Turtle Retina Research Articles

Mentors: Doing science with Alan Hodgkin

Baylor recollects the time he spent with his mentor, Alan Hodgkin, working on phototransduction in turtle retinas.

Interactions between the Serotoninergic System and the Ionotropic GABA Receptors in Frog and Turtle Retina: An Immunofluorescent Study

Interactions between the Serotoninergic System and the Ionotropic GABA Receptors in Frog and Turtle Retina: An Immunofluorescent Study

Correction to: Ultrastructural study of retinal development in the turtle Trachemys scripta elegans

The present study was conducted by using light and transmission electron microscopy to examine the morphologic development of turtle retina from embryonic stage 18 (S18) to S26 (hatching). Particular attention was paid to the formation of functional structures such as neurites, synapses, photoreceptors, among others, and the moment that chemical synapses appear in the outer and inner plexiform layers. The results show that retinal differentiation in the turtle follows the vitreal to scleral morphological differentiation of retinal cells. Moreover, the central region of the optic cup is most advanced compared to the peripheral parts. Early functional plexiform layers, based on appearance of synapses, precede the complete differentiation of photoreceptors. The first synaptic structures occur in the inner plexiform layer before the outer plexiform layer. Receptor outer segments and first synaptic ribbon in receptor synaptic terminals initiate the differentiation at the same time, but final maturation includes dendritic invaginations of bipolar and horizontal cells in the receptor terminals. We assume that at birth, the turtle retina has achieved the ability to see.

Dopamine D2 receptor blockade differentially affects the light-adapted turtle and frog electroretinogram

The effects of dopamine D2-class receptor blockade by sulpiride on the electroretinographic (ERG) b-wave (ON response) and d-wave (OFF response) were investigated in light-adapted turtle (Trachemys scripta elegans) and frog (Rana ridibunda) eyecups. For turtle ERG, sulpiride (240 μM) produced an amplitude increase of the b- and d-waves, while the 40 μM and 120 μM of sulpiride were ineffective. Alternatively, for frog ERG, a well-developed and dose-dependent b- and d-wave amplitude decrease was obtained with 40 μM and 240 μM sulpiride. In both species, 240 μM sulpiride significantly affected the maximal voltage range of the ERG responses without altering their relative sensitivity (determined by the semi-saturation point). The absolute sensitivity of the ON and OFF responses (evaluated by threshold estimation) was not significantly altered for turtle ERG, but it was decreased for frog ERG. The time characteristics of the ERG responses were unchanged in both species. Our results show important differences between dopamine D2-class receptor-mediated pathways in turtle and frog retina (revealed by ERG).

The role of nitric oxide in spectral information processing in the distal turtle retina

Chromaticity type horizontal cells (C-type HCs) are the first retinal neurons exhibiting spectral information processing in cold-blooded vertebrates. The simple input of hyperpolarizing responses of cone photoreceptors is transformed in the C-type HCs into spectral opponent output. Nitric oxide (NO), a known background neuromodulator in the distal retina, was tested here for its effects upon spectral information processing by C-type HCs in the retina of turtle. Photoresponses were intracellularly recorded from C-type HCs, using light stimuli of different wavelength, applied over backgrounds of different wavelengths, and changing retinal NO level. Raising retinal level of NO in darkness by adding the precursor for its synthesis (l-Arginine) augmented the depolarizing photoresponses elicited by long-wavelength light stimuli, and reduced the hyperpolarizing photoresponses elicited by short-wavelength light stimuli. Lowering retinal level of NO by l-NAME, an inhibitor of NO synthesis, induced the opposite effects. However, the total voltage range of operation remained constant regardless of the level of NO. Qualitatively similar effects were observed under background illuminations regardless of background strength and wavelength. Altering retinal level of NO exerted a small effect upon the null wavelength. Our findings are consistent with the known effects of NO upon turtle distal retinal neurons, with the addition of NO strengthening the negative feedback pathway from L-type horizontal cells onto medium-wavelength cones.

