Cellular and subcellular localization of PDE10A, a striatum-enriched phosphodiesterase.

PDE10A is a recently identified phosphodiesterase that is highly expressed by the GABAergic medium spiny projection neurons of the mammalian striatum. Inhibition of PDE10A results in striatal activation and behavioral suppression, suggesting that PDE10A inhibitors represent a novel class of antipsychotic agents. In the present studies we further elucidate the localization of this enzyme in striatum of rat and cynomolgus monkey. We find by confocal microscopy that PDE10A-like immunoreactivity is excluded from each class of striatal interneuron. Thus, the enzyme is restricted to the medium spiny neurons. Subcellular fractionation indicates that PDE10A is primarily membrane bound. The protein is present in the synaptosomal fraction but is separated from the postsynaptic density upon solubilization with 0.4% Triton X-100. Immuno-electron microscopy of striatum confirms that PDE10A is most often associated with membranes in dendrites and spines. Immuno-gold particles are observed on the edge of the postsynaptic density but not within this structure. Our studies indicate that PDE10A is associated with post-synaptic membranes of the medium spiny neurons, suggesting that the specialized compartmentation of PDE10A enables the regulation of intracellular signaling from glutamatergic and dopaminergic inputs to these neurons.

Subcellular localization of neuronal nitric oxide synthase in turtle retina: electron immunocytochemistry.

Recent studies imaging nitric oxide (NO) production in the retina have indicated a much wider distribution of NO production than would be suggested by previous light-microscopic localizations of neuronal nitric oxide synthase (nNOS). To help resolve this discrepancy, the present study analyzed the ultrastructural localization of nNOS-like immunoreactivity (-LI) in all layers of the retina. In the ellipsoids of rod photoreceptors and the accessory elements of double cones, nNOS-LI was associated with some atypical mitochondria. In the outer plexiform layer, nNOS-LI was in some postsynaptic horizontal and bipolar cell processes at photoreceptor ribbon synapses. In some amacrine and ganglion cell somata, nNOS-LI was diffusely localized in the cytoplasm and associated with the endoplasmic reticulum. In the inner plexiform layer, nNOS-LI diffusely filled some amacrine cell processes, while in other amacrine cells nNOS-LI was selectively localized at the presynaptic specializations of conventional synapses. Neuronal NOS-LI was also found at membrane specializations in bipolar cell terminals that were distinct from their normal ribbon synapses. Finally, some nNOS-LI was found in mitochondria in Muller cells. The diverse subcellular localizations of nNOS-LI indicates that NO may play distinct functional roles in many retinal cells, which correlates well with the widespread NO production found in previous NO imaging studies.

Direct imaging of NMDA-stimulated nitric oxide production in the retina.

In the retina, nitric oxide (NO) functions in network coupling, light adaptation, neurotransmitter receptor function, and synaptic release. Neuronal nitric oxide synthase (nNOS) is present in the retina of every vertebrate species investigated. However, although nNOS can be found in every retinal cell type, little is known about the production of NO in specific cells or about the diffusion of NO within the retina. We used diaminofluorescein-2 (DAF-2) to image real-time NO production in turtle retina in response to stimulation with N-methyl-D-aspartate (NMDA). In response to NMDA, NO was produced in somata in the ganglion cell and inner nuclear layers, in synaptic boutons and processes in the inner plexiform layer, in processes in the outer plexiform layer, and in photoreceptor inner segments. This NO-dependent fluorescence production quickly reached transient peaks and declined more slowly toward baseline levels at different rates in different cells. In some cases, the NO signal was primarily confined to within 10 microm of the source, which suggests that NO may not diffuse freely through the retina. Such limited spread was not predicted and suggests that NO signal transduction may be more selective than suggested, and that NO may play significant intracellular roles in cells that produce it. Because NO-dependent fluorescence within amacrine cells can be confined to the soma, specific dendritic sites, or both with distinct kinetics, NO may function at specific synapses, modulate gene expression, or coordinate events throughout the cell.

Localization of natriuretic peptides and their activation of particulate guanylate cyclase and nitric oxide synthase in the retina.

