Odorant specificity of single olfactory bulb neurons to amino acids in the channel catfish

Odorant specificity to l-alpha-amino acids was determined for 245 olfactory bulb (OB) neurons recorded from 121 channel catfish. The initial tests included 4 amino acids representing acidic [monosodium glutamate (Glu)], basic [arginine (Arg)], and neutral [possessing short: alanine (Ala) and long: methionine (Met) side chains] amino acids that were previously indicated to bind to independent olfactory receptor sites. Ninety-one (37%) units (Group I) tested at 1, 10, and 100 microM showed high selectivity and were excited by only one of the 4 amino acids. Odorant specificity for the vast majority of Group I units did not change over the 3 s of response time analyzed. A total of 154 OB units (63%) (Group II) were excited by a second amino acid, but only at >/=10x odorant concentration. An additional 69 Group I units were tested with related amino acids and derivatives from 10(-9) to 10(-5) M to determine their excitatory odorant thresholds and selectivities. Two groups of units originally selective for Met were evident: those most sensitive to neutral amino acids having branched and linear side chains, respectively. OB units originally selective for Ala responded at low concentration to other similar amino acids. Units originally selective for Arg were excited at low concentration by amino acids possessing in their side chains at least 3 methylene groups and a terminal amide or guanidinium group. The specificities of the OB units determined electrophysiologically are sufficient to account for many of the previous results of behavioral discrimination of amino acids in this and related species.     

Highly specific olfactory receptor neurons for types of amino acids in the channel catfish

Odorant specificity to l-alpha-amino acids was determined electrophysiologically for 93 single catfish olfactory receptor neurons (ORNs) selected for their narrow excitatory molecular response range (EMRR) to only one type of amino acid (i.e., Group I units). These units were excited by either a basic amino acid, a neutral amino acid with a long side chain, or a neutral amino acid with a short side chain when tested at 10(-7) to 10(-5) M. Stimulus-induced inhibition, likely for contrast enhancement, was primarily observed in response to the types of amino acid stimuli different from that which activated a specific ORN. The high specificity of single Group I ORNs to type of amino acid was also previously observed for single Group I neurons in both the olfactory bulb and forebrain of the same species. These results indicate that for Group I neurons olfactory information concerning specific types of amino acids is processed from receptor neurons through mitral cells of the olfactory bulb to higher forebrain neurons without significant alteration in unit odorant specificity.     

The functional organization of the fish olfactory system

Recent developments in the functional anatomy and physiology of the fish olfactory system reveal three parallel pathways from the sensory epithelium, via the olfactory bulb to the telencephalon. There are three morphological types of sensory neurones spread in a seemingly overlapping arrangement in the olfactory epithelium. The axons of each type of sensory neurones converge to a specific region of the olfactory bulb and connect to separate sets of relay neurones. The axons of these relay neurones leave in three bundles to the telencephalon. Each bundle conveys specific information that elicits sets of characteristic behaviour in response to odours involved in essential life processes in the fish. One pathway is tuned to social cues, another to sex pheromones, and the third to food odours.     

Specificities of olfactory receptor neuron responses to amino acids in the black bullhead catfish (Ameiurus melas)

In vivo investigations of catfish olfactory receptor neurons (ORNs) were previously limited to studying responses of spontaneously active cells. The olfactory organ, however, also contains ORNs that lack spontaneous activity and respond to amino acids. To record electrical activity of ORNs that were inactive prior to stimulation, we bathed the olfactory organ with low conductive, highly purified water that reduces shunting and enables detection of action potentials from ORNs distant to the electrode. After stimulation with amino acids, these ORNs elicited either phasic–tonic or tonic only activities. The spike frequency of the phasic activity consisted of transient frequencies up to 108 Hz that lasted <450 ms. All tonic activities saturated at action potential frequencies of 17–21 Hz. Their durations were dose dependent over several log units of concentration as they closely followed that of the suprathreshold amino acid stimulation. Specificities of 44 ORNs were investigated with ten different amino acids tested at 10^?4?M. Thirteen ORNs were excited by only one amino acid, l-norvaline, and 22 additional ORNs were excited by l-norvaline and l-methionine. Nine ORNs were excited by >2 amino acids that included l-norvaline. In 29 of 31 neurons responding to >1 amino acid, the duration of the responses to the most stimulatory amino acid was at least double compared to that to the other amino acids. The amplitude of electro-olfactogram (EOG) correlated significantly with the number of ORNs activated by the same amino acids confirming that the EOG represents the sum of ORN receptor potentials.     

