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.     

Intracellular recordings from spinal neurons during 'swimming' in paralysed amphibian embryos

Intracellular microelectrode recordings have been made from probable motoneurons in the spinal cord of Xenopus laevis embryos during fictive 'swimming' in preparations paralysed with the neuromuscular blocking agent tubocurarine. These cells had resting potentials of -50 mV or more. During spontaneous or stimulus-evoked 'swimming' episodes: (a) the cells were tonically excited; the level of tonic synaptic excitation and the conductance increase underlying it were both inversely related to the 'swimming' cycle period; (b) the cells usually fired one spike per cycle in phase with the motor root burst on the same side; spikes did not overshoot zero and were evoked by phasic excitatory synaptic input on each cycle, superimposed on the tonic excitation; (c) in phase with motor root discharge on the opposite side of the body, the cells were hyperpolarized by a chloride-dependent inhibitory postsynaptic potential. The nature of synaptic potentials during 'swimming' was evaluated by means of intracellular current injections. The 'swimming' activity could be controlled by natural stimuli. The results provide clear evidence on the relation of tonic excitation to rhythmic locomotory pattern generation, and indirect evidence for reciprocal inhibitory coupling between antagonistic motor systems.     

Intracellular recordings from nonspiking interneurons in a semiintact,tethered walking insect

Nonspiking interneurons were investigated in a tethered, walking insect, Carausius morosus, that was able to freely perform walking movements. Experiments were carried out with animals walking on a lightweight, double-wheel treadmill. Although the animal was opened dorsally, the walking system was left intact. Intracellular recordings were obtained from the dorsal posterior neuropil of the mesothoracic ganglion. Nonspiking inter-neurons, in which modulations of the membrane potential were correlated with the walking rhythm, were described physiologically and stained with Lucifer Yellow. Interneurons are demonstrated in which membrane potential oscillations mirror the leg position or show correlation with the motoneuronal activity of the protractor and retractor coxae muscles during walking. Other interneurons showed distinct hyperpolarizations at certain important trigger points in the step cycle, for example, at the extreme posterior position. Through electrical stimulation of single, nonspiking interneurons during walking, the motoneuronal activity in two antagonistic muscles—protractor and retractor coxae—could be reversed and even the movement of the ipsilateral leg could be influenced. The nonspiking interneurons described appear to be important premotor elements involved in walking. They receive, integrate, and process information from different leg proprioceptors and drive groups of leg motoneurons during walking.     

Intracellular calcium recordings from isolated cells of the mammalian central nervous system

Measurements made with two different techniques of intracellular calcium levels from small isolated cells of the mammalian central nervous system are described and compared. Recordings in cultured mouse embryo spinal cord and dorsal root ganglion neurons, made with double-barrelled borosilicate Ca2+-selective microelectrodes yielded a mean Ca2+ level of 2.3 (SE +/- 0.54) microM for the lowest values recorded in 24 out of 46 cells. Intracellular Ca2+ dependence on membrane potential was apparent with levels of calcium greater than or equal to 4 microM (r = 0.371, n = 29). Both cyclic fluctuations induced by tetraethylammonium and an apparent increase in Ca2+ evoked by the depolarizing excitatory amino acid, L-aspartate, were observed. In contrast, estimates of intracellular Ca2+ obtained by spectrofluorimetry of suspensions of mouse embryo brain cells, loaded with the intracellular Ca-binding fluorescent probe, quin2 provided a approximately equal to 10-fold lower value, 152 (SE +/- 7) nM. This more closely resembles levels reported for large neurons where large-tip microelectrodes with greater sensitivity were used, and in spite of the heterogeneity of the cells this value is presumed to be a more accurate estimate of intraneuronal Ca2+ concentration. In these fluorescence studies KCl readily evoked increases in intracellular Ca2+ which could be blocked by verapamil and Cd2+ and were not induced in the absence of Ca2+. Increases were also produced by N-methyl-D-aspartate, but not by the kainate-like Lathyrus neurotoxin, L-3-oxalylamino-2-aminopropionic acid. These results provide preliminary evidence for both voltage-sensitive and receptor-activated Ca channels in embryonic brain cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular recordings from nonspiking interneurons in a semiintact, tethered walking insect.

Nonspiking interneurons were investigated in a tethered, walking insect, Carausius morosus, that was able to freely perform walking movements. Experiments were carried out with animals walking on a lightweight, double-wheel treadmill. Although the animal was opened dorsally, the walking system was left intact. Intracellular recordings were obtained from the dorsal posterior neuropil of the mesothoracic ganglion. Nonspiking interneurons, in which modulations of the membrane potential were correlated with the walking rhythm, were described physiologically and stained with Lucifer Yellow. Interneurons are demonstrated in which membrane potential oscillations mirror the leg position or show correlation with the motoneuronal activity of the protractor and retractor coxae muscles during walking. Other interneurons showed distinct hyperpolarizations at certain important trigger points in the step cycle, for example, at the extreme posterior position. Through electrical stimulation of single, nonspiking interneurons during walking, the motoneuronal activity in two antagonistic muscles--protractor and retractor coxae--could be reversed and even the movement of the ipsilateral leg could be influenced. The nonspiking interneurons described appear to be important premotor elements involved in walking. They receive, integrate, and process information from different leg proprioceptors and drive groups of leg motoneurons during walking.     