In the vertebrate retina, cyclic guanosine monophosphate (cGMP) mediates photoreceptor signal transduction and modulates ion channel and gap junction conductivity. Although most previous studies have focused on its synthesis by nitric oxide (NO)-sensitive soluble guanylate cyclase, cGMP is also synthesized by NO-insensitive particulate guanylate cyclases (pGC). Natriuretic peptides and their associated pGC-coupled receptors have been reported in retina, but few studies have localized these natriuretic peptides or pGCs to specific retinal cells or demonstrated that activation of pGCs by natriuretic peptides increases cGMP synthesis. In this study, we immunocytochemically localized atrial, brain, and C-type natriuretic peptide-like immunoreactivity (ANP-LI, BNP-LI, and CNP-LI, respectively) in turtle retina by using isoform specific antisera, and determined the ability of each natriuretic peptide isoform to increase cGMP-like immunoreactivity (cGMP-LI) in retinal cells. ANP-LI and CNP-LI were localized in sparsely distributed amacrine cells with thin, intermittently varicose processes in the inner plexiform layer. BNP-LI was localized to abundant somata in the inner nuclear and ganglion cell layers and in specific amacrine and horizontal cells. Stimulation of turtle eyecups with each of these natriuretic peptides increased cGMP-LI in multistratified amacrine cells by means of NO-independent mechanisms in the central retina, and in select amacrine and bipolar cells in the peripheral retina by a nitric oxide-dependent mechanism. These results indicate that natriuretic peptides can modulate the synthesis of cGMP in select retinal neurons by two distinct signal transduction pathways in a regionally specific manner.

Localization of heme oxygenase-2 and modulation of cGMP levels by carbon monoxide and/or nitric oxide in the retina.

Heme oxygenase-2 (HO-2) synthesizes carbon monoxide (CO), a modulator of soluble guanylate cyclase (sGC). To examine this signal transduction pathway in the retina, we immunocytochemically localized HO-2, and investigated the effects of CO on cGMP levels. In turtle, HO-2-like immunoreactivity (-LI) was in all photoreceptors, some amacrine cells, and in numerous bipolar and ganglion cells. HO-2-LI colocalized with sGC activity in many cells. In rat, HO-LI was found only in the inner retina, in ganglion and amacrine cells. In turtle, stimulation with CO alone primarily increased cGMP-LI in bipolar cells in the visual streak. Stimulation with a combination of CO and nitric oxide (NO) dramatically increased cGMP-LI throughout the retina, in comparison to the smaller increases seen with NO or CO alone. These data suggest that CO is an endogenous modulator of the sGC/cGMP signaling pathway in many retinal neurons, and can dramatically amplify NO-stimulated increases in cGMP.

Gamma-atrial natriuretic peptide 1-25 is found in bipolar cells in turtle and rat retinas.

Immunocytochemistry was used to reveal a population of bipolar cells that contain gamma-atrial natriuretic peptide 1-25 (gamma-ANP) in turtle retina. This same antibody was also used in rat retina as a comparative control. The retinas were examined by both conventional light microscopy and confocal microscopy with double-labeling to determine whether protein kinase C-alpha-like immunoreactivity (PKC-alpha-LI) was colocalized with the gamma-ANP-LI. Some thick sections of turtle retina immunostained with only the gamma-ANP antibody were also examined by electron microscopy. In rat, a subpopulation of bipolar cells with axons terminating close to the ganglion cell layer was labeled. Double-labeling experiments indicated that the gamma-ANP-LI and PKC-alpha-LI were colocalized in rat retina, and thus all the bipolar cells with gamma-ANP-LI were rod bipolar cells. In turtle, the gamma-ANP antibody labeled certain bipolar cells that were characterized by bistratified axon terminals arborizing on the borders of strata S2/3 and S3/4 in the inner plexiform layer (IPL). Double labeling with PKC-alpha antibody indicated that bipolar cells with gamma-ANP-LI were not the same bipolar cell types with PKC-alpha-LI. Thus, gamma-ANP-LI appears to be a new marker for a distinct type of bipolar cell in turtle retina. At the ultrastructural level, the gamma-ANP-LI was visible throughout the cytoplasm of the bipolar cells from dendrites to axon terminals. In the outer plexiform layer (OPL), labeled dendrites contacted photoreceptor pedicles almost exclusively at narrow-cleft basal junctions, but infrequently formed the central element at a photoreceptor ribbon synapse. In the IPL, axon terminals with gamma-ANP-LI made ribbon synapses onto a combination of amacrine and ganglion cells. Since narrow-cleft basal junctions and photoreceptor ribbon-related junctions are known to be associated with ON-center bipolar cells in turtle, and since the axon terminals of bipolars with gamma-ANP-LI stratify primarily in the ON-strata of the IPL, we suggest that these cells are likely to be ON-center cells. It is possible that the gamma-ANP may be involved in regulating the activity of Na+/K+ ATPase or in the modulation of cGMP levels.

