Depolarization without calcium can release gamma-aminobutyric acid from a retinal neuron.

Calcium influx is often an essential intermediate step for the release of neurotransmitter. However, some retinal neurons appear to release transmitter by a mechanism that does not require calcium influx. It was uncertain whether depolarization released calcium from an intracellular store or released transmitter by a mechanism that does not require calcium. The possibility that voltage, and not calcium, can regulate the release of transmitter was studied with pairs of solitary retinal neurons. Horizontal and bipolar cells were isolated from fish retinas and juxtaposed in culture. Communication between them was studied with electrophysiological methods. A horizontal cell released its neurotransmitter, gamma-aminobutyric acid, when depolarized during conditions that buffered the internal calcium concentration and prohibited calcium entry. The speed and amount of material released were sufficient for a contribution to synaptic transmission.

L-glutamate conditionally modulates the K+ current of Müller glial cells.

L-Glutamate inhibits the K+ conductance that dominates the electrical behavior of a Müller glial cell. The effect of glutamate is enhanced by simultaneous exposure to dopamine. L-Glutamate acts at a metabotropic receptor that controls the K+ conductance through two pathways. A rapid pathway produces a partial inhibition in less than 2 s. Thereafter, a slow pathway progressively inhibits the conductance with a half-time of minutes. Pathways initiated by L-glutamate and dopamine appear to converge on and stimulate adenylyl cyclase. A subsequent step is the activation of a cAMP-dependent protein kinase, PKA. The local overflow of L-glutamate from active synapses may functionally remove K+ channels from nearby glial membranes. A uniform rise in extracellular L-glutamate concentration, as might occur during pathological conditions, should suppress a glial cell's K+ conductance and allow other voltage-dependent processes to be influenced by depolarization.

A cGMP-gated current can control exocytosis at cone synapses.

The voltage-gated Ca2+ current in cone photoreceptors operates over only a small part of the physiological voltage range produced by light and, consequently, appears insufficient for controlling transmitter release. We have used a whole-cell voltage clamp to measure membrane current and the capacitance change produced by exocytosis in solitary cone and rod photoreceptors isolated from the salamander retina. In both types of photoreceptor, Ca2+ influx through voltage-gated Ca2+ channels initiated exocytosis. In addition, Ca2+ influx through a cGMP-gated channel in the inner segment and synaptic processes of cones also initiated exocytosis. The cGMP-gated current sustained exocytosis over the entire physiological voltage range.

A GABA transporter operates asymmetrically and with variable stoichiometry.

Membrane currents produced by the expression of a rat GABA transporter (GAT-1) stably transfected into HEK293 cells were characterized with a whole-cell voltage clamp. Three modes of function were identified: ex-gated currents produced by extracellular GABA, in-gated currents produced by intracellular GABA, and uncoupled currents produced in the absence of GABA. The ex-gated current was not the reversal of the in-gated current; moreover, the stoichiometry between GABA and co-ions was not always fixed. Each mode of function required a different set of ions on the two sides of the membrane. We made rapid solution changes and observed an allosteric effect of Na+ that only occurred at the extracellular surface. Thus, the GAT-1 transporter does not behave like a recirculating carrier but may be described as a pore with ion gates at either end that are controlled in part by allosteric sites.

Characterization of deoxyribonucleic acid (DNA) obtained from teeth subjected to various environmental conditions.

This study was designed to determine the effects of various environmental factors on the deoxyribonucleic acid (DNA) obtained from dental pulp. Extracted teeth were subjected to the following conditions: varying pH (3,7,10); temperature (4 degrees C, 25 degrees C, 37 degrees C, incineration); humidity (20%, 66%, 98%); various types of soil (sand, potting soil, garden soil); seawater; burying the teeth outdoors, and aging (one week to six months). In addition, teeth that had been extracted and held at room temperature for 16 and 19 years were also examined. Following isolation of DNA, the samples were analyzed on yield gels to determine the concentration and integrity of the recovered DNA. Restriction digestion with Pst I was followed by electrophoresis of the generated fragments, Southern transfer to nylon membranes, and hybridization to both human and bacterial probes. It was determined that, aside from soil, the environmental conditions examined did not affect the ability to obtain high-molecular-weight human DNA from dental pulp. Restriction fragment length polymorphic (RFLP) analysis of selected samples was performed. Dental pulp patterns were compared with bloodstain exemplars, revealing matching patterns, although an increase in band-shifting was observed with extended exposure to elevated temperatures.

