Charles A. Greer

Dynamic Contribution of Nestin-Expressing Stem Cells to Adult Neurogenesis

Understanding the fate of adult-generated neurons and the mechanisms that influence them requires consistent labeling and tracking of large numbers of stem cells. We generated a nestin-CreERT2/R26R-yellow fluorescent protein (YFP) mouse to inducibly label nestin-expressing stem cells and their progeny in the adult subventricular zone (SVZ) and subgranular zone (SGZ). Several findings show that the estrogen ligand tamoxifen (TAM) specifically induced recombination in stem cells and their progeny in nestin-CreERT2/R26R-YFP mice: 97% of SGZ stem-like cells (GFAP/Sox2 with radial glial morphology) expressed YFP; YFP+ neurospheres could be generated in vitro after recombination in vivo, and maturing YFP+ progeny were increasingly evident in the olfactory bulb (OB) and dentate gyrus (DG) granule cell layer. Revealing an unexpected regional dissimilarity in adult neurogenesis, YFP+ cells accumulated up to 100 d after TAM in the OB, but in the SGZ, YFP+ cells reached a plateau 30 d after TAM. In addition, most SVZ and SGZ YFP+ cells became neurons, underscoring a link between nestin and neuronal fate. Finally, quantification of YFP+ cells in nestin-CreERT2/R26R-YFP mice allowed us to estimate, for example, that stem cells and their progeny contribute to no more than 1% of the adult DG granule cell layer. In addition to revealing the dynamic contribution of nestin-expressing stem cells to adult neurogenesis, this work highlights the utility of the nestin-CreERT2/R26R-YFP mouse for inducible gene ablation in stem cells and their progeny in vivo in the two major regions of adult neurogenesis.

Adult Neurogenesis and the Olfactory System

Though initially described in the early 1960s, it is only within the past decade that the concept of continuing adult neurogenesis has gained widespread acceptance. Neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) into the olfactory bulb, where they differentiate into interneurons. Neuroblasts from the subgranular zone (SGZ) of the hippocampal formation show relatively little migratory behavior, and differentiate into dentate gyrus granule cells. In sharp contrast to embryonic and perinatal development, these newly differentiated neurons must integrate into a fully functional circuit, without disrupting ongoing performance. Here, after a brief historical overview and introduction to olfactory circuitry, we review recent advances in the biology of neural stem cells, mechanisms of migration in the RMS and olfactory bulb, differentiation and survival of new neurons, and finally mechanisms of synaptic integration. Our primary focus is on the olfactory system, but we also contrast the events occurring there with those in the hippocampal formation. Although both SVZ and SGZ neurogenesis are involved in some types of learning, their full functional significance remains unclear. Since both systems offer models of integration of new neuroblasts, there is immense interest in using neural stem cells to replace neurons lost in injury or disease. Though many questions remain unanswered, new insights appear daily about adult neurogenesis, regulatory mechanisms, and the fates of the progeny. We discuss here some of the central features of these advances, as well as speculate on future research directions.

A putative pheromone receptor gene expressed in human olfactory mucosa

Pheromones elicit specific behavioural responses and physiological alterations in recipients of the same species. In mammals, these chemical signals are recognized within the nasal cavity by sensory neurons that express pheromone receptors. In rodents, these receptors are thought to be represented by two large multigene families, comprising the V1r and V2r genes, which encode seven-transmembrane proteins. Although pheromonal effects have been demonstrated in humans, V1R or V2R counterparts of the rodent genes have yet to be characterized.

