3.1. Light microscopic localization of nNOS
In these studies we used an older lot of Santa Cruz nNOS (SC-648) primary antiserum for the immunocytochemical staining. Unfortunately, the presently available lot of this same antiserum does not produce this same high quality staining. When we used our lot of the SC-648 antiserum in turtle it was possible to clearly distinguish three distinct amacrine cell types with nNOS-like immunoreactivity (nNOS-LI, ). The type 1 nNOS amacrine cells () were relatively common and in retinal cross-sections they had large vertically oriented darkly staining pyriform shaped somata with relatively symmetric dendritic arborizations. Their processes in the deeper portions of the inner plexiform layer (IPL) were studded with boutons. The type 2 amacrine cells () were the most common of the three nNOS amacrine cell types in the turtle retina. These cells had smallish, moderately stained, rounded somata that gave rise to several delicate processes, which ran a short distance in the outer IPL, before branching extensively in the central IPL and giving rise to terminal processes deep in the IPL or among the somata in the ganglion cell layer (GCL). Finally, the type 3 amacrine cells () had large, flattened, oval somata that often gave rise to thick, faintly labeled primary processes that arborized in the outer IPL. The type 1 and 2 amacrine cell types in turtle were very similar to the ND1 and ND2 (Vaney & Young, 1988
) or type 1 and type 2 (Sagar, 1990
) NADPH-diaphorase positive amacrine cell types in the rabbit, and to the NDa and NDb NADPH-diaphorase positive amacrine cell types in the guinea pig (Cobcroft, Vaccaro, & Mitrofanis, 1989
Fig. 1 At the LM level in turtle retina, strong nNOS-LI was present in at least three amacrine cell types (vertical arrows, type 1 in A; type 2 in B; and type 3 in C), their processes in the IPL (asterisks in A-C), some somata in the GCL (vertical arrowheads (more ...)
In contrast, when the primary antiserum directed against porcine cerebellar nNOS (Mayer et al., 1990
) was used there was much more extensive labeling. With this antiserum there was nNOS-LI in photoreceptor ellipsoids, many amacrine cell somata, near the outer limiting membrane, in many boutons and processes in the IPL, and in numerous somata in the GCL (). It is possible that the differences in labeling seen with these two antisera indicate that they may be recognizing alternate expressions of nNOS such as the natural variants of nNOS produced in the central nervous system by selective alternative splicing of the nNOS gene (Iwasaki et al., 1999
3.2. Ultrastructural localization of nNOS-LI
No significant labeling was seen in the bipolar or horizontal cell somata at the ultrastructural level, although patches of nNOS-LI have been associated with horizontal cell membranes in the OPL and there is nNOS-LI in the tips of some bipolar and horizontal cell processes that are postsynaptic to photoreceptor ribbon synapses and at basal junctions between photoreceptors (Haverkamp & Eldred, 1998
). The lack of stronger somatic labeling in horizontal and bipolar cells at the ultrastructural level is probably due to the decreased antigenicity caused by the fixation protocol used for electron versus light microscopy.
Neuronal NOS-LI was found associated with the outer membranes of the nucleus and the endoplasmic reticulum, and as diffuse flocculent immunoreactivity in the cytoplasm in some amacrine cell somata (). In the IPL many amacrine cell processes had strong nNOS-LI. These processes could be identified as amacrine cell processes by their lack of synaptic ribbons and the presence of synaptic vesicles at conventional synapses. In some labeled amacrine cells, the electron-dense reaction product diffusely filled the entire cell process ().
Fig. 2 (A) In the INL in turtle, nNOS-LI was found associated with the nuclear membrane (black arrow), the endoplasmic reticulum (white arrow), and as diffuse flocculent nNOS-LI (white arrowhead) in the cytoplasm in some amacrine cell somata. (B) In the IPL, (more ...)
