Frédérique Datiche

Expression of Fos in the piriform cortex after acquisition of olfactory learning: an immunohistochemical study in the rat.

The piriform cortex (PCx), the main area of the primary olfactory cortex, is assumed to play a role in olfactory memory. Involvement of this paleocortex in mnesic processes was investigated by using Fos immunocytochemistry after acquisition of a two-odor discrimination task. Trained rats had to associate one odor of a pair with water reward while pseudo-trained rats were randomly rewarded. We further used non-trained rats and home cage control animals to determine the effect of manipulation and basal Fos level respectively. Except in control rats, Fos immunoreactivity was mainly distributed in brain areas involved in olfactory processing, learning and arousal. The trained, pseudo-trained, and non-trained rats showed a high Fos labeling in the entire PCx. However, quantitative analysis demonstrated a statistically higher Fos immunoreactivity in the anterior PCx in comparison with the posterior PCx for these rats. Furthermore, behavioral data allowed us to distinguish two groups of trained rats according to the number of days required to acquire the task. Rats with slow acquisition showed a higher Fos immunoreactivity in the whole PCx in comparison with the rats exhibiting a fast acquisition. Our findings support the assumption of a PCx rostro-caudal heterogeneity which could sustain differential information processing.

Optical recording of the rat piriform cortex activity

The piriform cortex (PCx) is a phylogenetically old brain structure which presents characteristics of a content-addressable memory. Taking into account its particular anatomo-functional organization, we hypothesized that this cortex could behave rather as an assembly of different functional units than as a functionally homogeneous structure. This hypothesis was tested by using both anatomical and functional approaches. Immunohistological and tracing experiments demonstrated that both the connections of the PCx with the higher nervous centres, and its monoaminergic and cholinergic modulatory afferents exhibited a heterogeneous distribution. Then, optical monitoring of its neuronal activity with a voltage-sensitive dye pointed out that the PCx is a functionally heterogeneous structure. Electrical stimulations of the olfactory bulb showed that the inhibitory processes which control the cortical responsiveness were not identical in all the PCx area. Two different functional areas at least could be distinguished: in the ventromedial PCx, the afferent activity is privileged since the level of inhibition of disynaptic activation remained large during repetitive stimuli. Contrarily, in the posterior PCx, the disynaptic activity remained unchanged in response to successive stimulations and the responses of neighbouring sites were statistically more synchronized than in its anterior part. Moreover, a late depolarization wave was significantly larger in the posterior PCx. These data are in good agreement with the results provided by computational models of the PCx. In the future, theoretical and experimental investigations of this cortex will be useful for understanding olfactory information processing and as a model of brain functioning at the neocortical level as well.

Catecholamine innervation of the piriform cortex: a tracing and immunohistochemical study in the rat

In order to determine the origin of the catecholamine innervation of the rat piriform cortex (PC), we combined retrograde transport of the B subunit of the cholera toxin (CTb) with tyrosine hydroxylase (TH) immunohistochemistry. A substantial number of CTb retrogradely labeled cells was found in the parabrachial pigmented, paranigral and interfascicular nuclei of the ventral tegmental area and the dorsal part of the locus coeruleus, whereas nearly no labeling was noted in the substantia nigra. Following TH immunohistochemistry on the same sections, most if not all of the CTb labeled cells were also TH immunoreactive. Occasional double-labeled cells were also observed in the anterior part of the raphe dorsal nucleus. As visualized with dopamine beta-hydroxylase, dopamine or TH immunohistochemistry, the noradrenaline fibers were homogeneously distributed whereas the dopamine fibers showed rostro-caudal and latero-medial differences. The distribution of TH fibers overlapped both patterns. Our report suggests that the heterogeneous distribution of the DA fibers could support a differential centrifugal modulation of the olfactory information processing throughout the PC.

