Leonardo Belluscio

Mice Deficient in Golf Are Anosmic

Abstract We have used gene targeting to examine the role of the Gα subunit, G olf , in olfactory signal transduction. Mice homozygous for a null mutation in G olf show a striking reduction in the electrophysiological response of primary olfactory sensory neurons to a wide variety of odors. Despite this profound diminution in response to odors, the topographic map of primary sensory projections to the olfactory bulb remains unaltered in G olf mutants. Greater than 75% of the G olf mutant mice are unable to nurse and die within 2 days after birth. Rare surviving homozygotes mate and are fertile, but mutant females exhibit inadequate maternal behaviors. Surviving homozygous mutant mice also exhibit hyperactive behaviors. These behavioral phenotypes, taken together with the patterns of G olf expression, suggest that G olf is required for olfactory signal transduction and may also function as an essential signaling molecule more centrally in the brain.

A Map of Pheromone Receptor Activation in the Mammalian Brain

In mammals, the detection of pheromones is mediated by the vomeronasal system. We have employed gene targeting to visualize the pattern of projections of axons from vomeronasal sensory neurons in the accessory olfactory bulb. Neurons expressing a specific receptor project to multiple glomeruli that reside within spatially restricted domains. The formation of this sensory map in the accessory olfactory bulb and the survival of vomeronasal organ sensory neurons require the expression of pheromone receptors. In addition, we observe individual glomeruli in the accessory olfactory bulb that receive input from more than one type of sensory neuron. These observations indicate that the organization of the vomeronasal sensory afferents is dramatically different from that of the main olfactory system, and these differences have important implications for the logic of olfactory coding in the vomeronasal organ.

Odorant receptors instruct functional circuitry in the mouse olfactory bulb

The mammalian olfactory system detects and discriminates thousands of odorants using many different receptors expressed by sensory neurons in the nasal epithelium. Axonal projections from these neurons to the main olfactory bulbs form reproducible patterns of glomeruli in two widely separated regions of each bulb, creating two mirror-symmetric maps of odorant receptor projections. To investigate whether odorant receptors organize neural circuitry in the olfactory bulb, we have examined a genetically modified mouse line, rI7 → M71, in which a functionally characterized receptor, rI7, has been substituted into the M71 receptor locus. Here we show that despite their ectopic location the resulting glomeruli are responsive to known ligands of the rI7 receptor, attract postsynaptic innervation by mitral/tufted cell dendrites, and endow these cells with responses that are characteristic of the rI7 receptor. External tufted cells receiving input from rI7 → M71 glomeruli form precise intrabulbar projections that link medial and lateral rI7 → M71 glomeruli anatomically, thus providing a substrate for coordinating isofunctional glomeruli. We conclude that odorant receptor identity in epithelial neurons determines not only glomerular convergence and function, but also functional circuitry in the olfactory bulb.

Activity-dependent plasticity in the olfactory intrabulbar map.

In mammals, each olfactory bulb contains two mirror-symmetric glomerular maps. Isofunctional glomeruli within each bulb are specifically linked through a set of reciprocal intrabulbar projections (IBPs) to form an intrabulbar map. We injected neural tracers into the glomerular layer on one side of the bulb and examined the resulting projection on the opposite side. In adult mice, the size of the projection tuft is directly proportional to the size of the injected region. Using this ratio as a measure of IBP maturity, we find an immature 5:1 projection to injection ratio at 1 week of age that gradually refines to a mature 1:1 by 7 weeks. Moreover, whereas the glomerular map is able to form despite the elimination of odorant-induced activity, the intrabulbar map shows clear activity dependence for its precise formation. Here we show through experiments with both naris-occluded and anosmic mice that odorant-induced activity is not required to establish IBPs but is crucial for projection refinement. In contrast, increased glomerular activation through exposure to distinct odorants during map development can accelerate the refinement of projections associated with the activated glomeruli. These findings illustrate a clear role for odorant-induced activity in shaping the internal circuitry of the bulb. Interestingly, activity deprivation can alter the organization of both the developing and the mature map to the same degree, demonstrating that intrabulbar map plasticity is maintained into adulthood with no discernible critical period.

