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.     

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.     

Thermal and Spectral Sensitivities of Discrete Slow Potentials in Limulus Eye

The discrete, subthreshold, slow potential fluctuations (SPF's) which can be recorded intracellularly in Limulus ommatidia are sensitive to temperature and light wavelength. SPF frequency increases with increasing temperature (Q₁₀ about 3.5) and light intensity. The effects are additive. SPF rise and decay time decrease with increasing temperature (Q₁₀ between 2 and 3). There is a peak, near 520 nm, in the spectral sensitivity of SPF frequency. This peak may correspond to the wavelength of maximum absorption by rhodopsin in the ommatidia. Hydroxylamine produces a rapid, irreversible reduction of SPF frequency and amplitude perhaps owing to its action on the photopigment. The cornea and crystalline cones fluoresce (peak about 445 nm) when excited by near-ultraviolet energy (380 nm peak) and this fluorescence may influence SPF spectral sensitivity measurements. These findings suggest that the SPF's are the results of photolytic and thermolytic reactions occurring in the ommatidial visual pigments and that they have a role in the mechanisms which transduce light to electrical activity in the visual receptors.     

Electrophysiological Properties of Cells in the Median Ocellus of Limulus

Two types of photoreceptors are found in the median ocellus of Limulus. One type is maximally sensitive to ultraviolet (UV) light, the other to green light; they are called UV and VIS cells, respectively. Biphasic receptor potentials, consisting of a small initial hyperpolarizing phase and a later slow depolarizing phase, can be recorded from both receptor types. These biphasic responses are elicited in UV cells in response to long-wavelength light, and in VIS cells in response to ultraviolet light. Another type of hyperpolarizing response can be recorded in UV cells: after a bright ultraviolet stimulus, the cell remains depolarized; long-wavelength light rapidly returns the membrane potential to its value preceding ultraviolet illumination (this long-wavelength-induced potential change is called a "repolarizing response"). Also, a long-wavelength stimulus superimposed during a UV stimulus elicits a sustained repolarizing response. A third cell type (arhabdomeric cell) found in the median ocellus generates large action potentials and is maximally sensitive to UV light. Biphasic responses and repolarizing responses also can be recorded from arhabdomeric cells. The retina is divided into groups of cells; both UV cells and VIS cells can occur in the same group. UV cells in the same group are electrically coupled to one another and to an arhabdomeric cell.     

The Ventral Photoreceptor Cells of Limulus : II. The basic photoresponse

The ventral photoreceptors of Limulus polyphemus are unipolar cells with large, ellipsoidal somas located long both "lateral olfactory nerves." As a consequence of their size and location, the cells are easily impaled with microelectrodes. The cells have an average resting potential of -48 mv. The resting potential is a function of the external concentration of K. When the cell is illuminated, it gives rise to the typical "receptor potential" seen in most invertebrate photoreceptors which consists of a transient phase followed by a maintained phase of depolarization. The amplitude of the transient phase depends on both the state of adaptation of the cell and the intensity of the illumination, while the amplitude of the maintained phase depends only on the intensity of the illumination. The over-all size of the receptor potential depends on the external concentration of Na, e.g. in sodium-free seawater the receptor potential is markedly reduced, but not abolished. On the other hand lowering the Ca concentration produces a marked enhancement of both components of the response, but predominantly of the steady-state component. Slow potential fluctuations are seen in the dark-adapted cell when it is illuminated with a low intensity light. A spike-like regenerative process can be evoked by either the receptor potential or a current applied via a microelectrode. No evidence of impulse activity has been found in the axons of these cells. The ventral photoreceptor cell has many properties in common with a variety of retinular cells and therefore should serve as a convenient model of the primary receptor cell in many invertebrate eyes.     

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.     