Neuronal NOS-LI was also present in the ellipsoid in the inner segments of rods (not shown) or the accessory elements of the double cones (’) in some atypical mitochondria that were interspersed among the conventional mitochondria composing the ellipsoid. These atypical mitochondria were characterized by having poorly developed cristae. The adjacent conventional mitochondria in the ellipsoid showed no such increases in electron density. In some presumptive amacrine cell processes, there were also conventional mitochondria with nNO-SLI in their intermembrane space (’). These labeled mitochondria were distinct from those in the photoreceptor ellipsoids, in that in the photoreceptors there was granular immunoreactivity in the matrix and they had poorly developed cristae. In contrast, the labeled mitochondria in the amacrine cell profiles had nNOS-LI which was more selectively confined to the intermembrane space and they had well defined cristae.
Fig. 3 (A´) In some turtle photoreceptor ellipsoids (E) there were typical mitochondria (white arrow) and also some atypical mitochondria (black arrows) in the ellipsoids of rods and accessory elements of double cones (A) have nNOS-LI which increased (more ...)
In some amacrine cell boutons in the IPL, the nNOSLI was selectively associated with the presynaptic specializations (’), with relatively little cytoplasmic nNOS-LI. Some amacrine cells made more than one conventional synaptic contact in a given bouton, only some of which had nNOS-LI at their presynaptic specialization (’). It was likely that this nNOS-LI was truly presynaptic because in more weakly labeled preparations, it was possible to clearly see the nNOSLI was associated with clusters of synaptic vesicles and that there were the conventional parallel membrane specializations.
Selective subcellular localization of nNOS-LI was also seen in some bipolar cell axon terminals (’). In these bipolar terminals, the immunoreactivity was present in a small cytoplasmic region associated with the plasma membrane a micron or less from the ribbon synapse. In this specialized region, the two plasma membranes ran parallel to one another forming a uniform extracellular space. Although the parallel membranes and the consistent extracellular space suggested the existence of a synapse, the density of the nNOS-LI in this region obscured any clusters of synaptic vesicles.
At the ultrastructural level, very few somata in the GCL contained nNOS-LI and the labeling was much weaker than found in amacrine cell somata. Most of the nNOS-LI was associated with the endoplasmic reticulum or was located diffusely in the cytoplasm. The existence of nNOS-LI in some axons in the ganglion cell axon layer supported that some of these labeled somata were ganglion cells. Neuronal NOS-LI was present in mitochondria in Müller cells near the outer limiting membrane and in patches in Müller processes in the IPL. There was also some nNOS-LI associated with Müller cell villi surrounding the photoreceptors.
3.3. Nitric oxide imaging
The selective localization of nNOS-LI at specific synapses or in select mitochondria seems at odds with it being a freely diffusible gas which has been suggested to potentially diffuse 100́s of microns (Wood & Garthwaite, 1994
). Furthermore, although the NOS immunocytochemistry suggested that there were a wide variety of potential sources for NO production, questions remained as to which sources were active under what circumstances and how the NO spread in retina.
Kojima et al. (1998a
) developed the NO specific fluorescent probe diaminofluorescein (DAF). When DAF is nitrosated by reactive nitrogen oxides such as N2
at one of its amino groups the reaction leads to the formation of the triazole DAF-2T. DAF is relatively non-fluorescent until it binds the NO oxidation product and forms DAF-2T which increases the quantum efficiency of its fluorescence by ~180-fold (Kojima et al., 1998b
). When this dye is used to label retinal slices, it is possible to localize NO production within specific retinal cell types.
3.4. Specificity controls for imaging NO
In cell-free solutions, the fluorescence increase produced by the combination of DAF and NO donor shows a linear relationship between the concentration of NO donor in the solution and the rate of increase of NO (Blute et al., 2000
; Nagata, Momose, & Ishida, 1999
). This occurs because in a cell-free solution, the NO donor is continuously liberating NO. Thus, the increase in NO-IF over time represents an integral of the NO production. These results suggested that the rate of increase in fluorescence can be correlated with the relative concentration of NO.