Tryptophan Hydroxylase and Serotonin Receptors 5-HT1A, 5-HT2A, 5-HT3A, 5-HT4, 5-HT5A, 5-HT6 and 5-HT7 in Frog and Turtle Retina: an Immunofluorescence Study

Tryptophan Hydroxylase and Serotonin Receptors 5-HT1A, 5-HT2A, 5-HT3A, 5-HT4, 5-HT5A, 5-HT6 and 5-HT7 in Frog and Turtle Retina: an Immunofluorescence Study

Ultrastructural study of retinal development in the turtle Trachemys scripta elegans

The present study was conducted by using light and transmission electron microscopy to examine the morphologic development of turtle retina from embryonic stage 18 (S18) to S26 (hatching). Particular attention was paid to the formation of functional structures such as neurites, synapses, photoreceptors, among others, and the moment that chemical synapses appear in the outer and inner plexiform layers. The results show that retinal differentiation in the turtle follows the vitreal to scleral morphological differentiation of retinal cells. Moreover, the central region of the optic cup is most advanced compared to the peripheral parts. Early functional plexiform layers, based on appearance of synapses, precede the complete differentiation of photoreceptors. The first synaptic structures occur in the inner plexiform layer before the outer plexiform layer. Receptor outer segments and first synaptic ribbon in receptor synaptic terminals initiate the differentiation at the same time, but final maturation includes dendritic invaginations of bipolar and horizontal cells in the receptor terminals. We assume that at birth, the turtle retina has achieved the ability to see.

NEV2lkit: A NEW OPEN SOURCE TOOL FOR HANDLING NEURONAL EVENT FILES FROM MULTI-ELECTRODE RECORDINGS

The analysis and discrimination of action potentials, or "spikes", is a central issue to systems neuroscience research. Here we introduce a free open source software for the analysis and discrimination of neural spikes based on principal component analysis and different clustering algorithms. The main objective is to supply a friendly user interface that links the experimental data to a basic set of routines for analysis, visualization and classification of spikes in a consistent framework. The tool has been tested on artificial data sets, on multi-electrode extracellular recordings from ganglion cell populations in isolated superfused mouse, rabbit and turtle retinas, and on electrophysiological recordings from mouse visual cortex. Our results show that NEV2lkit is very reliable and able to satisfy the experimental demands in terms of accuracy, efficiency and consistency across experiments. It performs fast unit sorting in single or multiple experiments and allows the extraction of spikes from over large time intervals in continuously recorded data streams. The tool is implemented in C++ and runs cross-platform on Linux, OS X and Windows systems. To facilitate the adaptation and extension as well as the addition of new routines, tools and algorithms for data analysis, the source code, binary distributions for different operating systems and documentation are all freely available at http://nev2lkit.sourceforge.net .

Ionotropic purinergic receptors P2X in frog and turtle retina: Glial and neuronal localization

Purinergic signaling is represented in both the peripheral and central nervous system (CNS), and in particular in the retina, which may be regarded as a part of the CNS. While purigenic signaling is relatively well studied in mammalian retinas, little is known about it in retinas of lower vertebrates. The aim of present study was to investigate, using immunocytochemistry, the distribution of purinoreceptors P2X in retinas of frog and turtle, which are appropriate models of the brain neuron-to-glia interactions. The results showed widespread expression of all seven ionotropic purinoreceptors (P2X1–P2X7) in both frog and turtle retinas. They were predominantly expressed in Müller cells, the principal glial cells in the retina. All structures typical of Müller cells: the outer and the inner limiting membranes, the cells bodies in the inner nuclear layer, the radial processes in the inner plexiform layer (IPL), and the so called endfeet (frog) or the orthogonal arrays of particles (turtle) in the ganglion cells layer were immunostained. Colocalizations between P2X1–P2X7 and the glial cell marker Vimentin proved that the immunostaining was in the Müller cells. In addition to the glial staining, neuronal staining was also seen as fine puncta in the inner plexiform layer and by small dots and patches in the outer plexiform layer. Some cell bodies of horizontal, amacrine and ganglion cells were also stained. The results obtained imply that the purinergic P2X receptors may significantly contribute to the neuron-to-glia signaling in retinas of the lower vertebrates.

