Specific mesenchymal/epithelial induction of olfactory receptor, vomeronasal, and gonadotropin-releasing hormone (GnRH) neurons.

We asked whether specific mesenchymal/epithelial (M/E) induction generates olfactory receptor neurons (ORNs), vomeronasal neurons (VRNs), and gonadotropin-releasing hormone (GnRH) neurons, the major neuron classes associated with the olfactory epithelium (OE). To assess specificity of M/E-mediated neurogenesis, we compared the influence of frontonasal mesenchyme on frontonasal epithelium, which becomes the OE, with that of the forelimb bud. Despite differences in position, morphogenetic and cytogenic capacity, both mesenchymal tissues support neurogenesis, expression of several signaling molecules and neurogenic transcription factors in the frontonasal epithelium. Only frontonasal mesenchyme, however, supports OE-specific patterning and activity of a subset of signals and factors associated with OE differentiation. Moreover, only appropriate pairing of frontonasal epithelial and mesenchymal partners yields ORNs, VRNs, and GnRH neurons. Accordingly, the position and molecular identity of specialized frontonasal epithelia and mesenchyme early in gestation and subsequent inductive interactions specify the genesis and differentiation of peripheral chemosensory and neuroendocrine neurons.

Human olfactory epithelial cells generated in vitro express diverse neuronal characteristics.

The olfactory epithelium constitutes the sole source of regenerating neural cells that can be obtained from a living human. As such, primary cultures derived from human olfactory epithelial biopsies can be utilized to study neurobiological characteristics of individuals under different conditions and disease states. Here, using such human cultures, we report in vitro generation of cells that exhibit a complex neuronal phenotype, encompassing receptors and signaling pathways pertinent to both olfaction and other aspects of CNS function. Using in situ hybridization, we demonstrate for the first time the native expression of olfactory receptors in cultured cells derived from human olfactory epithelial tissue. We further establish the presence and function of olfactory transduction molecules in these cells using immunocytochemistry, calcium imaging and molecular methods. Western blot analysis revealed the expression of neurotransmitter receptors for dopamine (D2R), 5-HT (5HT2C) and NMDA subtypes 1 and 2A/2B. Stimulation with dopamine or 5-HT enhanced receptor G protein activation in a subtype specific manner, based on 35S-guanosine triphosphate incorporation assay. Functional characteristics of the cultured cells are demonstrated through enhanced tyrosine phosphorylation of NMDAR 2A/2B and recruitment of signaling partners in response to NMDA stimulation. The array of neuronal characteristics observed here establishes that proliferating cells derived from the human olfactory epithelium differentiate in vitro to express functional and molecular attributes of mature olfactory neurons. These cultured neural cells exhibit neurotransmitter pathways important in a number of neuropsychiatric disorders. Their ready availability from living humans thus provides a new tool to link functional and molecular features of neural cells with clinical characteristics of individual living patients.

Immunolocalization of retinoic acid receptors in the mammalian olfactory system and the effects of olfactory denervation on receptor distribution.

All-trans retinoic acid (ATRA), a metabolite of vitamin A, binds to retinoic acid receptors (RARs) to mediate gene transcription in target cells. We previously found that an ATRA supplement enhanced olfactory recovery rate in adult mice after olfactory bulb deafferentation. In this study, we examined the cellular localization of RARalpha, RARbeta, and RARgamma and the effects of surgery and ATRA treatment using immunocytochemistry. Mice received a left olfactory nerve transection with the right side serving as internal control. One day after surgery, the mice were given either ATRA mixed with sesame oil or just sesame oil. In the unoperated olfactory bulb, only RARalpha immunoreactivity (ir) was observed. In the unoperated right olfactory epithelium, RARalpha-ir was found in flask-shaped cells located in the supporting cell layer, in cell clusters above the basal cell layer, in cells in the lamina propria, in some respiratory cells and in the olfactory bulb. The flask-shaped cells did not immunostain for either neurons or sustentacular cells. RARbeta-ir was localized only in the respiratory cells while no RARgamma-ir was observed in the olfactory epithelium. The density of RARalpha-ir cells was higher in the operated left olfactory epithelium and highest after ATRA treatment. This study demonstrates the presence of RARs in the olfactory system, provides additional support that the ATRA-signaling pathway may be involved in the recovery of the olfactory epithelium after injury, and suggests a role for an unstudied cell type in that process.

Odorant exposure increases olfactory sensitivity: olfactory epithelium is implicated.

