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First published online June 1, 2005
doi: 10.1242/10.1242/dev.01855

Development 132, 2873-2882 (2005)
Published by The Company of Biologists 2005
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Neuronal death induced by endogenous extracellular ATP in retinal cholinergic neuron density control

Valentina Resta1, Elena Novelli1, Francesco Di Virgilio2 and Lucia Galli-Resta1,*

1 Istituto di Neuroscienze CNR-56100 Pisa, Italy
2 Department of Experimental and Diagnostic Medicine, Section of General Pathology, and Interdisciplinary Center for the Study of Inflammation (ICSI), University of Ferrara, 44100 Ferrara, Italy



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  		Fig. 1. Blocking e-ATP signaling in vivo increases the density of retinal cholinergic neurons in neonatal rats. (A) A sample of a control array of cholinergic (ChAT immunoreactive) neurons in the ganglion cell layer (GCL) of a P2 neonatal rat. (B) The density of cholinergic cells is increased 24 hours after in vivo treatment with oATP. Scale bar: 50 µm. (C,D) The density of cholinergic neurons in both the GCL (C) and the INL (D) is increased in vivo 24 hours after intraocular injection of oATP (an irreversible blocker of the P2X receptors), suramine (a blocker of the purinergic receptors), or apyrase (an eATP-degrading enzyme). Eight retinas were sampled per treatment. Data are displayed as means ± standard deviation. In all cases the increase in cholinergic cell density was statistically significant (P<0.0001, t-test). ctrl, normal control; sham, vehicle injected; oATP, oxidized-ATP injected; sura, suramine injected; apy, apyrase injected.

 


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  		Fig. 2. e-ATP normally induces cell death in the developing population of cholinergic neurons. (A,B) Total number of cholinergic (ChAT) cells in the GCL (A) and the INL (B) 24 hours after in vivo treatment with apyrase (filled squares), suramine (filled triangles), or oATP (filled circles); control non-injected (open circles); vehicle-injected (open squares). The abscissa gives the age of analysis. The cholinergic cell number is increased if the treatment is administered between P1 and P3, but not on P5. Between 4 and 8 retinas were analyzed for each treatment and age point. (C) New cell genesis does not contribute to the increased number of cholinergic neurons, since BrdU (red) administered after the treatment does not label any cholinergic cells, identified here with an antibody to Islet-1/2 (green), which also labels retinal ganglion cells (Galli-Resta et al., 1997Go). Confocal image of a P2 retinal section. (D) Cellular debris immunoreactive for choline acetyltransferase can be observed in P2 whole-mount retinas 90 minutes after a shot of 5 mM ATP in the eye (left and central panel), while they are very rarely seen in normal P2 retinas (right panel). Scale bar: 50 µm in C; 20 µm in D.

 


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  		Fig. 3. Direct application of ATP to isolated neonatal retinas induces death of cholinergic neurons. (A-C) A P7 rat central retina cholinergic cell labeled with Alexa Fluor 488 dextran is illustrated at different times after application of 1 mM ATP. Small blebs in the dendritic tree are already apparent after a few minutes of ATP application (A), and become more conspicuous within 30 minutes. The cell soma (located in a different focal plane) displayed blebbing (left inset in C), and exposed phosphatidylserine on the outer membrane, as evidenced by Cy3-Annexin labeling (right inset in C). (D,E) Extensive soma blebbing was apparent in cholinergic neurons 30 minutes after application of 1 mM ATP (D) or 0.1 mM ATP (E). (F) In addition to blebbing (left) the cholinergic cells became permeable to propidium iodide (right), which displays a stronger fluorescence in the cell nucleus as a consequence of DNA binding (30 minutes after application of 1 mM ATP). (G) Between 2 hours and 12 hours after ATP application the soma of most cholinergic neurons lost their blebs and appeared shrunken (right panel) with respect to their original size (left panel). The small dark spot visible in D-G is the gene-gun bullet. Scale bar: 30 µm (A-C); 15 µm (insets in C); 10 µm (D-G). Images were acquired in black and white. By convention acquisition with the 488 nm emission filter (Alexa Fluo 488 dextran) are shown in yellow, acquisitions with the 568 nm emission filter (propidium iodide, Cy3-annexin) in red.

