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Effects of excitatory amino acids and their antagonists on the light response of luminosity and color-opponent horizontal cells in the turtle (Pseudemys scripta elegans) retina.

Both kainic acid (KA) and N-methyl-d-aspartatic acid (NMDA) depolarize luminosity-type horizontal cells (L-type H cells) in normal turtle retina. The presence of both NMDA and non-NMDA receptors for excitatory amino acids (EAAs) on these cells was highlighted by an unusual effect of the noncompetitive NMDA-antagonist, MK-801. In retinas that had been exposed to MK-801, the action of NMDA was irreversibly altered to one of hyperpolarization, while the depolarizing effect of KA was unaltered. The aim of the present study was to further characterize these receptors on L-type H cells and to extend the investigation to color-opponent H cells (C-type H cells). Intracellular recording was used to study the effects of KA, NMDA, MK-801, the competitive NMDA antagonists, 2-amino-5-phosphonopentanoic acid (AP5) and 2-amino-7-phosphonoheptanoic acid (AP7), and the nonspecific EAA antagonist, kynurenic acid (KYN) on the light responses of L-type and C-type H cells in turtle retina. The effects of combinations of these drugs were also studied. In L-type H cells the agonists caused depolarization and loss of light response, KYN caused hyperpolarization and loss of light response, and MK-801, AP5 or AP7 had no direct effect. However, application of NMDA following MK-801, AP5 or AP7, but not KYN, caused hyperpolarization and loss of light response. The depolarizing effect of KA was unaltered by these antagonists. These data confirm the presence of an unusual NMDA receptor on L-type H cells. In the case of red/green C-type H cells, application of KA caused loss of responses to both red and green light, with loss of green responses preceding loss of red responses. NMDA initially removed responses to both red and green light. The most striking effect of NMDA was seen during early washout where the responses to red were reversed (hyperpolarizing). These responses eventually recovered their normal polarity. These results suggest that the depolarizing response of C-type H cells to red light is mediated by L-type H cells, but not via inhibition of the excitatory input from green cones to C-type H cells.

2-Amino-5-phosphonovalerate↗

Glutamate antagonists that block hyperpolarizing bipolar cells increase the release of dopamine from turtle retina.

Some neurochemical features of the neuronal circuitry regulating dopamine release were examined in the retina of the turtle, Pseudemys scripta elegans. Glutamate antagonists that block hyperpolarizing bipolar cells, such as 2,3 piperidine dicarboxylic acid (PDA), produced dose-dependent dopamine release. In contrast, the glutamate agonist 2-amino-4-phosphonobutyric acid (APB), which blocks depolarizing bipolar cell responses with high specificity, had no effect on the release of dopamine. The gamma-aminobutyric acid (GABA) antagonist, bicuculline, also produced potent dose-dependent release of dopamine. The release of dopamine produced by PDA was blocked by exogenous GABA and muscimol, suggesting that the PDA-mediated release process was polysynaptic and involved a GABAergic synapse interposed between the bipolar and dopaminergic amacrine cells. The only other agents that produced dopamine release were chloride-free media and high extracellular K+; in particular, kainic acid and glutamate itself were ineffective. These results suggest that the primary neuronal chain mediating dopamine release in the turtle retina is: cone----hyperpolarizing bipolar cell----GABAergic amacrine cell----dopaminergic amacrine cell.

Aminobutyrates↗

Dynamic changes in the receptive fields of L1-type horizontal cells in the retina of the turtle Mauremys caspica.

The resistances of the horizontal cell syncytium in the vertebrate retina are modulated in a time-dependent fashion during light stimulation. Therefore, the spatial properties of horizontal cells are expected to change with time after the illumination conditions are altered. This study was designed to investigate time- and intensity-dependent changes in the receptive-field properties of L1-type horizontal cells in the turtle Mauremys caspica. Photoresponses were elicited by monochromatic (650 nm) light stimuli of 2-s duration covering retinal spots of different radii. The length constants were derived from the relationships between amplitude and spot radius that were constructed for different time intervals after onset of the light stimulus. For a given stimulus intensity, the length constant transiently increased to a peak value and then slowly recovered to a plateau level. When the length constant was compared to the amplitude of the response to full-field illumination for the entire duration of the light stimulus, an ellipse-like curve was obtained indicating that for a given membrane potential, two different values of the length constant could be obtained. Dopamine considerably reduced the size of the receptive fields but did not affect the time-dependent changes in the length constant. These results indicate that changes in the membrane resistance underlie short-term modulation of the receptive-field properties of turtle L1-type horizontal cells after onset of a light stimulus.