Exposure-induced shifts in sensitivity to odors may involve peripheral and/or central components of the olfactory system. The ability to disconnect the olfactory epithelium from the bulbs provides a unique opportunity to examine how odorant exposure affects each component. In one experiment, odor thresholds were established for either amyl acetate or androstenone. The mice were then exposed for 10 days to the same test odorant for which a threshold was obtained. After exposure, sensitivity to the odorant increased relative to preexposure levels. The mice then underwent bilateral olfactory nerve transection (BNX). When both groups of mice were tested 45-50 days after recovery from surgery and return of olfactory function, increased sensitivity to the exposed odorant persisted; however, 121-203 days after surgery, sensitivity returned to preexposure levels. Another experiment was similar to the first except that mice were exposed to an odorant, either amyl acetate or androstenone, for 10 days beginning 1 day after BNX or sham surgery. When the mice were tested 45-50 days after surgery, sensitivity to the exposed odorant was increased relative to preexposure levels, whereas sensitivity to the nonexposed odorant remained at preexposure levels. Although further work is needed to determine the precise mechanism(s) underlying shifts in sensitivity to odors, these studies provide additional evidence for peripheral involvement in exposure-induced sensitization to odorants and demonstrate the remarkable capacity of the olfactory system to maintain or even regain sensitivity after injury.

Retinoic acid enhances the rate of olfactory recovery after olfactory nerve transection.

In the olfactory system, retinoic acid (RA) plays an important role in development and may affect growth in the adult animal. To explore the potential effects of RA on recovery after injuries, adult mice were trained in a buried food paradigm and were given a single oral supplement of RA after olfactory nerve transection. Results demonstrate that RA accelerates the recovery of olfactory functions after injury.

Changes in odor quality discrimination following recovery from olfactory nerve transection.

Following recovery from olfactory nerve transection, animals regain their ability to discriminate between odors. Odor discrimination is restored after new neurons establish connections with the olfactory bulb. However, it is not known if the new connections alter odor quality perception. To address this question, 20 adult hamsters were first trained to discriminate between cinnamon and strawberry odors. After reaching criterion (> or = 90% correct response), half of the animals received a bilateral nerve transection (BTX) and half a surgical sham procedure. Animals were not tested again until day 40, a point in recovery when connections are re-established with the bulb. When BTX animals were tested without food reinforcement, they could not perform the odor discrimination task. Sham animals, however, could discriminate, demonstrating that the behavioral response had not been extinguished during the 40 day period. When reinforcement was resumed, BTX animals were able to discriminate between cinnamon and strawberry after four test sessions. In addition, their ability to discriminate between these two familiar odors was no different than that of BTX and sham animals tested with two novel odors, baby powder and coffee. These findings suggest that, after recovery from nerve transection, there are alterations in sensory perception and that restoration of odor quality discrimination requires that the animal must again learn to associate individual odor sensations with a behavioral response.

Restoration of olfactory mediated behavior after olfactory bulb deafferentation.

Transection of olfactory nerve fibers leads to deafferentation of olfactory bulbs and a loss of olfactory mediated behavior. Nerve transection studies have shown that during recovery, olfactory nerve fibers can reestablish connections with the olfactory bulbs. Two groups of experimental animals were studied to determine if olfactory mediated behavior returns after recovery. One group (n = 18) received bilateral olfactory nerve transection (BTX), while the second group (n = 4) received a sham surgical procedure. Performance on odor detection and discrimination tasks was measured during recovery periods ranging from 1-120 days. Return of olfactory mediated behavior was first observed 19 days after nerve transection. Performance levels improved with recovery time and by day 40 animals returned to criterion level (> or = 90% correct response). Sham animals maintained a criterion level of performance throughout the recovery period. Horseradish peroxidase (HRP) was used to trace reconnection of olfactory nerve fibers. The absence of HRP label in the bulbs of animals examined one day after BTX, verified the completeness of the nerve transection procedure. After 10 days of recovery, a few HRP labeled axons were observed and the amount of HRP in the bulb increased with recovery time. The results of this study demonstrate that olfactory receptor axons can reestablish functional connections with the deafferented olfactory bulb and these connections are sufficient to restore olfactory mediated behavior.

Ellipsometric investigation of quadratic electrooptic and electrostrictive effects with anodic niobium pentoxide films.

Anodic oxide films on niobium were studied in situ during formation using a computer-controlled ellipsometer at a wavelength of 6328 A. They were found to be optically homogeneous, isotropic, and nonabsorbing with no field applied but to become anisotropic (optically uniaxial with optic axis normal to the film surface) with field applied. The ordinary and extraordinary indices decreased, and the thickness increased quadratically with applied field. Values are reported for the refractive index of niobium and of the oxide, and these are compared with previous reports. The quadratic electrooptic coefficients for the oxide are reported and are of the same order of magnitude as those for oxygen-octahedra ferroelectrics and somewhat greater than the values for tantalum pentoxide films.