Anne-Marie Mouly

The RUB Cage: Respiration–Ultrasonic Vocalizations–Behavior Acquisition Setup for Assessing Emotional Memory in Rats

In animals, emotional memory is classically assessed through pavlovian fear conditioning in which a neutral novel stimulus (conditioned stimulus) is paired with an aversive unconditioned stimulus. After conditioning, the conditioned stimulus elicits a fear response characterized by a wide range of behavioral and physiological responses. Despite the existence of this large repertoire of responses, freezing behavior is often the sole parameter used for quantifying fear response, thus limiting emotional memory appraisal to this unique index. Interestingly, respiratory changes and ultrasonic vocalizations (USV) can occur during fear response, yet very few studies investigated the link between these different parameters and freezing. The aim of the present study was to design an experimental setup allowing the simultaneous recording of respiration, USV, and behavior (RUB cage), and the offline synchronization of the collected data for fine-grain second by second analysis. The setup consisted of a customized plethysmograph for respiration monitoring, equipped with a microphone capturing USV, and with four video cameras for behavior recording. In addition, the bottom of the plethysmograph was equipped with a shock-floor allowing foot-shock delivery, and the top received tubing for odor presentations. Using this experimental setup we first described the characteristics of respiration and USV in different behaviors and emotional states. Then we monitored these parameters during contextual fear conditioning and showed that they bring complementary information about the animals anxiety state and the strength of aversive memory. The present setup may be valuable in providing a clearer appraisal of the physiological and behavioral changes that occur during acquisition as well as retrieval of emotional memory.

Neonatal odor-shock conditioning alters the neural network involved in odor fear learning at adulthood

Adult learning and memory functions are strongly dependent on neonatal experiences. We recently showed that neonatal odor-shock learning attenuates later life odor fear conditioning and amygdala activity. In the present work we investigated whether changes observed in adults can also be observed in other structures normally involved, namely olfactory cortical areas. For this, pups were trained daily from postnatal (PN) 8 to 12 in an odor-shock paradigm, and retrained at adulthood in the same task. (14)C 2-DG autoradiographic brain mapping was used to measure training-related activation in amygdala cortical nucleus (CoA), anterior (aPCx), and posterior (pPCx) piriform cortex. In addition, field potentials induced in the three sites in response to paired-pulse stimulation of the olfactory bulb were recorded in order to assess short-term inhibition and facilitation in these structures. Attenuated adult fear learning was accompanied by a deficit in 2-DG activation in CoA and pPCx. Moreover, electrophysiological recordings revealed that, in these sites, the level of inhibition was lower than in control animals. These data indicate that early life odor-shock learning produces changes throughout structures of the adult learning circuit that are independent, at least in part, from those involved in infant learning. Moreover, these enduring effects were influenced by the contingency of the infant experience since paired odor-shock produced greater disruption of adult learning and its supporting neural pathway than unpaired presentations. These results suggest that some enduring effects of early life experience are potentiated by contingency and extend beyond brain areas involved in infant learning.

A Physiological Increase of Insulin in the Olfactory Bulb Decreases Detection of a Learned Aversive Odor and Abolishes Food Odor-Induced Sniffing Behavior in Rats

Insulin is involved in multiple regulatory mechanisms, including body weight and food intake, and plays a critical role in metabolic disorders such as obesity and diabetes. An increasing body of evidence indicates that insulin is also involved in the modulation of olfactory function. The olfactory bulb (OB) contains the highest level of insulin and insulin receptors (IRs) in the brain. However, a role for insulin in odor detection and sniffing behavior remains to be elucidated. Using a behavioral paradigm based on conditioned olfactory aversion (COA) to isoamyl-acetate odor, we demonstrated that an intracerebroventricular (ICV) injection of 14 mU insulin acutely decreased olfactory detection of fasted rats to the level observed in satiated animals. In addition, whereas fasted animals demonstrated an increase in respiratory frequency upon food odor detection, this effect was absent in fasted animals receiving a 14 mU insulin ICV injection as well as in satiated animals. In parallel, we showed that the OB and plasma insulin levels were increased in satiated rats compared to fasted rats, and that a 14 mU insulin ICV injection elevated the OB insulin level of fasted rats to that of satiated rats. We further quantified insulin receptors (IRs) distribution and showed that IRs are preferentially expressed in the caudal and lateral parts of the main OB, with the highest labeling found in the mitral cells, the main OB projection neurons. Together, these data suggest that insulin acts on the OB network to modulate olfactory processing and demonstrate that olfactory function is under the control of signals involved in energy homeostasis regulation and feeding behaviors.

