Dong-Jing Zou

A G protein/cAMP signal cascade is required for axonal convergence into olfactory glomeruli

The mammalian odorant receptors (ORs) comprise a large family of G protein-coupled receptors that are critical determinants of both the odorant response profile and the axonal identity of the olfactory sensory neurons in which they are expressed. Although the pathway by which ORs activate odor transduction is well established, the mechanism by which they direct axons into proper glomerular relationships remains unknown. We have developed a gain-of-function approach by using injection of retroviral vectors into the embryonic olfactory epithelium to study the ORs′ contribution to axon guidance. By ectopically expressing ORs, we demonstrate that functional OR proteins induce axonal coalescence. Furthermore, ectopic expression of Gα mutants reveals that activation of the signal transduction cascade is sufficient to cause axonal convergence into glomeruli. Analysis of Gα subunit expression indicates that development and odorant transduction use separate transduction pathways. Last, we establish that the generation of cAMP through adenylyl cyclase 3 is necessary to establish proper axonal identity. Our data point to a model in which axonal sorting is accomplished by OR stimulation of cAMP production by coupling to Gαs.

Absence of Adenylyl Cyclase 3 Perturbs Peripheral Olfactory Projections in Mice

A remarkable feature of peripheral olfactory projections in mammals is the convergence of axons from olfactory sensory neurons (OSNs) expressing the same odorant receptor (OR) into the same glomeruli. There is mounting evidence that the ORs play critical roles in glomerular formation. However, it remains unclear how the OR exerts its function of sorting axons into homogeneity. We and others have shown previously that activation of the G-protein/cAMP signaling cascade underlies glomerular formation. Here, we further investigated whether establishment of the mature glomerular array requires adenylyl cyclase 3 (AC3), a key component of the OR-mediated cAMP-dependent signaling cascade. We found robust AC3 expression in both OSN cilia and axons during the period of active glomerular formation in neonatal mice. Examination of OR-tagged mice in an AC3 knock-out background revealed that the absence of AC3 drastically and differentially perturbed the formation of several representative glomeruli. Furthermore, heterogeneous glomeruli innervated by axons of multiple OSN populations persisted in such mice well into adulthood. In addition, reproducible aberrations in axonal projections in AC3−/− mice appeared to correlate with the activation of specific OR loci, regardless of the expressed receptor sequence, suggesting that OR expression is but one factor in determining OSN axonal projections. Together, our results indicate that cAMP signaling is critical for axonal sorting and the establishment of axonal identity.

Selective Gene Expression by Postnatal Electroporation during Olfactory Interneuron Neurogenesis

Neurogenesis persists in the olfactory system throughout life. The mechanisms of how new neurons are generated, how they integrate into circuits, and their role in coding remain mysteries. Here we report a technique that will greatly facilitate research into these questions. We found that electroporation can be used to robustly and selectively label progenitors in the Subventicular Zone. The approach was performed postnatally, without surgery, and with near 100% success rates. Labeling was found in all classes of interneurons in the olfactory bulb, persisted to adulthood and had no adverse effects. The broad utility of electroporation was demonstrated by encoding a calcium sensor and markers of intracellular organelles. The approach was found to be effective in wildtype and transgenic mice as well as rats. Given its versatility, robustness, and both time and cost effectiveness, this method offers a powerful new way to use genetic manipulation to understand adult neurogenesis.

Expression pattern of αCaMKII in the mouse main olfactory bulb

Multifunctional Ca2+/calmodulin‐dependent protein kinase II (CaMKII) is highly enriched at synapses and has been implicated in regulating the formation and function of several sensory systems, including the visual and the somatosensory systems. Although there is evidence for CaMKII expression in the olfactory system, the cellular localization of CaMKII has not been well studied and its function remains unknown. In this study, we examined the normal expression patterns of the predominant αCaMKII in the mouse main olfactory bulb. We showed that αCaMKII expression levels were high in the olfactory bulb and were developmentally regulated. Immunoreactivity to αCaMKII was heavy in the external plexiform layer and the granule cell layer but minimal in the olfactory nerve layer and the glomerular layer. At the cellular level, αCaMKII was selectively expressed in the γ‐aminobutyric acid (GABA)ergic granule cells but not in the GABAergic periglomerular cells. Unexpectedly, αCaMKII was not detected in the glutamatergic mitral/tufted cells. At the ultrastructural level, αCaMKII immunoreactivity was positive in granule cell spines and dendrites, but negative in mitral/tufted cell dendrites. In contrast, in the piriform cortex, as in the majority of cortical regions, αCaMKII was expressed in the glutamatergic neurons but not in the GABAergic neurons. Our results set the stage for ongoing investigations of the roles of CaMKII in the formation and function of the olfactory system. J. Comp. Neurol. 443:226–236, 2002.