In slices loaded with DAF and illuminated with 490 nm light, there was a slight fluorescence throughout the unstimulated control retinas which allowed the visualization of some labeled somata and the plexiform layers (). These weakly labeled somata are probably a reflection of the endogenous levels of NO in control tissue. When illuminated over time, there was a small steady decrease in this baseline fluorescence probably due to bleaching (). This indicates that the light stimulation itself does not activate DAF. The addition of the NO-donor, SNAP, caused an immediate overall increase in fluorescence throughout the slice which confirmed that all of the cells were loaded with DAF and that there was no obvious subcellular compartmentalization of the DAF (). Addition of depleted SNAP resulted in no such change in fluorescence.
Fig. 4 Effects of NO-donor on a turtle retinal slice loaded with DAF. (A) In these contt reversed images of a retinal slice loaded with DAF and illuminated with 488 nm light, there was some faint NO-IF apparent in the ONL and INL. (B) After 180 s of illumination (more ...)
All NMDA-stimulated increases in fluorescence were eliminated by inhibiting NOS. Slices that were pre-incubated and imaged in the presence of the potent irreversible NOS inhibitor, 100 μM L-NMMA, exhibited no increases in fluorescence in response to 250 μM NMDA (). This control is consistent with the fluorescence being due to the production of NO by NOS. Adding exogenous arginine to the loading Ringer enhanced the increase in fluorescence in response to NMDA stimulation, but did not evoke an increase in NO-IF by itself or alter the cell types responding to NMDA. A similar arginine-stimulated enhancement of DAF fluorescence is reported in several previous studies (Kojima et al., 1998a
; Nakatsubo et al., 1998
Fig. 5 Effects of NOS inhibitor on the levels of NMDA stimulated increases in NO-IF. (A) A contrast reversed image of a DAF loaded retinal slice that was pre-incubated with the NOS inhibitor 100 μM LNMMA. In the absence of stimulation there was some (more ...)
In total, these control experiments confirmed that the increased fluorescence we saw in response to NMDA stimulation was correlated with increased NO production resulting from NMDA receptor activation. With the results of these controls in mind, we use the term NO-IF.
3.5. Anatomical localization of NO production
In the turtle retina, at the light microscopic level, nNOS-LI is present in three amacrine cell types and their processes in the IPL, in ganglion cell somata, in photoreceptor inner segments, and in Müller cell processes near the outer limiting membrane (Blute et al., 1997
). Moreover, detailed ultrastructural investigations find nNOS-LI in photoreceptors, horizontal cells, and bipolar cells in the outer retina (Cao & Eldred, 2001
; Haverkamp & Eldred, 1998
). NMDA stimulation is able to increase NO-IF in all of these locations in select cells throughout the retina (Blute et al., 2000
). This NMDA-stimulated increase in NO production is consistent with the previous results of Blute et al. (1998)
showing that NMDA can increase levels of cGMP by activating NOS in turtle retina.
All of the images in were captured real-time from live slices of turtle retina. In some but not all photoreceptors, NMDA-stimulated NO-IF increased throughout the entire inner segment, soma and synaptic terminal (). The increased NO-IF was first observed in the ellipsoid, and the strongest overall increases in NO-IF in photoreceptors were in the ellipsoid. Within 30 s, lower levels of increased NO-IF were seen in photoreceptor somata and synaptic terminals. The presence of oil droplets indicated that at least some of these photoreceptors were cones. The high levels of NO-IF seen in the ellipsoid, agree with the immunocytochemical and histochemical localization of NOS in this region (Blute et al., 1997
; Cao & Eldred, 2001
; Koch, Lambrecht, Haberecht, Redburn, & Schmidt, 1994
)in many species. It is likely that the NO-IF seen in photo receptors seen response to NMDA may relate to the NMDA receptor subunits that have been previously localized presynaptically within both rod and cone photoreceptor terminals (Fletcher, Hack, Brandstatter, & Waässle, 2000
Fig. 6 Contrast-reversed digital images of NMDA (100 lM) stimulated increases in NO-IF that was imaged real-time in living slices of turtle retina. (A) Strong NO-IF was concentrated in the ellipsoid region (horizontal arrows) of the inner segment of many photoreceptors. (more ...)
