The 2000 Ogura Lecture: Olfactory neural cells: An untapped diagnostic and therapeutic resource

Objective. This is an over-view of the cellular biology of upper nasal mucosal cells that have special characteristics that enable them to be used to diagnose and study congenital neurological diseases and to aid neural repair. Study Design: After mapping the distribution of neural cells in the upper nose, the authors' investigations moved to the use of olfactory neurones to diagnose neurological diseases of development, especially schizophrenia. Olfactory-ensheating glial cells (OEGs) from the cranial cavity promote axonal penetration of the central nervous system and aid spinal cord repair in rodents. The authors sought to isolate these cells from the more accessible upper nasal cavity in rats and in humans and prove they could likewise promote neural regeneration, making these cells suitable for human spinal repair investigations. Methods: The schizophrenia-diagnosis aspect of the study entailed the biopsy of the olfactory areas of 10 schizophrenic patients and 10 control subjects. The tissue samples were sliced and grown in culture medium. The ease of cell attachment to fibronectin (artificial epithelial basement membrane), as well as the mitotic and apoptotic indices, was studied in the presence and absence of dopamine in those cell cultures. The neural repair part of the study entailed a harvesting and insertion of first rat olfactory lamina propria rich in OEGs between cut ends of the spinal cords and then later the microinjection of an OEG-rich suspension into rat spinal cords previously transected by open laminectomy. Further studies were done in which OEG insertion was performed up to 1 month after rat cord transection and also in monkeys. Results: Schizophrenic patients' olfactory tissues do not easily attach to basement membrane compared with control subjects, adding evidence to the theory that cell wall anomalies are part of the schizophrenic lesion of neurones. Schizophrenic patient cell cultures had higher mitotic and apoptotic indices compared with control subjects. The addition of dopamine altered these indices enough to allow accurate differentiation of schizophrenics from control patients, leading to, possibly for the first time, an early objective diagnosis of schizophrenia and possible assessment of preventive strategies. OEGs from the nose were shown to be as effective as those from the olfactory bulb in promoting axonal growth across transected spinal cords even when added I month after injury in the rat. These otherwise paraplegic rats grew motor and proprioceptive and fine touch fibers with corresponding behavioral improvement. Conclusions. The tissues of the olfactory mucosa are readily available to the otolaryngologist. Being surface cells, they must regenerate (called neurogenesis). Biopsy of this area and amplification of cells in culture gives the scientist a window to the developing brain, including early diagnosis of schizophrenia. The Holy Grail of neurological disease is the cure of traumatic paraplegia and OEGs from the nose promote that repair. The otolaryngologist may become the necessary partner of the neurophysiologist and spinal surgeon to take the laboratory potential of paraplegic cure into the day-to-day realm of clinical reality.

Effects of polyunsaturated fatty acids on voltage-gated K+ and Na+ channels in rat olfactory receptor neurons

Although the polyunsaturated fatty acids arachidonic acid (AA) and docosahexaenoic acid (DHA) are enriched in the olfactory mucosa, their possible contribution to olfactory transduction has not been investigated. This study characterized their effects on voltage-gated K+ and Na+ channels of rat olfactory receptor neurons. Physiological (3-10 mum) concentrations of AA and DHA potently and irreversibly inhibited the voltage-gated K+ current in a voltage-independent manner. In addition, both compounds significantly reduced the inhibitory potency of the odorants acetophenone and amyl acetate at these channels. By comparison, the steady-state effects of both AA and DHA on the voltage-gated Na+ channel were relatively weak, with half-maximal inhibition requiring approximate to 35 mum of either compound. However, a surprising finding was that the initial application of 3 mum AA to a naive neuron caused a strong but transient inhibition of the Na+ current. The channels became almost completely resistant to this inhibition within 1 min, and a 2-min wash in control solution was insufficient to restore the strong inhibitory effect. These observations suggest that polyunsaturated fatty acids have the potential to strongly influence the coding of odorant information by olfactory receptor neurons.

