Implantation and Extraction of Penetrating Electrode Arrays in Minipig Retinas.

Purpose: This work was motivated by the goals of demonstrating methods to fabricate and implant large numbers of penetrating arrays into the retina and the feasibility of extraction. Methods: Arrays of inactive, three-dimensional (3D) SU-8 structures were microfabricated onto 13-µm polyimide substrates. Standard vitreoretinal surgical techniques were used with an ab externo approach for subretinal implantation of arrays in 12 mini-pigs. In the first three surgeries, different post-geometries were explored, while a preferred design (128-µm tall, 30-µm diameter, 200-µm spacing) was used for the remaining nine implantations. Two arrays were extracted. Funduscopy, optical coherence tomography (OCT) and immunohistochemistry of the retinae were performed. The unoperated eyes and tissue far from implantation served as controls. A thirteenth pig was implanted with a planar array. Results: Ten implant surgeries had no significant complication, and two arrays were successfully extracted. One retinal tear occurred after implantation due to too long posts in an early surgery. In "successful" cases, OCT showed close apposition of the arrays to the retina and integration of the posts, the tops of which were positioned at the junction of the inner plexiform and ganglion cells, without significant gliosis. Conclusions: These results provide a proof-of-concept that relatively large numbers of 3D posts can be implanted into, and extracted from, the retina of mini-pigs. Our surgical numbers were relatively small, especially for the extractions, and our conclusions must be viewed with that limitation. Our methods are applicable for human surgeries. Translational Relevance: This study provides results of implantation and extraction of relatively large numbers of 3D posts from the retina of minipig eyes. If similar technology were used in humans, a 3D array of this type should lower perceptual thresholds, provide safer long-term stimulation, and perhaps provide better perceptual outcomes.

Rapid Changes in Synaptic Strength After Mild Traumatic Brain Injury.

Traumatic brain injury (TBI) affects millions of Americans annually, but effective treatments remain inadequate due to our poor understanding of how injury impacts neural function. Data are particularly limited for mild, closed-skull TBI, which forms the majority of human cases, and for acute injury phases, when trauma effects and compensatory responses appear highly dynamic. Here we use a mouse model of mild TBI to characterize injury-induced synaptic dysfunction, and examine its progression over the hours to days after trauma. Mild injury consistently caused both locomotor deficits and localized neuroinflammation in piriform and entorhinal cortices, along with reduced olfactory discrimination ability. Using whole-cell recordings to characterize synaptic input onto piriform pyramidal neurons, we found moderate effects on excitatory or inhibitory synaptic function at 48 h after TBI and robust increase in excitatory inputs in slices prepared 1 h after injury. Excitatory increases predominated over inhibitory effects, suggesting that loss of excitatory-inhibitory balance is a common feature of both mild and severe TBI. Our data indicate that mild injury drives rapidly evolving alterations in neural function in the hours following injury, highlighting the need to better characterize the interplay between the primary trauma responses and compensatory effects during this early time period.

Mild Blast Injury Produces Acute Changes in Basal Intracellular Calcium Levels and Activity Patterns in Mouse Hippocampal Neurons.

Mild traumatic brain injury (mTBI) represents a serious public health concern. Although much is understood about long-term changes in cell signaling and anatomical pathologies associated with mTBI, little is known about acute changes in neuronal function. Using large scale Ca2+ imaging in vivo, we characterized the intracellular Ca2+ dynamics in thousands of individual hippocampal neurons using a repetitive mild blast injury model in which blasts were directed onto the cranium of unanesthetized mice on two consecutive days. Immediately following each blast event, neurons exhibited two types of changes in Ca2+ dynamics at different time scales. One was a reduction in slow Ca2+ dynamics that corresponded to shifts in basal intracellular Ca2+ levels at a time scale of minutes, suggesting a disruption of biochemical signaling. The second was a reduction in the rates of fast transient Ca2+ fluctuations at the sub-second time scale, which are known to be closely linked to neural activity. Interestingly, the blast-induced changes in basal Ca2+ levels were independent of the changes in the rates of fast Ca2+ transients, suggesting that blasts had heterogeneous effects on different cell populations. Both types of changes recovered after ∼1 h. Together, our results demonstrate that mTBI induced acute, heterogeneous changes in neuronal function, altering intracellular Ca2+ dynamics across different time scales, which may contribute to the initiation of longer-term pathologies.

Region-specific alterations in astrocyte and microglia morphology following exposure to blasts in the mouse hippocampus.

