Specificity of odorant-evoked inhibition in lobster olfactory receptor neurons

Lobster olfactory receptor neurons, like those of many animals, use two modes of olfactory signaling, excitation and inhibition to code olfactory information. Inhibition appears to act through two distinct ionic mechanisms. Here we show that neither ionic mechanism is odor-specific, providing further support for the emerging understanding that there are no inhibitory odorants per se, but rather that the action of a particular odorant is inherent in the olfactory receptor cell on which an odorant acts.     

Plasma membrane inositol 1,4,5-trisphosphate-activated channels mediate signal transduction in lobster olfactory receptor neurons.

Inositol 1,4,5-trisphosphate (IP3) selectively evokes an inward (excitatory) current in cultured lobster olfactory receptor neurons (ORNs) and directly activates two types of channels in cell-free patches of plasma membrane from the ORNs. The IP3-activated channels have kinetic properties of odor-activated channels in the ORNs and pharmacological properties of intracellular IP3-activated channels in other systems. An antibody directed against an intracellular, cerebellar IP3 receptor recognizes a protein with a molecular weight similar to the mammalian receptor in the ORNs. The antibody selectively increases odor-evoked inward currents and IP3-activated unitary currents in the ORNs. The data provide further evidence for IP3 as an olfactory second messenger and implicate at least one and possibly two novel plasma membrane IP3 receptors in olfactory transduction.     

Properties of transient K+ currents and underlying single K+ channels in rat olfactory receptor neurons

The transient potassium current, IK(t), of enzymatically dissociated rat olfactory receptor neurons was studied using patch-clamp techniques. Upon depolarization from negative holding potentials, IK(t) activated rapidly and then inactivated with a time course described by the sum of two exponential components with time constants of 22.4 and 143 ms. Single-channel analysis revealed a further small component with a time constant of several seconds. Steady-state inactivation was complete at -20 mV and completely removed at -80 mV (midpoint -45 mV). Activation was significant at -40 mV and appeared to reach a maximum conductance at +40 mV (midpoint -13 mV). Deactivation was described by the sum of two voltage-dependent exponential components. Recovery from inactivation was extraordinarily slow (50 s at -100 mV) and the underlying processes appeared complex. IK(t) was reduced by 4-aminopyridine and tetraethylammonium applied externally. Increasing the external K+ concentration ([K+]o) from 5 to 25 mM partially removed IK(t) inactivation, usually without affecting activation kinetics. The elevated [K+]o also hyperpolarized the steady-state inactivation curve by 9 mV and significantly depolarized the voltage dependence of activation. Single transient K+ channels, with conductances of 17 and 26 pS, were observed in excised patches and often appeared to be localized into large clusters. These channels were similar to IK(t) in their kinetic, pharmacological, and voltage-dependent properties and their inactivation was also subject to modulation by [K+]o. The properties of IK(t) imply a role in action potential repolarization and suggest it may also be important in modulating spike parameters during neuronal burst firing. A simple method is also presented to correct for errors in the measurement of whole-cell resistance (Ro) that can result when patch-clamping very small cells. The analysis revealed a mean corrected Ro of 26 G omega for these cells.     

Cyclic AMP-sensitive ion channels in olfactory receptor cells

Membranes from rat olfactory epithelial homogenates were incorporatedinto planar bilayers by a tip-dipping method. Analysis of single-channelcurrents indicate the existence of a cation channel activatedby the addition of adenosine 3',5'monophosphate (cAMP). Theactivity did not require exogenous ATP or GTP. The current-voltagerelationship for the single-channel fluctuations gave a slopeconductance of 32 ± 5 pS and a reversal potential of–5 ±4 mV. Forskolin elicited an increase in patchconductance similar to that produced by cAMP. This responserequired the presence of ATP and could be enhanced by theophylline.     

Sustained primary culture of lobster (Panulirus argus) olfactory receptor neurons

Appropriate conditions were developed for primary sustained culture of olfactory neurons of the spiny lobster Panulirus argus. Neurons were cultured in a modified Liebowitz L15 media supplemented with Panulirus salts, basic minimal essential (BME) vitamins, L-glutamine, low dextrose, and either fetal calf serum (FCS) or lobster haemolymph. Neurite outgrowth and cell viability was strongly affected by choice of adherent substratum, presence of serum, and length of animal captivity. Neither nerve growth factor 7s (NGF-7s), HEPES, nor preconditioned media from the target organ, the olfactory lobe, had any gross effect on either longevity or neurite outgrowth. Five morphologically distinct neuronal cell types (8-16 mum soma diameter) could be defined based on their number and type of processes. All of these cells were electrically excitable (N = 50), and many (56%) produced either inward or outward currents in response to stimulation with single odors. The proportion of cells responding to odors increased (80%) when 10 cells were sequentially presented with a series of 3-5 odors. The finding that cultured cells maintain responsiveness to odors yet are morphologically more compact than their counterparts in situ, argues for the prospect of using these dissociated cultured olfactory receptor neurons to study signal transduction in olfaction.     

