Higher-order trace conditioning in newborn rabbits.

Temporal contingency is a key factor in associative learning but remains weakly investigated early in life. Few data suggest simultaneous presentation is required for young to associate different stimuli, whereas adults can learn them sequentially. Here, we investigated the ability of newborn rabbits to perform sensory preconditioning and second-order conditioning using trace intervals between odor presentations. Strikingly, pups are able to associate odor stimuli with 10- and 30-sec intervals in sensory preconditioning and second-order conditioning, respectively. The effectiveness of higher-order trace conditioning in newborn rabbits reveals that very young animals can display complex learning despite their relative immaturity.

Afterhyperpolarization Promotes the Firing of Mitral Cells through a Voltage-Dependent Modification of Action Potential Threshold.

In the olfactory bulb, mitral cells (MCs) display a spontaneous firing that is characterized by bursts of action potentials (APs) intermixed with silent periods. Intraburst firing frequency and duration are heterogeneous among MCs and increase with membrane depolarization. By using patch-clamp recording on rat slices, we dissected out the intrinsic properties responsible for this bursting activity. We showed that the threshold of AP generation dynamically changes as a function of the preceding trajectory of the membrane potential. In fact, the AP threshold became more negative when the membrane was hyperpolarized and had a recovery rate inversely proportional to the membrane repolarization rate. Such variations appeared to be produced by changes in the inactivation state of voltage-dependent Na+ channels. Thus, AP initiation was favored by hyperpolarizing events, such as negative membrane oscillations or inhibitory synaptic input. After the first AP, the following fast afterhyperpolarization (AHP) brought the threshold to more negative values and then promoted the emission of the following AP. This phenomenon was repeated for each AP of the burst making the fast AHP a regenerative mechanism that sustained the firing, AHP with larger amplitudes and faster repolarizations being associated with larger and higher-frequency bursts. Burst termination was found to be because of the development of a slow repolarization component of the AHP (slow AHP). Overall, the AHP characteristics appeared as a major determinant of the bursting properties.

Protocol of controlled odorant stimulation for reducing apnoeic episodes in premature newborns: a randomised open-label Latin-square study with independent evaluation of the main endpoint (PREMODEUR).

INTRODUCTION: Apnoea affects 85% of premature infants under 34 weeks of age and would be an important risk factor for subsequent neuropsychological disorders. Currently, premature children with life-threatening apnoeas receive stimulants such as methylxanthines (mainly, caffeine) or doxapram (an analeptic unlicensed in children under 15). However, these products have undesirable effects (hyperarousal, irritability, sleep disorders, tachycardia) and are not always effective because apnoea does persist in some premature newborns. Previous studies have indicated that odorant stimulation, a non-invasive intervention, may stimulate the respiratory rhythm. The objective of the present protocol is to reduce the occurrence of apnoeic episodes in premature newborns by controlled odorant stimulation added to current pharmacological treatments. METHODS AND ANALYSIS: The project is a randomised open-label Latin-square trial with independent evaluation of the main endpoint. It will include 60 preterm neonates from two university hospital neonatal intensive care units over 2 years (2021-2023). Each newborn will receive no (S0), sham (S1) or real olfactory stimulation (S2) in random order. During S2, three distinct odorants (mint, grapefruit and vanilla) will be delivered successively, in puffs, over 24 hours. Mint and grapefruit odours stimulate the main and the trigeminal olfactory pathways, whereas vanilla odour stimulates only the main olfactory pathway. A statistical analysis will compare the incidence of apnoeic episodes during S1 versus S2 using a mixed effects Poisson model. ETHICS AND DISSEMINATION: Ethical approval was obtained from the Comité de Protection des Personnes Île-de-France XI (# 2017-AO13-50-53). The results will be disseminated through various scientific meetings, specialised peer-reviewed journals and, whenever possible, posted on appropriate public websites. TRIAL REGISTRATION NUMBER: NCT02851979; Pre-results.

Brief olfactory learning drives perceptive sensitivity in newborn rabbits: New insights in peripheral processing of odor mixtures and induction.