Traumatic brain injury (TBI) is a serious public health concern, especially injuries from repetitive insults. The main objective of this study was to immunocytochemically examine morphological alterations in astrocytes and microglia in the hippocampus 48h following a single blast versus multiple blasts in adult C57BL/6 mice. The effects of ketamine and xylazine (KX), two common anesthetic agents used in TBI research, were also evaluated due to the confounding effect of anesthetics on injury outcome. Results showed a significant increase in hypertrophic microglia that was limited to the outer molecular layer of the dentate gyrus, but only in the absence of KX. Although the presence or absence of KX had no effect on astrocytes following a single blast, a significant decrease in astrocytic immunoreactivity was observed in the stratum lacunosum moleculare following multiple blasts in the absence of KX. The morphological changes in astrocytes and microglia reported in this study reveal region-specific differences in the absence of KX that could have significant implications for our interpretation of glial alterations in animal models of injury.

Visual system pathology in humans and animal models of blast injury.

Injury from blast exposure is becoming a more prevalent cause of death and disability worldwide. The devastating neurological impairments that result from blasts are significant and lifelong. Progress in the development of effective therapies to treat injury has been slowed by its heterogeneous pathology and the dearth of information regarding the cellular mechanisms involved. Within the last decade, a number of studies have documented visual dysfunction following injury. This brief review examines damage to the visual system in both humans and animal models of blast injury. The in vivo use of the retina as a surrogate to evaluate brain injury following exposure to blast is also highlighted.

Evidence for a functional adrenomedullin signaling pathway in the mouse retina.

PURPOSE: Adrenomedullin (ADM) is a small, secreted peptide often associated with vasodilation. However, ADM can also function as a neurotransmitter/neuromodulator, and studies suggest ADM is upregulated in the eye in several ocular diseases. However, no studies to date have described an ADM signaling pathway in the retina. METHODS: PCR, immunocytochemistry, nitric oxide imaging, western blots, and a nitrite assay were used to determine the localization of the components of the ADM signaling pathway in the mouse retina. RESULTS: We used reverse-transcriptase polymerase chain reaction to show that ADM and its primary receptor, calcitonin-receptor-like receptor, along with its associated receptor activity modifying proteins 2 and 3 are expressed in the retina. Using immunocytochemistry, we detected ADM staining throughout the retina in the photoreceptor outer segments, the outer nuclear layer, Müller and amacrine cell somata in the inner nuclear layer, and some somata in the ganglion cell layer. We found that calcitonin-receptor-like receptor and receptor activity modifying protein 2 had localization patterns similar to ADM, especially in somata in the inner nuclear and ganglion cell layers. Finally, we showed that the ADM receptor was functional in the retina. Stimulation of isolated retinas with ADM increased cyclic adenosine monophosphate- and cyclic guanosine monophosphate-like immunoreactivity, as well as nitric oxide production. CONCLUSIONS: These results are the first to show that ADM and functional ADM receptors are present in the retina. Since ADM is increased in eyes with ocular pathologies such as diabetic retinopathy, glaucoma, retinitis pigmentosa, and uveitis, the ADM signaling pathway may provide a new target for ameliorating these retinal pathologies.

Characterization of nitric oxide signaling pathways in the mouse retina.

Nitric oxide (NO) is a gaseous neuromodulator with physiological functions in every retinal cell type. NO is synthesized by several nitric oxide synthases (NOS) and often functions through its second messenger, cyclic guanosine monophosphate (cGMP), and protein kinase G (PKG). This study combined NO imaging, immunocytochemistry, biochemistry, and molecular biology to localize NO and its downstream signaling pathways in the mouse retina. Neuronal NOS (nNOS) was localized primarily in puncta in the inner plexiform layer, in amacrine cells, and in somata in the ganglion cell layer. Endothelial NOS was in blood vessels. Light-stimulated NO production imaged with diaminofluorescein was present in somata in the inner nuclear layer and in synaptic boutons in the inner plexiform layer. The downstream target of NO, soluble guanylate cyclase (sGC), was in somata in the inner and outer nuclear layers and in both plexiform layers. Cyclic GMP immunocytochemistry was used functionally to localize sGC that was activated by an NO donor in amacrine, bipolar, and ganglion cells. Cyclic GMP-dependent protein kinase (PKG) Iα was found in bipolar cells, ganglion cells, and both plexiform layers, whereas PKG II was found in the outer plexiform layer, amacrine cells, and somata in the ganglion cell layer. This study shows that the NO/cGMP/PKG signaling pathway is functional and widely distributed in specific cell types in the outer and inner mouse retina. A better understanding of these signaling pathways in normal retina will provide a firm basis for targeting their roles in retinal pathology.

