Localization of N-type Ca channels in ganglion cells in rat retina.

<p><b>R</b> at wholemount retina labelled with RBPMS (red), α1B VGCC subunit (green), and NF-M (white) antibodies. α1B colocalized with RBPMS in RGC somata (filled arrow), NF-M in RGC axons (arrowhead) and putative displaced amacrine cells (open arrow). Stack of six optical sections each of 0.3 µm thickness. Scale bar is 20 µm.</p

L-type VGCC subtypes contribute to calcium signalling in ganglion cell axons.

<p>A. Fluo-4 labelling of RGC axons in the wholemount retina. Scale bar is 20 µm. B. Application of nifedipine (NIF; 10 µM), an L-type Ca channel antagonist, reduced the second high K⁺-evoked calcium signal. C. Summary of Ca²⁺ imaging results from RGC axons showing the following changes in paired pulse Ca²⁺ signal in response to drugs (applied during the second K⁺ pulse) compared to their control paired K⁺ pulses (K): 10 µM nifedipine (20%±6%; p = 0.0053; n = 12), 100 µM verapamil (VPM; 52%±9%; p<0.0001; n = 9) and 200 nM TTX (52%±11%; p = 0.0009; n = 8). 400 nM ω-agatoxin IVA (AGT; n = 9), 3 µM ω-conotoxin-GVIA (CTX; n = 7) and 3 µM mibefradil (MIB; n = 12) did not change the calcium signal in a statistically significant manner.</p

P/Q-type Ca channel expression in ganglion cells in rat retina.

<p>Retina labelled with RBPMS (red) and α1A VGCC subunit (green) antibodies. α1A was colocalized with RBPMS in RGC somata (filled arrow). Additional cell bodies in the GCL staining for α1A and not RBPMS are likely to be displaced amacrine cells (open arrow). Scale bar is 20 µm. Stack of 10 optical sections each of 0.3 µm thickness.</p

Localization of L-type Ca channels in rat retina.

<p>(<b>Top row</b>) Rat wholemount retina triple labelled with RBPMS (red), α1C VGCC subunit (green), and NF-M (white) antibodies. α1C colocalized with RBPMS, which labels RGC somata (filled arrow), and NF-M, which labels RGC axons (arrowhead), as well as putative Müller cell endfeet. Stack of five z-axis optical sections each of 0.3 µm thickness. (<b>Lower row</b>) Rat wholemount retina labelled with RBPMS (red), α1D VGCC subunit (green), and NF-M (white) antibodies. α1D was localized to RGC somata (RBPMS) (filled arrow), RGC axons (NF-M) (arrowhead) and putative displaced amacrine cells (open arrow). Stack of four optical sections each of 0.3 µm thickness. Scale bar is 20 µm.</p

Many VGCC subtypes contribute to calcium signalling in ganglion cell bodies.

<p>A. Fluo-4 labelling of RGC somata in the wholemount retina. Scale bar is 20 µm. B. Application of nifedipine (NIF; 10 µM), an L-type Ca channel antagonist, reduced the second high K⁺-evoked calcium signal. C. Summary of Ca²⁺ imaging results in RGC somata showing the following changes in paired pulse Ca²⁺ signal in response to drugs (applied during the second K⁺ pulse) compared to their control paired K⁺ pulses (K): 10 µM nifedipine (29%±7%; p = 0.0003; n = 20), 100 µM verapamil (VPM; 39%±5%; p<0.0001; n = 17), 400 nM ω-agatoxin IVA (AGT; 35%±14%; p = 0.0364; n = 9), 3 µM ω-conotoxin-GVIA (CTX; 23%±10%; p = 0.0423; n = 15), 3 µM mibefradil (MIB; 21%±6%; p = 0.0011; n = 16) and 200 nM TTX (40%±9%; p = 0.0004; n = 14).</p

Depolarization-induced calcium signals in RGCs and their axons.

