Species specificity in the extrinsic absorption changes exhibited by some invertebrates stained with voltage-sensitive dyes

Summary The absorption changes of several invertebrate neuronal preparations stained by the potentiometric dyes (WW 375, WW 433, WW 401 and RGA 84) in response to electrical nerve stimulation were examined. The dyes did not penetrate the connective sheath of insect preparations, but stained it. Only a decremental spreading of optical signals was seen onPeriplaneta americana, Gryllus bimaculatus and GitGryllus campestris ganglia and nerves. In contrast to insect preparations, pond snail and leech neurons were well stained by these dyes. The dye WW 375 behaved somewhat distinctly on insect and pond snail preparations than had been previously reported on other invertebrates. Like the signals from vertebrate neurons, they usually had triphasic action spectra. Therefore, this kind of action spectrum is not found only in membranes of vertebrate neurons. The main conclusion of this work is that the species-specific effects of the dye on different invertebrate preparations have a common feature: the existence of three peaks in the change of absorption (at 575, 675 and 750 nm) in both kinds of WW 375 action spectra (monophasic or triphasic). The wavelength dependence of the change in absorption was not affected by concentration, staining time, pH, osmolarity or ionic composition of physiological saline.     

Dichroic behavior of the absorbance signals from dyes NK2367 and WW375 in skeletal muscle fibers

Absorbance signals were recorded from voltage-clamped single muscle fibers stained with the nonpenetrating potentiometric dyes NK2367 and WW375 and illuminated with quasimonochromatic light from 560 to 800 nm, linearly polarized either parallel (0 degree) or perpendicular (90 degrees) to the fiber long axis. The signals from both dyes depend strongly on the incident polarization. At any wavelength and/or polarization condition, the total absorbance signal is a superposition of the same two signal components previously identified with unpolarized light (Heiny, J. A., and J. Vergara, 1982, J. Gen. Physiol., 80:203)--namely, a fast step signal from the voltage-clamped surface membrane and a signal reflecting the slower T-system potential changes. The 0 degree and 90 degrees spectra of both membranes have similar positive and negative absorbance peaks (720 and 670 nm, respectively, for dye NK2367; 740 and 700 nm for dye WW375); in addition, they have the same dichroic maxima (670 for NK2367; 700 for WW375). However, for the surface membrane, the 0 degrees spectra are everywhere more positive than the 90 degrees spectra, whereas the reverse is true for the T-system, which results in a dichroism of opposite sign for the two membranes. These spectral characteristics were analyzed using a general model for the potential-dependent response of an absorbing dye (Tasaki, I., and A. Warashina, 1976, Photochem. Photobiol., 24:191), which takes into account both the dye response and the membrane geometries. They are consistent with the proposal that the dye responds via a common mechanism in both membranes that consists of a dye reorientation and a change in the absorption maxima.     

Membrane-potential-sensitive dyes for optical monitoring of activity in Aplysia neurons

Two membrane-associated dyes (WW375 and NK2367) which change their absorption of light when the membrane potential changes have been studied using several preparations from Aplysia. Action potentials are easily observed in nerve trunks (from a number of axons), in bag cell clusters, in some of the larger single cells of the parietovisceral ganglion, and in the optic nerve. Physiological effects of the dyes on the circadian rhythm of activity in the eye are described.     

Action potentials of isolated single muscle fibers recorded by potential-sensitive dyes

Light transmission changes upon massive stimulation of single muscle fibers of Xenopus were studied with the potential-sensitive nonpermeant dyes, merocyanine rhodanine (WW375) and merocyanine oxazolone (NK2367). Upon stimulation an absorption change (wave a) occurred, which probably represents the sum of action potentials in the transverse tubules and surface membrane. In WW375-stained fibers wave a is a decrease in transmission over the range of 630 to 730 nm (with NK2367, over the range of 590 to 700 nm) but becomes an increase outside this range, thus showing a triphasic spectral pattern. This spectrum differs from that of the squid axon, in which depolarization produces only an increase in transmission over the whole range of wavelengths (Ross et al. 1977. J. Membr. Biol. 33:141-183). When wave a was measured at the edge of the fiber to obtain more signal from the surface membrane, the spectrum did not seem to differ markedly from that obtained from the entire width of the fiber. Thus, the difference in the spectrum between the squid axon and the vertebrate muscle cannot be attributed to the presence of the tubular system.     