Optical analyses of eyeglass lens fragments and the unexpected detection of oral sperm in a homicide case.

A homicide case in which intact spermatozoa were found in the oral cavity of the deceased forty days after his disappearance is reported. The victim's partially frozen body was found outdoors in a wooded area of upstate New York during the month of January. During a subsequent investigation, pieces of eyeglass lens fragments and bloodstains were found in the suspect's house and vehicle. Chemical and optical analyses of the lens fragments are presented as well as results of the serological tests.

Responses of bipolar cells in the retina of the turtle.

The responses of bipolar cells in the retina of the turtle have been studied by intracellular recording. Two types of bipolar cell have been identified: one gave graded depolarizing and the other graded hyperpolarizing responses to small circles of light (100 mum diameter). The responses of both types of cell were similar in the following respects.1. Both were extremely sensitive to dim light; the amplitude of response to a small circle of light increased with light intensity more steeply than the cone response.2. Enlarging the diameter of a spot added an antagonistic effect which decreased response amplitude. This decrease in response amplitude was more apparent at dim than at bright light. Stimulating only distant areas of retina with an annulus produced a response of polarity opposite to that normally produced by a central spot. However, the responses of bipolar cells did not appear to be due to a simple summation of opposite polarity signals contributed from central and peripheral parts of their receptive fields.3. When small spots or annuli of light were turned off there frequently occurred an overshooting OFF transient. The occurrence of OFF transients depended on the duration of the stimulus. Cones recorded under similar conditions produced an OFF depolarization. The size of cone OFF depolarizations increased with increasing duration of the preceding light; following approximately 3 sec of illumination their maximum amplitude was roughly 1/10 the amplitude of the preceding hyperpolarization. The size of OFF responses in both cone and bipolar cells was increased when horizontal cells were hyperpolarized by light. It is concluded that bipolar cells produce large responses for very small cone responses, and, as a consequence, a small depolarization in cones following illumination produces large OFF transients in bipolar cells. Furthermore, the responses of bipolar cells do not appear to represent a simple summation of opposite polarity input from receptor and horizontal cells.

Synaptic transmission in amphibian retinae during conditions unfavourable for calcium entry into presynaptic terminals.

Toad (Bufo marinus) retinae were peeled from the pigment epithelium and superfused over the photoreceptor surface with a calcium-poor, cobalt-rich medium. The shape of the electroretinogram indicated that post-synaptic neurones received synaptic input. Adding the putative transmitters glutamate and N-acetylhistidine changed the shape of the electroretinogram. The change suggests that an excess of the putative transmitters blocked a component of synaptic transmission that persisted when a retina was bathed in cobalt. Salamander (Ambystomatigrinum) retinae in hemisected eye cups were superfused over their vitreal surface. Intracellular responses were recorded from photoreceptors. Reducing the calcium concentration in the superfusing medium from 1 mM to less than 10 microM slowly changed responses produced by light. The change indicates that the calcium concentration in the extracellular space surrounding photoreceptors fell to less than 100 microM. When retinae were superfused with a medium containing 1 mM-calcium, 3 mM-barium, and 10 mM-tetraethylammonium (TEA), rods produced action potentials that were later blocked by adding 1 mM-cobalt. Blocking calcium channels with cobalt and lowering the extracellular calcium concentration should together block calcium-dependent synaptic transmission. Intracellular responses were recorded from horizontal cells. After replacing external calcium with cobalt the membrane potential hyperpolarized and responses produced by light became smaller but did not entirely disappear. The responses that remained were less sensitive to light and had an altered shape. The change was reversible. Similar responses could be recorded after prolonged (30-120 min) exposure to cobalt. Electrical synapses between horizontal cells were uncoupled by adding 10 microM-forskolin to the cobalt medium. The polarity of a response could then be reversed if a cell was depolarized by injecting current. The observation of a reversal potential demonstrates that the response was produced by a conductance change. Intracellular responses were recorded from depolarizing and hyperpolarizing bipolar cells while retinae were superfused with cobalt-rich medium. After changing to a cobalt-free medium containing 1 mM-calcium, responses produced by light were slightly smaller. Large responses were recorded after superfusing with cobalt-rich, calcium-poor medium for 30-120 min. The results indicate that synaptic transmission by photoreceptors continues during conditions expected to block the entry of calcium into their presynaptic terminals.

Responses of single rods in the retina of the turtle.