Animals↗

Light adaptation and color opponency of horizontal cells in the turtle retina.

Chromaticity-type (C-type) horizontal cells of the turtle retina receive antagonistic inputs from cones of different spectral types, and therefore their response to background illumination is expected to reflect light adaptation of the cones and the interactions between their antagonistic inputs. Our goal was to study the behavior of C-type horizontal cells during background illumination and to evaluate the role of wavelength in background adaptation. The photoresponses of C-type horizontal cells were recorded intracellularly in the everted eyecup preparation of the turtle Mauremys caspica during chromatic background illuminations. The voltage range of operation was either reduced or augmented, depending upon the wavelengths of the background and of the light stimuli, while the sensitivity to light was decreased by any background. The response-intensity curves were shifted to brighter intensities and became steeper as the background lights were made brighter regardless of wavelength. Comparing the effects of cone iso-luminant backgrounds on the Red/Green C-type horizontal cells indicated that background desensitization in these cells could not solely reflect background adaptation of cones but also depend upon response compression/expansion and changes in synaptic transmission. This leads to wavelength dependency of background adaptation in C-type horizontal cells, that is expressed as increased light sensitivity (smaller threshold elevation) and improved suprathreshold contrast detection when the wavelengths of the background and light stimuli were chosen to exert opponent effects on membrane potential.

Adaptation, Ocular↗

Morphology of the turtle accessory optic system.

Neural signals of the moving visual world are detected by a subclass of retinal ganglion cells that project to the accessory optic system in the vertebrate brainstem. We studied the dendritic morphologies and direction tuning of these brainstem neurons in turtle (Pseudemys scripta elegans) to understand their role in visual processing. Full-field checkerboard patterns were drifted on the contralateral retina while whole-cell recordings were made in the basal optic nucleus in an intact brainstem preparation in vitro. Neurobiotin diffused into the neurons during the recording and was subsequently localized in brain sections. Neuronal morphologies were traced using appropriate computer software to analyze their position in the brainstem. Most labeled neurons were fusiform in shape and had numerous varicosities along their processes. The majority of dendritic trees spread out in a transverse plane perpendicular to the rostrocaudal axis of the nucleus. Neurons near the brainstem surface were often oriented tangential to that surface, whereas more cells at the dorsal side of the nucleus were oriented radial to the brainstem surface. Further analysis of Nissl-stained neurons revealed the largest neurons are located in the rostral and medial portions of the nucleus although neurons are most densely packed in the middle of the nucleus. The preferred directions of the visual responses of the neurons in this sample did not correlate with their morphology and position in the nucleus. Therefore, the morphology of the cells in the turtle accessory optic system appears dependent on its position within the nucleus while its visual responses may depend on the synaptic inputs that contact each cell.

Animals↗

The role of potassium conductance in the generation of light responses in Müller cells of the turtle retina.

Müller cells are highly permeable to potassium ions and play a major role in maintaining potassium homeostasis in the vertebrate retina during light-evoked neuronal activity. Potassium fluxes across the Müller cell's membrane are believed to underlie the light-evoked responses of these cells. We studied the potassium currents of turtle Müller cells in the retinal slice and in dissociated cell preparations and their role in the genesis of the light-evoked responses of these cells. In either preparation, the I-V curve, measured under voltage-clamp conditions, consisted of inward and outward currents. A mixture of cesium ions, TEA, and 4-AP blocked the inward current but had no effect on the outward current. Extracellular cesium ions alone blocked the inward current but exerted no effect on the photoresponses. Extracellular barium ions blocked both inward and outward currents, induced substantial depolarization, and augmented the light-evoked responses, especially the OFF component. Exposing isolated Müller cells to a high potassium concentration did not cause any current or voltage responses when barium ions were present. In contrast, application of glutamate in the presence of barium ions induced a small inward current that was associated with a substantially augmented depolarizing wave relative to that observed under control conditions. This observation suggests a role for an electrogenic glutamate transporter in generating the OFF component of the turtle Müller cell photoresponse.

4-Aminopyridine↗

Photoreceptor coupling in turtle retina.

