Turtle Cones Research Articles

Functional Mechanisms Shaping Lateral Geniculate Responses to Artificial and Natural Stimuli

Functional models of the early visual system should predict responses not only to simple artificial stimuli but also to sequences of complex natural scenes. An ideal testbed for such models is the lateral geniculate nucleus (LGN). Mechanisms shaping LGN responses include the linear receptive field and two fast adaptation processes, sensitive to luminance and contrast. We propose a compact functional model for these mechanisms that operates on sequences of arbitrary images. With the same parameters that fit the firing rate responses to simple stimuli, it predicts the bulk of the firing rate responses to complex stimuli, including natural scenes. Further improvements could result by adding a spiking mechanism, possibly one capable of bursts, but not by adding mechanisms of slow adaptation. We conclude that up to the LGN the responses to natural scenes can be largely explained through insights gained with simple artificial stimuli.

Field sensitivity action spectra of cone photoreceptors in the turtle retina.

1. The Stiles two-colour increment threshold technique was applied to turtle cone photoreceptors in order to derive their field sensitivity action spectra. 2. Photoresponses of cone photoreceptors were recorded intracellularly. Flash sensitivities were calculated from small amplitude (< 1 mV) responses. The desensitizing effects of backgrounds of different wavelengths were measured and the background irradiance needed to desensitize the cone by a factor of 10 (1 log unit) was defined as threshold. The reciprocals of these thresholds were used to construct the field sensitivity action spectrum. 3. The field sensitivity action spectra of long-wavelength-sensitive (L) and medium-wavelength-sensitive (M) cones depended upon the wavelength of the test flash used to measure them. This excludes the possibility that turtle cones can function as single-colour mechanisms in the Stiles sense. 4. In fourteen L-cones, the average wavelength of peak sensitivity of the field sensitivity action spectrum was 613.7 +/- 7.7 nm for the 500 nm test and 635.6 +/- 9.6 nm for the 700 nm test. For six M-cones, these values were 558.5 +/- 6.8 and 628.8 +/- 10.6 nm for the 500 and 700 nm tests, respectively. 5. Two physiological mechanisms are suggested as contributing to the dependency of the field sensitivity action spectrum upon test wavelength. One is based upon the transmissivity properties of the coloured oil droplets, while the other hypothesizes excitatory interactions between cones of different spectral type. 6. Computer simulations of the field sensitivity action spectra indicate that both mechanisms are needed in order to account for the dependency of the field sensitivity action spectrum upon the wavelength of the test flash.

Spectral properties of turtle cones.

Microelectrodes were used to record from red and green cones of the turtle Pseudemys scripta elegans. The purpose of this study was to determine the action spectra of the red and green cone photopigments, and to look closely for direct interactions between the two cone classes. An isolated retina preparation was employed so that cones could be stimulated from the outer segment side, thereby avoiding the oil droplets that reside in the inner segments of many cones and normally filter incident light. In agreement with some previous electrophysiological studies, we found little evidence for significant direct connections between red and green cones. Exceptions to this rule are noted and discussed. Measurements indicate that this result does not appear to be due to a general loss of cone connectivity in the isolated retina preparation. Action spectra of the cone photopigments differed markedly from action spectra reported for cones in the eyecup preparation. In contrast to cones in the eyecup, cones in the isolated retina showed higher short-wavelength sensitivity and had action spectra that were adequately described by photopigment nomograms. A model of cone optical properties suggests that in the eyecup up to about 40% of the light that reaches a cone outer segment may do so without first passing through an oil droplet.

The action spectra of cone photoreceptors in the turtle (Mauremys caspica) retina.