NR2C and NR2D subunits of NMDA receptors in frog and turtle retina

Glutamate NMDA (N-methyl-d-aspartate) receptors are widely distributed in the central nervous system where they are involved in cognitive processes, motor control and many other functions. They are also well studied in the retina, which may be regarded as a biological model of the nervous system. However, little is known about NR2C and NR2D subunits of NMDA receptors, which have some specific features as compared to other subunits. Consequently the aim of the present study was to investigate their distribution in frog (Rana ridibunda) and turtle (Emys orbicularis) retinas which possess mixed and cone types of retina respectively. The experiments were performed using an indirect immunofluorescence method. Four antibodies directed to NR2C and NR2D subunits of NMDA receptor, as well as three antibodies directed to different splice variants of NR1 subunit, which is known to be obligatory for proper functioning of the receptor, were applied. All antibodies caused well expressed labeling in frog and turtle retinas. The NR2C and NR2D subunits were localized in glial Müller cells, while the NR1 subunit had both neuronal and glial localization. Our results show that glial NMDA receptors differ from neuronal ones in their subunit composition. The functional significance of the NMDA receptors and their NR2C and NR2D subunits, in particular for the neuron–glia interactions, is discussed.

Detection of Motion Direction Using a Model of Turtle Retinal Patch

AbstractUsing a model of the turtle retina, in this paper we show how the model can be used to discriminate between targets that are moving, with a constant speed, along several different directions. Model of retinal cells are chosen from an earlier work of the authors, described in the PhD thesis of the first author, and a patch has been constructed on the visual streak of the retina. Distributions of intensity sensitive ‘A Cells’ and direction sensitive ‘B Cells’ are chosen from data already reported in the literature and our model of the retinal patch follows this distribution. The model patch is exposed to point targets moving at constant speed but along several directions that are separated by 2°. The obtained patch response is analyzed by two different methods discussed in this paper. In the first method the patch responses are low pass filtered and projected onto a vector on the principal component space. Detection is performed using maximum likelihood method assuming that the vector is distributed as a Gaussian random process conditioned on the direction of the target. In the second method, the spike rate is computed as an intensity function of a vector Poisson process. The detection algorithm is based on minimizing the distance between the intensity function of an unknown input to the intensity functions of inputs computed for an array of targets. The detection algorithms presented in this paper have been implemented using ‘a moving time window of detection’ and a window of ‘20 ms’ is presented for the purpose of illustration. Detection results are presented using a root mean square error criterion.

A mammalian melanopsin in the retina of a fresh water turtle, the red-eared slider ( Trachemys scripta elegans)

A mammalian-like melanopsin (Opn4m) has been found in all major vertebrate classes except reptile. Since the pupillary light reflex (PLR) of the fresh water turtle takes between 5 and 10 min to achieve maximum constriction, and since photosensitive retinal ganglion cells (ipRGCs) in mammals use Opn4m to control their slow sustained pupil responses, we hypothesized that a Opn4m homolog exists in the retina of the turtle. To identify its presence, retinal tissue was dissected from seven turtles, and total RNA extracted. Reverse transcriptase-polymerase chain reactions (RT-PCRs) were carried out to amplify gene sequences using primers targeting the highly conserved core region of Opn4m, and PCR products were analyzed by gel electrophoresis and sequenced. Sequences derived from a 1004-bp PCR product were compared to those stored in GenBank by the basic local alignment search tool (BLAST) algorithm and returned significant matches to several Opn4ms from other vertebrates including chicken. Quantitative real-time PCR (qPCR) was also carried out to compare expression levels of Opn4m in different tissues. The normalized expression level of Opn4m in the retina was higher in comparison to other tissue types: iris, liver, lung, and skeletal muscle. The results suggest that Opn4m exists in the retina of the turtle and provides a possible explanation for the presence of a slow PLR. The turtle is likely to be a useful model for further understanding the photoreceptive mechanisms in the retina which control the dynamics of the PLR.

