Renewing olfactory receptor neurons in goldfish do not require contact with the olfactory bulb to develop normal chemical responsiveness

This study investigated whether contact with the olfactory bulb was necessary for developing and renewing olfactory receptor neurons (ORNs) to attain normal odorant responsiveness, and whether the anatomical and functional recoveries of the olfactory epithelium were similar in both bulbectomized (BE) and bilaterally axotomized (AX) preparations. In vivo electrophysiological recordings were obtained in response to amino acids, a bile acid [taurolithocholic acid sulfate(TLCS)] and a pheromonal odorant [17α, 20β,-dihydroxy-4-pregnen-3-one (17,20P)] from sexually immature goldfish. Both transmission and scanning electron microscopy indicated that the olfactory epithelium degenerated in BE and AX goldfish. Within 1–2 weeks subsequent to the respective surgeries, responses to high concentrations (>0.1 mmol · l⁻¹) of the more stimulatory amino acids remained, whereas responses were no longer obtainable to TLCS and 17,20P. At 4 weeks, responses to amino acid stimuli recovered to control levels, while responses to TLCS and 17,20P were minimal. By 7 weeks post bilateral axotomy, the olfactory epithelium recovered to a condition similar to control sensory epithelium; however, the rate of degeneration and proliferation of receptor neurons in BE preparations appeared to remain in balance, thus blocking further recovery of the olfactory epithelium. At 7 weeks post surgery, odorant responses of AX and BE goldfish to TLCS and 17,20P were still recovering. Accepted: 14 June 1997     

Responses of Xenopus laevis water nose to water-soluble and volatile odorants.

Using the whole-cell mode of the patch-clamp technique, we recorded action potentials, voltage-activated cationic currents, and inward currents in response to water-soluble and volatile odorants from receptor neurons in the lateral diverticulum (water nose) of the olfactory sensory epithelium of Xenopus laevis. The resting membrane potential was -46.5 +/- 1.2 mV (mean +/- SEM, n = 68), and a current injection of 1-3 pA induced overshooting action potentials. Under voltage-clamp conditions, a voltage-dependent Na+ inward current, a sustained outward K+ current, and a Ca2+-activated K+ current were identified. Application of an amino acid cocktail induced inward currents in 32 of 238 olfactory neurons in the lateral diverticulum under voltage-clamp conditions. Application of volatile odorant cocktails also induced current responses in 23 of 238 olfactory neurons. These results suggest that the olfactory neurons respond to both water-soluble and volatile odorants. The application of alanine or arginine induced inward currents in a dose-dependent manner. More than 50% of the single olfactory neurons responded to multiple types of amino acids, including acidic, neutral, and basic amino acids applied at 100 microM or 1 mM. These results suggest that olfactory neurons in the lateral diverticulum have receptors for amino acids and volatile odorants.     

Inhibition of protein synthesis does not block physiological responses to odors in the frog over the short term

Frog olfactory mucosae were stimulated with an odorant every 60 sec and electrophysiological responses (electroolfactograms or EOGs) were monitored continuously--before, during, and after treatment with puromycin, a protein synthesis inhibitor. Repeated administration of puromycin at 4 hr intervals over a 36 hr period failed to suppress EOG responses. In an alternative approach, olfactory responses were inhibited with ethyl bromoacetate, a vaporous alkylating agent. EOG responses failed to reappear over a 72 hr period. We conclude that receptor turnover is not readily influenced by stimulation, and that the turnover time of frog olfactory receptor proteins is of the order of days or longer.     

Intracellular trafficking of a tagged and functional mammalian olfactory receptor.

Tagged G-protein-coupled receptors (GPCRs) have been used to facilitate intracellular visualization of these receptors. We have used a combination of adenoviral vector gene transfer and tagged olfactory receptors to help visualize mammalian olfactory receptor proteins in the normal olfactory epithelium of rats, and in cell culture. Three recombinant adenoviral vectors were generated carrying variously tagged versions of rat olfactory receptor I7. The constructs include an N-terminal Flag epitope tag (Flag:I7), enhanced green fluorescent protein (EGFP) fusion protein (EGFP:I7), and a C-terminal EGFP fusion (I7:EGFP). These receptor constructs were assayed in rat olfactory sensory neurons (OSNs) and in a heterologous system (HEK 293 cell line) for protein localization and functional expression. Functional expression of the tagged receptor proteins was tested by electroolfactogram (EOG) recordings in the infected rat olfactory epithelium, and by calcium imaging in single cells. Our results demonstrate that the I7:EGFP fusion protein and Flag:I7 are functionally expressed in OSNs while the EGFP:I7 fusion is not, probably due to inappropriate processing of the protein in the cells. These data suggest that a small epitope tag (Flag) at the N-terminus, or EGFP located at the C-terminus of the receptor, does not affect ligand binding or downstream signaling. In addition, both functional fusion proteins (Flag:I7 and I7:EGFP) are properly targeted to the plasma membrane of HEK 293 cells.     