Imaging Brain Activity With Voltage- and Calcium-Sensitive Dyes

This paper presents three examples of imaging brain activity with voltage- or calcium-sensitive dyes and then discusses the methodological aspects of the measurements that are needed to achieve an optimal signal-to-noise ratio.Internally injected voltage-sensitive dye can be used to monitor membrane potential in the dendrites of invertebrate and vertebrate neurons in in vitro preparations.Both invertebrate and vertebrate ganglia can be bathed in voltage-sensitive dyes to stain all of the cell bodies in the preparation. These dyes can then be used to follow the spike activity of many neurons simultaneously while the preparations are generating behaviors.Calcium-sensitive dyes that are internalized into olfactory receptor neurons in the nose will, after several days, be transported to the nerve terminals of these cells in the olfactory bulb. There they can be used to measure the input from the nose to the bulb.Three kinds of noise are discussed. a. Shot noise from the random emission of photons from the preparation. b. Vibrational noise from external sources. c. Noise that occurs in the absence of light, the dark noise.Three different parts of the light measuring apparatus are discussed: the light sources, the optics, and the cameras.The major effort presently underway to improve the usefulness of optical recordings of brain activity are to find methods for staining individual cell types in the brain. Most of these efforts center around fluorescent protein sensors of activity.     

Voltage-sensitive dye recording of action potentials and synaptic potentials from sympathetic microcultures.

Given the appropriate multicell electrophysiological techniques, small networks of cultured neurons (microcultures) are well suited to long-term studies of synaptic plasticity. To this end, we have developed an apparatus for optical recording from cultured vertebrate neurons using voltage-sensitive fluorescent dyes (Chien, C.-B., and J. Pine. 1991. J. Neurosci. Methods. 38:93-105). We evaluate here the usefulness of this technique for recording action potentials and synaptic potentials in microcultures of neurons from the rat superior cervical ganglion (SCG). After extensive dye screening and optimization of conditions, we chose the styryl dye RH423, which gave fast linear fluorescence changes of approximately 1%/100 mV for typical recordings. The root mean square noise of the apparatus (limited by shot noise) was typically 0.03%, equivalent to 3 mV of membrane potential. Illumination for at least 100 flashes of 100 ms each caused no noticeable photodynamic damage. Our results show that voltage-sensitive dyes can be used to record from microcultures of vertebrate neurons with high sensitivity. Dye signals were detected from both cell bodies and neurites. Signals from presumptive dendrites showed hyperpolarizations and action potentials simultaneous with those in the cell body, while those from presumptive axons showed delayed propagating action potentials. Subthreshold synaptic potentials in the cell body were occasionally detectable optically; however, they were usually masked by signals from axons passing through the same pixel. This is due to the complex anatomy of SCG microcultures, which have many crisscrossing neurites that often pass over cell bodies. Given a simpler microculture system with fewer neurites, it should be possible to use dye recording to routinely measure subthreshold synaptic strengths.     

Quantitative analysis of hemoglobin oxygenation state of rat brain in situ by near-infrared spectrophotometry

The light in the near-infrared region (700-900 nm) was illuminated on the rat head, and absorption spectra were measured with the transmitted light under various conditions. The absorbance changes less than 780 nm were attributable to hemoglobin in the brain tissue, whereas those greater than 780 nm were associated with both hemoglobin and cytochrome oxidase. The changes of oxy- and total (oxy- plus deoxy-) hemoglobin content in the rat head could be monitored quantitatively by expressions of delta A700--1.20 delta A730 and delta A700--1.52 delta A730, respectively. The oxyhemoglobin content in the tissue was decreased as the O2 tension in inspired gas was lowered. At 10% O2 approximately 50% of hemoglobin was deoxygenated. The total hemoglobin content was increased under anoxic conditions. Inhalation of 5% CO2 and intravenous injection of a Ca2+ blocker nicardipine increased the O2 saturation of hemoglobin in the brain. These conclusions were confirmed by measuring the difference absorption spectra in the near-infrared region.     

Qualitative changes in the fluorescence spectra of intact pea leaves after photoinhibition

In high light (1400 W m-2) treated, intact pea leaves, a decrease in the ratio of fluorescence emission at 685 to 730 nm and an increase in fluorescence intensity between 500 and 600 nm were observed. Furthermore, photoacoustically monitored heat emission increased slightly, and O2 evolution decreased significantly. These findings are interpreted as effects of a photoprotective mechanism separating the carotenoid pool from the chlorophylls. This is supported by fluorescence excitation measurements and the results of a study on the reversibility of the process.     