Inhibition of the adrenomedullin/nitric oxide signaling pathway in early diabetic retinopathy.

The nitric oxide (NO) signaling pathway is integrally involved in visual processing and changes in the NO pathway are measurable in eyes of diabetic patients. The small peptide adrenomedullin (ADM) can activate a signaling pathway to increase the enzyme activity of neuronal nitric oxide synthase (nNOS). ADM levels are elevated in eyes of diabetic patients and therefore, ADM may play a role in the pathology of diabetic retinopathy. The goal of this research was to test the effects of inhibiting the ADM/NO signaling pathway in early diabetic retinopathy. Inhibition of this pathway decreased NO production in high-glucose retinal cultures. Treating diabetic mice with the PKC ß inhibitor ruboxistaurin for 5 weeks lowered ADM mRNA levels and ADM-like immunoreactivity and preserved retinal function as assessed by electroretinography. The results of this study indicate that inhibiting the ADM/NO signaling pathway prevents neuronal pathology and functional losses in early diabetic retinopathy.

Transduction of the inner mouse retina using AAVrh8 and AAVrh10 via intravitreal injection.

Adeno-associated virus (AAV) is a proven, safe and effective vector for gene delivery in the retina. There are over 100 serotypes of AAV, and AAV2 through AAV9 have been evaluated in the retina. Each AAV serotype has different cell tropism and transduction efficiency. Intravitreal injections of AAV into the eye tend to transduce cells in the ganglion cell layer (GCL), while subretinal injections tend to transduce retinal pigment epithelium and photoreceptors. Efficient transduction of the inner retina beyond the GCL is not well established with the current methodologies and serotypes used to date. In this study, we compared the cellular tropism of AAVrh8 and AAVrh10 vectors encoding enhanced green fluorescent protein (EGFP) using intravitreal injections. We found that AAVrh8 largely transduced cells in the GCL and also amacrine cells in the inner nuclear layer (INL), as well as Müller and horizontal cells. Inner retinal transduction with AAVrh10 was similar to AAVrh8, but AAVrh10 appeared to also transduce bipolar cells. The transduction efficiency as measured by the intensity of EGFP signal was 3.5 fold higher in horizontal cells transduced with AAVrh10 than AAVrh8. Glial fibrillary accessory protein (GFAP) levels were increased in Müller cells in transduced areas for both serotypes. The results of this study suggest that AAVrh8 and AAVrh10 may be excellent vector candidates to deliver genetic material to the INL, particularly for amacrine and horizontal cells, however they may also cause cellular stress as shown by increased glial GFAP expression.

Increased neuronal nitric oxide synthase activity in retinal neurons in early diabetic retinopathy.

PURPOSE: There are increased levels of nitric oxide (NO) in diabetic retinas. The purpose of this study was to determine the extent that neuronal nitric oxide synthase (nNOS) contributes to the increased levels of retinal NO in early diabetic retinopathy by examining the expression and activity of nNOS in retinal neurons after 5 weeks of diabetes. METHODS: Changes in NO levels were measured using NO imaging of retinal neurons in mice with streptozotocin-induced diabetes for five weeks. NO imaging was compared to nNOS localization using immunocytochemistry, and nNOS message and protein levels were measured using quantitative real-time PCR and western blots. RESULTS: There was a close anatomic correlation between the localization of the increased NO production and the nNOS immunoreactivity in the retinal plexiform layers of diabetic retinas. There was no change in nNOS message, but nNOS protein was decreased and its subcellular localization was altered. Treatment with insulin or aminoguanidine partially ameliorated the increase in NO in diabetic retinas. CONCLUSIONS: These results suggest that increased nNOS activity is responsible for the majority of increased NO in retinal neurons in early diabetic retinopathy. This supports a role for increased nNOS activity in the early neuronal dysfunction in the diabetic retina.

Functional localization of the nitric oxide/cGMP pathway in the salamander retina.