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.     

Electrophysiological evidence for acidic, basic, and neutral amino acid olfactory receptor sites in the catfish

Electrophysiological experiments indicate that olfactory receptors of the channel catfish, Ictalurus punctatus, contain different receptor sites for the acidic (A), basic (B), and neutral amino acids; further, at least two partially interacting neutral sites exist, one for the hydrophilic neutral amino acids containing short side chains (SCN), and the second for the hydrophobic amino acids containing long side chains (LCN). The extent of cross-adaptation was determined by comparing the electro-olfactogram (EOG) responses to 20 "test" amino acids during continuous bathing of the olfactory mucosa with water only (control) to those during each of the eight "adapting" amino acid regimes. Both the adapting and test amino acids were adjusted in concentrations to provide approximately equal response magnitudes in the unadapted state. Under all eight adapting regimes, the test EOG responses were reduced from those obtained in the unadapted state, but substantial quantitative differences resulted, depending upon the molecular structure of the adapting stimulus. Analyses of the patterns of EOG responses to the test stimuli identified and characterized the respective "transduction processes," a term used to describe membrane events initiated by a particular subset of amino acid stimuli that are intricately linked to the origin of the olfactory receptor potential. Only when the stimulus compounds interact with different transduction processes are the stimuli assumed to bind to different membrane "sites." Four relatively independent L-alpha-amino acid transduction processes (and thus at least four binding sites) identified in this report include: (a) the A process for aspartic and glutamic acids; (b) the B process for arginine and lysine; (c) the SCN process for glycine, alanine, serine, glutamine, and possibly cysteine; (d) the LCN process for methionine, ethionine, valine, norvaline, leucine, norleucine, glutamic acid-gamma-methyl ester, histidine, phenylalanine, and also possibly cysteine. The specificities of these olfactory transduction processes in the catfish are similar to those for the biochemically determined receptor sites for amino acids in other species of fishes and to amino acid transport specificities in tissues of a variety of organisms.     

Intracellular trafficking of a tagged and functional mammalian olfactory receptor

Tagged G‐protein‐coupled receptors (GPCRs) have been used to facilitate intracellular visualization of these receptors. We have used a combination of adenoviral vector gene transfer and tagged olfactory receptors to help visualize mammalian olfactory receptor proteins in the normal olfactory epithelium of rats, and in cell culture. Three recombinant adenoviral vectors were generated carrying variously tagged versions of rat olfactory receptor I7. The constructs include an N‐terminal Flag epitope tag (Flag:I7), enhanced green fluorescent protein (EGFP) fusion protein (EGFP:I7), and a C‐terminal EGFP fusion (I7:EGFP). These receptor constructs were assayed in rat olfactory sensory neurons (OSNs) and in a heterologous system (HEK 293 cell line) for protein localization and functional expression. Functional expression of the tagged receptor proteins was tested by electroolfactogram (EOG) recordings in the infected rat olfactory epithelium, and by calcium imaging in single cells. Our results demonstrate that the I7:EGFP fusion protein and Flag:I7 are functionally expressed in OSNs while the EGFP:I7 fusion is not, probably due to inappropriate processing of the protein in the cells. These data suggest that a small epitope tag (Flag) at the N‐terminus, or EGFP located at the C‐terminus of the receptor, does not affect ligand binding or downstream signaling. In addition, both functional fusion proteins (Flag:I7 and I7:EGFP) are properly targeted to the plasma membrane of HEK 293 cells. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 56–68, 2002     

Characterization of electro-olfactogram oscillations and their computational reconstruction

