In the OSN culture system reported here, several modifi- cations were made to enhance the survivability and mor- phology of the OSNs compared to other published studies [15,28]. Dissociated OE cells were plated on a feeder layer of confluent astrocytes instead of directly on matrix coated coverglass to enhance their attachment, survivabil- ity and differentiation. Defined, serum-free media sup- ported the survival of neurons and inhibited cell proliferation and neurogenesis. Consistent with previous reports, OMP expression was present immediately after the plating and was undetectable at 3 DIV . A rapid decrease in OMP positive cells indicated that the culture condition does not support the survival of mature OSNs dissociated from the OE. In addition, a larger numbers of multipolar neurons were present at 1 DIV. The percentage of bipolar neurons increased almost 100% from 1 DIV to 6 DIV, while the number of multipolar neurons decreased. This change in the cell population in the OSN cultures suggests that mature OMP positive neurons and multipolar neurons were not well supported under our culture conditions. We do not have evidence at this point as to whether the multipolar neurons represent certain populations of OSNs. All multipolar neurons were GAP43 and NCAM positive in the cultures. Since all OSNs in the cultures were positive for GAP43 staining at 3 DIV, the OSNs in the cultures are most likely immature neurons. Expression of neuronal progenitor markers Mash1 and Ngn1 was not detected at 3 DIV in the culture. This obser- Expression of two exogenous odorant receptors in cultured olfactory sensory neurons
10 Read more
gave no hybridization signals (Fig. 4B), confirming the specificity of the signals obtained with the antisense RNA probe. At higher magnification (Fig. 4C and 4D) staining can be assigned to single long trichoid hairs, which have been reported to generally contain two olfactory sensory neurons [6, 34]. In agreement with this number of OSNs, on the slices that were made from antennae after WM-ISH, regularly two labelled cells in close vicinity to each other were visible, indi- cating co-localization in the same sensillum. In ex- periments using a probe for the pheromone receptor HR13 single cells were labelled under many but not all long trichoid hairs (Fig. 4E), thus confirming and extending previous results [16, 35]. Cells under sen- silla chaetica, which contain mechanosensory and gustatory sensory neurons , were not labelled (Fig. 4F); this result indicates that the expression of GABA B -R1 is restricted to olfactory sensory neurons.
The olfactory system is one of the most basic sensory systems in vertebrates (Mori et al., 2000; Mori and Yoshihara, 1995; Yoshihara and Mori, 1997). Olfactory sensory neurons (OSNs) reside in the olfactory epithelium (OE) and vomeronasal organs, where they express seven transmembrane-type odorant or pheromone receptors, respectively. Their axons extend through the lamina cribrosa and reach the olfactory bulbs (OBs), which protrude from the anterior telencephalon. The OB is a laminar structure that contains limited types of neurons and glia. The outermost layer is the olfactory sensory nerve layer (ONL), which consists of olfactory afferent axons and ensheathing glia. Beneath the ONL, OSNs converge and synapse onto the dendrites of glutamatergic projection neurons (mitral and tufted cells) in structures called glomeruli [the glomerular layer (GL)]. Between the glomeruli in the GL, local circuit interneurons (periglomerular cells) send their dendrites into the glomeruli. Below the GL, the external plexiform layer (EPL) contains the cell bodies of the tufted cells, and the secondary dendrites of the mitral and tufted cells. Beneath the EPL is the mitral cell layer (MCL), which contains the mitral cell bodies. The projection neurons send their axons into the fibrous plexiform layer (IPL). Below the IPL lie the cell bodies of granule cells, which are interneurons [granule cell layer (GCL)]. Beneath the GCL,
11 Read more
