Electroretinogram Characteristics and the Spectral Mechanisms of the Median Ocellus and the Lateral Eye in Limulus polyphemus

Electrical responses (ERG) to light flashes of various wavelengths and energies were obtained from the dorsal median ocellus and lateral compound eye of Limulus under dark and chromatic light adaptation. Spectral mechanisms were studied by analyzing (a) response waveforms, e.g. response area, rise, and fall times as functions of amplitude, (b) slopes of amplitude-energy functions, and (c) spectral sensitivity functions obtained by the criterion amplitude method. The data for a single spectral mechanism in the lateral eye are (a) response waveforms independent of wavelength, (b) same slope for response-energy functions at all wavelengths, (c) a spectral sensitivity function with a single maximum near 520 mµ, and (d) spectral sensitivity invariance in chromatic adaptation experiments. The data for two spectral mechanisms in the median ocellus are (a) two waveform characteristics depending on wavelength, (b) slopes of response-energy functions steeper for short than for long wavelengths, (c) two spectral sensitivity peaks (360 and 530–535 mµ) when dark-adapted, and (d) selective depression of either spectral sensitivity peak by appropriate chromatic adaptation. The ocellus is 200–320 times more sensitive to UV than to visible light. Both UV and green spectral sensitivity curves agree with Dartnall's nomogram. The hypothesis is favored that the ocellus contains two visual pigments each in a different type of receptor, rather than (a) various absorption bands of a single visual pigment, (b) single visual pigment and a chromatic mask, or (c) fluorescence. With long duration light stimuli a steady-state level followed the transient peak in the ERG from both types of eyes.     

The Spectral Sensitivities of Single Cells in the Median Ocellus of Limulus

The spectral sensitivities of single Limulus median ocellus photoreceptors have been determined from records of receptor potentials obtained using intracellular microelectrodes. One class of receptors, called UV cells (ultraviolet cells), depolarizes to near-UV light and is maximally sensitive at 360 nm; a Dartnall template fits the spectral sensitivity curve. A second class of receptors, called visible cells, depolarizes to visible light; the spectral sensitivity curve is fit by a Dartnall template with λ_max at 530 nm. Dark-adapted UV cells are about 2 log units more sensitive than dark-adapted visible cells. UV cells respond with a small hyperpolarization to visible light and the spectral sensitivity curve for this hyperpolarization peaks at 525–550 nm. Visible cells respond with a small hyperpolarization to UV light, and the spectral sensitivity curve for this response peaks at 350–375 nm. Rarely, a double-peaked (360 and 530 nm) spectral sensitivity curve is obtained; two photopigments are involved, as revealed by chromatic adaptation experiments. Thus there may be a small third class of receptor cells containing two photopigments.     

Neural Organization of the Median Ocellus of the Dragonfly : II. Synaptic structure

Two types of presumed synaptic contacts have been recognized by electron microscopy in the synaptic plexus of the median ocellus of the dragonfly. The first type is characterized by an electron-opaque, button-like organelle in the presynaptic cytoplasm, surrounded by a cluster of synaptic vesicles. Two postsynaptic elements are associated with these junctions, which we have termed button synapses. The second synaptic type is characterized by a dense cluster of synaptic vesicles adjacent to the presumed presynaptic membrane. One postsynaptic element is observed at these junctions. The overwhelming majority of synapses seen in the plexus are button synapses. They are found most commonly in the receptor cell axons where they synaptically contact ocellar nerve dendrites and adjacent receptor cell axons. Button synapses are also seen in the ocellar nerve dendrites where they appear to make synapses back onto receptor axon terminals as well as onto adjacent ocellar nerve dendrites. Reciprocal and serial synaptic arrangements between receptor cell axon terminals, and between receptor cell axon terminals and ocellar nerve dendrites are occasionally seen. It is suggested that the lateral and feedback synapses in the median ocellus of the dragonfly play a role in enhancing transients in the postsynaptic responses.     