The previous ultrastructural localization of nNOS-LI at specific contacts in the OPL (Haverkamp & Eldred, 1998
) is consistent with the strong NMDA-stimulated increase in NO-IF seen in the OPL. Horizontal () and bipolar cell () somata with NOIF were observed only occasionally, but fluorescent processes in the OPL were common (). Previous physiological studies support NO production by horizontal cells in the turtle retina (Miyachi et al., 1990
) and in the hybrid bass retina (McMahon & Ponomareva,1996
).Although NMDA receptors have not been localized on turtle horizontal or bipolar cells, it is possible that the NMDA stimulated NO-IF in these cells may also relate to the NMDA receptors localized on both rod and cone photoreceptor terminals (Fletcher et al., 2000
). If NMDA activates these receptors to increase glutamate release from photoreceptors, this glutamate could activate the kainate receptors which have been shown to activate bipolar and horizontal cells (Marc, 1999
A prominent feature of retinal nNOS-LI would be the numerous labeled amacrine cell somata (Blute et al., 1997
). Consistent with the strong nNOS-LI observed in multiple amacrine cell types, NMDA dramatically increased NO-IF in a wide variety of amacrine cells (). Amacrine cells stimulated with NMDA exhibited several different forms of NO-IF. Many amacrine cells had increased NO-IF that was confined to their somata (not shown), while in other cases the NO-IF increased in both their somata and processes (). Some of the labeled amacrine cells had swellings on their processes that resembled synaptic boutons (), while others did not (). When present, the active boutons often had more intense NOIF than the rest of the adjacent process, although the NO-IF in boutons was still often weaker than seen in the somata of the same cells. There were also activated processes in the IPL that were not directly associated with labeled somata (). These isolated processes with NO-IF usually had boutons. In addition, there were isolated boutons with high NO-IF found in the IPL that were not associated with any labeled processes ().
Immunocytochemistry indicates that there is nNOS confined within many somata in the GCL in turtle (Blute et al., 1997
). In response to NMDA, some somata in the GCL exhibited some of the strongest increases in NO-IF seen in the retina (). Interestingly, although the NO-IF appeared to diffuse out of these somata in the GCL, it did not fill the processes of these cells as it did in many of the adjacent amacrine cells.
Previous studies have described the presence of nNOS in Müller cells, although they usually have much less nNOS-LI than other retinal cell types (Kurenni et al., 1995
; Liepe, Stone, Koistinaho, & Copenhagen, 1994
). In turtle, there is little apparent nNOS-LI in Müller cells (Blute et al., 1997
). Considerable NO-IF appeared in Müller cells after several minutes of NMDA stimulation. These increases in NO-IF in Müller cells would often begin in their processes near the outer or inner limiting membranes and spread centrally to their soma (). In the GCL and ganglion cell axon layer (GCAL), Müller cell end feet had small varicosities (less than 5 μm in diameter) with strong NO-IF (). It is likely that the NMDA stimulated increases in NO-IF in Müller cells may relate to the NMDA receptors which have been shown to be present on Müller cells in human retina (Puro, Yuan, & Sucher, 1996
) and the nNOS-LI which has been reported in Müller cells in turtle retina (Cao & Eldred, 2001
In conclusion, every retinal cell type, but not every cell of a given type can show increases in NO-IF. It is interesting that all of the cells of a specific anatomical type, i.e., H1 horizontal cells, do not show a uniform response in that only some show increased NO-IF in response to a given stimulus. This raises the possibility that there may be neurochemical differences between anatomically similar retinal cells in that only some may utilize the NO/cGMP signaling pathway. This idea of selective localization is supported by the localization of nNOS-LI in only some rod bipolar cell dendrites in rat retina (Haverkamp & Eldred, 1998
3.6. Kinetics of NO-IF production in response to blocking GABAergic inhibition or stimulation with NMDA