1. The responses of rods in the retina of the turtle, Chelydra serpentina, have been studied by intracellular recording.2. The identification of rods as the origin of the recorded responses has been confirmed by marking with Procion Yellow.3. The response to a small spot of light was a hyperpolarization which increased with increasing light intensity. For dim, small diameter stimuli, the shape of the rod response was similar to that of cones but 2x slower and 2x larger in amplitude. The time integral of the rod response to a dim, small diameter flash is, therefore, approximately 4x greater than the integral of the cone response.4. The shape of the rod response depended on the pattern of retinal illumination as well as stimulus intensity. Enlarging the area of illumination increased the peak amplitude and delayed repolarization following a light step. The area of retina which influenced the response was approximately 200 mum in radius.5. It is concluded that for dim light the responses of rods are larger than those of cones because of (i) a greater response to direct illumination and (ii) an enhancement of response by interaction from a large retinal area.

Organization of on-off cells in the retina of the turtle.

1. The organization of central and peripheral responses for one type of spike-producing cell in the retina of the turtle (Pseudemys scripta elegans) has been studied. These cells produced a short burst of spikes following the onset and offset of a small spot of illumination (i.e. ON-OFF cells). The effect of increasing the area of illumination was to include a peripheral inhibition. Intracellular ON and OFF responses were, however, affected differently.2. ON-OFF cells marked intracellularly with Procion Yellow M4R had somata in the inner nuclear and ganglion cell layers.3. Peripheral inhibition could be evoked without a change in the membrane potential of horizontal cells.4. Passing 5 nA hyperpolarizing or depolarizing current through a micropipette within a horizontal cell elicited in ON-OFF cells a transient excitation following the onset and offset of current.5. It is concluded that inhibition from the periphery of an ON-OFF cell receptive field is not mediated by luminosity horizontal cells but more probably by peripheral bipolar cells.

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This article offers the nurse information needed for planning the care of Jewish patients and their families during the dying and mourning process - whether that patient is Orthodox, Conservative, or Reform.

Transport-mediated synapses in the retina.

Most synapses rely on regulated exocytosis for determining the concentration of transmitter in the synaptic cleft. However, this mechanism may not be universal. Several synapses in the retina appear to use a synaptic machinery in which transmitter transporters play an essential role. Two types of transport-mediated synapses have been proposed. These synapses have been best observed in horizontal cells and cones of nonmammalian retinas. Horizontal cells use a transporter to mediate a bidirectional shuttle, whose balance point is set by ion concentrations and voltage. Nonmammalian cones combine exocytosis and the activity of a transporter. Because exocytosis is voltage independent over most of a cone's physiological voltage range, a voltage-dependent transporter determines the concentration of transmitter in the synaptic cleft. These two synapses may be models for transport-mediated synapses that operate in other parts of the brain.

Voltage noise observed in rods of the turtle retina.

1. Intracellular voltage was recorded from rods in isolated retinae of the snapping turtle, Chelydra serpentina. The voltage was observed during darkness or during uniform illumination of a large retinal area. During darkness the voltage fluctuated continuously about a mean level. The spontaneous fluctuation is termed ;noise'. During illumination the amplitude of the noise was reduced.2. The noise observed during darkness could also be reduced by injecting a hyperpolarizing current into the impaled rod. The noise could be increased by a depolarizing current. The component of the noise that could be altered by polarizing the rod is termed ;voltage-sensitive noise'.3. When voltage-sensitive noise was first minimized by a continuous hyperpolarizing current, bright light produced an additional decrease in the noise. The component of the noise that was eliminated by light, but not eliminated by the injection of current, is termed ;light-sensitive noise'.4. The power density spectrum of voltage-sensitive noise, G(v)(f), could be described by an equation of the form [Formula: see text] tau(M) was approximately 7 msec, which is in good agreement with an apparent membrane time constant of 5-8 msec. The largest value of alpha(v) was 2.1 x 10(-9) V(2) sec.5. The power density spectrum of light-sensitive noise could be described by an equation of the form [Formula: see text] tau(L) was approximately 200-300 msec. The largest value of alpha(L) was 8.0 x 10(-9) V(2) sec.6. The potential maintained during darkness could be altered by superfusing a retina with artificial media of different compositions. Depolarizing the rods by changing the extracellular calcium concentration from 1 to 5 mM increased the voltage-sensitive noise. A similar effect was observed after adding 2 mM lanthanum.7. In contrast, 5 mM cobalt produced a small hyperpolarization and suppressed the voltage-sensitive noise. Injecting a depolarizing current, after exposure to cobalt, re-initiated the voltage-sensitive noise. The ability to elicit voltage-sensitive noise in the presence of cobalt indicates that it was not of synaptic origin.8. The results are consistent with the noise present during dark being produced by two types of channel in the rod membrane. One is controlled by the phototransduction process; each individual channel of this type may be described as having a mean open time of 200-300 msec and a conductance of approx. 6 x 10(-13) Omega(-1). The absorption of one photon closes approx. 100-300 of these channels. The other type of channel is controlled by membrane potential; each individual channel of this type has a mean open time which is less than the membrane time constant of 8 msec.