Neural Organization of the Median Ocellus of the Dragonfly : I. Intracellular electrical activity

Intracellular responses from receptors and postsynaptic units have been recorded in the median ocellus of the dragonfly. The receptors respond to light with a graded, depolarizing potential and a single, tetrodotoxin-sensitive impulse at "on." The postsynaptic units (ocellar nerve dendrites) hyperpolarize during illumination and show a transient, depolarizing response at "off." The light-evoked slow potential responses of the postsynaptic units are not altered by the application of tetrodotoxin to the ocellus. It appears, therefore, that the graded receptor potential, which survives the application of tetrodotoxin, is responsible for mediating synaptic transmission in the ocellus. Comparison of pre- and postsynaptic slow potential activity shows (a) longer latencies in postsynaptic units by 5–20 msec, (b) enhanced photosensitivity in postsynaptic units by 1–2 log units, and (c) more transient responses in postsynaptic units. It is suggested that enhanced photosensitivity of postsynaptic activity is a result of summation of many receptors onto the postsynaptic elements, and that transients in the postsynaptic responses are related to the complex synaptic arrangements in the ocellar plexus to be described in the following paper.     

Leishmania amazonensis Infection Induces Changes in the Electrophysiological Properties of Macrophage-like Cells

Whole cell patch-clamp recordings were used to study the electrical properties of the macrophage-like cell line J774.1, after infection with Leishmania amazonensis. Infection induced a significant increase in cell size and membrane capacitance, suggesting that parasite invasion leads to the addition of plasma membrane to the host cell. By 24 hr after infection, the host cell membrane potential was significantly more hyperpolarized than control cells, and this difference remained for the subsequent 72 hr post-infection. The hyperpolarization was paralleled by an increase in the density of inward rectifying K⁺ currents. The shape of the conductance vs. voltage curve, the kinetic properties and the pharmacological profile of these currents were not significantly altered by infection. These results suggest that infection by L. amazonensis causes an increase in the number of functional inward rectifying K⁺ channels, leading to hyperpolarization of the host cell membrane. Received: 19 January 1999/Revised: 20 April 1999     

Electrophysiological measurement of the number of rhodopsin molecules in single Limulus photoreceptors

Two partly independent electrophysiological methods are described for measuring the number of rhodopsin molecules (R) in single ventral photoreceptors. Method 1 is based on measurements of the relative intensity required to elicit a quantal response and the relative intensity required to half-saturate the early receptor potential (ERP). Method 2 is based on measurements of the absolute intensity required to elicit a quantal response. Both methods give values of R approximately equal to 10(9). From these and other measurements, estimates are derived for the surface density of rhodopsin (8,000/micrometer2), the charge movement during the ERP per isomerized rhodopsin (20 X 10(-21) C), and the half-time for thermal isomerization of rhodopsin (36yr).     

The Spectral Sensitivities of Single Receptor Cells in the Lateral, Median, and Ventral Eyes of Normal and White-Eyed Limulus

Spectral sensitivity curves can be distorted by screening pigments. We have determined whether this is true for Limulus polyphemus by determining, from receptor potentials recorded using intracellular microelectrodes, spectral sensitivity curves for normal animals and for white-eyed animals (which lack screening pigment). Our results show: (a) In median ocelli, the curve for UV-sensitive receptor cells peaks at 360 nm and does not depend on the presence of screening pigment, (b) The curve for ventral eye photoreceptors is identical to that for retinular cells from the lateral eyes of white-eyed animals and peaks at 520–525 nm. (c) In normal lateral eyes, when the stimulating light passes through screening pigment, the curve indicates relatively more sensitivity in the red region of the spectrum than does the curve for white-eyed animals. Therefore, the screening pigment is probably red-transmitting, (d) In median ocelli, the curve for visible-sensitive cells peaks at 525 nm and is approximately the same whether the ocelli are from normal or white-eyed animals. However, the curve is significantly broader than that for ventral eyes and for lateral eyes from white-eyed animals.     

