ER stress transforms random olfactory receptor choice into axon targeting precision.

Olfactory sensory neurons (OSNs) convert the stochastic choice of one of >1,000 olfactory receptor (OR) genes into precise and stereotyped axon targeting of OR-specific glomeruli in the olfactory bulb. Here, we show that the PERK arm of the unfolded protein response (UPR) regulates both the glomerular coalescence of like axons and the specificity of their projections. Subtle differences in OR protein sequences lead to distinct patterns of endoplasmic reticulum (ER) stress during OSN development, converting OR identity into distinct gene expression signatures. We identify the transcription factor Ddit3 as a key effector of PERK signaling that maps OR-dependent ER stress patterns to the transcriptional regulation of axon guidance and cell-adhesion genes, instructing targeting precision. Our results extend the known functions of the UPR from a quality-control pathway that protects cells from misfolded proteins to a sensor of cellular identity that interprets physiological states to direct axon wiring.

Maturation arrest in early postnatal sensory receptors by deletion of the miR-183/96/182 cluster in mouse.

The polycistronic miR-183/96/182 cluster is preferentially and abundantly expressed in terminally differentiating sensory epithelia. To clarify its roles in the terminal differentiation of sensory receptors in vivo, we deleted the entire gene cluster in mouse germline through homologous recombination. The miR-183/96/182 null mice display impairment of the visual, auditory, vestibular, and olfactory systems, attributable to profound defects in sensory receptor terminal differentiation. Maturation of sensory receptor precursors is delayed, and they never attain a fully differentiated state. In the retina, delay in up-regulation of key photoreceptor genes underlies delayed outer segment elongation and possibly mispositioning of cone nuclei in the retina. Incomplete maturation of photoreceptors is followed shortly afterward by early-onset degeneration. Cell biologic and transcriptome analyses implicate dysregulation of ciliogenesis, nuclear translocation, and an epigenetic mechanism that may control timing of terminal differentiation in developing photoreceptors. In both the organ of Corti and the vestibular organ, impaired terminal differentiation manifests as immature stereocilia and kinocilia on the apical surface of hair cells. Our study thus establishes a dedicated role of the miR-183/96/182 cluster in driving the terminal differentiation of multiple sensory receptor cells.

Magnetic resonance imaging of odorant activity-dependent migration of neural precursor cells and olfactory bulb growth.

Neural progenitors or neuroblasts are produced by precursor cells in the subventricular zone (SVZ) and migrate along the rostral migratory stream (RMS) to the olfactory bulbs (OB) throughout life. In the OB, these adult born neurons either die or replace existing olfactory interneurons, playing a critical role in the stabilization of OB circuitry. Although several aspects of the addition of new neurons into the OB have been studied, it is unclear whether long-distance activity from the OB can regulate the influx of migrating neuroblasts along the RMS. In this study, iron oxide-assisted MRI was used to track the migration of neuroblasts in combination with reversible naris occlusion to manipulate odorant-induced activity. It was found that decreasing olfactory activity led to a decrease in the rate of neuroblast migration along the RMS. Removal of the naris occlusion led to an increase in migratory rate back to control levels, indicating that olfactory activity has regulatory function on neuroblast migration in the RMS. Blocking odorant activity also led to an arrest in OB growth and re-opening the block led to a rapid re-growth returning the bulb size to control levels. Furthermore, pharmacogenetic elimination of the neuroblasts demonstrated that they were required for re-growth of the bulb following sensory deprivation. Together, these results show that sensory activity, neural migration and OB growth are tightly coupled in an interdependent manner.

Adult neurogenesis is necessary to refine and maintain circuit specificity.

The circuitry of the olfactory bulb contains a precise anatomical map that links isofunctional regions within each olfactory bulb. This intrabulbar map forms perinatally and undergoes activity-dependent refinement during the first postnatal weeks. Although this map retains its plasticity throughout adulthood, its organization is remarkably stable despite the addition of millions of new neurons to this circuit. Here we show that the continuous supply of new neuroblasts from the subventricular zone is necessary for both the restoration and maintenance of this precise central circuit. Using pharmacogenetic methods to conditionally ablate adult neurogenesis in transgenic mice, we find that the influx of neuroblasts is required for recovery of intrabulbar map precision after disruption due to sensory block. We further demonstrate that eliminating adult-born interneurons in naive animals leads to an expansion of tufted cell axons that is identical to the changes caused by sensory block, thus revealing an essential role for new neurons in circuit maintenance under baseline conditions. These findings show, for the first time, that inhibiting adult neurogenesis alters the circuitry of projection neurons in brain regions that receive new interneurons and points to a critical role for adult-born neurons in stabilizing a brain circuit that exhibits high levels of plasticity.

Laminar specific detection of APP induced neurodegeneration and recovery using MEMRI in an olfactory based Alzheimer's disease mouse model.

