Neuronal nitric oxide synthase (nNOS) is implicated in some developmental processes, including neuronal survival, differentiation, and precursor proliferation. To define the roles of nNOS in neuronal development, we utilized the olfactory system as a model. We hypothesized that the role of nNOS may be influenced by its localization. nNOS expression was developmentally regulated in the olfactory system. During early postnatal development, nNOS was expressed in developing neurons in the olfactory epithelium (OE), while in the adult its expression was restricted to periglomerular (PG) cells in the olfactory bulb (OB). At postnatal week 1 (P1W), loss of nNOS due to targeted gene deletion resulted in a decrease in immature neurons in the OE due to decreased proliferation of neuronal precursors. While the pool of neuronal precursors and neurogenesis normalized in the nNOS null mouse by P6W, there was an overgrowth of mitral or tufted cells dendrites and a decreased number of active synapses in the OB. Cyclic GMP (cGMP) immunostaining was reduced in the OE and in the glomeruli of the OB at early postnatal and adult ages, respectively. Our results suggest that nNOS appears necessary for neurogenesis in the OE during early postnatal development and for glomerular organization in the OB in the adult. Thus, the location of nNOS, either within cell bodies or perisynaptically, may influence its developmental role.
The mechanism by which growing neurites sense and respond to small applied electrical fields is not known, but there is some evidence that the entry of Ca 2؉ from the external medium, with the subsequent formation of intracellular Ca 2؉ gradients, is important in this process. We have employed two approaches to test this idea. Xenopus spinal neurites were exposed to electrical fields in a culture medium in which Ca 2؉ was chelated to very low levels compared to the normal extracellular concentration of 2 mM. In other experiments, loading the neurites with the calcium buffer, 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA), disrupted the putative internal Ca 2؉ gradients, and the effects on the electrical response were determined. Fields of 100 mV/mm were applied for 12 h, and no difference was detected in the cathodal turning response between the treated neurites and the untreated controls. Using the Differential Growth Index (DGI), an asymmetry index, to quantitate the turning response, we recorded DGIs of ؊0.64, ؊0.65, and ؊0.62 for control cells, cells in Ca 2؉ -free medium, and cells preloaded with BAPTA, respectively. Furthermore, we detected an increase in neurite length for those neurons cultured in Ca 2؉ -free medium; they were 1.5-1.7 times as long as neurites from neurons cultured in normal Ca 2؉ medium. Likewise, we found that BAPTA-loaded neurites were longer than control neurites. Our data indicate that neuronal galvanotropism is independent of the entry of external Ca 2؉ or of internal Ca 2؉ gradients. Both cell-permeant agonistic and antagonistic analogs of cyclic 3,5-adenosine monophosphate (cAMP) increased the response to applied electrical fields.
Rett Syndrome (RTT) is an autism spectrum disorder caused by mutation in the gene encoding methyl CpG binding protein 2 (MECP2). Evidence to date suggests that these disorders display defects in synaptic organization and plasticity. A hallmark of the pathology in RTT has been identified as decreased dendritic arborization, which has been interpreted to represent abnormal dendritic formation and pruning during development. Our previous studies revealed that olfactory axons display defective pathfinding and targeting in the setting of Mecp2 mutation. In the present work, we use Mecp2 mutant mouse models and the olfactory system to investigate dendritic development. Here, we demonstrate that mitral cell dendritic development proceeds normally in mutant mice, resulting in typical dendritic morphology at early postnatal ages. We also failed to detect abnormalities in dendritic inputs at symptomatic stages when glomeruli from mutant mice appear smaller in area than the WT (6 weeks postnatally). Collectively, these findings suggest that the initial defects in glomeruli impairment seen with Mecp2 mutation do not result from abnormal dendritic development. Our results using the olfactory system indicate that dendritic abnormalities are not an early feature in the abnormalities incurred by Mecp2 mutation.
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