Stimulation with N-methyl-D-aspartate or kainic acid increases cyclic guanosine monophosphate-like immunoreactivity in turtle retina: involvement of nitric oxide synthase.

In brain and retina, stimulation with excitatory amino acids (EAA) can generate nitric oxide (NO) and increase levels of cyclic guanosine monophosphate (cGMP). Because nitric oxide synthase (NOS) has been found in retinas of all species examined to date, an NO signal-transduction pathway is likely to be present in all retinas. We tested the hypothesis that stimulation of ionotropic glutamate receptors in turtle retina would result in increases in cGMP through an NOS/NO/cGMP pathway. Following in vitro incubations of turtle eye cups with the glutamate receptor agonists, N-methyl-D-aspartate (NMDA) or kainic acid (KA), we quantified the increases in cGMP-like immunoreactivity (cGMP-LI) by using enzyme-linked immunosorbant assay (ELISA) and localized the increased cGMP-LI by using an antibody against cGMP. Stimulation with NMDA or KA increased cGMP-LI in bipolar and amacrine cells as well as in some somata in the ganglion cell layer. Either KA or NMDA produced statistically significant increases in total retinal cGMP-LI by ELISA. To test the involvement of NO, we used the NOS inhibitors 7-nitroindazole and L-nitroarginine. Both inhibitors blocked virtually all of the KA- or NMDA-stimulated increases in cGMP-LI. These results indicate that activation of ionotropic glutamate receptors can increase cGMP in select retinal neurons. Differences between the agonist-evoked increases of retinal cGMP-LI suggest that there can be specificity in the activation of the NOS/NO/cGMP signal-transduction pathway by glutamate. This suggests that, in addition to short-term electrical changes, activation of ionotropic glutamate receptors also may produce longer term modulatory or metabolic effects involving NO/cGMP.

Localization of nNOS in photoreceptor, bipolar and horizontal cells in turtle and rat retinas.

Neuronal nitric oxide synthase (nNOS), an enzyme that synthesizes NO, has been found in the outer retina using light microscopic immunocytochemistry, but its subcellular localization is unknown. We used electron immunocytochemistry to examine nNOS-like immunoreactivity (nNOS-LI) in the outer plexiform layer of turtle and rat retinas. In turtle, nNOS-LI was present in some bipolar and horizontal cell processes at photoreceptor ribbon synapses and at basal junctions between photoreceptors. In rat, nNOS-LI was present in some rod bipolar and B-type horizontal cell axon terminals at rod ribbon synapses. These results indicate that in vertebrates, NO is produced by all of the major nerve cell types in the outer retina at specific synaptic contacts.

Functional localization of soluble guanylate cyclase in turtle retina: modulation of cGMP by nitric oxide donors.

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.

Localization of the origin of retinal efferents in the turtle brain and the involvement of nitric oxide synthase.

