A. Duchamp

Single olfactory sensory neurons simultaneously integrate the components of an odour mixture

Most odours are complex mixtures. However, the capacities of olfactory sensory neurons (OSNs) to process complex odour stimuli have never been explored in air‐breathing vertebrates. To face this issue, the present study compares the electrical responses of single OSNs to two odour molecules, delivered singly and mixed together, in rats in vivo. This work is the first aimed at demonstrating that single OSNs simultaneously integrate several chemical signals and which, furthermore, attempts to describe such processes for the whole concentration range over which single OSNs can work. The results stress that complex interactions occur between components in odour mixtures and that OSN responses to such mixtures are not simply predictable from the responses to their components. Three types of interactions are described. They are termed suppression, hypoadditivity and synergy, in accord with psychophysical terminology. This allows us to draw links between peripheral odour reception and central odour coding. Indeed, events occurring in single OSNs may account for the dominating or even the masking effects of odour molecules in complex mixtures, i.e. for the prevailing action of a minor component in the final qualitative perception of a mixture. We conclude that our observations with binary mixtures anticipate the complexity of processes which may rise at the level of a single OSN in physiological conditions. Following this hypothesis, a natural odour would induce a multi‐chemical integration at the level of single OSNs which may result in refining their individual odour‐coding properties, leading them to play a crucial role in the final performance of the olfactory system.

5-Hydroxytryptamine action in the rat olfactory bulb: in vitro electrophysiological patch-clamp recordings of juxtaglomerular and mitral cells.

The olfactory bulb, first relay of olfactory pathways, is densely innervated by serotoninergic centrifugal fibers originating from the raphe nuclei. Although serotonin innervation was reported to be involved in olfactory learning in mammals, the action of this neurotransmitter on its putative cellular targets has been never described through unitary recordings. This lack of data initiated the present study where the effects of 5HT on juxtaglomerular and mitral cells are analyzed using whole-cell recordings on olfactory bulb slices. Serotonin depolarizes 34% of 525 JG cells. A multivariate statistical analysis of juxtaglomerular cells characteristics shows that the serotonin responsive cell group can be individualized regarding their tonic discharge-mode in response to a direct current injection, their lower expression of hyperpolarization-activated cation current and their low membrane capacities. The use of ion channel blockers and ramp voltage protocol indicate that serotoninergic depolarization of juxtaglomerular cells may be due to a nonselective cation current with a reversal potential of -44 mV. Pharmacological tests with serotonin receptor antagonists and agonists reveal that 5HT action on juxtaglomerular cells would be mainly mediated by 5HT2C receptors. In mitral cells, serotonin acts on 49.1% of the 242 tested cells, inducing two types of responses. A first subset of mitral cells (26.8%, n=65) were hyperpolarized by serotonin. This response would be indirect and mediated by action of GABA on GABAA receptors since it was antagonized by bicuculline. The involved GABAergic neurons are hypothesized to be juxtaglomerular and granular cells, on which serotonin would act mainly via 5HT2C and via 5HT2A receptors respectively. The second subset of mitral cells (22.3%, n=54) were directly depolarized by serotonin acting through 5HT2A receptors. Our data on serotonin action on juxtaglomerular cells and mitral cells reveal a part of functional mechanisms whereby serotonin can act on olfactory bulb network. This is expected to enrich the understanding of its determining role in olfactory learning.

Spontaneous activity of first- and second-order neurons in the frog olfactory system

The spontaneous activity of first-order neurons (neuroreceptors of the mucosa) and second-order neurons (mitral cells of the bulb) was recorded extracellularly in the frog olfactory system. To assess the influence of peripheral inputs upon mitral cells, the bulb was either normally connected or partially deafferented. Our first set of findings concern the firing behavior. We found that most neurons generated interspike intervals (ISIs) that were stationary in mean and variance, and were not serially correlated at first and second order. Individual spikes in mitral cells and bursts of spikes in neuroreceptors were found to be generated by a Poisson process. Stochastic modeling suggests that the Poissonian behavior depends on the mean value of the membrane potential at the axon hillock. In these models, the mean potential in mitral cells would be far below the firing threshold and in neuroreceptors it would fluctuate at random between two states, one close to resting potential (between bursts) and the other close to the firing threshold with occasional crossings (within bursts). Secondly, partially deafferented mitral cells had significantly higher activity and lower variance than mitral cells receiving normal afferent input. This effect gives evidence that peripheral inputs influence mitral cells at rest not only through direct excitation but also through indirect inhibition exerted by local neurons. Thus, the unstimulated state of the olfactory bulb would not be qualitatively different from its stimulated state in the sense that both states involve the same types of synaptic interactions. Consequently, understanding the synaptic relationships that take place in the bulb network can benefit from studies of its spontaneous activity.