Intrinsic association fiber system of the piriform cortex: A quantitative study based on a cholera toxin B subunit tracing in the rat

By using retrograde and anterograde transport of the B subunit of cholera toxin (CTb), we examined quantitatively the association fiber systems, i.e., the collaterals of pyramidal cell axons, that reciprocally connect both the rostral and the caudal parts of the piriform cortex (PC). Well‐defined CTb injections were obtained in layers Ib or II‐III of the rostral and the caudal parts of the PC. Using precision counting, we determined the proportion of cellular profiles in layers II and III that gave rise to association fibers and thus demonstrated a predominance of rostrocaudal fibers over the caudorostral ones. Our data also support a precise laminar organization of the PC in which the rostrocaudal fibers originated mainly from layer II and the caudorostral fibers primarily from layer III. Cholera toxin injections into layer Ib produced a peak of labeled profiles 2 mm from the site, indicating that a large proportion of the association fibers from layer II travel for at least 2 mm and then synapse in layer Ib. At either end of the PC, the association projections are concentrated laterally. The functional significance of these anatomical features is discussed with respect to olfactory processing, propagation of the activity within the PC, and the possible role of intrinsic fibers in olfactory memory.

Cell activity in the anterior piriform cortex during an olfactory learning in the rat.

Several studies have shown that the piriform cortex is involved in learning processes and pyramidal cell activity does not only encode the odour quality but is also related to contextual information about past experience and future action. To study how odour-specific patterns in neuronal activity are established we used an odour discrimination go/no go task with water reinforcement for analysing extracellular single cell activity in anterior piriform cortex in freely moving rats. During conditioning single cells responded to different task events. Of the cells 52% participate in odour sampling and 87% were involved in odour discrimination. More than half of the responses to odours were inhibitory responses. Seventeen percent changed their activity for nose-poke only. The activity of 33% was related to reinforcement. Once established the pattern of reaction to the odour was preserved for several days. It is suggested that the anterior part of the piriform cortex is not involved in odour coding only. However, learning-related plasticity was not observed in this area.

Olfactory discrimination ability and brain expression of c-fos, Gir and Glut1 mRNA are altered in n-3 fatty acid-depleted rats.

The long-chain polyunsaturated n-3 fatty acids (n-3 PUFA), particularly docosahexaenoic acid (DHA), are abundantly present in the central nervous system and play an important role in cognitive functions such as learning and memory. We, therefore, investigated the effects of n-3 PUFA-depletion in rats (F2 generation) on the learning of an olfactory discrimination task, progressively acquired within a four-arm maze, and on the mRNA expression of some candidate genes, i.e., c-fos, Gir and glucose transporter (Glut1), which could reflect the level of cerebral activity. We observed that DHA contents were dramatically decreased in the olfactory bulb, the piriform cortex and the neocortex of n-3-depleted rats. Furthermore, the n-3 deficiency resulted in a mild olfactory learning impairment as these rats required more days to master the olfactory task compared to control rats. Real-time RT-PCR experiments revealed that the training induced the expression of c-fos mRNA in all the three regions of the brain whereas Gir and Glut1 mRNA were induced only in olfactory bulb and neocortex. However, such an increase was less marked in the n-3-deficient rats. Taken together, these results allow us to assume that the behavioural impairment in n-3-deficient rats is linked to the depletion of n-3 fatty acids in brain regions processing olfactory cues. Data are discussed in view of the possible role of some of these genes in learning-induced neuronal olfactory plasticity.

Does taste or odor activate the same brain networks after retrieval of taste potentiated odor aversion

When simultaneous presentation of odor and taste cues precedes illness, rats acquire robust aversion to both conditioned stimuli. Such a phenomenon referred to as taste-potentiated odor aversion (TPOA) requires information processing from two sensory modalities. Whether similar or different brain networks are activated when TPOA memory is retrieved by either the odor or the taste presentation remains an unsolved question. By means of Fos mapping, we investigated the neuronal substrate underlying TPOA retrieval elicited by either the odor or the taste conditioned stimulus. Whatever the sensory modality used to reactivate TPOA memory, a significant change in Fos expression was observed in the hippocampus, the basolateral nucleus of amygdala and the medial and the orbito-frontal cortices. Moreover, only the odor presentation elicited a significantly higher Fos immunoreactivity in the piriform cortex, the entorhinal cortex and the insular cortex. Lastly, according to the stimulus tested to induce TPOA retrieval, the BLA was differentially activated and a higher Fos expression was induced by the odor than by the taste in this nucleus. The present study indicates that even if they share some brain regions, the cerebral patterns induced by either the odor or the taste are different. Data are discussed in view of the relevance of each conditioned stimulus to reactivate TPOA memory and of the involvement of the different labeled brain areas in information processing and TPOA retrieval.