Cone photoreceptors in the turtle retina are involved in intricate neuronal interactions with other retinal neurons that modify the responses of the cones to photons absorbed in their outer segments. Therefore, the action spectra of cones strongly depend upon the conditions of measurements. This study describes an attempt to derive the action spectra of turtle cones which are the least distorted by neuronal interactions. To achieve this goal, the photoresponses of cones and horizontal cells were recorded from the turtle retina under different conditions of adaptation using different patterns of the stimulating test flashes. The sensitivity action spectra, derived from small-amplitude (<1 mV) photoresponses, were strongly affected by the recording conditions indicating the contributions of multiple neuronal inputs. Action spectra, constructed from large criterion photoresponses, were less distorted by neuronal interactions and better described the spectral properties of the "isolated" cones. The action spectra of the hyperpolarizing inputs to chromaticity-type horizontal cells were derived by stimulating these cells with mixtures of a saturating red light and a monochromatic light of different wavelength and intensity. The action spectra were constructed from the intensity of the addend component needed to "pull down" the depolarizing response to the red component by a fixed criterion. These spectra, measured in red/green and yellow/blue C-type horizontal cells, are suggested to best represent the "isolated" M-cones and S-cones, respectively.

Extrinsic current and flash sensitivity in turtle cones

The effects of hyperpolarizing current and background light on intracellular responses of red cones in turtle were compared. Even though a background light always reduced response amplitude, hyperpolarizing current did so in only 25% of the cells studied. When hyperpolarizing current reduced response amplitude it also produced changes in response kinetics and the intensity-response relationships, but these changes differed from those produced by background light. Considerably greater hyperpolarization was required with current than with light to produce equivalent reductions in amplitude. The results suggest that current reduces amplitude by activating a membrane conductance, while background light acts through a different mechanism.

Field sensitivity action spectra of turtle cones

Field sensitivity action spectra of turtle cones

Cholinergic and GABAergic neurons occur in both the distal and proximal turtle retina

Turtle retinas were processed immunocytochemically and histochemically to detect the presence of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and glutamate decarboxylase (GAD). We observed cholinergic and γ-aminobutyric acid (GABA)ergic neurons in the proximal retina, as expected, and in the distal retina as well. ChAT immunoreactivity in the distal retina was observed within the axons and pedicles of numerous cone photoreceptors, suggesting that a population of turtle cone photoreceptors uses ACh as a neurotransmitter. Type L2 horizontal cells were immunoreactive for GAD, and their dendrites invaginated into come pedicles. AChE histochemistry revealed processes within the outer plexiform layer which formed a loosely organized lattice. In the proximal retina, labeling for ChAT and GAD was similar to that reported by previous investigators. Processes from ChAT-labeled amacrine cells in the inner nuclear layer formed a stratum within the distal inner plexiform layer (IPL) (at 16–21% relative IPL depth), and processes from ChAT-labeled amacrines in the ganglion cell layer formed a proximal ChAT stratum (at 55–58% relative IPL depth). In addition, six AChE-labeled bands and five GAD-labeled bands were observed within the IPL of stained retinas. Therefore, we determined that the two broadest AChE-labeled bands and the two broadest GAD-labeled bands overlapped the two labeled ChAT strata. The evidence for cholinergic and GABAergic processes in both the inner plexiform layer and the outer plexiform layer, combined with electrophysiological evidence from other investigators, raises the possibility that distal retinal neurons may be involved in the encoding of directional information.

Light adaptation in turtle cones. Testing and analysis of a model for phototransduction

Light adaptation in cones was characterized by measuring the changes in temporal frequency responses to sinusoidal modulation of light around various mean levels spanning a range of four log units. We have shown previously that some aspects of cone adaptation behavior can be accounted for by a biochemical kinetic model for phototransduction in which adaptation is mediated largely by a sigmoidal dependence of guanylate cyclase activity on the concentration of free cytoplasmic Ca2+, ([Ca2+]i) (Sneyd and Tranchina, 1989). Here we extend the model by incorporating electrogenic Na+/K+ exchange, and the model is put to further tests by simulating experiments in the literature. It accounts for (a) speeding up of the impulse response, transition from monophasic to biphasic waveform, and improvement in contrast sensitivity with increasing background light level, I0; (b) linearity of the response to moderate modulations around I0; (c) shift of the intensity-response function (linear vs. log coordinates) with change in I0 (Normann and Perlman, 1979); the dark-adapted curve adheres closely to the Naka-Rushton equation; (d) steepening of the sensitivity vs. I0 function with [Ca2+]i fixed at its dark level, [Ca2+]i dark; (Matthews et al., 1988, 1990); (e) steepening of the steady-state intensity-response function when [Ca2+]i is held fixed at its dark level (Matthews et al., 1988; 1990); (f) shifting of a steep template saturation curve for normalized photocurrent vs. light-step intensity when the response is measured at fixed times and [Ca2+]i is held fixed at [Ca2+]i dark (Nakatani and Yau, 1988). Furthermore, the predicted dependence of guanylate cyclase activity on [Ca2+] closely matches a cooperative inhibition equation suggested by the experimental results of Koch and Stryer (1988) on cyclase activity in bovine rods. Finally, the model predicts that some changes in response kinetics with background light will still be present, even when [Ca2+]i is held fixed at [Ca2]i dark.