New photoresponses of phycomyces

The influence of light on asexual fruiting and mycelial growth of Phycomyces blakesleeanus Burgeff was studied by means of fruiting body counts and size measurements in cultures on solid media under varied incubation conditions. Five types of photoresponses were shown by ATCC Strain 8743a: (a) photoinduction of giant sporangiophores; (b) interference by light with an endogenous system that otherwise induces fruiting when the mycelium approaches the rim of the Petri dish; (c) inhibition of mycelial growth rates by light; (d) inhibition of dwarf sporangiophore induction by light; and (e) postponement by light of death in clones maintained by serial transfer at low temperature. A second strain, designated G5, showed responses comparable to (a), (b), and (d). The magnitudes of the responses were greatly affected by temperature of incubation and available nitrogen (asparagine) supply. The photoinduction of giant sporangiophores could be demonstrated with light of wavelengths between 380 and 480 nanometers but not with 520 nanometers or above. At 480 nanometers, light doses as small as 40 ergs per square centimeter were effective in inducing giant sporangiophores in strain 8743a.     

Intracellular voltage recordings in the opercular epithelium of Fundus heteroclitus

Opercular epithelial cells of Fundus heteroclitus were investigated using conventional microelectrodes. The area of interest was the cells lining the inside of the opercular epithelium closest to the gill arches, an area with a high density of chloride cells. Only one cell type could be discerned from the values of 60 opercular cells measured with the opercular epithelium in open circuit conditions. A mean apical voltage of -18.0 +/- 0.6 mV was observed with intracellular values ranging from -10 to -30 mV. The predicted intracellular chloride content was 59 mM/liter. Apical fractional resistance (faR) was 0.78 +/- 0.02. The intracellular potential measurements were typically difficult to maintain for extended periods (longer than 3 min). The opercular cells depolarize with serosal isoproterenol treatment (10(-6) M) corresponding to the increase in opercular transepithelial potential. The opercular cell apical fR decreased with isoproterenol treatment. These data indicate the observed opercular cells were involved in opercular chloride transport.     

Intersexual partial diploids of phycomyces

Sexual interaction between strains of opposite sex in many fungi of the order Mucorales modifies hyphal morphology and increases the carotene content. The progeny of crosses of Phycomyces blakesleeanus usually include a small proportion of anomalous segregants that show these signs of sexual stimulation without a partner. We have analyzed the genetic constitution of such segregants from crosses that involved a carF mutation for overaccumulation of beta-carotene and other markers. The new strains were diploids or partial diploids heterozygous for the sex markers. Diploidy was unknown in this fungus and in the Zygomycetes. Random chromosome losses during the vegetative growth of the diploid led to heterokaryosis in the coenocytic mycelia and eventually to sectors of various tints and mating behavior. The changes in the nuclear composition of the mycelia could be followed by selecting for individual nuclei. The results impose a reinterpretation of the sexual cycle of Phycomyces. Some of the intersexual strains that carried the carF mutation contained 25 mg beta-carotene per gram of dry mass and were sufficiently stable for practical use in carotene production.     

Alternative fruiting pathways in phycomyces

Developmental distinctions between giant and dwarf fruiting bodies of Phycomyces blakesleeanus (Burgeff) were studied by means of size measurements and growth analyses. Histograms of fruiting body lengths showed a bimodal distribution, with peaks around 0.3 millimeter (dwarfs) and 30 millimeters (giants). Individual cultures contain both giants and dwarfs. Differences between giants and dwarfs appear in the first phase of development; the apex of the giant is tapered, whereas the dwarf apex is dome-shaped. Probable cytological distinctions at this stage are cited in discussion. The dwarfs terminate enlargement upon expansion of the sporangium, thus lacking the subsequent phase of rapid elongation (stage IV) that contributes 90% of the length in the case of giants. It was concluded that P. blakesleeanus maintains two developmental patterns for asexual fruiting, with dwarfs and giants differing radically in growth regulation.     

Local metabolic autonomy in phycomyces sporangiophores

The degree of dependence of the growing zone of the Phycomyces sporangiophore upon other parts of the stalk was tested by inhibiting glycolysis and oxidative phosphorylation in the stalk below the growing zone. Initially the growing zone is capable of nearly normal growth when the metabolism in the rest of the stalk is inhibited in this way. However, the growing zone appears to become depleted of something normally supplied from below, because after 3 to 4 hr its growth rate slows down and the growth stops much sooner than in normal sporangiophores. The rest of the sporangiophore appears to have a similar degree of local autonomy because isolated sections from below the growing zone can support protoplasmic streaming for 10 to 20 hr.     