Differential dynamics of amino acid release in the amygdala and olfactory cortex during odor fear acquisition as revealed with simultaneous high temporal resolution microdialysis

Although the amygdala seems to be essential to the formation and storage of fear memories, it might store only some aspects of the aversive event and facilitate the storage of more specific sensory aspects in cortical areas. We addressed the time course of amygdala and cortical activation in the context of odor fear conditioning in rats. Using high temporal resolution (1-min sampling) intracerebral microdialysis, we investigated the dynamics of glutamate and GABA fluctuations simultaneously in basolateral amygdala (BLA) and posterior piriform cortex (pPCx) during the course of the acquisition session, which consisted of six odor (conditioned stimulus)-footshock (unconditioned stimulus) pairings. In BLA, we observed a transient increase in amino acid concentrations following the first odor-shock pairing, after which concentrations returned to baseline levels or slightly below. In pPCx, transient increases were seen after each pairing and were also observed after the last odor-shock pairing, corresponding to the predicted times of anticipated trials. Furthermore, we observed that for the first pairing, the increase in BLA occurred earlier than the increase in pPCx. These data suggest that the amygdala is engaged early during acquisition and precedes the activation of the olfactory cortex, which is maintained until the end of the session. In addition, our data raise the challenging idea that the olfactory cortex might store certain aspects of fear conditioning related to the timing of the associations.

It's time to fear! Interval timing in odor fear conditioning in rats

Time perception is crucial to goal attainment in humans and other animals, and interval timing also guides fundamental animal behaviors. Accumulating evidence has made it clear that in associative learning, temporal relations between events are encoded, and a few studies suggest this temporal learning occurs very rapidly. Most of these studies, however, have used methodologies that do not permit investigating the emergence of this temporal learning. In the present study we monitored respiration, ultrasonic vocalization (USV) and freezing behavior in rats in order to perform fine-grain analysis of fear responses during odor fear conditioning. In this paradigm an initially neutral odor (the conditioned stimulus, CS) predicted the arrival of an aversive unconditioned stimulus (US, footshock) at a fixed 20-s time interval. We first investigated the development of a temporal pattern of responding related to CS-US interval duration. The data showed that during acquisition with odor-shock pairings, a temporal response pattern of respiration rate was observed. Changing the CS-US interval duration from 20-s to 30-s resulted in a shift of the temporal response pattern appropriate to the new duration thus demonstrating that the pattern reflected the learning of the CS-US interval. A temporal pattern was also observed during a retention test 24 h later for both respiration and freezing measures, suggesting that the animals had stored the interval duration in long-term memory. We then investigated the role of intra-amygdalar dopaminergic transmission in interval timing. For this purpose, the D1 dopaminergic receptors antagonist SCH23390 was infused in the basolateral amygdala before conditioning. This resulted in an alteration of timing behavior, as reflected in differential temporal patterns between groups observed in a 24 h retention test off drug. The present data suggest that D1 receptor dopaminergic transmission within the amygdala is involved in temporal processing.

Rearing with artificially scented mothers attenuates conditioned odor aversion in adulthood but not its amygdala dependency.

The aim of the present study was to investigate whether neonatal odor experience associated with the mother affects food avoidance learning and basolateral amygdala (BLA) involvement in adulthood. Odorization of mothers nipples with banana or almond solutions from birth to weaning resulted in an impairment at adulthood of conditioned odor aversion (COA). These effects were specific to the early-experienced odor since no deficit was observed for COA to a novel odor (Experiment 1). In contrast, mere exposure to an odor in the home cage instead of on mothers nipples induced no deficit in COA at adulthood (Experiment 2). Finally, transitory inactivation of the BLA during COA acquisition in adult animals impaired the normal COA of naïve animals but also the attenuated COA of rats with early odor experience on the mother (Experiment 3). These results demonstrate that neonatal odor experience associated with the mother promotes the acquisition of appetitive memories which can interfere with food avoidance learning in adulthood. They also suggest that this early experience did not modify the BLA involvement in learned aversion.

