Johannes Reisert

Response properties of isolated mouse olfactory receptor cells were investigated using the suction pipette technique. Cells were exposed to the odour cineole or to solutions of modified ionic content by rapidly changing the solution superfusing the cilia. All experiments were performed at 37°C. Mouse olfactory receptor cells displayed a steep dependence of action potential frequency on stimulus concentration, a 3-fold increase in stimulus concentration often saturating the firing frequency at 200-300 Hz. The receptor current increased more gradually with increasing cineole concentration and did not saturate within the 100-fold range of cineole concentrations applied. When stimulated for 30 s with a low odour concentration, cells responded with sporadic spike firing. Higher concentrations led to the generation of a large receptor current at the onset of stimulation which returned to baseline levels within a few seconds, accompanied during its rising phase by a short burst of action potentials. Thereafter an oscillating response pattern was observed during the remainder of the stimulus, consisting of repetitive increases in receptor current of around 1 s duration accompanied by short bursts of action potentials. Olfactory adaptation was studied by comparing the responses to two closely spaced odour stimuli. The response to the second odour stimulus recovered to 80% of its original magnitude when the cell was superfused with Ringer solution during the 5 s interval between odour exposures. In contrast, exposure to a choline-substituted low Na+ solution between odour stimuli had two effects. First, the receptor current response to the first odour stimulus did not terminate as quickly as in the presence of Na+, suggesting the presence of a Na+-Ca2+ exchanger. Second, the response to the second stimulus only recovered to 55% of its original magnitude, demonstrating the involvement of Na+-Ca2+ exchange in the recovery of sensitivity in mouse olfactory receptor cells following stimulation.

The suction pipette technique was used to study simultaneously the odour-induced action potential and receptor current responses in frog olfactory receptor cells, which were exposed to the odour cineole for 1 s by rapidly exchanging the solution bathing their cilia. The frequency of action potential firing increased as the odour concentration was raised and saturated within a 15-fold elevation above the odour threshold, while the number of spikes fired initially grew at low-to-intermediate concentrations but then declined at higher concentrations. The receptor current response rose steadily and showed no clear sign of saturation over the 300-fold range of cineole concentration employed. The effect of adaptation on the sensitivity of olfactory receptor cells was investigated by first exposing the cell for 4 s to an adapting pre-pulse and then stimulating with a 1 s test pulse. As the pre-pulse concentration was increased, adaptation led to a progressive shift of the dose–response relationships towards higher test pulse concentrations. This resulted in a steep decline in the sensitivity of the receptor current response, combined with an even more dramatic fall in the sensitivity of the spiking responses, since the higher pre-pulse concentrations prevented the generation of action potentials at test pulse concentrations which still evoked a receptor current response.

1. Receptor current and spiking responses were recorded simultaneously from isolated frog olfactory receptor cells using the suction pipette technique. Cells were stimulated with the odour cineole by rapid exchange of the solution bathing the olfactory cilia. 2. The receptor current response to a 1 s odour stimulus increased in a graded manner over a 300-fold range of odour concentration without clear saturation, and was accompanied by a train of action potentials. As the concentration of the odour stimulus increased, the frequency of firing increased also, until it saturated at the highest concentrations. The number of spikes evoked by the stimulus first increased and then decreased with increasing concentration, reaching a maximum at intermediate odour concentrations. The dose-response relation for spike firing rose at lower odour concentrations than the dose-response relation for the receptor current response. 3. Adaptation to steady odour stimuli was investigated by exposing the cilia to a 4 s odour pre-pulse and then to a 1 s odour test pulse. As the pre-pulse concentration was increased the dose-response relations derived from the receptor current and spiking responses shifted to higher absolute test pulse concentrations. However the number of spikes fired in response to a given test pulse was little affected by the pre-pulse until, at the highest pre-pulse concentrations spike firing was abolished despite the continued presence of a receptor current response. 4. The sensitivity of the receptor-current response to incremental stimuli fell with increasing pre-pulse concentration, declining with a limiting slope of 2.4 in double logarithmic co-ordinates. The sensitivity determined from the spiking responses declined to zero at a lower pre-pulse concentration, reflecting the abolition of spike firing at pre-pulse concentrations which still evoked a graded receptor-current response.

To study the mechanism by which Ca²⁺, which enters during the odor response, is extruded during response recovery, recordings were made from isolated frog olfactory receptor cells using the suction pipette technique, while superfusing the olfactory cilia with solutions of modified ionic composition. When external Na⁺ was substituted with another cation, the response to odor was greatly prolonged. This prolongation of the response was similar irrespective of whether Na⁺ was replaced with Li⁺, which permeates the cyclic nucleotide-gated conductance, or choline, which does not. The prolonged current was greatly reduced by exposure to 300 μM niflumic acid, a blocker of the calcium-activated chloride channel, indicating that it is carried by this conductance, and abolished if Ca²⁺ was omitted from the external solution, demonstrating that Ca²⁺ influx is required for its generation. When the cilia were exposed to Na⁺-free solution after odor stimulation, the recovery of the response to a second stimulus from the adaptation induced by the first was greatly reduced. We conclude that a Na⁺-dependent Ca²⁺ extrusion mechanism is present in frog olfactory cilia and that it serves as the main mechanism that returns cytoplasmic Ca²⁺ concentration to basal levels after stimulation and mediates the normally rapid recovery of the odor response and the restoration of sensitivity after adaptation.