Temperature-induced reversals of rotation in phycomyces

The steady state extension and rotation rates of the Phycomyces sporangiophore were measured as a function of temperature. Maximum growth occurred at 27°C; maximum rotation at 28°C. The rotation to extension ratio, a qualitative parameter of cell wall structure, is affected differently by high and low temperatures. Steady state counterclockwise rotation, as opposed to the normal clockwise rotation, was found at both high and low temperatures. The extensional and rotational responses to step changes in temperature were also measured. The conclusions are drawn that a relative decrease in the lysis rate of wall polymer is responsible for the decrease in growth rate at low temperatures, and that a relative increase in the rate of wall synthesis and cross-linking is responsible for the decrease in growth rate at high temperatures. It is suggested that reversals in rotation result from changes in the handedness of the wall's helical structure.     

Growth rate fluctuations in phycomyces sporangiophores

The growth rate of the Phycomyces sporangiophore fluctuates under constant environmental conditions. These fluctuations underlie the well-characterized sensory responses to environmental changes. We compared growth fluctuations in sporangiophores of unstimulated wild type and behavioral mutants by use of maximum entropy spectral analysis, a mathematical technique that estimates the frequency and amplitude of oscillations in a time series. The mutants studied are believed to be altered near the input (“night-blind”) or output (“stiff” and “hypertropic”) of the photosensory transduction chain. The maximum entropy spectrum of wild type shows a sharp drop-off in spectral density above 0.3 millihertz, several minor peaks between 0.3 and 10 millihertz, and a broad maximum near 10 millihertz. Similar spectra were obtained for a night-blind mutant and a hypertropic mutant. In contrast, the spectra of three stiff mutants, defective in genes madD, madE, or madG, had distinctive peaks near 1.6 mHz and harmonics of this frequency. A madF stiff mutant, which is less stiff than madD, madE, and madG mutants, had a spectrum intermediate between wild type and the three other stiff mutants. Our results indicate that alterations in one or more steps associated with growth regulation output cause the Phycomyces sporangiophore to express a rhythmic growth rate.     

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.     

Genetic recombination in sexual crosses of phycomyces

Sexual crosses between strains of Phycomyces blakesleeanus , involving three auxotrophic and one color marker and yielding a high proportion of zygospore germination, are described. Samples of 20–40 germ spores from 311 individual fertile germ sporangia originating from five two-factor and three three-factor crosses were characterized. The results show: (1) absence of any contribution of apogamic nuclei to the progeny, (2) confirmation of Burgeff's conjecture that the germ spores of any germ sporangium in most cases derive from one meiosis. In a cross involving two allelic markers the analysis of 175 pooled germ sporangia suggests an intragenic recombination frequency of 0.6%. All other factor combinations tested are unlinked. The bulk of the germ spores are homokaryotic. However, a small portion (4%) are heterokaryotic with respect to mating type.     

Electromyogram recordings from freely moving animals

Electromyography can be used to record activity from sets of muscles in awake, freely moving animals using implanted intramuscular electrodes. As a tool, EMG has a wide range of applications ranging from inferring neural processes to analyzing movement. The amplitude of the rectified and filtered electromyogram (EMG) can be used as an indirect measure of muscle activity. Although it is often tempting to correlate the EMG with muscle force, the fact that force varies more with different activation strategies than with EMG estimates must be taken into account. The purpose of this article is to provide the researcher wishing to introduce the technique of recording EMGs from conscious animals using intramuscular electrodes with a step-by-step guide. It includes details on the manufacture of electromyograph electrodes, recording, and analysis considerations along with a section on solving common problems. For the sake of clarity, this article focuses on using the cat as a model and on the implantation of hindlimb muscles with intramuscular wire electrodes. However, the procedures can be adapted for use on other striated muscles and species.     

Pressure probe technique to study transpiration in phycomyces sporangiophores

The growth equation for the rate of water uptake is augmented with a transpiration term. The obtained augmented growth equations are used to develop methodology which employs the pressure probe to measure transpiration rates from single plant cells. Experiments are conducted on the sporangiophores of Phycomyces blakesleeanus to demonstrate this technique.     

Crystalloids of phycomyces sporangiophores: nature and photosensitive accumulation

Crystalloids accumulate in the vacuoles of the giant sporangiophores of Phycomyces blakesleeanus Burgeff during growth. On the basis of solubility in alkaline solutions, cytochemical staining reactions, trypsin sensitivity, optical absorption and response in the Lowry protein test, the crystalloids have been judged to consist principally of an acidic protein. In assays by Lowry test and by reference to optical absorption at 280 mμ, dark-grown sporangiophores were consistently found to contain from 2 to 4 times as much crystalloid material as light-grown counterparts. Concurrent assays of soluble phenolic materials revealed no significant effect of culture illumination, while carotene content of sporangiophores and mycelium was found to be raised from 2 to 4-fold by illumination during growth.