Electrophysiological basis for the spatial dependence of the inhibitory coupling in the Limulus retina

A technique for measuring, with total optical isolation, the inhibition between two individual receptor units in the Limulus lateral eye is described. The extracellular responses of pairs of units were recorded, using light piping microelectrodes. The inhibitory coupling between two units was found to be nonlinear and describable by a simple hyperbolic equation written in terms of saturation rate (S), half saturation (H), and threshold (ft). By plotting reciprocal frequencies, the data could be linearized and compared for different pairs of units. The magnitude of inhibition (in terms of S and H) was found to decrease monotonically as the anatomical distance between receptors increased. An electrical model of the inhibitory system was developed which accounts for many of the properties of the observed inhibitory interactions. Using the equations from the model and the experimental data, it is shown that the "electrical distances" (which are computed in terms of space constants lambda) of the inhibitory synapses from the impulse-generating region of the test unit are directly related to the anatomical distance between receptors. It is also shown that "synaptic strength" is relatively constant with separation. The electrical distances of the inhibitory synapses range from about 0.1lambda to 0.25lambda for adjacent units to greater than 0.5lambda for units seven to nine receptors away. It is concluded that the nonlinear character of the inhibitory coupling is attributable to synaptic effects, and that the decrease of inhibition with distance between receptors is caused primarily by an increase in the electrical distance of the inhibitory synapses from the test unit.     

Fluctuations of the Impulse Rate in Limulus Eccentric Cells

Fluctuations in the discharge of impulses were studied in eccentric cells of the compound eye of the horseshoe crab, Limulus polyphemus. A theory is presented which accounts for the variability in the response of the eccentric cell to light. The main idea of this theory is that the source of randomness in the impulse rate is "noise" in the generator potential. Another essential aspect of the theory is that the process which transforms the generator potential "noise" into the impulse rate fluctuations may be treated as a linear filter. These ideas lead directly to Fourier analysis of the fluctuations. Experimental verification of theoretical predictions was obtained by calculation of the variance spectrum of the impulse rate. The variance spectrum of the impulse rate is shown to be the filtered variance spectrum of the generator potential.     

Spectral Sensitivity of the Planarian Ocellus

The ocellar potential (OP) of planaria was recorded using microelectrode techniques. The action spectrum and spectral sensitivity of the OP are described. Maximum OP sensitivity was found with 508 nm light. A moderate increase in sensitivity to blue light was observed. This is typical of many invertebrate photoreceptors and was shown, by selective chromatic adaptation, not to indicate the presence of a second pigment.     

Two Light-Induced Processes in the Photoreceptor Cells of Limulus Ventral Eye

The dark-adapted current-voltage (I-V) curve of a ventral photoreceptor cell of Limulus, measured by a voltage-clamp technique, has a high slope-resistance region more negative than resting voltage, a lower slope-resistance region between resting voltage and zero, and a negative slope-resistance region more positive than 0 v. With illumination, we find no unique voltage at which there is no light-induced current. At the termination of illumination, the I-V curve changes quickly, then recovers very slowly to a dark-adapted configuration. The voltage-clamp currents during and after illumination can be interpreted to arise from two separate processes. One process (fast) changes quickly with change in illumination, has a reversal potential at +20 mv, and has an I-V curve with positive slope resistance at all voltages. These properties are consistent with a light-induced change in membrane conductance to sodium ions. The other process (slow) changes slowly with changes in illumination, generates light-activated current at +20 mv, and has an I-V curve with a large region of negative slope resistance. The mechanism of this process cannot as yet be identified.     

Stochastic Properties of Discrete Waves of the Limulus Photoreceptor

In the dark-adapted photoreceptor of the horseshoe crab, Limulus, transient discrete depolarizations of the cell membrane, discrete waves, occur in total darkness and their rate of occurrence is increased by illumination. The individual latencies of the discrete waves evoked by a light stimulus often cannot be resolved because the discrete waves overlap in time. The latency of the first discrete wave that follows a stimulus can be determined with reasonable accuracy. We propose a model which allows us to make an estimate of the distribution of the latencies of the individual light-evoked discrete waves, and to predict the latency distribution of the first discrete wave that follows a stimulus of arbitrary intensity-time course from the latency distribution of the first discrete wave that follows a brief flash of light. For low intensity stimuli, the predictions agree well with the observations. We define a response as the occurrence of one or more discrete waves following a stimulus. The distribution of the peak amplitudes of responses suggests that the peak amplitude of individual discrete waves sometimes has a bimodal distribution. The latencies of the two types of discrete waves, however, follow similar distributions. The area under the voltage-time curve of responses that follow equal energy long (1.25 sec) and short (10 msec) light stimuli follows similar distributions, and this suggests that discrete waves summate linearly.     

