Behavioural response of sexually naïve and experienced male rats to the smell of 6-methyl-5-hepten-2-one and female rat faeces.

Sexually experienced male rats display penile erections when exposed to faeces from mammalian females in oestrus (Rampin et al., Behav Brain Res, 172:169, 2006), suggesting that specific odours indicate female receptiveness across species. However, it is unknown to what extent the sexual response observed results from an odorous conditioning acquired during sexual experience. We tested the behavioural response of male Brown Norway rats both when sexually naïve and experienced to four odours, including oestrous rat faeces and 6-methyl-5-hepten-2-one (methylheptenone; a molecule found in higher concentrations during oestrus in female rats, foxes and horses). Odour had a significant effect on the sexual response of the naïve rats, with oestrus faeces provoking significantly more erections than herb odour, and with methylheptenone and di-oestrus faeces being intermediate. This indicates that sexually naïve male rats have an unconditioned ability to detect oestrous mediated via odour. After gaining sexual experience, the response to methylheptenone, di- and oestrus faeces was significantly higher than that observed with herb odour. These results strongly suggest that methylheptenone is part of the odorous bouquet of oestrus and contributes to the olfactory determination of female receptiveness.

Brain processing of biologically relevant odors in the awake rat, as revealed by manganese-enhanced MRI.

BACKGROUND: So far, an overall view of olfactory structures activated by natural biologically relevant odors in the awake rat is not available. Manganese-enhanced MRI (MEMRI) is appropriate for this purpose. While MEMRI has been used for anatomical labeling of olfactory pathways, functional imaging analyses have not yet been performed beyond the olfactory bulb. Here, we have used MEMRI for functional imaging of rat central olfactory structures and for comparing activation maps obtained with odors conveying different biological messages. METHODOLOGY/PRINCIPAL FINDINGS: Odors of male fox feces and of chocolate flavored cereals were used to stimulate conscious rats previously treated by intranasal instillation of manganese (Mn). MEMRI activation maps showed Mn enhancement all along the primary olfactory cortex. Mn enhancement elicited by male fox feces odor and to a lesser extent that elicited by chocolate odor, differed from that elicited by deodorized air. This result was partly confirmed by c-Fos immunohistochemistry in the piriform cortex. CONCLUSION/SIGNIFICANCE: By providing an overall image of brain structures activated in awake rats by odorous stimulation, and by showing that Mn enhancement is differently sensitive to different stimulating odors, the present results demonstrate the interest of MEMRI for functional studies of olfaction in the primary olfactory cortex of laboratory small animals, under conditions close to natural perception. Finally, the factors that may cause the variability of the MEMRI signal in response to different odor are discussed.

Electrophysiological responses of rat olfactory tubercle neurons to biologically relevant odours.

Biologically relevant odours were used to stimulate olfactory tubercle neurons in anaesthetized male rats. Among 120 recorded neurons, 118 showed spontaneous activity (mean firing rate, 15.0 ± 1.4 spikes/s). Ninety-eight neurons were exposed to at least one of the four following odour sources: an empty vial, or a vial containing food pellets (familiar odour), a sample of oestrous rat faeces (conspecific sexual odour), or a sample of male fox faeces (predator odour). The proportion of neurons responding with a change in activity was significantly linked to the odour applied. Repetition of the stimulation with the same odour elicited the same activity change. Between 50 and 70% of neuronal activity changes were not accompanied by respiration changes. Fifty-six neurons were exposed successively to all four odours, and 38 of them showed an activity change in response to at least one. The response of a neuron to an odour was not affected by its response to the previous one, and no neuron responded in the same manner to all odours. Conversely, no odour elicited a unique response in this population of neurons. However, the proportions of excited, inhibited and insensitive neurons depended significantly on the odour applied, suggesting that the recruitment of olfactory tubercle neurons is directly dependent on the biological significance of the odour.

Effects of manganese injected into rat nostrils: implications for in vivo functional study of olfaction using MEMRI.

