Search

Retinal progenitors in the circumferential marginal zone (CMZ) and Müller glia-derived progenitors have been well described for the eyes of fish, amphibians, and birds. However, there is no information regarding a CMZ and the nature of retinal glia in species phylogenetically bridging amphibians and birds. The purpose of this study was to examine the retinal glia and investigate whether a CMZ is present in the eyes of reptilian species. We used immunohistochemical analyses to study retinal glia, neurons that could influence CMZ progenitors, the retinal margin, and the nonpigmented epithelium of ciliary body of garter snakes, queen snakes, anole lizards, snapping turtles, and painted turtles. We compare our observations on reptile eyes to the CMZ and glia of fish, amphibians, and birds. In all species, Sox9, Pax6, and the glucocorticoid receptor are expressed by Müller glia and cells at the retinal margin. However, proliferating cells were found only in the CMZ of turtles and not in the eyes of anoles and snakes. Similar to eyes of chickens, the retinal margin in turtles contains accumulations of GLP1/glucagonergic neurites. We find that filamentous proteins, vimentin and GFAP, are expressed by Müller glia, but have different patterns of subcellular localization in the different species of reptiles. We provide evidence that the reptile retina may contain nonastrocytic inner retinal glial cells, similar to those described in the avian retina. We conclude that the retinal glia, glucagonergic neurons, and CMZ of turtles appear to be most similar to those of fish, amphibians, and birds., (© 2015 Wiley Periodicals, Inc.)

In spite of the numerous reports on the optic fiber distribution in the optic nerve and tract of vertebrates, there have been few studies of the visual pathway in reptiles. The arrangement of fibers in the optic nerve and tract of the turtle Mauremys leprosa was studied by placing a small granule of carbocyanine dye (DiI or DiA) in one of the four quadrants of the retina. The labeled fibers were traced through transverse sections of the retinofugal pathway with confocal microscopy. Retinal axons displayed a quadrant-specific order along the optic nerve. However, retinal ganglion cell axons were re-organized as they passed through the chiasmatic region of the optic pathway. In the optic tract, the nasal and temporal fibers remained intermingled, but there was segregation of dorsal from ventral fibers. This re-ordering is similar to that described in other vertebrates, suggesting the existence of an evolutionarily conserved mechanism.

In the intact vertebrate central nervous system, the quantal nature of synaptic transmission is difficult to measure because the postsynaptic sites may be distributed along a tortuous dendritic tree that cannot be readily clamped spatially to a uniform potential. Titrating the intact brain's extracellular concentration of calcium ions is also challenging because of its strong buffering mechanisms. In this study, using a whole brain with eye attached preparation, quantal neurotransmission was examined in the turtle brainstem in vitro, by recording from accessory optic system neurons that receive direct input from visually responsive retinal ganglion cells. Unitary EPSPs, evoked by microstimulation of a single ganglion cell, were measured during whole cell current-clamp recordings. In this preparation, the neurons exhibit direction-selectivity, despite the hypoxic conditions. Bath application of cadmium to reduce calcium influx also reduced evoked EPSP amplitudes to that of the spontaneous synaptic events. Statistical analyses indicated that these evoked response amplitudes could be well fitted to a Poisson distribution for most brainstem neurons. Therefore, the spontaneous miniature excitatory synaptic events of approximately 1 mV, as also observed during spike blockade of the retina [Kogo, N., Ariel, M., 1997. Membrane properties and monosynaptic retinal excitation of neurons in the turtle accessory optic system. Journal of Neurophysiology 78, 614-627], are likely responses to the neurotransmitter of single vesicles release by retinal axon terminals.

The histochemistry of reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) and immunoreactivity of neuronal nitric oxide synthase (nNOS-IR) can be demonstrated in various cell types of the vertebrate retina. In this study, we have focused on characterizing the different NADPH-d-positive amacrine cell types in turtle retina. Cryostat sections were examined by confocal laser scanning microscopy for double immunofluorescence with antibodies against nNOS and either GABA or glycine, or by combining histochemistry with immunocytochemistry to obtain triple labeling with NADPH-d, GABA, and glycine. Forty-eight percent of the NADPH-d-labeled amacrine cells colocalized GABA, 52% glycine. Here we show that two morphologically different types of amacrine cell are nNOS/glycine-IR and three types are nNOS/GABA-IR. Antibodies against calretinin, parvalbumin, somatostatin, tyrosine hydroxylase, and choline acetyltransferase did not colocalize with nNOS-IR or NADPH-d-labeled amacrine cells, but 15% of the NOS-labeled amacrine cells showed immunoreactivity against calbindin. Only GABA has been seen to colocalize with NADPH-d in amacrine cells in previous reports in other species. The finding here of glycine colocalizing with NO-containing cells is novel. We suggest that NO, apart from its well known function in gap junction regulation, can also modulate the release of both GABA and glycine in the turtle 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., (Copyright 2000 Wiley-Liss, Inc.)

Microspectrophotometry, electroretinography and behavioural studies have indicated that ultraviolet (UV) light contributes to functional vision in various vertebrate species. Based on behavioural evidence, this was also suggested for turtle vision. In order to reveal the interactions underlying detection of UV light in the distal retina, we recorded intracellularly the photoresponses of cones and horizontal cells in retinas of Pseudemys scripta elegans and Mauremys caspica and calculated the action spectra of these cells under different conditions of adaptation. In the dark-adapted retina, all three types of horizontal cells; luminosity-type, red/green chromaticity-type and yellow/blue chromaticity-type exhibited increased sensitivity in the UV region of the spectrum. However, chromatic adaptation indicated that only the yellow/blue chromaticity-type horizontal cells received excitatory input from UV-sensitive cones with peak sensitivity approximately 360 nm. The enhanced UV sensitivity of luminosity-type horizontal cells probably reflected the beta-band of the long-wavelength sensitive visual pigment as indicated by the action spectra of dark-adapted L-cones. It is suggested that the enhanced UV sensitivity of red/green chromaticity-type horizontal cells reflects the beta-band of the medium-wavelength sensitive visual pigment. Transmission measurements of the optical media (cornea, lens and vitreous) indicated that UV vision can be functional under normal circumstances.

