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Processing of olfactory cues in the brain

The question as to how the brain produces useful information from the electrical stimuli coming from the sensory organs is a key issue in the neurosciences. Researchers from Heidelberg are investigating how mice discriminate odours and have shown that complex behaviour emerges from the properties of cells and molecules. Using highly specific genetic manipulations for their experiments, the researchers have been able to show that inhibitory interneurons in the olfactory bulb play an important role in shaping the neuronal representation of odours.

In humans, the nose plays a less important role than the eyes and ears when it comes to processing sensory cues. It is perhaps the aesthetically most prominent structure on the human face. However, compared to other mammals, humans are relatively weak smellers. For the majority of animals, including most mammals, the sense of smell is the most important instrument for communicating with the environment; their sense of smell helps animals to find food as well as identify enemies and mating partners. In vertebrate brains, the rhinencephalon with the olfactory bulb, which is directly linked with the axons of the olfactory receptor neurons of the olfactory epithelium, is a vast, rather prominent part of the forebrain. In humans, the rhinencephalon is relatively small and almost completely covered by the cerebral cortex.

Lateral view of the brain of a shrew (A) and a human (B), both are drawn to the same scale. The olfactory brain (olfactory bulb and olfactory lobe) is labelled red, the acoustic centre blue and the visual centre black. © A.S. Romer: Vergleichende Anatomie der Wirbeltiere (1959); slightly modified by EJ

Many genes for all odours

The epithelium contains a large number of specialised nerve cells (humans have around 10 million, other animals a lot more) known as olfactory receptors, whose axons reach into the olfactory bulb. The odorant-sensitive tips of the olfactory receptors protrude into the nasal mucosa where they convert the chemical signals of odour molecules into electrical signals. The olfactory receptors belong to the large family of G protein coupled receptors (GPCRs) that sense molecules outside the cell and activate signal transduction pathways inside the cell.

Prof. Dr. Richard Axel was awarded the Nobel Prize in Physiology or Medicine in 2004. © Nobel Committee

The neurobiologists Richard Axel and Linda Buck from Columbia University in New York, who were awarded the Nobel Prize in Physiology or Medicine in 2004 for “their discoveries of odorant receptors and the organisation of the olfactory system”, identified around 1,300 mouse GPCR genes that give rise to an equivalent number of olfactory receptor types (or molecule class). This corresponds to about five per cent of all protein-coding genes in the mouse. Around 500 GPCR genes, with which thousands of different odours can be perceived, have been identified in humans. Even simple invertebrates such as the threadworm Caenorhabditis elegans (which only has 302 nerve cells, including 16 olfactory neurons) have as many as 1000 or so olfactory receptor genes.

The functional principle of the sense of smell is therefore quite simple: a large number of sensory neurons with receptors interact with a small number of specific olfactory molecules. The electrical signals that are generated are transported by the olfactory receptor neurons directly into the olfactory centre of the brain.
This leads to the question Richard Axel focused on in his Nobel Lecture: “How does the brain know what the nose is smelling?” This is one of the key questions dealt with by the neurosciences: how is a sensible picture translated into the brain from environmental stimuli to which the brain can react? In order to answer this question, it is crucial to understand the mechanisms and the connections of neurons in the brain. 


How mice discriminate odours

Dr. Andreas Schaefer, head of a group of researchers at the Max Planck Institute for Medical Research in Heidelberg, is concentrating on the question as to how olfactory stimuli are processed in the olfactory bulb. In 2010, he accepted the position of research professor in the field of neuroscience at the Institute of Anatomy and Cell Biology at Universität Heidelberg, where he established the “Functional Neuroanatomy of Behaviour” research group. Schaefer uses the mouse as model organism, which, in genetic terms, is the best known mammal.

Prof. Dr. Andreas Schaefer © MPImF

Using computer-based conditioning experiments in which the mice had learned to specifically associate odours with rewards, Schaefer and his team were able to measure how long the mice took to process the odour cues. This allowed the researchers to determine the time frame for their examinations of the olfactory system. They found that the mice only took around 0.25 seconds to discriminate perfectly between two monomolecular odours (amyl acetate with a banana-like smell and ethyl butyrate with a pineapple-like smell). The mice were also able to discriminate between mixtures of the same two substances that differed to a lesser extent (e.g. 40/60% against 60/40%), but they took slightly longer to do so. The researchers interpreted the results as an indication of the effects of the inhibitory neuron network in the olfactory bulb. 

The axons of the receptor neurons in the olfactory bulb are connected with so-called mitral cells by way of synapses. The mitral cells (projection neurons) relay the signals into many other brain structures, including the hippocampus, which is responsible with associative learning, and the amygdala, which plays a key role in processing emotion (e.g., fear). The transmission of the signals is inhibited by the large number of inhibitory granule cells found in the olfactory bulb. Granule cells are small multipolar neurons that form inhibitory synapses. It is assumed that such inhibitory connections are, amongst other things, required to deal with, select and suppress some of the large number of sensory impressions to which humans and animals are exposed.

Specific genetic manipulations to cancel the inhibitory circuitry of the olfactory bulb

In order to test their hypothesis, the researchers used transgenic mice with specific genetic alterations in the neurons of the olfactory bulb. The experiments were carried out in cooperation with Prof. Peter Seeburg and Dr. Rolf Sprengel (Max Planck Institute for Medical Research), Professor Thomas Kuner (Institute of Anatomy and Cell Biology at Universität Heidelberg) and Dr. Veronika Egger (now at the University of Munich). The researchers were unable to use the well-known knock-out technology which allows individual genes to be switched off in all of an animal’s cells. However, the technology might lead to widely varying phenotypes and make it difficult to infer the probable function of a gene under investigation. The scientists therefore used the “Cre-lox” technology, which is currently the best experimental technique to link genotypes with phenotypes. Cre-lox recombination enables researchers to irreversibly switch off a specific gene flanked by so-called loxP cleavage sites. The gene under investigation is then excised with the enzyme Cre recombinase (ed. note: Cre protein - encoded by the locus originally named as "causes recombination”).

How odors are perceived © A. Schaefer, MPImF

Cre recombinase is a bacteriophage enzyme whose gene was introduced into the granule cells in the centre of the olfactory bulb of young mice using adeno-associated viruses as gene shuttles. In the mouse genome, the gene coding for the glutamate receptor GluA2 was excised by way of the Cre recombinase loxP recognition sites that were introduced with genetic methods. The removal of this receptor led to the increased influx of calcium into the granule cells and as a result to the greater inhibition of the mitral cells. When the researchers analysed the transgenic mice’s ability to discriminate odours, they found no change in the learning of odours and odour memory compared to control animals. However, they discovered differences in the speed with which the animals were able to discriminate different odours. While the control and transgenic mice needed approximately the same time to differentiate between simple odours, the transgenic mice were much quicker than the control animals in discriminating very similar odour mixtures. The researchers observed the opposite in mice lacking the GluA1 receptor, which led to a reduced influx of calcium ions and lesser inhibition. Although these mice did not differ in their ability to learn and memorise the odours, they needed a lot more time to differentiate between similar odours.

In combination with other electrophysiological measurements, imaging methods and behaviour experiments, these experiments show that the inhibitory circuitry in the olfactory bulb is of crucial importance for the processing of olfactory cues. Within a limited area in the neurosciences, the researchers have thus been able to establish a link between molecules and behaviour as well as answer the question as to how the brain knows what the nose is smelling.

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