Manganese-enhanced magnetic resonance imaging (MEMRI) is a powerful tool for visualizing neuronal pathways and mapping brain activity modulation. A potential drawback of MEMRI lies in the toxic effects of manganese (Mn), which also depend on its administration route. The aim of this study was to analyze the effects of Mn doses injected into the nostrils of rats on both olfactory perception and MRI contrast enhancement. For this purpose, doses in the range 0-8 µmol MnCl(2) were tested. Behavioral items were quantified with and without odor stimulation during the first 2 h following Mn injection. The MRI study was performed after 16 h of intermittent olfactory stimulations. Behavioral results showed that, during the early period following Mn administration, spontaneous motor activity was not affected, while odor-related behaviors were dose-dependently reduced. MRI results showed that, in the primary olfactory cortex, contrast was rapidly enhanced for Mn doses up to 0.3 µmol and very slowly above. This dose of 0.3 µmol Mn can thus be taken as the optimal dose for injection into rat nostrils to ensure a reproducible contrast in MRI studies while sparing olfactory perception.

A mixture of odorant molecules potentially indicating oestrus in mammals elicits penile erections in male rats.

A common set of odorous molecules may indicate female receptiveness across species, as male rats display sexual arousal when exposed to the odour of oestrous faeces from rats, vixens and mares. More than 900 different compounds were identified by GC-MS analyses performed on faeces samples from di-oestrous and oestrous females and from males of the three species. Five carboxylic acids were found in lower concentrations in faeces from all oestrous females. We subjected 12 sexually trained male rats to a 30 min exposure to different dilutions of a mixture of these five molecules in the same proportions as found in female oestrous faeces. The behavioural responses of the rats were compared to those displayed when exposed to water (negative control) and faeces from oestrous female rats (positive control). Frequency of penile erections were found to be significantly dependent on mixture dilution, with two intermediate dilutions eliciting frequencies of penile erections that did not differ from those obtained during exposure to oestrous female rat faeces. Higher and lower dilutions did not elicit more penile erections than observed with water. These results support our hypothesis that a small set of odorous molecules may indicate sexual receptiveness in mammalian females.

Iterative algorithm for spatial and intensity normalization of MEMRI images. Application to tract-tracing of rat olfactory pathways.

Manganese (Mn)-enhanced magnetic resonance imaging (MEMRI) is an emerging technique for visualizing neuronal pathways and mapping brain activity modulation in animal models. Spatial and intensity normalizations of MEMRI images acquired from different subjects are crucial steps as they can influence the results of groupwise analysis. However, no commonly accepted procedure has yet emerged. Here, a normalization method is proposed that performs both spatial and intensity normalizations in a single iterative process without the arbitrary choice of a reference image. Spatial and intensity normalizations benefit from this iterative process. On one hand, spatial normalization increases the accuracy of region of interest (ROI) positioning for intensity normalization. On the other hand, improving the intensity normalization of the different MEMRI images leads to a better-averaged target on which the images are spatially registered. After automatic fast brain segmentation and optimization of the normalization process, this algorithm revealed the presence of Mn up to the posterior entorhinal cortex in a tract-tracing experiment on rat olfactory pathways. Quantitative comparison of registration algorithms showed that a rigid model with anisotropic scaling is the best deformation model for intersubject registration of three-dimensional MEMRI images. Furthermore, intensity normalization errors may occur if the ROI chosen for intensity normalization intersects regions where Mn concentration differs between experimental groups. Our study suggests that cross-comparing Mn-injected animals against a Mn-free group may provide a control to avoid bias introduced by intensity normalization quality. It is essential to optimize spatial and intensity normalization as the detectability of local between-group variations in Mn concentration is directly tied to normalization quality.

Statistical analysis of 3D images detects regular spatial distributions of centromeres and chromocenters in animal and plant nuclei.