<p><b>A</b>. Multiple RGC somata and axon bundles in fluo-4 labelled retinal wholemount. Scale bar is 20 µm. <b>B</b>. A 33 s application of 60 mM K⁺ was applied at the time indicated (bar below the rising phases of the traces) and the simultaneous responses of 23 RGC somata (lower panel) and 13 RGC axon bundles (upper panel) was recorded and fit with exponential functions for comparison. Both the axon bundle and the cell body responses were complex with sequential and unsynchronized rising phases but both the axon bundle and somatic responses relaxed with similar time courses. The falling phase of the axon bundles was best fit with a single exponential having a time course of 25.7 s (overlay trace in dots) while that of the cell body required a double exponential fit with time constants of 11.5 and 65.8 s (overlay trace in dots).</p

Opioid-induced MAPK activation in naïve (A) and chronically treated (B) enteric neurons.

<p>DAMGO (1 µM, black bars) induced a transient MAPK/ERK1/2 activation in naïve (A) and chronic (B) neurons at 5 and 10 minutes, whereas morphine (grey bars) induced MAPK/ERK1/2 activation only in chronic (B) neurons. **p<0.01 compared to controls (white bars). N = 4–7 experiments in triplicate. Representative gels of pERK1/2 and tERK are shown at the bottom of each graph. tERK was used to verify that the treatment did not affect the total level of this protein and to confirm equal gel loading.</p

Effect of opioids on CREB phosphorylation in enteric neurons.

<p>Naïve (A) and chronic (B) neurons were stimulated with 1 µM DAMGO, 10 µM morphine or medium (control) for 0–20 minutes. DAMGO and morphine induced a significant, transient CREB activation in chronic, but not naïve enteric neurons. CREB phosphorylation in chronic neurons was blocked by the MEK1/2 inhibitor (U0126) treatment. *p<0.05 and **p<0.01 vs. control; n = 4–7 experiments in triplicate per group. Representative gels of pCREB and CREB are shown at the bottom of the figure. Total CREB was used to verify that the treatment did not affect the total level of this protein and to confirm equal gel loading.</p

Desensitization of μOR signaling in enteric neurons.

<p>A: Single exposure to DAMGO (1 µM, 5 min) induced significant MAPK activation in naïve enteric neurons, whereas a second exposure to the same DAMGO dose following 2 hours DAMGO pretreatment abolished DAMGO-mediated MAPK response, indicating desensitization. B: Single exposure to DAMGO (1 µM) or morphine (10 µM) activated MAPK in chronic neurons. A second exposure to the same dose of DAMGO or morphine following 2 hours DAMGO or morphine pretreatment induced the same effect in chronic neurons as single exposures, indicating suppression of desensitization. (** p<0.01 vs. control in A and B). C and D: DAMGO and morphine inhibit forskolin-stimulated cAMP in naïve (C) and chronic (D) enteric neurons. This effect was not observed in naïve enteric neurons (C) with a second opioid stimulation following a prior 2 hour exposure, indicative of desensitization. D: Note the over 2 fold increase in cAMP in unstimulated chronic neurons (cAMP superactivation or “overshooting”) vs. naïve control; DAMGO and morphine inhibition of cAMP was not prevented by 2 hours DAMGO or morphine pretreatment in chronic neurons, indicating suppression of desensitization. **p<0.01 vs. controls. N = 5–7 experiments performed in duplicate per group.</p

Morphological comparison of M1 ganglion cells in normal and optic nerve transected retina.

<p>(a) M1 ganglion cell immunostaining in the control eye. Bottom panel shows a side view with the typical stratification in the OFF sublamina of the inner plexiform layer. (b) M1 ganglion cell immunostaining 60 days after optic nerve transection. A displaced M1 ganglion cell is included (arrow). Bottom panel shows that in the injured retina the M1 ganglion cell dendrites remain in the same lamina of the IPL as in the control retina. Scale bar = 50 μm.</p