A clockwork hypothesis: synaptic release by rod photoreceptors must be regular

We can see at light intensities much lower than an average of one photon per rod photoreceptor, demonstrating that rods must be able to transmit a signal after absorption of a single photon. However, activation of one rhodopsin molecule (Rh*) hyperpolarizes a mammalian rod by just 1 mV. Based on the properties of the voltage-dependent Ca2+ channel and data on [Ca2+] in the rod synaptic terminal, the 1 mV hyperpolarization should reduce the rate of release of quanta of neurotransmitter by only 20%. If quantal release were Poisson, the distributions of quantal count in the dark and in response to one Rh* would overlap greatly. Depending on the threshold quantal count, the overlap would generate too frequent false positives in the dark, too few true positives in response to one Rh*, or both. Therefore, quantal release must be regular, giving narrower distributions of quantal count that overlap less. We model regular release as an Erlang process, essentially a mechanism that counts many Poisson events before release of a quantum of neurotransmitter. The combination of appropriately narrow distributions of quantal count and a suitable threshold can give few false positives and appropriate (e.g., 35%) efficiency for one Rh*.     

Signal transmission through the dark-adapted retina of the toad (Bufo marinus). Gain, convergence, and signal/noise

Responses to light were recorded from rods, horizontal cells, and ganglion cells in dark-adapted toad eyecups. Sensitivity was defined as response amplitude per isomerization per rod for dim flashes covering the excitatory receptive field centers. Both sensitivity and spatial summation were found to increase by one order of magnitude between rods and horizontal cells, and by two orders of magnitude between rods and ganglion cells. Recordings from two hyperpolarizing bipolar cells showed a 20 times response increase between rods and bipolars. At absolute threshold for ganglion cells (Copenhagen, D.R., K. Donner, and T. Reuter. 1987. J. Physiol. 393:667-680) the dim flashes produce 10-50-microV responses in the rods. The cumulative gain exhibited at each subsequent synaptic transfer from the rods to the ganglion cells serves to boost these small amplitude signals to the level required for initiation of action potentials in the ganglion cells. The convergence of rod signals through increasing spatial summation serves to decrease the variation of responses to dim flashes, thereby increasing the signal-to-noise ratio. Thus, at absolute threshold for ganglion cells, the convergence typically increases the maximal signal-to-noise ratio from 0.6 in rods to 4.6 in ganglion cells.     

Visual Adaptation in the Retina of the Skate

The electroretinogram (ERG) and single-unit ganglion cell activity were recorded from the eyecup of the skate (Raja erinacea and R. oscellata), and the adaptation properties of both types of response compared with in situ rhodopsin measurements obtained by fundus reflectometry. Under all conditions tested, the b-wave of the ERG and the ganglion cell discharge showed identical adaptation properties. For example, after flash adaptation that bleached 80% of the rhodopsin, neither ganglion cell nor b-wave activity could be elicited for 10–15 min. Following this unresponsive period, thresholds fell rapidly; by 20 min after the flash, sensitivity was within 3 log units of the dark-adapted level. Further recovery of threshold was slow, requiring an additional 70–90 min to reach absolute threshold. Measurements of rhodopsin levels showed a close correlation with the slow recovery of threshold that occurred between 20 and 120 min of dark adaptation; there is a linear relation between rhodopsin concentration and log threshold. Other experiments dealt with the initial unresponsive period induced by light adaptation. The duration of this unresponsive period depended on the brightness of the adapting field; with bright backgrounds, suppression of retinal activity lasted 20–25 min, but sensitivity subsequently returned and thresholds fell to a steady-state value. At all background levels tested, increment thresholds were linearly related to background luminance.     