Olfactory memory networks: from emotional learning to social behaviors

Odors are powerful stimuli that can evoke emotional states, and support learning and memory. Decades of research have indicated that the neural basis for this strong “odor-emotional memory” connection is due to the uniqueness of the anatomy of the olfactory pathways. Indeed, unlike the other sensory systems, the sense of smell does not pass through the thalamus to be routed to the cortex. Rather, odor information is relayed directly to the limbic system, a brain region typically associated with memory and emotional processes. This provides olfaction with a unique and potent power to influence mood, acquisition of new information, and use of information in many different contexts including social interactions. Indeed, olfaction is crucially involved in behaviors essential for survival of the individual and species, including identification of predators, recognition of individuals for procreation or social hierarchy, location of food, as well as attachment between mating pairs and infant-caretaker dyads. Importantly, odors are sampled through sniffing behavior. This active sensing plays an important role in exploratory behaviors observed in the different contexts mentioned above. Odors are also critical for learning and memory about events and places and constitute efficient retrieval cues for the recall of emotional episodic memories. This broad role for odors appears highly preserved across species. In addition, the consistent early developmental emergence of olfactory function across diverse species also provides a unique window of opportunity for analysis of myriad behavioral systems from rodents to nonhuman primates and humans. This, when combined with the relatively conserved organization of the olfactory system in mammals, provides a powerful framework to explore how complex behaviors can be modulated by odors to produce adaptive responses, and to investigate the underlying neural networks. The present research topic brings together cutting edge research on diverse species and developmental stages, highlighting convergence and divergence between humans and animals to facilitate translational research. It is composed of 25 articles and encompasses 5 sections: human olfaction, odor preferences and aversions, odors and social behavior, olfaction and sniffing, and olfactory memory.

Infant rats can learn time intervals before the maturation of the striatum: evidence from odor fear conditioning

Interval timing refers to the ability to perceive, estimate and discriminate durations in the range of seconds to minutes. Very little is currently known about the ontogeny of interval timing throughout development. On the other hand, even though the neural circuit sustaining interval timing is a matter of debate, the striatum has been suggested to be an important component of the system and its maturation occurs around the third post-natal (PN) week in rats. The global aim of the present study was to investigate interval timing abilities at an age for which striatum is not yet mature. We used odor fear conditioning, as it can be applied to very young animals. In odor fear conditioning, an odor is presented to the animal and a mild footshock is delivered after a fixed interval. Adult rats have been shown to learn the temporal relationships between the odor and the shock after a few associations. The first aim of the present study was to assess the activity of the striatum during odor fear conditioning using 2-Deoxyglucose autoradiography during development in rats. The data showed that although fear learning was displayed at all tested ages, activation of the striatum was observed in adults but not in juvenile animals. Next, we assessed the presence of evidence of interval timing in ages before and after the inclusion of the striatum into the fear conditioning circuit. We used an experimental setup allowing the simultaneous recording of freezing and respiration that have been demonstrated to be sensitive to interval timing in adult rats. This enabled the detection of duration-related temporal patterns for freezing and/or respiration curves in infants as young as 12 days PN during odor fear conditioning. This suggests that infants are able to encode time durations as well as and as quickly as adults while their striatum is not yet functional. Alternative networks possibly sustaining interval timing in infant rats are discussed.

Long-term effects of infant learning on adult conditioned odor aversion are determined by the last preweaning experience.

We recently showed that odorizing mothers nipples from birth to weaning attenuated adult conditioned odor aversion (COA). The present study evaluated whether shorter durations of preweaning olfactory experiences could induce similar long-term effects. We first showed that late preweaning odorization (PN13-PN25) impaired adult COA similarly to odorization from birth to weaning (PN0-PN25) whereas early odorization (PN0-PN12) had no effect on adult COA. As early odorization was followed by an odorless suckling period, we evaluated whether this odorless suckling could have interfered with early associative learning. We therefore weaned the animals either immediately after early odorization or 7 days later. Early odorization (PN0-PN18) followed by late odorless suckling (PN19-PN25) had no effect on adult COA. However, pups with early odorization (PN0-PN18) but without late odorless suckling (weaned at PN18) showed attenuated COA. These results support the hypothesis that interference between early and late preweaning experiences with the mother determines the long-term impact on adult COA.

Differential involvement of amygdala and cortical NMDA receptors activation upon encoding in odor fear memory

Although the basolateral amygdala (BLA) plays a crucial role for the acquisition of fear memories, sensory cortices are involved in their long-term storage in rats. However, the time course of their respective involvement has received little investigation. Here we assessed the role of the glutamatergic N-methyl-d-aspartate (NMDA) receptors in the BLA and olfactory cortex at discrete moments of an odor fear conditioning session. We showed that NMDA receptors in BLA are critically involved in odor fear acquisition during the first association but not during the next ones. In the cortex, NMDA receptor activation at encoding is not necessary for recent odor fear memory while its role in remote memory storage needs further investigation.