The Electrical Response of the Planarian Ocellus

The planarian ocellar potential (OP), an action potential evoked from the planarian ocellus by a light flash, was recorded with microelectrodes. OP amplitude, latency, and peak delay varied as a function of stimulus intensity and state of adaptation in a manner similar to the responses of other photoreceptors. Changes in the OP that occurred with different directions of incident light are described and attributed to screening effects of the ocellar pigment cells. The temperature coefficient (Q₁₀) of OP latency was 1.5; latency decreased continuously as temperature was increased to destructive levels. The energy of activation of the rate of OP formation was calculated to approximate 10 kcal. These findings suggest dependence of OP latency on ionic diffusion and of OP formation on a biocatalytic process.     

Transient Responses to Sudden Illumination in Cells of the Eye of Limulus

Responses recorded from visual cells of Limulus (presumably eccentric cells) following abrupt and maintained illumination consist of depolarization with superimposed spikes. Both the depolarization and the frequency of firing are greater at the beginning of the response than later on. Frequency of firing decreases with time also during stimulation with constant currents, but the decay is then less than it is during constant illumination. Early and steady-state responses do not increase in the same proportion following illumination at different intensities. Membrane conductance is higher during the early peak of the response than in steady state. Early and late potential changes appear to tend to the same equilibrium value. The results support the assumptions that: (a) discharge of impulses is the consequence of depolarization of a specialized "pacemaker region" in the axon; (b) depolarization induced by light is the consequence of increase of membrane conductance. The major conductance changes occurring during constant illumination may be due to corresponding changes of the "stimulus" supplied by the photoreceptor or to changes of sensitivity of the eccentric cell's membrane to this stimulus. Some accessory phenomena may be the consequence of regenerative properties of the nerve cell itself.     

Properties of visual cells in the lateral eye of Limulus in situ.

Excitatory properties of visual cells in the lateral eye of Limulus, investigated by optic nerve recordings in situ, differ significantly from the properties of cells in the classical, excised eye preparation. The differences suggest the possibility that two receptor mechanisms function in the eye in situ: one mechanism encodes low light intensities and the other responds to high intensities. The two mechanisms enable each ommatidium to respond over an intensity range of approximately 10 log units. This hypothesis was tested by measuring the increment threshold and the spectral sensitivity, by studying light and dark adaptation, and by analyzing the variability of the impulse discharge. Although the results do not conclusively identify two receptor mechanisms, they indicate that a process or a part of a process that functions in the eye in situ is abolished by excising the eye or cutting off its blood supply.     

蛙皮素对豚鼠肠系膜下神经节细胞的生物电影响

应用离休细胞内记录技术,观察了蛙皮素（BOM）对豚鼠离体肠系膜下神经节（IMG）细胞膜电位和膜电阻的影响,结果表明,181个IMG细胞在压力注射BOM（10^-5mol/L,1-15pulse,3-15ms)时呈现缓慢去极化（84．0％）,先超极化后去极化（8．3％）,和无明显反应（7．7％）,在10个细胞上灌流BOM（10^-7-10^-6mol/L,60s),90％的细胞亦缓慢去极化,该去极化反应受低钙／高镁溶液的影响,但不为胆碱和肾上腺素受体阻断剂所阻断;膜电阻表现为减小（60．0％）,不变（35．0％）和增大（5．0％）,说明BOM可能存在于豚鼠IMG细胞上且发挥易化作用。     

The Intrinsic Electrophysiological Characteristics of Fly Lobula Plate Tangential Cells: II. Active Membrane Properties