Control of Retinal Sensitivity : I. Light and Dark Adaptation of Vertebrate Rods and Cones

Rods and cones in Necturus respond with graded hyperpolarization to test flashes spanning about 3.5 log units of intensity. Steady background levels hyperpolarize the rods, and the rod responses become progressively smaller as background level is increased. In cones, higher background levels reduce the effectiveness of test flashes, so higher ranges of test intensities are required to elicit the full range of graded responses. When backgrounds are terminated, cones return rapidly, but rods return slowly to the dark potential level. The effects of backgrounds on both rods and cones can be observed at intensities that cause negligible bleaching as determined by retinal densitometry. During dark adaptation, changes are observed in the rods and cones that are similar to those produced by backgrounds. Receptor sensitivities, derived from these results, show that rods saturate, cones obey Weber's law, and sensitization during dark adaptation follows a two-phase time-course.     

Control of Retinal Sensitivity : III. Lateral Interactions at the Inner Plexiform Layer

Both the "on" and the "on-off" ganglion cells in the mudpuppy retina generate graded responses over a narrow range of log test intensities. Sustained full field or surround backgrounds change the range of center log test intensities that elicits the graded response for both cell types. The on-off, but not the on ganglion cells are further affected by moving or flashing surround backgrounds. These cells are hyperpolarized, threshold is elevated, and the entire graded range of response is elicited by a higher range of log center test intensities. Depolarizing activity is elicited in amacrine cells by moving backgrounds that affect the on-off ganglion cells, but bipolar activity is unaffected. These results suggest that the amacrine cells at the inner plexiform layer mediate a third stage of sensitivity control in the retina, increasing threshold for response to change specifically in the on-off ganglion cells.     

Cellular mechanisms that underlie bleaching and background adaptation.

Experiments were performed on rod photoreceptors isolated from the eye of the larval tiger salamander to determine if the same or different mechanisms underlie the desensitization produced by dim background light (background adaptation) and that which persists in the steady state in darkness after a significant fraction of the photopigment is bleached (bleaching adaptation). We have examined adaptational effects after light that bleached between approximately 50% and 95% of the photopigment under conditions which preclude pigment regeneration. The steady-state desensitization, far greater than that predicted by quantum-catch loss, is relieved upon regeneration of the visual pigment with 11-cis retinal. We measured the spread of desensitization along the long axis of the rod after a local bright bleach at one end by comparing responses to dim local test flashes elicited in different regions of the outer segment, before and after bleaching. The space constant for this spread was less than 2.5 microns. We have previously measured the space constant for the longitudinal spread of desensitization during a local dim background in Ambystoma rods to be 7 microns. This is similar to a space constant of 6 microns measured under similar conditions in Bufo rods by Lamb et al. (1981. J. Physiol. 319:463-496). If calcium carries the signal for background desensitization, this difference in space constant for background and bleaching adaptation precludes it as the messenger for the steady component of bleaching adaptation. Experiments with isobutylmethyl xanthine (IBMX) also indicate that Ca2+ as well as c-GMP are unlikely regulators of bleaching desensitization, since elevation of cytosolic levels of both of these internal messengers by IBMX has little effect on sensitivity in bleach-adapted cells. All of our findings are consistent with the notion that bleaching adaptation is not mediated by a freely diffusible cytoplasmic messenger.     

S-Potentials in the Skate Retina : Intracellular recordings during light and dark adaptation

The S-potentials recorded intracellularly from the all-rod retina of the skate probably arise from the large horizontal cells situated directly below the layer of receptors. These cells hyperpolarize in response to light, irrespective of stimulus wavelength, and the responses in photopic as well as scotopic conditions were found to be subserved by a single photopigment with λ_max = 500 nm. The process of adaptation was studied by recording simultaneously the threshold responses and membrane potentials of S-units during both light and dark adaptation. The findings indicate that the sensitivity of S-units, whether measured upon steady background fields or in the course of dark adaptation, exhibits changes similar to those demonstrated previously for the ERG b-wave and ganglion cell discharge. However, the membrane potential level of the S-unit and its sensitivity to photic stimulation varied independently for all the adapting conditions tested. It appears, therefore, that visual adaptation in the skate retina occurs before the S-unit is reached, i.e., at the receptors themselves.     