Gabaergic control of odor-induced activity in the frog olfactory bulb: Electrophysiological study with picrotoxin and bicuculline

In the olfactory bulb, the first relay of the olfactory pathways, GABA, could be largely involved in the information processing since the two main populations of interneurons, periglomerular and granular cells, use it as neurotransmitter through reciprocal synapses with second-order neurons. This study planned to clarify the role of GABAergic inhibition in odor coding and, more precisely, the role of glomerular GABAergic inhibition. To do so, we attempted to specifically block in vivo GABAA receptors with either picrotoxin or bicuculline. The drug was applied at the level of the glomerular layer so that the antagonist could act primarily via periglomerular cells. The analysis of the effects of blocking GABAA on the coding was studied by recording the second-order neuron responses to odor stimuli delivered in a wide concentration range. Under drug treatment, the second-order neuron properties were deeply changed: response thresholds to odors were often lowered and spike bursts were more sustained in frequency and in duration. Thus, the GABAergic control on second-order neurons might be carried out by limiting the neuron excitability. GABAA antagonists applied in this manner could act to suppress the inhibitory effect of either the periglomerular cells or of the granule cells, both of which have been shown to contain enzymes for GABA production. The placement of the drug suggests to us that the action is primarily at the glomerulus. The results are consistent with periglomerular cells exerting a tonic inhibition on second-order neurons, an inhibition whose strength would be modulated by stimulus intensity. As a result, the amplifying role of glomerular convergence might be partly counterbalanced by input inhibition. Nevertheless, due to our procedure of drug application, one cannot rule out the possibility that the effects observed may partly reflect granular cell blocking. It can be concluded that the whole GABAergic inhibition, through GABAA receptors, permits a wide dynamic range of intensity coding.

Olfactory discrimination over a wide concentration range. Comparison of receptor cell and bulb neuron abilities

Until now, olfactory discrimination had never been investigated using stimuli delivered over a wide concentration range. However, the fact that intensity variations might influence qualitative discrimination has been suggested in numerous physiological and psychophysical studies. The aim of the present work was to investigate qualitative coding mechanisms when stimulus intensity varies. For this purpose, receptor cell and olfactory bulb neuron unit activities were recorded in response to 2-s pulse delivery of 4 odorants available at 20 discrete concentration values over a range from 1 x 10(-6) to 5.62 x 10(-2) of saturation. Two types of mathematical analyses, Pearsons r correlation coefficient calculation and principal component factor analysis, were applied to odor-evoked discharge frequencies. In both receptor cells and bulb neurons, qualitative discrimination abilities were found to increase with stimulus concentration. Furthermore, the results suggest that the olfactory bulb can send a discriminant and specific message at lower concentrations than the olfactory mucosa. The amplifying role of convergence of primary afferences onto olfactory glomeruli could account for this ability of the bulb neurons.

Gabaergic control of odour-induced activity in the frog olfactory bulb: Possible gabaergic modulation of granule cell inhibitory action