Responses of olfactory forebrain units to amino acids in the channel catfish

A paucity of information exists concerning the processing of odorant information by single neurons in any vertebrate above the level of the olfactory bulb (OB). In this report, odorant specificity to four types of L-alpha-amino acids (neutral with long side-chains, neutral with short side-chains, basic and acidic), known biologically relevant odorants for teleosts, was determined for 217 spontaneously active forebrain (FB) neurons in the channel catfish. Group I FB units were identified that were excited by only one of four types of amino acids; no Group I unit was encountered that was excited by an acidic amino acid. The Group I FB units exhibited similar preferences as described previously for OB neurons, suggesting that no major modifications of olfactory information for at least some of these units occurred between the OB and FB. Evidence, however, for the convergence of odor information between the OB and FB was suggested by Group II FB units that exhibited a broader excitatory molecular receptive range (EMRR) than those of previously recorded types of OB units or the Group I FB units. Group II FB units were excited by both neutral and basic amino acids and a few also by acidic amino acids, EMRRs not observed previously in OB units. Stimulus-induced inhibition, likely for contrast enhancement, was also often observed for the many of the FB units encountered. The observed EMRRs of the FB units presently identified and those of the OB units previously studied are consistent with the ability of catfish to behaviorally discriminate these compounds.     

The spatial distribution of odour induced potentials in the olfactory bulb of char and trout (Salmonidae)

Monopolar DC-recordings were made of the gross responses from the olfactory bulb of char (Salvelinus alpinus syn. Salmo alpinus L.) and trout (Salmo trutta L.) during stimulation with different odours. The response features studied were: the magnitude and polarity of the slow potential shift, the amplitude of the induced waves and their asymmetrical waveform. Amino acids elicited the largest responses in the lateral part of the bulb. Water containing “crude fish odour” caused the largest responses in the rostral and medial parts. The results demonstrate odour specific differences in the localization of the bulb responses and the separate origin of the slow potential and the induced waves.     

Functional anatomy of the peripheral olfactory system of the african lungfish Protopterus annectens owen: Development of the primary olfactory pathway during postembryonic growth

Seven specimens of the African lungfish ranging in size from 4 to 350 gm were used for the study of changes in the olfactory nerve and bulb during postembryonic growth as expressed by the increasing number of lamellae of the olfactory organ. Serial semithin sections were used, and the data were studied mathematically. The number of axons of the olfactory nerve, the surface area of the bulb, and the number of mitral cells increase exponentially; the mitral cell density decreases exponentially; and the calculated average convergence (counted axons/counted mitral cells) of axons onto the cells of the bulbar relay increases exponentially. Convergence figures obtained are smaller than those cited by other authors; two factors might explain this: first the mode of calculation, and second the fact that every year this fish undergoes a 6-month starvation stage marked by degeneration of the primary olfactory structure. A preliminary view of the primary olfactory pathway using serial-section reconstructions is proposed: An orderly projection of the mucosa onto the bulb takes place after a 90° rotation of the system of two perpendicular axes which apply to both the olfactory organ and the olfactory bulb. This study suggests an original attempt at relating the postembryonic change in convergence and projection to the primary processing of the olfactory message.     

cAMP-independent olfactory transduction of amino acids in Xenopus laevis tadpoles

