Primary culture of lobster (Homarus americanus) olfactory sensory neurons

Lobster olfactory sensory neurons have contributed to a number of advances in our understanding of olfactory physiology. To facilitate further study of their function, we have developed conditions allowing primary culture of the olfactory sensory neurons in a defined medium. The most common cells in the culture were round cell bodies with diameters of 10-15 micro m that often extended fine processes, features resembling olfactory sensory neurons. We discovered that acetylcholinesterase acted as a growth factor for these cells, improving their survival in culture. We also confirmed previous evidence from spiny lobsters that poly-D-lysine was a superior substrate for olfactory cells of this size and morphology. We then identified olfactory sensory neurons in the culture in two ways. Almost half the cells tested responded to application of a complex odorant with an inward current. An even more rigorous test was made possible by the development of an antiserum to OET-07, an ionotropic glutamate receptor homolog specifically expressed by Homarus americanus olfactory sensory neurons. It labeled a majority of the round cells in the culture, unequivocally identifying them as olfactory sensory neurons.     

Analysis of the functional maturation of olfactory neurons in chicks before and after birth

There has been indirect evidence that the olfactory system of mammals could be functional shortly before birth. Taking advantage of the accessibility of bird embryos, we studied the functional maturation of the olfactory mucosa during embryonic development in birds. Using the combination of electrophysiological EOG recordings and immunohistochemical studies, it was possible to directly demonstrate for the first time that the olfactory system is functional during embryogenesis from embryonic day (ED) 13 and that the beginning of olfactory function coincides with the first localization of the calcium dependent calmodulin kinase II (CaMKIIalpha) in the dendrites of the olfactory receptor neurons. CaMKII and olfactory receptor genes are expressed much earlier in olfactory neurons, both involved in the sensory transduction, but the pattern of expression of CaMKIIalpha changes during the ontogenesis. The increase of EOG amplitude between ED13 and ED15 also coincides with the increase of the number of neurons presenting the dendritic localization of CaMKIIalpha. These results suggest that the enzyme CaMKII might play a role in the functional maturation of the olfactory mucosa.     

The influence of training on chemosensory event-related potentials and interactions between the olfactory and trigeminal systems

It is not possible to accurately predict the perceptual response to odorants and odorant mixtures without understanding patterns of suppression and facilitation that result from interactions between the olfactory and trigeminal systems. The current study extends previous findings by exploring the effect of intensive training on the interaction between these systems and also by using a different mixed chemosensory stimulus to examine whether the principles established in earlier studies generalize to different odorants. Stimuli were chosen so as to selectively activate the olfactory (H2S) and trigeminal (CO2) nerves. In addition, linalool was included as a stimulus that activated both systems. Thirty-five participants (19 men, 16 women) rated the intensity of each stimulus when presented both alone and in binary mixtures (linalool + H2S, and linalool + CO2). Chemosensory event-related potentials were obtained from three recording positions. Analysis of intensity ratings showed that linalool was significantly less intense than the other stimuli when presented alone. In binary mixtures, H2S was strongly suppressed by linalool. One week of intensive odor training produced significant and specific reductions in the intensity of linalool and H2S, both alone and in their mixture. Training with a different odor (champignol) had no effect. Chemosensory event-related potential data confirmed previous findings showing changes in topographical distribution that reflected the degree of trigeminal activity. Binary mixtures generally produced larger amplitudes than single stimuli. Latencies clearly differentiated between the three single stimuli and the binary mixtures. Changes were observed in event-related potentials that reflected those obtained for intensity ratings in that they were observed for linalool and H2S in the linalool trained group only. The amplitude of the late 'endogenous' component (P3) was significantly decreased for these odors at frontal recording sites. In summary, strong and specific training effects were observed in intensity ratings for participants trained with the test odor (linalool), but not for those trained with a different odor. This was supported by a significant decrease of amplitudes of the event-related potentials at frontal recording sites following training with the test odor only     

Characterization of the synaptic properties of olfactory bulb projections

The olfactory bulb directly projects to several diverse telencephalic structures, but, to date, few studies have investigated the physiological characteristics of most of these areas. As an initial step towards understanding the odor processing functions of these secondary olfactory structures, we recorded evoked field potentials in response to lateral olfactory tract stimulation in vivo in urethane-anesthetized Sprague-Dawley rats in the following brain structures: anterior olfactory nucleus, ventral and dorsal tenia tecta, olfactory tubercle, anterior and posterior piriform cortex, the anterior cortical nucleus of the amygdala, and lateral entorhinal cortex. Using paired-pulse stimulation with interpulse intervals of 25-1000 ms, we observed facilitation of the response to the second pulse in every structure examined, although the degree of facilitation varied among the target structures. Additionally, pulse train stimulation at three different frequencies (40, 10 and 2 Hz) produced facilitation of evoked field potentials that also varied among target structures. We discuss the potential utility of such short-term facilitation in olfactory processing.     

