors that regulate taste organ development and innervation.

ABSTRACT NOT PROVIDED

DESCRIPTION (provided by applicant): During development, gustatory innervation is a tightly regulated process in which specific numbers of primary taste afferents project to discrete regions of the oral cavity. Brain-derived neurotrophic factor (BDNF) regulates both the number and location of gustatory innervation during embryonic development. Our current research is designed to determine if and/or how these connections are important for central gustatory development or taste function and whether BDNF regulates development of the postnatal taste system. The proposed studies combine cell counting, tract tracing, immunohistochemistry, electrophysiology, and behavioral testing in conditional and inducible transgenic mice to: 1) determine if BDNF influences postnatal changes in geniculate ganglion and taste bud development, 2) determine if embryonic or postnatal BDNF is important for central terminal field organization, and 3) determine if embryonic or postnatal BDNF is important for adult gustatory function. Together, these studies test the hypotheses that: 1) embryonic BDNF determines the number of neurons innervating a taste bud, and taste buds increase in size to meet that number postnatally. 2) CNS terminal field formation is regulated by BDNF and the specificity of peripheral connections. 3) Gustatory sensitivity is reduced but that the taste system still functions despite wide-scale reorganization. These studies will determine how early peripheral innervation patterns influence continued development and function of the taste system. In addition, we will determine whether BDNF is required for postnatal gustatory development. Because these experiments examine how neurotrophin regulation of sensory innervation influences continued development and adult function, this project has important implications for the potential therapeutic use of these powerful signaling molecules in re-establishing gustatory function following neuronal impairment.

Description (provided by applicant): During development, sensory innervation to taste buds is tightly regulated by the actions of two neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4). Although both of these factors regulate gustatory innervation, their specific functions are different from one another. BDNF is expressed in gustatory epithelium and determines whether gustatory neurons will locate and innervate taste placodes during development, while NT4 does not. Both BDNF and NT4 regulate gustatory neuron and taste bud number, but they have their influences at different times during development. The long range goal of this research is to understand the cellular and molecular mechanisms that regulate the neural innervation to and the maintenance of taste buds during development. The proposed studies focus on mechanisms by which these two neurotrophins, BDNF and NT4, can influence various aspects of neural innervation through the same receptors, TrkB and p75. The proposed studies use cell counting, tract tracing, immunohistochemistry and scanning electron microscopy in gene knockout mice to determine when TrkB and p75 are required for specific aspects of gustatory development, including axonal extension into the tongue, target selection, and geniculate neuron/taste bud survival. Furthermore, these studies combine these anatomical approaches with sophisticated genetic tools allowing the function of single phosphorylation sites on the TrkB receptor protein to be blocked in vivo. These experiments will determine which TrkB intracellular signaling pathways are required for axonal extension into the tongue, target selection, and geniculate neuron/taste bud survival. Together these studies test the hypotheses that TrkB mediates the effects of both BDNF and NT4, but that each function of these neurotrophins requires a different intracellular signaling pathway. While p75's, more subtle role, is to modulate the regulation of gustatory development by neurotrophins. Because these experiments examine the fundamental mechanisms of how neurotrophins regulate neuronal survival and axon targeting via Trk receptors, this project has important implications for the therapeutic use of these signaling mechanisms in controlling neuron survival and axon targeting during regeneration. PUBLIC HEALTH RELEVANCE: The taste system is an ideal and commonly used system for examining axon regeneration following injury. We are examining the signaling mechanisms for two factors (neurotrophins) that influence axon targeting and neuron survival during development. By understanding of how these signaling mechanisms normally function, techniques can be developed to manipulate these important signaling factors during nerve regeneration which will improve reinnervation and function following nervous system injury.