Optical properties of Lactuca and Taraxacum seed and fruit coats: Their role as light filters

The optical properties of seed and fruit coats were examined from several varieties of light-sensitive achenes. Taraxacum vulgare L. and Lactuca sativa L. cv. Grand Rapids achenes with dark fruit coats and L. sativa cvs Huvudsallat and Issallat with white fruit coats were examined. Transmission spectra varied among the different achenes: white fruit coats of Lactuca acted as neutral density filters between 450 and 780 nm, whereas Taraxacum transmitted 2–36% in this region. The ribbed fruit coat structure greatly affected transmission so that at different locations in the same coat, transmission varied between 20 to 80% at 660 and 730 nm. Fruit coats of Grand Rapids lettuce and Taraxacum transmitted more far-red than red light with T₆₆₀/T₇₃₀ ratios of 0.8 and 0.4, respectively. The relationship between the optical properties of fruit coats and light-stimulated germination is discussed.     

Comparison of Two Voltage-Sensitive Dyes and Their Suitability for Long-Term Imaging of Neuronal Activity

One of the key approaches for studying neural network function is the simultaneous measurement of the activity of many neurons. Voltage-sensitive dyes (VSDs) simultaneously report the membrane potential of multiple neurons, but often have pharmacological and phototoxic effects on neuronal cells. Yet, to study the homeostatic processes that regulate neural network function long-term recordings of neuronal activities are required. This study aims to test the suitability of the VSDs RH795 and Di-4-ANEPPS for optically recording pattern generating neurons in the stomatogastric nervous system of crustaceans with an emphasis on long-term recordings of the pyloric central pattern generator. We demonstrate that both dyes stain pyloric neurons and determined an optimal concentration and light intensity for optical imaging. Although both dyes provided sufficient signal-to-noise ratio for measuring membrane potentials, Di-4-ANEPPS displayed a higher signal quality indicating an advantage of this dye over RH795 when small neuronal signals need to be recorded. For Di-4-ANEPPS, higher dye concentrations resulted in faster and brighter staining. Signal quality, however, only depended on excitation light strength, but not on dye concentration. RH795 showed weak and slowly developing phototoxic effects on the pyloric motor pattern as well as slow bleaching of the staining and is thus the better choice for long-term experiments. Low concentrations and low excitation intensities can be used as, in contrast to Di-4-ANEPPS, the signal-to-noise ratio was independent of excitation light strength. In summary, RH795 and Di-4-ANEPPS are suitable for optical imaging in the stomatogastric nervous system of crustaceans. They allow simultaneous recording of the membrane potential of multiple neurons with high signal quality. While Di-4-ANEPPS is better suited for short-term experiments that require high signal quality, RH795 is a better candidate for long-term experiments since it has only minor effects on the motor pattern.     

Optical signals from surface and T system membranes in skeletal muscle fibers. Experiments with the potentiometric dye NK2367

Absorbance signals were recorded from cut single skeletal muscle fibers stained with the nonpenetrating potentiometric dye NK2367 and mounted in a three-vaseline-gap voltage clamp. The characteristics of the optical signals recorded under current and voltage-clamp conditions were studied at various wavelengths between 500 and 800 nm using unpolarized light. Our results indicate that the absorbance signals recorded with this dye reflect potential changes across both the surface and T system membranes and that the relative contribution of each of these membrane compartments to the total optical change is strongly wavelength dependent. A peak intensity change was detected at 720 nm for the surface membrane signal and at 670 nm for the T system. Evidence for this wavelength-dependent separation derives from an analysis of the kinetics and voltage dependence of the optical signals at different wavelengths, and results obtained in detubulated fibers. The 670-nm optical signal was used to demonstrate the lack of potential control in the T system by the voltage clamp and the effect of a tetrodotoxin (TTX)-sensitive sodium conductance on tubular depolarization.     

Postsynaptic potentials from a single monopolar neuron of the ganglion opticum I of the blowfly Calliphora

Summary Hyperpolarizing potentials were recorded from second order monopolar neurons of the ganglion opticum I of Calliphora by means of glass microcapillaries. The eye was stimulated with a spotlike light source. Potentials were recorded in relation to the intensity of the light stimulus. The recording site was identified by marking the cell with a dye and by localizing the tip of the electrode.This work was supported by the Deutsche Forschungsgemeinschaft.     

Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates.