In response to pharmacological stimulation, somatic increases in NO-IF all followed a similar general time-course, when intensity levels were plotted against time (). During the rising phase, the intensity of NOIF increased exponentially from baseline levels to a transient peak, which was immediately followed by a slower exponential decrease. For each of these pharmacological stimuli the different sources of NO-IF had characteristic time-courses. Though some ganglion cells differed in their peak responses, their response kinetics were similar. The same kinetic similarities were seen for other sources such as amacrine cell somata or boutons. Interestingly, ganglion cells showed a more sharply peaked response to 100 μM NMDA, while the boutons showed a more sharply peaked response to picrotoxin/bicuculline. However, amacrine cell somata had less steep rising and falling phases than did ganglion cells. Boutons in the IPL displayed less stereotypic responses and more variability, with a slower, more linear (less exponential) rising phase, a lower relative peak, and often a slower decay. In some cases, the increases in NO-IF in some boutons and ganglion cells had shorter onset latency in response to blockade of GABAergic inhibition than those seen in response to NMDA.
Fig. 7 Quantitative analysis of increases in NO-IF in turtle in response to 100 μM NMDA (A) or a combination of 50 μM bicuculline and 50 lM picrotoxin (B). In all cases the drugs were added at timeT = 0. Each of the curves represents the levels (more ...)
3.7. Quantification of the apparent diffusion of NO-IF
In some cells, the NO-IF spread well beyond their cell boundaries (), as would be predicted by the previous models of NO diffusion (Wood & Garthwaite, 1994
). However, in many cells, even in cells with very high levels of NO-IF, there was relatively little spread of NO-IF beyond the cell boundary () and the NOIF did not spread more than 10 μm from the source. In contrast, in some other cells, particularly somata in the GCL, there was considerable spread beyond the site of production (). These differences in spread were often seen between two different cell types within the same retinal slice, which would support there being a cell specific control over the spread of NO.
It is possible to use small NO-selective electrodes to measure the NO levels near a single retinal neuron. shows the NO-electrode placed near a soma in the GCL, while shows the change in the concentration of NO in response to stimulation with 100 μM NMDA. In this case the NO concentration rose from the preexisting background concentration of ~96 to ~174 nM which indicates that DAF is able to easily detect changes in nanomolar levels of NO. Given that there may be some retention of NO within the soma, it is possible that the intracellular levels of NO were higher than 174 nM. Such quantitative measurements of NO levels can then be used to quantify the NO levels within a given digital image because there is a linear relationship between the NO concentration and NO-IF (Blute et al., 2000
). Surprisingly, even if an intracellular NO concentration of 174 nM is assumed, there are NO concentrations below 10 nM within 10-20 lm away from relatively strong sources of NO production. This further supports a rather limited spread of NO in some cases.
Fig. 8 (A) Image of a NO selective microelectrode (M) with its sensor tip (arrow) placed adjacent to a ganglion cell with NO-IF in turtle. (B) Time-course of the increase in NO concentration measured with this microprobe. Before the application of NMDA the existing (more ...)
3.8. Aldehyde fixation of DAF
Previous studies have shown that aldehydes can be used to stabilize activated DAF in tissues (Kasim et al., 2001
; Sugimoto et al., 2000
). We reasoned that aldehyde fixation could stabilize the activated DAF to visualize NO produced in the dark using conventional confocal microscopy. However, this DAF fixation protocol has not been applied previously to examine retinal slices. To validate this method, control fixation experiments were performed to show that the fixation did not non-specifically increase NO-IF and that fixation did not alter the localization of NO-IF.