COMPARTMENTAL ORGANIZATION OF THE OLFACTORY BULB GLOMERULUS

Olfactory receptor cell (ORC) axons terminate in the olfactory bulb glomerular neuropil, where they synapse with dendrites of mitral, tufted, and periglomerular neurons. We investigated the organization of the glomerular neuropil by using antibodies to both single‐ and double‐label constituents for analyses with confocal microscopy. Electron microscopy (EM) was employed to assess the distribution of synaptic appositions within the glomerulus. Adult Sprague‐Dawley rats were processed for immunocytochemistry with olfactory marker protein (OMP), synaptophysin, synapsin 1, glial fibrillary acidic protein (GFAP), and/or microtubule‐associated protein 2 (MAP2). Equivalent rats were processed for transmission EM. Double labeling for OMP and MAP2 revealed two distinctive subcompartments within glomeruli: an axonal compartment containing predominately primary afferent axons with individual dendritic inserts and a complementary dendritic compartment that excluded primary afferent axons. Areas not occupied by OMP or MAP2 immunoreactivity were either immunoreactive for GFAP, indicating a glial process, or were blood vessels. Synaptophysin and synapsin 1 also showed differential labeling within the glomerulus. Synaptophysin strongly colocalized with OMP, whereas synapsin 1 was associated most strongly with MAP2. Reconstructions of glomeruli from EM montages revealed interdigitating axonal and dendritic subcompartments. The axonal subcompartments were composed primarily of ORC processes with individual or small groups of dendrites interspersed. Dendritic subcompartments were composed predominately of dendritic processes. Primary afferent axodendritic and local‐circuit dendrodendritic synapses segregated within the glomerulus into the axonal and dendritic subcompartments, respectively. The results support the hypothesis of subcompartmental organization within olfactory bulb glomeruli. J. Comp. Neurol. 407:261–274, 1999.

Increased fitness of rice plants to abiotic stress via habitat adapted symbiosis: A strategy for mitigating impacts of climate change

Climate change and catastrophic events have contributed to rice shortages in several regions due to decreased water availability and soil salinization. Although not adapted to salt or drought stress, two commercial rice varieties achieved tolerance to these stresses by colonizing them with Class 2 fungal endophytes isolated from plants growing across moisture and salinity gradients. Plant growth and development, water usage, ROS sensitivity and osmolytes were measured with and without stress under controlled conditions. The endophytes conferred salt, drought and cold tolerance to growth chamber and greenhouse grown plants. Endophytes reduced water consumption by 20–30% and increased growth rate, reproductive yield, and biomass of greenhouse grown plants. In the absence of stress, there was no apparent cost of the endophytes to plants, however, endophyte colonization decreased from 100% at planting to 65% compared to greenhouse plants grown under continual stress (maintained 100% colonization). These findings indicate that rice plants can exhibit enhanced stress tolerance via symbiosis with Class 2 endophytes, and suggest that symbiotic technology may be useful in mitigating impacts of climate change on other crops and expanding agricultural production onto marginal lands.

Loss-of-function mutations in sodium channel Nav1.7 cause anosmia.

Loss of function of the gene SCN9A, encoding the voltage-gated sodium channel Nav1.7, causes a congenital inability to experience pain in humans. Here we show that Nav1.7 is not only necessary for pain sensation but is also an essential requirement for odour perception in both mice and humans. We examined human patients with loss-of-function mutations in SCN9A and show that they are unable to sense odours. To establish the essential role of Nav1.7 in odour perception, we generated conditional null mice in which Nav1.7 was removed from all olfactory sensory neurons. In the absence of Nav1.7, these neurons still produce odour-evoked action potentials but fail to initiate synaptic signalling from their axon terminals at the first synapse in the olfactory system. The mutant mice no longer display vital, odour-guided behaviours such as innate odour recognition and avoidance, short-term odour learning, and maternal pup retrieval. Our study creates a mouse model of congenital general anosmia and provides new strategies to explore the genetic basis of the human sense of smell.

Functional expression of the olfactory signaling system in the kidney

Olfactory-like chemosensory signaling occurs outside of the olfactory epithelium. We find that major components of olfaction, including olfactory receptors (ORs), olfactory-related adenylate cyclase (AC3) and the olfactory G protein (Golf), are expressed in the kidney. AC3 and Golf colocalize in renal tubules and in macula densa (MD) cells which modulate glomerular filtration rate (GFR). GFR is significantly reduced in AC3−/− mice, suggesting that AC3 participates in GFR regulation. Although tubuloglomerular feedback is normal in these animals, they exhibit significantly reduced plasma renin levels despite up-regulation of COX-2 expression and nNOS activity in the MD. Furthermore, at least one member of the renal repertoire of ORs is expressed in a MD cell line. Thus, key components of olfaction are expressed in the renal distal nephron and may play a sensory role in the MD to modulate both renin secretion and GFR.