In eukaryotes, the interphase nucleus is organized in morphologically and/or functionally distinct nuclear "compartments". Numerous studies highlight functional relationships between the spatial organization of the nucleus and gene regulation. This raises the question of whether nuclear organization principles exist and, if so, whether they are identical in the animal and plant kingdoms. We addressed this issue through the investigation of the three-dimensional distribution of the centromeres and chromocenters. We investigated five very diverse populations of interphase nuclei at different differentiation stages in their physiological environment, belonging to rabbit embryos at the 8-cell and blastocyst stages, differentiated rabbit mammary epithelial cells during lactation, and differentiated cells of Arabidopsis thaliana plantlets. We developed new tools based on the processing of confocal images and a new statistical approach based on G- and F- distance functions used in spatial statistics. Our original computational scheme takes into account both size and shape variability by comparing, for each nucleus, the observed distribution against a reference distribution estimated by Monte-Carlo sampling over the same nucleus. This implicit normalization allowed similar data processing and extraction of rules in the five differentiated nuclei populations of the three studied biological systems, despite differences in chromosome number, genome organization and heterochromatin content. We showed that centromeres/chromocenters form significantly more regularly spaced patterns than expected under a completely random situation, suggesting that repulsive constraints or spatial inhomogeneities underlay the spatial organization of heterochromatic compartments. The proposed technique should be useful for identifying further spatial features in a wide range of cell types.

Three-dimensional macronutrient-associated Fos expression patterns in the mouse brainstem.

BACKGROUND: The caudal brainstem plays an important role in short-term satiation and in the control of meal termination. Meal-related stimuli sensed by the gastrointestinal (GI) tract are transmitted to the area postrema (AP) via the bloodstream, or to the nucleus tractus solitarii (NTS) via the vagus nerve. Little is known about the encoding of macronutrient-specific signals in the caudal brainstem. We hypothesized that sucrose and casein peptone activate spatially distinct sub-populations of NTS neurons and thus characterized the latter using statistical three-dimensional modeling. METHODOLOGY/PRINCIPAL FINDINGS: Using immunolabeling of the proto-oncogene Fos as a marker of neuronal activity, in combination with a statistical three-dimensional modeling approach, we have shown that NTS neurons activated by sucrose or peptone gavage occupy distinct, although partially overlapping, positions. Specifically, when compared to their homologues in peptone-treated mice, three-dimensional models calculated from neuronal density maps following sucrose gavage showed that Fos-positive neurons occupy a more lateral position at the rostral end of the NTS, and a more dorsal position at the caudal end. CONCLUSION/SIGNIFICANCE: To our knowledge, this is the first time that subpopulations of NTS neurons have be distinguished according to the spatial organization of their functional response. Such neuronal activity patterns may be of particular relevance to understanding the mechanisms that support the central encoding of signals related to the presence of macronutrients in the GI tract during digestion. Finally, this finding also illustrates the usefulness of statistical three-dimensional modeling to functional neuroanatomical studies.

Modeling the 3D functional architecture of the nucleus in animal and plant kingdoms.

Compartmentalization is one of the fundamental principles which underly nuclear function. Numerous studies describe complex and sometimes conflicting relationships between nuclear gene positioning and transcription regulation. Therefore the question is whether topological landmarks and/or organization principles exist to describe the nuclear architecture and, if existing, whether these principles are identical in the animal and plant kingdoms. In the frame of an agroBI-INRA program on nuclear architecture, we set up a multidisciplinary approach combining biological studies, spatial statistics and 3D modeling to investigate spatial organization of a nuclear compartment in both plant and animal cells in their physiological contexts. In this article, we review the questions addressed in this program and the methodology of our work.

3D fluorescent in situ hybridization using Arabidopsis leaf cryosections and isolated nuclei.

BACKGROUND: Fluorescent hybridization techniques are widely used to study the functional organization of different compartments within the mammalian nucleus. However, few examples of such studies are known in the plant kingdom. Indeed, preservation of nuclei 3D structure, which is required for nuclear organization studies, is difficult to fulfill. RESULTS: We report a rapid protocol for fluorescent in situ hybridization (FISH) performed on 3D isolated nuclei and thin cryosectioned leaves of Arabidopsis thaliana. The use of direct labeling minimized treatment steps, shortening the overall procedure. Using image analysis, we measured different parameters related to nucleus morphology and overall 3D structure. CONCLUSION: Our work describes a 3D-FISH protocol that preserves the 3D structure of Arabidopsis interphase nuclei. Moreover, we report for the first time FISH using cryosections of Arabidopsis leaves. This protocol is a valuable tool to investigate nuclear architecture and chromatin organization.

