Vomeronasal organ and human pheromones.

For many organisms, pheromonal communication is of particular importance in managing various aspects of reproduction. In tetrapods, the vomeronasal (Jacobson's) organ specializes in detecting pheromones in biological substrates of congeners. This information triggers behavioral changes associated, in the case of certain pheromones, with neuroendocrine correlates. In human embryos, the organ develops and the nerve fibers constitute a substrate for the migration of GnRH-secreting cells from the olfactory placode toward the hypothalamus. After this essential step for subsequent secretion of sex hormones by the anterior hypophysis, the organ regresses and the neural connections disappear. The vomeronasal cavities can still be observed by endoscopy in some adults, but they lack sensory neurons and nerve fibers. The genes which code for vomeronasal receptor proteins and the specific ionic channels involved in the transduction process are mutated and nonfunctional in humans. In addition, no accessory olfactory bulbs, which receive information from the vomeronasal receptor cells, are found. The vomeronasal sensory function is thus nonoperational in humans. Nevertheless, several steroids are considered to be putative human pheromones; some activate the anterior hypothalamus, but the effects observed are not comparable to those in other mammals. The signaling process (by neuronal detection and transmission to the brain or by systemic effect) remains to be clearly elucidated.

[Exploration of olfactory difficulties]. / Exploration des troubles de l'odorat.

Sense-of-smell disorders are a frequent occurrence at ORL clinics. The clinical exploration must include a thorough and comprehensive interview to discover the etiology. The clinical examination is easily performed using cold-light endoscopes, which can reveal anatomical or mucous membrane pathologies, even in far- situated anatomical parts. The access to olfactometric examinations is limited by the small number of health centers which have the proper equipment and trained staff. Guided by the initial interview and clinical exploration, the diagnosis of smell disorder can benefit from the conclusive and factual contribution of the CAT scan and MRI.

Photolysis of caged inositol 1,4,5-trisphosphate induces action potentials in frog vomeronasal microvillar receptor neurones.

To study the effect of inositol 1,4,5-trisphosphate (IP(3)) in isolated frog vomeronasal microvillar receptor neurones, whole-cell recordings were performed with 0.5 microM caged IP(3) dissolved in the pipette solution. IP(3) was released by photolysis of caged IP(3) initiated by a 0.8-ms ultraviolet flash from a xenon flash lamp 70 s after the start of dialysis of caged IP(3) into the cell. Flash illuminating the whole receptor neurone with caged IP(3) triggered action potentials when the current was clamped at zero and a series of transient inward currents of 12-55 pA at a holding potential of -70 mV. The average number of spikes during the first 40 s after release of IP(3) was 7.2+/-2.5 (n=6, mean+/-S.E.M.). The average maximum current and the total inward transport of charge during the first 40 s after photolysis of caged IP(3) were -24+/-8.0 pA and -1.7+/-0.8 pC, respectively (n=5, mean+/-S.E.M.). Inward membrane currents of 12-55 pA after release of IP(3) were not observed with 50 microM La(3+) in the bath. Notably, flash focused on the terminal vesicle also triggered action potentials. No action potentials were observed following flash focused on the soma or outside the dendrite. The average number of spikes during the first 40 s after release of IP(3) initiated by flash spatially restricted to the terminal vesicle was 5.0+/-2.0 (n=4, mean+/-S.E.M.).The present study indicates that local release of IP(3) in the terminal vesicle of the vomeronasal neurones triggers transient depolarizations and induces action potentials. We suggest that IP(3) might be a second messenger in the vomeronasal microvillar receptor neurones.

The vomeronasal cavity in adult humans.