Intracellular recordings from gecko photoreceptors during light and dark adaptation

Intracellular recordings were obtained from rods in the Gekko gekko retina and the adaptation characteristics of their responses studied during light and dark adaptation. Steady background illumination induced graded and sustained hyperpolarizing potentials and compressed the incremental voltage range of the receptor. Steady backgrounds also shifted the receptor's voltage-intensity curve along the intensity axis, and bright backgrounds lowered the saturation potential of the receptor. Increment thresholds of single receptors followed Weber's law over a range of about 3.5 log units and then saturated. Most of the receptor sensitivity change in light derived from the shift of the voltage-intensity curve, only little from the voltage compression. Treatment of the eyecup with sodium aspartate at concentrations sufficient to eliminate the beta-wave of the electroretinogram (ERG) abolished initial transients in the receptor response, possibly indicating the removal of horizontal cell feedback. Aspartate treatment, however, did not significantly alter the adaptation characteristics of receptor responses, indicating that they derive from processes intrinsic to the receptors. Dark adaptation after a strongly adapting stimulus was similarly associated with temporary elevation of membrane potential, initial lowering of the saturation potential, and shift of the voltage-intensity curve. Under all conditions of adaptation studied, small amplitude responses were linear with light intensity. Further, there was no unique relation between sensitivity and membrane potential suggesting that receptor sensitivity is controlled at least in part by a step of visual transduction preceding the generation of membrane voltage change.     

Membrane current responses of skate photoreceptors

Light-evoked membrane currents were recorded with suction electrodes from the outer segments of individual photoreceptors enzymatically dissociated from the skate retina. The intensity-response relation of dark-adapted cells closely followed a Michaelis function for which a half-saturating response was elicited by a flash intensity that produced about 36 photoisomerizations. Dim-light responses, as well as the early rising phase of the responses to a wide range of flash intensities, could be described by a reaction scheme that involved a series of four first-order delay stages. The number of delay stages required to model the rising phase of the photocurrents did not change in light adaptation. However, background illumination that reduced sensitivity by 1.5 log units, or a bleaching exposure that resulted in a nearly equivalent desensitization, shortened significantly the time scale of the responses. In both instances there were two- to threefold increases in the rate constants of the transitional delays, and almost complete suppression of the tail current that characterized the response of the dark-adapted cell. These findings suggest that although light adaptation alters the gain and kinetics of the transduction mechanism, the nature of the intervening processes is the same in dark- and light-adapted photoreceptors. Moreover, the results show clearly that there is no need to postulate the existence of a second class of cone-like rods to account for the remarkable ability of skate photoreceptors to respond to incremental stimuli presented on "saturating" background fields or after exposure to an intense bleaching light.     

The cGMP-phosphodiesterase and its contribution to sensitivity regulation in retinal rods

We have used the truncated outer segment preparation to measure rod cGMP-phosphodiesterase activity, as well as its modulation by Ca2+, in darkness and in light. The basal enzyme activity in darkness was approximately 0-3 s-1, and was largely independent of Ca2+ concentration from 10 nM to 10 microM. The steady state activity elicited by a step of light (lambda = 520 nm) was strongly enhanced by Ca2+, increasing from approximately 0.005 s-1/(h nu micron-2 s-1) at 10 nM Ca2+ to approximately 0.16 s-1/h nu micron-2 s-1) at 10 microM Ca2+. Based on these measurements, as well as previous measurements on the effects of Ca2+ on rod guanylate cyclase and the cGMP-gated channel, we have calculated the step response-intensity relation for the rod cell in steady state. This relation agrees reasonably well with the relation directly measured from intact rods. We have also evaluated the relative contributions from the three Ca2+ effects to rod sensitivity. At low background light intensities, the Ca2+ modulation of the guanylate cyclase appears to be the most important for sensitivity regulation. At higher light intensities, especially above half-saturation of the response, the Ca2+ modulation of the light-stimulated phosphodiesterase shows a progressively important influence on the light response; it also extends the Weber-Fechner behavior of the cell to higher intensities. The contribution of the Ca2+ modulation of the cGMP-gated channel is slight throughout.     

