Adam C. Puche

Decoupling of erosion and precipitation in the Himalayas

Centre–surround inhibition—the suppression of activity of neighbouring cells by a central group of neurons—is a fundamental mechanism that increases contrast in patterned sensory processing. The initial stage of neural processing in olfaction occurs in olfactory bulb glomeruli, but evidence for functional interactions between glomeruli is fragmentary. Here we show that the so-called ‘short axon’ cells, contrary to their name, send interglomerular axons over long distances to form excitatory synapses with inhibitory periglomerular neurons up to 20–30 glomeruli away. Interglomerular excitation of these periglomerular cells potently inhibits mitral cells and forms an on-centre, off-surround circuit. This interglomerular centre–surround inhibitory network, along with the well-established mitral–granule–mitral inhibitory circuit, forms a serial, two-stage inhibitory circuit that could enhance spatiotemporal responses to odours.

Ultrasensitive pheromone detection by mammalian vomeronasal neurons.

The vomeronasal organ (VNO) is a chemoreceptive organ that is thought to transduce pheromones into electrical responses that regulate sexual, hormonal and reproductive function in mammals. The characteristics of pheromone signal detection by vomeronasal neurons remain unclear. Here we use a mouse VNO slice preparation to show that six putative pheromones evoke excitatory responses in single vomeronasal neurons, leading to action potential generation and elevated calcium entry. The detection threshold for some of these chemicals is remarkably low, near 10-11 M, placing these neurons among the most sensitive chemodetectors in mammals. Using confocal calcium imaging, we map the epithelial representation of the pheromones to show that each of the ligands activates a unique, nonoverlapping subset of vomeronasal neurons located in apical zones of the epithelium. These neurons show highly selective tuning properties and their tuning curves do not broaden with increasing concentrations of ligand, unlike those of receptor neurons in the main olfactory epithelium. These findings provide a basis for understanding chemical signals that regulate mammalian communication and sexual behaviour.

Quantitative analysis of neuronal diversity in the mouse olfactory bulb

Olfactory sensory information is processed and integrated by circuits within the olfactory bulb. Golgi morphology suggests the olfactory bulb contains several major neuronal classes. However, an increasingly diverse collection of neurochemical markers have been localized in subpopulations of olfactory bulb neurons. While the mouse is becoming the animal model of choice for olfactory research, little is known about the proportions of neurons expressing and coexpressing different neurochemical markers in this species. Here we characterize neuronal populations in the mouse main olfactory bulb, focusing on glomerular populations. Immunofluorescent labeling for: 1) calretinin, 2) calbindin D‐28K (CB), 3) parvalbumin, 4) neurocalcin, 5) tyrosine hydroxylase (TH), 6) the 67‐kDa isoform of GAD (GAD67), and 7) the neuronal marker NeuN was performed in mice expressing green fluorescent protein under the control of the glutamic acid decarboxylase 65kDa (GAD65) promoter. Using unbiased stereological cell counts we estimated the total numbers of cells and neurons in the bulb and the number and percentage of neurons expressing and coexpressing different neurochemical populations in each layer of the olfactory bulb. Use of a genetic label for GAD65 and immunohistochemistry for GAD67 identified a much larger percentage of GABAergic neurons in the glomerular layer (55% of all neurons) than previously recognized. Additionally, while many glomerular neurons expressing TH or CB coexpress GAD, the majority of these neurons preferentially express the GAD67 isoform. These data suggest that the chemospecific populations of neurons in glomeruli form distinct subpopulations and that GAD isoforms are preferentially regulated in different neurochemical cell types. J. Comp. Neurol. 501:825–836, 2007.

Blood Vessels Form a Scaffold for Neuroblast Migration in the Adult Olfactory Bulb

New cells are continuously added to the rodent olfactory bulb (OB), throughout development and in adults. These cells migrate tangentially from the subventricular zone along the rostral migratory stream to the OB, where they migrate radically from the center to periphery of the OB. Although different modalities of radial migration have been described in other brain regions, the mechanisms governing radial migration in the OB are still mostly unknown. Here, we identify a new modality of migration in which neuronal precursors migrate along blood vessels toward their destination. Our results show that half of the radially migrating cells associate with the vasculature in the granule cell layer of the OB, and in vivo time-lapse imaging demonstrates that they use blood vessels as a scaffold for their migration through an interaction with the extracellular matrix and perivascular astrocyte end feet. The present data provide evidence that a new modality of migration, vasophilic migration, is occurring in the adult brain and reveals a novel role of brain vasculature.