Hypothalamus-Olfactory System Crosstalk: Orexin A Immunostaining in Mice

It is well known that olfaction influences food intake, and conversely, that an individual’s nutritional status modulates olfactory sensitivity. However, what is still poorly understood is the neuronal correlate of this relationship, as well as the connections between the olfactory bulb and the hypothalamus. The goal of this report is to analyze the relationship between the olfactory bulb and hypothalamus, focusing on orexin A immunostaining, a hypothalamic neuropeptide that is thought to play a role in states of sleep/wakefulness. Interestingly, orexin A has also been described as a food intake stimulator. Such an effect may be due in part to the stimulation of the olfactory bulbar pathway. In rats, orexin positive cells are concentrated strictly in the lateral hypothalamus, while their projections invade nearly the entire brain including the olfactory system. Therefore, orexin appears to be a good candidate to play a pivotal role in connecting olfactory and hypothalamic pathways. So far, orexin has been described in rats, however, there is still a lack of information concerning its expression in the brains of adult and developing mice. In this context, we revisited the orexin A pattern in adult and developing mice using immunohistological methods and confocal microscopy. Besides minor differences, orexin A immunostaining in mice shares many features with those observed in rats. In the olfactory bulb, even though there are few orexin projections, they reach all the different layers of the olfactory bulb. In contrast to the presence of orexin projections in the main olfactory bulb, almost none have been found in the accessory olfactory bulb. The developmental expression of orexin A supports the hypothesis that orexin expression only appears post-natally.

Learning-stage dependent Fos expression in the rat brain during acquisition of an olfactory discrimination task.

By using Fos immunocytochemistry, we investigated the activation in olfactory-related areas at three stages (the first and fourth days of conditioning and complete acquisition) of an olfactory discrimination learning task. The trained rats (T) had to associate one odour of a pair with water-reward within a four-arm maze whereas pseudo-trained (P) rats were only submitted to the olfactory cues without any reinforcement. In the piriform cortex, both T and P rats exhibited a higher immunoreactivity on the first day, which seemed to indicate a novelty-related Fos expression in this area, but whatever the learning-stage, no significant difference in Fos expression between T and P rats was observed. In hippocampus, Fos expression was significantly different between T and P rats in CA1 and CA3 on the first and fourth days respectively. Thus we showed a differential activation of CA1 and CA3 subfields which might support a possible functional heterogeneity. In the orbitofrontal cortex, Fos immunoreactivity was significantly higher in T rats compared to P rats when mastery of the discrimination task was complete. In contrast, no learning-related Fos expression was found in infralimbic and prelimbic cortices. The present data suggest an early implication of the hippocampal formation and a later involvement of neocortical areas throughout different stages of a progressively acquired olfactory learning task.

Cue valence representation studied by Fos immunocytochemistry after acquisition of a discrimination learning task

The piriform cortex (PCx) and related structures such as hippocampus and frontal cortex could play an important role in olfactory memory. We investigated their involvement in learning the biological value of an odor cue, i.e. predicting reward or non-reward in a two-odor discrimination task. Rats were sacrificed after stimulation by either rewarded or non-rewarded odor and Fos immunocytochemistry was performed. The different experimental groups of rats did not show strongly differentiated Fos expression pattern in either the PCx or the hippocampus. A few differences were noted in frontal areas. In the ventro-lateral orbital cortex, rats, ramdomly rewarded during the conditionning had a higher Fos level in comparison with other groups. In infralimbic cortex, rats, which learned the reward value of the olfactory cue and were water-reinforced the day of sacrifice, showed a higher Fos expression. Data are discussed in view of the olfactory learning paradigm and of the accuracy of the control groups used in the present experimental design. The behavioural conditions leading to Fos expression are further discussed since Fos is a marker of learning-induced plasticity as well as a general activity marker which can be activated by a wide range of stimuli not directly linked to memory.