Sorting and convergence of primary olfactory axons are independent of the olfactory bulb

Primary olfactory axons expressing the same odorant receptor gene sort out and converge to fixed sites in the olfactory bulb. We examined the guidance of axons expressing the P2 odorant receptor when they were challenged with different cellular environments in vivo. In the mutant extratoes mouse, the olfactory bulb is lacking and is replaced by a fibrocellular mass. In these animals, primary olfactory axons form glomerular-like loci despite the absence of normal postsynaptic targets. P2 axons are able to sort out from other axons in this fibrocellular mass and converge to form loci of like axons. The sites of these loci along mediolateral and ventrodorsal axes were highly variable. Similar convergence was observed for larger subpopulations of axons expressing the same cell surface carbohydrates. The sorting out and convergence of like axons also occurred during regeneration following bulbectomy. Olfactory axon behaviour in these models demonstrates that sorting and convergence of axons are independent of the target, which instead provides distinct topographic cues for guidance. (C) 2003 Wiley-Liss, Inc.

A Lower Olfactory Capacity Is Related to Higher Circulating Concentrations of Endocannabinoid 2-Arachidonoylglycerol and Higher Body Mass Index in Women.

The endocannabinoid (eCB) system can promote food intake by increasing odor detection in mice. The eCB system is over-active in human obesity. Our aim is to measure circulating eCB concentrations and olfactory capacity in a human sample that includes people with obesity and explore the possible interaction between olfaction, obesity and the eCB system. The study sample was made up of 161 females with five groups of body mass index sub-categories ranging from under-weight to morbidly obese. We assessed olfactory capacity with the "Sniffin´Sticks" test, which measures olfactory threshold-discrimination-identification (TDI) capacity. We measured plasma concentrations of the eCBs 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine or anandamide (AEA), and several eCB-related compounds, 2-acylglycerols and N-acylethanolamines. 2-AG and other 2-acylglycerols fasting plasma circulating plasma concentrations were higher in obese and morbidly obese subjects. AEA and other N-acylethanolamine circulating concentrations were lower in under-weight subjects. Olfactory TDI scores were lower in obese and morbidly obese subjects. Lower TDI scores were independently associated with higher 2-AG fasting plasma circulating concentrations, higher %body fat, and higher body mass index, after controlling for age, smoking, menstruation, and use of contraceptives. Our results show that obese subjects have a lower olfactory capacity than non-obese ones and that elevated fasting plasma circulating 2-AG concentrations in obesity are linked to a lower olfactory capacity. In agreement with previous studies we show that eCBs AEA and 2-AG, and their respective congeners have a distinct profile in relation to body mass index. The present report is the first study in humans in which olfactory capacity and circulating eCB concentrations have been measured in the same subjects.

Visualizing olfactory receptor expression and localization in Drosophila.

Odor detection and discrimination by olfactory systems in vertebrates and invertebrates depend both on the selective expression of individual olfactory receptor genes in subpopulations of olfactory sensory neurons, and on the targeting of the encoded proteins to the exposed, ciliated endings of sensory dendrites. Techniques to visualize the expression and localization of olfactory receptor gene products in vivo have been essential to reveal the molecular logic of peripheral odor coding and to permit investigation of the developmental and cellular neurobiology of this sensory system. Here, we describe methods for detection of olfactory receptor transcripts and proteins in the antennal olfactory organ of the fruit fly, Drosophila melanogaster, an important genetic model organism. We include protocols both for antennal cryosections and whole-mount antennae. These methods can be adapted for detection of receptor expression in other olfactory and gustatory tissues in Drosophila, as well as in the chemosensory systems of other insects.