Manganese enhanced MRI (MEMRI) was used to detect specific laminar changes in the olfactory bulb (OB) to follow the progression of amyloid precursor protein (APP)-induced neuronal pathology and its recovery in a reversible olfactory based Alzheimer's disease (AD) mouse model. Olfactory dysfunction is an early symptom of AD, which suggests that olfactory sensory neurons (OSNs) may be more sensitive to AD related factors than neurons in other brain areas. Previously a transgenic mouse model was established that causes degeneration of OSNs by overexpressing humanized APP (hAPP), which results in a disruption of the olfactory circuitry with changes in the glomerular structure. In the present work, OB volume and manganese enhancement of the glomerular layer in the OB were decreased in mutant mice. Turning off APP overexpression with doxycycline produced a significant increase in manganese enhancement of the glomerular layer after only 1week, and further recovery after 3weeks, while treatment with Aß antibody produced modest improvement with MRI measurements. Thus, MEMRI enables a direct tracking of laminar specific neurodegeneration through a non-invasive in vivo measurement. The use of MRI will enable assessment of the ability of different pharmacological reagents to block olfactory neuronal loss and can serve as a unique in vivo screening tool to both identify potential therapeutics and test their efficacy.

Olfactory functions scale with circuit restoration in a rapidly reversible Alzheimer's disease model.

Neural circuits maintain a precise organization that is vital for normal brain functions and behaviors, but become disrupted during neurological disease. Understanding the connection between wiring accuracy and function to measure disease progression or recovery has been difficult because of the complexity of behavioral circuits. The olfactory system maintains well-defined neural connections that regenerate throughout life. We previously established a reversible in vivo model of Alzheimer's disease by overexpressing a humanized mutated amyloid precursor protein (hAPP) in olfactory sensory neurons (OSNs). Using this model, we currently show that hAPP is present in the OSN axons of mutant mice, which exhibit strong caspase3 signal and reduced synaptic protein expression by 3 weeks of age. In the olfactory bulb, we show that glomerular structure is distorted and OSN axonal convergence is lost. In vivo functional imaging experiments further demonstrate disruption of the glomerular circuitry, and behavioral assays reveal that olfactory function is significantly impaired. Because OSNs regenerate, we also tested if the system could recover from hAPP-induced disruption. We found that after 1 or 3 weeks of shutting-off hAPP expression, the glomerular circuit was partially restored both anatomically and functionally, with behavioral deficits similarly reversed. Interestingly, the degree of functional recovery tracked directly with circuit restoration. Together, these data demonstrate that hAPP-induced circuit disruption and subsequent recovery can occur rapidly and that behavior can provide a measure of circuit organization. Thus, olfaction may serve as a useful biomarker to both follow disease progression and gauge potential recovery.

Critical role of GFRα1 in the development and function of the main olfactory system.

Glial cell line-derived neurotrophic factor (GDNF) and its receptor GFRα1 are prominently expressed in the olfactory epithelium (OE) and olfactory bulb (OB), but their importance for olfactory system development is completely unknown. We have investigated the consequences of GFRα1 deficiency for mouse olfactory system development and function. In the OE, GFRα1 was expressed in basal precursors, immature olfactory sensory neurons (OSNs), and olfactory ensheathing cells (OECs), but was excluded from mature OSNs. The OE of newborn Gfra1 knock-out mice was thinner and contained fewer OSNs, but more dividing precursors, suggesting deficient neurogenesis. Immature OSN axon bundles were enlarged and associated OECs increased, indicating impaired migration of OECs and OSN axons. In the OB, GFRα1 was expressed in immature OSN axons and OECs of the nerve layer, as well as mitral and tufted cells, but was excluded from GABAergic interneurons. In newborn knock-outs, the nerve layer was dramatically reduced, exhibiting fewer axons and OECs. Bulbs were smaller and presented fewer and disorganized glomeruli and a significant reduction in mitral cells. Numbers of tyrosine hydroxylase-, calbindin-, and calretinin-expressing interneurons were also reduced in newborn mice lacking Gfra1. At birth, the OE and OB of Gdnf knock-out mice displayed comparable phenotypes. Similar deficits were also found in adult heterozygous Gfra1(+/-) mutants, which in addition displayed diminished responses in behavioral tests of olfactory function. We conclude that GFRα1 is critical for the development and function of the main olfactory system, contributing to the development and allocation of all major classes of neurons and glial cells.

Odorant receptors in the formation of the olfactory bulb circuitry.

In mammals, smell is mediated by odorant receptors expressed by sensory neurons in the nose. These specialized receptors are found both on olfactory sensory neurons' cilia and axon terminals. Although the primary function of ciliary odorant receptors is to detect odorants, their axonal role remains unclear but is thought to involve axon guidance. This review discusses findings that show axonal odorant receptors are indeed functional and capable of modulating neural connectivity.