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.

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.

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.

The study of neural interactions in the vertebrate retina carried out after the pioneering studies of Svaetichin has provided important information on the functioning of nerve circuits in the central nervous system. Recently we have investigated the effects of changes of divalent cation concentration on the synaptic transmission between cones and horizontal cells of the turtle retina. Our results seemed apparently in contrast with the classical Ca2(+)-hypothesis of chemical synaptic transmission. Application of low Ca2+ media resulted in a recovery of synaptic transmission after application of divalent cations such as Ca2+, Zn2+ and Ni2+ traditionally considered as Ca2+ channel antagonists. Moreover, in the absence of exogenous divalent cations, low Ca2+ could result in an increase of transmitter release particularly if Mg2+ was omitted from the perfusing medium. These apparently paradoxical results can be reconciled with the postulates of the Ca2(+)-hypothesis of synaptic transmission by taking into account the effects of divalent cations on the fixed charges present at the external surface of cell membrane. It is possible that a similar interpretation could also account for the so-called "Ca2(+)-independent" transmission in other structures of the nervous system.

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.

Pseudo-3-dimensional models and surface renderings of a small group of neighboring cone pedicles in the turtle retina, in which the 5 different spectral types are represented, have been made from scanned serial electron micrographs using a Macintosh personal computer, Autocad and Studio Pro software. The resultant computer generated images clarify the morphological differences between the spectral types and show how they are related to each other via telodendrial connections and different levels of ending in the neuropil of the outer plexiform layer (OPL). The double cone pedicles end highest in the neuropil and the single red and green pedicles lie slightly lower into the OPL with the single blue and ultraviolent (UV)-sensitive cone pedicles coming in from obliquely angled axons to assume positions between the others much more vitread in the OPL. Telodendria interconnect the double and single red and green cones but not the blue and UV-sensitive cones.

Color coding and directional selectivity (DS) of retinal neurons were studied in the Pseudemys turtle by using similar intracellular recording and staining techniques as in the preceding paper (J. Ammermüller and H. Kolb, 1995, J. Comp. Neuronal. 358:1-34). Color-coded responses were elicited by red (621 or 694 nm), green (525 or 514 nm), and blue (455 nm) light flashes. In addition to red/green and yellow/blue types of chromaticity horizontal cells, in our sample of 305 identified cells we found that 17% of bipolar cells, 6.5% of amacrine cells, and 18% of ganglion cells exhibit color-coded responses. DS responses were found in 37% of the tested ganglion cells and 41% of the tested amacrine cells. Two morphologically identified bipolar cell types, B10 and B11, were red-ON/blue-OFF and red-OFF/green, blue-ON, respectively. Of five identified amacrine cell types, three were red-OFF/blue-ON center (A1, A3, A23b), one was red-OFF/green-ON center (A32), and one (A33) was double color-opponent of red-ON/blue-OFF center:red-OFF/blue-ON surround. Five ganglion cell types had variously color-coded centers (G14 and G24) or surrounds (G3 and G18), including one type, G6, that was double color-opponent (red-OFF/green-ON center:red-ON/green-OFF surround). Responses to colors were found primarily in sustained responses of bipolar and ganglion cells. However, in amacrine cells, transient components of the response also showed color dependence. Red-OFF-center responses were found in ganglion cells that were in a position to make connections at the strata 2/3 border with the red-OFF bipolar cell (B11); red-ON-center responses occurred in ganglion cells with branches in stratum 4 of the IPL where the red-ON-center bipolar (B10) ended. Blue-ON-center signals appeared to be processed mainly in strata 1-2/3, and blue-OFF-center signals in strata 3-5 of the IPL, with contributions of amacrine cells and bipolar cells. Labeled DS amacrine cells could be identified as A9, A20, and A22, and ganglion cells as G19, G20, and G24. The latter type (G24) showed DS and color coding. All response types (ON-center, OFF-center, ON-OFF) were encountered. DS amacrine cells were monostratified near the middle of the IPL, whereas DS ganglion cells were mono-, bi-, and multistratified, although all DS ganglion cells had one feature in common: they had dendrites in stratum 1 of the IPL.(ABSTRACT TRUNCATED AT 400 WORDS)

Intracellular recordings and dye injections of Lucifer yellow, horseradish peroxidase, or Neurobiotin were made in bipolar, amacrine, and ganglion cells of the Pseudemys turtle retina. By using a standard light-stimulation protocol in a sample of 375 labeled neurons, we were able to identify morphological and physiological characteristics of 11 types of bipolar cell, 37 types of amacrine cell, and 24 types of ganglion cell. To make sense of these data, we have chosen to group the 72 essentially different neuron types into traditional, functionally significant pathways. In this paper we look at the neuronal types in the inner plexiform layer (IPL) in terms of their contribution to generalized luminosity responses such as sustained ON- or OFF-center and transient ON-OFF ganglion cells; in the companion paper (J. Ammermüller, J.F. Muller, and H. Kolb, 1995, J. Comp. Neurol. 358:35-62) we look at them in terms of their involvement in color opponency and directional selectivity. A functional organization of the turtle IPL into OFF sublaminae (strata 1 and 2) and ON sublaminae (strata 3, 4, and 5), as has been described for other vertebrate retinas, was quite clear for two varieties of OFF-center bipolar cells (B4 and B5) and for all four types of sustained ON-center bipolar cell (B1, B2, B6, and B7). Thus, we found no sustained ON-center bipolar cell terminating in strata 1 and 2. We did, however, see three varieties of sustained OFF-center bipolar cells (B3, B9, and B10) having axon terminals in strata 3-5 (the ON sublamina) in addition to their terminations in stratum 1 or 2 (the OFF sublamina). Monostratified sustained ON- and OFF-center amacrine and ganglion cells rigidly obeyed the border of ON and OFF sublaminae. However, multistratified and diffuse sustained amacrine and ganglion cells could be either ON-center or OFF-center, and they did not strictly obey the border: such ON-center cells always had processes in one of the ON sublaminae (strata 3-5), and the equivalent OFF-center cells always had processes in one of the OFF sublaminae (strata 1 and 2). Monostratified transient amacrine and ganglion cells were concentrated in the middle of the IPL (around stratum 3), whereas bi-, tri-, or multistratified transient amacrine or ganglion cells always had processes in both the ON and the OFF sublaminae.(ABSTRACT TRUNCATED AT 400 WORDS)