Electro-olfactogram (EOG) oscillations induced by odorant stimulation have been often reported in various vertebrates from fishes to mammals. However, the mechanism of generation of EOG oscillations remains unclear. In the present study, we first characterized the properties of EOG oscillations induced by amino acid odorants in the rainbow trout and then performed a computer simulation based on the main assumption that olfactory receptor neurons (ORNs) have intrinsic oscillatory properties due to two types of voltage-gated ion channels, which have not yet been reported in vertebrate ORNs. EOG oscillations appeared mostly on the peak and decay phases of negative EOG responses, when odorant stimuli at high intensity flowed regularly anterior to posterior olfactory lamellae in the olfactory organ. The appearance of EOG oscillations was dependent on the odorant intensity but not on the flow rate. The maximum amplitude and the maximum power frequency of EOG oscillations were 3.51 +/- 3.35 mV (mean +/- SD, n = 232, range 0.12-16.79 mV) and 10.59 +/- 5.05 Hz (mean +/- SD, n = 232, range 3.51-40.03 Hz), respectively. The simulation represented sufficiently well the characteristics of EOG oscillations; occurrence at high odorant concentration, odorant concentration-dependent amplitude and the maximum power frequency range actually observed. Our results suggest that EOG oscillations are due to the intrinsic oscillatory properties of individual ORNs, which have two novel types of voltage-gated ion channels (resonant and amplifying channels). The simulation program for Macintosh ('oscillation 3.2.4' for MacOS 8.6 or later) is available on the world wide web (http://bio2.sci.hokudai.ac.jp/bio/chinou1/noriyo_home.html).     

Neuropeptide Y Enhances Olfactory Mucosa Responses to Odorant in Hungry Rats

Neuropeptide Y (NPY) plays an important role in regulating appetite and hunger in vertebrates. In the hypothalamus, NPY stimulates food intake under the control of the nutritional status. Previous studies have shown the presence of NPY and receptors in rodent olfactory system, and suggested a neuroproliferative role. Interestingly, NPY was also shown to directly modulate olfactory responses evoked by a food-related odorant in hungry axolotls. We have recently demonstrated that another nutritional cue, insulin, modulates the odorant responses of the rat olfactory mucosa (OM). Therefore, the aim of the present study was to investigate the potential effect of NPY on rat OM responses to odorants, in relation to the animal''s nutritional state. We measured the potential NPY modulation of OM responses to odorant, using electro-olfactogram (EOG) recordings, in fed and fasted adult rats. NPY application significantly and transiently increased EOG amplitudes in fasted but not in fed rats. The effects of specific NPY-receptor agonists were similarly quantified, showing that NPY operated mainly through Y1 receptors. These receptors appeared as heterogeneously expressed by olfactory neurons in the OM, and western blot analysis showed that they were overexpressed in fasted rats. These data provide the first evidence that NPY modulates the initial events of odorant detection in the rat OM. Because this modulation depends on the nutritional status of the animal, and is ascribed to NPY, the most potent orexigenic peptide in the central nervous system, it evidences a strong supplementary physiological link between olfaction and nutritional processes.     

Internalization of G Protein-Coupled Receptors in Single Olfactory Receptor Neurons

Abstract : Desensitization of many G protein-coupled receptors after ligand binding generally involves phosphorylation of the receptors and internalization of the ligandbound, phosphorylated receptors by a clathrin-mediated endocytic pathway. Olfactory receptor neurons from the channel catfish ( Ictalurus punctatus ) express the G protein-coupled odorant receptors and metabotropic glutamate receptors. To determine whether a clathrin-dependent receptor internalization pathway exists in olfactory receptor neurons, western blotting and immunocytochemistry were used to identify and localize clathrin and dynamin in isolated olfactory neurons. Clathrin and dynamin immunoreactivity was found in the cell bodies, dendrites, and dendritic knobs of the neurons. Using the activity-dependent fluorescent dye FM1-43 to monitor receptor internalization, we show that single olfactory neurons stimulated with the odorant amino acid l -glumate internalized the dye. Odorant-stimulated neurons showed a consistent pattern of internalized FM1-43 fluorescence localized in the cell bodies and dendritic knobs. Odorant-stimulated internalization was unaffected by the caveolae activator okadaic acid and was significantly decreased by a metabotropic glutamate receptor antagonist, suggesting that a functional, clathrindependent, receptor-mediated internalization pathway exists in olfactory receptor neurons.     