Previous studies have used selective neurochemical markers or retrograde tracers to localize the cells in the brain giving rise to efferents to the turtle retina. Because of the relative selectivity of the neurochemical markers or the lack of sensitivity of the previously employed retrograde tracers, these studies did not locate all the efferent cell bodies, or they could not describe the anatomy of the efferent cells. In the present study, cholera toxin B was used as a highly sensitive retrograde tracer to investigate the distribution, number, and morphology of the retinal efferent or centrifugal cell system in turtle brain. Previous studies of the turtle retina have indicated that nitric oxide synthase may be found in some retinal efferents. Therefore, we also did colocalization studies of the retrograde tracer with reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry to investigate nitric oxide as a possible transmitter used by efferent fibers and to localize these NADPH-diaphorase-positive efferent cell bodies in the turtle brain. We found that each eye received projections from approximately 40 efferent cell bodies that were located primarily in the contralateral midbrain. The majority of efferent cell bodies were centered in the isthmic tegmentum; other efferent cells extended more rostrally into the substantia nigra, and some efferent cells extended more caudally into the nucleus raphes superior. The double-label results showed that 30% of the cholera toxin B-like immunoreactive cells were also positive for NADPH-diaphorase. The location of these double-labeled cells around the locus coeruleus corresponded to the NADPH-diaphorase-positive efferent cells in the avian isthmo-optic field. The localization of NADPH-diaphorase in these efferents indicated that they may use nitric oxide to modulate retinal function.

Specialized neuropeptide Y- and glucagon-like immunoreactive amacrine cells in the peripheral retina of the turtle.

There are many regional differences in cell morphology and neurochemistry in the retina. This study examined a specialized population of neuropeptide Y- and glucagon-like immunoreactive amacrine cells in the peripheral retina of the turtle. Some of the dendritic processes from these peptidergic amacrine cells formed a dense circumferentially oriented nerve fiber plexus which ran parallel to the ora serrata. Collaterals from this plexus projected into and innervated the nonpigmented ciliary epithelium in the pars plana region of the ciliary body. Electron microscopy revealed that the neuropeptide Y- and glucagon-like immunoreactive processes in the ciliary epithelium contained many labeled, large dense-cored vesicles. Small crystals of lipid-soluble fluorescent dye were implanted in the retina near the ora serrata in fixed retinal tissue to search for other peripheral retinal specializations. Numerous thick and thin cell processes oriented parallel to the ora serrata were labeled in the retina by the dye. In addition, many dye-labeled somata with circumferentially oriented dendritic arborizations were seen in the extreme periphery of the retina. Many of these dye-labeled cells and processes were clearly not associated with the neuropeptide Y- and glucagon-like immunoreactive cells described above. This study has shown that some peptidergic neurons in the peripheral retina have a unique morphology in comparison to more centrally located cells. The function of these specialized peripheral cells is not established, but the innervation of the ciliary epithelium by peptidergic amacrine cells suggests that they may be involved in control of aqueous inflow.

Immunocytochemical and histochemical localization of nitric oxide synthase in the turtle 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.

Synaptic inputs to identified color-coded amacrine and ganglion cells in the turtle retina.

Previous studies have proposed models of the specific synaptic circuitry responsible for color processing in the turtle retina. To determine the accuracy of these models of the circuits underlying color opponency in the inner retina of the turtle (Pseudemys scripta), we have studied the physiology, morphology, and synaptic connectivity of identified amacrine and ganglion cells. These cells were first characterized electrophysiologically and were then stained with horseradish peroxidase. Postembedding electron immunocytochemistry for gamma-aminobutyric acid (GABA) and glycine was used to reveal the neurochemical identity of their synaptic inputs. The red-ON/green, blue-OFF small-field ganglion cell, classified as G24, branched primarily in strata S1, S4, and S5 of the inner plexiform layer (IPL). Ganglion cell G24 showed a complex receptive field organized into a red-ON center surrounded by an inhibitory region, which, in turn, was surrounded by a second excitatory region. Only the center responses were color opponent. The red-OFF/green, blue-ON large-field, stellate amacrine cell, classified as A23b, stratified exclusively in stratum S2, near the S2/S3 border. The color-coded center was surrounded by a luminosity, red-sensitive surround. Synaptic input to G24 and A23b was dominated by amacrine cells (89% and 87%, respectively). G24 received significant input from amacrine cell profiles with GABA (13% of total) as well as glycine (11% of total) immunoreactivity, mostly in the proximal stratum S5 of the IPL (64% and 67% of the total GABA- and glycine-immunoreactive input, respectively). Bipolar cell synaptic input was also found predominantly in S4 and S5 (89%). In contrast, we found no glycine-immunoreactive input to A23b, and the density of the GABA-immunoreactive amacrine cell synaptic input revealed a central (15%) to peripheral (3%) gradient within the dendritic tree. The results of the present study support the previous models of the synaptic circuitry responsible for color-opponent signal processing in the inner retina of the turtle.