Perception of the wide, complex and moving odor world requires that the olfactory system engages processing mechanisms ensuring detection, discrimination and environment adaptation, as early as the peripheral stages. Odor items are mainly elicited by odorant mixtures which give rise to either elemental or configural perceptions. Here, we first explored the contribution of the peripheral olfactory system to configural and elemental perception through odorant interactions at the olfactory receptor (OR) level. This was done in newborn rabbits, which offer the opportunity to pair peripheral electrophysiology and well characterized behavioral responses to two binary mixtures, AB and A'B', which differ in their component ratio (A: ethyl isobutyrate, B: ethyl maltol), and that rabbit pups respectively perceived configurally and elementally. Second, we studied the influence on peripheral reactivity of the brief but powerful learning of one mixture component (odorant B), conditioned by association with the mammary pheromone (MP), which allowed us to assess the possible implication of the phenomenon called induction in neonatal odor learning. Induction is a plasticity mechanism expected to alter both the peripheral electrophysiological responses to, and perceptual detection threshold of, the conditioned stimulus. The results reveal that perceptual modes are partly rooted in differential peripheral processes, the AB configurally perceived mixture mirroring odorant antagonist interactions at OR level to a lesser extent than the A'B' elementally perceived mixture. Further, the results highlight that a single and brief MP-induced odor learning episode is sufficient to alter peripheral responses to the conditioned stimulus and mixtures including it, and shifts the conditioned stimulus detection threshold towards lower concentrations. Thus, MP-induced odor learning relies on induction phenomenon in newborn rabbits.

Microdialysis Unveils the Role of the α2-Adrenergic System in the Basolateral Amygdala during Acquisition of Conditioned Odor Aversion in the Rat.

Previous work has shown that ß-adrenergic and GABAergic systems in the basolateral amygdala (BLA) are involved in the acquisition of conditioned odor aversion (COA) learning. The involvement of α2-adrenoreceptors, however, is poorly documented. In a first experiment, male Long-Evans rats received infusions of 0.1 µg of the selective α2-antagonist dexefaroxan (Dex) in the BLA before being exposed to COA learning. In a second experiment, levels of norepinephrine (NE) were analyzed following Dex retrodialysis into the BLA. While microdialysis data showed a significant enhancement of NE release in the BLA with Dex, behavioral results showed that pre-CS infusion of Dex impaired, rather than facilitated, the acquisition of COA. Our results show that the NE system in the BLA is involved in the acquisition of COA, including a strong α2-receptor modulation until now unsuspected. Supported by the recent literature, the present data suggest moreover that the processes underlying this learning are probably mediated by the balanced effects of NE excitatory/inhibitory signaling in the BLA, in which interneurons are highly involved.

In Vivo Electrophysiological Recordings of Olfactory Receptor Neuron Units and Electro-olfactograms in Anesthetized Rats.

In vivo recordings of single olfactory receptor neurons and electro-olfactograms (EOG, field potentials from the olfactory mucosa) provide insights into the olfactory processing properties of the olfactory peripheral stage. Because the olfactory receptor neurons are very small electrical generators, it is not easy to unitarily record them in amphibians, reptilians, and fishes. In mammals such recordings are even more difficult to obtain: primarily due to the anatomical configuration in complex turbinates of the olfactory mucosa and its propensity to hemorrhage during surgery; secondarily due to the fact that olfactory receptor neurons are held in closely packed clusters in the olfactory mucosa and are difficult to isolate, from the electrophysiological recording point of view. Here we describe the material and methods we used in vivo, in rats-occasionally, also tested in mice-to get simultaneously receptor neuron single and electro-olfactogram recordings, from septal region or the endoturbinate II, in freely breathing or tracheotomized anesthetized animals. Recording EOG in parallel with receptor neuron units provide, by reflecting the population response to the olfactory stimulus, the continuous assurance of the good physiological state and reactivity of the olfactory epithelium. This configuration will ensure that when a single ORN does not respond to a stimulus it resulted from its qualitative selectivity and not from the olfactory mucosa damaged status.

Odorant-odorant metabolic interaction, a novel actor in olfactory perception and behavioral responsiveness.

In the nasal olfactory epithelium, olfactory metabolic enzymes ensure odorant clearance from the olfactory receptor environment. This biotransformation of odorants into deactivated polar metabolites is critical to maintaining peripheral sensitivity and perception. Olfactory stimuli consist of complex mixtures of odorants, so binding interactions likely occur at the enzyme level and may impact odor processing. Here, we used the well-described model of mammary pheromone-induced sucking-related behavior in rabbit neonates. It allowed to demonstrate how the presence of different aldehydic odorants efficiently affects the olfactory metabolism of this pheromone (an aldehyde too: 2-methylbut-2-enal). Indeed, according to in vitro and ex vivo measures, this metabolic interaction enhances the pheromone availability in the epithelium. Furthermore, in vivo presentation of the mammary pheromone at subthreshold concentrations efficiently triggers behavioral responsiveness in neonates when the pheromone is in mixture with a metabolic challenger odorant. These findings reveal that the periphery of the olfactory system is the place of metabolic interaction between odorants that may lead, in the context of odor mixture processing, to pertinent signal detection and corresponding behavioral effect.