Prolonged depolarization in rods in situ

Intracellular recordings were made from rods in the superfused retina of the marine toad (Bufo marinus). It was found that injection of a brief depolarizing current pulse (0.04-1 nA) evoked a distinctive, long-lasting response, here called "the prolonged depolarization." The response appears to be regenerative, has a stereotypical waveform, is typically about 6 mV in amplitude and 3 s in duration, and has a relatively long recovery period (10-60 s). As a rule, the response cannot be directly evoked by light but the current-evoked response is significantly enhanced in the presence of steady illumination. The light-evoked hyperpolarization and the depolarizing spikes of the rod are both attenuated in the presence of the prolonged depolarization. The prolonged depolarization is not an altered manifestation of the depolarizing spikes of toad rods since both can be recorded simultaneously and steady illumination suppresses the spikes while enhancing the prolonged depolarization. The response is enhanced in chloride-free superfusate and also appears to be enhanced by the use of electrodes containing chloride. The response is markedly shortened in superfusates that lack calcium or contain 1-5 mM cobalt. On this and other evidence, it is suggested that the response may be generated by the sequential action of calcium channels and calcium-activated chloride channels. Although rarely evoked by light, the prolonged depolarization of toad rods is otherwise remarkably similar to the prolonged depolarization of turtle cones. It is proposed that the prolonged depolarization, in contrast to the feedback depolarization of cones, arises from mechanisms common to both rods and cones.

Ionic influences on the prolonged depolarization of turtle cones in situ

1. The effects of ion channel blockers and ion substitutions on the prolonged depolarization of cones in the retina of the turtle (Pseudemys scripta elegans) were studied by intracellular recording. 2. The results of current injection experiments indicate that the prolonged depolarization is regenerative and accompanied by a reduction in the cone's input resistance. 3. The addition of cobalt (5-10 mM) or the removal of extracellular calcium suppressed the prolonged depolarization. Raising extracellular calcium or adding strontium (10 mM) lowered the threshold and increased the duration of the response. 4. Unlike the feedback spikes of turtle cones studied by Piccolino and Gerschenfeld, the prolonged depolarization was not blocked by the organic calcium channel blocker, D600. 5. Adding a calcium chelator, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), to the electrolyte caused a progressive shortening of the prolonged depolarization until it was ultimately abolished. 6. Lowering extracellular sodium or use of the potassium channel blockers tetraethylammonium (TEA) and 4-aminopyridine (4-AP) had little effect on the prolonged depolarization. 7. Removing chloride from the superfusate induced a significant enhancement of the prolonged depolarization. In normal superfusate, the response tended to be of larger amplitude when recorded with electrodes containing chloride [1.5 M KCl + 1.5 M potassium acetate (KA)] rather than KA or potassium methylsulfate (KM) alone. 8. The results suggest that the prolonged depolarization is initiated by the regenerative activation of voltage-sensitive calcium channels and sustained by a calcium-dependent chloride efflux. The present findings are also discussed in relation to the functional significance of the prolonged depolarization and mechanisms for the surround antagonism of cones in situ.