Mechanisms of Regulation of Olfactory Transduction and Adaptation in the Olfactory Cilium

Olfactory adaptation is a fundamental process for the functioning of the olfactory system, but the underlying mechanisms regulating its occurrence in intact olfactory sensory neurons (OSNs) are not fully understood. In this work, we have combined stochastic computational modeling and a systematic pharmacological study of different signaling pathways to investigate their impact during short-term adaptation (STA). We used odorant stimulation and electroolfactogram (EOG) recordings of the olfactory epithelium treated with pharmacological blockers to study the molecular mechanisms regulating the occurrence of adaptation in OSNs. EOG responses to paired-pulses of odorants showed that inhibition of phosphodiesterases (PDEs) and phosphatases enhanced the levels of STA in the olfactory epithelium, and this effect was mimicked by blocking vesicle exocytosis and reduced by blocking cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) and vesicle endocytosis. These results suggest that G-coupled receptors (GPCRs) cycling is involved with the occurrence of STA. To gain insights on the dynamical aspects of this process, we developed a stochastic computational model. The model consists of the olfactory transduction currents mediated by the cyclic nucleotide gated (CNG) channels and calcium ion (Ca²⁺)-activated chloride (CAC) channels, and the dynamics of their respective ligands, cAMP and Ca²⁺, and it simulates the EOG results obtained under different experimental conditions through changes in the amplitude and duration of cAMP and Ca²⁺ response, two second messengers implicated with STA occurrence. The model reproduced the experimental data for each pharmacological treatment and provided a mechanistic explanation for the action of GPCR cycling in the levels of second messengers modulating the levels of STA. All together, these experimental and theoretical results indicate the existence of a mechanism of regulation of STA by signaling pathways that control GPCR cycling and tune the levels of second messengers in OSNs, and not only by CNG channel desensitization as previously thought.     

Metabotropic glutamate receptor expression in olfactory receptor neurons from the channel catfish,Ictalurus punctatus

Metabotropic glutamate receptors (mGluRs) were identified in olfactory receptor neurons of the channel catfish, Ictalurus punctatus, by polymerase chain reaction. DNA sequence analysis confirmed the presence of two subtypes, mGluR1 and mGluR3, that were coexpressed with each other and with the putative odorant receptors within single olfactory receptor neurons. Immunocytochemical data showed that both mGluR subtypes were expressed in the apical dendrites and some cilia of olfactory neurons. Pharmacological analysis showed that antagonists to each mGluR subtype significantly decreased the electrophysiological response to odorant amino acids. α-Methyl-L -CCG1/(2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG), a known antagonist to mGluR3, and (S)-4-carboxyphenylglycine (S-4CPG), a specific antagonist to mGluR1, each significantly reduced olfactory receptor responses to L -glutamate. S-4CPG and MCCG reduced the glutamate response to 54% and 56% of control, respectively, which was significantly greater than their effect on a neutral amino acid odorant, methionine. These significant reductions of odorant response by the antagonists, taken with the expression of these receptors throughout the dendritic and ciliated portions of some olfactory receptor neurons, suggest that these mGluRs may be involved in olfactory reception and signal transduction. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 94–104, 1998     

Olfactory signal modulation by molluscan cardioexcitatory tetrapeptide (FMRFamide) in axolotls (Ambystoma mexicanum)

The terminal nerve, which innervates the nasal epithelia of most jawed vertebrates, is believed to release neuropeptides that modulate activity of sensory receptor neurons. The terminal nerve usually contains gonadotropin-releasing hormone as well as at least one other peptide that has not been characterized, but which bears some structural similarity to molluscan cardioexcitatory tetrapeptide (FMRFamide) and neuropeptide tyrosine (NPY). We investigated the effects of FMRFamide on both voltage-gated currents and odorant responses in the olfactory epithelium of axolotls (Ambystoma mexicanum), using whole-cell patch clamp and electro-olfactogram (EOG) recording techniques. In the presence of FMRFamide, the magnitude of a voltage-gated inward current was dramatically increased, reaching an average of 136% of the initial (pre-exposure) magnitude in neurons that showed a response to the peptide. This increase is detectable within approximately 1-2 min of exposure to FMRFamide and is sustained for at least 10 min. In EOG experiments, odorant responses are not affected during FMRFamide application, but are sometimes increased or decreased during the subsequent wash period. On average, the largest single EOG response in each trial was detected approximately 25 min after initial FMRFamide application, and ranged from 110 to 147% of baseline. These results suggest that a compound similar to FMRFamide, if released from the terminal nerve, may function in peripheral olfactory signal modulation.     