An enhanced olfactory marker protein immunoreactivity in individual olfactory receptor neurons following olfactory bulbectomy may be related to increased neurogenesis

Olfactory marker protein (OMP) is a 19-kD acidic protein found throughout the cytoplasm of mature olfactory receptor neurons (ORNs). Its function remains unknown. Following olfactory bulbectomy, the proportion of ORNs mature enough to express OMP declines greatly. However, in the few remaining mature ORNs, it has been observed that the intensity of OMP immunoreactivity (IR) appears to increase over that of ORNs on the unoperated side. We have now investigated this phenomenon quantitatively in rats subjected to unilateral olfactory bulbectomy. Results show that at all postbulbectomy survival periods examined quantitatively (3 days to 6 months), a significant decrease (19–37%) occurs in the transmission of incident light through OMP(+)-ORNs in bulbectomized versus unoperated olfactory epithelium (OE). Further, we also observed a consistent side-to-side difference in OMP IR in control unoperated animals. Possible explanations for these observations and their relation to the still unknown function of OMP are discussed. To test the possibility that OMP might serve a mitogenic role in the OE, recombinant OMP was added to organotypic explant cultures of fetal olfactory mucosa. Addition of OMP resulted in a dose-dependent increase in the density of bromodeoxyuridine-positive cells in the cultures, with a 50% increase occurring at the plateau OMP concentration of 25 nM. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 377–390, 1998     

Neurodevelopmental involvement in schizophrenia: the olfactory epithelium as an alternative model for research

It has been suggested that disturbances during neurodevelopment may play a crucial role in the etiology of schizophrenia (SZ). This premise is supported by brain imaging, epidemiological, and pathological studies as well as the discovery of susceptibility genes for SZ that appear to be implicated in development of the central nervous system. Here, we discuss the limitations of the current methods and models for studying the neurodevelopmental implications in SZ. We agree with the proposal that the olfactory epithelium, in which neurodevelopment continues throughout life, might represent an alternative model for understanding the pathophysiology of the disorder.     

Axon mis-targeting in the olfactory bulb during regeneration of olfactory neuroepithelium

During development, primary olfactory axons typically grow to their topographically correct target zone without extensive remodelling. Similarly, in adults, new axons arising from the normal turnover of sensory neurons essentially project to their target without error. In the present study we have examined axon targeting in the olfactory pathway following extensive chemical ablation of the olfactory neuroepithelium in the P2-tau:LacZ line of mice. These mice express LacZ in the P2 subpopulation of primary olfactory neurons whose axons target topographically fixed glomeruli on the medial and lateral surfaces of the olfactory bulb. Intraperitoneal injections of dichlobenil selectively destroyed the sensory neuroepithelium of the nasal cavity without direct physical insult to the olfactory neuron pathway. Primary olfactory neurons regenerated and LacZ staining revealed the trajectory of the P2 axons. Rather than project solely to their topographically appropriate glomeruli, the regenerating P2 axons now terminated in numerous inappropriate glomeruli which were widely dispersed over the olfactory bulb. While these errors in targeting were refined over time, there was still considerable mis-targeting after four months of regeneration.     

Membrane and junctional properties of the isolated frog lens epithelium

Summary The isolated frog lens epithelium can be maintained intact in both appearance and electrical properties for more than 24 hours. The mean resting membrane potential was –80 mV and the cells were depolarized by both high potassium and low calcium Ringer's solution in a manner very similar to that of the whole lens. The epithelial cells were found to be well coupled using both electrical and dye-injection techniques. Electrical coupling was measured using separate current-injection and voltage-measuring electrodes and the relationship between the induced voltage and distance from the current-passing electrode could be well fitted by a Bessel Function solution to the cable equation. The values obtained from the fit for the membrane and internal resistances were 1.95 m² and 25 m, respectively. Exposure to octanol (500m) or low external Ca²⁺ (<1m) failed to disrupt significantly the intercellular flow of current. There was evidence to suggest thatraised intracellular calcium does, however, uncouple the cells. Dye coupling was investigated by microinjecting Lucifer Yellow CH into single epithelial cells. Diffusion into surrounding cells was rapid and, in control medium, occurred in a radially symmetrical manner. In contrast to the electrical coupling data, dye transfer appeared to be blocked by exposure to 500 m octanol and was severely restricted on perfusing with low external calcium. Differences between the electrical and dye-coupling experiments indicate either that there are two types of junction within the cell and only the larger type, permeable to Lucifer Yellow, is capable of being uncoupled or that there is only one large type of junction which can be partially closed by uncoupling agents.     