Whether odorants are transduced by only one or more than one second messenger has been a long-standing question in olfactory research. In a previous study we started to address this question mainly by using calcium imaging in the olfactory bulb. Here, we present direct evidence for our earlier conclusions using the calcium imaging technique in the mucosa slice. The above question can now unambiguously be answered. We show that some olfactory receptor neurons (ORNs) respond to stimulation with amino acids with an increase of the intracellular calcium concentration [Ca²⁺]_i. In order to see whether or not these responses were mediated by the cAMP transduction pathway we applied forskolin or the membrane-permeant cAMP analogue pCPT-cAMP to the olfactory epithelium. The ensemble of ORNs that was activated by amino acids markedly differed from the ensemble of neurons activated by forskolin or pCPT-cAMP. Less than 6 % of the responding ORNs showed a response to both amino acids and the pharmacological agents activating the cAMP transduction pathway. We conclude that ORNs of Xenopus laevis tadpoles have both cAMP-dependent and cAMP-independent olfactory transduction pathways and that most amino acids are transduced in a cAMP-independent way.     

cAMP-independent responses of olfactory neurons in Xenopus laevis tadpoles and their projection onto olfactory bulb neurons

We report on responses of olfactory receptor neurons (ORNs) upon application of amino acids and forskolin using a novel slice preparation of the olfactory epithelium of Xenopus laevis tadpoles. Responses were measured using the patch-clamp technique. Both amino acids and forskolin proved to be potent stimuli. Interestingly, a number of ORNs that responded to amino acids did not respond to forskolin. This suggests that some amino acids activate transduction pathways other than the well-known cAMP-mediated one. The differential processing of cAMP-mediated stimuli on the one hand and amino acid stimuli on the other was further elucidated by calcium-imaging of olfactory bulb neurons using a novel nose-olfactory bulb preparation of Xenopus laevis tadpoles. The projection pattern of amino acid-sensitive ORNs to olfactory bulb neurons differed markedly from the projection pattern of forskolin-sensitive ORNs. Olfactory bulb neurons activated by amino acids were located laterally compared to those activated by forskolin, and only a small proportion responded to both stimuli. The ensemble of neurons activated by forskolin was also activated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and the membrane-permeant cAMP analogue 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (pCPT-cAMP). We therefore conclude that sensory transduction of a number of amino acids is cAMP independent, and amino acid- and cAMP-mediated responses are processed differentially at the level of the olfactory bulb.     

Olfaction and gustation in fish: an overview

Living in an aquatic environment, often devoid of light but rich in dissolved compounds, fish have highly developed chemosensory and chemical signalling systems. The olfactory and gustatory systems comprise the major chemosensory pathways. Despite considerable variations in structural organization of the peripheral olfactory organ throughout fish species, ultrastructural organization of the olfactory sensory epithelium is extremely consistent. The olfactory receptor cell is a bipolar neurone which is directly exposed to the external environment and sends information to the brain by its own axon (cranial nerve I). Four major classes of chemicals have been identified as specific olfactory stimuli and their stimulatory effectiveness characterized: amino acids, sex steroids, bile acids/salts and prostaglandins. Olfactory signals such as those involved in reproduction and feeding may be processed independently through two distinct subsystems: the lateral and medial olfactory systems. The taste buds constitute the structural basis of the gustatory organ. Taste buds may occur not only in the oropharyngeal cavity, but on the whole body surface. Chemical information detected by specialized epithelial cells, gustatory cells, is transmitted to the central nervous system by cranial nerve VII (facial), IX (glossopharyngeal), and X (vagal). Besides diverse sensitivities and specificities for amino acids, fish gustatory receptors detect various organic acids, nucleotides and bile salts. Putative receptors, molecular mechanisms of transduction and the role played by olfaction and gustation in feeding, reproduction, migration and other fish behaviours are discussed.     

Responses of olfactory bulb neurones to odour stimulation of small nasal areas in the salamander