We observed the surface of the anterior part of the nasal septum of living subjects using an endoscope. In approximately 13% of 1842 patients without pathology of the septum, the vomeronasal pit was clearly observed on each side of the septum, and in 26% it was observed only on one side. The remaining observations indicated either the presence of putative pits or no visible evidence of a pit. However, repetitive observations on 764 subjects depicted changes over time, from nothing visible to well-defined pits and vice versa. Based on 130 subjects observed at least four times, we estimate that approximately 73% of the population exhibits at least one clearly defined pit on some days. By computer tomography, the vomeronasal cavities were located at the base of the most anterior part of the nasal septum. Histological studies indicated that the vomeronasal cavities consisted of a pit generally connected to a duct extending in a posterior direction under the nasal mucosa. Many glands were present around the duct, which contained mucus. There was no sign of the pumping elements found in other mammalian species. Most cells in the vomeronasal epithelium expressed keratin, a protein not expressed by olfactory neurons. Vomeronasal epithelial cells were not stained by an antibody against the olfactory marker protein, a protein expressed in vomeronasal receptor neurons of other mammals. Moreover, an antibody against protein S100, expressed in Schwann cells, failed to reveal the existence of vomeronasal nerve bundles that would indicate a neural connection with the brain. Positive staining was obtained with the same antibodies on specimens of human olfactory epithelium. The lack of neurons and vomeronasal nerve bundles, together with the results of other studies, suggests that the vomeronasal epithelium, unlike in other mammals, is not a sensory organ in adult humans.

Odorant and pheromone binding by aphrodisin, a hamster aphrodisiac protein.

Aphrodisin is a soluble glycoprotein of hamster vaginal discharges, which stimulates male copulatory behavior. Natural aphrodisin was purified and its post-translational modifications characterized by MALDI-MS peptide mapping. To evaluate its ability to bind small volatile ligands, the aphrodisiac protein was expressed in the yeast Pichia pastoris as two major isoforms differing in their glycosylation degree, but close in conformation to the natural protein. Dimeric recombinant aphrodisins were equally able to efficiently bind odors (2-isobutyl-3-methoxypyrazine and methyl thiobutyrate) and a pheromone (dimethyl disulfide), suggesting that they could act as pheromone carriers instead of, or in addition to, direct vomeronasal neuron receptor activators.

Structure and function of the vomeronasal organ.

Many animals use their vomeronasal organs to gain direct and specific contact with chemical cues released by congeners and in biological fluids. These cues provide information about the physiological status of the emitter and facilitate or regulate social interactions such as sexual relationships. The present review gives a short description of the discovery of the vomeronasal organ and the pivotal findings of Jacobson. The distribution of the organ and its anatomy in some vertebrates are described. The mechanisms for stimulus entry and egress are discussed, and the findings that led to the appreciation of the vomeronasal organ in mammals as a main chemosensory organ for pheromones mediating reproductive status and inducing sexual behaviour are reported. The anatomical, biochemical and functional properties of the receptor neurones are described.

Electrophysiological properties of frog olfactory supporting cells.

Cells, identified as supporting cells by Lucifer Yellow injection, were recorded from slices of frog olfactory epithelium using patch-clamp recordings. Cell-attached single-channel recordings indicated that the intracellular potential (IP) was -68 +/- 7 mV (n = 22) with 4 mM K+ in the bath ([K+]o). IP was -67 +/- 4 mV (n = 32) in whole-cell conditions with 100 mM KCl inside the cell, suggesting a low membrane permeability for Cl-. IP depended on [K+]o in a manner described by the Goldman-Hodgkin-Katz equation with a permeability ratio pk+:PNa+ of 40. The input resistance was 32 +/- 14 M omega (n = 15), indicating a high membrane conductance at rest. Odorant stimulations evoked passive membrane depolarizations, probably reflecting an increase in [K+]o due to the neuronal activation. Whole-cell recordings with 100 mM CsCl instead of KCl in the pipette, together with the block of gap-junctions with octanol, indicated the existence of an electrical coupling between supporting cells. The electrical coupling between these glial-like cells could facilitate the clearance of K+ ions released by olfactory receptor neurons during odorant stimulation.

Scanning electron microscopy and gramicidin patch clamp recordings of microvillous receptor neurons dissociated from the rat vomeronasal organ.