Amine-reactive N-hydroxysuccinimidyl esters of Alexa Fluor fluorescent dyes with principal absorption maxima at about 555 nm, 633 nm, 647 nm, 660 nm, 680 nm, 700 nm, and 750 nm were conjugated to antibodies and other selected proteins. These conjugates were compared with spectrally similar protein conjugates of the Cy3, Cy5, Cy5.5, Cy7, DY-630, DY-635, DY-680, and Atto 565 dyes. As N-hydroxysuccinimidyl ester dyes, the Alexa Fluor 555 dye was similar to the Cy3 dye, and the Alexa Fluor 647 dye was similar to the Cy5 dye with respect to absorption maxima, emission maxima, Stokes shifts, and extinction coefficients. However, both Alexa Fluor dyes were significantly more resistant to photobleaching than were their Cy dye counterparts. Absorption spectra of protein conjugates prepared from these dyes showed prominent blue-shifted shoulder peaks for conjugates of the Cy dyes but only minor shoulder peaks for conjugates of the Alexa Fluor dyes. The anomalous peaks, previously observed for protein conjugates of the Cy5 dye, are presumably due to the formation of dye aggregates. Absorption of light by the dye aggregates does not result in fluorescence, thereby diminishing the fluorescence of the conjugates. The Alexa Fluor 555 and the Alexa Fluor 647 dyes in protein conjugates exhibited significantly less of this self-quenching, and therefore the protein conjugates of Alexa Fluor dyes were significantly more fluorescent than those of the Cy dyes, especially at high degrees of labeling. The results from our flow cytometry, immunocytochemistry, and immunohistochemistry experiments demonstrate that protein-conjugated, long-wavelength Alexa Fluor dyes have advantages compared to the Cy dyes and other long-wavelength dyes in typical fluorescence-based cell labeling applications.     

Photobiomodulation at a wavelength of 633 nm leads to faster functional recovery than 804 nm after facial nerve injury

We analyzed the effects of photobiomodulation (PBM) of various wavelengths on regeneration of the facial nerve using in vitro and in vivo experimental models. We assessed the antioxidative effect of PBM in geniculate ganglion neurons irradiated with a diode laser at 633 nm, 780 nm and 804 nm. Wavelengths of 633 and 780 nm but not 804 nm inhibited cell death by oxidative stress. We assessed the effects of PBM on functional and morphologic recovery in rats divided into control, facial nerve damage (FND) and FND irradiated with a 633 nm or 804 nm lasers. Injured rats treated with 633-nm light had better facial palsy scores, larger axon diameter and higher expression of Schwann cells compared with the FND group. No positive results were observed in rats irradiated at 804-nm light. These findings indicate that 633-nm PBM promotes accelerated nerve regeneration and improved functional recovery in an injured facial nerve.     

In vitro imaging using laser photostimulation with flavoprotein autofluorescence

Imaging of 300-500 μm mouse brain slices by laser photostimulation with flavoprotein autofluorescence (LFPA) allows the rapid and sensitive mapping of neuronal connectivity. It is accomplished using UV laser-based photo-uncaging of glutamate and imaging neuronal activation by capturing changes in green light (～520 nm) emitted under blue light (～460 nm) excitation. This fluorescence is generated by the oxidized form of flavoprotein and is a measure of metabolic activity. LPFA offers several advantages over imaging techniques that rely on dye loading. First, as flavoprotein imaging measures endogenous signals, it avoids the use of heterogeneously loaded and potentially cytotoxic dyes. Second, flavoprotein signals are large (1-20% above baseline), obviating the need for averaging. Third, the use of photostimulation ensures orthodromic neuronal activation and permits the rapid interrogation of multiple stimulation sites of the slice with a high degree of precision (～50 μm). Here we describe a step-by-step protocol for the incorporation of LPFA into virtually any slice rig, as well as how to do the experiment.     

CCD imaging of the electrical activity in the leech nervous system

A single ganglion of the nervous system of the leechHirudo medicinalis was isolated. One or both roots emerging from each side of the ganglion were sucked into suction pipettes used either for extracellular stimulation or for recording the gross electrical activity. The ganglion was stained with the fluorescence voltage sensitive dye Di-4-Anepps. The fluorescence was measured with a nitrogen cooled CCD camera. Our recording system allowed us to measure in real time slow optical signals corresponding to changes in light intensity of at least 5. These signals were caused by the direct polarization of neuronal structures, the afterhyperpolarization or the afterdischarge induced by a prolonged stimulation. When images were acquired at fixed times, several of them could be averaged and optical signals of at least 2 could be reliably measured. These optical signals originated from well identified neurons, such as T, P and N sensory neurons. By taking images at different times and at different focal planes, electrical events could be followed at a temporal resolution of 50 Hz. The three dimensional dynamics of electrical events, initiated by a specific stimulation, was imaged and the spread of excitation among leech neurons was followed. When two roots were selectively stimulated, their neuronal interactions could be imaged and the linear and non-linear terms of the interaction could be characterized.     