Inositol-trisphosphate-dependent calcium currents precede cation currents in insect olfactory receptor neurons in vitro

Specialized olfactory receptor neurons in insects respond to species-specific sex pheromones with transient rises in inositol trisphosphate and by opening pheromone-dependent cation channels. These channels resemble cation channels which are directly or indirectly Ca²⁺-dependent. But there appear to be no internal Ca²⁺ stores in the outer dendrite where the olfactory transduction cascade is thought to start. Hence, it remains to be determined whether an influx of external Ca²⁺ precedes pheromone-dependent cation currents. Patch clamp measurements in cultured olfactory receptor neurons from Manduca sexta reveal that a transient inward current precedes pheromone-dependent cation currents. A transient inositol trisphosphate-dependent Ca²⁺ current, also preceding cation currents with the characteristics of pheromone-dependent cation currents, shares properties with the transient pheromone-dependent current. These results match the biochemical measurements with the electrophysiological data obtained in insect olfactory receptor neurons.Abbreviations ORNs Olfactory receptor neurons - IP₃ Inositol-1,4,5-trisphosphate - I_t Transient pheromone-dependent current - I_ir Transient IP₃-dependent current     

Nonselective cation channel activated by patch excision from lobster olfactory receptor neurons

Summary A nonselective cation channel activated by patch excision was characterized in inside-out patches from spiny lobster olfactory receptor neurons. The channel, which was permeable to Na⁺, K⁺ and Cs⁺, had a conductance of 320 pS and was weakly voltage dependent in the presence of micromolar divalent cations. Millimolar internal divalent cations caused a voltage-and concentration-dependent block of Na⁺ permeation. Analysis of the voltage dependence indicated that the proportion of the membrane's electric field sensed by Mg²⁺ was >1, suggesting that the channel contains a multi-ion pore. Internal divalent cations also reduced the frequency of channel opening in a concentration-dependent, but not voltage-dependent, manner, indicating that different cation binding sites affect gating and conductance. While block of gating prevented determining if internal divalent cations permeate the channel, a channel highly permeable to external divalent cations was observed upon patch excision to the inside-out configuration. The monovalent and divalent cation conductances shared activation by patch excision, weak voltage dependence, and steady-state activity, suggesting that they are the same channel. These data extend our understanding of this type of channel by demonstrating permeation by monovalent cations, detailing Mg²⁺ block of Na^– permeation, and demonstrating the channel's presence in arthropods.     

Transient and persistent tetrodotoxin-sensitive sodium currents in squid olfactory receptor neurons

Squid olfactory receptor neurons are primary bipolar sensory neurons capable of transducing water-born odorant signals into electrical impulses that are transmitted to the brain. In this study, we have identified and characterized the macroscopic properties of voltage-gated Na⁺ channels in olfactory receptor neurons from the squid Lolliguncula brevis. Using whole-cell voltage-clamp techniques, we found that the voltage-gated Na⁺ channels were tetrodotoxin sensitive and had current densities ranging from 5 to 169 pA pF⁻¹. Analyses of the voltage dependence and kinetics revealed interesting differences from voltage-gated Na⁺ channels in olfactory receptor neurons from other species; the voltage of half-inactivation was shifted to the right and the voltage of half-activation was shifted to the left such that a “window-current” occurred, where 10–18% of the Na⁺ channels activated and did not inactivate at potentials near action potential threshold. Our findings suggest that in squid olfactory neurons, a subset of voltage-gated Na⁺ channels may play a role in generating a pacemaker-type current for setting the tonic levels of electrical activity required for transmission of hyperpolarizing odor responses to the brain. Accepted: 1 October 1998     

Acid-sensing ion channels in mouse olfactory bulb M/T neurons

The olfactory bulb contains the first synaptic relay in the olfactory pathway, the sensory system in which odorants are detected enabling these chemical stimuli to be transformed into electrical signals and, ultimately, the perception of odor. Acid-sensing ion channels (ASICs), a family of proton-gated cation channels, are widely expressed in neurons of the central nervous system. However, no direct electrophysiological and pharmacological characterizations of ASICs in olfactory bulb neurons have been described. Using a combination of whole-cell patch-clamp recordings and biochemical and molecular biological analyses, we demonstrated that functional ASICs exist in mouse olfactory bulb mitral/tufted (M/T) neurons and mainly consist of homomeric ASIC1a and heteromeric ASIC1a/2a channels. ASIC activation depolarized cultured M/T neurons and increased their intracellular calcium concentration. Thus, ASIC activation may play an important role in normal olfactory function.     