Nitric oxide (NO) is a gaseous neuromodulator that has physiological functions in every cell type in the retina. Evidence indicates that NO often plays a role in the processing of visual information in the retina through the second messenger cyclic guanosine monophosphate (cGMP). Despite numerous structural and functional studies of this signaling pathway in the retina, none have examined many of the elements of this pathway within a single study in a single species. In this study, the NO/cGMP pathway was localized to specific regions and cell types within the inner and outer retina. We have immunocytochemically localized nitric oxide synthase, the enzyme that produces NO, in photoreceptor ellipsoids, four distinct classes of amacrine cells, Müller and bipolar cells, somata in the ganglion cell layer, as well as in processes within both plexiform layers. Additionally, we localized NO production in specific cell types using the NO-sensitive dye diaminofluorescein. cGMP immunocytochemistry was used to functionally localize soluble guanylate cyclase that was activated by an NO donor in select amacrine and bipolar cell classes. Analysis of cGMP and its downstream target, cGMP-dependent protein kinase II (PKGII), showed colocalization within processes in the outer retina as well as in somata in the inner retina. The results of this study showed that the NO/cGMP signaling pathway was functional and its components were widely distributed throughout specific cell types in the outer and inner salamander retina.

Identification of alternate transcripts of neuronal nitric oxide synthase in the mouse retina.

Nitric oxide (NO) is a major signaling molecule in the retina and CNS, with physiological roles in every cell type in the retina. Previous work shows that neuronal nitric oxide synthase (nNOS) is an important source of NO in the vertebrate retina. There are distinct, active alternative transcripts of nNOS observed in many tissues, including testes and brain, that may differ in both localization and enzyme kinetics. The present study characterized nNOS and the NO production from nNOS in the mouse retina in terms of its alternate transcripts, namely, nNOS alpha, nNOS beta, and nNOS gamma. We examined both basal and light-stimulated NO production as imaged using the NO-sensitive dye 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate-FM (DAF-FM), and we compared the NO production with the immunocytochemical localization of nNOS using antisera that recognize nNOS alpha/beta or nNOS alpha/beta/gamma. Western blots suggested the presence of NOS alpha/gamma protein in retina, but not nNOS beta, and we confirmed this at the message level by using a combination of RT-PCR and quantitative real-time PCR. Our findings indicated that the primary source of NO in the mammalian retina is nNOS alpha and that nNOS gamma may contribute to NO production as well.

Interactions of the gaseous neuromodulators nitric oxide, carbon monoxide, and hydrogen sulfide in the salamander retina.

The three gaseous neuromodulators nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) are endogenously produced in vertebrate retinas. The NO/cyclic guanosine monophosphate (cGMP) and CO/cGMP pathways have been previously shown to interact synergistically in the turtle retina to increase cGMP levels. In this study, we examined H2S as a modulator of cGMP-like immunoreactivity (-LI) and its interactions with the NO/CO/cGMP signaling pathways in the tiger salamander retina. Stimulation with NO donor or CO significantly increased cGMP-LI from basal levels in bipolar and amacrine cells and in stratified arborizations in the inner plexiform layer. Stimulation with a combination of NO donor and CO significantly increased cGMP-LI above that seen with either stimulation alone. Nitric oxide synthase inhibitors reduced CO-induced cGMP-LI, suggesting that CO-induced cGMP-LI is not produced from direct activation of soluble guanylate cyclase. Exogenous H2S alone, from the donor NaHS, did not significantly modify cGMP-LI in dosages ranging from 2 to 1,200 microM NaHS, but there was a significant decrease in NO-induced cGMP-LI in the presence of 200 muM NaHS. This reduction of NO-induced cGMP-LI was not significantly affected by the addition of CuCl2, suggesting that the decrease was not a result of H2S and NO sequestering to form a novel nitrosothiol. NaHS did not have any significant effect on CO-induced cGMP-LI levels. Our results concur with previous studies showing synergistic interactions between NO and CO/cGMP retinal signaling pathways. We now show that H2S inhibits NO-induced cGMP-LI but not CO-induced cGMP-LI. In conclusion, all three gaseous neuromodulators have interactive roles in modulating retinal cGMP signaling.

Role of acetylcholine in nitric oxide production in the salamander retina.

Although acetylcholine is one of the most widely studied neurotransmitters in the retina, many questions remain about its downstream signaling mechanisms. In this study we initially characterized the cholinergic neurotransmitter system in the salamander retina by localizing a variety of cholinergic markers. We then examined the link between both muscarinic and nicotinic receptor activation and nitric oxide production by using immunocytochemistry for cyclic guanosine monophosphate (cGMP) as an indicator. We found a large increase in cGMP-like immunoreactivity (cGMP-LI) in the inner retina in response to muscarinic (but not nicotinic) receptor activation. Based on the amplification of mRNA transcripts, receptor immunocytochemistry, and the use of selective antagonists, we identified these receptors as M2 muscarinic receptors. Using double-labeling techniques, we established that these increases in cGMP-LI were seen in GABAergic but not cholinergic amacrine cells, and that the increases were blocked by inhibitors of nitric oxide production. The creation of nitric oxide in response to cholinergic receptor activation may provide a mechanism for modulating the well-known mutual interactions of acetylcholine-glycine-GABA in the inner retina. As GABA and glycine are the primary inhibitory neurotransmitters in the retina, signaling pathways that modulate their levels or release will have major implications for the processing of complex stimuli by the retina.