Insulin modulates network activity in olfactory bulb slices: impact on odour processing.

Odour perception depends closely on nutritional status, in animals as in humans. Insulin, the principal anorectic hormone, appears to be one of the major candidates for ensuring the link between olfactory abilities and nutritional status, by modifying processing in the olfactory bulb (OB), one of its main central targets. The present study investigates whether and how insulin can act in OB, by evaluating its action on the main output neurons activities, mitral cells (MCs), in acute rat OB slices. Insulin was found to act at two OB network levels: (1) on MCs, by increasing their excitability, probably by inhibiting two voltage-gated potassium (K(+)) channels; (2) on interneurons by modifying the GABAergic and on glutamatergic synaptic activity impinging on MCs, mainly reducing them. Insulin also altered the olfactory nerve (ON)-evoked excitatory postsynaptic currents in 60% of MCs. Insulin decreased or increased the ON-evoked responses in equal proportion and the direction of its effect depended on the initial neuron ON-evoked firing rate. Indeed, insulin tended to decrease the high and to increase the low ON-evoked firing rates, thereby reducing inter-MC response firing variability. Therefore, the effects of insulin on the evoked firing rates were not carried out indiscriminately in the MC population. By constructing a mathematical model, the impact of insulin complex effects on OB was assessed at the population activity level. The model shows that the reduction of variability across cells could affect MC detection and discrimination abilities, mainly by decreasing and, less frequently, increasing them, depending on odour quality. Thus, as previously proposed, this differential action of insulin on MCs across odours would allow this hormone to put the olfactory function under feeding signal control, given the discerning valence of an odour as a function of nutritional status.

The orexin component of fasting triggers memory processes underlying conditioned food selection in the rat.

To test the selectivity of the orexin A (OXA) system in olfactory sensitivity, the present study compared the effects of fasting and of central infusion of OXA on the memory processes underlying odor-malaise association during the conditioned odor aversion (COA) paradigm. Animals implanted with a cannula in the left ventricle received ICV infusion of OXA or artificial cerebrospinal fluid (ACSF) 1 h before COA acquisition. An additional group of intact rats were food-deprived for 24 h before acquisition. Results showed that the increased olfactory sensitivity induced by fasting and by OXA infusion was accompanied by enhanced COA performance. The present results suggest that fasting-induced central OXA release influenced COA learning by increasing not only olfactory sensitivity, but also the memory processes underlying the odor-malaise association.

The speed of learning instructed stimulus-response association rules in human: experimental data and model.

Humans can learn associations between visual stimuli and motor responses from just a single instruction. This is known to be a fast process, but how fast is it? To answer this question, we asked participants to learn a briefly presented (200ms) stimulus-response rule, which they then had to rapidly apply after a variable delay of between 50 and 1300ms. Participants showed a longer response time with increased variability for short delays. The error rate was low and did not vary with the delay, showing that participants were able to encode the rule correctly in less than 250ms. This time is close to the fastest synaptic learning speed deemed possible by diffusive influx of AMPA receptors. Learning continued at a slower pace in the delay period and was fully completed in average 900ms after rule presentation onset, when response latencies dropped to levels consistent with basic reaction times. A neural model was proposed that explains the reduction of response times and of their variability with the delay by (i) a random synaptic learning process that generates weights of average values increasing with the learning time, followed by (ii) random crossing of the firing threshold by a leaky integrate-and-fire neuron model, and (iii) assuming that the behavioural response is initiated when all neurons in a pool of m neurons have fired their first spike after input onset. Values of m=2 or 3 were consistent with the experimental data. The proposed model is the simplest solution consistent with neurophysiological knowledge. Additional experiments are suggested to test the hypothesis underlying the model and also to explore forgetting effects for which there were indications for the longer delay conditions. This article is part of a Special Issue entitled Neural Coding 2012.

Responses of single neurons and neuronal ensembles in frog first- and second-order olfactory neurons.