The Ventral Photoreceptor Cells of Limulus : III. A voltage-clamp study

In the dark, the ventral photoreceptor of Limulus exhibits time-variant currents under voltage-clamp conditions; that is, if the membrane potential of the cell is clamped to a depolarized value there is an initial large outward current which slowly declines to a steady level. The current-voltage relation of the cell in the dark is nonlinear. The only ion tested which has any effect on the current-voltage relation is potassium; high potassium shifts the reversal potential towards zero and introduces a negative slope-conductance region. When the cell is illuminated under voltage-clamp conditions, an additional current, the light-induced current, flows across the cell membrane. The time course of this current mimics the time course of the light response (receptor potential) in the unclamped cell; namely, an initial transient phase is followed by a steady-state phase. The amplitude of the peak transient current can be as large as 60 times the amplitude of the steady-state current, while in the unclamped cell the amplitude of the peak transient voltage never exceeds 4 times the amplitude of the steady-state voltage. The current-voltage relations of the additional light-induced current obtained for different instants of time are also nonlinear, but differ from the current-voltage relations of the dark current. The ions tested which have the greatest effect on the light-induced current are sodium and calcium; low sodium decreases the current, while low calcium increases the current. The data strongly support the hypothesis that two systems of electric current exist in the membrane. Thus the total ionic current which flows in the membrane is accounted for as the sum of a dark current and a light-induced current.     

Dual Sensitivities of Cells in Wolf Spider Eyes at Ultraviolet and Visible Wavelengths of Light

Intracellular recordings have been made from visual cells in principal and secondary eyes of in vitro wolf spider preparations. The responses of all cells to all wavelengths of light were graded depolarizations; no hyperpolarizations or nerve discharges were seen. Cells in a secondary eye, the anterior lateral eye, had a maximum sensitivity in the visible at 510 nm and a secondary maximum, or shoulder, of sensitivity in the near ultraviolet at 380 nm. Cells in principal eyes, the anterior median eyes, all responded maximally both in the visible at 510 nm and in the ultraviolet at 360–370 nm or less. However, there was no typical ratio of ultraviolet to visible sensitivities; the differences in log sensitivities (log UV/VIS) varied from 3.3 to -0.5. Each principal eye had a population of cells with different ratios. These populations varied with the time of the year, possibly due to changes in light upon the animals. Chromatic adaptations of cells in anterior median (but not anterior lateral) eyes resulted in small, selective changes in spectral sensitivities, and there was some facilitation of responses from cells repeatedly stimulated. It is concluded that cells of secondary eyes contain only a visual pigment absorbing maximally in the visible, while cells of principal eyes probably contain variable amounts of both this pigment and one absorbing in the ultraviolet as well.     

Profile and Determinants of Retinal Optical Intensity in Normal Eyes with Spectral Domain Optical Coherence Tomography

Purpose

To investigate the profile and determinants of retinal optical intensity in normal subjects using 3D spectral domain optical coherence tomography (SD OCT).

Methods

A total of 231 eyes from 231 healthy subjects ranging in age from 18 to 80 years were included and underwent a 3D OCT scan. Forty-four eyes were randomly chosen to be scanned by two operators for reproducibility analysis. Distribution of optical intensity of each layer and regions specified by the Early Treatment of Diabetic Retinopathy Study (ETDRS) were investigated by analyzing the OCT raw data with our automatic graph-based algorithm. Univariate and multivariate analyses were performed between retinal optical intensity and sex, age, height, weight, spherical equivalent (SE), axial length, image quality, disc area and rim/disc area ratio (R/D area ratio).

Results

For optical intensity measurements, the intraclass correlation coefficient of each layer ranged from 0.815 to 0.941, indicating good reproducibility. Optical intensity was lowest in the central area of retinal nerve fiber layer, ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer and photoreceptor layer, except for the retinal pigment epithelium (RPE). Optical intensity was positively correlated with image quality in all retinal layers (0.553<β<0.851, p<0.01), and negatively correlated with age in most retinal layers (-0.362<β<-0.179, p<0.01), except for the RPE (β = 0.456, p<0.01), outer nuclear layer and photoreceptor layer (p>0.05). There was no relationship between retinal optical intensity and sex, height, weight, SE, axial length, disc area and R/D area ratio.

Conclusions

There was a specific pattern of distribution of retinal optical intensity in different regions. The optical intensity was affected by image quality and age. Image quality can be used as a reference for normalization. The effect of age needs to be taken into consideration when using OCT for diagnosis.     