Comparative localization of cystathionine beta-synthase and cystathionine gamma-lyase in retina: differences between amphibians and mammals.

Hydrogen sulfide (H(2)S) is a gaseous neuromodulator that can be synthesized by the transsulfuration enzymes cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CGL). In this study we examined H(2)S as a potential neuromodulator in vertebrate retina. CBS-like immunoreactivity (LI) was found in somas in the inner nuclear layer and as punctate staining in the inner and outer plexiform layers in the salamander retina. CGL-LI was most clearly characterized in salamander, where it was localized in Müller cells. Western blots indicated proteins with the correct molecular weights for both enzymes in both species for liver and cerebellum. Correct molecular weight proteins were identified for both CGL and CBS in salamander retina. The CBS antiserum did not recognize the correct molecular weight protein in mouse retina but the CGL antiserum recognized the correct molecular weight protein for mouse retina. Enzyme assays indicated both CGL and CBS enzyme activity in all three tissues in the salamander. There was good CBS activity in the liver and cerebellum of the mouse but no activity in the retina. CGL activity was clearly present only in the mouse liver, with only trace activity in the cerebellum and retina. In conclusion, both CBS and CGL are present in the amphibian retina, which suggests either a potential role for H(2)S as a gaseous neuromodulator in both neurons and glia in the retina or a requirement for cysteine and glutathione synthesis via the transsulfuration pathway as a defense against oxidative stress.

Imaging of nitric oxide in the retina.

Nitric oxide (NO) is the most widespread signaling molecule found in the retina in that it can be made by every retinal cell type. NO is able to influence a wide variety of synaptic mechanisms ranging from increasing or decreasing neurotransmitter release to the modulation of gap junction conductivity. Although biochemical methods can analyze overall levels of NO, such methods cannot indicate the specific cell types involved. In the last few years, fluorescent imaging methods utilizing diaminofluorescein have allowed the real-time visualization of neurochemically or light stimulated NO-induced fluorescence (NO-IF) in specific retinal cells. Recent experiments have shown that this NO-IF can be stabilized using paraformaldehyde fixation. This aldehyde stabilization has allowed the imaging of NO production in the dark and in response to light, as well as the neurochemical modulation of light stimulated NO production. The results of these studies indicate that NO is not always freely diffusible and that NO is largely retained in many cells which make it. The NO production in retina is highly damped in that in the absence of stimulation, the endogenous levels of NO production are extremely low. Finally, different neurochemical or light stimulation protocols activate NO production in specific cells and subcellular compartments. Therefore, although the NO signaling is widespread in retina, it is very selectively activated and has different functions in specific retinal cell types. The use of NO imaging will continue to play a critical role in future studies of the function of NO in retina and other neural systems.

Nitric oxide stimulates gamma-aminobutyric acid release and inhibits glycine release in retina.

Nitric oxide (NO) modulates the uptake and/or release of neurotransmitters through a variety of cellular mechanisms. However, the pharmacological and biochemical processes underlying these neurochemical effects of NO often remain unclear. In our study, we used immunocytochemical methods to study the effects of NO, cyclic guanosine monophosphate (cGMP), and peroxynitrite on the uptake and release of gamma-aminobutyric acid (GABA) and glycine in the turtle retina. In addition, we examined the involvement of glutamate receptors, calcium, and the GABA transporter in this GABA uptake and release. We also tested for interactions between the GABAergic and glycinergic systems. In general, we show that NO stimulated GABA release and inhibited glycine release. The NO-stimulated GABA release involved calcium-dependent or calcium-independent synaptic release or reversal of the GABA transporter. Some effects of NO on GABA release involved glutamate, cGMP, or peroxynitrite. NO promoted glycine uptake and inhibited its release, and this inhibition of glycine release was influenced by GABAergic modulation. These findings indicate that NO modulates the levels of the inhibitory transmitters GABA and glycine through several specific biochemical mechanisms in different retinal cell types and layers. Thus it appears that some of the previously described reciprocal interactions between GABA and glycine in the retina function through specific NO signaling pathways.