Photoreceptors in the isolated turtle retina of two species of turtle, Chelydra serpentina and Pseudemus scripta elegans, were penetrated with double-barrel electrodes. Physiological responses were recorded through one barrel and Neurobiotin tracer was injected from the other. Intracellular injection of Neurobiotin revealed patterns of tracer-coupled photoreceptors. Both the patterns of tracer coupling and the electrophysiology suggest a high degree of specificity of connections. Rods seem to be coupled only to rods and green and red cones seem to be coupled to cones of the same spectral type. Receptive-field profiles, measured with a thin, sharply focused slit of light, often had well-defined peaks and troughs in sensitivity. We have taken advantage of this observation and used the position of a peak in sensitivity to locate the position on the retina of a coupled cell. In one rod, it was possible to correlate physiological and morphological data and to show that the peaks in the physiological receptive field occurred at positions on the retina where there were dye-coupled cells. This provides direct evidence that gap junctions produce the physiological coupling between rods.

Animals↗

UV responses in the retina of the turtle.

To study processing of UV stimuli in the retina of the turtle, Trachemys dorbignii, we recorded intracellular responses to spectral light from 89 cells: 54 horizontal (47 monophasic, five (R/G) biphasic and two (Y/B) triphasic), 14 bipolar, 12 amacrine, and nine ganglion cells. Spectral sensitivities were measured with monochromatic flashes or with the dynamic constant response method in dark or chromatic adapted states. Stray light and second-order harmonics were also measured. (1) All cells responded to UV stimuli, although none had maximum sensitivity in the UV. (2) Most horizontal, bipolar, and amacrine cells had red-peaked spectral sensitivities. (3) Red adaptation of all monophasic horizontal cells indicated a single red input, except one that had additional peaks in the blue and UV. (4) Responses of biphasic and triphasic horizontal cells to UV light were always hyperpolarizing. Opposition between hyperpolarizing and depolarizing responses at long wavelengths indicates that UV responses were not due to the beta band of red receptors. (5) An unstained spectrally opponent bipolar cell hyperpolarized in the center to green light and antagonistically depolarized in the surround to UV, blue, and green flashes, but hyperpolarized to red. (6) All dark-adapted amacrine cells were red-peaked monophasic cells, but red adaptation broadened their spectral-sensitivity curves or displaced their peaks. An A15, an A18, and an A24 wide-field amacrine cell were stained. (7) A G15 bistratified ganglion cell is shown here for the first time to be spectrally opponent. This UVB/RG cell depolarized to UV and blue and hyperpolarized to red and green. It differs from previously reported turtle ganglion cells in being color opponent in the entire field, not only in the surround, and in showing spatial opponency.

Animals↗

Different types of synapses with different spectral types of cones underlie color opponency in a bipolar cell of the turtle retina.

Electrophysiologically, color-opponent retinal bipolar cells respond with opposite polarities to stimulation with different wavelengths of light. The origin of these different polarities in the same bipolar cell has always been a mystery. Here we show that an intracellularly recorded and HRP-injected, red-ON, blue/green-OFF bipolar cell of the turtle retina made invaginating (ribbon associated) synapses exclusively with L-cones. Non-invaginating synapses resembling wide-cleft basal junctions were made exclusively with M-cones. Input from S-cones was not seen. From these results we suggest sign-inverting transmission from L-cones at invaginating synapses via metabotropic glutamate receptors, and sign-conserving transmission from M-cones at wide-cleft basal junctions via ionotropic receptors. To explain the pronounced blue sensitivity of the bipolar cell, computer simulations were performed using a sign-conserving input from a yellow/blue chromaticity-type (H3) horizontal cell. The response properties of the red-ON, blue/green-OFF bipolar cell could be quantitatively reproduced by this means. The simulation also explained the asymmetry in L- and M-cone inputs to the bipolar cell as found in the ultrastructural analysis and assigned a putative role to H3 horizontal cells in color processing in the turtle retina.

Animals↗

Propagating waves in visual cortex: a large-scale model of turtle visual cortex.

This article describes a large-scale model of turtle visual cortex that simulates the propagating waves of activity seen in real turtle cortex. The cortex model contains 744 multicompartment models of pyramidal cells, stellate cells, and horizontal cells. Input is provided by an array of 201 geniculate neurons modeled as single compartments with spike-generating mechanisms and axons modeled as delay lines. Diffuse retinal flashes or presentation of spots of light to the retina are simulated by activating groups of geniculate neurons. The model is limited in that it does not have a retina to provide realistic input to the geniculate, and the cortex and does not incorporate all of the biophysical details of real cortical neurons. However, the model does reproduce the fundamental features of planar propagating waves. Activation of geniculate neurons produces a wave of activity that originates at the rostrolateral pole of the cortex at the point where a high density of geniculate afferents enter the cortex. Waves propagate across the cortex with velocities of 4 microm/ms to 70 microm/ms and occasionally reflect from the caudolateral border of the cortex.