野外条件下光强对盾叶薯蓣影响的初步研究

通过在野外栽培条件下的笼罩实验(4个光强等级1855～2104,913～1004,525～615,141～215μmol*m-2*s-1),发现光强影响盾叶薯蓣的根状茎发芽率、叶面积、叶片丙二醛(MDA)含量、叶片过氧化物酶活性(POD)、叶片含水量以及整个植株的生物量.弱光可能因带入的热能少而对根状茎发芽不利.叶片含水量随光照强度的降低而增多.叶面积随光照强度的减小而增加,在525～615μmol*m-2*s-1光强下,盾叶薯蓣叶片的MDA含量最低,POD活性最低,地上生物量最高,对于地下部分而言,最适光强是913～1004μ*mol*m-2*s-1,在此光强下,根状茎生物量增加近3倍.故在生产中,一定程度的强光逆境是有利的.     

Equivalence of background and bleaching desensitization in isolated rod photoreceptors of the larval tiger salamander

Psychophysical experiments have shown an equivalence between sensitivity reduction by background light and by bleaches for the human scotopic system. We have compared the effects of backgrounds and bleaches on the light-sensitive membrane-current responses of isolated rod photoreceptors from the salamander Ambystoma tigrinum. The quantum catch loss was factored out from the desensitization due to bleaching to give the fraction of "extra" desensitization due to adaptation. For backgrounds, desensitization is well described by the Weber/Fechner equation. The extra desensitization after bleaches can also be described by the Weber/Fechner equation, if an "equivalent" background produced by bleaching is made linearly proportional to the fraction of pigment bleached. A background which produces an extra desensitization of a factor of two is equivalent to a fractional bleach of approximately 6%. Equivalent background and bleaching desensitizations were associated with similar reductions in circulating current. There is a linear relation between log flash sensitivity and decrease in circulating current. Equivalent background and bleaching desensitizations were associated with similar increases in cGMP phosphodiesterase and guanylate cyclase activity. These were inferred from membrane current changes after steps into lithium or IBMX solutions. There were also similar reductions in the integration times of dim flash responses for equivalent desensitizations produced by backgrounds and bleaches. These results suggest that the equivalence between background and bleaching found psychophysically may arise at the very earliest stages of visual processing and that these two processes of desensitization have similar underlying mechanisms.     

Melanopsin-Derived Visual Responses under Light Adapted Conditions in the Mouse dLGN

A direct projection from melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) reaches the primary visual thalamus (dorsal lateral geniculate nucleus; dLGN). The significance of this melanopsin input to the visual system is only recently being investigated. One unresolved question is the degree to which neurons in the dLGN could use melanopsin to track dynamic changes in light intensity under light adapted conditions. Here we set out to address this question. We were able to present full field steps visible only to melanopsin by switching between rod-isoluminant ‘yellow’ and ‘blue’ lights in a mouse lacking cone function (Cnga3^-/-). In the retina these stimuli elicited melanopsin-like responses from a subset of ganglion cells. When presented to anaesthetised mice, we found that ~25-30% of visually responsive neurones in the contralateral dLGN responded to these melanopsin-isolating steps with small increases in firing rate. Such responses could be elicited even with fairly modest increases in effective irradiance (32% Michelson contrast for melanopsin). These melanopsin-driven responses were apparent at bright backgrounds (corresponding to twilight-daylight conditions), but their threshold irradiance was strongly dependent upon prior light exposure when stimuli were superimposed on a spectrally neutral ramping background light. While both onset and offset latencies were long for melanopsin-derived responses compared to those evoked by rods, there was great variability in these parameters with some cells responding to melanopsin steps in <1 s. These data indicate that a subset of dLGN units can employ melanopsin signals to detect modest changes in irradiance under photopic conditions.     

