Stuart Firestein

How the olfactory system makes sense of scents

The human nose is often considered something of a luxury, but in the rest of the animal world, from bacteria to mammals, detecting chemicals in the environment has been critical to the successful organism. An indication of the importance of olfactory systems is the significant proportion — as much as 4% — of the genomes of many higher eukaryotes that is devoted to encoding the proteins of smell. Growing interest in the detection of diverse compounds at single-molecule levels has made the olfactory system an important system for biological modelling.

The olfactory receptor gene superfamily of the mouse

Olfactory receptor (OR) genes are the largest gene superfamily in vertebrates. We have identified the mouse OR genes from the nearly complete Celera mouse genome by a comprehensive data mining strategy. We found 1,296 mouse OR genes (including ∼20% pseudogenes), which can be classified into 228 families. OR genes are distributed in 27 clusters on all mouse chromosomes except 12 and Y. One OR gene cluster matches a known locus mediating a specific anosmia, indicating the anosmia may be due directly to the loss of receptors. A large number of apparently functional fish-like Class I OR genes in the mouse genome may have important roles in mammalian olfaction. Human ORs cover a similar receptor space as the mouse ORs, suggesting that the human olfactory system has retained the ability to recognize a broad spectrum of chemicals even though humans have lost nearly two-thirds of the OR genes as compared to mice.

The molecular receptive range of an odorant receptor

An odor perception is the brains interpretation of the activation pattern of many peripheral sensory neurons that are differentially sensitive to a wide variety of odors. The sensitivity of these neurons is determined by which of the thousand or so odor receptor proteins they express on their surface. Understanding the odor code thus requires mapping the receptive range of odorant receptors. We have adopted a pharmacological approach that uses a large and diverse pool of odorous compounds to characterize the molecular receptive field of an odor receptor. We found a high specificity for certain molecular features, but high tolerance for others—a strategy that enables the olfactory apparatus to be both highly discriminating, and able to recognize several thousand odorous compounds.

THE RELATION BETWEEN STIMULUS AND RESPONSE IN OLFACTORY RECEPTOR CELLS OF THE TIGER SALAMANDER

1. Olfactory receptor cells were isolated from the adult tiger salamander Ambystoma tigrinum and the current in response to odorant stimuli was measured with the whole‐cell voltage‐clamp technique while odorants at known concentrations were rapidly applied for controlled exposure times. 2. Three odorants, cineole, isoamyl acetate and acetophenone, were first applied at 5 x 10(‐4) M. Out of forty‐nine cells tested, 53% responded to one odorant only, 22% to two odorants and 25% to all three odorants. 3. The amplitude of the current in response to a given odorant concentration was found to be dependent on the duration of the odorant stimulus and reached a saturating peak value at 1.2 s of stimulus duration. 4. The current measured at the peak of the response for odorant steps of 1.2 s as a function of odorant concentration was well described by the Hill equation for the three odorants with Hill coefficients higher than 1 and K1/2 (odorant concentration needed to activate half the maximal current) ranging from 3 x 10(‐6) to 9 x 10(‐5) M. 5. It is concluded that olfactory receptor cells are broadly tuned and have a low apparent affinity for odorants, integrate stimulus information over time, and have a narrow dynamic range.

Direct Activation of the Olfactory Cyclic Nucleotide–Gated Channel through Modification of Sulfhydryl Groups by NO Compounds

The activation of a cyclic nucleotide-gated channel is the final step in sensory transduction in olfaction. Normally, this channel is opened by the intracellular cyclic nucleotide second messenger cAMP or cGMP. However, in single channel recordings we found that donors of nitric oxide, a putative intercellular messenger, could directly activate the native olfactory neuron channel. Its action was independent of the presence of the normal ligand and did not involve the cyclic nucleotide binding site, suggesting an alternate site on the molecule that is critical in channel gating. The biochemical pathway appears to utilize nitric oxide in one of its alternate redox states, the nitrosonium ion, transnitrosylating a free sulfhydryl group belonging to a cysteine residue tentatively identified as being in the region linking the S6 transmembrane domain to the ligand binding domain.

Activation of the sensory current in salamander olfactory receptor neurons depends on a G protein-mediated cAMP second messenger system

Olfactory receptor neurons respond to odor stimulation with an inward cationic current. Under whole-cell patch clamp, individual, isolated olfactory receptors were exposed to pharmacological agents known to interact with distinct enzymes in a putative second messenger cascade, and their response to odors was measured. IBMX prolonged the odor-evoked current and also reduced its amplitude. cAMP and cGMP induced a current electrically identical to the odor current, but the current showed desensitization only with cAMP. GTP-gamma-s prolonged and GDP-beta-s interfered with the odor-evoked current. The long latency seen in the odor response appears to be mainly due to the loading of the G protein and secondarily to the requirement for cAMP accumulation. The main source of the response decay appears to be cyclic nucleotide hydrolysis.

Time course of the membrane current underlying sensory transduction in salamander olfactory receptor neurones.