The afferent and efferent cortical projections of the dorsal lateral geniculate nucleus (GLD) of adult specimens of the turtle Emys orbicularis were investigated after intraocular or intracortical injections of horseradish peroxidase (HRP), and the distribution of gamma aminobutyric acid (GABA) immunoreactivity in the nucleus was carried out by immunocytochemical techniques, both techniques being combined with light and electron microscopy. In addition, some specimens were prepared for double-labeling of HRP and GABA immunoreactivity, and additional samples impregnated by a rapid Golgi technique. On purely morphological grounds, four types of neurons can be distinguished by light microscopy: two types of large cells in the cell plate which project to the cortex, and two types of smaller cells in the neuropil and optic tract which do not. The small cells are consistently GABA-immunoreactive, while the former are, with extremely rare exceptions, immunonegative for GABA. The supposition that the small neurons of the neuropil are interneurons is supported by electron microscopic observations; these strongly GABA-immunoreactive cells have large plicated nuclei surrounded by a thin layer of cytoplasm poorly endowed with organelles. The dendrites of these cells may contain pleomorphic synaptic vesicles (DCSVs) and appear to be presynaptic to other dendritic profiles. These DCSVs are occasionally contacted by GABA-immunoreactive axon terminals, and more frequently by retinal terminals consistently immunonegative for GABA. The latter, frequently organized in glomeruli, also make synaptic contacts with immunonegative dendrites arising from corticopetal neurons of the cell plate. Two major categories of GABA-immunoreactive axon terminals can be distinguished, and we are led to the conclusion that one of these represents an intrinsic GABAergic innervation of the GLD, while the second is tentatively interpreted as an extrinsic source of GABA to the nucleus, possibly from ventral thalamic structures. The fine structure of the dorsal lateral geniculate nucleus of Emys orbicularis thus shows many similarities with that of mammals.

An immunoreaction against glutamate was used to visualize photoreceptors, bipolar, and ganglion cells in the turtle retina. Incubation of the retina prior to fixation in kainic acid (9 microM) led to selective loss of glutamate-like immunoreactivity in OFF-centre bipolar cells, as judged by the loss of staining in the distal half of the inner plexiform layer. In addition, displaced bipolar cells and ganglion cells lost their immunoreactivity. Incubation of the retina in 2,3-cis piperidine dicarboxylate (1 mM) did not result in noticeable glutamate depletion in any cell but enhanced labelling in displaced bipolar cells. These findings suggest that all displaced bipolar cells in the turtle retina are depolarized by kainic acid and hyperpolarized by 2,3-cis piperidine dicarboxylate.

Networks of physiologically identified H2 horizontal cells in the turtle retina were labeled by intracellular injection of Neurobiotin. We obtained a quantitative description of the neighbourhood relations in the dye-coupled cell mosaics by using the somata as centers for the Voronoi-Delaunay construction. Computational models simulating the experimental data are presented.

A combination of immunocytochemical and biochemical methods was used to study histamine in the turtle retina. Histamine-like immunoreactivity was localized within paraboloids of certain cone photoreceptors by use of two different antisera directed against histamine. Preincubation of eyecups in Ringer's containing 10 microM histamine selectively increased the immunoreactivity of these photoreceptor paraboloids. The present localization of histamine in paraboloids indicated that, although histamine is in photoreceptors of the turtle retina, it may play some metabolic or neuromodulatory role, and not function as a neurotransmitter.

This study examined amacrine cells in the retina of the turtle Pseudemys scripta elegans, which were labelled using an antiserum directed against tyrosine hydroxylase (an enzyme participating in catecholamine synthesis). These cells were investigated using both light and electron microscopy. Labelled somata were located in the inner nuclear layer near the border of the inner plexiform layer. The dendritic arborizations of these neurons were tristratified and arborized in strata 1 and 3 and near the border between strata 4 and 5. Serial tangential sections taken through the entire inner plexiform layer of a 1 mm-2 region in mid-peripheral retina were examined. All of the synapses associated with labelled profiles were counted and classified. The majority (84%) of the synapses involving labelled processes represented output, while the remaining 16% represented synaptic input. The synaptic output of the labelled processes was as follows: 87% onto unlabelled amacrine cells, 4% onto ganglion cells, 9% onto unidentified cell processes. None of the synaptic output from labelled processes was onto bipolar cells. The synaptic input to these labelled cells was from bipolar cells (29%) and from unlabelled amacrine cells (71%). A well labelled amacrine cell was serially sectioned and examined at the ultrastructural level to analyze its synaptic connectivity. Immunoreaction product was located diffusely throughout the cytoplasm and in large vesicles. The synaptic organization of the cell was directed primarily toward output. The labelled processes were postsynaptic and presynaptic to unlabelled amacrine cell processes in strata 1 and 3 and at the border between strata 4 and 5. Synaptic input from bipolar cells was seen exclusively near the border between strata 4 and 5. Labelled processes were presynaptic to ganglion cell processes in stratum 1 and at the border between strata 4 and 5, but not in stratum 3. Quantitative studies suggested that amacrine cell inputs and outputs were evenly distributed across the dendritic arborization, while bipolar cell inputs and outputs to ganglion cells were concentrated on the distal parts of the dendritic arborization. No labelled processes were seen in the outer plexiform layer, indicating that the cells with tyrosine hydroxylase-like immunoreactivity in the turtle retina were true amacrine cells and not interplexiform cells.