2013) and has been shown to enhance the activity of olfactory sensory neurons (OSNs) that express ORs (Olsson et al., 2011; Getahun et al., 2013). Insect OSNs possess the cellular machinery required to produce cAMP (Iourgenko and Levin, 2000; Boto et al., 2010) and disruption of this signaling cascade has been reported to affect the functional properties of OSNs (Martín et al., 2001; Gomez-Diaz et al., 2004; Deng et al., 2011). However, insect ORs show an inverted topology with respect to their mammalian counterparts (Benton et al., 2006), they are not related to any known G protein-coupled receptor and there is no proof of a direct interaction between insect ORs and G proteins. The current consensus model suggests that the slow metabotropic regulation of ORs by cAMP or 3 ′ ,5 ′ -cyclic guanosine monophosphate (cGMP) is achieved indirectly by as yet undescribed membrane receptors co-stimulated by the ORs or by the influx of Ca 2+ through the
Odour detection in vertebrates is achieved by large families of olfactory receptor genes that are expressed mutually exclusively in a large number of primary sensory neurons (Mombaerts, 2004; Serizawa et al., 2005). Thus, the ligand specificity of olfactory sensory neurons should reflect that of the odorant receptors they express, and may allow investigation of odorant receptor ligand sensitivity in vivo as well as in vitro. Visualization of the olfactory sensory neurons responding to a particular odorant has been achieved in the peripheral region (Ma and Shepherd, 2000), and in the terminal fields of the olfactory bulb (e.g. Friedrich and Korsching, 1998; Johnson and Leon, 2007; Lancet et al., 1982; Leveteau and MacLeod, 1966). However, it has not been possible to visualize the entire shape of activated olfactory sensory neurons, from their dendritic processes to the axonal terminals in the olfactory bulb, using conventional methods of imaging odour-induced neuronal activation.
Temporal response characteristics of olfactory receptors The results of this study show that olfactory sensory neurons in the antennae of the American cockroach Periplaneta americana that are sensitive to general odors are able to resolve pulsatile stimuli of 1-hexanol up to rates of 40 pulses s −1 (25 ms pulses) and are able to resolve pulsatile stimuli of coconut oil up to rates of 20 pulses s −1 (50 ms pulses). These maximum rates appeared to be relatively independent of odor concentration, provided that the concentration and pulse duration produced a stimulus that was above threshold. These resolution frequencies are substantially faster than those previously reported for pheromone-sensitive neurons in moths. Rumbo and Kaissling (1989) found that pheromone receptor cells in male Antheraea polyphemus were able to resolve pulsatile stimuli at rates up to 10 stimuli s −1 (100 ms pulses), and Christensen and Hildebrand (1988) found that projection neurons in the central nervous system of Manduca sexta were also able to resolve 10 stimuli s −1 . Our results suggest that insects orienting towards a source of plant-type odors are able to evaluate fluctuations in concentration on a finer time scale than they are able to evaluate fluctuations in pheromone concentration. This difference may be because pheromone receptors are adapted to respond to much lower concentrations of odors than are general odor receptors (Fujimura et al. 1991; Sass, 1983).
11 Read more
Background: The b-secretase, b-site amyloid precursor protein cleaving enzyme 1 (BACE1), is a prime therapeutic target for lowering cerebral b-amyloid (Ab) levels in Alzheimer’s disease (AD). Clinical development of BACE1 inhibitors is being intensely pursued. However, little is known about the physiological functions of BACE1, and the possibility exists that BACE1 inhibition may cause mechanism-based side effects. Indeed, BACE1 -/- mice exhibit a complex neurological phenotype. Interestingly, BACE1 co-localizes with presynaptic neuronal markers, indicating a role in axons and/or terminals. Moreover, recent studies suggest axon guidance molecules are potential BACE1 substrates. Here, we used a genetic approach to investigate the function of BACE1 in axon guidance of olfactory sensory neurons (OSNs), a well-studied model of axon targeting in vivo.