One particular advantage of this method is that if the DAF is activated before it is fixed with paraformaldehyde, it will remain activated after fixation. shows the NO-IF in a living turtle retinal slice that has been stimulated with 20 μM picrotoxin. The NO-IF was present in what appears to a bipolar cell terminal. shows a comparable slice that was also stimulated with picrotoxin before being fixed with paraformaldehyde. The labeling in this fixed slice closely resembled that seen in the live slice. The fixation also provided several other beneficial results. There was an apparent increase in NO-IF in response to aldehyde fixation as has been described previously (Sugimoto et al., 2000
). This increase in NO-IF was often so substantial as to require a decrease in the gain of the CCD camera to successfully capture the image. The fixation and subsequent increase in NO-IF also made it much easier to use confocal microscopy to obtain high resolution images. Finally, fixation seems to optically clear the tissue which makes it easier to obtain confocal images from deeper portions of thick retinal slices. shows a three dimensional rendering of NO-IF in a retinal slice that was stimulated with 660 nm flashing light before being fixed with aldehydes.
Fig. 9 (A) Image of picrotoxin stimulated NO-IF in a slice of turtle retina. (B) A lower magnification image of NO-IF in a slice that was fixed with paraformaldehyde following stimulation with picrotoxin. The NO-IF seen in both the living and tissue is quite (more ...)
Fig. 10 This is an image of NO-IF in a retinal slice made from a collapsed confocal stack that has been three-dimensionally rendered using the three-dimensional rendering software on a Zeiss LSM510NLO two-photon confocal microscope. This slice was stimulated (more ...)
To confirm that fixation did not non-specifically increase NO-IF and that fixation did not alter the localization of NO-IF, retinal slices were stimulated with 50 μM nicotine and then imaged before and following fixation. In the red color indicates the NO-IF that was present following 10 min of stimulation before fixation. The green color indicates the NO-IF that was present following 30 min of paraformaldehyde fixation. The yellow color indicates the NO-IF that was present before and after fixation. There is virtually no pure green in the image indicating that fixation did not induce or create any NO-IF that was not there prior to fixation. There is however extensive red over the photoreceptors which indicates that much of the NO-IF in the photoreceptors was lost during fixation. It is possible that photoreceptors lack some binding element that paraformaldehyde can cross-link with DAF to retain activated DAF within the photoreceptors. The increased NO-IF in horizontal cells may be related to the uncoupling effects of cholinergic agonists that have been reported in horizontal cells in mudpuppy retina (Myhr & McReynolds, 1996
). Thus there is no indication that fixation produces any artifactual production of NOIF or DAF activation and that in some cell types there can be a decrease in NO-IF in response to fixation. This aldehyde fixation method is thus ideal for studying light stimulated NO production in retina.
Fig. 11 The red indicates the NO-IF that was present in salamander following 10 min of stimulation with 50 μm nicotine before fixation. The green indicates the NO-IF that was present following 30 min of paraformaldehyde fixation. The yellow indicates (more ...)
3.9. Light stimulated NO-IF
The ability to use paraformaldehyde fixation to stabilize NO-IF allows the analysis of NO production in the dark and in response to various light stimulation protocols. It is also possible to use a multi-photon confocal microscope to image NO-IF real-time using an 810-nm excitation wavelength without activating or bleaching the photoreceptors (Eldred, personal communication). However, this method is expensive and logistically challenging. It is much simpler to use aldehyde fixed DAF. If the dark adapted retinal slices are made in the dark using infrared imaging equipment, then loaded with DAF and washed in the dark, it is possible to image the levels of NO-IF that are present in the dark. In salamander retina in the dark, there was strong NO-IF in many horizontal cells, some somata in the INL, and some photoreceptors (’). As described above, there may be a potential loss of NO-IF from photoreceptors so this photoreceptor labeling may be underrepresented. We examined the light-driven NO-IF in dark adapted retinal slices. In steady light, there was still NO-IF in some photoreceptors and horizontal cells, but there were also some somata in the GCL, many more somata in the INL, and many boutons in the IPL with strong NO-IF (’). In flashing light there was still strong NO-IF in the same sources seen with steady light, but there was much stronger NO-IF in more amacrine cell somata, in somata in the GCL, and in processes and numerous boutons in the IPL (’). These results indicate that NO is part of a light activated signaling pathway.