Electrical properties of the rod syncytium in the retina of the turtle.

1. Intracellular responses were recorded from rods in isolated eye-cups of the snapping turtle. Chelydra serpentina. Responses to flashes of small (less than 100 mum diameter) and large (1000 mum diameter) spots of 500 nm light were studied. 2. Responses produced by small and large diameter spots which delivered less than 0-3 photons mum-2 had the same shape. The responses produced by large spots were, however, nearly ten times greater in amplitude. The difference in amplitude is termed enhancement. 3. Perfusing an eye-cup with a Co2+-containing medium blocked synaptic transmission from receptors to horizontal cells but did not affect the responses of rods. 4. The membrane conductance of a single rod, estimated by three independent methods, was approximately 1-2 X 10(-9) MHo. 5. Enhancement can be predicted by a mathematical model which treats rods as an electrical syncytium. The space coefficient describing the spread of current is approximately 65 mum indicating that the coupling conductance between rods was relatively high. 6. When the intensity of a small spot was increased from 0-3 photons mum-2 up to 6 photons mum-2, the shape of the response was unchanged. When the intensity of a large spot was increased to more than 0-3 photons mum-2, the voltage during the recovery phase was decreased. This decrease is termed disenhancement. 7. The voltages produced by bright, large and small diameter spots which delivered the same quantity of light to the impaled rod were compared. The voltage produced by a large diameter spot became for a short period during the recovery phase less than the voltage produced by a small diameter spot. This observation indicates that the response to a large spot included during recovery an active process which is not apparent in the response to a small spot.

Rod-rod interaction in the retina of the turtle.

Intracellular responses were recorded from rods in isolated eye-cups of the snapping turtle, Chelydra serpentina. Responses to small and large diameter spots of 500 nm light were studied. 1. The peak amplitudes of responses smaller than approximately 2 mV were directly proportional to irradiance. Small spots (less than 100 mum diameter) produced approximately 30 muV/rhodopsin molecule bleached. Increasing stimulus diameter to 400-500 mum increased this five to seven times to about 200 muV/rhodopsin molecule bleached in the impaled receptor. The difference is attributed to a neural "enhancement" produced by stimulating neighbouring rods. 2. Enlarging the diameter of a spot altered the shape of responses produced by very dim lights. 3. The variance of responses to a small spot was only slightly less than the mean. The variance of responses to a large spot was much less than the mean. 4. Responses evoked by a small spot of dim light obeyed the superposition principle in that the response to a very dim step of light was the integral of the response of a very dim flash. Responses evoked by a large spot did not obey the superposition principle. The response to a step of dim light covering a spot of large diameter was less than predicted from the integral of the response to a flash. The difference is attributed to a neural "disenhancement" produced by stimulating neighbouring rods. 5. The time course of this disenhancement could be observed by presenting two large diameter, dim flashes within a short interval. The time course of disenhancement did not coincide with that of the voltage response but was delayed such that its maximum occurred after the peak amplitude. 6. Dim background lights of different diameter, which delivered the same quantity of light to the impaled cell but very different quantities of light to neighbouring cells, left the response produced by a small diameter test spot unaltered. It is concluded that rod-rod interaction can modify the intracellular responses of rods in two ways; it produces an early enhancement which increases response amplitude nearly tenfold and also a delayed disenhancement which replaces the wave of enhancement that follows a flash.

Cones excite rods in the retina of the turtle.

