Person:

Whitman, Mary

In adult mice, new neurons born in the subventricular zone (SVZ), lining the lateral ventricles, migrate tangentially into the olfactory bulb along a well-delineated path, the Rostral Migratory Stream (RMS). Neuroblasts in the RMS migrate tangentially in chains, without a recognized migratory scaffold. Here, we quantitatively examine the distribution of, and relationships between, cells within the RMS, throughout its rostral-caudal extent. We show that there is a higher density of blood vessels in the RMS than in other brain regions, including areas with equal cell density, and that the orientation of blood vessels parallels the RMS throughout the caudal to rostral path. Of particular interest, migratory neuroblast chains are longitudinally aligned along blood vessels within the RMS, with over 80% of vessel length in rostral areas of the RMS apposed by neuroblasts. Electron micrographs show direct contact between endothelial cells and neuroblasts, although intervening astrocytic processes are often present. Within the RMS, astrocytes arborize extensively, extending long processes which are parallel to blood vessels and the direction of neuroblast migration. Thus, the astrocytic processes establish a longitudinal alignment within the RMS, rather than a more typical stellate shape. This complementary alignment suggests that blood vessels and astrocytes may cooperatively establish a scaffold for migrating neuroblasts, as well as provide and regulate migratory cues.

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.

Olfactory sensory neurons (OSN) in mice express only 1 of a possible 1,100 odor receptors (OR) and axons from OSNs expressing the same odor receptor converge into ,2 of the 1,800 glomeruli in each olfactory bulb (OB) in mice; this yields a convergence ratio that approximates 2:1, 2 glomeruli/OR. Because humans express only 350 intact ORs, we examined human OBs to determine if the glomerular convergence ratio of 2:1 established in mice was applicable to humans. Unexpectedly, the average number of human OB glomeruli is .5,500 yielding a convergence ratio of ,16:1. The data suggest that the initial coding of odor information in the human OB may differ from the models developed for rodents and that recruitment of additional glomeruli for subpopulations of ORs may contribute to more robust odor representation.

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.

The adult mammalian olfactory bulb (OB) receives a continuing influx of new interneurons. Neuroblasts from the subventricular zone (SVZ) migrate into the OB and differentiate into granule cells and periglomerular cells that are presumed to integrate into the synaptic circuits of the OB. We have used retroviral infection into the SVZ of mice to label adult-generated granule cells and follow their differen- tiation and integration into OB circuitry. Using synaptic markers and electron microscopy, we show new granule cells integrating into the reciprocal circuitry of the external plexiform layer (EPL), beginning at 21 d postinfection (dpi). We further show that synapses are formed earlier, beginning at 10 dpi, on the somata and basal dendrites of new cells in the granule cell layer (GCL), before dendritic elaboration in the EPL. In the EPL, elaborate dendritic arbors with spines are first evident at 14 dpi. The density of spines increases from 14 to 28 dpi, and then decreases by 56 dpi. Despite the initial appearance of dendritic spines at 14 dpi in the EPL, no expression of presynaptic or postsynaptic markers is seen until 21 dpi. These data suggest that adult-generated granule cells are first innervated by centrifugal or mitral/tufted cell axon collaterals in the GCL and that these inputs may contribute to their differentiation, maturation, and synaptic integration into the dendrodendritic local circuits found in the EPL.

HLA-G is a nonclassical class I MHC molecule with an unknown function and with unusual characteristics that distinguish it from other class I MHC molecules. Here, we demonstrate that HLA-G forms disulfide-linked dimers that are present on the cell surface. Immunoprecipitation of HLA-G from surface biotinylated transfec- tants using the anti-[]2-microglobulin mAb BBM.1 revealed the presence of an []78-kDa form of HLA-G heavy chain that was reduced by using DTT to a 39-kDa form. Mutation of Cys-42 to a serine completely abrogated dimerization of HLA-G, suggesting that the disulfide linkage formed exclusively through this residue. A possible interaction between the HLA-G monomer or dimer and the KIR2DL4 receptor was also investigated, but no interaction between these molecules could be detected through several approaches. The cell-surface expression of dimerized HLA-G molecules may have implications for HLA-G[]receptor interactions and for the search for specific receptors that bind HLA-G.

Ly49A, an inhibitory C-type lectin-like mouse natural killer cell receptor, functions through interaction with the major histocompatibility complex class I molecule, H-2Dd. The x-ray crystal structure of the Ly49A[]H-2Dd complex revealed that homodimeric Ly49A interacts at two distinct sites of H-2Dd: Site 1, spanning one side of the []1 and []2 helices, and Site 2, involving the []1, []2, []3, and []2 m domains. Mutants of Ly49A, H-2Dd , and 2 mi- croglobulin at intermolecular contacts and the Ly49A dimer interface were examined for binding affinity and kinetics. Although mutations at Site 1 had little affect, several at Site 2 and at the dimer interface hampered the Ly49A[]H-2Dd interaction, with no effect on gross structure or T cell receptor interaction. The region sur- rounding the most critical residues (in H-2Dd, Asp122; in Ly49A, Asp229, Ser236, Thr238, Arg239, and Asp241; and in 􏰊2-microglobulin, Gln29 and Lys58) of the Ly49A[]H-2Dd interface at Site 2 includes a network of water mole- cules, suggesting a molecular basis for allelic specificity in natural killer cell recognition.

Objective: To present and evaluate a new technique that allows the second-stage suture adjustment in strabis- mus surgery to be skipped or delayed if the immediate postoperative alignment is satisfactory. Methods: The “short tag noose” technique replaces long sutures with short tags that can be left under the con- junctiva after adjustment. Retrospectively, the medical records of all patients treated by a single surgeon with this approach between January 1, 2005, and December 31, 2008, were evaluated for success rate ([]10 prism di- opters [PD] horizontal and []6 PD vertical), reopera- tion rate, and complications. Results: Of 120 procedures reviewed, 27 (22.5%) were performed in children; 97 procedures (80.8%) were per- formed in patients with complex strabismus. Mean fol- low-up was 6 months. The success rate was 81.0% for horizontal strabismus and 70.7% for vertical strabismus at 2 months. The reoperation rate was 10.0% for hori- zontal strabismus and 19.0% for vertical strabismus. The noose slipped in 1 patient (0.8%) and was corrected by readjustment on day 4. Two patients (1.7%) required in- office excision of cysts or granulomas. Conclusions: The short tag noose technique simplifies the logistics of suture adjustment and avoids the need for sedation in children who do not require adjustment. It provides the ability to defer adjustment for days after surgery.