Vomeronasal organs from female rats were dissociated and isolated microvillous receptor neurons were studied. The isolated receptor neurons kept the typical bipolar shape which they have in situ as observed by scanning electron microscopy. We applied the perforated patch-clamp technique using the cation-selective ionophore gramicidin on freshly isolated and well differentiated receptor neurons. The mean resting potential was -58+/-14 mV (n=39). The contribution of the sodium pump current to the resting potential was demonstrated by lowering the K+ concentration in the bath or by application of 100 microM dihydro-ouabain. The input resistance was in the range of 1-6 GOmega and depolarizing current pulses of a few pA were sufficient to trigger overshooting action potentials. In voltage clamp conditions a fast transient sodium inward current and a sustained outward potassium current were activated by membrane depolarization. These observations indicate that freshly isolated vomeronasal receptor neurons of rats can be recorded, using gramicidin, with little modification of the intracellular content. Their electrophysiological properties are very similar to those observed in situ. Four out of eight female vomeronasal receptor cells were depolarized by diluted rat male urine.

Functional role of receptor neurons in encoding olfactory information.

In the present review we have considered the properties of the olfactory receptor neurons and discuss the strategy these cells use to perform their signaling task. Special emphasis is laid on the mechanisms for setting the membrane potential at rest and the mechanisms that the cell can use to respond with action potentials to significant stimuli only. We demonstrate that the firing properties of the receptor neurons depend upon the initial level of the membrane potential. We present the idea that the olfactory glomerulus can function as a unit in olfactory processing. In this perspective the olfactory receptor neuron is a subunit of the olfactory glomerulus.

Direct influence of the sodium pump on the membrane potential of vomeronasal chemoreceptor neurones in frog.

1. Whole-cell measurements were made from microvillous receptor neurones isolated from the frog vomeronasal organ. We examined the mechanisms that determined the value of the resting membrane potential. 2. Cells recorded in Ringer solution containing 4 mM K+ showed a resting membrane potential of -88 +/- 20 mV (mean +/- 1 S.D., n = 56). Sixty-six per cent of the cells had stable resting potentials more negative than the calculated equilibrium potentials for K+ (EK, -82 mV) indicating the presence of a hyperpolarizing outward pump current. 3. Cells recorded with an intracellular solution containing Na+ instead of K+, to set EK at 0 mV, presented stable membrane potentials in the range -65 to -119 mV when bathed in a normal Ringer solution. 4. Ouabain, a specific inhibitor of the Na+,K(+)-ATPase, blocked the outward sodium pump current (Ip) and depolarized the membrane. 5. The sodium pump current, measured as the current blocked by 0.5 mM dihydro-ouabain, was linearly related to the membrane potential in the range -60 to -120 mV. The reversal potential measured with a calculated free energy of ATP hydrolysis of -36.2 kJ mol-1 was estimated to be -143 mV. 6. Reduction of the external K+ concentration to 0 mM depolarized the membrane to less than -40 mV. Voltage-clamp observations in this condition indicated a reduction of Ip. Ouabain added to the bath reduced the blocking effect of low external K+. The addition of external K+ activated Ip and induced a rapid hyperpolarization of the cell membrane. 7. At membrane potentials more negative than -80 mV, an inward rectifying depolarizing current characterized as Ih was activated. When Ih was blocked by 5 mM external Cs+ the resting membrane potential increased. 8. These data indicate that the membrane potential of the vomeronasal receptor neurones is not generated by a passive diffusion of K+ ions but by the hyperpolarizing current created by the Na+,K(+)-ATPase. We propose that the resting potential is set by a balance between Ip and Ih. The physiological implications of these mechanisms for setting the resting potential are discussed.

[The vomeronasal organ--a rediscovered sensory organ]. / Vomeronasale--et gjenoppdaget sanseorgan.