Cyclic nucleotide signaling mediates an odorant-suppressible chloride conductance in lobster olfactory receptor neurons

We have previously shown that lobster olfactory receptor neurons (ORNs) express an odorant-suppressible Cl(-) conductance that modulates the output of the cells. Here, we develop a more complete pharmacological profile of this conductance, showing it is blockable by the Cl(-) channel blockers DIDS, 9-AC and flufenamic acid, but not by niflumic acid. We then show that a conductance with this pharmacological profile is mediated by cyclic nucleotide signaling. These findings further our understanding of the cellular mechanisms through which odorants can modulate the output of lobster ORNs.     

Slowly activating K+ channels in rat olfactory receptor neurons.

Patch-clamp techniques were used to investigate slowly activating, Ca(2+)-insensitive K+ channels of isolated rat olfactory receptor neurons. These channels had a unitary conductance of 135 pS and were only found in a small proportion (less than 5%) of membrane patches. Upon depolarization to voltages more positive than -50 mV, the channels activated gradually over a period of at least 10 s. When hyperpolarized to negative voltages, channel activity deactivated in a slow but voltage-dependent manner. These channels may underlie a slowly activating K+ current that is observed in approximately 30% of whole-cell recordings. Similar single channels have been reported in smooth muscle cells, but this is the first demonstration of these channels in any type of neuron. The channels may contribute to the spike frequency adaptation and post-stimulus hyperpolarization that are observed during the excitatory response to odorants. They may also contribute to cell repolarization following large odorant-stimulated receptor currents.     

Pharmacological investigation of protein kinase C- and cGMP-dependent ion channels in cultured olfactory receptor neurons of the hawkmoth Manduca sexta

In the hawkmoth Manduca sexta, pheromone stimuli of different strength and duration rise the intracellular Ca2+ concentration in olfactory receptor neurons (ORNs). While second-long pheromone stimuli activate protein kinase C (PKC), which apparently underlies processes of short-term adaptation, minute-long pheromone stimuli elevate cyclic guanosine monophosphate (cGMP) concentrations, which correlates with time courses of long-term adaptation. To identify ion channels involved in the sliding adjustment of olfactory sensitivity, inside-out patch clamp recordings on cultured ORNs of M. sexta were performed to characterize Ca2+-, PKC-, and cGMP-dependent ion channels. Stepping to positive holding potentials in high intracellular Ca2+ elicits different Ca2+-dependent ion channels, namely small-conductance channels (2-20 ps), medium-conductance channels (20-100 ps), and large-conductance channels (>100 ps). Ion channels of 40, 60, and 70 ps opened after PKC activation, whereas 10- and >100-ps channels were observed less frequently. Application of 8-bromo cyclic guanosine monophosphate opened 55- and 70-ps channels and increased the open probability of >100-ps channels, whereas even in the presence of phorbol ester 40-ps channels were inhibited. Thus, cGMP elevations activate a different set of ion channels as compared with PKC and suppress at least one PKC-dependent ion channel.     

Voltage-dependent and odorant-regulated currents in isolated olfactory receptor neurons of the channel catfish.

The electrical properties of olfactory receptor neurons, enzymatically dissociated from the channel catfish (Ictalurus punctatus), were studied using the whole-cell patch-clamp technique. Six voltage-dependent ionic currents were isolated. Transient inward currents (0.1-1.7 nA) were observed in response to depolarizing voltage steps from a holding potential of -80 mV in all neurons examined. They activated between -70 and -50 mV and were blocked by addition of 1 microM tetrodotoxin (TTX) to the bath or by replacing Na+ in the bath with N-methyl-D-glucamine and were classified as Na+ currents. Sustained inward currents, observed in most neurons examined when Na+ inward currents were blocked with TTX and outward currents were blocked by replacing K+ in the pipette solution with Cs+ and by addition of 10 mM Ba2+ to the bath, activated between -40 and -30 mV, reached a peak at 0 mV, and were blocked by 5 microM nimodipine. These currents were classified as L-type Ca2+ currents. Large, slowly activating outward currents that were blocked by simultaneous replacement of K+ in the pipette with Cs+ and addition of Ba2+ to the bath were observed in all olfactory neurons examined. The outward K+ currents activated over approximately the same range as the Na+ currents (-60 to -50 mV), but the Na+ currents were larger at the normal resting potential of the neurons (-45 +/- 11 mV, mean +/- SD, n = 52). Four different types of K+ currents could be differentiated: a Ca(2+)-activated K+ current, a transient K+ current, a delayed rectifier K+ current, and an inward rectifier K+ current. Spontaneous action potentials of varying amplitude were sometimes observed in the cell-attached recording configuration. Action potentials were not observed in whole-cell recordings with normal internal solution (K+ = 100 mM) in the pipette, but frequently appeared when K+ was reduced to 85 mM. These observations suggest that the membrane potential and action potential amplitude of catfish olfactory neurons are significantly affected by the activity of single channels due to the high input resistance (6.6 +/- 5.2 G omega, n = 20) and low membrane capacitance (2.1 +/- 1.1 pF, n = 46) of the cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Contribution of cyclic-nucleotide-gated channels to the resting conductance of olfactory receptor neurons