Gycine and GABA interact to regulate the nitric oxide/cGMP signaling pathway in the turtle retina.

Nitric oxide (NO) is a free radical that is important in retinal signal transduction and cyclic guanosine monophosphate (cGMP) is a critical downstream messenger of NO. The NO/cGMP signaling pathway has been shown to modulate neurotransmitter release and gap junction coupling in horizontal cells and amacrine cells, and increase the gain of the light response in photoreceptors. However, many of the mechanisms controlling the production of NO and cGMP remain unclear. Previous studies have shown activation of NO/cGMP production in response to stimulation with N-methyl-d-aspartate (NMDA) or nicotine, and the differential modulation of cGMP production by GABA(A) and GABA(C) receptors (GABA(A)Rs and GABA(C)Rs). This study used cGMP immunocytochemistry and NO imaging to investigate how the inhibitory GABAergic and glycinergic systems modulate the production of NO and cGMP. Our data show that blocking glycine receptors (GLYR) with strychnine (STRY) produced moderate increases in cGMP-like immunoreactivity (cGMP-LI) in select types of amacrine and bipolar cells, and strong increases in NO-induced fluorescence (NO-IF). TPMPA, a selective GABACR antagonist, greatly reduced the increases in cGMP-LI stimulated by STRY, but did not influence the increase in NO-IF stimulated by STRY. Bicuculline (BIC), a GABA(A)R antagonist, however, enhanced the increases in both the cGMP-LI and NO-IF stimulated by STRY. CNQX, a selective antagonist for alpha-Amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid hydrobromide/kainic acid (AMPA/KA) receptors, eliminated both the increases in cGMP-LI and NO-IF stimulated by STRY, while MK801, a selective antagonist for NMDA receptors, slightly increased the cGMP-LI and slightly decreased the NO-IF stimulated by STRY. Finally, double labeling of NO-stimulated cGMP and either GLY or GABA indicated that cGMP predominantly colocalized with GLY. Taken together, these findings support the hypothesis that GLY and GABA interact in the regulation of the NO/cGMP signaling pathway, where GLY primarily inhibits NO production and GABA has a greater effect on cGMP production. Such interacting inhibitory pathways could shape the course of signal transduction of the NO/cGMP pathway under different physiological situations.

Inhibitors of nitric oxide synthase block carbon monoxide-induced increases in cGMP in retina.

Previous studies indicate that the gaseous messengers carbon monoxide (CO) and nitric oxide (NO) can interact to cause robust increases in intracellular cGMP levels in the retina. The purpose of the present study was to investigate the biochemical basis of the interactions between NO and CO for these increases. Turtle retinas were incubated in vitro with CO to stimulate cGMP production in the presence or absence of the nitric oxide synthase inhibitors N-omega-nitro-L-arginine methyl ester and S-methyl-thiocitrulline. Cyclic GMP immunocytochemistry was then used to evaluate the changes in cGMP levels in response to these stimuli. The results indicated that CO itself stimulated increases in cGMP in bipolar and amacrine cells, and that the increases were completely blocked by SMTC and L-NAME. We postulate that the increases of cGMP in response to CO might be mediated, at least partly, by CO displacing and releasing NO from its intracellular storage pool(s).

Activation of the cGMP/nitric oxide signal transduction system by nicotine in the retina.

Acetylcholine is one of the primary excitatory neurotransmitters/neuromodulators in the retina, but little is known about the downstream signaling pathways it can activate. The present study immunocytochemically examines the potential sources of acetylcholine and the location of the nicotinic cholinergic receptors in the turtle retina. It also examines how activation of these receptors can influence the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signal-transduction pathways. Photoreceptors, amacrine cells, and potentially ganglion cells contain choline acetyltransferase-like immunoreactivity (LI). Nicotinic acetylcholine receptors are immunocytochemically localized on photoreceptors, horizontal, bipolar, and ganglion cells. Nitric oxide imaging indicates that stimulation with nicotine increases NO production primarily in photoreceptors, horizontal, Muller, bipolar, and ganglion cells. In turn, very select populations of amacrine cells respond to this NO with increased levels of cGMP-LI. Selective inhibitors reveal that nitric oxide synthase is involved in most, but not all, of these increases in cGMP-LI. These results show that acetylcholine can activate the NO/cGMP signal-transduction pathways in both the inner and outer retina. This indicates that both of the major excitatory retinal transmitters, glutamate and acetylcholine, can stimulate NO production that increases levels of cGMP-LI in overlapping populations of retinal cells.