Effects of synaptic blocking agents on the depolarizing responses of turtle cones evoked by surround illumination

The effects of synaptic blocking agents on the antagonistic surround of the receptive field of cone photoreceptors were studied by intracellular recording in the retina of the turtle (Pseudemys scripta elegans). Illumination of a cone's receptive-field surround typically evoked a hybrid depolarizing response composed of two components: (1) the graded synaptic feedback depolarization and (2) the prolonged depolarization, a distinctive, intrinsic response of the cone. The locus of action of synaptic blocking agents was analyzed by comparing their effects on the light-evoked response of horizontal cells, the hybrid cone depolarization evoked by surround illumination, and the pure prolonged depolarization evoked by intracellular current injection. The excitatory amino-acid antagonists, d-O-phosphoserine (DOS) and kynurenic acid (KynA), suppressed the light responses of horizontal cells and eliminated the surround-evoked, hybrid cone depolarization without affecting the prolonged depolarization evoked by current injection. Cobalt at 5-10 mM suppressed horizontal cell responses and thereby eliminated surround-evoked cone depolarizations. Cobalt (5-10 mM) also blocked the current-evoked prolonged depolarization, suggesting that the intrinsic cone mechanisms responsible for the prolonged depolarization are likely to be calcium-dependent. Various GABA agonists and antagonists were found to have no effect on the surround-evoked depolarizations of cones. In contrast, a very low concentration of cobalt (0.5 mM) selectively suppressed the light-evoked feedback depolarization of cones without affecting horizontal cell responses or the current-evoked prolonged depolarization. Cobalt at 0.5 mM thus blocks the light-evoked action of the cone feedback synapse while sparing feedforward synaptic transmission from cones to horizontal cells. The implications of the present work for the possible neurotransmitters used at these synapses is discussed.

Square root intensity coding in turtle cones: Physiological mechanisms

The effect of coupling on the intensity-response functions for full field and for slit stimuli was studied by comparing the shapes of the curves obtained from several strongly coupled red cones of turtle with that of one very weakly coupled cone. The full field V-log I curves could be fitted by a Michaelis-Menten relationship, regardless of the strength of the coupling. For the weakly coupled cone the slit V-log I could also be fitted by a Michaelis-Menten curve. For strongly coupled cones a major portion of the curve (1.2–2 log units of intensity) was better fitted by V ∝ I m. For centered slits “ m” was 0.5. With increased distance between the slit and the center of the receptive field “ m” was found to increase slightly. The results were analyzed in terms of a theory in which the shape of the slit intensity-response curves arises from scattered light progressively recruiting neighboring cone responses. An analytical formulation of this idea is presented and a plausible light distribution function which supports the recruitment hypothesis is derived. A numerical model of the cone network, which includes the effect of scattering and transduction saturation, accounts well for all of the experimental data obtained with single and paired slits.

Signal integration at the pedicle of turtle cone photoreceptors: An anatomical and electrophysiological study

The morphology of the axon which connects the cell body and pedicle of turtle cone photoreceptors was studied by light and electron microscopy. The axon which contains numerous synaptic vesicles, some endoplasmic reticulum, and a few cisternae is basically filled with cytoplasm. The length of the axon is related to the class of cone and varies slightly with retinal location, with axons as short as 3-6 microns found in red cones, and as long as 60 microns in cones containing colorless oil droplets. By simultaneously voltage clamping the cell body and pedicle regions of single isolated cones, we measured the longitudinal axonal resistance and the cell body and pedicle membrane resistances. For each cell studied, the axonal resistance of cones with short axons was lower than the cell and pedicle membrane resistances. Thus, the cell can be considered to be an isopotential structure. However, in some cones with long axons, the axonal, cell body, and pedicle resistances were comparable. The pedicles of these cones, therefore, could act like summing points and may provide a locus for spatial signal integration. Electrical coupling between the principal and accessory members of double cones was also studied. Electron-microscopic observation of the membrane junction between the apposed inner segments of the double cones in the intact retina show narrow segments which resemble gap junctions. However, in every double cone studied in culture, passing currents into one member of the double cone did not result in measurable current flow in the adjacent cell. Thus, the two members of the double cone, isolated from the turtle retina, are not electrically coupled.

