stage 6, at the height of glial migration, and then display an influx that declines to about half maximum by stage 9, indicating a strong temporal correlation between acetylcholine-induced glial calcium influx and glial cell migration to surround protoglomeruli. In the context of our results that FGFR activation is coupled to NP glial cell migration, phH3+ nuclei per 1000 glia 4.6 2.0 5.3 2.1 60% 57% Treatment 1XDMSO 1XPD 2XDMSO 2XPD Totals Vibratome sections examined 6 6 8 11 31 Optical sections examined 107 108 128 170 513 Optical sections quantitated 13 12 16 21 62 Avg ” glia/optical Avg phH3+ section nuclei/op sec 210.3 167.5 358.0 183.3 0.95 0.27 1.85 0.4 Reduction in glial division with PD173074 Total glial nuclei counted: 14,428. p-values obtained using Student’s t-test. doi:10.1371/journal.pone.0033828.t002 13 
Glial FGFRs in Glia-Neuron Signaling Number of Apoptag-positive AL+AN nuclei per frozen section. Stage Control PBTZ 169 Median Avg PD173074 Median Avg mid-5 n=5 2 1.2 n = 18 16.5 18 early-6 n=7 0 0.3 n = 16 40 38.9 12 n=6 0 0 n = 12 12.5 14.7 Note: Cells undergoing apoptosis were limited to regions normally occupied solely by NP glia at stages mid-5 and early-6. At stage 12, apoptotic nuclei were found in the sorting zone and antennal nerve. “n”= number of frozen sections examined. doi:10.1371/journal.pone.0033828.t003 the above observations raise the intriguing possibility for future study that glial FGFR activation, modulated by arrival of ORN axons, leads to expression or functionality of voltage-gated calcium channels and/or nicotinic acetylcholine receptors ” on NP glial cells. Alternatively, pathways downstream of calcium influx and FGFR activation could intersect to produce glial cell migration via, for example, activation of doublecortin, src-family kinases, and focal adhesion kinases. In contrast to the effect on NP glial cells, pharmacologic blockade of FGFR activation did not prevent the migration of SZ or AN glial cells. Blockade of ORN-mediated nitric oxide signaling or disruption of sterol-rich membrane subdomains with methyl-b-cyclodextrin also failed to block SZ glial cell migration. Our inability to block SZ glial migration by these various methods may be due to the fact that the initial contact between ORN growth cones and the glial cells that become SZ glia occurs late in stage 3, and thus the signaling necessary for SZ glial migration may have occurred before the various drug treatments could take effect. Injecting drugs at earlier stages generally results in developmental arrest a short distance into the sorting zone. B: PD173074-treated animals exhibited unchanged fasciculation in traveling through the sorting zone, although they did show increased fasciculation on exiting the sorting zone. Projection depths = 35 mm in A, 45 mm in B. doi:10.1371/journal.pone.0033828.g011 lished). Another possibility is that redundancy in the signaling pathways that elicit SZ glial cell migration ensures formation of this critical region in the olfactory pathway. As for the continued migration of AN glia in PD173074-treated animals in the Manduca system, similar results have been reported in Drosophila antennal nerves in which glial cells express a dominant-negative form of Heartless. We have found AN glia to express EGFRs as well as FGFRs; it is possible that they depend on EGFR activation for migration and FGFR activation for survival. Survival. Activation of FGFRs is known to be essential for survival of many cell types, althou
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