Rewiring the olfactory bulb: changes in odor maps following recovery from nerve transection

Recent studies have shown that axons from olfactory receptor subtypes converge onto glomeruli in fixed positions within the olfactory bulb. Different receptor subtypes project to different glomeruli, forming a spatial distribution of odor information or 'odor maps'. Olfactory receptor neurons are continuously replaced throughout the life span of an animal, yet they preserve this highly localized mapping of receptor subtypes. In this study we used a transgenic mouse (P2-IRES-tau-lacZ) to map axons from a single receptor subtype (P2 receptors) in order to determine if regenerating axons were able to re-establish the P2 receptor map following nerve transection. Results confirm that P2 receptor axons retain their capacity to grow back to the olfactory bulb and converge onto glomeruli following nerve transection. However, the location and number of convergence sites was significantly altered compared to the control map. This change in the spatial distribution of axons alters the topography of odor mapping and has important implications for the processing of olfactory information. Findings from this study may explain why animals recovering from nerve injury require odor training before odor discrimination is restored. Future studies of olfactory receptor mapping could prove helpful in planning strategies to rewire connections in the brain and to restore function following injury or neurological disease.     

Identification of carbonic anhydrase activity in bullfrog olfactory receptor neurons: histochemical localization and role in CO2 chemoreception

The objectives of this study were to determine: (1) the frequency and distribution of carbonic anhydrase (CA) activity in the bullfrog nasal cavities, and (2)␣whether inhibition of nasal CA affects the olfactory receptor response to CO₂ or other odorants. It was found, using Hansson's staining technique, that some olfactory receptor neurons exhibited CA activity and that these CA-positive receptors were distributed throughout the nasal cavity with peak densities in the dorsal and ventral sensory epithelial regions. To test for the role of CA in olfactory transduction, electro-olfactograms (EOGs) were recorded from the surface of the ventral sensory epithelium in response to 2-s pulses of 5% CO₂ and amyl acetate before and after topical CA inhibition with acetazolamide (10⁻³mol · l⁻¹). In 52 bullfrogs, 1222 sites on the ventral epithelium were tested resulting in 23 locations that exhibited a response to 5% CO₂. Inhibition of CA caused an immediate 65% reduction in the EOG response to CO₂ while the response to amyl acetate was not affected. These results, along with the histochemical localization of CA in some olfactory receptor neurons, indicate that CA plays a role in the detection of CO₂ in frog olfactory neurons and that only a small population of olfactory receptor neurons are CO₂ sensitive. Accepted: 31 July 1997     

Electro-olfactogram and multiunit olfactory receptor responses to complex mixtures of amino acids in the channel catfish, Ictalurus punctatus

In vivo electrophysiological recordings from populations of olfactory receptor neurons in the channel catfish, Ictalurus punctatus, clearly showed that both electro-olfactogram and integrated neural responses of olfactory receptor cells to complex mixtures consisting of up to 10 different amino acids were predictable with knowledge of (a) the responses to the individual components in the mixture and (b) the relative independence of the respective receptor sites for the component stimuli. All amino acid stimuli used to form the various mixtures were initially adjusted in concentration to provide approximately equal response magnitudes. Olfactory receptor responses to both multimixtures and binary mixtures were recorded. Multimixtures were formed by mixing equal aliquots of 3-10 different amino acids. Binary mixtures were formed by mixing equal aliquots of two equally stimulatory solutions. Solution 1 contained either one to nine different neutral amino acids with long side-chains (LCNs) or one to five different neutral amino acids with short side-chains (SCNs). Solution 2, comprising the binary mixture, consisted of only a single stimulus, either a LCN, SCN, basic, or acidic amino acid. The increasing magnitude of the olfactory receptor responses to mixtures consisting of an increasing number of neutral amino acids indicated that multiple receptor site types with highly overlapping specificities exist to these compounds. For both binary mixtures and multimixtures composed of neutral and basic or neutral and acidic amino acids, the receptor responses were significantly enhanced compared with those mixtures consisting of an equal number of only neutral amino acids. These results demonstrate that receptor sites for the basic and acidic amino acids, respectively, are highly independent of those for the neutral amino acids, and suggest that a mechanism for synergism is the simultaneous activation of relatively independent receptor sites by the components in the mixture. In contrast, there was no evidence for the occurrence of mixture suppression.     