The formation of olfactory maps in the olfactory bulb (OB) is crucial for the control of innate and learned mouse behaviors. Olfactory sensory neurons (OSNs) expressing a specific odorant receptor project axons into spatially conserved glomeruli within the OB and synapse onto mitral cell dendrites. Combinatorial expression of members of the Kirrel family of cell adhesion molecules has been proposed to regulate OSN axonal coalescence; however, loss-of-function experiments have yet to establish their requirement in this process. We examined projections of several OSN populations in mice that lacked either Kirrel2 alone, or both Kirrel2 and Kirrel3. Our results show that Kirrel2 and Kirrel3 are dispensable for the coalescence of MOR1-3-expressing OSN axons to the most dorsal region (DI) of the OB. In contrast, loss of Kirrel2 caused MOR174-9- and M72-expressing OSN axons, projecting to the DII region, to target ectopic glomeruli. Our loss-of-function approach demonstrates that Kirrel2 is required for axonal coalescence in subsets of OSNs that project axons to the DII region and reveals that Kirrel2/3- independent mechanisms also control OSN axonal coalescence in certain regions of the OB.
Stochastically dispersed throughout the teleost olfactory epithelium are ciliated, microvillous, crypt and kappe morphotypes of olfactory sensory neurons (OSNs), that bind diverse dissolved odorants (Friedrich and Korsching 1997, 1998, Braubach et al. 2013, Kerman et al. 2013) to mediate different behaviours such as prey avoidance, foraging and finding mates (Sorensen and Caprio 1998). These OSN morphotypes display distinctive morphological, molecular and physiological properties (Gayoso et al. 2011), with their nuclei situated at different depths of the pseudostratified olfactory epithelium. The cell bodies of ciliated OSNs are found in the basal layer of the olfactory epithelium (Hansen et al. 2004, Sato et al. 2005), microvillous OSN cell bodies at intermediate depths (Hansen et al. 2004, Sato et al. 2005), and crypt and Kappe cells are situated in the superficial layers (Morita and Finger 1996, Hansen and Zeiske 1998, Ahuja et al. 2014). Axons from different OSN morphotypes project to specific and consistent regions of the olfactory bulb (Sato et al. 2005), where they terminate on discrete units of odour discrimination called glomeruli, creating a highly stereotyped organization of glomerular regions in the olfactory bulb (Baier and Korsching 1994, Riddle and Oakley 1992, Braubach et al. 2013).
156 Read more
Reproductive adult (N=16, June-October, 2006) and metamorphic stage VII (N=10, November 2005-April 2006) sea lampreys used in this study were obtained from the Hammond Bay Biological Research Station. All sea lampreys were collected from wild populations in the Great Lakes region. Initial experiments were conducted on metamorphic lampreys in the autumn and winter, when the reproductive phase was not available for study. In accordance with the Canadian Council on Animal Care, fish were anaesthetised in 0.05 g/l MS222 (tricaine methanesulfonate; Argent Laboratories, WA, USA), decapitated and the olfactory epithelium and brains exposed. Initially, two retrograde labelling techniques were used on the metamorphic phase lampreys: 1) the post mortem lipophilic tracer 1,1-dioctadecyl-3,3,3,3- tetramethylindocarbocyanineperchlorate (DiI), and 2) biocytin dye loading of live tissue. Both DiI (N=5) and biocytin (N=5) neuronal labelling strategies from these initial experiments in the metamorphic phase animals yielded identical polymorphic characteristics. We chose to stay with the biocytin labelling for the remaining metamorphic and adult samples because tissue was available for analysis for an extended period with this technique.