The intracellular responses of rods in the retina of the turtle, Chelydra serpentina, were studied with brief flashes of monochromatic light. 1. Flashes of red or green light applied over an area 25 mum in diameter produce responses with the same shape. With such restricted stimuli, the spectral sensitivity of a rod agrees well with the absorption spectrum of the porphyropsin pigment contained in its outer segment. 2. With stimulating spots more than 500 mum in diameter, dim flashes of red or green light produce responses having different shapes. When the spectral sensitivity of a rod is tested using dim lights of large diameter, the sensitivity to red light is much greater than predicted by the absorption spectrum of porphyropsin. 3. The shape of the response produced by large diameter spots of dim, red light resembles that of cones. 4. Increasing the diameter of a dim, red spot beyond 500 mum markedly alters the amplitude and shape of responses from horizontal cells but does not significantly affect the response of rods. It is concluded that rods receive an excitation from neighbouring cones. This interaction is unlikely to be mediated by type I luminosity horizontal cells but may be mediated by either direct connexions between cones and rods or by an interneurone with a small receptive field.

Calcium-independent release of GABA from isolated horizontal cells of the toad retina.

1. When toad retinae were incubated first with veratrine, then with antibodies that reacted with the outer segments of photoreceptors, and finally with complement, horizontal cells survived and most other neurones died. This preparation of 'isolated' horizontal cells accumulated radioactive GABA from the incubation medium. The subsequent release of radioactive GABA could then be measured. 2. The efflux of GABA was increased by exposure to an elevated potassium concentration or added glutamate. Both procedures are known to depolarize horizontal cells. 3. GABA in the external medium also increased the efflux of GABA. 4. The increase in GABA efflux produced by an elevated potassium concentration was unaffected with calcium in the external medium was replaced with cobalt and when sodium was replaced with choline or lithium. 5. The increase in GABA efflux produced by glutamate was unaffected when calcium was replaced with cobalt and when sodium was replaced with lithium, but was inhibited when sodium was replaced with choline. 6. The increase in GABA efflux produced by external GABA was unaffected when calcium was replaced with cobalt but required sodium. Neither choline nor lithium would substitute for sodium. 7. An increase in GABA efflux was accompanied by an increase in sodium efflux. 8. After a high concentration of GABA (2-20 mM) had produced a maximal increase in GABA efflux, the addition of glutamate produced no further effect. Conversely, after a high concentration of glutamate (2-20 mM) had produced a maximal increase in efflux, the addition of external GABA produced only a small further increase. These and the preceding results could occur if GABA release were mediated by a carrier system which could be activated by either depolarization or homoexchange.

Electrophysiology of glutamate and sodium co-transport in a glial cell of the salamander retina.

1. Müller cells were isolated from salamander retinas and their membrane voltage was controlled with a whole-cell voltage clamp. External D-aspartate, L-aspartate and L-glutamate each induced a membrane current. D-Glutamate, kainate, quisqualate and N-methyl-D-aspartate were more than 100x less effective than L-aspartate. Kynurenic acid had no effect on the current produced by L-glutamate, L-aspartate or D-aspartate. 2. The current induced by an acidic amino acid (AAA) was completely dependent on the presence of external Na+. Neither Li+, Cs+, choline nor TEA+ were able to substitute for Na+. The relationship between external Na+ concentration and current amplitude can be explained if the binding of three Na+ ions enabled transport. The apparent affinity constant for Na+ binding was 41 mM. Altering K+, H+ and Cl- concentrations demonstrated that these ions are not required for transport. 3. The shape of the current-voltage relation did not depend on the external amino acid concentration. The relationship between D-aspartate concentration and current amplitude can be described by the binding of D-aspartate to a single site with an apparent affinity constant of 20 microM. 4. Influx and efflux of AAA were not symmetric. Although influx was electrogenic, efflux did not produce a current. Moreover, influx stimulated efflux; but efflux inhibited influx. 5. Removing external Na+ demonstrated that Na+ carried a current in the absence of an AAA. Li+ was a very poor substitute for Na+. This current may be due to the uncoupled movement of Na+ through the transporter. The relationship between the external Na+ concentration and the amplitude of the uncoupled current can be explained if the binding of two or three Na+ ions enabled the translocation of Na+ in the absence of an AAA. The apparent affinity constant for Na+ binding was approximately 90 mM. 6. The temperature dependence of the AAA-induced current had a Q10 between 8 and 18 degrees C of 1.95. The Q10 is consistent with a rate constant for influx of 10(4) s-1 (at -70 mV and 20 degrees C). The maximum rate of influx was measured following a concentration jump produced by the photolysis of 'caged' L-glutamate. The onset of the observed current was limited by the 1.3 ms resolution of the recording system. Hence, the rate constant for influx must be faster than 10(3) s-1.(ABSTRACT TRUNCATED AT 400 WORDS)