Synaptic organization of the internal plexiform layer (IPL) of the retina has been studied electron microscopically in two aquatic and two land Chelonia species. Certain signs in common of the IPL structure have been revealed, they are similar with those in other Vertebrata, as well as common peculiarities inherent in Chelonia of all the four species studied. Any species-specific differences are not detected. In the land Chelonia, unlike the aquatic Chelonia, a relative content of the amacrinal cell synapses is higher in serial combinations. The ratio of the amacrinal cell synapses to all bipolar neurocyte synapses is within the limits 3.1-4.1.

Injections of 125I wheat-germ agglutinin or horseradish peroxidase into the eyes of turtles labeled retrogradely cells in a mesencephalic reticular area lying between the trochlear and the isthmic nuclei. Their number was small and they were found predominantly contralateral to the injected eye. These reticular neurons were not labeled following control injections into the orbital cavity and therefore are considered to project to the retina similar to correspondingly located neurons in some other vertebrates.

The morphology of the neurons that contribute to the inner plexiform layer of the retina of the turtle Pseudemys scripta elegans has been studied by light microscopy of whole-mount material stained by the method of Golgi. Cells have been distinguished on the basis of criteria that include dendritic branching patterns, dendritic morphology, dendritic tree sizes and stratification of processes in the inner plexiform layer. Many of the neurons have dendritic trees oriented parallel to and a few exhibit an orthogonal orientation with the linear visual streak present in the retina of this species. The neurons of the turtle retina have been compared, where possible, with the neurons of the lizard retina as described by Cajal. The findings are discussed in relation to other vertebrate retinas, and correlations are made with recent electrophysiological recordings of the turtle retina. Comments are made with regard to the significance of orientation of neurons relative to the linear visual streak.

The morphology of amacrine cells has been studied in Golgi-stained retinae of the salt water sparid Boops boops. The great variety of size and shapes of amacrine cells has been organized into five classes according to the qualitative parameter 'dendritic architecture', which describes the number, size, course, degree of dichotomy, and varicosity of dendrites. Extent of dendritic field and depth of stratification in the inner plexiform layer, viewed as quantitative variations of a given cell type morphology, complete the criteria for classification. Each amacrine cell is unequivocally defined by a three element code according to the values assumed by the parameters used for the classification. The possible functional implications of dendritic morphology are briefly discussed.

Retinofugal pathways in the painted turtle were examined with autoradiographic and HRP methods. The majority of the retinal fibers decussate at the optic chiasm and course caudally to terminate in 12 regions of the diencephalon and mesencephalon. The pars dorsalis of the lateral geniculate nucleus is the densest target in the thalamus. Two nuclei dorsal to pars dorsalis--the dorsal optic and dorsal central nuclei--receive optic input. Three nuclei ventral to pars dorsalis and retinal targets--the ventral geniculate nucleus, nucleus ventrolateralis pars dorsalis, and nucleus ventrolateralis pars ventralis. Contralateral fibers course through the pretectum where they terminate in nucleus geniculatis pretectalis, nucleus lentiformis mesencephali, nucleus posterodorsalis, and the external pretectal nucleus. Retinal fibers also terminate within the superficial zone of the optic tectum. HRP material demonstrates three optic fiber layers--laminae 9, 12, and 14. Optic fibers leave the main optic tract as a distinct accessory tegmental optic pathway and terminate in the basal optic nucleus. Ipsilateral retinal terminals occur in a pars dorsalis and a pars ventralis of the lateral geniculate nucleus, the dorsal optic nucleus, nucleus posterodorsalis, the basal optic nucleus, and in laminae 9 and 12 of the optic tectum. Rostrally, the ipsilateral tectal fibers occupy two zones along the medial and lateral tectal roof; these zones converge caudally and are continuous along the caudal wall of the tectum.

We have performed a serial-section electron microscopic study of the inner plexiform layer (IPL) of the retina of the turtle Pseudemys scripta elegans. A qualitative and quantitative assessment of the neuropil of the IPL has been done from photomontages taken from the linear visual streak area and peripheral retina. Counts of conventional, ribbon, serial, and reciprocal synapses, of ganglion cell dendrites, and of profiles containing large, dense-cored vesicles were made in five equal-thickness strata in each montage. Averages of these different features were plotted for each stratum in the linear visual streak and compared with peripheral retina. The trend was for stratum 2 to have the highest overall absolute number of amacrine and bipolar interactions, and also of serial synapses, both in the linear visual streak and in peripheral regions. Stratum 4 tended to have the second-highest number of synapses. The total number of synapses for the entire thickness of the IPL, regardless of stratification, is higher in the streak than in the periphery. The total amacrine-to-bipolar-synapse ratio in the IPL is the highest of any vertebrate studied to date (11.0 in the streak and 14.5 in the periphery) but the number of synapses/micron 2 was found to be similar to that reported for other vertebrates. Amacrine-to-amacrine synaptic contacts greatly outnumber other types of synapses; amacrines constitute the principal input to ganglion cells, whereas bipolar output is mainly onto amacrines. The trend for higher numbers of synaptic interactions in strata 2 and 4 of the streak region of the turtle IPL can be correlated with the branching of small-field amacrine and ganglion cells described in Golgi studies (Kolb: Philos. Trans. R. Soc. Lond. B 298:355-393, '82). In peripheral retina, branching of large-field amacrines and a lower number of bipolar pathways may account for the trend for larger numbers of amacrine synapses in strata 2 and 4. Profiles having large, dense-cored vesicles tend to occur most frequently in strata 1 and 5, which correlates well with the stratification in the IPL of the processes of immunoreactive amacrine cells described in other studies.

