Tracing of mono- and polysynaptic afferent connections between the main olfactory bulb and higher-order brain regions in the mouse.

Tracing of mono- and polysynaptic afferent connections between the main olfactory bulb and higher-order brain regions in the mouse Processing of odors in the main olfactory bulb (MOB) is modulated by higher brain afferents depending on the internal state, motivation, memory and emotions. For example satiety or hunger are known to change the perception of food odors. To shed light on this modulation a greater understanding of the underlying circuitry is required. To this aim we conducted tracing experiments in mice. First, stereotaxic injections of monosynaptic retrograde DiI and choleratoxin subunit B (CTb) into the dorsal olfactory bulb (focusing on the granular layer) were performed. In a second approach, we injected the pseudorabies virus 152 (PRV152) (kindly provided by Prof L. Enquist; Princeton University). As a neuronal tracer, this neurotropic virus can spread in synaptically connected neurons, dissecting the entire circuitry. The temporal analysis of the viral distribution allows to determine the number of synapses crossed. Both DiI and CTb confirm all the main centrifugal afferents to the MOB which were previously described in the literature. Thus, except the olfactory tubercle, all regions belonging to the olfactory primary cortex were labeled. Moreover, in the piriform cortex, labeling was mainly located in its dorsal part, confirming the topographical anatomical organization of cortico-bulbar projections. Direct projections arising from orexinergic neurons in the lateral hypothalamus were also observed. Regarding polysynaptic tracing, mice were sacrificed one, two and three days after PRV152 injections. After one day, only some of the direct neuromodulatory afferents were labelled. Thus, the locus coeruleus, which has a very caudal location in the brain close to the forth ventricle, showed already staining. In contrast, other neuromodulatory afferents (arising from raphe nuclei, ventral tegmental area, basal forebrain) were not labeled at this stage. Two days after injection, comparison with DiI/CTb results indicate that all the first-order connections were labeled. At this stage, second-order projections started to appear (for example, the midline thalamic nuclei as reuniens and rhomboid nuclei). Lastly, after three days, an extensive brain labeling occurred although some brain regions still showed a lack of staining. In conclusion, using DiI/CTb tracers that do not cross synapses allow to stain all primary afferents. In contrast, viral tracer migration depends on timing as well as number of connecting synapses. Thus, labeling occurring already one day after injection indicates a strong connection. Accordingly, our results suggest that the stained cells in the locus coeruleus send strong projections to the MOB. Furthermore this method allows to follow up the circuits involved in olfactory modulation. Data analysis of all the other labeled regions, focusing on hypothalamic nuclei and brain areas involved in arousal and food intake, are in progress.