Albert Irving Farbman - US grants

For several years, the focus of research in this laboratory has been the study of olfactory receptor cell differentiation with attention to the extrinsic influences that modulate differentiation, particularly those influences originating in the olfactor bulb. We have pioneered the development and utilization of in vitro techniques for these studies. We will continue this research on receptor-target interaction, and, three other goals. First, modulating effects on olfactory receptor cell differentiation originating from neighboring cells within the olfactory epithelium will be studied. The plan is to examine, in detail, the morphology of disaggregated olfactory epithelial cell cultures, and of reaggregates made from the latter. Preliminary data indicate that differentiation proceeds more expeditiously when reaggregation occurs, thus suggesting that intercellular contact within the epithelium enhances differentiation. It is possible that supporting cells play a role. Monoclonal antibodies to supporting cells will be made in order to provide a means to do immunosurgical experiments in vitro, i.e., to specifically eliminage supporting cells from the cultures by using the antibody (with complement), and thus produce a culture (or reaggregate) containing no supporting cells. Second, the glycoconjugates on axonal surfaces of growing olfactory neurons will be studied with lectin-colloidal gold complexes. Glycoconjugates are important components of cell membranes and presumably play a role in both guidance of olfactory axons as they grow to their synaptic target, and in synaptogenesis. Third, the effect of antidepressant drugs on differentiation will be studied. These studies may lead to a better understanding of the cellular physiology associated with differentiation, specifically the role of calmodulin, cyclic AMP and phosphodiesterase.

The sensory tissues of the nose contain cells that have microsopic hairlike processes called cilia. The membranes of these sensory cilia are unlike those of cilia from areas that are not sensory, in showing higher densities of ultramicroscopic particles. It is believed that these particles seen in the electron microscope represent large molecular species such as proteins involved in binding important odorant molecules, or involved in control of excitation of the cell membrane in response to odorant binding. This work will utilize unusual high-resolution electron microscopical techniques to explore the nature of these particles. Specific molecular probes will be used that are known to bind to certain kinds of proteins. The localization of the bound probe molecules will be used to infer the identity of the membrane components, and study their distribution. These morphological properties will be compared data from biochemical and physiological studies, to clarify how chemosensory signals are produced in response to the presence of odors. This issue of transduction, where an stimulus is converted into a nerve signal to the brain, is fundamental to the operation of sensory systems. The very demanding techniques in the hands of these particularly skilled investigators promise advances in understanding such transduction processes.

taste; tongue; parotid gland; parasympathetic nervous system; lipase; axon; digestion; apical membrane; secretion; amylases; sucrose; fructose; saliva; electron microscopy; monoclonal antibody; histochemistry /cytochemistry;

The experiments in this proposal are designed to investigate the interactions between sensory nerves and their target organs during development. The model system for these studies is the fungiform papillae (FP) and taste buds (TBs) on the embryonic rat tongue. Each papilla is innervated by two sensory nerves, the chorda tympani (CT), which innervates TBs exclusively, and the lingual, a branch of the trigeminal nerve, which provides somatosensory innervation to the papilla and the entire mucosa of the anterior part of the tongue, but is essentially excluded from the TB. We plan to use a novel approach to study the development of TBs and papillae with minimum perturbation of the tissue. 1) We shall use in utero surgery to interrupt the CT before it reaches the tongue epithelium, and immunological means to inhibit development of the trigeminal nerve, to determine how removal of either or both nerves affects differentiation of the FP and their TB. 2) We shall do immunohistochemical studies on the extracellular matrix of the tongue before and during nerve growth to determine whether the matrix is organized in a way that might provide a pathway for the nerves to follow as they grow through the body of the tongue toward the mucosa. 3) We shall do immunohistochemical studies on the FP of rat tongue during development to determine whether nerve growth factor (NGF) or NGF-receptors are differentially present in the FP epithelium, TB and nerves. The rationale for this is based on the observation that trigeminal ganglion cells require NGF for their maintenance, whereas CT ganglion cells do not. It is possible that the reason why the nerves are differentially distributed to different parts of the FP is that the respective neurotrophic factors, such as NGF, required by each nerve are made by those epithelial cells that they innervate, i.e., perigemmal cells for the lingual and TB cells for the CT nerves.