Prolonged depolarization in turtle cones evoked by current injection and stimulation of the receptive field surround.

1. Responses evoked by stimulation of the receptive field surround were recorded intracellularly from cone photoreceptors in the retina of the turtle (Pseudemys scripta elegans). 2. A distinctive depolarizing response was evoked by flashing an annulus of light while steadily illuminating the centre of the receptive field. The response, here called 'the prolonged depolarization', was found in 67% of a sample of 125 cones and could reach some 20 mV in amplitude. 3. The prolonged depolarization is characterized by a set of properties which include: the capacity to persist up to 17 s after the flash, a stereotypical waveform, a long period of temporal facilitation, a very narrow dynamic range, and a long refractory period (30-45 s). 4. Depolarizing current pulses (0.01-0.1 nA) evoke a prolonged depolarization which is similar to and functionally interchangeable with that evoked by light. The prolonged depolarization is thus apparently generated by a voltage-sensitive mechanism intrinsic to the cone. 5. Brief depolarizing spikes were recorded in a small fraction of cones. The spikes appear to be dissociable from the prolonged depolarization although both might arise for similar regenerative mechanisms. 6. The prolonged depolarization is typically preceded by a graded, stimulus-locked depolarization which can also be recorded in isolation by flashing annuli of low intensity. The graded depolarization is probably a manifestation of the depolarizing influence arising from synaptic feed-back from horizontal cells first described by Baylor, Fuortes & O'Bryan (1971). 7. It is suggested that the graded depolarization triggers the prolonged depolarization and that complex responses arise from the interaction of these disparate components.

Retinal bipolar cells receive negative feedback input from GABAergic amacrine cells.

Bipolar cells make reciprocal synapses with amacrine cells in the inner plexiform layer; both feedforward connections and feedback connections are present. The physiological properties of the feedback synapse have not been well described. Since some amacrine cells are thought to be GABAergic, we examined bipolar cells for feedback input from gamma-aminobtyric acid (GABA)ergic amacrine cells. Solitary bipolar cells were dissociated enzymatically from the goldfish retina. Cells were voltage clamped with a patch pipette and their GABA sensitivity was examined. GABA evoked responses in all bipolar cells with a large axon terminal, which were identified to be the rod dominant ON type, and in some bipolar cells with a small axon terminal. The highest GABA sensitivity was located at the axon terminal. The least effective dose was as low as 100 nM. A small insignificant response of high threshold was evoked when GABA was applied to the dendrite and soma. GABA increased the Cl conductance and caused membrane hyperpolarization. The bipolar cells had the GABAA receptor coupled with a benzodiazepine receptor. The GABA-evoked response was not susceptible to Co ions, which suppressed the GABA-induced responses in turtle cones by 50% at 5 microM concentration. Incomplete desensitization was observed, suggesting that the GABAergic pathway seems capable of transmitting signals tonically. The present results strongly indicate that the rod-dominant ON-type bipolar cells and some bipolar cells with a small axon terminal receive negative feedback inputs from GABAergic amacrine cells.

Effects of gamma-aminobutyric acid on isolated cone photoreceptors of the turtle retina.