Olfactory mediated interactions between Citrus aurantium Toxoptera citricida and Lysiphlebus testaceipes

The objective of this study was to establish whether there are olfactory interactions in the Lysiphlebus testaceipes Toxoptera citricida and Citrus aurantium tritrophic system. The response of male and female L. testaceipes to different odour sources of the host plant C. aurantium, the aphid host T. citricida and aphid-plant complex were investigated using a Y-tube olfactometer. Laboratory experiments were conducted by exposing individually aged male and female L. testaceipes to eight different odour treatments. Response of the parasitoids was taken after 15 min exposure to the volatiles from the different odour sources and based on their orientation to the particular chamber. Seventy percent of both male and female L. testaceipes showed high attractivity to aphid infested leaves. There was no significant difference based on age and sex of the parasitoid on their choice of odour. The organic compounds released by these combinations acted as semiochemicals in the tritrophic interactions and it is suggested that insect feeding induced attraction of the parasitoid L. testaceipes.

Myelin-associated proteins block the migration of olfactory ensheathing cells: an in vitro study using single cell tracking and traction force microscopy

Newly generated olfactory receptor axons grow from the peripheral to the central nervous system aided by olfactory ensheathing cells (OECs). Thus, OEC transplantation has emerged as a promising therapy for spinal cord injuries and for other neural diseases. However, these cells do not present a uniform population, but, instead, a functionally heterogeneous population that exhibits a variety of responses including adhesion, repulsion and crossover during cell-cell and cell-matrix interactions. Some studies report that the migratory properties of OECs are compromised by inhibitory molecules and potentiated by chemical gradients. Here, we demonstrated that rodent OECs express all the components of the Nogo Receptor complex and that their migration is blocked by Myelin. Next, we used cell tracking and traction force microscopy to analyze OEC migration and its mechanical properties over Myelin. Our data relate the absence of traction force of OEC with lower migratory capacity, which correlates with changes in the F-Actin cytoskeleton and focal adhesion distribution. Lastly, OEC traction force and migratory capacity is enhanced after cell incubation with the Nogo Receptor inhibitor NEP1-40.

Dynamic characterization of olfactory receptors in the fruit fly Drosophila melanogaster

Temporal changes in odor concentration are vitally important to many animals orienting and navigating in their environment. How are such temporal changes detected? Within the scope of the present work an accurate stimulation and analysis system was developed to examine the dynamics of physiological properties of Drosophila melanogaster olfactory receptor organs. Subsequently a new method for delivering odor stimuli was tested and used to present the first dynamic characterization of olfactory receptors at the level of single neurons. Initially, recordings of the whole antenna were conducted while stimulating with different odors. The odor delivery system allowed the dynamic characterization of the whole fly antenna, including its sensilla and receptor neurons. Based on the obtained electroantennogram data a new odor delivery method called digital sequence method was developed. In addition the degree of accuracy was enhanced, initially using electroantennograms, and later recordings of odorant receptor cells at the single sensilla level. This work shows for the first time that different odors evoked different responses within one neuron depending on the chemical structure of the odor. The present work offers new insights into the dynamic properties of olfactory transduction in Drosophila melanogaster and describes time dependent parameters underlying these properties.

Umami: a delicious flavor formed by convergence of taste and olfactory pathways in the human brain

Umami taste is produced by glutamate acting on a fifth taste system. However, glutamate presented alone as a taste stimulus is not highly pleasant, and does not act synergistically with other tastes (sweet, salt, bitter and sour). We show here that when glutamate is given in combination with a consonant, savory, odour (vegetable), the resulting flavor can be much more pleasant. Moreover, we showed using functional brain imaging with fMRI that the glutamate taste and savory odour combination produced much greater activation of the medial orbitofrontal cortex and pregenual cingulate cortex than the sum of the activations by the taste and olfactory components presented separately. Supralinear effects were much less (and significantly less) evident for sodium chloride and vegetable odour. Further, activations in these brain regions were correlated with the pleasantness and fullness of the flavor, and with the consonance of the taste and olfactory components. Supralinear effects of glutamate taste and savory odour were not found in the insular primary taste cortex. We thus propose that glutamate acts by the nonlinear effects it can produce when combined with a consonant odour in multimodal cortical taste-olfactory convergence regions. We propose the concept that umami can be thought of as a rich and delicious flavor that is produced by a combination of glutamate taste and a consonant savory odour. Glutamate is thus a flavor enhancer because of the way that it can combine supralinearly with consonant odours in cortical areas where the taste and olfactory pathways converge far beyond the receptors.