Sarthy and Lam (1978) described a method for isolation of glial (Müller) cells from turtle retina which involves papain treatment, mechanical dissociation of the retinae, and subsequent separation of the dispersed cells by velocity sedimentation at unit gravity. In order to establish the cytologic integrity and the extent of contamination, we have carried out ultrastructural studies on the isolated cells using scanning and transmission electron microscopy. During the course of these studies we have also examined the effect of papain on the morphology of the retina. Scanning electron microscopic studies show that the isolated cells have a free surface with limited extraneous material attached. The basal portion of the cell is organized as end bulbs with a convoluted surface while the apical region is specialized with long microvilli. Transmission electron microscopy shows that the isolated cells have intact plasma membranes with very few interruptions. The cytoplasm is dense and contains the normal complement of smooth endoplasmic reticulum, microtubules, and filaments. The extent of contamination as estimated from serial sections of a single cell is about 10-15% of the total cell volume. These studies indicate that Müller cells isolated by this technique should be useful for metabolic studies and for characterizing the Müller cell surface.

Cryoultramicroscopy was carried out in the retina of lower vertebrates. Myeloid bodies of the pigment epithelium of turtles and synaptic vesicles and ribbons of frogs were stained negatively. Numerous altered, shaped, dense bodies, containing granular and myelinlike matrices, were found in the retinal pigment epithelium of turtle, and were thought to be lysosome. The advantages and disadvantages of this method are discussed.

The dorsal lateral geniculate complex in turtles receives a bilateral, topographic projection from the retina and projects to the telencephalon. This study examined the morphology of individual retinogeniculate terminals that were filled with horseradish peroxidase by injections in the optic tract or optic tectum. A large number of retinogeniculate terminals were successfully filled and detailed drawings were prepared of 87 terminals. Terminals were classified into three types based on the size and number of varicosities in the terminal, and (if a terminal formed a spatially restricted arbor) the volume of the arbor. Type I retinogeniculate terminals form spatially restricted, large-volume arbors with a low density of large varicosities. Type II retinogeniculate terminals form small volume arbors with a high density of small varicosities. Type III retinogeniculate terminals, in contrast to types I and II, do not form spatially restricted arbors. Rather, they consist of sparsely branched axons that parallel the optic tract and contain scattered en passant varicosities. Plots of the distribution of different terminal types throughout the geniculate complex show that all three terminal types occur throughout the rostrocaudal and mediolateral extents of the complex. However, each terminal type has a preferential distribution with type II terminals being concentrated in the outer half of the neuropile, type I terminals in the inner half of the neuropile, and type III terminals in the cell plate. All three types can arise from axons that continue caudally to terminate in the tectum. These findings raise the possibility that various classes of retinal ganglion cells differ in their mode of termination within the geniculate complex, but the precise relation between the three types of retinogeniculate terminals and the classes of ganglion cells remains to be determined.

Criteria were established whereby the chief and accessory members of double cones, red-, green-, and blue-sensitive single cones, and rods could be distinguished from one another in 1-micrometer sections through the retinas of Pseudemys scripta elegans and Chelydra serpentina. Criteria included the presence of absence of an oil droplet, the size, color, and radial position of the oil droplet, the size and shape of the inner and outer segments, and the shape orientation of the connection between the perikaryon and synaptic terminal of each receptor cell. Using these criteria, the color-specific interconnections between horizontal cells and receptor cells were analyzed using 1-micrometer serial sections through Golgi-stained examples of horizontal cells of each type. The results of this analysis are: (a) H1 cells bodies contact both the red- and green-sensitive members of double cones, as well as red- and green-sensitive single cones. (b) H1 axon terminals contact only the red-sensitive chief members of double cones, red-sensitive single cones, and rods. (c) H2-type cells contact only green- and blue-sensitive single cones. (d) H3-type cells contact only blue-sensitive single cones. (e) H4-type cells contact only the green-sensitive accessory members of double cones. With the exception of H1 cell bodies, all horizontal cells generally contacted every appropriate receptor cell type within their receptive field. A model for the generation of complex horizontal cell light responses is proposed and discussed.

A light microscopical study of the cell types that stain by immunohistochemistry for the synthesizing enzyme for dopamine, tyrosine hydroxylase, has been performed on the retina of the turtle Pseudemys scripta elegans. The immunostain can be localized to a single morphological type of amacrine cell. The cells are like A28 cells of a Golgi classification. They have medium sized dendritic fields that range in diameter from 200 to 700 micron with eccentricity from the visual streak. The amacrines have a tri-stratified dendritic tree with tiers of fine, curved dendrites ramifying in strata S1, lower S2 and the S4/5 border of the inner plexiform layer. We, like others, can find no good evidence that these cells are interplexiform cells. The dopaminergic amacrine cells have a low frequency (approximately 1300-1500 total cells in 130 mm2 retina), with their highest density occurring in the visual streak (60 cells per mm2). The density profiles fall in elliptical isodensity rings from the visual streak towards the peripheral retina. At all points on the retina the dendritic fields maintain a constant coverage factor independent of eccentricity. A comparison of the dopaminergic amacrine cells in the turtle and other vertebrate retinae is made.

The centrifugal innervation of the retina was reinvestigated in albino and pigmented rats with intraocular injections of horseradish peroxidase (HRP), radioactive wheat germ agglutinin (WGA) and proline. No labeled cells were found in the brains injected with HRP and proline, except some eye muscle motoneurons in one case apparently involving orbital contamination from the injection. In the cases injected with WGA and having a survival time of at least two days cells were labeled in the lateral mesencephalic tegmentum, ventral to the parabigeminal nucleus and in the periaqueductal gray. Both these findings are most likely due to transneuronal anterograde-retrograde transport of the tracer through the superior colliculus. The results yielded no compelling evidence for the existence of a direct retinopetal pathway in the rat, which is in contrast to a recently claimed retinal projection originating from the pretectum. Special attention was paid to the labeling in the lateral mesencephalic tegmentum, an area giving rise to retinal projections in various submammalian species. This finding is discussed with regard to the possibility that also in the rat the lateral tegmentum exerts an early influence on visual input, but at the "higher" collicular level and not at the "original" retinal one.