The role of steady phosphodiesterase activity in the kinetics and sensitivity of the light-adapted salamander rod photoresponse

We investigated the kinetics and sensitivity of photocurrent responses of salamander rods, both in darkness and during adaptation to steady backgrounds producing 20-3,000 photoisomerizations per second, using suction pipet recordings. The most intense backgrounds suppressed 80% of the circulating dark current and decreased the flash sensitivity approximately 30-fold. To investigate the underlying transduction mechanism, we expressed the responses as a fraction of the steady level of cGMP-activated current recorded in the background. The fractional responses to flashes of any fixed intensity began rising along a common trajectory, regardless of background intensity. We interpret these invariant initial trajectories to indicate that, at these background intensities, light adaptation does not alter the gain of any of the amplifying steps of phototransduction. For subsaturating flashes of fixed intensity, the fractional responses obtained on backgrounds of different intensity were found to "peel off" from their common initial trajectory in a background-dependent manner: the more intense the background, the earlier the time of peeling off. This behavior is consistent with a background-induced reduction in the effective lifetime of at least one of the three major integrating steps in phototransduction; i.e., an acceleration of one or more of the following: (1) the inactivation of activated rhodopsin (R*); (2) the inactivation of activated phosphodiesterase (E*, representing the complex G(alpha)-PDE of phosphodiesterase with the transducin alpha-subunit); or (3) the hydrolysis of cGMP, with rate constant beta. Our measurements show that, over the range of background intensities we used, beta increased on average to approximately 20 times its dark-adapted value; and our theoretical analysis indicates that this increase in beta is the primary mechanism underlying the measured shortening of time-to-peak of the dim-flash response and the decrease in sensitivity of the fractional response.     

VA opsin,melanopsin, and an inherent light response within retinal interneurons

BACKGROUND: Although photoreception is best understood in rods and cones, it is increasingly clear that these are not the only photoreceptive cells of the vertebrate retina. While considerable attention has been paid to the role of melanopsin in the generation of intrinsic light sensitivity in the retinal ganglion cells of mammals, nothing is known about the photoreceptive capacity of the horizontal cells of the fish retina in which both VA opsin and melanopsin are expressed. As yet, there has been little more than speculation as to the physiological function of these opsins within local retinal circuit neurons. RESULTS: VA opsin and melanopsin have been isolated and localized within the well-characterized cyprinid retina of the roach (Rutilus rutilus). Parallel electrophysiological studies identified a novel subtype of horizontal cell (HC-RSD) characterized by a depolarizing response that fits an opsin photopigment with a lambda(max) of 477 nm. The HC-RSD cells mediate responses to light that are characterized by long integration times, well beyond those observed for rods and cones. Significantly, HC-RSD responses persist when the conventional photoreceptor inputs are saturated by background light. CONCLUSIONS: The syncytium of coupled horizontal cells has long been considered to provide a signal of overall retinal irradiance. Our data suggest that this light information is, at least in part, derived from a population of intrinsically photosensitive VA opsin and/or melanopsin horizontal cells.     

Control of Retinal Sensitivity : II. Lateral Interactions at the Outer Plexiform Layer

Test stimuli, presented at the center of the bipolar cell receptive field, spanning less than 2 log units of intensity, elicit the full range of graded response. The intensity range of test stimuli that elicits the graded response depends upon the background conditions. A higher range of log test intensities is required to elicit the graded bipolar response in the presence of surround backgrounds. But surround backgrounds can also serve to unsaturate the bipolar response and thereby increase sensitivity under certain conditions. The results suggest that a second stage of sensitivity-control is mediated by the horizontal cell system at the outer plexiform layer, concatenated with the effects of adaptation in the photoreceptors.     