Distribution of neurons in the major pelvic ganglion of the rat which supply the bladder,colon or penis

Summary In male rats a large number of the postganglionic neurons which innervate the pelvic organs are located in the major pelvic ganglion. In the present study we have identified the location within this ganglion of neurons which project to either of three pelvic organs, the penis, colon or urinary bladder. Two fluorescent retrogradely-transported dyes, Fast Blue and Fluoro-Gold, were used. For most animals one dye was injected into the cavernous space of the penis, the wall of the distal colon or the wall of the urinary bladder. In a small number of animals two organs were injected, each with a different dye. One to six weeks after injection the major pelvic ganglia were fixed in buffered formaldehyde. The distribution of fluorescent dye-labelled cells was observed in whole mounts of complete ganglia and, in most cases, also in small accessory ganglia located between the ureter and the prostate. The studies showed a unique pattern of distribution for each organ-specific group of neurons. Most of the colon neurons are located in the major pelvic ganglion near the entrance of the pelvic nerve, whereas almost all of the penis neurons are near or within the penile nerve. Bladder neurons are relatively evenly distributed throughout the ganglion. These results demonstrate a distinct topographical organization of organ-specific neurons of the major pelvic ganglion of the male rat, a phenomenon which has also been observed in other peripheral ganglia.     

The behavioral response of amphipods harboring Corynosoma constrictum (Acanthocephala) to various components of light

Many studies have shown that photic behavior of amphipods is subject to parasitic manipulation. However, all these investigations have focused on but one property of light (i.e., intensity). This study investigated the possibility that variable wavelength sensitivity, as a potentially important component of amphipod ecology, is subject to parasitic manipulation. The photic behavior of freshwater amphipods Hyalella azteca, infected with the duck acanthocephalan Corynosoma constrictum, was tested. The phototactic responses of infected and uninfected amphipods to various wavelengths in the visible spectrum were compared, and to delineate the effects of intensity and wavelength on behavior, the preferences of amphipods for environments characterized by various combinations of light intensity and wavelength were determined. Response to blue light (400-450 nm) was little affected by infection. Amphipod response to higher red region wavelengths (600-700 nm) was altered by infection. Infected amphipods were significantly less responsive to green region light (500-550 nm), which could lead to increased wandering throughout the water column, thereby facilitating increased parasite transmission through increased predation risk. This study reinforces the subtlety with which parasites can alter their host's behavior, presumably resulting in an increased probability of being transmitted from the intermediate host to a definitive host.     

VEGF activates divergent intracellular signaling components to regulate retinal progenitor cell proliferation and neuronal differentiation

During vertebrate neurogenesis, multiple extracellular signals influence progenitor cell fate choices. The process by which uncommitted progenitor cells interpret and integrate signals is not well understood. We demonstrate here that in the avascular chicken retina, vascular endothelial growth factor (VEGF) secreted by postmitotic neurons acts through the FLK1 receptor present on progenitor cells to influence cell proliferation and commitment. Augmenting VEGF signals increases progenitor cell proliferation and decreases retinal ganglion cell genesis. Conversely, absorbing endogenous VEGF ligand or disrupting FLK1 activity attenuates cell proliferation and enhances retinal ganglion cell production. In addition, we provide evidence that VEGF signals transmitted by the FLK1 receptor activate divergent intracellular signaling components, which regulate different responses of progenitor cells. VEGF-induced proliferation is influenced by the MEK-ERK pathway, as well as by the basic helix-loop-helix factor HES1. By contrast, VEGF-dependent ganglion cell suppression does not require MEK-ERK activation, but instead relies on VEGF-stimulated HES1 activity, which is independent of NOTCH signaling. Moreover, elevated HES1 expression promotes progenitor cell proliferation and prevents overproduction of retinal ganglion cells owing to the loss of VEGF or sonic hedgehog (SHH), another signal that suppresses ganglion cell development. Based on previous and current findings, we propose that HES1 serves as a convergent signaling node within early retinal progenitor cells to integrate various cell-extrinsic cues, including VEGF and SHH, in order to control cell proliferation and neuronal specification.