Progressive Spread of Beta-amyloid Pathology in an Olfactory-driven Amyloid Precursor Protein Mouse Model.

Years before Alzheimer's disease (AD) is diagnosed, patients experience an impaired sense of smell, and ß-amyloid plaques accumulate within the olfactory mucosa and olfactory bulb (OB). The olfactory vector hypothesis proposes that external agents cause ß-amyloid to aggregate and spread from the OB to connected downstream brain regions. To reproduce the slow accumulation of ß-amyloid that occurs in human AD, we investigated the progressive accumulation of ß-amyloid across the brain using a conditional mouse model that overexpresses a humanized mutant form of the amyloid precursor protein (hAPP) in olfactory sensory neurons. Using design-based stereology, we show the progressive accumulation of ß-amyloid plaques within the OB and cortical olfactory regions with age. We also observe reduced OB volumes in these mice when hAPP expression begins prior-to but not post-weaning which we tracked using manganese-enhanced MRI. We therefore conclude that the reduced OB volume does not represent progressive degeneration but rather disrupted OB development. Overall, our data demonstrate that hAPP expression in the olfactory epithelium can lead to the accumulation and spread of ß-amyloid through the olfactory system into the hippocampus, consistent with an olfactory system role in the early stages of ß-amyloid-related AD progression.

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.

In vivo olfactory model of APP-induced neurodegeneration reveals a reversible cell-autonomous function.

Amyloid precursor protein (APP) has long been linked to the neurodegeneration of Alzheimer's disease (AD), but the associated cell death has been difficult to capture in vivo, and the role of APP in effecting neuron loss is still unclear. Olfactory dysfunction is an early symptom of AD with amyloid pathology in the olfactory epithelium correlating well to the brain pathology of AD patients. As olfactory sensory neurons (OSNs) regenerate continuously with immature and mature OSNs coexisting in the same olfactory epithelium, we sought to use this unique system to study APP-induced neurodegeneration. Here we have developed an olfactory-based transgenic mouse model that overexpresses humanized APP containing familial AD mutations (hAPP) in either mature or immature OSNs, and found that despite the absence of extracellular plaques a striking number of apoptotic neurons were detected by 3 weeks of age. Importantly, apoptosis was restricted to the specific population overexpressing hAPP, either mature or immature OSNs, sparing those without hAPP. Interestingly, we observed that this widespread neurodegeneration could be rapidly rescued by reducing hAPP expression levels in immature neurons. Together, these data argue that overexpressing hAPP alone could induce cell-autonomous apoptosis in both mature and immature neurons, challenging the notion that amyloid plaques are necessary for neurodegeneration. Furthermore, we show that hAPP-induced neurodegeneration is reversible, suggesting that AD-related neural loss could potentially be rescued. Thus, we propose that this unique in vivo model will not only help determine the mechanisms underlying AD-related neurodegeneration but also serve as a platform to test possible treatments.

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.

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 Ggamma8-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 Ggamma8 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 Ggamma8-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.

The influence of stimulus duration on olfactory perception.

The duration of a stimulus plays an important role in the coding of sensory information. The role of stimulus duration is extensively studied in the tactile, visual, and auditory system. In the olfactory system, temporal properties of the stimulus are key for obtaining information when an odor is released in the environment. However, how the stimulus duration influences the odor perception is not well understood. To test this, we activated the olfactory bulbs with blue light in mice expressing channelrhodopsin in the olfactory sensory neurons (OSNs) and assessed the relevance of stimulus duration on olfactory perception using foot shock associated active avoidance behavioral task on a "two-arms maze". Our behavior data demonstrate that the stimulus duration plays an important role in olfactory perception and the associated behavioral responses.

Functional Relevance of Dual Olfactory Bulbs in Olfactory Coding.

Bilateral convergence of external stimuli is a common feature of vertebrate sensory systems. This convergence of inputs from the bilateral receptive fields allows higher order sensory perception, such as depth perception in the vertebrate visual system and stimulus localization in the auditory system. The functional role of such bilateral convergence in the olfactory system is unknown. To test whether each olfactory bulb (OB) contributes a separate piece of olfactory information, and whether information from the bilateral OB is integrated, we synchronized the activation of OBs with blue light in mice expressing ChIEF in the olfactory sensory neurons (OSNs) and behaviorally assessed the relevance of dual OBs in olfactory perception. Our findings suggest that each OB contributes separate components of olfactory information, and the mice integrate the bilaterally synchronized olfactory information for olfactory identity.

In vivo detection of individual glomeruli in the rodent olfactory bulb using manganese enhanced MRI.