The basal conductance of unstimulated frog olfactory receptor neurons was investigated using whole-cell and perforated-patch recording. The input conductance, measured between -80 mV and -60 mV, averaged 0.25 nS in physiological saline. Studies were conducted to determine whether part of the input conductance is due to gating of neuronal cyclic-nucleotide-gated (CNG) channels. In support of this idea, the neuronal resting conductance was reduced by each of five treatments that reduce current through CNG channels: external application of divalent cations or amiloride; treatment with either of two adenylate cyclase inhibitors; and application of AMP-PNP, a competitive substrate for adenylate cyclase. The current blocked by divalent cations or by a cyclase inhibitor reversed near 0 mV, as expected for a CNG current. Under physiological conditions, gating of CNG channels contributes approximately 0.06 nS to the resting neuronal conductance. This implies a resting cAMP concentration of 0.1-0.3 micro M. A theoretical model suggests that a neuron containing 0.1-0.3 micro M cAMP is poised to give the largest possible depolarization in response to a very small olfactory stimulus. Although having CNG channels open at rest decreases the voltage change resulting from a given receptor current, it more substantially increases the receptor current resulting from a given increase in [cAMP].     

Ion channels from chemosensory olfactory neurons

The olfactory epithelium has the ability to respond to a large number of volatile compounds of small molecular weight. Ultimately, such a property lies on a specialized type of neuron, the olfactory receptor cell. In the presence of odorants, the olfactory receptor neuron responds with action potentials whose frequency depends on odorant concentration. The primary events in the process of olfactory transduction are thought to occur at the cilia of olfactory receptor neurons and involve the binding of odorants to receptor molecules followed by the opening of ion channels. A crucial step in understanding olfactory transduction requires identifying the mechanisms that regulate the electrical activity of olfactory cells. In the last couple of years, patch-clamp recording from isolated olfactory cells and reconstitution of olfactory membranes in planar lipid bilayers have begun to shed light on some of these mechanisms. Although the information emerging from such studies is still preliminary, there are already well-defined hypotheses on the molecular events that might underlie the primary events in olfactory transduction. Currently, attention is being focused on the notions that second messengers might be involved in the activation of ion channels in olfactory cilia, and that odorant binding to a receptor molecule might lead directly to the gating of ion channels in chemosensory olfactory membranes. The coming years promise to be exciting ones in the field of olfactory transduction. We have now the necessary tools to be able to confront hypotheses and experimental facts.     