Cognitive Deficits in Machado-Joseph Disease Correlate with Hypoperfusion of Visual System Areas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cognitive and olfactory deficits in Machado-Joseph disease: A dopamine transporter study

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Astrocyte intermediate filaments are important markers in olfactory bulb of bovine herpesvirus type 5 natural infections

Astroglial cells are the most abundant cells in the mammalian central nervous system, yet our knowledge about their function in bovine Herpesvirus type 5 (BoHV-5) has been limited. The aim of this study was to detect by immunohistochemistry assay the reactive astrocytes for glial fibrilary acidic protein (GFAP) and vimentin (VIM), considered intermediate filaments of the cytoskeleton, localized in olfactory bulb from natural acute cases of BoHV-5 infection. All samples were submitted to virus isolation, real-time polymerase chain reaction (RT-PCR) and in situ hybridization (ISH) technique to confirm the virus transcription and respective genome. Samples were classified into four groups according to the severity of histological lesions. Groups III and IV, which histological lesions were classified as alacia, gliosis, satellitosis, neuronophagia and neuronal necrosis, 35% (± 1.8-2.1) of the inflammatory mononuclear cells, corresponded to CD3 positive lymphocytes. In the same group, 35% (± 1.8) of astrocytes were described as reactive to GFAP and VIM proteins. An agreement of r = 1.0 (P<0.0001) was found between histological lesions, intermediate filaments expression, viral DNA and transcription and CD3 lymphocytes. However, samples with mild histological lesions, 10.8 to 14.2% of astrocytes were classified as reactive to GFAP and VIM filaments. Our findings suggest that GFAP and VIM reactive astrocytes, in primary site of virus replication, seems to play an important role in neurovirulence, in spite of many questions concerning the virus immunopathology remains unclear.

optomotor-blind and the horizontal and vertical system cells of the drosophila optic lobes

The horizontal and vertical system neurons (HS and VS cells) are part of a conserved set of lobula plate giant neurons (LPGNs) in the optic lobes of the adult brain. Structure and physiology of these cells are well known, predominantly from studies in larger Dipteran flies. Our knowledge about the ontogeny of these cells is limited and stems predominantly from laser ablation studies in larvae of the house fly Musca domestica. These studies suggested that the HS and VS cells stem from a single precursor, which, at least in Musca, has not yet divided in the second larval instar. A regulatory mutation (In(1)omb[H31]) in the Drosophila gene optomotor-blind (omb) leads to the selective loss of the adult HS and VS cells. This mutation causes a transient reduction in omb expression in what appears to be the entire optic lobe anlage (OLA) late in embryogenesis. Here, I have reinitiated the laser approach with the goal of identifying the presumptive embryonic HS/VS precursor cell in Drosophila. The usefulness of the laser ablation approach which has not been applied, so far, to cells lying deep within the Drosophila embryo, was first tested on two well defined embryonic sensory structures, the olfactory antenno-maxillary complex (AMC) and the light-sensitive Bolwing´s organ (BO). In the case of the AMC, the efficiency of the ablation procedure was demonstrated with a behavioral assay. When both AMCs were ablated, the response to an attractive odour (n-butanol) was clearly reduced. Interestingly, the larvae were not completely unresponsive but had a delayed response kinetics, indicating the existence of a second odour system. BO will be a useful test system for the selectivity of laser ablation when used at higher spatial resolution. An omb-Gal4 enhancer trap line was used to visualize the embryonic OLA by GFP fluorescence. This fluorescence allowed to guide the laser beam to the relevant structure within the embryo. The success of the ablations was monitored in the adult brain via the enhancer trap insertion A122 which selectively visualizes the HS and VS cell bodies. Due to their tight clustering, individual cells could not be identified in the embryonic OLA by conventional fluorescence microscopy. Nonetheless, systematic ablation of subdomains of the OLA allowed to localize the presumptive HS/VS precursor to a small area within the OLA, encompassing around 10 cells. Future studies at higher resolution should be able to identify the precursor as (an) individual cell(s). Most known lethal omb alleles do not complement the HS/VS phenotype of the In(1)omb[H31] allele. This is the expected behaviour of null alleles. Two lethal omb alleles that had been isolated previously by non-complementation of the omb hypomorphic allele bifid, have been reported, however, to complement In(1)omb[H31]. This report was based on low resolution paraffin histology of adult heads. Four mutations from this mutagenesis were characterized here in more detail (l(1)omb[11], l(1)omb[12], l(1)omb[13], and l(1)omb[15]). Using A122 as marker for the adult HS and VS cells, I could show, that only l(1)omb[11] can partly complement the HS/VS cell phenotype of In(1)omb[H31]. In order to identify the molecular lesions in these mutants, the exons and exon/intron junctions were sequenced in PCR-amplified material from heterozygous flies. Only in two mutants could the molecular cause for loss of omb function be identified: in l(1)omb[13]), a missense mutation causes the exchange of a highly conserved residue within the DNA-binding T-domain; in l(1)omb[15]), a nonsense mutation causes a C-terminal truncation. In the other two mutants apparently regulatory regions or not yet identified alternative exons are affected. To see whether mutant OMB protein in the missense mutant l(1)omb[13] is affected in DNA binding, electrophoretic shift assays on wildtype and mutant T-domains were performed. They revealed that the mutant no longer is able to bind the consensus palindromic T-box element.