Pheromone-binding proteins contribute to the activation of olfactory receptor neurons in the silkmoths antheraea polyphemus and Bombyx mori

The sensilla trichodea of the silkmoth Antheraea polyphemus are innervated by three types of receptor neurons each responding specifically to one of three pheromone components. The sensillum lymph of these sensilla surrounding the sensory dendrites contains three different types of pheromone-binding proteins (PBPs) in high concentrations. The sensilla trichodea of the silkmoth Bombyx mori are supplied by two receptor neurons each tuned specifically to one of the two pheromone components bombykol and bombykal, but only one type of PBP has been found so far in these sensilla. Recombinant PBPs of both silkmoth species in various combinations with pheromone components were applied to the receptor neurons via tip-opened sensilla during electrophysiological recordings. Over a fairly broad range of pheromone concentrations the responses of the receptor neurons depended on both, the pheromone component and the type of the PBP. Therefore, the PBPs appear to contribute to the excitation of the receptor neurons. Furthermore, bombykal in combination with the expressed PBP of B. mori failed to activate the corresponding receptor neuron of B. mori, but did so if combined with one of the PBPs of A. polyphemus. Therefore, a still unknown binding protein involved in bombykal transport might be present in B. mori.     

The septal organ of the rat during postnatal development

The septal organ of Masera (SO) is a small, isolated patch of olfactory epithelium, located in the ventral part of the nasal septum. We investigated in this systematic study the postnatal development of the SO in histological sections of rats at various ages from the day of birth (P1) to P666. The SO-area increases to a maximum at P66-P105, just as the animals reach sexual maturity, and decreases thereafter, significantly however only in males, indicating a limited neurogenetic capacity for regeneration. In contrast, the main olfactory epithelium area continues to expand beyond P300. The modified respiratory epithelium ('zwischen epithelium') separating the SO and the main olfactory epithelium contains a few olfactory neurons up to age P66. Its length increases postnatally so that the SO becomes more ventral to the OE. Although the position of the SO relative to other anatomical landmarks changes with development it is consistently located just posterior to the opening of the nasopalatine duct (NPAL). Thus, a possible function of the SO is in sensing chemicals in fluids entering the mouth by licking and then delivered to the nasal cavity via the NPAL; therefore the SO may be involved in social/sexual behavior as is the vomeronasal organ (VNO). We suggest that the SO is a separate accessory olfactory organ with properties somewhat different from both OE and VNO and may exist only in species where the NPAL does not open into the VNO.     

Odorant-dependent, spatially restricted induction of c-fos in the olfactory epithelium of the mouse

Volatile odorous chemicals are detected by around a thousand different G protein-coupled odorant receptors in the mouse. We demonstrated that exposure of the behaving mouse to odorant for a few minutes led to induction of the immediate early gene c-fos for several hours in a fraction of the olfactory sensory neurones in the nasal cavity. Associated with this odorant-specific induction event was activation of extracellular-regulated kinase (ERK)1/2 that preceded increased c-fos expression. The distribution of odorant-activated neurones mimicked the scattered and spatially limited distribution of neurones expressing a single odorant receptor gene. A small change in odorant chemical structure caused a zonal shift in the spatial distribution of activated neurones, suggesting that the gene expression change resulted from specific receptor interaction. Repeated exposure to odorant or use of different concentrations did not change the pattern of c-fos induction. These results indicate that odorant-induced c-fos expression can be used to visualize odorant representations in the olfactory epithelium that reflect late cellular events regulated by adequate odorant receptor stimulation.     

Communicating (gap) junctions between chloride cells in the gill epithelium of the lamprey,Geotria australis

Summary Freeze-fracture replicas show that communicating (gap) junctions are present between chloride cells in the gill epithelium of young adults of the Southern Hemisphere lamprey, Geotria australis, acclimated to full-strength sea water. The junctions, which were already present when these lampreys were migrating downstream, may help coordinate the secretory activities of the chloride cells during the marine phase of the lamprey life cycle.     