Ionic Dependence of Reversal Voltage of the Light Response in Limulus Ventral Photoreceptors

The light-induced current as measured using a voltage clamp (holding voltage at resting potential) is attenuated when sodium ions in the bathing solution, Na_o, are replaced by Tris, choline, or Li or when NaCl is replaced by sucrose. After replacement of NaCl by sucrose, the reversal voltage, V_rev, for the light response becomes more negative. In this case, the slope of the V_rev vs. log Na_o near Na_o = 425 mM is approximately 55 mV/decade increase of Na_o (mean for 13 cells). The slope decreases at lower values of Na_o. Choline is not impermeant and partially substitutes for Na; the slope of V_rev vs. log Na_o is 20 mV/decade (mean for three cells). V_rev does not change when Na is replaced by Li. Decreases in the bath concentrations of Ca, Mg, Cl, or K do not affect V_rev. When Na_o = 212 mM, V_rev becomes more positive when K_o is increased. Thus, light induces a change in membrane permeability to Na and probably also to K.     

Different Sensitivities of Granulocyte-Macrophage and Erythroid Progenitor Cells of Normal Human Bone Marrow to S-Phase-Specific Agents

The extraordinary sensitivity of early erythroid progenitor cells (BFU-e) of normal human bone marrow to tritiated thymidine ([³H]TdR) was studied. While exposure of bone-marrow cells to [³H]TdR for 1 hr resulted in the death of only 40% of the granulocyte-macrophage progenitor cells (CFU-c), 90% of BFU-e were killed. Experiments in which normal bone-marrow cells were mixed with bone-marrow cells which had been exposed to [³H]TdR demonstrated that the excessive killing of BFU-e by [³H]TdR reflected carry-over of the [³H]TdR by the exposed cells. A carry-over effect was not observed for CFU-c, suggesting the presence of a fundamental difference in the metabolism of TdR between CFU-c and BFU-e. There was a suggestion of a carry-over effect regarding two other S-phase-specific agents, hydroxyurea and 1-β-D-arabinofuranosylcytosine.     

Adaptation in the Ventral Eye of Limulus is Functionally Independent of the Photochemical Cycle, Membrane Potential, and Membrane Resistance

The early receptor potential (ERP), membrane potential, membrane resistance, and sensitivity were measured during light and/or dark adaptation in the ventral eye of Limulus. After a bright flash, the ERP amplitude recovered with a time constant of 100 ms, whereas the sensitivity recovered with an initial time constant of 20 s. When a strong adapting light was turned off, the recovery of membrane potential and of membrane resistance had time-courses similar to each other, and both recovered more rapidly than the sensitivity. The receptor depolarization was compared during dark adaptation after strong illumination and during light adaptation with weaker illumination; at equal sensitivities the cell was more depolarized during light adaptation than during dark adaptation. Finally, the waveforms of responses to flashes were compared during dark adaptation after strong illumination and during light adaptation with weaker illumination. At equal sensitivities (equal amplitude responses for identical flashes), the responses during light adaptation had faster time-courses than the responses during dark adaptation. Thus neither the photochemical cycle nor the membrane potential nor the membrane resistance is related to sensitivity changes during dark adaptation in the photoreceptors of the ventral eye. By elimination, these results imply that there are (unknown) intermediate process(es) responsible for adaptation interposed between the photochemical cycle and the electrical properties of the photoreceptor.     

A Nonlinearity in the Inhibitory Interactions in the Lateral Eye of Limulus

Receptor units in the eye of the horseshoe crab are more sensitive to lateral inhibition at some levels of excitation than they are at others. As a result, the steady-state inhibition of the response of a given unit is not directly proportional to the response levels of neighboring units. This effect may be represented by the introduction of a nonlinearity in the Hartline-Ratliff system of equations. The nonlinear inhibitory effect appears to increase the operating range of the receptor units.     

The Embryonic Septum and Ventral Pallium, New Sources of Olfactory Cortex Cells

The mammalian olfactory cortex is a complex structure located along the rostro-caudal extension of the ventrolateral prosencephalon, which is divided into several anatomically and functionally distinct areas: the anterior olfactory nucleus, piriform cortex, olfactory tubercle, amygdaloid olfactory nuclei, and the more caudal entorhinal cortex. Multiple forebrain progenitor domains contribute to the cellular diversity of the olfactory cortex, which is invaded simultaneously by cells originating in distinct germinal areas in the dorsal and ventral forebrain. Using a combination of dye labeling techniques, we identified two novel areas that contribute cells to the developing olfactory cortices, the septum and the ventral pallium, from which cells migrate along a radial and then a tangential path. We characterized these cell populations by comparing their expression of calretinin, calbindin, reelin and Tbr1 with that of other olfactory cell populations.     