Ionic requirements for bursting activity in lobster stomatogastric neurons

Summary The ionic requirements for bursting activity have been investigated in the electrically coupled PD-AB cells group of the Stomatogastric ganglion in the lobster.The passive electrical properties and coupling parameters have been determined in either current or voltage clamp conditions. In voltage clamped cells, the current displayed slow inward transients with superimposed fast transients corresponding respectively to the slow waves and spikes of the coupled undamped cells. The amplitude and frequency of the slow transients were reduced upon hyperpolarization.Cyclic conductance changes were observed with short current pulses, the coupling ratio also changes cyclically being lower during the bursts and slowly increasing during the interburst interval.The slow wave amplitude increased in low K-saline. The post-burst hyperpolarization but not the top level of the wave behaved like a potassium electrode for [K]_o higher than 10 mM/l.TEA at low concentration (1 to 5 mM/l) increased the slow wave amplitude by lifting its top level by 10 to 20 mV. The post-burst hyperpolarization remained almost unchanged and its K-dependence was not altered by TEA.Low Na-saline reduced the slow wave amplitude (6 to 11 mV per decade). The Na-dependence increased in the presence of TEA. Slow waves devoid of spikes persisted in 10% Na saline containing TEA. 10^–9 M/1 TTX blocked the spikes. 10^–7 M/1 TTX blocked the slow waves.Mg-free saline had no effect on the slow wave. In Ca-free saline the cells depolarized and the bursting activity tended to vanish. Repolarization with current led to long lasting slow waves deprived of post-burst hyperpolarization. The bursting ceased when EDTA was added to the Ca-free saline.Cobalt (up to 10 mM/l) was similar to Ca-free saline in its effects; lengthening of the wave and blockage of the repolarization. Replacing Ba for Ca produced large (up to 70 mV) slow waves which were reduced by Co and Ca.Bistable states were observed in various experimental conditions. It is concluded that the slow waves are produced by activation of sodium and calcium currents. The amplitude of the slow wave is modulated by the simultaneous activation of a TEA-sensitive K current. The repolarization is caused by increased K current activated by the inward calcium current. The slow pacemaker potential in the interburst interval corresponds to the slow disappearance of the K current.This work was supported by N.I.H. grant no. 09322, NSF grant no. 00250, and a Guggenheim Foundation Fellowship to A.D.S. and by the CNRS and a DGRST grant no. 16501891 to M. Gola. We are grateful to Stuart Thompson and Felix Strumwasser for helpful comments and to Barbara McLean for technical assistance.     

Outward currents in olfactory receptor neurons activated by odorants and by elevation of cyclic AMP

We studied the outward currents elicited by an odorous compound, isoamyl acetate, in isolated olfactory receptor neurons of the grass frog under whole-cell perforated-patch voltage-clamp recording. Odorant-induced outward currents were relatively rare, occurring in about 16% of the responding cells. Responses had smaller amplitudes and shorter time courses when compared to the more commonly found odorant-induced inward currents. There was a high correlation between odorant-induced outward current and responses evoked by either 8-(4-chlorophenylthio) adenosine 3':5'-cyclic monophosphate, a membrane-permeant cyclic adenosine monophosphate analog, or 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor. The outward current responses to all three substances increased in amplitude when the membrane potential was more negative than -60 mV and decreased in amplitude when the membrane potential was more positive. Responses were still present when the potential was held at -100 mV, indicating that the responses are not the result of a potassium conductance. Removal of external calcium from the perfusion medium abolished the outward currents. Our results indicate that the odorant-induced outward current is a calcium-dependent event that may be mediated by cyclic adenosine monophosphate.     

The permeation of organic cations through cAMP-gated channels in mammalian olfactory receptor neurons

The permeation of monovalent organic cations through adenosine 3,5-cyclic monophosphate-(cAMP) activated channels was studied by recording macroscopic currents in excised inside-out membrane patches from the dendritic knobs of isolated mammalian olfactory receptor neurons (ORNs). Current-voltage relations were measured when bathing solution Na⁺ was replaced by monovalent organic cations. Permeability ratios relative to Na⁺ ions were calculated from changes in reversal potentials. Some of the small organic cations tested included ammonium (NH ₄ ⁺ ), hydroxylammonium and formamidinium, with relative permeability ratios of 1.41, 2.3 and 1.01 respectively. The larger methylated and ethylated ammonium ions studied included: DMA (dimethylammonium), TMA (tetramethylammonium) and TEA (tetraethylammonium) and they all had permeability ratios larger than 0.09. Even large cations such as choline, arginine and tris(hydroxymethyl)aminomethane (Tris) were appreciably permeant through the cAMP-activated channel with permeability ratios ranging from 0.19 to 0.7. The size of the permeating cations, as assessed by molecular weight, was a good predictor of the permeability. The permeability sequence of the cAMP-activated channel in our study was P_NH4 > P_Na > p_DMA > p_TMA > P_Choline > P_TEA. Higher permeability ratios of hydroxylammonium, arginine and tris(hydroxymethyl)aminomethane cannot be explained by ionic size alone. Our results indicate that: (i) cAMP-activated channels poorly select between monovalent cations; (ii) the pore dimension must be at least 6.5 × 6.5 Å, in order to allow TEA and Tris to permeate and (iii) molecular sieving must be an important mechanism for the permeation of large organic ions through the channels with specific ion binding playing a smaller role than in other structurally similar channels. In addition, the results clearly indicate that cyclic nucleotide-gated (CNG) channels in different cells are not the same, the olfactory CNG channel being different from that of the photoreceptors, particularly with respect to the permeation of large organic cations, which the ORN channels allow to permeate readily.This work was supported by the Australian Research Council of Australia.