Multifunctional nanocarriers encapsulating anti-Alzheimer drug for nasal delivery to central nervous system

Alzheimer's disease (AD) is a fatal neurodegenerative condition characterized clinically by progressive memory loss and irreversible cognitive deterioration. It has been shown that there is a progressive degeneration of the brain cholinergic neurons which leads to the appearance of cognitive symptoms of the disease. The aim of this work was the formulation of multifunctional nanocarriers for nasal administration of tacrine-HCl (THA). This route has many advantages; in particular is possible to convey the drug directly to the Central Nervous System, through the olfactory bulb. In particular, were prepared Albumin nanoparticles carrying beta cyclodextrin and two different beta cyclodextrin derivatives (hydroxypropyl beta cyclodextrin and sulphobutylether beta cyclodextrin), and Multifunctional liposomes, prepared using traditional excipients (cholesterol and phosphatidylcholine), partly enriched with α-tocopherol (Toc) and/or polyunsaturated fatty acids (eicosapentaenoic acid and docosahexaenoic acid) (Ω3). Both nanosystems were characterized in terms of size, Zeta potential and encapsulation efficiency. Were also evaluated their functional properties such as mucoadhesion and permeability, using an ex-vivo assay based on nasal sheep mucosa. On Liposomes were also assessed drug neuronal uptake, cell toxicity, antioxidant and, cytoprotective activity in the human neuronal cell line SH-SY5Y and finally tocopherol trans-membrane diffusion. Both the nanocarriers produced presented excellent properties and a high potential as new systems for CNS-delivery of anti-Alzheimer drugs via the nasal route.

Expression of adenosine A2a receptors gene in the olfactory bulb and spinal cord of rat and mouse

The expression of adenosine A2a receptors (A2aR) in the mammalian striatum is well known. In contrast the exact distribution of A2aR in other regions of the central nervous system remains unclear. The aim of this study was to investigate the A2aR gene expression in the rat olfactory bulb and spinal cord, two regions which are seldom included in mapping studies. Secondly, we compared the A2aR expression in the rat and in the mouse brain. Hybridization histochemistry was performed with an S35-labelled radioactive oligonucleotide probe. The results show strong expression of A2aR in the mouse and rat striatum in accordance with previous reports. In the olfactory bulb a weak but specific expression of A2aR was found in the granular cell layer in both species. In contrast, no significant expression of the A2aR gene was observed in other parts of the brain or the rat spinal cord. The presence of the A2aR in the mammalian olfactory bulb suggests a functional role for this receptor in olfaction.