1. Previous experiments have suggested that one way odours may be discriminated is by different spatial patterns of response at both the olfactory bulb and receptor level. The present experiments were designed to test to what extent the position of an odour on the receptor mucosa can influence the activity of olfactory bulb neurones.2. To deliver odours to small areas on the nasal receptor sheet a new method for local application of odour was developed. The flow rate, concentration, and time course of the odour were controlled using the olfactometer described in the preceding paper.3. In thirty olfactory bulb units in the salamander it was found that if the response of a unit to odour delivered to the entire exposed receptor epithelium were suppression (type S), then the unit tended to be suppressed when odour was delivered to a number of localized epithelial regions. If the response were excitation (type E) to stimulation of the entire epithelium, then stimulation to only one or two localized regions would elicit the maximum response.4. Different epithelial regions had the ability to cause excitation in the same bulbar unit depending on the odour being used. Two odours, camphor and amyl acetate, elicited maximum excitation when they were presented to different mucosal areas. The areas at which presentation of these odours gave excitation were surprisingly consistent from unit to unit and animal to animal.5. The data presented here suggest the presence of restricted excitatory receptive fields for some olfactory bulb neurones for a particular odour.6. The presence of spatial response patterns using odour delivery to small nasal receptor regions and thus the presence of receptive fields is discussed with reference to bulbar neuronal circuitry.     

Olfactory experience decreases responsiveness of the olfactory bulb in the adult rat

We recently reported the existence of dramatic modifications of the olfactory bulb reactivity following a very simple manipulation of the olfactory input as an exposure to an odorant. The present study aimed at testing the possibility that such effects could depend on the nature of the exposure odour. For this purpose, rats were exposed 20 min per day during six consecutive days to cineole, methyl-amyl ketone, isoamyl acetate or with no odour in the control group. On day 7, rats were anaesthetized and the spontaneous activity of mitral/tufted cells was recorded along with their responses to the familiar odour and to four novel odours. Results revealed that: (i) the firing frequencies were not significantly different in the four groups; (ii) the proportion of excitatory responses was considerably decreased in the exposed groups while the number of non-responses was significantly enhanced; (iii) excitatory responses were decreased not only to the familiar odour but also to four other novel odours; (iv) this lower responsiveness was long lasting at least for isoamyl acetate exposure; and (v) increasing concentration of test odours was not enough to allow mitral/tufted cells to recover control responsiveness. All of these effects have a differential importance according to the exposure odour. In particular, the more powerful an odour is in activating control cells, the more non-specific the decrease in mitral/tufted cell reactivity is. Hypotheses on the underlying mechanisms are advanced.     

Olfactory experience decreases responsiveness of the olfactory bulb in the adult rat

We recently reported the existence of dramatic modifications of the olfactory bulb reactivity following a very simple manipulation of the olfactory input as an exposure to an odorant. The present study aimed at testing the possibility that such effects could depend on the nature of the exposure odour. For this purpose, rats were exposed 20 min per day during six consecutive days to cineole, methyl-amyl ketone, isoamyl acetate or with no odour in the control group. On day 7, rats were anaesthetized and the spontaneous activity of mitral/tufted cells was recorded along with their responses to the familiar odour and to four novel odours. Results revealed that: (i) the firing frequencies were not significantly different in the four groups; (ii) the proportion of excitatory responses was considerably decreased in the exposed groups while the number of non-responses was significantly enhanced; (iii) excitatory responses were decreased not only to the familiar odour but also to four other novel odours; (iv) this lower responsiveness was long lasting at least for isoamyl acetate exposure; and (v) increasing concentration of test odours was not enough to allow mitral/tufted cells to recover control responsiveness.All of these effects have a differential importance according to the exposure odour. In particular, the more powerful an odour is in activating control cells, the more non-specific the decrease in mitral/tufted cell reactivity is. Hypotheses on the underlying mechanisms are advanced.     

Immunocytochemical characterization of olfactory ensheathing cells in fish

In the olfactory system of vertebrates, neurogenesis occurs throughout life. The regenerating activities of the olfactory receptor neurons are connected to particular glial cells in the olfactory pathway: the olfactory ensheathing cells. A considerable number of studies are available in literature regarding mammalian olfactory ensheathing cells; this is due to their potential role in cell-based therapy for spinal cord injury repair. But very little is known about these cells in non-mammalian vertebrates. In this study we examined the immunocytochemical characteristics of the olfactory ensheathing cells in fish, which provide a good model for the study of glial cells in the olfactory pathway of non-mammalian vertebrates. Paraffin sections from decalcified heads of Poecilia reticulata (microsmatic fish) and Carassius auratus (macrosmatic fish) were processed to immunocytochemically detect ensheathing cell markers used in research on mammals: GFAP, S100, NCAM, PSA-NCAM, vimentin, p75NTR and galectin-1. GFAP, S100 and NCAM were clearly detected in both fish, though the intracranial tract of the primary olfactory pathway of Carassius appears more S100 stained than the extracranial tract. P75NTR staining is more evident in Poecilia, PSA-NCAM positivity in Carassius. A slight vimentin immunostaining was detected only in Carassius. No galectin-1 staining appeared in the olfactory pathways of either fish. This study shows that some markers for mammalian olfactory ensheathing cells also stain the olfactory pathway in fish. Immunocytochemical staining differs in the two fish under examination, even along the various tracts of the olfactory pathway in the same species.     