150 Read more
The sense of smell is not just crucial for animal survival ; it also assumes importance for human health, emotional connections, and social interactions [2-4]. The initial events of olfaction take place within the olfactory neuroepithelium situated in the posterior nasal cavity. Olfactory sensory neurons (OSNs) are bipolar neurons with a single axonal end extending basally and a dendritic end that extends apically towards the nasal cavity airway. OSNs direct their unbranched axons to the olfactory bulb (OB), passing through the cribriform plate. Within the OB, the OSNs axons form contact with secondary neurons (mitral and other cells) in spherical neuropils, called glomeruli. OSNs have a few dozen hair-like cellular structures, referred to as olfactory cilia. Cilia harbor the sensory apparatus (olfactory receptor (OR) proteins and other components) that converts and amplifies the physical-chemical signal of the odorant
11 Read more
The topographic projection along the D–V axis of the OB is maintained by axon-axon and axon-target interac- tions. Several axon guidance molecules have been pro- posed to be involved in OSN axonal projections along the D – V axis [17 – 20]. We have previously demonstrated that Neuropilin-2 (Nrp2)/Sema3F repulsive interactions between OSN axons play important roles in preserving the topographic order along the D – V axis of the OB . Nrp2 and Sema3F show complementary, gradient expressions in the OE. More specifically, the expression of Sema3F is high in the D-zone and low in the V-zone, while Nrp2 shows the opposite gradient. We also showed through gain and loss of function experiments that these molecules are necessary for the dorsal – ventral topographic projection in the OB. Further, we found a temporal difference in axonal extensions , whereby OSN axons project to the OB from in sequential dorsal to ventral order. These sequential projections are quite important to maintain the topographic order of the ol- factory map because early-arriving D-zone OSN axons guide late-arriving V-zone OSN axons by secreting Sema3F. However, it is not clear how the differential timing of these axonal projections is regulated. We hy- pothesized that the timing of OSN production would be different depending on the location of OSNs in the OE. To test this, we examined spatiotemporal patterns of OSN neurogenesis during olfactory development.
The behaviour of the desert locust, Schistocera gregaria, is largely directed by volatile olfactory cues. The relevant odorants are detected by specialized antennal sensory neurons which project their sensory dendrites into hair-like structures, the sensilla. Generally, the responsiveness of the an- tennal chemosensory cells is determined by specific receptors which may be either odorant re- ceptors (ORs) or variant ionotropic receptors (IRs). Previously, we demonstrated that in locust the co-receptor for ORs (ORco) is only expressed in cells of sensilla basiconica and sensilla trichodea, suggesting that cells in sensilla coeloconica may express different types of chemosen- sory receptors. In this study, we have identified the genes of S. gregaria which encode homologues of co-receptors for the variant ionotropic receptors, the subtypes IR8a and IR25a. It was found that both subtypes, SgreIR8a and SgreIR25a, are expressed in the antennae of all five nymphal stages and in adults. Attempts to assign the relevant cell types by means of in situ hybridization revealed that SgreIR8a and SgreIR25a are expressed in cells of sensilla coeloconica. Double flu- orescence in situ hybridization experiments disclosed that the two IR-subtypes are co-expressed in some cells of this sensillum type. Expression of SgreIR25a was also found in some of the sensilla chaetica, however, neither SgreIR25a nor SgreIR8a was found to be expressed in sensilla basiconica and sensilla trichodea. This observation was substantiated by the results of double FISH experiments demonstrating that cells expressing SgreIR8a or SgreIR25a do not express ORco. These results support the notion that the antenna of the desert locust employs two different populations of OSNs to sense odors; cells which express IRs in sensilla coeloconica and cells which express ORs in sensilla basiconica and sensilla trichodea.