The activity of retinal ganglion cell ensembles in the turtle retina encode velocity of moving objects

The activity of retinal ganglion cell ensembles in the turtle retina encode velocity of moving objects

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

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

Effect of visual experience on the maturation of ON–OFF direction selective ganglion cells in the rabbit retina

Activity-dependent neural plasticity is well known in the development of the visual cortical circuitry. However, the role of neural plasticity in the developing retina is less well understood. In the light of recent findings that light deprivation alters the development of synaptic pathway in the mouse and turtle retinas, we studied whether visual experience is required for the maturation of the ON–OFF direction selective ganglion cells (DSGCs) in the rabbit retina. The DSGCs of rabbits raised under a normal light–dark cycle and in the constant darkness were recorded extracellularly at various postnatal stages. Receptive field properties, such as direction selectivity, velocity tuning, classical center–surround interaction and motion-induced surround inhibition were examined. Recorded cells were subsequently injected with Neurobiotin in order to characterize their morphological features and tracer coupling patterns. Our results revealed that visual experience is not critical for the maturation of the classical receptive field properties of the DSGCs, such as direction selectivity and velocity tuning. However, the dark-reared rabbits showed altered surround inhibition, which is mediated by the amacrine cells of the inner retina. In addition, the DSGCs of both normal- and dark-reared rabbits showed similar dendritic features and tracer coupling patterns. Taken together, this study indicates that visual experience plays a less significant role on the DS circuitry maturation in the retina than in the cortex.

Twelve chromatically opponent ganglion cell types in turtle retina

The turtle retina has been extensively used for the study of chromatic processing mechanisms. Color opponency has been previously investigated with trichromatic paradigms, but behavioral studies show that the turtle has an ultraviolet (UV) channel and a tetrachromatic visual system. Our laboratory has been working in the characterization of neuronal responses in the retina of vertebrates using stimuli in the UV-visible range of the electromagnetic spectrum. In the present investigation, we recorded color-opponent responses from turtle amacrine and ganglion cells to UV and visible stimuli and extended our previous results that UV color-opponency is present at the level of the inner nuclear layer. We recorded from 181 neurons, 36 of which were spectrally opponent. Among these, there were 10 amacrine (5%), and 26 ganglion cells (15%). Morphological identification of color-opponent neurons was possible for two ganglion cell classes (G17 and G22) and two amacrine cell classes (A22 and A23b). There was a variety of cell response types and a potential for complex processing of chromatic stimuli, with intensity- and wavelength-dependent response components. Ten types of color opponency were found in ganglion cells and by adding previous results from our laboratory, 12 types of opponent responses have been found. The majority of the ganglion cells were R+UVBG- and RG+UVB-color-opponents but there were other less frequent types of chromatic opponency. This study confirms the participation of a UV channel in the processing of color opponency in the turtle inner retina and shows that the turtle visual system has the retinal mechanisms to allow many possible chromatic combinations.

Choline acetyltransferase‐immunoreactive neurons in the retina of normal and dark‐reared turtle

Visual deprivation alters retinal-ganglion-cell response properties through changes in spontaneous wave-like activity (Sernagor and Grzywacz [1996] Curr Biol 6:1503-1508). This activity depends on cholinergic synaptic transmission in the turtle retina (ibid; Sernagor and Mehta [ 2001] J Anat 199:375-383). We studied the expression of choline acetyltransferase (ChAT) by immunocytochemistry and Western blot in developing retinas of control and dark-reared turtles. At postnatal day 0 (P0), right after hatching, ChAT-immunoreactivity was present in the ganglion cell layer (GCL), in the inner nuclear layer (INL), and in two distinct bands of the inner plexiform layer (IPL). In P14- and P28-control, and P14- and P28-dark-reared retinas, ChAT-immunoreactivity showed similar patterns to those in P0. However, in P14- and P28-dark-reared retinas the density of ChAT-immunoreactive cells was higher in both the INL and GCL than in P14- and P28-control retinas, respectively. Moreover, Western blotting showed that ChAT protein levels were significantly increased in the dark-reared retina compared to those of the control. TUNEL studies indicated that the difference between normal and dark-reared conditions was not due to extra apoptosis in the former. In turn, proliferating-cell nuclear antigen immunocytochemistry showed no extra proliferating cells in the latter. Finally, nearest-neighbor analysis revealed that the denser population of cholinergic cells in dark-reared turtles formed a mosaic as regular as the normal ones in the GCL. Thus, light deprivation increases the expression of ChAT, increasing the apparent density of cholinergic neurons in the developing turtle retina.