The voltage-gated currents in the fly lobula plate tangential cellswere examined using the switched electrode voltage clamp technique. InCH cells, two currents were identified (Figs. 1, 2): a slow calciuminward current and a delayed rectifying, noninactivating potassiumoutward current. HS and VS cells appear to possess similar currentsto CH cells, but in addition, exhibit a fast-activating sodium inwardcurrent and a sodium-activated potassium outward current(Figs. 3, 4). While the delayed rectifying potassium current in allthree cell classes is responsible for the observed outwardrectification described previously (Borst and Haag, 1996), the sodiuminward current produces the fast and irregular spikelikedepolarizations found in HS and VS cells but not in CH cells: Whenthe sodium current is blocked by either TTX or intracellular QX314,no more action potentials can be elicited in HS cells undercurrent-clamp conditions (Fig. 5). As is demonstrated in HS cells,space clamp conditions are sufficient to suppress synapticallyinduced action potentials (Fig. 6).The currents described above were incorporated with the appropriatecharacteristics into compartmental models of the cells (Figs. 7, 8).The anatomical and electrically passive membrane parameters of thesecells were determined in a preceding paper (Borst and Haag,1996). After fitting the current parameters to the voltage-clamp data(Fig. 9), the model cells qualitatively mimicked the fly tangentialcells under current clamp conditions in response to current injection(Fig. 10). The simulations demonstrated that the electricalcompactness seen in the HS and VS cells, either in passive models orin active models during continuous hyperpolarization, decreasedsignificantly in the active models during continuous depolarization(Fig. 11). Active HS models reproduce the frequency-dependentamplification of current injected into their axon (Fig. 12).     

The Intrinsic Electrophysiological Characteristics of Fly Lobula Plate Tangential Cells: III. Visual Response Properties

In this last paper in a series (Borst and Haag, 1996; Haag et al., 1997) about the lobula plate tangential cells of the fly visual system (CH, HS, and VS cells), the visual response properties were examined using intracellular recordings and computer simulations. In response to visual motion stimuli, all cells responded mainly by a graded shift of their axonal membrane potential. While ipsilateral motion resulted in a graded membrane potential shift, contralateral motion led to distinct EPSPs. For HS cells, simultaneous extracellular recorded action potentials of a spiking interneuron, presumably the H2 cell, corresponded to the EPSPs in the HS cell in a one-to-one fashion. When HS cells were hyperpolarized during ipsilateral motion, they mainly produced action potentials, but when they were hyperpolarized during contralateral motion only a slight increase of EPSP amplitude, could be observed. Intracellular application of the sodium channel blocker QX 314 abolished action potentials of HS cells while having little effect on the graded membrane response to ipsilateral motion. HS and CH cells were also studied with respect to their spatial integration properties. For both cell types, their graded membrane response was found to increase less than linearly with the size of the ipsilateral motion pattern. However, while for HS cells various amounts of hyperpolarizing current injected during motion stimulation led to different saturation levels, this was not the case for CH cells. In response to a sinusoidal velocity modulation, CH cells followed pattern motion only up to 10 Hz modulation frequency, but HS cells still revealed significant membrane depolarizations up to about 40 Hz.In the computer simulations, the compartmental models of tangential cells, as derived in the previous papers, were linked to an array of local motion detectors. The model cells revealed the same basic response features as their natural counterparts. They showed a response saturation as a function of stimulus size. In CH-models, however, the saturation was less pronounced than in real CH-cells, indicating spatially nonuniform membrane resistances with higher values in the dendrite. As in the experiments, HS models responded to high-frequency velocity modulation with a higher amplitude than did CH models.     

An Electrophysiological Study of Photomixotrophically Cultured Tobacco Cells

Membrane potential properties of photomixotrophically culturedgreen tobacco cells with chloroplasts were studied in comparisonwith white tobacco cells without chloroplasts. In the dark therewas almost no difference in their membrane potential properties.In the light some of the green cells showed a light-inducedpotential change (LPC), but other green cells did not, nor didthe white cells. Our results indicate that the green cells arecomposed of two kinds of cells, one that shows the LPC and onethat does not. (Received October 5, 1983; Accepted May 10, 1984)