BMCB-Faculty Research, Alan Nighorn

Associate Professor of Arizona Research Laboratories - Division of Neurobiology and Molecular & Cellular Biology
Ph.D., Baylor College of Medicine

Understanding signal transduction in the adult and developing olfactory system.

Research Interests

Neurons function by integrating multiple external stimuli and responding to those stimuli with changes in activity and gene expression. Thus, understanding how neurons respond to external stimuli is central to understanding their function both individually and in networks. Neurons respond to a wide variety of external stimuli including neurotransmitters, modulatory peptides, growth factors, and environmental signals such as odorants in the case of the olfactory system. These external stimuli are interpreted within the cell by changes in the levels or actions of a relatively small number of intercellular messengers. Although much research has been focused on the actions of molecules such as cAMP, relatively little effort has been made to understand the role of the cyclic nucleotide, cGMP. This has changed recently with the discovery of nitric oxide (NO) as a gaseous neurotransmitter since NO is known to exert its effects partially through the actions of cGMP. We are using the olfactory system of the moth, Manduca sexta, as a model system in which to study the physiological role of cGMP. This is an excellent model system in which cGMP plays a large role in its function through both NO-sensitive and NO-insensitive mechanisms. For example, pheromone stimulation of antennae is known to cause a slow rise in cGMP that is likely to be involved in adaptation (Zeigelberger et al. 1990). More centrally, in the antennal lobe, the NO/sGC signaling system is thought to play a role in the processing of odorant information (Breer and Shepherd 1993). Our goals are first, to gain a better understanding of signal transduction via cGMP within the olfactory system, and second, to better understand the physiological roles of cGMP modulation and the mechanisms by which those roles may be effected in the nervous system in general.

Our initial efforts have focused on understanding the regulation of guanylyl cyclases (GCs), the enzymes that generate cGMP, in the Manduca sexta olfactory system. The GCs are usually classified as being either soluble GCs (sGCs) or receptor GCs (rGCs). sGC is an obligate heterodimer comprised of an alpha and a beta subunit. Upon heterodimer formation, sGC can be strongly stimulated by NO to generate cGMP. The rGCs are single transmembrane polypeptides with a extracellular ligand binding domain. They act as homodimers and are activated either through ligand binding or interactions with guanylyl cyclase activating proteins (GCAPs). We have cloned cDNA fragments of eight Manduca sexta GCs, including three with sequence similarity to sGCs and five with similarity to rGCs. We have investigated the expression patterns and obtained full-length clones of one alpha and two beta sGC subunits as well as the enzyme that generates NO, nitric oxide synthase (NOS). We have also obtained full-length clones of two GCs with significant sequence similarity to rGCs including MsGC-I, a novel form of GC, and MsGC-II, a GC with similarity to mammalian GC-E.

The expression patterns of these GCs gives us some insight into their possible functions. In the antennae, we find that the cell bodies and dendrites of the olfactory receptor cells express MsGC-I and don't express either NOS or sGC subunits, suggesting that the slow rise in cGMP seen after odor stimulation is due to the activation of this unique guanylyl cyclase. In addition, we find that this unique GC is highly expressed in higher order neuropils of the brain such as the mushroom bodies and the central complex. When we examine the antennal lobe, the first synaptic neuropil of the olfactory system, we find that NOS is expressed in the axons of the olfactory receptor neurons and MsGCa1 is expressed in a subset of antennal lobe neurons including projection neurons that carry olfactory information to higher brain regions and interneurons that mediate communication within the antennal lobe.