The broad goal of the research described in this proposal is to understand (i) how developing and regenerating chemosensory nerves navigate their paths to their appropriate targets; (ii) how, when they reach the targets, they stop growing and (iii) how they alter their gene expression from a growth mode to a mature mode in which they form a functional relationship with the target. In both the taste and the olfactory systems we suggest that both the environment through which the nerve growth and the target structures provide signals that regulate the behavior of growing and mature nerve. These signals are likely to include navigational guidance cues, "stop growth" signals, and signals that induce changes in gene expression. In the rat tongue, 2 sensory nerves, the lingual proper (LP) branch of the trigeminal, and the chorda tympani (CT) provide innervation to fungiform papillae. During development they enter the tongue simultaneously but are directed to different targets. The CT innervates the apices of the fungiform papillae whereas the LP innervates other regions of these structures, as well as the remainder of the anterior tongue. We propose to identify the navigational signals directing these nerves to different targets in the fungiform papilla by; (1) mapping the distribution of extracellular matrix (ECM) molecules that might promote (or inhibit) nerve growth; (2) identifying surface molecules on growing and regenerating sensory nerves in the tongue that might act as receptors of signals present in the environment or on the synaptic target; (3) identifying positive and negative tropic signals and stop signals for axonal growth in the developing tongue; (4) identifying signals that alter gene expression in gustatory neurons. The taste bud containing circumvallate papilla is innervated by the glossopharyngeal nerve. Because gustatory and somatosensory nerves in the glossopharyngeal nerve arise from two distinct ganglia in the rat we will also be able to examine some of these issues in the innervation o the circumvallate papilla. In the olfactory system we propose (1) to identify the "stop growth" signal for olfactory axons; (2) to identify soluble or membrane-associated bulbar signals that act to alter gene expression in olfactory neurons. Both in vivo and in vitro methods will be used.

Neurons in the geniculate ganglion provide sensory innervation to: 1) taste buds on the anterior region of the tongue, via the chorda tympani nerve; 2) taste buds on the incisive papilla and the soft palate, via the greater superficial petrosal nerve; and 3) the skin near the ear via a cutaneous nerve. Preliminary studies in our laboratory showed that three high affinity neurotrophin receptor tyrosine kinases (trkA, trkB and trkC) are found in neurons of the geniculate ganglion. The ligands for these receptors are a family of secreted neurotrophic factors: trkA binds Nerve Growth Factor, trkB binds Brain-Derived Neurotrophic Factor and Neurotrophin-4, and trk C binds Neurotrophin-3. In pilot studies on identified single neurons of the rat geniculate ganglion we have found that they can be divided into subsets according to the trk genes they express. Moreover, we have evidence suggesting that trk expression by rat geniculate ganglion neurons changes during development. This research will test two hypotheses: 1) The division of geniculate ganglion neurons into sub-populations based on differences in trk gene expression is related to one or more of the following: a) the projection of 3 main nerve branches from the ganglion, b) the taste and other modalities associated with neurons in the geniculate ganglion, or c) possible differences in central projections of the neurons. We shall use tracer molecules and electrophysiology to identify specific neurons, and subject these to RNA amplification and PCR methodology to look for functional correlates of the differences in trk expression. Hypothesis 2: The changes in trk gene expression in geniculate ganglion neurons reflect major developmental events, such as axon outgrowth, innervation of the peripheral target tissue, naturally occurring cell death, or innervation of the central target. We shall use in vitro and in vivo methods to relate changes in trk expression during development to other major developmental events.