In Golgi preparations of turtle retina, four types of horizontal cells were observed and their morphological characteristics determined in vertical thick sections, whole mount preparations, and reconstructions from serial 1-micrometer sections. H1 consists of a nucleated, stellate cell body (H1CB) and an irregular, tuberous axon terminal (H1AT) connected by a slender axon. Both parts of these cells make contact with receptor cells. H1CB's appear to correspond to "L2-type cells" while H1AT's correspond to "L1-type cells" described in the physiological literature. H2 and H3 are axonless stellate cells which are similar to one another in vertical profile and may occasionally appear similar in horizontal view. In general, the dendritic tree is more densely branched and the density of receptor cell contacts is higher for H2 than for H3. H2-type cells may correspond to "R/G C-type cells." H4 is also an axonless stellate cell type which is smaller than H2 or H3 at equivalent retinal locations. The dendritic fields of H1CB's vary widely, but systematically, in size and shape over the retina. Their size is inversely related to receptor cell density, and the shape of the dendritic tree varies from roughly circular in the central area to elliptical in the periphery of the retina.

Müller cells are the major type of glial cell in the vertebrate retina, and appear to participate in important structural and metabolic functions. Although the morphological features of Müller cells have been extensively studied, their topographic distribution across the retina has not been previously reported. We have used a Müller cell-specific monoclonal antibody, 19-33, to study the distribution of Müller cells in turtle retina. The antibody was obtained during a search for cell type-specific monoclonal antibodies in the rat retina. Immunoblotting studies show that 19-33 reacts with a 58 KDa protein that is present in Müller cells. Immunocytochemical studies with en face sections of turtle retina show that the density of Müller cells is fairly uniform across the retina although there are small regional differences. We estimate that the mean Müller cell density is about 1600 cells mm-2 of turtle retina and that each turtle retina contains about 54,000 Müller cells.

Bistratified amacrine cells of the turtle retina containing enkephalin-like immunoreactivity were examined with the electron microscope with the aid of peroxidase immunocytochemical techniques. Our goal was to determine the nature and the location of the synaptic contacts of these cells and the intracellular localization of the immunoreactivity. There was a diffuse reaction product throughout the cytoplasm which coated the surfaces of all the organelles and a dense reaction product which filled the core of some large cytoplasmic vesicles (130 nm in dia.). These labeled amacrine cells received conventional synaptic contacts from other unlabeled amacrine cells and ribbon synaptic contacts from unlabeled bipolar cells, in both the proximal and distal inner plexiform layer. These enkephalin-positive amacrine cells made conventional synaptic contacts containing unlabeled synaptic vesicles (60 nm in dia.), with ganglion cells in the proximal inner plexiform layer and with bipolar cells in the distal inner plexiform layer. These results suggest that enkephalin-like material coexists with another neurotransmitter within these neurons and that these amacrine cells are able to integrate information from both amacrine cells and bipolar cells and provide synaptic input to bipolar cells, ganglion cells, and possibly other amacrine cells.

Multiple pathways for the transmission of visual information from retina to brain have been described in reptiles, but little is known about their functional organization. These parallel channels begin at the retina, and we have therefore begun to study the functional organization of retinal ganglion cells in the turtle, Pseudemys scripta elegans. This paper describes the numbers and distribution of cells in the ganglion cell layer. To develop criteria for the identification of ganglion cells, we labelled them retrogradely by applying horseradish peroxidase (HRP) to the optic nerve. Ganglion cells were found to vary substantially in size and cytology. In low density areas of the retina, ganglion cells typically have cytoplasm with well developed Nissl substance, a distinct, pale nucleus, and a large nucleolus. In high density areas of retina, ganglion cells are small, densely staining, and gliaform. The average minimum proportion of ganglion cells in the ganglion cell layer is 75--80% of total profiles. No more than five or six percent of profiles in the ganglion cell layer are neurons which do not send an axon into the optic nerve (displaced amacrine cells or intraretinal association cells). The ganglion cell layer of P. s. elegans can be divided into a number of regions on the basis of cell density. Isodensity maps constructed from Nissl-stained, wholemounted retinas indicate that there is an elongated region of high ganglion cell density, the visual streak, which extends from nasal to temporal retina and is oriented such that its long axis follows the horizontal axis of the eye. The streak is aligned with the externally visible iris line. Seen in cross-section, the ganglion cell layer in the streak is three to four cells thick; in nonstreak retina, ganglion cells form only a monolayer of somas. Ganglion cell density drops off more rapidly above the streak than below it. The temporal arm of the streak is both shorter and broader than the nasal arm. There is a peak in ganglion cell density at the midpoint of the streak, in the approximate center of the retina. Here, ganglion cell densities exceed 20,000 cells mm-2. The total number of ganglion cells in the retina is 350,000--390,000.

Using combined physiological and anatomical methods, we have shown that triphasic chromaticity horizontal cells (THC) in the turtle retina make ribbon synaptic contacts with about 20 cones, two-thirds were red-sensitive, while the rest were blue-sensitive. The response of THC to red flashes appeared to be generated via synapses from red-sensitive cones; this interpretation differs from those of the previous studies where only blue-sensitive cones were thought to make synaptic contacts with the THC.