Isolated cones dissociated from the retina of the freshwater turtle were voltage clamped using a single 'patch' pipette electrode. gamma-Aminobutyric acid (GABA) applied ionophoretically to the axon terminal evoked an inward current in cells held at -66 mV when they were recorded with patch pipettes filled with the 'control' pipette solution containing 120 mM-Cl-. Polarity of the GABA-induced current reversed near 0 mV when examined with the pipette filled with the control pipette solution. The reversal potential depended strongly on both external and intrapipette Cl- concentrations ([Cl-]o, and [Cl-]p). The reversal potential agreed with the equilibrium potential for Cl- calculated by the Nernst equation with given [Cl-]o and [Cl-]p. The reversal potential was not affected by concentrations of either external Na or K ions. Voltage responses evoked by GABA were hyperpolarizing from its resting level of about -50 mV immediately after the rupture of the patch membrane. The response polarity reversed into depolarization in a few seconds when [Cl-]p was greater than 24 mM, while hyperpolarizing responses persisted when [Cl-]p was less than 12 mM. Thus, the intracellular Cl- concentration of undisturbed isolated cones was estimated to be between 12 and 24 mM. Cones were desensitized to GABA in the presence of GABA (greater than 100 nM) in the medium, or by a prolonged ionophoretic application. The maximum reduction in response amplitude was about 70% in both experiments. Muscimol was as potent as GABA, while beta-p-chlorophenyl-GABA (baclofen) was ineffective even at 100 microM. GABA was antagonized by bicuculline competitively, and by picrotoxin non-competitively. These observations suggest that turtle cones have GABAA receptors which associate with chloride channels. The present results suggest that GABA, presumably released continuously from monophasic horizontal cells in the dark, would exert a tonic hyperpolarization in red-sensitive and green-sensitive cones. Suppression by light of tonic GABA release would depolarize these types of cones by disinhibition. Disinhibitory depolarization in cones may contribute to the centre surround antagonism in retinal neurones, and to the biphasic colour responses recorded in a subtype of horizontal cells.

The blocking of GABA-induced responses in turtle retinal cones by co ions and related agents

The blocking of GABA-induced responses in turtle retinal cones by co ions and related agents

Mixing of color signals by turtle cone photoreceptors.

The direct mixing of red and green cone signals in the outer plexiform layer of the turtle retina was studied by using intracellular recordings from red cone photoreceptors. Cone photoresponses were a function of the wavelength of the photons that stimulated them, even when small-diameter stimuli were used. The intensity response curves measured with red and green test flashes had different shapes. The kinetics of approximately equal amplitude red and green responses also differed. To quantify the short wavelength input onto red cones, differential chromatic adaptation was used. The relative sensitivity of the red cone to red and green test flashes was a function of the color and intensity of the background illumination; red backgrounds decreased relative red sensitivity, and green backgrounds increased relative red sensitivity. The spectral sensitivity of the additional short wavelength input onto red cones was determined by using differential chromatic adaptation, and was found to peak approximately 550 nm. We conclude that red cones receive an additional excitatory input from green cones (and possibly blue cones). A model of the cone mosaic suggests that approximately 50% of the red cone response (linear range) to a dim green test flash arises from neighboring green cones.

Temporal information processing in cones: Effects of light adaptation on temporal summation and modulation

We have studied the temporal information processing of turtle cones in steady states of light adaptation using intracellular recording techniques. We measured the linear range incremental sensitivity of cones as a function of the stimulus duration. Linear range incremental sensitivity is a function of the background intensity. It is also proportional to the duration of short duration stimuli but is independent of duration for long duration stimuli. The plot of log sensitivity versus log stimulus duration displays two straight line asymptotes; a slope of one for short durations and a slope of zero for long durations. These asymptotes intersect at a time, the critical duration, which decreases with increasing background intensity. Linear systems theory was used to predict these results in addition to the interdependence of critical duration, response kinetics, and sensitivity for any state of adaptation. We have also calculated cone sensitivity as a function of sinusoidal frequency for a variety of background intensities. Correlations between these results and psychophysical studies suggest that the limits on temporal summation established by the cones appear not to be substantially altered by the rest of the retina.

Direct excitatory interactions between cones of different spectral types in the turtle retina.

Cone linear sensitivities to red and green stimuli were measured intracellularly in the dark- and light-adapted turtle retina. Test flashes of small diameter were used to minimize horizontal cell feedback. Light adaptation was achieved with either green or red background illumination. The ratio of cone sensitivities to red and green light depended on the color of the background light and differed from the ratio measured in the dark. Electron microscope studies of Golgi-stained turtle cones revealed direct synaptic connections between red and green cones mediated by cone telodendria. These data indicate that the red cone photoresponse is not univariant as has been previously supposed and suggest that mixing of signals from different spectral classes of cones can occur via direct excitatory connections between cones.