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Electron microscopic immunohistochemistry, although generally providing good localization, has often failed to produce satisfying ultrastructural preservation. Techniques that result in well-preserved tissue ultrastructure often hinder penetration of immunological reagents or render antigens non-immunoreactive. These are particularly serious limitations in studies of central nervous system and retina. We have evaluated several fixatives, including picric acid, high pH paraformaldehyde, and glutaraldehyde with subsequent sodium borohydride treatment, and penetration enhancement techniques, including buffered-ethanolic treatment and freeze--thaw, for their applicability in the retina. Our best fixation was achieved with 1 hr in 4% paraformaldehyde and 0.2% glutaraldehyde (pH 7.4) followed by overnight fixation in 4% paraformaldehyde (pH 10.4). Treatment with sodium borohydride after glutaraldehyde fixation restores much of the immunoreactivity that would otherwise be undetectable. The penetration of immunological reagents can be greatly increased by using either a buffered-ethanolic series or by freezing the tissue after careful cryoprotection. Using these methods we have been able to achieve specific immunological staining throughout the full thickness of retinal slices, up to 500 microns across, while preserving good ultrastructure. The methods should prove useful in the immunocytochemical localization of many different antigens in a variety of tissues.

Although there is a growing interest in the application of fractal analysis in neurobiology, questions about the methodology have restricted its wider application. In this report we discuss some of the underlying principles for fractal analysis. we propose the cumulative-mass method as a standard method and we extend the applicability of fractal analysis to both 2 and 3 dimensions. We have examined the relationship between the method of log-log Sholl analysis and fractal analysis and have found that they correlate well. Measurements of physiologically characterized retinal ganglion cells indicate that different cell types can have significantly different fractal dimensions. Such differences may allow the correlation of the physiological type of a neuron with its morphological fractal dimension.

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.

The second messenger cyclic guanosine monophosphate (cGMP) plays a role in many aspects of retinal processing. cGMP-gated channels function in photoreceptors, Müller, bipolar, and ganglion cells; and cGMP can modulate gap-junction conductivity. In the inner retina, both particulate and soluble guanylate cyclases can elevate levels of cGMP. The soluble isoform of guanylate cyclase is activated by nitric oxide (NO). In turtle retina, nitric oxide synthase, the enzyme that synthesizes NO, has been previously localized in discrete amacrine cells, somata in the ganglion cell layer, and in many processes in the inner plexiform layer. However, there have been no studies localizing soluble guanylate cyclase in the turtle retina. To functionally localize soluble guanylate cyclase, we stimulated retinas with the NO donors (+/-)-S-nitroso-N-acetylpenicillamine or spermine (nitric oxide) adduct, and then used immunocytochemistry to localize increases in cGMP-like immunoreactivity (cGMP-LI). The cells containing soluble guanylate cyclase should show cell autonomous increases in cGMP-LI in response to stimulation with NO. NO-stimulated increases in cGMP-LI occurred in many distinct amacrine cell types, select bipolar cells, some somata in the ganglion cell layer, and in discrete bands of processes in the inner plexiform layer. The pattern of cGMP-LI demonstrated qualitative dose response differences to the NO donors. This is the first localization of soluble guanylate cyclase in specific retinal neurons in the turtle; and the first functional activation of soluble guanylate cyclase in the amacrine cells of any species. The broad neuronal distribution of NO-stimulated cGMP-LI suggests that the NO/soluble guanylate cyclase/cGMP cascade is involved at several levels of visual processing in the inner retina.

Recent interest in nitric oxide and its relationship to cGMP has produced many attempts to anatomically localize the enzyme synthesizing nitric oxide, nitric oxide synthase. In the retina, numerous previous studies have used the NADPH-diaphorase enzyme activity of nitric oxide synthase as a histochemical method to localize nitric oxide synthase. However, all NADPH-diaphorase activity is not necessarily nitric oxide synthase, because several enzymes have similar biochemical activity. Additionally, various histochemical methods have been used to demonstrate NADPH-diaphorase activity, which makes comparisons between studies difficult. The purpose of this study was twofold. First, we wanted to examine the histochemical labeling of NADPH-diaphorase in the turtle retina to allow comparisons to previous studies. Second, we wanted to compare the histochemical localization of NADPH-diaphorase activity to the immunocytochemical localization of nitric oxide synthase in the turtle retina. Our histochemical localization of NADPH-diaphorase activity and our localization of nitric oxide synthase-like immunoreactivity in the turtle retina both produced similar results. Both the histochemistry and immunocytochemistry consistently labeled photoreceptor inner segments, at least three amacrine cell types, and processes in the inner plexiform layer. In optimized double-labeled preparations, all cells with NADPH-diaphorase activity were also positive for nitric oxide synthase-like immunoreactivity, although some somata in the ganglion cell layer only had nitric oxide synthase-like immunoreactivity. The immunocytochemical localization of nitric oxide synthase in photoreceptors, amacrine cells, and putative ganglion cells indicates that nitric oxide may function at several levels of visual processing in the turtle retina.

We show that-over a range of length scales r-the shapes of quasi-two-dimensional retinal neurons are fractal objects, and hence may be quantitatively characterized in part by their fractal dimension d/.We analyze the shapes of numerous retinal neurons, both in vivo and in vitro. The neurons in vivo are found to have a fractal dimension df of 1.68 ±0.15. We also propose an explanation of certain stages of neuronal shape development in terms of a diffusion-limited-aggregation model, which predicts

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