A major challenge in sensory neuroscience is to elucidate the coding and processing of stimulus representations in successive populations of neurons. Here we recorded the spiking activity of receptor neurons (RNs) and mitral/tufted cells (MCs) in the frog olfactory epithelium and olfactory bulb respectively, in response to four odorants applied at precisely controlled concentrations. We compared how RN responses are translated in MCs. We examined the time course of the instantaneous firing frequency before and after stimulation in neuron ensembles and the dependency on odorant concentration of the number of action potentials fired in a preselected 5-s time window (dose-response curves) in both single neurons and neuron ensembles. In RNs and MCs, the dose-response curves typically increase then decrease and are well described by alpha functions. We established the main quantitative properties of these curves, including the distributions of concentrations at threshold and maximum responses. We showed that the main transformations occurring in the transition from RNs to MCs is the lowering of the firing threshold and a large decrease in the total number of spikes fired. We also found that the number of action potentials fired by recorded neurons and hence their energy consumption is independent of odorant concentration, and that this is a consequence of their time- and concentration-dependent activities. This article is part of a Special Issue entitled Neural Coding 2012.

Modulation of spontaneous and odorant-evoked activity of rat olfactory sensory neurons by two anorectic peptides, insulin and leptin.

In mammals, the sense of smell is modulated by the status of satiety, which is mainly signaled by blood-circulating peptide hormones. However, the underlying mechanisms linking olfaction and food intake are poorly understood. Here we investigated the effects of two anorectic peptides, insulin and leptin, on the functional properties of olfactory sensory neurons (OSNs). Using patch-clamp recordings, we analyzed the spontaneous activity of rat OSNs in an in vitro intact epithelium preparation. Bath perfusion of insulin and leptin significantly increased the spontaneous firing frequency in 91.7% (n = 24) and 75.0% (n = 24) of the cells, respectively. When the activity was electrically evoked, both peptides shortened the latency to the first action potential by approximately 25% and decreased the interspike intervals by approximately 13%. While insulin and leptin enhanced the electrical excitability of OSNs in the absence of odorants, they surprisingly reduced the odorant-induced activity in the olfactory epithelium. Insulin and leptin decreased the peak amplitudes of isoamyl acetate-induced electroolfactogram (EOG) signals to 46 and 38%, respectively. When measured in individual cells by patch-clamp recordings, insulin and leptin decreased odorant-induced transduction currents and receptor potentials. Therefore by increasing the spontaneous activity but reducing the odorant-induced activity of OSNs, an elevated insulin and leptin level (such as after a meal) may result in a decreased global signal-to-noise ratio in the olfactory epithelium, which matches the smell ability to the satiety status.

Competitive and noncompetitive odorant interactions in the early neural coding of odorant mixtures.

Most olfactory receptor neurons (ORNs) express a single type of olfactory receptor that is differentially sensitive to a wide variety of odorant molecules. The diversity of possible odorant-receptor interactions raises challenging problems for the coding of complex mixtures of many odorants, which make up the vast majority of real world odors. Pure competition, the simplest kind of interaction, arises when two or more agonists can bind to the main receptor site, which triggers receptor activation, although only one can be bound at a time. Noncompetitive effects may result from various mechanisms, including agonist binding to another site, which modifies the receptor properties at the main binding site. Here, we investigated the electrophysiological responses of rat ORNs in vivo to odorant agonists and their binary mixtures and interpreted them in the framework of a quantitative model of competitive interaction between odorants. We found that this model accounts for all concentration-response curves obtained with single odorants and for about half of those obtained with binary mixtures. In the other half, the shifts of curves along the concentration axis and the changes of maximal responses with respect to model predictions, indicate that noncompetitive interactions occur and can modulate olfactory receptors. We conclude that, because of their high frequency, the noncompetitive interactions play a major role in the neural coding of natural odorant mixtures. This finding implies that the CNS activity caused by mixtures will not be easily analyzed into components, and that mixture responses will be difficult to generalize across concentration.

Patterns of spontaneous activity in single rat olfactory receptor neurons are different in normally breathing and tracheotomized animals.