MRI contrast based on relaxation times, proton density, or signal phase have been applied to delineate neural structures in the brain. However, neural units such as cortical layers and columns have been difficult to identify using these methods. Manganese ion delivered either systemically or injected directly has been shown to accumulate specifically within cellular areas of the brain enabling the differentiation of layers within the hippocampus, cortex, cerebellum, and olfactory bulb in vivo. Here we show the ability to detect individual olfactory glomeruli using manganese enhanced MRI (MEMRI). Glomeruli are anatomically distinct structures ( approximately 150 microm in diameter) on the surface of the olfactory bulb that represent the first processing units for olfactory sensory information. Following systemic delivery of MnCl(2) we used 3D-MRI with 50 microm isotropic resolution to detect discrete spots of increased signal intensity between 100 and 200 microm in diameter in the glomerular layer of the rat olfactory bulb. Inflow effects of arterial blood and susceptibility effects of venous blood were suppressed and were evaluated by comparing the location of vessels in the bulb to areas of manganese enhancement using iron oxide to increase vessel contrast. These potential vascular effects did not explain the contrast detected. Nissl staining of individual glomeruli were also compared to MEMRI images from the same animals clearly demonstrating that many of the manganese enhanced regions corresponded to individual olfactory glomeruli. Thus, MEMRI can be used as a non-invasive means to detect olfactory glomeruli for longitudinal studies looking at neural plasticity during olfactory development or possible degeneration associated with disease.

Olfactory experience accelerates glomerular refinement in the mammalian olfactory bulb.

Primary olfactory sensory neurons (OSNs) impart both molecular and functional organization to the olfactory bulb. Because OSNs can be selectively activated by odorants in vivo, we sought to determine whether odorant experience alters the cellular dynamics of specific OSNs or their axonal projections. Using mice, we found that odorant stimulation associated with behavioral conditioning influenced OSN wiring by accelerating glomerular refinement independent of OSN number; furthermore, this wiring was strongly associated with olfactory learning.

The unfolded protein response is activated in the olfactory system in Alzheimer's disease.

Olfactory dysfunction is an early and prevalent symptom of Alzheimer's disease (AD) and the olfactory bulb is a nexus of beta-amyloid plaque and tau neurofibrillary tangle (NFT) pathology during early AD progression. To mitigate the accumulation of misfolded proteins, an endoplasmic reticulum stress response called the unfolded protein response (UPR) occurs in the AD hippocampus. However, chronic UPR activation can lead to apoptosis and the upregulation of beta-amyloid and tau production. Therefore, UPR activation in the olfactory system could be one of the first changes in AD. In this study, we investigated whether two proteins that signal UPR activation are expressed in the olfactory system of AD cases with low or high amounts of aggregate pathology. We used immunohistochemistry to label two markers of UPR activation (p-PERK and p-eIF2α) concomitantly with neuronal markers (NeuN and PGP9.5) and pathology markers (beta-amyloid and tau) in the olfactory bulb, piriform cortex, entorhinal cortex and the CA1 region of the hippocampus in AD and normal cases. We show that UPR activation, as indicated by p-PERK and p-eIF2α expression, is significantly increased throughout the olfactory system in AD cases with low (Braak stage III-IV) and high-level (Braak stage V-VI) pathology. We further show that UPR activation occurs in the mitral cells and in the anterior olfactory nucleus of the olfactory bulb where tau and amyloid pathology is abundant. However, UPR activation is not present in neurons when they contain NFTs and only rarely occurs in neurons containing diffuse tau aggregates. We conclude that UPR activation is prevalent in all regions of the olfactory system and support previous findings suggesting that UPR activation likely precedes NFT formation. Our data indicate that chronic UPR activation in the olfactory system might contribute to the olfactory dysfunction that occurs early in the pathogenesis of AD.

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.

Manganese enhanced MRI reveals functional circuitry in response to odorant stimuli.

To investigate the circuitry involved in detecting odorants in the rodent brain, we developed a method using manganese-enhanced MRI (MEMRI) to map the flow of neural information from the olfactory sensory neurons (OSNs) to the central layers of the olfactory bulb. Studies have shown that Mn(2+) enters active neurons and is transported anterogradely to axon terminals where it can cross synapses to functionally trace neural networks. Thus, by delivering MnCl(2) directly into the nasal cavity of mice and then exposing them to defined odorants, Mn(2+) is preferentially taken up by activated OSNs. Using the time course of the MRI signal, we generated maps of Mn(2+) accumulation in the olfactory bulb for both glomerular and mitral cell layers. Results demonstrated that overlapping yet distinct enhancement patterns were produced by exposure to either octanal, acetophenone, or carvone. Notably, areas of Mn(2+) accumulation in the mitral cell layer were similar to those in the glomerular layer consistent with neural information that passes from specific OSNs to specific mitral cells. Finally, by correlating specific Mn(2+) signal peaks to genetically labeled glomeruli that are known to be activated by the odorant octanal, we show that MEMRI maps can be resolved at the level of individual glomeruli.