Viral tracing identifies distributed columnar organization in the olfactory bulb

Olfactory sensory neurons converge onto glomeruli in the olfactory bulb (OB) to form modular information processing units. Similar input modules are organized in translaminar columns for other sensory modalities. It has been less clear in the OB whether the initial modular organization relates to a columnar structure in the deeper layers involved in local circuit processing. To probe synaptic connectivity in the OB, we injected a retrograde-specific strain of the pseudorabies virus into the rat OB and piriform cortex. The viral-staining patterns revealed a striking columnar organization that extended across all layers of the OB from the glomeruli to the deep granule cell layer. We hypothesize that the columns represent an extension of the glomerular unit. Specific patterning was observed, suggesting selective, rather than distance-dependent, center-surround connectivity. The results provide a previously undescribed basis for interpreting the synaptic connections between mitral and granule cells within the context of a columnar organization in the OB and have implications for olfactory coding and network organization.

Glomerular formation in the developing rat olfactory bulb

Using the confocal microscope together with markers for the cellular components of glomeruli, we examined the spatiotemporal cellular interactions that occur between the axons of olfactory receptor cells, their dendritic targets, and glial cells during the critical period of glomerular formation. We have employed markers of immature and mature olfactory receptor cell axons, mitral/tufted cell dendrites, and glial cells as well as a synapse‐associated protein for double‐ and triple‐label immunocytochemistry. Axons of olfactory receptor cells grew into a dense dendritic zone of the olfactory bulb (comprising the dendrites of both mitral and tufted cells) between E17 and E18. At E19, these axons coalesced into protoglomeruli, which continued to develop until birth, when the basic anatomical structure of adult glomeruli emerged. Neither mitral/tufted cell dendrites nor olfactory bulb astrocytes became specifically associated with these protoglomeruli until E21. Ensheathing cells remained restricted to the outer nerve fiber layer and did not appear to contribute to glomerular formation. Finally, the synaptophysin staining has shown that synaptic constituents are expressed as early as E17, prior to the appearance of mature olfactory receptor cell axons. Based on these data, we have established a time line detailing the temporal and spatial interactions that occur between cell types during late embryonic rat olfactory bulb development. We conclude that the initial event in the formation of glomeruli is the penetration of the mitral/tufted cell dendritic zone by olfactory receptor cell axons. The coalescence of dendritic and glial processes into glomerular structures appears secondary to the arrival of the olfactory receptor cell axons. J. Comp. Neurol. 413:289–304, 1999.

Sublaminar organization of the mouse olfactory bulb nerve layer

Olfactory sensory neuron (OSN) axons coalesce to form the olfactory nerve (ON) and then grow from the olfactory epithelium to the olfactory bulb (OB), enter the olfactory nerve layer (ONL), reorganize extensively, and innervate specific glomeruli. Within the ON and ONL a population of glial cells, the olfactory ensheathing cells (OECs), surround OSN axon fascicles. To better understand the relationship between OECs and axon fascicles in the ONL of the adult mouse, we used confocal microscopy and antibodies to the low affinity nerve growth factor receptor p75 (p75), glial fibrillary acidic protein (GFAP), neuropeptide Y (NPY), and S‐100 to identify glia. Antibodies to olfactory marker protein (OMP) and neuronal cell adhesion molecule (NCAM) were used to identify OSN axons. Electron microscopy characterized the ONL ultrastructure. We found that glial processes were not uniformly distributed in the ONL of the mouse. The p75+ OEC processes were restricted to the ON and the outer ONL sublamina, and oriented parallel to the plane of the OB layers. In the inner ONL NPY+ OEC‐like processes were seen. GFAP+ processes were restricted to the inner ONL sublamina, the ONL/GL boundary, and the GL, where they delineated loosely aggregated axon fascicles that entered the glomeruli obliquely. S‐100+ processes and somata were distributed throughout the ONL; the outer and inner ONL were equivalent in their S‐100 staining. Ultrastructural studies showed that, although OECs could be identified in both the outer and inner ONL, in the latter, their relationship to bundles of OEC axons appeared less orderly than seen in the outer ONL. Our data demonstrate a differential organization of the ONL that could subserve distinct functions; axon extension may occur predominately in the outermost ONL, whereas glomerular targeting occurs in the inner sublamina of the ONL. J. Comp. Neurol. 446:68–80, 2002.