AMPA and kainate receptors in turtle retina: an immunocytochemical study.

1. Glutamate is one of the main neurotransmitters in the retina. Its effects are mediated by a large number of ionotropic and metabotropic receptors.2. The distribution of ionotropic AMPA receptor subunits GluR1-4, kainate receptor subunits GluR5-7 and KA2, as well as delta receptors 1-2 was studied in turtle retina. Indirect immunofluorescence was used to localize the different receptor subunits viewed using light microscopy.3. Results show that all subunits, with exception of GluR1 and GluR5, are widely distributed in the turtle retina.4. They are mainly located in the both plexiform layers of the retina where punctate staining, a sign for synaptic localization, is observed.5. The vast majority of the subunits possess specific pattern of staining that allow to suppose that they are involved in different retinal circuits.6. It can be assumed that the GluR2/3 and GluR6/7 subunits are expressed on the dendrites of a subpopulation of bipolar cells that are immunopositive for alpha-isoform of protein kinase C (PKCalpha). The GluR2/3 and GluR6/7 subunits are most probably used by the same PKCalpha immunopositive bipolar cells in their synaptic contacts with the third-order retinal neurons, the amacrine and ganglion cells.

Heterogeneity of horizontal cells in the chicken retina

Despite numerous reports that different markers are expressed by horizontal cells in the avian retina, it remains unknown whether different types of horizontal cells can be defined by differences in their immunocytochemical profiles. The purpose of this study was to rectify this deficiency. We identified horizontal cells by indirect immunofluorescence with antibodies to calretinin, trkA, GABA, Prox1, AP2alpha, Pax6, islet1, and Lim1 + 2. We found two major groups of horizontal cells, those that express trkA and those that express calretinin. The trkA-immunoreactive (-IR) horizontal cells had small, round somata and robust, bulbous dendritic endings, whereas calretinin-IR horizontal cells had large, polygonal cell bodies and fine, diffuse dendritic endings, both contacting the calbindin-IR pedicles of double cones. Weak gamma-aminobutyric acid (GABA) immunoreactivity was observed only in a few of the trkA-IR horizontal cells, whereas the overlap of calretinin and GABA immunoreactivities was 100%. The majority of trkA-IR horizontal cells expressed islet1, and the majority of calretinin-IR horizontal cells expressed Lim1 + 2, AP2alpha, and Pax6. Islet1 immunoreactivity was observed in a small fraction of calretinin-IR/non-trkA-IR cells. In agreement with previous reports, we detected Prox1 immunoreactivity in all types of horizontal cells. These immunolabeling profiles suggest that there are four immunochemically distinct subtypes of horizontal cells in the postnatal chick retina, which may match the four types that have been observed in Golgi-impregnated pigeon and turtle retinas.

Complex Spike-Event Pattern of Transienton-offRetinal Ganglion Cells

ON-OFF transient ganglion cells of the turtle retina show distinct spike-event patterns in response to abrupt intensity changes, such as during saccadic eye movements. These patterns consist of two main spike events, with the latency of each event showing a systematic dependency on stimulus contrast. Whereas the latency of the first event decreases monotonically with increasing contrast, as expected, the second event shows the shortest latency for intermediate contrasts and a longer latency for high and low contrasts. These spike-event patterns improve the discrimination of different light-intensity transitions based on ensemble responses of the ON-OFF transient ganglion cell subpopulation. Although the discrimination results are far better than chance using either spike counts or latencies of the first spikes, they are further improved by using properties of the second spike event. The best classification results are obtained when spike rates and latencies of both events are considered in combination. Thus spike counts and temporal structure of retinal ganglion cells carry complementary information about the stimulus condition, and thus spike-event patterns could be an important aspect of retinal coding. To investigate the origin of the spike-event patterns in retinal ganglion cells, two computational models of retinal processing are compared. A linear-nonlinear model consisting of separate filters for on and off response components fails to reproduce the spike-event patterns. A more complex cascade filter model, however, accurately predicts the timing of the spike events by using a combination of gain control loop and spike rate adaptation.