Raman Spectral Dynamics of Single Cells in the Early Stages of Growth Factor Stimulation

Cell fates change dynamically in response to various extracellular signals, including growth factors that stimulate differentiation and proliferation. The processes underlying cell-fate decisions are complex and often include large cell-to-cell variations, even within a clonal population in the same environment. To understand the origins of these cell-to-cell variations, we must detect the internal dynamics of single cells that reflect their changing chemical milieu. In this study, we used the Raman spectra of single cells to trace their internal dynamics during the early stages of growth factor stimulation. This method allows nondestructive and inclusive time-series analyses of chemical compositions of the same single cells. Applying a Gaussian mixture model to the major principal components of the single-cell Raman spectra, we detected the dynamics of the chemical states in MCF-7 cancer-derived cells in the absence and presence of differentiation and proliferation factors. The dynamics displayed characteristic variations according to the functions of the growth factors. In the differentiation pathway, the chemical composition changed directionally between multiple states, including both reversible and irreversible state transitions. In contrast, in the proliferation pathway, the chemical composition was homogenized into a single state. The differentiation factor also stimulated fluctuations in the chemical composition, whereas the proliferation factor did not.     

Function of Ventral and Lateral Processes in Larvae of Chironomus (Diptera, Chironomidae)

Oxygen consumption by larvae of five Chironomus species from the Plumosus group belonging to different larval forms was studied. Larvae of C. agilis ( f. l. reductus) were unable to utilize oxygen at its concentrations lower than 1 mg/l. Larvae of C. plumosus ( f. l. plumosus) used oxygen from water at concentrations higher than 0.7 mg/l; pupas of this species and larvae of C. borokensis ( f. l. plumosus) utilized oxygen at concentrations lower than 0.4 mg/l. C. entis and C. muratensis (both-- f. l. semireductus) utilized oxygen from water at its concentrations up to 0.05 mg/l. The lack of correlation between the rate and ability of larvae to use oxygen at low concentrations and the degree of development of processes allows suggesting that these structures are not additional respiratory organs, as is commonly accepted. At the same time, ability of species to inhabit under conditions of oxygen deficiency correlates directly with the size of the processes. Therefore, ventral and lateral processes are suggested to perform a function of excretion of anaerobic metabolism metabolites.     

Autocrine Growth Loop of The Epidermal Growth Factor Receptor in Normal and Immortalized Human Bronchial Epithelial Cells

Epidermal growth factor (EGF) is a potent growth factor for human normal bronchial epithelial (HBE) cells and lung cancer cells, which often demonstrate an EGF receptor (EGFR) autocrine loop. We have found that HBE cells are capable of proliferating in basal medium without EGF supplementation, and this suggests the probable presence of an active EGFR autocrine loop in non-neoplastic HBE cells. Northern blot hybridization shows that the parental and immortalized HBE cells express comparable and high levels of mRNA for EGFR, transforming growth factor-alpha (TGF-α), and amphiregulin (AR), but not EGF. Incubation with neutralizing monoclonal antibodies (mAb) against EGFR partially inhibits the growth of these cells. Immunohistochemistry shows that HBE cells express the TGF-α peptidein vitroandin vivo,however, neutralizing mAbs against TGF-α fail to inhibit their proliferation. In contrast, AR stimulates the growth of HBE cells. Thus, several EGF-family ligands appear to be involved functionally in the EGFR autocrine growth loop in HBE cells.     

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.     

Independence of Location of Light Absorption and Discrete Wave Latency Distribution in Limulus Ventral Nerve Photoreceptors

Discrete waves of depolarization evoked by dim pulses of light in dark-adapted ventral nerve photoreceptors in Limulus show fluctuation in their latency. To a resolution of 5–10 µm the latency distribution function appears to be independent of where in the receptor light is absorbed. Also, there is apparent local adaptation to bright light pulses.