Spontaneous firing of olfactory receptor neurons (ORNs) was recently shown to be required for the survival of ORNs and the maintenance of their appropriate synaptic connections with mitral cells in the olfactory bulb. ORN spontaneous activity has never been described or characterized quantitatively in mammals. To do so we have made extracellular single unit recordings from ORNs of freely breathing (FB) and tracheotomized (TT) rats. We show that the firing behavior of TT neurons was relatively simple: they tended to fire spikes at the same average frequency according to purely random (Poisson) or simple (Gamma or Weibull) statistical laws. A minority of them were bursting with relatively infrequent and short bursts. The activity of FB neurons was less simple: their firing rates were more diverse, some of them showed trends or were driven by breathing. Although more of them were regular, only a minority could be described by simple laws; the majority displayed random bursts with more spikes than the bursts of TT neurons. In both categories bursts and isolated spikes (outside bursts) occurred completely at random. The spontaneous activity of ORNs in rats resembles that of frogs, but is higher, which may be due to a difference in body temperature. These results suggest that, in addition to the intrinsic thermal noise, spontaneous activity is provoked in part by mechanical, thermal, or chemical (odorant molecules) effects of air movements due to respiration, this extrinsic part being naturally larger in FB neurons. It is suggested that spontaneous activity may be modulated by respiration. Because natural sampling of odors is synchronized with breathing, such modulation may prepare and keep olfactory bulb circuits tuned to process odor stimuli.

Orexin A modulates mitral cell activity in the rat olfactory bulb: patch-clamp study on slices and immunocytochemical localization of orexin receptors.

Orexin A and B are involved in feeding behaviors, and recently fibers containing these peptides were found in the rat olfactory bulb. These fibers, which originate from the lateral and posterior hypothalamus and the perifornical area, are distributed in the glomerular, mitral cell, and granule cell layers. Orexin receptors are mainly expressed by mitral cells. In the present study, RT-PCR experiments were done to determine orexin receptor expression during the early postnatal life of rats, and immunocytochemical experiments were performed to further clarify the structural and ultrastructural localization of orexin receptors in the olfactory bulb. Furthermore, a functional electrophysiological approach examined the action of orexin A on mitral cell excitability and spontaneous activity using in vitro patch-clamp techniques. RT-PCR results show that mRNA of the two type receptors, type 1 orexin receptors and type 2 orexin receptors, are expressed in the olfactory bulb of rat from 10 d to the adult stage. At the same ages, immunocytochemical data show that orexin 1 receptors are localized in the cell bodies of periglomerular, mitral/tufted, and granule cells. Immunoreactivity was also demonstrated in mitral/tufted cell dendrites arborizing in the glomerulus and mitral/tufted and granule cell processes running in the external plexiform layer. Functionally, orexin A produced either a direct, tetrodotoxin-insensitive depolarization in one group of mitral cells (7%), or, in another group (30%), an indirect, tetrodotoxin-sensitive hyperpolarization. Both actions were mediated by type 1 orexin receptors because the response was antagonized by SB-334867-A, a selective antagonist. Mitral cell recordings performed under bicuculline [gamma-aminobutyric acid (GABA)A receptor antagonist], indicate that the orexin-induced indirect hyperpolarization was partly mediated through GABA(A) receptors. Because granule cells and periglomerular cells express orexin receptors and are GABAergic cells, they could be both involved in this hyperpolarization. Other mechanisms, which could support an indirect hyperpolarization of mitral cells through dopamine interneuron solicitation, are proposed. Our results provide data that should allow us to better understand neural communication and regulation mechanisms between the hypothalamic feeding centers and the olfactory bulb.

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.

Relation between stimulus and response in frog olfactory receptor neurons in vivo.

The spiking activity of receptor neurons was recorded extracellularly in the frog olfactory epithelium in response to four odourants applied at precisely controlled concentrations. A set of criteria was formulated to define the spikes in the response. Four variables - latency, duration, number of interspike intervals and frequency - were determined to quantify the responses. They were studied at the single neuron, neuron population and ciliary membrane levels. The dose-response curves were determined using specific functions and their characteristics were evaluated. The characteristic molar concentrations at threshold or at maximum duration and the characteristics of variables, e.g. minimum latency or maximum frequency, have asymmetric histograms with peaks close to the origin and long tails. Dynamic ranges have even more asymmetric histograms, so that a significant fraction of neurons presents a much wider range than their one-decade peak. From these histograms, response properties of the whole neuron population can be inferred. In general, location along the concentration axis (thresholds), width (dynamic ranges) and heights of dose-response curves are independent, which explains the diversity of curves, prevents their global categorization and supports the qualitative coding of odourants. No evidence for odourant-independent types of neurons was found. Finally, receptor activation and ciliary membrane conductance were reconstructed in the framework of a model based on firing data, known mucus biochemical and neuron morpho-electrical characteristics. It is in agreement with independent determinations of Kd of odourant-receptor interaction and of conductance characteristics, and describes their statistical distributions in the neuron population.