The expression pattern strongly suggests that NO/sCG signaling plays a part in shaping the responses of antennal lobe neurons. A large focus of the lab is now on understanding the role of NO signaling using a variety of histochemical and electrophysiological methods. We are investigating the function of NO in the olfactory system using the NO-sensitive dye, DAF-2. Imaging the antennal lobes after loading the cells with DAF-2 we find that odor stimulation of the antennae results in the production of NO in the antennal lobe. We are also investigating the consequences of odor induced NO production using both intracellular and multiunit extracellular recording methods. We find that NO has a profound effect on the excitability of a subset of neurons including inhibitory local interneurons and projection neurons that carry olfactory information to higher brain centers. In addition to measuring the elctrophsiological conseqeunces we are also examining the possibility that No signaling is important for olfactory learning. In collaboration with Dr. Brian Smith and Dr. Tom Christensen we are examining the effects of inhibition of NO signaling on the ability of Manduca to perform the proboscis extension reflex - an olfactory learning paradigm.

NO is not likely to be the only regulator of cGMP in the antennal lobe. MsGC-I, for example, is highly expressed in the cell bodies and processes of inhibitory interneurons within the antennal lobe. These expression patterns suggest that both NO-sensitive and NO-insensitive GCs are important for the regulation of cGMP in the olfactory system. Moreover, we have identified two different sGC beta subunits in Manduca. The first, MsGCb1, is expressed at a low level in all of the antennal lobe neurons and forms NO-sensitive heterodimers with MsGCa1. The second, MsGCb3, is expressed in a subset of antennal lobe neurons. MsGCb3 may function as a homodimer as well as a relatively NO-insensitive heterodimer with MsGCa1. We are currently establishing the exact distribution of these proteins and possible biochemical implications of co-expression of all three sGC subunits. Developmental Studies. In the developing olfactory system, axons from olfactory receptor neurons (ORNs) scattered throughout the epithelium extend into the primary olfactory neuropil to converge in spherical neuropil structures called glomeruli. This represents a very complex axon guidance task. In the moth Manduca sexta, for example, ORNs of a particular odor tuning are scattered throughout the antennal epithelium but converge to project to distinct glomeruli. Thus, throughout most of the length of the antennal nerve, unrelated axons are bundled together until they reach an area just outside of the brain. Within this area, termed the sorting zone, axons defasciculate and re-sort before entering the brain. In addition, the development of glomeruli requires not only the proper axon guidance of the ORNs, but also proliferation and extension of processes from the antennal lobe neurons including glial cells, projection neurons, and interneurons that interact with ORN axons to produce a functional unit. While many of the cellular interactions that govern these processes have been determined, the identities of the molecules that underlie those cellular interactions are unclear. Our lab is focused on understand the potential developmental roles of two different signaling systems: the nitric oxide/soluble guanylyl cyclase signaling pathway and the Eph receptor/ephrin signaling pathway. Eph receptor/ephrin signaling. Members of the Eph family of receptor tyrosine kinases and their ligands, the ephrins, have been suggested to mediate direct cell-cell communication during development. The Eph/ephrin signaling pathway has been shown to function in axon pathway selection, regionalization of the nervous system, and in neural precursor migration in many different systems. Although Eph receptors and ephrins are expressed in the developing olfactory system of both vertebrates and invertebrates, their role in mediating cell-contact communication during developmental events in the olfactory system is unknown. Our lab utilizes the complementary strengths of two insect model systems, Drosophila melanogaster and Manduca sexta, to examine the role(s) of the Eph receptor/ephrin signaling system in the development of the olfactory system. For example, we have shown that Dek, the Drosophila Eph receptor homolog, is expressed in the 3rd antennal segment; and MsEph, the Manduca Eph receptor homolog is expressed on developing ORN axons with an increase in expression at the sorting zone and within the developing glomeruli. We are building upon these observations and other preliminary data to design a series of experiments to test the following two hypotheses. First, we will test the hypothesis that Eph receptor/ephrin interactions mediate/regulate fasciculation within at least a subset of developing ORNs. Second, we will test the hypothesis that Eph receptor/ephrin interactions mediate target recognition and/or synapse formation within developing glomeruli.