Analyses of the photoresponses and morphologies of ganglion cells in the turtle retina suggests that these cells are of two types: type A ganglion cells receive predominantly bipolar inputs, while type B ganglion cells receive a mixture of inputs from bipolar and amacrine cells. According to these classification criteria, ganglion cells may reflect either activities of the outer or of the inner plexiform layer, respectively. To corroborate the validity of this classification a number of basic properties of ganglion cells were analysed. It was found that double colour opponency was characteristic of type A cells and directional selectivity was performed only by type B cells. In addition, the absolute sensitivity and the firing rate of type A cells were significantly higher than those of type B cells.

Organization of retinal projections to the dorsal lateral geniculate complex in turtles has been studied by means of light and electron microscopic axon tracing techniques. Orthograde degeneration studies with Fink-Heimer methods following restricted retinal lesions show the entire retina has a topologically organized projection to the contralateral dorsal lateral geniculate complex. The nasotemporal axis of the retina projects along the rostrocaudal axis of the geniculate complex; the dorsoventral axis of the retina projects along the dorsoventral axis of the geniculate complex. The projection to the ipsilateral dorsal lateral geniculate complex originates from the ventral, temporal and nasal edges of the retina. The nasotemporal axis of the ipsilateral retina projects along the rostrocaudal axis of the geniculate complex. It was not possible to determine the orientation of the dorsoventral axis of the ipsilateral retina on the geniculate complex. Light microscopic autoradiographic tracing experiments and electron microscopic degeneration experiments show the retinogeniculate projection has a laminar organization. Retinogeniculate terminals are found in both the neuropile and cell plate throughout all three subnuclei of the dorsal lateral geniculate complex but have a distinctive distribution in each subnucleus. In the subnucleus ovalis, they are frequent in both the neuropile and cell plate which forms the rostral pole of the complex. In the dorsal subnucleus, they are most prevalent in the outer part of the neuropile layer, less frequent in the inner part of the neuropile, and rare in the cell plate. In the ventral subnucleus, they are frequent in the outer part of the neuropile but are also common in the inner part of the neuropile and cell plate. These observations point to several principles of geniculate organization in turtles. First, the complex receives projections from the entire contralateral retina and a segment of the ipsilateral retina. It thus has monocular and binocular segments that together receive a topologically organized representation of the binocular visual space and the contralateral monocular visual space. Second, the three geniculate subnuclei receive information from different, specialized regions of the retina and visual space. Subnucleus ovalis receives information from the frontal binocular visual field. The ventral subnucleus receives information from the caudal binocular field. The dorsal subnucleus receives input from the contralateral monocular field. Third, there is a lamination of retinal inputs in the geniculate complex which differs in character within the three subnuclei.(ABSTRACT TRUNCATED AT 400 WORDS)

The entire population of retinal serotoninergic bipolar cells in the turtle Pseudemys scripta elegans was labeled by immunocytochemical methods. This allowed a systematic analysis to be made of the morphological variabilities among a functionally homogeneous neuronal population. The analyzed morphological characteristics included: size of the Landolt club, size of the soma, lateral extension of the ramification within the outer plexiform layer, course of the axon across the inner nuclear layer, pattern of the axonal ramification within the inner plexiform layer, lateral extension of these ramifications, and density of the cells. Whereas characteristics 1-4 and 7 show a morphological variability strictly related to the location of the bipolar cell with respect to the visual streak, a fovealike structure, characteristics 5 and 6 show no such correlation. The size of the soma increases by a factor of 4 from the visual streak toward the periphery. The area covered by the ramification in the OPL increases from 330 micron 2 at the visual streak to 50,000 microns 2 at the dorsal and ventral edges of the retina. The coverage factor remains the same throughout the retina, as well as the area covered by the ramifications in the IPL, which is about 2,000 microns 2. At the visual streak and at 110 degrees ventral and 68 degrees dorsal of the visual streak, the bipolar axons cross the INL perpendicularly. In between the axons take an oblique course leading to an axon-induced shift between input and output of up to 250 microns.(ABSTRACT TRUNCATED AT 250 WORDS)

An electron microscopic histochemical study has been carried out on the localization of acetylcholinesterase (AChE) in the optic tectum of a lizard and a turtle. Several types of AChE-synthetizing elements have been described. In both species these neurons constitute only a fraction of the total neuronal popultio- of the optic tectum. The area of maximum accumulation of AChE activity is in both cases at level of the neuron pil of the stratum fibrosum et griseum superficiale, where most retinal afferents are known to terminate. There is however a peculiar difference in the ultrastructural localization of the enzyme activity in the stratum et fibrosum et griseum superficiale of the two species. While in the turtle most activity is localized inside dendritic structures, a definite prevalence of extracellular localization, including synaptic spaces, has been noticed of the lizard. The main effect of eye deprivation on AChE localization in the optic tectum has been noticed for the lizard, in which a remarkable decrease of extracellular AChE occurs 2 months after contralateral enucleation. In the lizard a peculiar localization of pseudocholinesterase AChE occurs 2 months after contralateral enucleation. In the lizard a peculiar localization of pseudocholinesterase (BuChE) has been in addition demonstrated in the spaces between axon bundles in the stratum opticum and stratum album centrale. The relevance of these findings is briefly discussed also in relation to the existence of cholinergic mechanisms of neurotransmission in the optic tectum.

Co-localization of retrograde transported Nuclear yellow and serotonin immunoreactivity revealed the existence of a central serotonergic projection to the retina in the turtle Pseudemys scripta elegans. This serotonergic retinopetal system consists of only one fiber originating in the contralateral caudal mesencephalon. In the retina, the fiber arborizes exclusively in the temporal hemisphere, covering about one third of the total retinal surface. Interestingly this projecting area lies in the part of the retina, to which binocular perception can be attributed.