Algorithms↗

Function of the dorsal and medial cortex of turtles in learning.

The effects of damage to the dorsal and medial cortex of turtles were investigated in two experiments. In the first, damage to the dorsal cortex disrupted acquisition and reversal of a go-no-go discrimination but had no effect on retention of the discrimination if it had been learned preoperatively. Medial cortex damage had no effect. In the second experiment, dorsal cortex damage impaired acquisition, but not extinction or reacquisition, of a discrete-trial keypress. Again, medial cortex damage had no effect. The results suggest that the dorsal cortex is involved in learning in turtles.

Afferent Pathways↗

Release of endogenous excitatory amino acids from turtle photoreceptors.

Responses to light are transmitted from photoreceptors to second-order retinal neurons by chemical synapses that may use an excitatory amino acid (EAA) as the neurotransmitter. This hypothesis is based primarily on the pharmacological actions of EAA agonists and antagonists on the membrane potentials and light responses of second-order neurons. But the release of endogenous EAAs, which is a critical criterion for the identification of EAAs as transmitters, has not been demonstrated. Here we report the use of outside-out membrane patches excised from rat hippocampal neurons to detect the release of EAAs from synaptic terminals of isolated turtle photoreceptors. Electrical stimulation of or application of lanthanum chloride to photoreceptors induced an increase in the frequency of opening of 50-pS channels in the patches. These channels were identified as the class of glutamate-activated channels that are also gated by aspartate and NMDA (N-methyl-D-aspartate). In several photoreceptor-patch pairs, spontaneous channel activity was observed near the synaptic terminals. These results provide strong evidence to support the hypothesis that both rods and cones of the turtle use an EAA as their neurotransmitter.

Acetylcholine↗

Evolutionary distinctiveness of the endangered Kemp's ridley sea turtle.

The endangered Kemp's ridley sea turtle (Lepidochelys kempi) nests almost exclusively at a single locality in the western Gulf of Mexico, whereas the olive ridley (L. olivacea) nests globally in warm oceans. Morphological similarities between kempi and olivacea, and a geographical distribution that "...makes no sense at all under modern conditions of climate and geography", raise questions about the degree of evolutionary divergence between these taxa. Analysis of mitochondrial (mt) DNA restriction sites shows that Kemp's ridley is distinct from the olive ridley in matriarchal phylogeny, and that the two are sister taxa with respect to other marine turtles. Separation of olive and the Kemp's ridley lineages may date to formation of the Isthmus of Panama, whereas the global spread of the olive ridley lineage occurred recently. In contrast to recent examples in which molecular genetic assessments challenged systematic assignments underlying conservation programmes, our mtDNA data corroborate the taxonomy of an endangered form.

Animals↗

Local genetic structure within two rookeries of Chelonia mydas (the green turtle).

We used multilocus minisatellite DNA fingerprinting to examine the local genetic structure within nesting populations of green turtles (Chelonia mydas) in Tortuguero, Costa Rica and Melbourne, Florida, USA. In the Tortuguero population, there was a significant negative correlation between genetic similarity of pairs of nesting females and the distance between their nest sites both within years (r2 = 0.273; P < 0.001) and between years (r2 = 0.578; P < 0.001). Of the 122 female pairs scored for Tortuguero, 12.3 per cent had genetic similarity values resembling those of mother-offspring pairs. In the Melbourne population, however, no relationship between genetic similarity and distance was found (r2 = 0.017; P = 0.075). The distance-related genetic structure of the Tortuguero population indicates that these females exhibit low levels of dispersal from natal sites, and that nestmates return independently to nest near their natal sites. The lack of a similar structure in the Melbourne population suggests that females from this population may not return to natal sites with comparable precision. High levels of mortality among nests, hatchlings or maturing turtles produced in the Melbourne rookery may also be responsible for the absence of distance-related local genetic structure.

Animals↗

Time course of anoxia-induced increase in cerebral blood flow rate in turtles: evidence for a role of adenosine.