 


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  		Fig. 4. Apyrase, oATP and BBG prevent ATP effects on individual cholinergic neurons in isolated retinas. (A,B) A P4 rat cholinergic cell before (A; cell soma in the inset) and 30 minutes after (B) application of 1 mM ATP. ATP induces extensive blebbing in the dendrites and soma (bottom inset in B), and permeability to PI (top inset in B). (C,D) A P5 rat cholinergic cell before (C, soma in inset), and 30 minutes after (D) application of 1 mM ATP in the presence of apyrase (30 U/ml). Apyrase totally prevented dendritic and somal blebbing (bottom inset in D), as well as PI permeability (top inset in D). (E,F) A P3 rat cholinergic cell preincubated with oATP (300 µM, 2 hours preincubation) is shown before (E) and 30 minutes after application of 1 mM ATP. Preincubation with oATP totally prevented dendritic and somal blebbing (bottom inset in F), as well as PI permeability (top inset in F). (G-I) A P5 rat cholinergic cell is shown just before (G, cell soma in the inset) and 30 minutes after (H) application of 1 mM ATP in the presence of 0.2 µM BBG. No blebbing is observed in the dendrites (H) or in the cell soma (bottom inset in H), nor was the cell permeable to PI (top inset in H; red dot is the gene gun bullet). The protective effect of BBG was reversible: after 10 minutes washing to remove BBG, application of 1 mM ATP induced blebbing of the cell dendrites (I) and soma (bottom inset in I), as well as cell permeability to PI (top inset in I) within 30 minutes. Scale bar: 15 µm (A-F); 8 µm (insets in A-F); 20 µm (G-I); 10 µm (insets in G-I).

 


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  		Fig. 5. The cholinergic neurons express the P2X7 receptors and accumulate ATP in granules, which suggests that they could be sources of e-ATP. (A) In the neonatal rat retina the cholinergic cells (red) express the P2X7 receptors (green), as do many retinal ganglion cells. Confocal image of a P4 retinal section. (B-D) Quinacrine, which labels high levels of ATP stored in granules, is found in the cholinergic cells. (B) Confocal image of a P8 rat cholinergic neuron labeled with Alexa Fluor 568 dextran. (C) A higher magnification of the same cell shows that its soma (red) contains quinacrine labeling (green), which is also shown in isolation in D. Scale bar: 20 µm (A); 40 µm (B); 10 µm (C,D).

 


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  		Fig. 6 . Blocking e-ATP signaling increases the frequency of cholinergic cells not obeying the minimal spacing characteristic of these cells. (A) Autocorrelation plot of a normal cholinergic array at P2. The autocorrelation plots all the distances between the cells in the sample analyzed. In the normal cholinergic arrays the autocorrelation is a uniform distribution with a central hole. This means that the only spatial constraint these cells obey is to avoid getting closer to one another than a minimal distance, as already described (Galli-Resta et al., 1997Go). (B) Autocorrelation plot of an apyrase-treated cholinergic array at P2. The autocorrelation is still a uniform distribution with a central hole, but this is smaller than normal. This indicates the reduced efficacy of a mechanism normally ensuring the minimal spacing typical of the cholinergic arrays. (C,D) Examples of density recovery profiles (DRPs) histograms, plotting the density of counts in the autocorrelation as a function of the distance from the center of the coordinates, are shown for control (C) and treated (D) cells. In both cases the DRP rises from zero to a constant density, but does so in much shorter distances in the treated (D) than in the normal case (C). Count density is normalized to the average plateau density; an arrow indicates the exclusion radius (ER) value. (E) The size of the central exclusion region in the autocorrelation is quantified using the ER. The average ER is plotted with its standard deviation for the GCL cholinergic mosaics in control (vehicle injected) and apyrase- or oATP-injected retinas. e-ATP signaling blockade induces a significant ER reduction with respect to control (t-test, apyrase P<10-7; oATP P<10-6). Data were derived from the autocorrelation of all sampled fields (for each treatment n=8 retinas, 4 samples per retina).

 

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© The Company of Biologists Ltd 2005