14 Read more
pheromone communication (Yambe et al., 2006) and kin recognition (Hinz et al., 2013). Amino acids activate olfactory sensory neurons that project to the rostral lateral olfactory bulb region, and sensory neurons projecting to the more medial bulbar regions respond to bile salts and pheromones (steroids or prostaglandins), except in zebrafish where a reproductive pheromone activates a central portion in the ventral olfactory bulb region (catfish – Nikonov and Caprio, 2001; Hansen et al., 2003; zebrafish – Friedrich and Korsching, 1997, 1998; and salmonids – Hara and Zhang, 1996, 1998). While odour responses take place in a single olfactory system in teleosts, many amphibians and terrestrial vertebrates process chemosensory information in the main and accessory (vomeronasal) olfactory subsystems (Eisthen and Polese, 2006). Within the main olfactory bulb of the larval stage of the amphibian Xenopus laevis , a lateral processing stream responds to amino acids, an intermediate stream responds to bile acids, amines and occasionally to amino acids, and the medial stream responds to amines and alcohols and infrequently to bile acids (Gliem et al., 2013). In the mammalian main olfactory bulb, glomeruli that respond to structurally similar compounds are congregated within a bulbar region, yet this grouping is rather coarse (Johnson and Leon, 2007; Soucy et al., 2009).
10 Read more
Unlike the development of the disease in humans, inocula- tion of mice with a low dose of VEE virus into the rear footpad results in a rapidly progressing disease culminating in 100% mortality due to encephalitis. Virus replication in the draining lymph node (DLN) is detectable within 3 h. By 12 h, viremia is at or near its peak level; by 18 h, virus is replicating in virtually every lymphoid tissue, and a marked leucopenia is evident. Virus replication in the brain is detectable in olfactory tracts by 30 h postinfection (hpi), presumably having crossed from the circulation into olfactory sensory neurons (OSN) in the nasal olfactory neuroepithelium and hence along olfactory nerve tracts to the central nervous system (CNS). Encephalitis and death ensues 7 to 10 days after infection (3, 6, 11, 13, 21, 25). Viral invasion and successful infection of the CNS is an important step in the life cycle of neurotropic viruses (14). The CNS is an immune-privileged site, protected by the blood-
Nasal epithelial cells can switch between a sensory and respiratory epithelial cell fate in response to Fgf and Bmp activity. The idea that Fgf and Bmp signals act in an opposing manner during cell specification has previously been revealed in various tissues (De Robertis and Kuroda, 2004; Minina et al., 2002; Neubuser et al., 1997; Rice et al., 2005; Sjödal et al., 2007; Wilson and Edlund, 2001; Yoon et al., 2006). Our results indicate that Fgf signals restrict the range of Bmp activity in the nasal epithelium, thereby allowing differentiated specification of sensory and respiratory cell fate. Both Fgf inhibition and Bmp activation upregulated pSmad1/5/8 expression, which resulted in induction of respiratory epithelial cells and inhibition of sensory epithelial cells. Accordingly, inhibition of Bmp activity resulted in a decrease of pSmad1/5/8, which promoted the generation of sensory epithelial cells at the expense of respiratory epithelial cells. In line with our findings, recent results have suggested that Fgf-induced Foxg1 (Storm et al., 2006) promotes the generation of olfactory sensory neurons via upregulation of the Tgfb antagonist follistatin, which antagonizes Gdf11 activity in the olfactory pit (Kawauchi et al., 2009). In summary, we find that at olfactory placodal and pit stages, Fgf and Bmp signals act in an opposing manner to regulate sensory versus respiratory epithelial cell fate decision. Our results reveal that Fgf signals are required for the generation of sensory epithelial cells and restrict the range of Bmp signals, and that Bmp activity promotes respiratory epithelial character.
11 Read more
Augmenting sea lamprey management with insights from olfactory communication provides a rare example of sensory-integrated control in vertebrates. Manipula- tion of olfactory systems is a widely used as tools to con- trol pest insect populations . Extension of olfactory integrated control of insects to invasive vertebrates is conceptually sound [16–19, 106, 118], however, after de- cades of research into fish olfaction, olfactory communi- cation is not integrated into control of any invasive fish. Developing olfactory-integrated management is a chal- lenging and costly endeavor , but offers a suite of potentially robust and environmentally benign tools. Olfactory-guided behaviors are not unique to sea lam- prey, and insights gained while developing olfactory- integrated control of sea lamprey can be extended to other species of concern throughout the world. Further- more, the sea lamprey model offers the opportunity to optimize olfactory-integrated control methods without the confounding interactions of other sensory modal- ities. For example, most organisms, including sea lam- prey, incorporate information from several sensory modalities while making reproductive decisions. How- ever, sensory-guided behaviors in sea lamprey are clearly biased towards olfaction [45, 46]. Similar to the insect model used as a conceptual foundation for sea lamprey olfaction research, the sea lamprey model can function as a model for more complex vertebrates. Likewise, the technologies and methods developed for studying sea lamprey olfaction provide a foundation that can be used to expedite future research into olfaction in other organ- isms that are invasive or in decline in the Laurentian Great Lakes and throughout the world.