Axon fasciculation in the developing olfactory nerve

Olfactory sensory neuron (OSN) axons exit the olfactory epithelium (OE) and extend toward the olfactory bulb (OB) where they coalesce into glomeruli. Each OSN expresses only 1 of approximately 1,200 odor receptors (ORs). OSNs expressing the same OR are distributed in restricted zones of the OE. However, within a zone, the OSNs expressing a specific OR are not contiguous - distribution appears stochastic. Upon reaching the OB the OSN axons expressing the same OR reproducibly coalesce into two to three glomeruli. While ORs appear necessary for appropriate convergence of axons, a variety of adhesion associated molecules and activity-dependent mechanisms are also implicated. Recent data suggest pre-target OSN axon sorting may influence glomerular convergence. Here, using regional and OR-specific markers, we addressed the spatio-temporal properties associated with the onset of homotypic fasciculation in embryonic mice and assessed the degree to which subpopulations of axons remain segregated as they extend toward the nascent OB. We show that immediately upon crossing the basal lamina, axons uniformly turn sharply, usually at an approximately 90° angle toward the OB. Molecularly defined subpopulations of axons show evidence of spatial segregation within the nascent nerve by embryonic day 12, within 48 hours of the first OSN axons crossing the basal lamina, but at least 72 hours before synapse formation in the developing OB. Homotypic fasciculation of OSN axons expressing the same OR appears to be a hierarchical process. While regional segregation occurs in the mesenchyme, the final convergence of OR-specific subpopulations does not occur until the axons reach the inner nerve layer of the OB.

Nonsensory target-dependent organization of piriform cortex

Significance The mammalian olfactory system is capable of detecting and discriminating a vast and diverse array of small organic molecules or odorants. Complex blends of these chemicals are finally perceived as a unified odor object—for example, a rose contains dozens of active compounds. The piriform cortex (PCX) is the largest component of the olfactory cortex and has been hypothesized to be the locus of odor object formation. However, the PCX shows a distributed odorant representation that contrasts sharply with the topographical representation typically seen in the other primary sensory cortices. In this article, we explore an alternative organizational principle for the PCX based on where neurons are sending their output, rather than where these neurons are receiving their input. The piriform cortex (PCX) is the largest component of the olfactory cortex and is hypothesized to be the locus of odor object formation. The distributed odorant representation found in PCX contrasts sharply with the topographical representation seen in other primary sensory cortices, making it difficult to test this view. Recent work in PCX has focused on functional characteristics of these distributed afferent and association fiber systems. However, information regarding the efferent projections of PCX and how those may be involved in odor representation and object recognition has been largely ignored. To investigate this aspect of PCX, we have used the efferent pathway from mouse PCX to the orbitofrontal cortex (OFC). Using double fluorescent retrograde tracing, we identified the output neurons (OPNs) of the PCX that project to two subdivisions of the OFC, the agranular insula and the lateral orbitofrontal cortex (AI-OPNs and LO-OPNs, respectively). We found that both AI-OPNs and LO-OPNs showed a distinct spatial topography within the PCX and fewer than 10% projected to both the AI and the LO as judged by double-labeling. These data revealed that the efferent component of the PCX may be topographically organized. Further, these data suggest a model for functional organization of the PCX in which the OPNs are grouped into parallel output circuits that provide olfactory information to different higher centers. The distributed afferent input from the olfactory bulb and the local PCX association circuits would then ensure a complete olfactory representation, pattern recognition capability, and neuroplasticity in each efferent circuit.