Quantitative anatomy, synaptic connectivity and physiology of amacrine cells with glucagon-like immunoreactivity in the turtle retina.

Although a wide variety of neuropeptides have been localized in vertebrate retinas, many questions remain about the function of these peptides and the amacrine cells that contain them. This is because many of these peptidergic amacrine cells have been studied using only immunocylochemical techniques. To address this limitation, the present study used a combination of quantitative anatomy, biochemistry and electrophysiology to examine amacrine cells in the turtle retina that contain the neuropeptide glucagon. In the turtle retina, there is a small population of 2500 glucagonergic amacrine cells, which probably represents < 1% of the total number of amacrine cells. Circular distribution statistics indicated that many of these tristratified amacrine cells had asymmetric dendritic arborizations that were radially oriented toward the retinal periphery. The cells were found to have similar dendritic coverage factors, to be distributed in a non-random arrangement in all regions of the retina, and to peak in density in the visual streak region. Electron microscopic studies indicated that glucagonergic amacrine cells made synaptic contacts primarily with other amacrine cells, and small numbers of bipolar cells. The synaptic inputs and outputs were balanced in the inner strata of the inner plexiform layer, and were biased toward synaptic outputs in the outer strata of the inner plexiform layer. These contacts involved small unlabelled synaptic vesicles, and not the large labelled dense core vesicles also found in these neurons. The biochemical studies indicated that glucagon could be released from the retina in a calcium dependent manner by high potassium stimulation. The electrophysiology found no color opponency, and the glucagonergic amacrine cells gave sustained hyperpolarizing responses to small stimulation spots and had antagonistic surrounds. The results of these studies suggest that there are significant regional specializations of glucagonergic amacrine cells, and that they may provide OFF-modulation in interactions between the ON-and OFF-centre visual pathways in the turtle retina.

Use of the sensitive anterograde tracer cholera toxin fragment B reveals new details of the central retinal projections in turtles.

Previous anterograde degeneration and autoradiographic studies have yielded inconsistent results on the extent and target areas of the central retinal projections in turtles. We used the highly sensitive anterograde and retrograde tracer, cholera toxin B fragment (CTB), to re-examine the central retinal projections in Pseudemys scripta elegans. In contrast to the results of the previous anterograde degeneration studies and autoradiographic studies, immunohistochemical detection of CTB-labeled central retinal axons and terminals revealed these projections with great morphological detail and clarity. In addition, the CTB labeling revealed much more extensive projections than previously realized to all known major retinorecipient areas, particularly in the dorsal and ventral lateral geniculate nuclei, the pretectal region, the nucleus of the basal optic root and the optic tectum. Although the contralateral projections were always much more extensive, ipsilateral projections were clearly present (and in some cases abundant) in all previously known retinorecipient areas. In addition, prominent contralateral (and lesser ipsilateral) retinal projections to several novel target, including the basal hypothalamus, the perirotundal nuclei, the suprapeduncular nucleus and the region between the pretectal nuclei and the tegmentum, were observed. Our results suggest that the use of CTB as a tracer to examine central retinal projections in diverse other species will show that central retinal projections are much more widespread and extensive than previously realized.

Neurotransmitter modulation of Fos- and Jun-like proteins in the turtle retina.