Continuous Neural Plasticity in the Olfactory Intrabulbar Circuitry

In the mammalian brain each olfactory bulb contains two mirror-symmetric glomerular maps linked through a set of reciprocal intrabulbar projections. These projections connect isofunctional odor columns through synapses in the internal plexiform layer (IPL) to produce an intrabulbar map. Developmental studies show that initially intrabulbar projections broadly target the IPL on the opposite side of the bulb and refine postnatally to their adult precision by 7 weeks of age in an activity-dependent manner (Marks et al., 2006). In this study, we sought to determine the capacity of intrabulbar map to recover its precision after disruption. Using reversible naris closure in both juvenile and adult mice, we distorted the intrabulbar map and then removed the blocks for varying survival periods. Our results reveal that returning normal olfactory experience can indeed drive the re-refinement of intrabulbar projections but requires 9 weeks. Since activity also affects olfactory sensory neurons (OSNs) (Suh et al., 2006), we further examined the consequence of activity deprivation on P2-expressing OSNs and their associated glomeruli. Our findings indicate that while naris closure caused a marked decrease in P2-OSN number and P2-glomerular volume, axonal convergence was not lost and both were quickly restored within 3 weeks. By contrast, synaptic contacts within the IPL also decreased with sensory deprivation but required at least 6 weeks to recover. Thus, we conclude that recovery of the glomerular map precedes and likely drives the refinement of the intrabulbar map while IPL contacts recover gradually, possibly setting the pace for intrabulbar circuit restoration.

Intrabulbar projecting external tufted cells mediate a timing-based mechanism that dynamically gates olfactory bulb output.

In the mammalian olfactory system, intrabulbar projections (IBPs) mediated by a class of external tufted cells (ET cells) specifically link isofunctional odor columns within the same olfactory bulb. To study the function of these ET cells within the glomerular network, we developed a “hemibulb” preparation that maintains IBPs intact enabling the select activation of ET cells associated with specific glomeruli. Using P2-GFP mice, a line in which the P2 glomeruli are labeled with green fluorescent protein, we recorded from P2 mitral cells (MT cells) while selectively stimulating P2 ET cells. Here, we show that ET-cell activity evokes a slow modulatory (SM) potential within MT cells, which is mediated by the glomerular network and consists of both excitatory and inhibitory components. Interestingly, the timing of the SM potential with respect to olfactory nerve (ON) stimulation can produce converse effects on MT-cell output. When ET-cell activity precedes ON stimulation, the MT-cell response is potentiated; however, when ET-cell activity follows ON stimulation, the MT-cell response is inhibited. Thus, intrabulbar projecting ET cells can shape olfactory bulb output through intraglomerular modulation of MT cells.

Early Expression of Odorant Receptors Distorts the Olfactory Circuitry

The odor response properties of a mammalian olfactory sensory neuron (OSN) are determined by the tightly regulated expression of a single member of a very large family of odorant receptors (ORs). The OR also plays an important role in focusing the central projections of all OSNs expressing that particular receptor to a pair of stereotypic locations (glomeruli) in each olfactory bulb (OB), thus creating a spatial map of odor responses in the brain. Here we show that when initiated late in neural development, transgenic expression of one OR in almost all OSNs has little influence on the architecture of the OB in mice. In contrast, early OR–transgene expression (mediated by the Gγ8-promoter) in 50–70% of OSNs grossly distorts the morphology of glomeruli and alters the projection patterns of many residual OSNs not expressing the transgene. Interestingly, this disruption of targeting persists in adult animals despite the downregulation of Gγ8 and transgenic OR expression that occurs as olfactory neurogenesis declines. Indeed, functional imaging studies reveal a dramatic decrease in the complexity of responses to odorants in adult Gγ8-transgenic OR mice. Thus, we show that initiation of transgenic OR expression early in the development of OSNs, rather than just the extent of transgene expression, determines its effectiveness at modifying OB anatomy and function. Together, these data imply that OR-expression timing needs to be very tightly controlled to achieve the precise wiring and function of the mammalian olfactory system.