The authors review the function and anatomy of the vomeronasal organ (organ of Jacobson), a chemosensory organ situated in the nose. In mammals, stimulation of the vomeronasal organ induces sexual behaviour and changes the hormonal status of males and females. Impairment of the vomeronasal organ in rats disrupts both the females' ultrasound calling during oestrus and their maternal behaviour. Stimulation of the vomeronasal organ promotes sexual maturation of juvenile females and induces abortion and production of pheromones. Earlier on, the vomeronasal organ in man was believed to be present only at the foetal stage; in 1991 its presence was reestablished in adults. Electrophysiological studies show that the vomeronasal organ of man is stimulated by steroids found in human skin. The physiological properties of the receptor cells of the vomeronasal organ are different from those of the olfactory organ.

A new clinical olfactory test to quantify olfactory deficiencies.

We have recently developed a computer-assisted olfactory test to measure detection thresholds for five pure odorants. The reference group consisted of 30 subjects without olfactory complaints. Statistical analysis was carried out to identify a statistical criterion for determining olfactory deficiencies. This criterion was applied to 54 subjects suspected to have an olfactory deficiency, either on the basis of their subjective complaints or on clinical examinations (e.g. scanner radiography, endoscopic investigations, rhinomanometric measurements). Nine aetiological groups were screened: trauma, nasal polyposis, nasal obstruction, allergic rhinitis, post-influenza, post-anaesthesia, endocrine dysfunction, hereditary hyposmia, and subjective olfactory loss without a clear aetiology. In each group, this method allowed us to discriminate between deficient and non-deficient patients, and the olfactory deficit could be quantified. This rapid procedure was well-accepted by all subjects and gave reproducible quantitative results. It can provide useful information about the relationship between olfactory acuity and a given aetiologic category.

Intensity coding in olfactory receptor cells.

Olfactory receptors code the concentration of stimulating molecules into an impulse frequency message. Patch-clamp recordings have now demonstrated, in the olfactory receptor cell membrane, a number of membrane conductances. Some of them are gated by odorants, in the cilia, and depolarize the cell through cAMP- or IP3-sensitive channels, depending on the species. Other conductances are activated by membrane depolarization and/or an increased intracellular Ca2+ concentration; they participate in oscillating membrane potential changes during impulses and post-spike after-polarizations, and control the repetitive firing. Original data relative to the resting potential and the impulse frequency coding of the odorant concentration are presented.

Voltage-dependent currents in microvillar receptor cells of the frog vomeronasal organ.

Vomeronasal receptor cells are differentiated bipolar neurons with a long dendrite bearing numerous microvilli. Isolated cells (with a mean dendritic length of 65 microns) and cells in mucosal slices were studied using whole-cell and Nystatin-perforated patch-clamp recordings. At rest, the membrane potential was -61 +/- 13 mV (mean +/- SD; n = 61). Sixty-four per cent of the cells had a resting potential in the range of -60 to -86 mV, with almost no spontaneous action potential. The input resistance was in the G omega range and overshooting repetitive action potentials were elicited by injecting depolarizing current pulses in the range of 2-10 pA. Voltage-dependent currents were characterized under voltage-clamp conditions. A transient fast inward current activating near -45 mV was blocked by tetrodotoxin. In isolated cells, it was half-deactivated at a membrane potential near -75 mV. An outward K+ current was blocked by internal Cs+ ions or by external tetraethylammonium or Ba2+ ions. A calcium-activated voltage-dependent potassium current was blocked by external Cd2+ ions. A voltage-dependent Ca2+ current was observed in an iso-osmotic BaCl2 solution. Finally, a hyperpolarization-activated inward current was recorded. Voltage-dependent currents in these microvillar olfactory receptor neurons appear qualitatively similar to those already described in ciliated olfactory receptor cells located in the principal olfactory epithelium.

Luminal non-specific cationic channels in cultured strial marginal cells of guinea pig and gerbil as determined by patch clamp technique.