Three-dimensional statistical modeling of neuronal populations: illustration with spatial localization of supernumerary neurons in the locus coeruleus of quaking mutant mice.

An algorithm for the three-dimensional statistical representation of neuronal populations was designed and implemented. Using this algorithm a series of 3D models, calculated from repeated histological experiments, can be combined to provide a synthetic vision of a population of neurons taking into account biological and experimental variability. Based on the point process theory, our algorithm allows computation of neuronal density maps from which isodensity surfaces can be readily extracted and visualized as surface models revealing the statistical organization of the neuronal population under study. This algorithm was applied to the spatial distribution of locus coeruleus (LC) neurons of 30- and 90-day-old control and quaking mice. By combining 12 3D models of the LC, a region of the nucleus in which a subpopulation of neurons loses its noradrenergic phenotype between 30 and 90 days postnatally was demonstrated in control mice but not in quaking mice, leading to the hyperplasia previously reported in adult mutants. Altogether, this algorithm allows computation of 3D statistical and graphical models of neuronal populations, providing a contribution to quantitative 3D neuroanatomical modeling.

Are oestrus odours species specific?

Adult male rats were exposed to faeces odours of three animal species (rat, fox and horse). They displayed erections in the presence of faeces from oestrous females (whatever the species). In addition, fox faeces (whatever the gender or hormonal status) elicited an expected freezing reaction. It is suggested that oestrous female faeces of these three species share common odorants which depend on the hormonal status and characterize female receptivity.

Possible neural mediation of the central effects of oxytocin on uterine motility.

The central nervous system contains the nuclei at the origin of autonomic and neuroendocrine pathways to the uterus. Although the anatomical basis of these pathways is known, the conditions of their recruitment and their interactions in the context of copulation remain to be explored. We tested the hypothesis that some central mechanisms could simultaneously recruit both pathways to the uterus. In this aim, we recorded intrauterine pressure changes in anesthetized female rats at the estrus stage after intracerebroventricular (ICV) administration of oxytocin (OT). Doses of 0.3-300 ng elicited increases of frequency and amplitude of uterine contractions. These effects were partly mimicked by the OT agonist [Thr(4),Gly(7)]OT but not by arginine vasopressin. They were blocked by the OT receptor antagonist atosiban delivered either ICV or intravenously. The latter suggests that ICV OT activated the systemic release of OT. The effects of OT were also blocked by hexamethonium, a ganglionic blocking agent, by atropine, a muscarinic receptor antagonist, and by N(omega)-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthesis. The results reveal that ICV OT recruits autonomic efferent pathways to the uterus. These results support our hypothesis that the activation of central nuclei can promote uterine contractility, and that OT may be a central coordinator of autonomic and neuroendocrine pathways. The hypothalamus, the source of direct OT-ergic projections to the pituitary, the brain stem, and the spinal cord, may be a target of central OT.

Free-D: an integrated environment for three-dimensional reconstruction from serial sections.

Three-dimensional (3D) reconstruction is a powerful tool to investigate complex neuroanatomical organizations. 3D models are often generated by piling up registered segmentations carried out on serial sections labeled by histological means. However, these models suffer limitations (incompleteness and lack of statistical representativity), which can be overcome by model averaging and fusion. These operations require an appropriate reconstruction environment allowing the simultaneous processing of several data sets. This paper describes the first release of Free-D, a software designed for the reconstruction of 3D models generated from stacks of serial sections, in the perspective of model averaging and fusion. A unique graphical user interface integrates the 3D reconstruction tools. Several large stacks (tens of gigabytes) including hundreds of images having heterogeneous characteristics (size, resolution, depth, etc.) can be simultaneously processed, thus complying to most encountered experimental situations. This first version of Free-D constitutes the required environment for the future integration of the averaging and fusion algorithms currently developed in our group and illustrated here with preliminary results.

Spinal effects of oxytocin on uterine motility in anesthetized rats.

