Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and <em>in situ</em> Hybridization

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of ...

University of Campinas

To date, oil-rich plants are the main source of biodiesel products. Because concerns have been voiced about the impact of oil-crop cultivation on the price of food commodities, the interest in oil plants not used for food production and amenable to cultivation on non-agricultural land has soared. As a non-food, drought-resistant and oil-rich crop, Jatropha curcas L. fulfils many of the requirements for biofuel production.

Transcriptome analysis of the oil-rich seed of the bioenergy crop Jatropha curcas L.

The nervous system is organized to detect, internally represent and process sensory information to generate appropriate behaviors. Despite the crucial importance of odors that elicit instinctive behaviors, such as pheromones and kairomones, their neural representation remains little characterized in the mammalian brain. Here we used expression of the immediate early gene product c-Fos as a marker of neuronal activity to find that a wide range of pheromones and kairomones produces activation in the medial nucleus of the amygdala, a brain area anatomically connected with the olfactory sensory organs. We see that activity in this nucleus depends on vomeronasal organ input, and that distinct vomeronasal stimuli activate a dispersed ensemble of cells, without any apparent spatial segregation. This activity pattern does not reflect the chemical category of the stimuli, their valence or the induced behaviors. These findings will help build a complete understanding of how odor information is processed in the brain to generate instinctive behaviors. 

Lack of spatial segregation in the representation of pheromones and kairomones in the mouse medial amygdala.

Olfaction is a fundamental sense through which most animals perceive the external world. The olfactory system detects odors via specialized sensory organs such as the main olfactory epithelium and the vomeronasal organ. Sensory neurons in these organs use G-protein coupled receptors to detect chemosensory stimuli. The odorant receptor (OR) family is expressed in sensory neurons of the main olfactory epithelium, while the adult vomeronasal organ is thought to express other types of receptors.

Detection of pup odors by non-canonical adult vomeronasal neurons expressing an odorant receptor gene is influenced by sex and parenting status.

The mouse olfactory sensory neuron (OSN) repertoire is composed of 10 million cells and each expresses one olfactory receptor (OR) gene from a pool of over 1000. Thus, the nose is sub-stratified into more than a thousand OSN subtypes. Here, we employ and validate an RNA-sequencing-based method to quantify the abundance of all OSN subtypes in parallel, and investigate the genetic and environmental factors that contribute to neuronal diversity. We find that the OSN subtype distribution is stereotyped in genetically identical mice, but varies extensively between different strains. Further, we identify -acting genetic variation as the greatest component influencing OSN composition and demonstrate independence from OR function. However, we show that olfactory stimulation with particular odorants results in modulation of dozens of OSN subtypes in a subtle but reproducible, specific and time-dependent manner. Together, these mechanisms generate a highly individualized olfactory sensory system by promoting neuronal diversity.

Variation in olfactory neuron repertoires is genetically controlled and environmentally modulated.

Territorial male mice are aggressive toward intruding males, but socially bonded males are not. Through manipulation of activity in a subset of neurons in the ventromedial hypothalamus, Yang et al. (2017) report that social and physiological factors non-linearly interact to control male aggression.

The Strange Case of Aggression and the Brain.

Olfaction is a fundamental sense in most animal species. In mammals, the olfactory system comprises several subpopulations of sensory neurons located throughout the nasal cavity, which detect a variety of chemostimuli, including odorants, intraspecies and interspecies chemical communication cues. Some of these compounds are important for regulating innate and learned behaviors, and endocrine changes in response to other animals in the environment. With a particular focus on laboratory rodent species, this chapter provides a comprehensive description of the most important behavioral assays used for studying the olfactory system, and is meant to be a practical guide for those who study olfaction-mediated behaviors or who have an interest in deciphering the molecular, cellular, or neural mechanisms through which the sense of smell controls the generation of adaptive behavioral outputs.

Behavioral Assays in the Study of Olfaction: A Practical Guide.

Very little is known about long non-coding RNAs (lncRNAs) in the mammalian olfactory sensory epithelia. Deciphering the non-coding transcriptome in olfaction is relevant because these RNAs have been shown to play a role in chromatin modification and nuclear architecture reorganization, processes that accompany olfactory differentiation and olfactory receptor gene choice, one of the most poorly understood gene regulatory processes in mammals. In this study, we used a combination of in silico and ex vivo approaches to uncover a comprehensive catalogue of olfactory lncRNAs and to investigate their expression in the mouse olfactory organs. Initially, we used a novel machine-learning lncRNA classifier to discover hundreds of annotated and unannotated lncRNAs, some of which were predicted to be preferentially expressed in the main olfactory epithelium and the vomeronasal organ, the most important olfactory structures in the mouse. Moreover, we used whole-tissue and single-cell RNA sequencing data to discover lncRNAs expressed in mature sensory neurons of the main epithelium. Candidate lncRNAs were further validated by in situ hybridization and RT-PCR, leading to the identification of lncRNAs found throughout the olfactory epithelia, as well as others exquisitely expressed in subsets of mature olfactory neurons or progenitor cells.