Charting Plasticity in the Regenerating Maps of the Mammalian Olfactory Bulb

The anatomical organization of a neural system can offer a glimpse into its functional logic. The basic premise is that by understanding how something is put together one can figure out how it works. Unfortunately, organization is not always represented purely at an anatomical level and is sometimes best revealed through molecular or functional studies. The mammalian olfactory system exhibits organizational features at all these levels including 1) anatomically distinct structural layers in the olfactory bulb, 2) molecular maps based upon odorant receptor expression, and 3) functional local circuits giving rise to odor columns that provide a contextual logic for an intrabulbar map. In addition, various forms of cellular plasticity have been shown to play an integral role in shaping the structural properties of most neural systems and must be considered when assessing each systems anatomical organization. Interestingly, the olfactory system invokes an added level of complexity for understanding organization in that it regenerates both at the peripheral and the central levels. Thus, olfaction offers a rare opportunity to study both the structural and the functional properties of a regenerating sensory system in direct response to environmental stimuli. In this review, we discuss neural organization in the form of maps and explore the relationship between regeneration and plasticity. NEUROSCIENTIST 14(3):251–263, 2008. DOI: 10.1177/1073858408315026

Fas-Associated Factor 1 as a Regulator of Olfactory Axon Guidance

Axon guidance is a crucial part of neural circuit formation. While precise axonal targeting forms the basis of accurate information delivery, the mechanisms that regulate this process are still unclear. Apoptotic signaling molecules have been identified in the axon terminal, but their specific role in axon guidance is not well understood. Here we use the mouse olfactory system as an in vivo model to demonstrate that by modulating Fas-associated factor 1 (FAF1), an apoptosis regulatory molecule, we can rewire axonal projections. Interestingly, FAF1 is highly expressed in the developing mouse olfactory system, but its expression is downregulated postnatally. Using a tetracycline-inducible promoter Tet-Off system, we generated transgenic mice in which FAF1 is specifically expressed in immature olfactory sensory neurons (OSNs) and show that overexpression of FAF1 not only misroutes OSN axons to deep layers of the olfactory bulb but also leads to widespread disruption of the glomerular layer. In addition, we also demonstrate that the specific convergence of P2 receptor OSN axons is completely distorted in the FAF1 mice. Strikingly, all of the mutant phenotypes can be recovered by shutting down FAF1 expression through the administration of doxycycline. Together, our study provides clear in vivo evidence that an apoptotic molecule can indeed regulate axon targeting and that OSNs can restore their organization even after broad disruption.

MouSensor: A Versatile Genetic Platform to Create Super Sniffer Mice for Studying Human Odor Coding

Typically, ∼0.1% of the total number of olfactory sensory neurons (OSNs) in the main olfactory epithelium express the same odorant receptor (OR) in a singular fashion and their axons coalesce into homotypic glomeruli in the olfactory bulb. Here, we have dramatically increased the total number of OSNs expressing specific cloned OR coding sequences by multimerizing a 21-bp sequence encompassing the predicted homeodomain binding site sequence, TAATGA, known to be essential in OR gene choice.xa0Singular gene choice is maintained in these MouSensors. Inxa0vivo synaptopHluorin imaging of odor-induced responses by known M71 ligands shows functional glomerular activation in an M71 MouSensor. Moreover, a behavioral avoidance task demonstrates that specific odor detection thresholds are significantly decreased in multiple transgenic lines, expressing mouse or human ORs. We have developed a versatile platform to study gene choice and axon identity, to create biosensors with great translational potential, and to finally decode human olfaction.