The exceptional ability of the turtle brain to survive prolonged anoxia makes it a unique model for studying anoxic survival mechanisms. We have used epi-illumination microscopy to record blood flow rate in venules on the cortical surface of turtles (Trachemys scripta). During anoxia, blood flow rate increased 1.7 times after 45-75 min, whereupon it fell back, reaching preanoxic values after 115 min of anoxia. Topical superfusion with adenosine (50 microM) during normoxia caused a 3.8-fold increase in flow rate. Superfusing the brain with the adenosine receptor blocker aminophylline (250 microM) totally inhibited the effects of both adenosine and anoxia, while aminophylline had no effect on normoxic flow rate. None of the treatments affected systemic blood pressure. These results indicate an initial adenosine-mediated increase in cerebral blood flow rate during anoxia, probably representing an emergency response before deep metabolic depression sets in.

Adenosine↗

On the mechanism of lithium-induced renal tubular acidosis: studies in the turtle bladder.

The mechanism whereby lithium impairs urinary acidification was investigated in vitro with the bladder of the fresh water turtle. The effect of lithium was compared to that of choline or cesium. In the presence of 1% carbon dioxide, lithium (40 mM in the mucosa) failed to alter hydrogen ion secretion. Lithium also caused a significant decrease in sodium transport and in potential difference. Lithium failed to increase the passive loss of hydrogen ions from the mucosal solution, indicating that it does not affect the permeability of the mucosa to hydrogen ions. Bicarbonate secretion also was unchanged in lithium-treated hemibladders. Under open-circuit conditions, lithium caused a significant decrease in potential difference and, hence, caused a significant decrease in hydrogen ion secretion. Restoration of the potential difference, in the presence of lithium, to control levels caused a return of hydrogen ion secretion to the original value. These data demonstrate that in the turtle baldder lithium inhibits hydrogen ion secretion solely by reducing the potential difference and suggest that in vivo lithium causes an acidification defect not by directly inhibiting the hydrogen ion pump or causing a hydrogen ion backleak but by inhibiting sodium transport, thereby decreasing the favorable electric gradient for hydrogen ion secretion.

Acidosis, Renal Tubular↗

Cholinergic inhibition of urinary acidification by the turtle bladder.

The effect of carbachol on urinary acidification by the turtle bladder in vitro was studied. Carbachol inhibited urinary acidification in a dose-dependent fashion, with half maximal inhibition occurring at 4.5 x 10(-5) M. The effect of carbachol on urinary acidification could be totally prevented by atropine, indicating that the inhibition is mediated through a muscarinic receptor. Carbachol inhibited hydrogen ion secretion by decreasing the active proton conductance and not by altering the proton motive force. Carbachol failed to increase passive loss of hydrogen ion from the mucosa. Carbachol increased calcium uptake by the turtle bladder; this increase in calcium uptake could be prevented by pretreatment with atropine, pentobarbital, or lanthanum. Pentobarbital or lanthanum blunted the inhibitory effect of carbachol on hydrogen ion secretion. In the presence of low extracellular calcium (0.2 mM), carbachol failed to increase calcium uptake but caused a significant inhibition of hydrogen ion secretion. In the presence of normal calcium concentration, carbachol caused a significant efflux of calcium. These data demonstrate that carbachol inhibits urinary acidification and suggest that the mechanism of this inhibition may be related, at least in part, to changes in cytosolic calcium.

Animals↗

Multiple paternity and female-biased mutation at a microsatellite locus in the olive ridley sea turtle (Lepidochelys olivacea).

Multiple paternity in the olive ridley sea turtle (Lepidochelys olivacea) population nesting in Suriname was demonstrated using two microsatellite loci, viz., Ei8 and Cm84. The large number of offspring sampled per clutch (70 on average, ranging from 15 to 103) and the number of alleles found at the two loci (18 and eight alleles, respectively) enabled unambiguous assessment of the occurrence of multiple paternity. In two out of 10 clutches analysed, the offspring had been sired by at least two males, which was confirmed at both loci. In both clutches, unequal paternity occurred: 73% and 92% of the offspring had been sired by the primary male. The probability of detecting multiple paternity was 0.903, and therefore there is a small chance that multiple paternity occurred but remained undetected in some of the eight clutches that appeared to be singly sired. Analysis of 703 offspring revealed a high mutation rate for locus Ei8 (micro = 2.3 x 10(-2)) with all 33 mutations occurring in maternal alleles. In particular, one allele of 274 bp mutated at a high frequency in a clutch to which the mother contributed the allele, but in another clutch where the father contributed the same allele, no such mutations were observed. Inferred allele-specific mutation rates for Ei8 and expected numbers of mutations per clutch confirmed that maternal alleles for Ei8 are more likely to mutate in the olive ridley sea turtle than paternal alleles. Possible explanations are discussed.

Alleles↗