Using primary cultures of marginal cells of stria vascularis from guinea pig and gerbil, ionic channels located on the luminal membrane were investigated by means of patch clamp technique. Recordings were performed in cell-attached and inside-out configurations. In cell-attached configuration, single channel activity was identified with a conductance of about 25 pS. I-V curve was linear. The probability of opening was increased upon depolarization. Up to 7 channels could be present in the same patch, indicating a rather high density. In inside-out configuration, the reversal potential was 0 mV, suggesting a non-specific cationic channel. These luminal non-specific cationic channels would allow the passive K+ efflux and Na+ influx across the apical membrane of marginal cells. This finding is consistent with the "one-pump" model of strial activity. The present study suggests that culture of strial marginal cells may be a suitable model for in-depth investigation of endolymph physiology.

Odorant-evoked potassium changes in the frog olfactory epithelium.

Electroolfactogram (EOG) and extracellular potassium activity (aK) measurements were carried out in frog olfactory epithelia in vivo. Odorant-evoked changes in aK were characterized on the basis of depth profile analysis. Following an olfactory stimulation with butanol vapours, an increase in aK was measured in the mucus and the proximal part of the epithelium; this response started after the beginning of the EOG and was proportional to the amplitude of the latter. In the deeper part of the epithelium, the aK response had complex waveforms showing an initial K decrease which was suppressed by local application of ouabain, suggesting the existence of a pumping mechanism at this level. The results are discussed in terms of extracellular accumulation of K ions following neuroreceptor activation with respect to EOG generation theories.

A patch-clamp analysis of membrane currents in salamander olfactory receptor cells.

Isolated olfactory receptor cells were obtained from salamander olfactory epithelium and kept in short term culture conditions. They were studied by means of the whole cell patch-clamp technique associated with ionic substitutions and channel blockers. Under physiological ionic gradients, these cells had a resting potential of -39 +/- 10 mV and an input resistance above 2 G omega. Using different channel blockers and ion substitutions, we could separate several distinct components in the overall whole cell current. In most cells, inward current reflected the activation of a TTX resistant conductance which was blocked by cobalt ions. This inward current lasted only for about 5 min of whole cell recording. In a minority of cells, a TTX sensitive sodium current was also observed. The outward K+ current was blocked when the cells were loaded with cesium and tetraethylammonium. It inactivated slowly and incompletely and could act as a depolarization limited in case of intense odour stimulations. Single channel analysis from outside out patches suggested that it corresponded to the activity of 34 pS channels. In some cells a rapidly inactivating K+ current was also present. Single channel activities (27 +/- 6 pS) were commonly recorded with KCl-filled pipettes, at resting or hyperpolarized membrane potentials but not at depolarized potentials. Membrane hyperpolarization increased the open-state probability. A preliminary study with odorant stimulations indicated the existence of a stimulus-induced current probably corresponding to the activation of the chemoreceptive membrane.

Intracellular recordings from salamander olfactory supporting cells.

Stable intracellular potentials were recorded just below the surface of the salamander olfactory epithelium. The site of recording corresponded to the zone of highest density of supporting cell perikarya. The electrophysiological properties of cells recorded in this zone included: neither spontaneous nor evoked spike activity, high resting potential (-96 +/- 10 mV, n = 113) and low input resistance (15 +/- 12 M omega, n = 64). The cells were depolarized to -9 +/- 8 mV when the extracellular potassium concentration was increased from 2 to 100 mM. The membrane potential also changed during activation of the olfactory receptor neurons. Antidromic stimulation of olfactory axons elicited both rapid and slow depolarizations. Odorant stimulation induced graded depolarizations which always lagged behind the electro-olfactogram by more than 1 s. In contrast to the responses of the olfactory receptor neurons, these responses were nearly identical from one cell to another. Compared with the concomitant electro-olfactogram, they had almost the same amplitude, with a reversed polarity. These findings are discussed in the context of the possible auxiliary functions of supporting cells in olfactory processes.