Sequential onset of presynaptic molecules during olfactory sensory neuron maturation

Differentiated olfactory sensory neurons express specific presynaptic proteins, including enzymes involved in neurotransmitter transport and proteins involved in the trafficking and release of synaptic vesicles. Studying the regulation of these presynaptic proteins will help to elucidate the presynaptic differentiation process that ultimately leads to synapse formation. It has been postulated that the formation of a synapse between the axons of the sensory neurons and the dendrites of second order neurons in the olfactory bulb is a critical step in the processes of sensory neuron maturation. One approach to study the relationship between synaptogenesis and sensory neuron maturation is to examine the expression patterns of synaptic molecules through the olfactory neuron lineage. To this end we designed specific in situ hybridization probes to target messengers for proteins involved in presynaptic vesicle release. Our findings show that, as they mature, mouse olfactory neurons sequentially express specific presynaptic genes. Furthermore, the different patterns of expression of these presynaptic genes suggest the existence of discrete steps in presynaptic development: genes encoding proteins involved in scaffolding show an early onset of expression, whereas expression of genes encoding proteins involved in the regulation of vesicle release starts later. In particular, the signature molecule for glutamatergic neurons vesicle glutamate transporter 2 shows the latest onset of expression. In addition, contact with the targets in the olfactory bulb is not controlling presynaptic protein gene expression, suggesting that olfactory sensory neurons follow an intrinsic program of development. J. Comp. Neurol. 516:187–198, 2009.

Exuberant growth and synapse formation of olfactory sensory neuron axonal arborizations

Neural connections in the adult nervous system are established with a high degree of precision. Several examples throughout the nervous system indicate that this precision is achieved by first establishing an initial exuberant immature pattern of connectivity that is then sculpted into the adult pattern via pruning. This often emerges as an activity‐dependent process. In the olfactory system, sensory axons from neurons expressing the same odorant receptor project with high precision to specific glomerular structures in the olfactory bulb. This process undergoes maturation‐dependent refinements that are not fully understood. Due to technical impediments that have made it difficult to focus on single axons, it is unknown whether olfactory sensory projections are established in an exuberant fashion. Here we developed a novel technique of electroporation that allowed us to simultaneously label single olfactory sensory neuron (OSN) axonal arbors and their presynaptic specializations. Using this method we were able to incorporate plasmids into OSNs at an immature stage, thereby allowing a time‐course study of axonal arbor development and synapse formation in single olfactory sensory axons. We observed that the number of branch points, the total branch length, and the number and density of presynaptic specializations peaked at postnatal day 8 and decreased afterwards. Our data demonstrate that olfactory sensory axons develop in an exuberant way, both in terms of branch growth and synaptic composition. We hypothesize that exuberant branches and synapses are eliminated to achieve the mature pattern in a process likely to be regulated by neural activity. J. Comp. Neurol.519:3713‐3726, 2011.

Applying medicinal chemistry strategies to understand odorant discrimination

Associating an odorants chemical structure with its percept is a long-standing challenge. One hindrance may come from the adoption of the organic chemistry scheme of molecular description and classification. Chemists classify molecules according to characteristics that are useful in synthesis or isolation, but which may be of little importance to a biological sensory system. Accordingly, we look to medicinal chemistry, which emphasizes biological function over chemical form, in an attempt to discern which among the many molecular features are most important for odour discrimination. Here we use medicinal chemistry concepts to assemble a panel of molecules to test how heteroaromatic ring substitution of the benzene ring will change the odour percept of acetophenone. This work allows us to describe an extensive rule in odorant detection by mammalian olfactory receptors. Whereas organic chemistry would have predicted the ring size and composition to be key features, our work reveals that the topological polar surface area is the key feature for the discrimination of these odorants.

Functional odor classification through a medicinal chemistry approach

Mechanistic approaches provide alternative solutions to in silico analyses of odorant molecules’ odor-structure relationships. Crucial for any hypothesis about odor coding is the classification and prediction of sensory qualities in chemical compounds. The relationship between perceptual quality and molecular structure has occupied olfactory scientists throughout the 20th century, but details of the mechanism remain elusive. Odor molecules are typically organic compounds of low molecular weight that may be aliphatic or aromatic, may be saturated or unsaturated, and may have diverse functional polar groups. However, many molecules conforming to these characteristics are odorless. One approach recently used to solve this problem was to apply machine learning strategies to a large set of odors and human classifiers in an attempt to find common and unique chemical features that would predict a chemical’s odor. We use an alternative method that relies more on the biological responses of olfactory sensory neurons and then applies the principles of medicinal chemistry, a technique widely used in drug discovery. We demonstrate the effectiveness of this strategy through a classification for esters, an important odorant for the creation of flavor in wine. Our findings indicate that computational approaches that do not account for biological responses will be plagued by both false positives and false negatives and fail to provide meaningful mechanistic data. However, the two approaches used in tandem could resolve many of the paradoxes in odor perception.