Field potentials in the olfactory bulb of the rainbow trout (Salmo gairdneri): Evidence for a dendrodendritic inhibitory pathway

Summary Field potentials have been recorded from the olfactory bulb of the teleost fish, Salmo gairdneri. Stimulation of the olfactory nerve generates a complex wave pattern, which when recorded near the surface in the centre of the bulb consists of one positive and three negative components, P, N₁, N₂ and N₃. The N₁ has been attributed to depolarization of mitral cell dendrites, N₂ to depolarization of the superficial dendrites of granule cells, and P to subsequent hyperpolarization of mitral cells. Possible origins of the N₃ wave are discussed. Spikes from single units recorded in the mitral cell layer usually occur superimposed on the N₁ wave, and single units recorded in the region of granule cells are usually found coincident with the training edge of the deep positive wave. With the exception of N₃ the patterns of activity observed are very similar to previously published results obtained from other vertebrates. It is suggested that the dendrodendritic inhibitory pathway proposed for the mammalian olfactory bulb (Rall et al., 1966; Rall and Shepherd, 1968) is common to all vertebrates.     

Immunohistochemical demonstration of salmon olfactory glutathione S-transferase class pi (N24) in the olfactory system of lacustrine sockeye salmon during ontogenesis and cell proliferation

In mammals, glutathione S-transferase (GST) in the olfactory epithelium is involved in assistance of the olfactory reception by the xenobiotic metabolism. We previously reported the protein and gene expressions of salmon olfactory GST class pi (soGST) in the olfactory receptor cells (ORCs) of the salmonid fish. However, the chronological appearances of soGST in ORCs during ontogeny and cell proliferation are still unknown in this species. In this study, we performed immunohistochemistry of soGST using an antibody specific to soGST in the olfactory system (olfactory placode, olfactory pit, olfactory epithelium, olfactory nerve and olfactory bulb) of lacustrine sockeye salmon (Oncorhynchus nerka) embryos and 5-bromo-2-deoxyuridine (BrdU) experimental fish. The projection of olfactory nerve bundles from the olfactory pit to the presumptive olfactory bulb was identified at embryonic day 28 after fertilization. The olfactory cilia were first detected on the apical surface of ORCs at day 43. soGST-immunoreactivity was first detected within the olfactory pit cells at day 55. At 58 day, the number of soGST-immunoreactive cells increased markedly in the olfactory epithelia, and soGST-immunoreactive fibers were observed in the olfactory nerves and olfactory bulbs. By in vivo uptake of BrdU in 1-year-old fish, we observed for the first time at day 7 after labeling that the olfactory epithelia showed ORCs in which both soGST-immunoreactivity and BrdU coexisted. These results indicate that soGST is synthesized in the mature ORCs of lacustrine sockeye salmon after cell formation and differentiation.     

Regeneration of olfactory sensory axons into transplanted segments of peripheral nerve

Isolated segments of cervical sympathetic trunk were transplanted onto the olfactory mucosa and bulb in adult rats, and olfactory nerve fascicles were sectioned in the region of the transplant. Olfactory axons, presumably arising from newly-formed sensory neurons, were observed to grow into the transplanted segments of peripheral nerve and were ensheathed by Schwann cells of the transplant. The axons were ensheathed as large bundles containing many axons in direct contact, an arrangement characteristic of the normal olfactory nerves, and not of the normal sympathetic trunk. However, single Schwann cell ‘units’ were each surrounded by basal lamina, an arrangement typical of the sympathetic trunk, not of olfactory nerves.The results indicate that olfactory axons, like other peripheral axons, are capable of directing some aspects of the manner in which they are ensheathed by Schwann cells.     