The expression of the Fos and Jun families of nuclear phosphoproteins can be induced by a variety of extracellular stimuli and is known to participate in the transcriptional regulation of target genes. To examine the role of these transcription factors in retinal function, we used polyclonal antisera to localize these protein families in the turtle retina. Fos-like immunoreactivity was in many somata in the inner nuclear and ganglion cell layers. In contrast, Jun-like immunoreactivity was in a smaller number of amacrine cells and many somata in the ganglion cell layer. The monostratified dendritic arbors of one prominent amacrine cell type with Jun-like immunoreactivity were also labeled. There were no dramatic differences in the levels of Fos-like immunoreactivity or Jun-like immunoreactivity between light- or dark-adapted retinas. We examined the effects of excitatory amino acids and gamma-aminobutyric acid (GABA) on the expression of these proteins in vitro. In some experiments, cobalt was used to block synaptic transmission. The excitatory amino acids increased both Fos- and Jun-like immunoreactivity, while GABA generally showed no such stimulatory effect. In cobalt-treated retinas, the same cell types had Jun-like immunoreactivity as seen in the controls, but overall levels of immunoreactivity were increased. In cobalt-treated dark-adapted retinas, some excitatory amino acids increased cytoplasmic Fos-like immunoreactivity in the somata and processes of large cells in the ganglion cell layer. Our results suggest that Fos- and Jun-related proteins may play an important role in the postsynaptic responses to amino acid transmitters in a wide variety of amacrine and ganglion cells.

Determination of fractal dimension of physiologically characterized neurons in two and three dimensions.

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.

Complexity and scaling properties of amacrine, ganglion, horizontal, and bipolar cells in the turtle retina.

In the present study we have evaluated the complexity and scaling properties of the morphology of retinal neurons using fractal dimension as a quantitative parameter. We examined a large number of cells from Pseudemys scripta and Mauremys caspica turtles that had been labeled using Golgi-impregnation techniques, intracellular injection of Lucifer Yellow followed by photooxidation, intracellular injection of rhodamine conjugated horseradish peroxidase, or intracellular injection of Lucifer Yellow or horseradish peroxidase alone. The fractal dimensions of two-dimensional projections of the cells were calculated using a box counting method. Discriminant analysis revealed fractal dimension to be a significant classification parameter among several other parameters typically used for placing turtle retinal neurons in different cell classes. The fractal dimension of amacrine cells was significantly correlated with dendritic field diameters, while the fractal dimensions of ganglion cells did not vary with dendritic field span. There were no significant differences between the same cell types in two different turtle species, or between the same types of neurons in the same species after labeling with different techniques. The application of fractal dimension, as a quantitative measure of complexity and scaling properties and as a classification criterion of neuronal types, appears to be useful and may have wide applicability to other parts of the central nervous system.

Anatomical characterization of retinal ganglion cells that project to the nucleus of the basal optic root in the turtle (Pseudemys scripta elegans).

There is little detailed information about retinal ganglion cells which project to specific central targets in the brain. The present study examined retinal ganglion cells projecting to the nucleus of the basal optic root, a major accessory retinal target in the turtle. These ganglion cells were first selectively labeled using retrograde transport of rhodamine injected stereotaxically into the nucleus of the basal optic root. The number and distribution of the retrogradely labeled cells in the retina was then determined. Some of these retrogradely labeled cells were then injected intracellularly with Lucifer Yellow, photoconverted using diaminobenzidine, and drawn in detail using a camera lucida attachment. There were approximately 1500 ganglion cells in each retina which projected to the nucleus of the basal optic root, of which 20% had cell bodies displaced to the inner nuclear layer. More than 50% of the total population was concentrated in the visual streak region. All ganglion cells projecting to the nucleus of the basal optic root, both normal and displaced, had monostratified dendritic arborizations in stratum 1 of the inner plexiform layer. About 41% of these ganglion cells had elongated dendritic arborizations with distinct orientations, which may suggest a correlation of morphology and function. There were similarities in the overall appearance, and in the type and stratification of the dendritic arborizations of all ganglion cells projecting to the nucleus of the basal optic root. These anatomical similarities are consistent with the previously demonstrated similarities in physiology and may reflect a common role for these ganglion cells in visual processing.