The rat uterus receives an innervation from the lumbosacral and thoracolumbar segments of the spinal cord. These segments receive descending oxytocinergic projections from the paraventricular nucleus of the hypothalamus. We tested the hypothesis that oxytocin regulates uterine motility through a spinal site of action. Oxytocin was administered in anesthetized female rats either intrathecally at the lumbosacral or thoracolumbar spinal cord levels or intravenously. Uterine activity was revealed by measuring changes of intrauterine pressure using an indwelling balloon placed in one caudal uterine horn. The uterus displayed a spontaneous activity characterized by intrauterine pressure rises, the frequency, amplitude, and duration of which were dependent on the stage of the estrous cycle. Oxytocin delivered at the lumbosacral level affected the frequency (during proestrus, estrus, and diestrus) and amplitude (during proestrus and estrus) of uterine activity. During estrus, oxytocin delivered at the thoracolumbar level affected the frequency, amplitude, and duration of the intrauterine pressure rises. Intravenous oxytocin not only affected intrauterine pressure rises (namely amplitude during proestrus and estrus and frequency and duration during estrus) but also increased the basal tone during estrus. The effects of lumbosacral oxytocin were partly mimicked by the oxytocin agonist [Thr(4),Gly(7)]-oxytocin blocked by the oxytocin receptor antagonist atosiban and by hexamethonium. Arginine vasopressin delivered at the lumbosacral level had no effect. These results support our hypothesis that oxytocin released by descending paraventriculo-spinal pathways and acting on spinal oxytocin receptors modulates the activity of the uterus. This regulation is cycle dependent.

Spinal control of erection by glutamate in rats.

The lumbosacral spinal network controlling penile erection is activated by information from peripheral and supraspinal origins. We tested the hypothesis that glutamate, released by sensory afferents from the genitals, activates this proerectile network. In anesthetized intact and T8 spinalized (i.e., freed from supraspinal inhibition) male rats, the parameters of electrical stimulation of the dorsal penile nerve (DPN) that elicited intracavernous pressure (ICP) rises were determined. In T8 spinalized rats, DPN stimulations were applied in the presence of d(-)-2-amino-5-phosphonopentanoic acid (d-AP5), a competitive NMDA receptor antagonist, or of 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulphonamide (NBQX), an AMPA-kainate receptor antagonist, injected intrathecally at the lumbosacral level. Both antagonists, alone or in combination, dose dependently decreased the ICP rise and increased its latency. In conscious rats, reflexive erections were depressed by d-AP5 and NBQX, as revealed by an increased latency of the first erection and by decreases of the number of rats displaying erections, of the number of erection clusters and of the number of erections per cluster. In anesthetized ats, the combined administration of the glutamatergic agonists NMDA and AMPA elicited ICP rises in the absence of DPN stimulation. In contrast, both agonists moderately decreased the ICP rise elicited by DPN stimulation but did not affect its latency. These results support our hypothesis that glutamate, released on stimulation of the genitals and acting at AMPA and NMDA receptors, is a potent reactivator of the spinal proerectile network.

Abnormal postnatal ontogeny of the locus coeruleus in the epileptic mutant mouse quaking.

The tonic-clonic convulsions of the quaking mutant mice have been shown to be associated with the hyperplasia of the nucleus locus coeruleus, the origin of most brain noradrenergic neurons. In the present study, the postnatal ontogeny of the locus coeruleus has been studied by tyrosine hydroxylase immunolabeling in the mutant mice quaking and their controls at postnatal days 1, 30 and 90. In the control mice, the number of immunoreactive neuronal cell bodies increased significantly in the rostral half of the locus coeruleus between birth and postnatal day 30, while it decreased significantly in the caudal half between birth and adulthood. Thus, during postnatal maturation, the distribution of locus coeruleus neurons was shifted in the rostral direction. In the quaking mutant mice, while the increase of immunolabeling between birth and postnatal day 30 was observed in the rostral half of the locus coeruleus, no diminution could be found in the caudal half between birth and adulthood. As a result, the rostral shift of tyrosine hydroxylase immunoreactivity was not observed. Consequently, in adult mice, the caudal part of the mutants locus coeruleus appeared to contain significantly more neurons than the corresponding region in the controls. These results indicate that the hyperplasia of the locus coeruleus of the quaking mice that we had previously reported results from an alteration of the postnatal maturation of this nucleus. This developmental abnormality might be a primary determinant of the inherited epilepsy of the quaking mutant mice.