? DESCRIPTION (provided by applicant: A general feature of sensory organization is that neurons can be divided into different types based on molecular, morphological, and functional characteristics. Whether this type of organization exists in the taste system is unclear. It is generally accepted that taste receptor cells exist as different types. The next critical step is to determine how these different taste receptor cell types can connect with the ganglion neurons that carry taste information to the brain. Because peripheral gustatory axons invade the tongue in fascicles, with individual axons intertwining at every level, past attempts at reconstructing single fibers have not been successful. This project will overcome this critical barrier by combining genetically-directed sparse labeling of nerve fibers with three-dimensional visualization and reconstruction in taste buds. Using these approaches, we will test the hypothesis that gustatory neurons can be divided into subtypes based on their morphology (i.e., branching) and connectivity with specific types of taste receptor cells. These experiments will reveal how connections between taste cells and nerve fibers are organized. The approaches established in this project can be used in future studies to understand 1) if morphological subtypes correspond to specific molecular and/or functional subtypes, 2) the branching of individual taste neurons in the central nervous system (CNS), 3) how branching patterns are established during development, and 4) whether normal branching patterns are reestablished following injury, disease, or chemotherapy.

Project Summary/Abstract: Taste receptor cells detect chemicals in our food and communicate this information to the nerve fibers innervating them. Our goal is to understand the structural and functional relationship between taste receptor cells and innervating neurons. Because taste receptor cells undergo continuous renewal and must constantly attract and connect to nerve fibers even in the adult, some mechanism exists to control this process. Since the neurotrophin BDNF controls initial innervation of taste buds and is present in adult taste receptor cells, it may have this role. BDNF also can function as a neuromodulator regulating function, so it is certainly possible that BDNF can regulate both innervation and/or function of adult taste buds. Here we are testing the hypothesis that BDNF signaling through its receptor, TrkB, regulates both the structural and functional connections between taste receptor cells and ganglion neurons. The proposed studies combine sparse cell genetic labeling with 3-dimensional analysis of the taste bud and electrophysiology, in mice where TrkB-signaling can be blocked using a combination of chemical and genetic approaches to: 1) determine the identity and connectivity of BDNF expressing taste receptor cells, 2) determine the role of BDNF-TrkB-signaling in maintaining this connectivity, and 3) determine if BDNF-TrkB signaling regulates taste function. Together, these experiments will determine how specific taste receptor cells (BDNF-expressing) become innervated by a subset of taste ganglion neurons (TrkB-expressing) during taste cell renewal and will determine if BDNF-TrkB-signaling is an important gustatory system neuromodulator of this pathway.   

Project Summary/Abstract Along with taste, we perceive the texture and location of food in the oral cavity and these modalities contribute to food choice. The first step leading to the perception of location and texture is activation of oral cavity mechanosensory neurons, most of which reside in the trigeminal ganglion. Some of these neurons project to gustatory regions and modulate taste responses beginning at the first gustatory relay (NST). Yet little is known about the sensitives, receptive field properties, or response specificity of these neurons. The goal of this application is to redirect the research program of a taste scientist through the acquisition of new research skills (genetic encoded calcium imaging of the trigeminal ganglion neurons) to answer questions concerning how these neurons function. With these tools in-hand the long-term goal is to 1) elucidate the classes of mechanosensitive neurons based on functional characteristics, genetic expression and structural characteristics. 2) to manipulate these neurons to determine their specific role in the central nervous system coding of oral cavity stimuli and contributions to behavioral food choice.

Project Summary/Abstract: A unique feature of the peripheral taste system is that the receptor cells die and are replaced. This requires a constant rewiring of the peripheral system. In order for a new taste receptor cell to form a synapse with an existing neuron it must first come into contact with the correct type of neuron. Here, our goal is to understand the cellular dynamics involved in this process. We hypothesize that since many receptor cells are replaced within 10 days, this cellular influx should be accompanied by cellular migration of taste bud cells and/or remodeling by the arbor. To test this hypothesis, we will combine genetic labeling with intra vital two-photon microscopy to examine plasticity in the taste bud over a 10 day period. Aim 1 will examine the migration of precursors into the taste bud as they mature. Aim 2 will measure the growth and retraction of individual nerve branches. These studies will be first to examine taste bud plasticity with direct observation of changes over time in a live animal. Future studies will use this approach to help identify roles of molecular factors underlying the formation of new connections in the taste bud.