1. Odour elicited currents in freshly isolated olfactory receptor neurones were analysed using the whole‐cell patch‐clamp technique. Brief pulses (35‐50 ms) and steps (100 ms‐5 s) of odour solution were delivered by pressure ejection from a nearby micropipette. 2. Pulses of odour solution directed at the cell induced an inward depolarizing current of 50‐750 pA leading to the generation of action potentials. The I‐V relation for this current was linear over the range ‐60‐(+)20 mV and showed a reversal potential of +5 mV. The magnitude of the current increased with stimulus strength, for a given pulse duration, over approximately one decade of concentration change. 3. Pulses of odour solution focally delivered to the cilia elicited a large response, but those directed toward the soma did not. Conversely pulses of K+ solution at the cilia failed to evoke any response while those directed at the dendrite and soma elicited an inward clamp current. This provides direct evidence that odour sensitivity is localized mainly to the cilia and possibly the distal dendrite. 4. The odour elicited current activated with a long latency of 150‐600 ms after the odour solution arrived at the cell. This latency, as well as the time‐to‐peak and the rise half‐time, were relatively independent of stimulus concentration, changing less than 25% over the entire concentration range of stimulus sensitivity. These observations are consistent with the participation of a second messenger system in olfactory transduction. 5. For brief stimulus pulses less than 100 ms, the stimulus diffused away before the odour response current reached its peak value, so that the peak and decay of the odour response occurred in the absence of significant odour stimulus. The time course of the current decay was fitted by a single exponential with a time constant that was concentration dependent, varying from 0.8 to 1.3 s. 6. For longer steps of stimulus presentation, up to 1 s, the magnitude of the response current became a function of the duration of the pulse as well as the stimulus concentration, indicating that the transduction process involved an integrating step. This is consistent with the idea that the odour elicited current is the result of the summation of many smaller unitary events. From responses to weak stimulation an integration period of 700‐1000 ms was calculated. 7. During prolonged steps of maintained stimulus presentation (greater than 5 s) the odour elicited current was transient.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of the olfactory cyclic nucleotide gated ion channel by intracellular calcium

When olfactory receptor neurons are exposed to sustained application of odours, the elicited ionic current is transient. This adaptation-like effect appears to require the influx of Ca2+ through the odour-sensitive conductance; in the absence of extracellular Ca2+ the current remains sustained. Odour transduction proceeds through a G-protein-based second messenger system, resulting finally in the direct activation of an ion channel by cyclic AMP. This channel is one possible site for a negative feedback loop using Ca2+ as a messenger. In recordings of single cyclic AMP gated channels from olfactory receptor neurons, the open probability of the channel in saturating cAMP concentrations was dependent on the concentration of intracellular Ca2+. It could be reduced from 0.6 in 100 nM C a2+ to 0.09 in 3 |4.m Ca2+. However, as neither the single channel conductance nor the mean open time were affected by Ca2+ concentration, this does not appear to be a mechanism of simple channel block. Rather, these results suggest that intracellular Ca2+ acts allosterically to stabilize a closed state of the channel.

A noseful of odor receptors

I t is estimated that the mammalian nose is able to detect and discriminate 10 000 different odorbearing molecules in the environment 1. Even lower vertebrates like amphibia can distinguish between thousands of different odors. Unquestionably, this constitutes the largest single class of ligands detected by any neuronal signal-transducing system. Indeed, only the immune system displays a more diverse recognition capability. Precisely how the olfactory system accomplishes these herculean tasks of detection and discrimination has been a tantalizing question 2. Is specificity a peripheral receptor property, requiring a large number of receptors with high affinity and specificity for odor ligands? Or does it result from central processing of the input from only a few receptors with overlapping specificities? A surprisingly direct answer to this long-standing issue has emerged from the main finding of a recently published report by Linda Buck and Richard Axel of Columbia University Medical Center 3. Their study, directed at identifying the genes responsible for coding olfactory receptors, enabled them to find a rnultigene family of startling size, the members of which bear similarity to other known receptors, but which nonetheless distinctly make up their own class of molecule. The family appears to contain at least 100, and possibly many more, distinct gene sequences. This suggests that the receptor neurons are the main site of odor discrimination and that even at the peripheral level considerable stimulus information is able to be encoded. Olfactory neurons are found in a thin layer of tissue called the olfactory epithelium, which lines the upper nasal passages in most mammals. They are bipolar neurons with a relatively simple geometry

Electrical signals in olfactory transduction

Olfactory transduction involves a G-protein-coupled second messenger system, which results in the odor-dependent production of cAMP. The direct activation of ion channels in the cilia membrane by cAMP is the final step in producing the slow depolarization that brings the membrane potential to threshold for spike generation. Because of the central role in the transduction cascade occupied by these channels considerable effort has been directed toward understanding their behavior at a molecular level. Alternative second messenger pathways have also been proposed in olfaction, but the physiological evidence for these is less well developed.