Uncovering the mouse olfactory long non-coding transcriptome with a novel machine-learning model.

C1ql3 protein and its receptor Bai3 are involved in synaptic organization and function. In this issue of Neuron, Wang et al. (2020) report that both are essential for synaptic function between the anterior olfactory nucleus and the olfactory bulb and for the generation, but not recall, of associative olfactory memories determining food preference in mice.

From Synapse to Supper: A Food Preference Recipe with Olfactory Synaptic Ingredients.

The internal representation of sensory information via coherent activation of specific pathways in the nervous system is key to appropriate behavioral responses. Little is known about how chemical stimuli that elicit instinctive behaviors lead to organized patterns of activity in the hypothalamus. Here, we study how a wide range of chemosignals form a discernible map of olfactory information in the ventromedial nucleus of the hypothalamus (VMH) and show that different stimuli entail distinct active neural ensembles. Importantly, we demonstrate that this map depends on functional inputs from the vomeronasal organ. We present evidence that the spatial locations of active VMH ensembles are correlated with activation of distinct vomeronasal receptors and that disjunct VMH ensembles exhibit differential projection patterns. Moreover, active ensembles with distinct spatial locations are not necessarily associated with different behavior categories, such as defensive or social, calling for a revision of the currently accepted model of VMH organization.

Representation of Olfactory Information in Organized Active Neural Ensembles in the Hypothalamus.

Behaviors are shaped by hormones, which may act either by changing brain circuits or by modifying sensory detection of relevant cues. Pup-directed behaviors have been previously shown to change via action of hormones at the brain level. Here, we investigated hormonal control of pup-induced activity in the vomeronasal organ, an olfactory sensory structure involved in the detection of non-volatile chemosignals. Vomeronasal activity decreases as males switch from a pup-aggressive state to a non-aggressive parenting state, after they socially contact a female. RNA sequencing, qPCR, and in situ hybridization were used to identify expression, in the vomeronasal sensory epithelium, of candidate GPCR hormone receptors chosen by in silico analyses and educated guesses. After identifying that oxytocin and vasopressin receptors are expressed in the vomeronasal organ, we injected the corresponding hormones in mice and showed that oxytocin administration reduced both pup-induced vomeronasal activity and aggressive behavior. Conversely, injection of an oxytocin receptor antagonist in female-primed male animals, which normally exhibit reduced vomeronasal activity, significantly increased the number of active vomeronasal neurons. These data link oxytocin to the modulation of olfactory sensory activity, providing a possible mechanism for changes in male behavior after social experience with females.

Peripheral oxytocin injection modulates vomeronasal sensory activity and reduces pup-directed aggression in male mice.

This protocol combines fluorescent  hybridization and immunostaining to simultaneously detect, in histological sections from the same animal, subpopulations of neurons activated after two episodes of sensory stimulation. It allows the identification of groups of cells singly activated by either stimulus or co-activated by both stimuli. Our method results in nuclear staining for  mRNA and c-Fos protein, allowing better spatial and temporal resolution than previously published protocols, although it requires quick brain fixation. For complete details on the use and execution of this protocol, please refer to Carvalho et al. (2015, 2020).

Detection of Activated Mouse Neurons with Temporal Resolution via Dual c-Fos Staining.

The human transcription factor 4 gene (TCF4) encodes a helix-loop-helix transcription factor widely expressed throughout the body and during neural development. Mutations in TCF4 cause a devastating autism spectrum disorder known as Pitt-Hopkins syndrome, characterized by a range of aberrant phenotypes including severe intellectual disability, absence of speech, delayed cognitive and motor development, and dysmorphic features. Moreover, polymorphisms in TCF4 have been associated with schizophrenia and other psychiatric and neurological conditions. Details about how TCF4 genetic variants are linked to these diseases and the role of TCF4 during neural development are only now beginning to emerge. Here, we provide a comprehensive review of the functions of TCF4 and its protein products at both the cellular and organismic levels, as well as a description of pathophysiological mechanisms associated with this gene.

Fabio Papes

Department of Genetics,
Evolution,
Microbiology and Immunology,
Institute of Biology,
Department of Genetics, Evolution, Microbiology and Immunology, Institute of Biology

University of Campinas

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

Fabio Papes

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Genetics,Evolution,Microbiology and Immunology,Institute of Biology,Department of Genetics, Evolution, Microbiology and Immunology, Institute of Biology

University of Campinas

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

Department of Genetics,
Evolution,
Microbiology and Immunology,
Institute of Biology,
Department of Genetics, Evolution, Microbiology and Immunology, Institute of Biology