Nitric oxide/soluble guanylyl cyclase signaling.
In addition to our efforts at understanding the regulation of cGMP in the adult olfactory system and its involvement in olfactory coding, our lab is interested in understanding the roles of this signaling pathway in the developing olfactory system. In a collaborative project with Dr. Leslie Tolbert's and Dr. John Hildebrand's laboratories, we are investigating the effects of pharmacological manipulation of NO signaling on the development of the Manduca antennal lobe. These ongoing studies, mainly performed by Dr. Nick Gibson, have shown that inhibition of NO signaling leads to profound effects on glial cell proliferation and axon guidance in the antennal lobe. We are now focusing on complete characterization of these effects and the biochemical pathways by which the effects are mediated.

Select Publications

Any link on the below references will take you off of the BMCB site and to an abstract of that particular paper.

Collmann, C., M.A. Carlsson, B.S. Hansson, and A. Nighorn. 2004. Odorant-evoked nitric oxide signals in the antennal lobe of Manduca sexta. Journal of Neuroscience 24: 6070-6077.

Kaneko, M., and A. Nighorn. 2003. Interaxonal Eph-ephrin signaling may mediate sorting of olfactory sensory axons in Manduca sexta. Journal of Neuroscience 23: 11523-11538.

Morton, D.B., and A. Nighorn. 2003. MsGC-II, a receptor guanylyl cyclase isolated from the CNS of Manduca sexta that is inhibited by calcium. Journal of Neurochemistry 84: 363-372.

Kumar, D.V., A. Nighorn, and P.A. St. John. 2002. Role of Nova-1 in regulating alpha2N, a novel glycine receptor splice variant, in developing spinal cord neurons. Journal of Neurobiology 52: 156-165.

Higgins, M.R., N.J. Gibson, P.A. Eckholdt, A. Nighorn, P.F. Copenhaver, J. Nardi, and L.P. Tolbert. 2002. Different isoforms of fasciclin II are expressed by a subset of developing olfactory receptor neurons and by olfactory-nerve glial cells during formation of glomeruli in the moth Manduca sexta. Developmental Biology 244: 134-154.

Nighorn, A., and J.G. Hildebrand. 2002. Dissecting the molecular mechanisms of olfaction in a malaria-vector mosquito. Proceedings of the National Academy of Sciences of the United States of America 99: 1113-1114.

Gibson, N.J., W. Rossler, A.J. Nighorn, L.A. Oland, J.G. Hildebrand, and L.P. Tolbert. 2001. Neuron-glia communication via nitric oxide is essential in establishing antennal-lobe structure in Manduca sexta. Developmental Biology 240: 326-339.

Dubuque, S.H., J. Schachtner, A.J. Nighorn, K.P. Menon, K. Zinn, and L.P. Tolbert. 2001. Immunolocalization of synaptotagmin for the study of synapses in the developing antennal lobe of Manduca sexta. Journal of Comparataive Neurology 441: 277-287.

Stengl, M., R. Zintl, J. De Vente, and A. Nighorn. 2001. Localization of cGMP immunoreactivity and of soluble guanylyl cyclase in antennal sensilla of the hawkmoth, Manduca sexta. Cell and Tissue Research 304: 409-421.

Nighorn, A., P.J. Simpson, and D.B. Morton. 2001. The novel guanylyl cyclase MsGC-I is strongly expressed in higher-order neuropils in the brain of Manduca sexta. Journal of Experimental Biology 204: 305-314.

Gibson, N.J., and A. Nighorn. 2000. Expression of nitric oxide synthase and soluble guanylyl cyclase in the developing olfactory system of Manduca sexta. Journal of Comparative Neurology 422: 191-205.

Contact Information

Mailing:
Alan Nighorn, Associate Professor
Arizona Research Laboratories -
Division of Neurobiology
University of Arizona
Gould-Simpson 626
P.O. Box 210077
Tucson AZ 85721-0077

Telephone:
520-621-9720 (Office)
520-626-6482 (Lab)

Fax:
520-621-8282

Email:
nighorn@neurobio.arizona.edu

Please e-mail any comments to:
bmcb@email.arizona.edu