Molecular Identity of Periglomerular and Short Axon Cells

Within glomeruli, the initial sites of synaptic integration in the olfactory pathway, olfactory sensory axons terminate on dendrites of projection and juxtaglomerular (JG) neurons. JG cells form at least two major circuits: the classic intraglomerular circuit consisting of external tufted (ET) and periglomerular (PG) cells and an interglomerular circuit comprised of the long-range connections of short axon (SA) cells. We examined the projections and the synaptic inputs of identified JG cell chemotypes using mice expressing green fluorescent protein (GFP) driven by the promoter for glutamic acid decarboxylase (GAD) 65 kDa, 67 kDa, or tyrosine hydroxylase (TH). Virtually all (97%) TH+ cells are also GAD67+ and are thus DAergic–GABAergic neurons. Using a combination of retrograde tracing, whole-cell patch-clamp recording, and single-cell three-dimensional reconstruction, we show that different JG cell chemotypes contribute to distinct microcircuits within or between glomeruli. GAD65+ GABAergic PG cells ramify principally within one glomerulus and participate in uniglomerular circuits. DAergic–GABAergic cells have extensive interglomerular projections. DAergic–GABAergic SA cells comprise two subgroups. One subpopulation contacts 5–12 glomeruli and is referred to as “oligoglomerular.” Approximately one-third of these oligoglomerular DAergic SA cells receive direct olfactory nerve (ON) synaptic input, and the remaining two-thirds receive input via a disynaptic ON→ET→SA circuit. The second population of DAergic–GABAergic SA cells also disynaptic ON input and connect tens to hundreds of glomeruli in an extensive “polyglomerular” network. Although DAergic JG cells have traditionally been considered PG cells, their interglomerular connections argue that they are more appropriately classified as SA cells.

Generation of Distinct Types of Periglomerular Olfactory Bulb Interneurons during Development and in Adult Mice: Implication for Intrinsic Properties of the Subventricular Zone Progenitor Population

The subventricular zone (SVZ) of the lateral ventricle develops from residual progenitors of the embryonic lateral ganglionic eminence (LGE) and maintains neurogenic activity throughout life. Precursors from LGE/SVZ migrate to the olfactory bulb (OB) where they differentiate into local interneurons, principally in the granule layer and glomerular layer (GL). By in situ dye labeling, we show that neonatal and adult SVZ progenitors differentially contribute to neurochemically distinct types of periglomerular interneurons in the GL. Namely, calbindin-positive periglomerular cells are preferentially generated during early life, whereas calretinin- and tyrosine hydroxylase-expressing neurons are mainly produced at later ages. Furthermore, homochronic/heterochronic transplantation demonstrates that progenitor cells isolated from the LGE or SVZ at different stages (embryonic day 15 and postnatal days 2 and 30) engraft into the SVZ of neonatal or adult mice, migrate to the OB, and differentiate into local interneurons, including granule and periglomerular cells as well as other types of interneurons. The total number of integrated cells and the relative proportion of granule or periglomerular neurons change, according to the donor age, whereas they are weakly influenced by the recipient age. Analysis of the neurochemical phenotypes acquired by transplanted cells in the GL shows that donor cells of different ages also differentiate according to their origin, regardless of the host age. This suggests that progenitor cells at different ontogenetic stages are intrinsically directed toward specific lineages. Neurogenic processes occurring during development and in adult OB are not equivalent and produce different types of periglomerular interneurons as a consequence of intrinsic properties of the SVZ progenitors.

Development of the olfactory bulb: Evidence for glia‐neuron interactions in glomerular formation

Olfactory bulb (OB) glomeruli have long been considered functional units in the processing of odor information. Recently, it has been shown that axons from olfactory receptor neurons (ORNs) expressing the same odorant receptor gene converge onto two or a few topographically fixed glomeruli in the OB. The interactions between ORN axons, mitral/tufted cell dendrites, juxtaglomerular (JG) cells, and glial cells during the development of glomeruli is of great importance in light of this receptor gene glomerular topography in the primary olfactory projection. To explore the development of mammalian olfactory glomeruli, we investigated the relationships among radial glia (RG), astrocytes, ORNs, JG cells, mitral/tufted cell dendrites, and olfactory Schwann cells throughout embryonic and early postnatal development. Our results indicate that glomeruli are formed through an invariant sequence of cellular events: (1) pioneering ORN axons contact the rostral telencephalon at approximately E11–14, which coincides with the onset of morphologic changes in telencephalic RG; (2) at E15–16, RG branch and begin to form two plexuses, one located in the subventricular layer and the other superficial to the presumptive mitral cell layer; (3) at E17–18, ORN axons accumulate in a dense band superficial to the outer radial glia plexus; (4) at E19–20, processes from RG and astrocytes begin to ramify to form glial tufts, or glial glomeruli. Coincident with the formation of these glial glomeruli, ORN axons intermingle with the glial processes and form proto‐glomeruli; (5) at E21 to P0, JG cells begin to migrate into position surrounding glomeruli, (6) and at P4, the apical tuft of mitral cells becomes restricted to a single glomerulus. Interestingly, glomerular development also occurs in a distinct rostral to caudal gradient. That is, glomeruli in the rostral OB develop earlier than those in the caudal OB, but the sequence of cellular events at any point in the bulb is invariant. These results demonstrate that glomeruli are formed in a specific spatiotemporal sequence beginning with ORN axon‐glia contacts, then JG cell arrival, and finally mitral cell apical dendrite restriction. J. Comp. Neurol. 415:423–448, 1999.