Taurine action on mitral cell activity in the frog olfactory bulb in vivo

Taurine (TAU) is a free amino acid that is particularly abundant in the olfactory bulb. In the frog, TAU is located in the terminations of the primary olfactory axons and in the granular cell layer. TAU action seems to be associated with gamma amino butyric acid (GABA), the main inhibitory neurotransmitter involved in the processing of the sensory signal. The present study was designed to assess the action of TAU in vivo during the olfactory network's stimulation by odors. It was performed by recording the single-unit activity of mitral cells, the main bulbar output neurons. TAU effects were tested on both their spontaneous and odor-induced firing activity. Interactions between TAU and GABA were examined by analyzing TAU effects under the selective blocking action of GABAA or GABAB antagonists. TAU was found to suppress the spontaneous firing of mitral cells, mainly without altering their odor response properties. By testing GABA antagonists, we further show that TAU action is associated with GABAergic inhibitory mechanisms mainly via GABAB receptors. Thus, TAU action clearly reduces background activity in favor of the emergence of the odor-induced activity in the same manner as GABA action does via GABAB receptors. As a conclusion, we propose that, in the frog olfactory bulb, the joint actions of TAU and GABA may favor the processing of the primary sensory information by increasing the signal to noise ratio.     

Ultrastructure of cell types of the olfactory epithelium in a catfish,Heteropneustesfossilis (Bloch)

Transmission electron microscopical study of olfactory epithelium of a mud-dwelling catfish,Heteropneustes fossilis (Bloch) shows receptor, supporting, goblet and basal cells. The receptor cells are of ciliated and microvillous type. Both ciliated and microvillous receptor cells are provided with olfactory knob. The dendrite of all the receptor cells bears many longitudinally arranged microtubules. Occurrence of the rod cell and its function is quite debatable. Specialized juctional complexes between the receptor and adjacent cells are clearly noted. The supporting cells are both ciliated and nonciliated. The ciliated supporting cells are responsible for water ventilation in the olfactory chamber as well as in the inter-lamellar spaces. This facilitates better perception of odours by the receptor cells. In addition to providing mechanical support to other cells, the nonciliated supporting cells also have a secretory function which is evident from the present study. The different stages of maturity of goblet cells are well documented. The presence of white cells in the olfactory epithelium is a very rare finding.     

IP3-and cAMP-induced responses in isolated olfactory receptor neurons from the channel catfish

Summary Olfactory receptor neurons enzymatically dissociated from channel catfish olfactory epithelium were depolarized transiently following dialysis of IP₃ or cAMP (added to the patch pipette) into the cytoplasm. Voltage and current responses to IP₃ were blocked by ruthenium red, a blocker of an IP₃-gated Ca²⁺-release channel in sarcoplasmic reticulum. In contrast, the responses to cAMP were not blocked by extracellularly applied ruthenium red, nor by l-cis-diltiazem or amiloride and two of its derivatives. The current elicited by cytoplasmic IP₃ in neurons under voltage clamp displayed a voltage dependence different from that of the cAMP response which showed marked outward rectification. A sustained depolarization was caused by increased cytoplasmic IP₃ or cAMP when the buffering capacity for Ca²⁺ of the pipette solution was increased, when extracellular Ca²⁺ was removed or after addition of 20–200 nm charibdotoxin to the bathing solution, indicating that the repolarization was caused by an increase in [Ca _i ] that opened Ca²⁺-activated K⁺ channels. The results suggest that different conductances modulated by either IP₃ or cAMP are involved in mediating olfactory transduction in catfish olfactory receptor neurons and that Ca²⁺-activated K⁺ channels contribute to the termination of the IP₃ and cAMP responses.Abbreviations ATP adenosine 5-triphosphate - BAPTA (bis-(o-aminophenoxy)-ethane-N-N-N-N)-tetraacetic acid - cAMP adenosine cyclic 3,5-monophosphate - cGMP guanosine cyclic 3,5-monophosphate - CTX charybdotoxin - DCB 3,4-dichlorobenzamil - EDTA ethylenediaminetetraacetic acid - EGTA ethylenglycol-bis-(b-aminoethyl)-N-N-N-N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - IP₃ inositol-1,4,5-triphosphate - NMDG N-methyl-d-glucamine We would like to thank the Tanabe Seiyaku Co., Ltd., for their gift of l-cis-diltiazem. This work was supported by National Institutes of Health grants DC00566 and BRSG S07RR05825.     