Immunohistochemical and Histochemical Characteristics of the Olfactory System of the Guppy,Poecilia reticulata (Teleostei,Poecilidae)

Olfaction in fish has been studied using preferentially macrosmatic species as models. In the present research, the labelling patterns of different neuronal markers and lectins were analyzed in the olfactory neurons and in their bulbar axonal endings in the guppy Poecilia reticulata, belonging to the group of microsmatic fish. We observed that calretinin immunostaining was confined to a population of olfactory receptor cells localized in the upper layers of the sensory mucosa, probably microvillous neurons innervating the lateral glomerular layer. Immunoreactivity for S100 proteins was mainly evident in crypt cells, but also in other olfactory cells belonging to subtypes projecting in distinct regions of the bulbs. Protein gene product 9.5 (PGP 9.5) was not detected in the olfactory system of the guppy. Lectin binding revealed the presence of N‐acetylglucosamine and α‐N‐acetylgalactosamine residues in the glycoconjugates of numerous olfactory neurons ubiquitously distributed in the mucosa. The low number of sugar types detected suggested a reduced glycosidic variability that could be an index of restricted odorant discrimination, in concordance with guppy visual‐based behaviors. Finally, we counted few crypt cells which were immunoreactive for S100 and calretinin. Crypt cells were more abundant in guppy females. This difference is in accordance with guppy gender‐specific responses to pheromones. Cells immunoreactive to calretinin showed no evidence of ventral projections in the bulbs. We assumed the hypothesis that their odorant sensitivity is not strictly limited to pheromones or sexual signals in general. Anat Rec, 2009. © 2009 Wiley‐Liss, Inc.     

Fine structure of three types of olfactory organs in Xenopus laevis

There is no report on the fine structure of three types of olfactory organs in Xenopus laevis. Their functional assignments in olfaction are not yet established. The fine structure of three types of olfactory organs, olfactory epithelium (OE), vomeronasal organ (VNO), and middle chamber epithelium (MCE), was examined in Xenopus laevis by light and electron microscopy. The olfactory cells of the OE and the sensory cells of the VNO were equipped with cilia and microvilli, respectively, similar to terrestrial animals that possess both the OE and the VNO. On the other hand, the sensory cells of the MCE were classified into two types, the sensory cells with cilia and the sensory cells with microvilli, like those of the OE in fish. These findings suggest that the OE and the VNO in Xenopus laevis detect different kinds of odoriferous molecules in air, whereas the MCE is involved in the perception of odorants in water. Anat. Rec. 252:301–310, 1998. © 1998 Wiley-Liss, Inc.     

Histopathological analysis of the olfactory epithelium of zebrafish (Danio rerio) exposed to sublethal doses of urea

Chronic renal disease is known to alter olfactory function, but the specific changes induced in olfactory organs during this process remain unclear. Of the uraemic toxins generated during renal disease, high levels of urea are known to induce hyposmic conditions. In this study, the effects of environmental exposure to elevated concentrations of urea (7, 13.5 and 20 g L^?1) on the sensory mucosa of zebrafish in acute toxicity and chronic toxicity tests were described. It was observed that lamellae maintained structural integrity and epithelial thickness was slightly reduced, but only following exposure to the highest concentrations of urea. Pan‐neuronal labelling with anti‐Hu revealed a negative correlation with levels of urea, leading to investigation of whether distinct neuronal subtypes were equally sensitive. Using densitometric analysis of immunolabelled tissues, numbers of G_{α olf}‐, TRPC2‐ and TrkA‐expressing cells were compared, representing ciliated, microvillous and crypt neurons, respectively. The three neuronal subpopulations responded differently to increasing levels of urea. In particular, crypt cells were more severely affected than the other cell types, and G_{α olf}‐immunoreactivity was found to increase when fish were exposed to low doses of urea. It can be concluded that exposure to moderate levels of urea leads to sensory toxicity directly affecting olfactory organs, in accordance with the functional olfactometric measurements previously reported in the literature.