In the olfactory bulb, the activity of the output neurons, the mitral cells, is under inhibitory control exerted by GABAergic interneurons, the granule cells. Although the mechanisms of this inhibition are well known from in vitro studies, its physiological role in controlling mitral cell activity in response to odours has never been investigated. This study planned to improve understanding of the involvement of granule cells. To do so, GABAA-synaptic mechanisms were altered using GABAA antagonists in order to observe the consequences on mitral cell electrophysiological responses to odours, delivered over a wide concentration range. Due to the laminar organization of bulbar cell populations, the antagonists picrotoxin or bicuculline were injected into the bulbar ventricle in order to block granule cell inhibitory action at first. Surprisingly, the early consequence of the antagonist injection was a decrease in cell responsivity: response spike frequencies were lowered while thresholds were occasionally shifted toward higher concentrations. This initial depressive effect was followed by a recovery of control excitability and, later, by an increase in excitability: spike bursts became more sustained in frequency and in duration. At the same time, in most of the cells studied, spontaneous activity became bursting. The early depressive effect of GABAA antagonists is discussed in terms of an enhancement of the inhibitory influence of granule cells on mitral cells. This might reflect a blocking action of the antagonists at the level of GABAergic synapses located on granule cells themselves. The late effect, an increase in excitability, is explained as the consequence of the alteration of the functioning of dendrodendritic synapses between granule and mitral cells leading to a disinhibition of the latter. The comparison of the present findings with others obtained when antagonists were applied on to glomerular layers led us to infer that granule cell inhibition would be devoted to limiting mitral cell responses in frequency and in duration rather than to adjusting their response threshold. The chronology of the effects observed strongly supports the fact that, following the intraventricular injection, the antagonists acted primarily in the deep layers of the bulb. Nevertheless, due to free diffusion starting from the injection site, the possibility that drugs act later in the glomerular layer can not be rejected. It can be concluded that, in addition to its extensive involvement through intrinsic interneurons, GABA might also control the strength of the inhibition exerted by granule cells on mitral cells via centrifugal fibres.

GABAB receptor-mediated inhibition of mitral/tufted cell activity in the rat olfactory bulb: A whole-cell patch-clamp study in vitro

GABA, the major inhibitory neurotransmitter involved in information processing in the olfactory bulb, is hypothesized to act through GABA(B) receptors by depressing primary neurotransmitter release at the level of olfactory nerve axon endings. The present study was designed to analyze GABA(B) receptor-mediated inhibition mechanisms by performing whole-cell patch-clamp recordings of mitral/tufted cell activity in the rat in vitro. To do so, GABA(B) receptor-mediated action was mimicked by baclofen and antagonized by saclofen. Our protocol led us to provide an original description of GABA(B) receptor-mediated inhibition exerted on mitral/tufted cells. First, their spontaneous activity was shown to be drastically abolished by baclofen. Second, their responses to olfactory nerve electrical stimulation were graded by GABA(B) receptor-mediated inhibition. Indeed, this inhibition may be described as inducing effects ranked from a slight increase in response latency to a complete response suppression.Altogether, our results corroborate the hypothesis of a presynaptic extrasynaptic GABA(B) receptor-mediated inhibition influencing mitral/tufted cell olfactory nerve responsivity. However, the involvement of postsynaptic receptors, with different properties or with different anatomical locations, cannot be ruled out, particularly in the control of spontaneous activity. In conclusion, we underline that, in the vertebrate olfactory bulb, GABA(B) receptor-mediated action appears to contribute to make mitral/tufted cell responses more salient by reducing their resting activity.

GABAB-MEDIATED ACTION IN THE FROG OLFACTORY BULB MAKES ODOR RESPONSES MORE SALIENT

In the olfactory bulb, GABA(B) receptors are selectively located in the glomerular layer. A current hypothesis is that GABAergic inhibition mediated through these receptors would be, at least partly, presynaptic and would exerted by decreasing the release of the olfactory receptor neuron excitatory neurotransmitter. Here, we assessed, in the frog, the in vivo action of baclofen, a GABA(B) agonist, on single-unit mitral cell activity in response to odors. Local application of baclofen in the glomerular region of the olfactory bulb was shown to drastically affect mitral cell spontaneous activity, since they became totally silent. Moreover, under baclofen, mitral cells still responded to odors and still specified odor concentration increases through their temporal response patterns. The pharmacological specificity of the GABA(B) agonist action was confirmed by showing that saclofen, a GABA(B) antagonist, partly prevented the inhibitory action of baclofen and restored the initial rate of mitral cell spontaneous activity. The results show that GABA(B)-mimicked inhibition suppressed mitral cell spontaneous activity while odor responses were maintained. This suggests that olfactory receptor neurons partly drive spontaneous mitral cell activity. Moreover, the effect of GABA(B)-mediated inhibition was seen to be very close to that described previously for dopamine D(2) receptor-mediated inhibition. In conclusion, we propose that these two inhibitory mechanisms would offer the possibility to reduce or suppress mitral cell spontaneous activity so as to make their responses to odor especially salient.