11 Read more
The Drosophila larva has turned into a particularly simple model system for studying the neuronal basis of innate behaviors and higher brain functions. Neuronal networks involved in olfaction, gustation, vision and learning and memory have been described during the last decade, often up to the single-cell level. Thus, most of these sensory networks are substantially defined, from the sensory level up to third-order neurons. This is especially true for the olfactory system of the larva. Given the wealth of genetic tools in Drosophila it is now possible to address the question how modulatory systems interfere with sensory systems and affect learning and memory. Here we focus on the serotonergic system that was shown to be involved in mammalian and insect sensory perception as well as learning and memory. Larval studies suggested that the serotonergic system is involved in the modulation of olfaction, feeding, vision and heart rate regulation. In a dual anatomical and behavioral approach we describe the basic anatomy of the larval serotonergic system, down to the single- cell level. In parallel, by expressing apoptosis-inducing genes during embryonic and larval development, we ablate most of the serotonergic neurons within the larval central nervous system. When testing these animals for naı¨ve odor, sugar, salt and light perception, no profound phenotype was detectable; even appetitive and aversive learning was normal. Our results provide the first comprehensive description of the neuronal network of the larval serotonergic system. Moreover, they suggest that serotonin per se is not necessary for any of the behaviors tested. However, our data do not exclude that this system may modulate or fine-tune a wide set of behaviors, similar to its reported function in other insect species or in mammals. Based on our observations and the availability of a wide variety of genetic tools, this issue can now be addressed.
23 Read more
The results of this paper strongly suggest that input from both antennae is crucial for initiating blocking in honeybees. While odor identity may be a product of each autonomous hemisphere (Masuhr and Menzel, 1972; Fig. 6), it is apparent from our results that inputs from both antennae are integrated when utilizing odor information in higher-order decisions. Anatomically, the connections between the brain hemispheres that help mediate blocking might pass through the mushroom bodies, since that is one of the points at which olfactory information decussates. Mushroom bodies clearly have a critical role in associative odor learning in Drosophila (de Belle and Heisenberg, 1994). In addition, there are interneurons that span both pairs of mushroom bodies and antennal lobes (Hammer, 1993; Hammer and Menzel, 1995), so it is certainly possible that the interaction we describe may not involve intrinsic mushroom body neurons. Such modulation by central associative areas also has a correlate in rats, where hippocampal lesions can interfere with an odor learning task similar to blocking (Schmajuk et al. 1983) as well as blocking in non-odor learning paradigms (Solomon, 1977; Rickert et al. 1978).
11 Read more
Background: The main olfactory epithelium (MOE) is a complex organ containing several functionally distinct subpopulations of sensory neurons. One such subpopulation is distinguished by its expression of the guanylyl cyclase GC-D. The axons of GC-D-expressing (GC-D+) neurons innervate 9–15 "necklace" glomeruli encircling the caudal main olfactory bulb (MOB). Chemosensory stimuli for GC-D+ neurons include two natriuretic peptides, uroguanylin and guanylin, and CO 2 . However, the biologically-relevant source of these chemostimuli is unclear: uroguanylin is both excreted in urine, a rich source of olfactory stimuli for rodents, and expressed in human nasal epithelium; CO 2 is present in both inspired and expired air.