Slow PIII component of the carp electroretinogram

The slow PIII component of the electroretinogram (ERG) was studied in the isolated, aspartate-treated carp retina. Although the latter is richly populated with cones, slow PIII appeared to reflect almost exclusively the activity of rods; e.g. the spectral sensitivity of the potential paralleled closely the rod pigment curve, its operating range (i.e. the V-log I curve) was limited to 3 log units above absolute threshold, and raising background intensities to photopic levels produced saturation of the increment threshold function without evidence of a cone-mediated segment. Only after bleaching away a significant fraction of the porphyropsin was it possible to unmask a small photopic contribution to slow PIII, as evidenced by a displacement in the action spectrum to longer wavelengths. The spatial distribution of the slow PIII voltage within the retina (Faber, D.S. 1969. Ph.D. Thesis. State University of New York. Buffalo, N.Y.; Witkovsky, P.J. Nelson, and H. Ripps. 1973. J. Gen Physiol. 61:401) and its ability to survive aspartate treatment indicate that this potential arises in the Muller (glial) fiber. Additional support for this conclusion is provided by the slow rise time (several seconds) and long temporal integration (up to 40s) of the response. In many respects the properties of slow PIII resemble those of the c-wave, a pigment epithelial response also subserved by rod activity. On the other hand, the receptoral (fast PIII) and the b-wave components of the ERG behave quite differently. Unlike slow PIII, response saturation could not be induced, since both potentials are subserved by cones when the stimulus conditions exceed the limits of the scotopic range. Receptors appear to govern light adaptation at photopic background levels; both fast PIII and b-wave manifest identical incremental threshold values over this range of intensities. However, under scotopic conditions, the sensitivity of the b-wave is affected by luminous backgrounds too weak to alter fast PIII threshold, indicating a postreceptoral stage of adaptation.     

Desensitization and recovery of phototropic responsiveness in Arabidopsis thaliana.

Phototropism is induced by blue light, which also induces desensitization, a partial or total loss of phototropic responsiveness. The fluence and fluence-rate dependence of desensitization and recovery from desensitization have been measured for etiolated and red light (669-nm) preirradiated Arabidopsis thaliana seedlings. The extent of desensitization increased as the fluence of the desensitizing 450-nm light was increased from 0.3 to 60 micromoles m-2 s-1. At equal fluences, blue light caused more desensitization when given at a fluence rate of 1.0 micromole m-2 s-1 than at 0.3 micromole m-2 s-1. In addition, seedlings irradiated with blue light at the higher fluence rate required a longer recovery time than seedlings irradiated at the lower fluence rate. A red light preirradiation, probably mediated via phytochrome, decreased the time required for recovery from desensitization. The minimum time for detectable recovery was about 65 s, and the maximum time observed was about 10 min. It is proposed that the descending arm of the fluence-response relationship for first positive phototropism is a consequence of desensitization, and that the time threshold for second positive phototropism establishes a period during which recovery from desensitization occurs.     

Non-image-forming ocular photoreception in vertebrates

It has been accepted for a hundred years or more that rods and cones are the only photoreceptive cells in the retina. The light signals generated in rods and cones, after processing by downstream retinal neurons (bipolar, horizontal, amacrine and ganglion cells), are transmitted to the brain via the axons of the ganglion cells for further analysis. In the past few years, however, convincing evidence has rapidly emerged indicating that a small subset of retinal ganglion cells in mammals is also intrinsically photosensitive. Melanopsin is the signaling photopigment in these cells. The main function of the inner-retina photoreceptors is to generate and transmit non-image-forming visual information, although some role in conventional vision (image detection) is also possible.     

On the role of horizontal cells in the mechanism of retinal adaptation

We recorded by intracellular means responses of horizontal cells of the turtle retina to light increase and decrease of different values against the starting adapting level. In measuring these responses, curves reflecting the dependence of membrane potential deflection on light intensity (amplitude characteristics — ACh) were plotted. It is demonstrated that the ACh of transitional processes (on- and off-peaks) is considerably steeper than ACh of the plateau of the potential, but embraces a much smaller range of light intensities (slightly more than 1 log. un.). During a change in intensity of the adapting background (up to 3 log. un.), the ACh of transitional processes shifts along the scale of light intensities in such a way that its steep part remains in the zone of adapting light. We followed the dynamics in time of ACh shift after the transition from one adapting brightness to another. The ACh of total impulse response was plotted for ganglionic cells of the turtle at different intensities of adapting light. Comparison of these curves with the ACh of horizontal cells shows that its peripheral components are responsible for adaptive shifts of ACh of the visual system and that horizontal cells play an important role in the mechanism of adaptation. It is hypothesized that adaptive ACh shifts are the consequence of positive feedback between the horizontal cells and receptors.Institute of Problems of Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 1, No. 2, pp. 210–218, September–October, 1969.