The ultrastructural features and synaptic contacts of two types of neurotensin-containing amacrine cells in turtle retina were examined by electron immunocytochemistry, and the retinal peptides were characterized using radioimmunoassay and high-pressure liquid chromatography. The two types of cell were distinguished on the basis of their sizes, dendritic arborizations, synaptic connections and cytoplasmic staining characteristics. Type A cells had lightly labeled cytoplasm and large vertically elongated cell bodies which gave rise to a single primary process which in turn branched and ramified as smooth tapering processes in stratum 3 of the inner plexiform layer. Type A cells received approximately equal synaptic input from amacrine and bipolar cells. Type A amacrines had much more overall synaptic input than synaptic output, and they made conventional synaptic contacts onto bipolar, amacrine, and ganglion cells. Type B cells had a much darker-staining cytoplasm and a smaller cell body which gave rise to numerous delicate beaded dendrites which arborized in strata 3, 4 and 5 of the inner plexiform layer. Type B cells received primarily amacrine and some bipolar cell input. Type B cells had equal amounts of synaptic input and output and they made conventional synaptic contacts onto amacrine, bipolar, and ganglion cells. Whereas there were numerous large vesicles (120 nm diameter) that stained for neurotensin in both types of cells, conventional synaptic vesicles (60 nm diameter) were not labeled. In several cases these large labeled vesicles appeared to fuse with the cell membrane in non-synaptic regions and release their contents into extracellular space, which suggested a non-synaptic release of the neurotensin from type A neurons. Immunochemical and chromatographic studies demonstrated that the neurotensin-related material in retina was indistinguishable from neurotensin found in brain. These results are consistent with a neuroactive role for the neurotensin present within the large vesicles. The differences in the synaptic contacts and dendritic arborizations of the two amacrine cell types suggest they play distinctive functions in visual processing.

Two types of Golgi-stained bipolar cells have been examined by light and electron microscopy to determine the ultrastructure of their synapses with photoreceptors and the spectral type of photoreceptor with which they connect in the retina of the turtle (Pseudemys scripta elegans). We have chosen bipolar cells that correspond in morphology to a center-hyperpolarizing type (H-bipolar) and a center-depolarizing type (D-bipolar) shown in Marchiafava and Weiler's paper (Proc. R. Soc. B 208:103-113, '80). The latter authors recorded intracellular responses and marked their cells with the fluorescent dye Procion yellow so that we have a clear picture of their morphology. These bipolars have been called B4 and B6, respectively, according to our recent classification scheme of Golgi-stained cells. Serial section electron microscopy of two B4 bipolars shows that they made wide-cleft, striated basal junctions with red and green cone pedicles. They connected to six or seven different cone pedicles. The three B6 bipolar cells studied made narrow-cleft, semi-invaginating basal junctions with cone pedicles. The dendrites of B6 bipolars did not become central elements at ribbon synapses although they invaginated toward the synaptic ribbon. Serial section electron microscopy indicated that B6 bipolars contacted three to five cone pedicles also of a red or green cone type. We suggest that in the case of these two varieties of bipolar cell in turtle retina, sign-conserving synapses with cones are wide-cleft basal junctions while sign-inverting synapses are narrow-cleft, semi-invaginating in morphology.

Types and densities of retinal cells with somata located in the outer nuclear layer of the turtle retina were studied with combined light and electron microscopic observations of serial sections. One third of the somata in the outer nuclear layer were found to be those of displaced bipolar cells, while the rest were of those of photoreceptors. Somata were 8-10 micrometers in horizontal diameter for both types of retinal cells.

Antiserum directed against neuropeptide Y selectively labeled certain amacrine cells in the turtle retina. The cell types, sizes, dendritic stratification, regional distribution, and degrees of immunolabeling were examined. The results indicated that three morphologically distinct cell types were labeled: types A, B, and C. Computer rotation of digitized data from camera lucida drawings was used to study dendritic stratification. The type A somata were large (11.5 micron in diameter), well-stained, and located in the third tier of the inner nuclear layer. Type A somata gave rise to well-stained processes which arborized within the inner plexiform layer in strata 1 and 3 and at the border between strata 4 and 5. Processes in stratum 1 were sparse and delicate with small boutons. Processes in stratum 3 were numerous and often coarse, with many large and small boutons. At the border between strata 4 and 5 the processes were frequently numerous but slender, with many small boutons. Occasional immunolabeled processes were found in the ganglion cell layer. The somata of the type B cells were smaller (9.0 micron in diameter) and gave rise to single labeled processes which descended into the inner plexiform layer and divided quickly into two secondary processes. These secondary processes gave rise to lightly labeled dendritic fields which arborized primarily in strata 2 and 4. The type C cells were usually observed at the periphery of the retina and had large somata (12.0 micron in diameter) with simple, but very elongated, dendritic arborizations in strata 1, 4, and 5. Observations also showed that type A and B cells were often found in close proximity to each other and suggested that dendrites of these cells made contact with each other. The labeled neurons were distributed relatively evenly throughout the retina except for the visual streak where they were sparse. This study indicates that neuropeptide Y-like immunoreactivity is found in more than one anatomically distinct class of amacrine cells in the turtle retina.

Directionally selective (DS) and other ganglion cells of the turtle retina were studied in in vitro eyecup preparations by using intracellular recording and staining techniques. DS ganglion cells responded to moving gratings with periodic, depolarizing synaptic potentials in both preferred and null directions. Although depolarizations were usually larger in the preferred than in the null directions, in a few cells spike discharges were directional but depolarizations were not. Directionality could disappear if inappropriate field sizes or grating spatial frequencies were used. Unlike non-DS cells, one-half of the DS cells penetrated showed two sizes of action potentials upon photostimulation. It is proposed that the smaller spikes originated at axonal initial segments and failed to invade the somas actively. DS cells also exhibited postspike depolarizations (PSDs). Three types of DS ganglion cells (ON-OFF, OFF center, and ON center) were identified morphologically with Lucifer yellow injection. Other ganglion cells, which were also recorded from and stained intracellularly, are compared to the DS cells.