Fig. 12 Imaging of NO-IF in response to different lighting conditions in salamander retina. (A´) In the dark, there was strong NO-IF in many horizontal cells (H), some photoreceptors (P), and some amacrine cell somata (A) in the INL. There may be a potential (more ...)
It is interesting that the production of NO-IF in horizontal cells is increased by light in that photoreceptor transmitter release is reduced in the light which should hyperpolarize the horizontal cells. This result suggests that activation of NOS is not purely by influx of extra-cellular calcium and that other sources of calcium may be involved. For instance, it is possible that metabotropic receptors, such as the mGluR5 receptors reported to be present on horizontal cells (Hartveit, Brandstatter, Enz, & Wassle, 1995
) may be activating release of calcium from intracellular stores to increase NO-IF. It is also important to point out that the dark adapted retinas had been dark adapted for several hours and the increased levels of NO-IF may be a reflection of this long adaptation time. The levels of NO-IF were highest in flashing light, versus steady light or darkness, which indicates the transition between light to dark may be the strongest activator of NO production.
3.10. Colocalization of nNOS-LI with NO-IF
The ability to fix NO-IF with paraformaldehyde allows imaging of NO-IF to be combined with conventional nNOS immunocytochemistry. This can serve as a control to confirm that increases in NO-IF are due to the activation of NOS; and by using isoform specific NOS antisera it is possible to associate these increases with particular isoforms of NOS. Moreover, it is possible to determine which of a population of NOS containing cells can be selectively activated by a particular stimulus.
shows NO-IF in retinal slices that have been stimulated with 50 μM nicotine and then immunocytochemically labeled using an antiserum directed against nNOS. In these images the NO-IF is green, the nNOSLI is red, and the yellow indicates that it was nNOS that was producing the NO-IF. Interestingly, the yellow double labeling indicated that nicotine primarily activated nNOS in the somata of amacrine cells and in not their processes in the IPL. In contrast, nicotine increased levels of NO-IF in both the somata and some primary processes in horizontal cells (). Finally, these images indicate that nicotine did not activate the nNOS in all of the cells that contained it (red somata in ) and further that nicotine increased NO-IF in cells which did not contain nNOS-LI (green somata in ). This suggests that another NOS isoform, perhaps eNOS was also activated by nicotine.
Fig. 13 Colocalization of NO-IF and nNOS-LI in salamander retinal slices stimulated with 50 μM nicotine. In these images the NO-IF is green, the nNOS-LI is red, and yellow indicates that it was nNOS that was producing the NO-IF. The yellow double labeling (more ...)
3.11. Imaging NO-IF and the downstream production of cGMP
The use of paraformaldehyde fixation also allows the simultaneous visualization of the cells which produce NO-IF and the cells in which soluble guanylate cyclase is activated to produce the synthesis of cGMP. When retinal slices were stimulated with 50 μM nicotine and imaged using a combination of NO-IF and cGMP immunocytochemistry, it was evident that in turtle retina some horizontal cells can have primarily have NO-IF alone (, green somata with horizontal arrow), while other horizontal cells can show an increase in NO-IF and also show a cell autonomous increase in cGMP-LI (, vertical arrowheads). These results indicate that the entire NO/cGMP signaling pathway can be functionally demonstrated using a combination of imaging of NO-IF and cGMP immunocytochemistry.
Fig. 14 Colocalization of NO-IF and cGMP-LI in a salamander retinal slice stimulated with 50 μM nicotine. The NO-IF is green and the cGMP-LI is red. The horizontal cells on the left and right (vertical arrows) had both strong NO-IF and cGMP-LI, while (more ...)