Neuronal degeneration and regeneration in the olfactory epithelium of pigeon following transection of the first cranial nerve.

The pigeon olfactory nerve has been sectioned to explore the course of retrograde degeneration of the sensory neurons' perikarya, which are located in the olfactory neuroepithelium. Both light- and electron-microscopic observations have shown that from 3 to 8 days after axotomy the sensory neurons undergo retrograde, irreversible degeneration. Following disappearance of the mature neurons, the basal cells of the neuroepithelium actively divide and differentiate into mature olfactory sensory neurons. Consequently, the basal cells represent true stem cells of the olfactory sensory neurons. The olfactory mucosa regains a structural organization close to normal in a period of 30-50 days after axotomy. These observations indicate that, when the primary olfactory neurons degenerate as a consequence of the experimental section of their axons, restitutio ad integrum of the sensory olfactory connections can be reestablished by new elements which differentiate from basal cells of the olfactory neuroepithelium.20     

Identification of a novel WD repeat-containing gene predominantly expressed in developing and regenerating neurons

In the present study, we have identified a novel gene, NDRP (for neuronal differentiation-related protein), which is predominantly expressed in developing and regenerating neurons. The predicted NDRP comprises 1,019 amino acid residues and has 6 WD repeats in the N-terminal half and multiple potential nuclear localization signals (NLSs) at the C-terminal part. This molecule shows no significant structural similarity with any other molecules in available databases. In situ hybridization and immunohistochemistry revealed the highest expression of NRDP in sensory neurons, for instance, olfactory epithelia and neural layer of retina during embryonic development, as well as in perinatal dorsal root ganglions. The expression of this gene in intact motor neurons such as in the hypoglossal nerve was undetectable but became obvious after axotomy. These results suggest that the product of this gene might be involved in the development of sensory neurons as well as the regeneration of motor neurons.     

GABAB receptor expression and function in olfactory receptor neuron axon growth

Neurotransmitters have been implicated in regulating growth cone motility and guidance in the developing nervous system. Anatomical and electrophysiological studies show the presence of functional GABAB receptors on adult olfactory receptor neuron (ORN) nerve terminals. Using antisera against the GABAB R1a/b receptor isoforms we show that developing mouse olfactory receptor neurons express GABAB receptors from embryonic day 14 through to adulthood. GABAB receptors are present on axon growth cones from both dissociated ORNs and olfactory epithelial explants. Neurons in the olfactory bulb begin to express glutamic acid decarboxylase (GAD), the synthetic enzyme for GABA, from E16 through to adulthood. When dissociated ORNs were cultured in the presence of the GABAB receptor agonists, baclofen or SKF97541, neurite outgrowth was significantly reduced. Concurrent treatment of the neurons with baclofen and the GABAB receptor antagonist CGP54626 prevented the inhibitory effects of baclofen on ORN neurite outgrowth. These results show that growing ORN axons express GABAB receptors and are sensitive to the effects of GABAB receptor activation. Thus, ORNs in vivo may detect GABA release from juxtaglomerular cells as they enter the glomerular layer and use this as a signal to limit their outgrowth and find synaptic targets in regeneration and development.     

Chemotopy of amino acids on the olfactory bulb predicts olfactory discrimination capabilities of zebrafish Danio rerio

Amino acids reliably evoke strong responses in fish olfactory system. The molecular olfactory receptors (ORs) are located in the membrane of cilia and microvilli of the olfactory receptor neurons (ORNs). Axons of ORNs converge on specific olfactory bulb (OB) glomeruli and the neural responses of ORNs expressing single Ors activate glomerular activity patterns typical for each amino acid. Chemically similar amino acids activate more similar glomerular activity patterns then chemically different amino acids. Differential glomerular activity patterns are the structural basis for amino acid perception and discrimination. We studied olfactory discrimination in zebrafish Danio rerio (Hamilton 1822) by conditioning them to respond to each of the following amino acids: L-Ala, L-Val, L-Leu, L-Arg, and L-Phe. Subsequently, zebrafish were tested for food searching activities with 18 nonconditioned amino acids. The food searching activity during 90 s of the test period was significantly greater after stimulation with the conditioned stimulus than with the nonconditioned amino acid. Zebrafish were able to discriminate all the tested amino acids except L-Ile from L-Val and L-Phe from L-Tyr. We conclude that zebrafish have difficulties discriminating amino acid odorants that evoke highly similar chemotopic patterns of activity in the OB.