A Divergent Pattern of Sensory Axonal Projections Is Rendered Convergent by Second-Order Neurons in the Accessory Olfactory Bulb

The mammalian vomeronasal system is specialized in pheromone detection. The neural circuitry of the accessory olfactory bulb (AOB) provides an anatomical substrate for the coding of pheromone information. Here, we describe the axonal projection pattern of vomeronasal sensory neurons to the AOB and the dendritic connectivity pattern of second-order neurons. Genetically traced sensory neurons expressing a given gene of the V2R class of vomeronasal receptors project their axons to six to ten glomeruli distributed in globally conserved areas of the AOB, a theme similar to V1R-expressing neurons. Surprisingly, second-order neurons tend to project their dendrites to glomeruli innervated by axons of sensory neurons expressing the same V1R or the same V2R gene. Convergence of receptor type information in the olfactory bulb may represent a common design in olfactory systems.

Control of RSV-induced lung injury by alternatively activated macrophages is IL-4R|[alpha]|-, TLR4-, and IFN-|[beta]|-dependent

Severe respiratory syncytial virus (RSV)-induced bronchiolitis has been associated with a mixed “Th1” and “Th2” cytokine storm. We hypothesized that differentiation of “alternatively activated” macrophages (AA-Mφ) would mediate the resolution of RSV-induced lung injury. RSV induced interleukin (IL)-4 and IL-13 by murine lung and peritoneal macrophages, IL-4Rα/STAT6-dependent AA-Mφ differentiation, and significantly enhanced inflammation in the lungs of IL-4Rα−/− mice. Adoptive transfer of wildtype macrophages to IL-4Rα−/− mice restored RSV-inducible AA-Mφ phenotype and diminished lung pathology. RSV-infected Toll-like receptor (TLR)4−/− and interferon (IFN)-β−/− macrophages and mice also failed to express AA-Mφ markers, but exhibited sustained proinflammatory cytokine production (e.g., IL-12) in vitro and in vivo and epithelial damage in vivo. TLR4 signaling is required for peroxisome proliferator-activated receptorγ expression, a DNA-binding protein that induces AA-Mφ genes, whereas IFN-β regulates IL-4, IL-13, IL-4Rα, and IL-10 expression in response to RSV. RSV-infected cotton rats treated with a cyclooxygenase-2 inhibitor increased expression of lung AA-Mφ. These data suggest new treatment strategies for RSV that promote AA-Mφ differentiation.

Toll-Like Receptor 2-Mediated Signaling Requirements for Francisella tularensis Live Vaccine Strain Infection of Murine Macrophages

ABSTRACT Francisella tularensis, an aerobic, non-spore-forming, gram-negative coccobacillus, is the causative agent of tularemia. We reported previously that F. tularensis live vaccine strain (LVS) elicited strong, dose-dependent NF-κB reporter activity in Toll-like receptor 2 (TLR2)-expressing HEK293T cells and proinflammatory gene expression in primary murine macrophages. Herein, we report that F. tularensis LVS-induced murine macrophage proinflammatory cytokine gene and protein expression are overwhelmingly TLR2 dependent, as evidenced by the abrogated responses of TLR2−/− macrophages. F. tularensis LVS infection also increased expression of TLR2 both in vitro, in mouse macrophages, and in vivo, in livers from F. tularensis LVS-infected mice. Colocalization of intracellular F. tularensis LVS, TLR2, and MyD88 was visualized by confocal microscopy. Signaling was abrogated if the F. tularensis LVS organisms were heat or formalin killed or treated with chloramphenicol, indicating that the TLR2 agonist activity is dependent on new bacterial protein synthesis. F. tularensis LVS replicates in macrophages; however, bacterial replication was not required for TLR2 signaling because LVSΔguaA, an F. tularensis LVS guanine auxotroph that fails to replicate in the absence of exogenous guanine, activated NF-κB in TLR2-transfected HEK293T cells and induced cytokine expression in wild-type macrophages comparably to wild-type F. tularensis LVS. Collectively, these data indicate that the primary macrophage response to F. tularensis LVS is overwhelmingly TLR2 dependent, requires de novo bacterial protein synthesis, and is independent of intracellular F. tularensis replication.