Six1 is essential for early neurogenesis in the development of olfactory epithelium

The olfactory epithelium (OE) is derived from the olfactory placode (OP) during mouse development. At embryonic day (E) 10.0-E10.5, “early neurogenesis” occurs in the OE, which includes production of pioneer neurons that emigrate out of the OE and other early-differentiated neurons. Around E12.5, the OE becomes organized into mature pseudostratified epithelium and shows “established neurogenesis,” in which olfactory receptor neurons (ORNs) are differentiated from basal progenitors. Little is known about the molecular pathway of early neurogenesis. The homeodomain protein Six1 is expressed in all OP cells and neurogenic precursors in the OE. Here we show that early neurogenesis is severely disturbed despite the unaltered expression of Mash1 at E10.5 in the Six1-deficient mice (Six1^−/−). Expression levels of neurogenin1 (Ngn1) and NeuroD are reduced and those of Hes1 and Hes5 are augmented in the OE of Six1^−/− at E10.5. Pioneer neurons and cellular aggregates, which are derived from the OP/OE and situated in the mesenchyme between the OE and forebrain, are completely absent in Six1^−/−. Moreover, ORN axons and the gonadotropin-releasing hormone-positive neurons fail to extend and migrate to the forebrain, respectively. Our study indicates that Six1 plays critical roles in early neurogenesis by regulating Ngn1, NeuroD, Hes1, and Hes5.     

Olfactory sensitivity for carboxylic acids in spider monkeys and pigtail macaques

Using a conditioning paradigm, the olfactory sensitivity of four spider monkeys and four pigtail macaques for a homologous series of carboxylic acids (n-propionic acid to n-heptanoic acid) was investigated. With only few exceptions, the animals of both species significantly discriminated concentrations <1 p.p.m. from the odorless solvent and in several cases individual monkeys even demonstrated thresholds <1 p.p.b. The results showed (i). both primate species to have a well-developed olfactory sensitivity for carboxylic acids, which for some substances matches or even is markedly better than that of species such as the rat or the dog and (ii). a significant correlation between perceptibility in terms of olfactory detection thresholds and carbon chain length of the carboxylic acids in both species tested. These findings lend further support to the growing body of evidence suggesting that between-species comparisons of the number of functional olfactory receptor genes or of neuroanatomical features are poor predictors of olfactory performance, and that general labels such as 'microsmat' or 'macrosmat'-which usually are based on allometric comparisons of olfactory brain structures-are inadequate to describe a species' olfactory capabilities.     

Gap-junction blocker carbenoxolone differentially enhances NMDA-induced cell death in hippocampal neurons and astrocytes in co-culture

The beneficial or detrimental role of gap junction communication in the pathophysiology of brain injury is still controversial. We used co-cultures of hippocampal astrocytes and neurons, where we identified homocellular astrocyte-astrocyte and heterocellular astrocyte-neuron coupling by fluorescence recovery after photobleaching, which was decreased by the gap junction blocker carbenoxolone (CBX). In these cultures, we determined the cell type-specific effects of CBX on the excitotoxic damage caused by N-methyl-D-aspartate (NMDA). We determined in both astrocytes and neurons the influence of CBX, alone or together with NMDA challenge, on cytotoxicity using propidium iodide labeling. CBX alone was not cytotoxic, but CBX treatment differentially accelerated the NMDA-induced cell death in both astrocytes and neurons. In addition, we measured mitochondrial potential using rhodamine 123, membrane potential using the oxonol dye bis(1,3-diethylthiobarbituric acid)trimethine oxonol, cytosolic Ca(2+) level using fura-2, and formation of reactive oxygen species (ROS) using dihydroethidium. CBX alone induced neither an intracellular Ca(2+) rise nor a membrane depolarization. However, CBX elicited a mitochondrial depolarization in both astrocytes and neurons and increased the ROS formation in neurons. In contrast, NMDA caused a membrane depolarization in neurons, coinciding with intracellular Ca(2+) rise, but neither mitochondrial depolarization nor ROS production seem to be involved in NMDA-mediated cytotoxicity. Pre-treatment with CBX accelerated the NMDA-induced membrane depolarization and prevented the repolarization of neurons after the NMDA challenge. We hypothesize that these effects are possibly mediated via blockage of gap junctions, and might be involved in the mechanism of CBX-induced acceleration of excitotoxic cell death, whereas the CBX-induced mitochondrial depolarization and ROS formation are not responsible for the increase in cytotoxicity. We conclude that both in astrocytes and neurons gap junctions provide protection against NMDA-induced cytotoxicity.