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C J Karwoski

Publications and source records attributed to C J Karwoski.

At least 19 recordsLinked to original sources
PubMed1998

Ultrastructure of the frog retina after high-pressure freezing and freeze substitution.

In many types of tissue, high-pressure freezing (HPF), followed by freeze substitution, can produce excellent ultrastructural preservation at depths over 10 times that obtained by other cryofixation techniques. However, in the case of neural tissue, the benefits of HPF have not been realized. In the present study, isolated frog (Rana pipiens) retina was sliced at a thickness of 150 or 350 microns, rapidly frozen in a Balzers HPM 010 high-pressure freezer, and freeze substituted with 1% OsO4 and 0.1% tannic acid in acetone. Specially designed HPF chambers and specific freezing media (35% high-MW dextran for 150-micron slices or 15% low-MW dextran for 350-micron slices) were required for adequate freezing. The quality of preservation after HPF was excellent throughout the retina in both the 150- and 350-micron slices, compared with chemically fixed slices. Specifically, HPF resulted in better preserved cellular, mitochondrial and nuclear membranes in all retinal layers. This is the first study to successfully cryofix all of the layers of the retina. The increased depths of adequate freezing achieved by HPF should facilitate various ultrastructural studies of retina, as well as of other CNS tissues, where preservation approaching that of the 'native' state is required.

Animals↗
PubMed1996

Current-source density analysis of the electroretinogram of the frog: methodological issues and origin of components.

The technique of current-source density (CSD) analysis for extracellular potentials is reviewed, along with some methodological features that are important for performing CSD analysis of the electroretinogram. In addition, three formulas for computing CSD's are examined on model circuits of resistors and current generators. Finally, CSD results from frog retina that bear on the origins of the b, d, and M waves, along with slow PIII, are presented. It is concluded that the b and d waves are generated primarily and directly by bipolar cells, whereas the M wave and the slow PIII are generated by Müller (glial) cells through the K+ spatial buffer mechanism.

Animals↗
PubMed1994

Current source density (CSD) analysis of retinal field potentials. I. Methodological considerations and depth profiles.

1. The technique of current source density (CSD) analysis was used to obtain depth profiles of light-evoked source/sink distributions in the retina of the frog. 2. The effects of a number of methodological considerations on the CSD profiles were explored. Adoption of best technique leads to minimal variability and noise in the CSD profiles. Several situations that give rise to artifactual source/sinks were identified. 3. At the time of the b-wave peak, there is a large sink near the outer plexiform layer (OPL), a source at the inner limiting membrane (ILM), and a complex response at the inner plexiform layer (IPL). 4. The IPL response at light onset consists of 1) an initial, sharp sink, followed by 2) a slower source with a time course similar to the b-wave, and last 3) an even slower sink with a time course similar to the M-wave. 5. The first component of the IPL response is the current sink for the proximal negative response (PNR) elicited by a diffuse light stimulus. The PNR has current sources both proximal and distal to this layer. 6. The OPL sink underlies the b-wave. The second component of the IPL response is a source for the b-wave. Part of the ILM source also might be a b-wave source. 7. A large part of the ILM source is the current source for the M-wave. The third component of the IPL response is the M-wave sink, but it is small because it subtractively interacts with the IPL b-wave source.

Animals↗
PubMed1994

Current source density analysis of retinal field potentials. II. Pharmacological analysis of the b-wave and M-wave.

1. The actions of two pharmacological agents, barium ions (Ba2+) and picrotoxin (PTX), were examined on components of the electroretinogram (ERG) in frog retina. Depth profiles of light-evoked field potentials were recorded, and current source densities (CSDs) were computed from these. 2. Ba2+ abolished the M-wave, slow PIII, and the c-wave, but only decreased b-wave amplitude down to approximately 65% of control amplitude. 3. Ba2+ abolished a slow current sink in the inner plexiform layer (IPL) and the source at the inner limiting membrane (ILM). This IPL sink/ILM source appears to generate the M-wave. 4. Ba2+ decreased the current sink at the outer plexiform layer (OPL) to approximately 70% of control amplitude, and it increased an IPL source. This Ba(2+)-resistant OPL sink/IPL source appears to generate a significant portion of the b-wave. The Ba(2+)-sensitive portion of the b-wave might be generated by Müller cells. 5. PTX enhanced retinal field potentials, particularly the M-wave in the proximal retina. This enhanced M-wave was shown to originate from an enhanced IPL sink/ILM source. 6. Our results suggest that the M-wave originates from Müller cells, through the spatial buffering of the light-evoked increase in [K+]o of the proximal retina. A portion of the b-wave may also originate from Müller cells, but a stronger direct contribution from depolarizing bipolar cells is suggested.

Animals↗
PubMed1993

Excitation failure in eccentric contraction-induced injury of mouse soleus muscle.

1. Histological evidence suggests that the force deficit associated with eccentric contraction-induced muscle injury is due to structural damage to contractile elements within the muscle fibre. Alternatively, the force deficit could be explained by an inability to activate the contractile proteins. It was the objective of this study to investigate the latter possibility. 2. Mouse soleus muscles were isolated, placed in an oxygenated Krebs-Ringer buffer at 37 degrees C, and baseline measurements were made. The muscle then performed one of three contraction protocols: (1) twenty eccentric (n = 10 muscles); (2) ten eccentric (n = 12); or (3) twenty isometric (n = 10) contractions. At the end of the injury protocol, measurements were made during performance of a passive stretch, twitch and tetanus. Next, force was recorded during exposure of the muscle to buffer containing 50 mM caffeine. 3. Decrements in maximal isometric tetanic force (P0) observed for muscles in the twenty eccentric, ten eccentric, and twenty isometric contraction protocols were 42.6 +/- 4.2, 20.0 +/- 2.3 and 3.9 +/- 2.4%, respectively. However, the caffeine-elicited forces in muscles from the three protocols were not different when corrected for initial differences in P0 (64.9 +/- 1.3, 64.2 +/- 2.1 and 68.9 +/- 2.5% of pre-injury P0). The peak caffeine-elicited force was 118.4 +/- 8.6% of post-injury P0 for the muscles in the twenty eccentric contraction protocol, which was significantly different from that observed for the other protocols (71.8-80.2% post-injury P0). These findings indicate that the force deficit in this muscle injury model results from a failure of the excitation process at some step prior to calcium (Ca2+) release by the sarcoplasmic reticulum. 4. In an attempt to locate the site of failure, intracellular measurements were made in injured muscles to test whether injury to the sarcolemma might have resulted in a shift of the resting membrane potential of the muscle fibre. However, microelectrode measurements of resting membrane potential for muscles in the twenty eccentric contraction protocol (-74.4 +/- 0.6 mV) were not different from muscles in the twenty isometric contraction protocol (-73.4 +/- 1.0 mV). These data suggest that membrane resting conductances were normal and are compatible with the idea that the ability of the injured fibres to conduct action potentials was probably not impaired.

Action Potentials↗
PubMed1993

Circadian rhythm in the visual system of the lizard Anolis carolinensis.

The electroretinogram (ERG) was recorded from free-moving Anolis lizards once per hour for 5 days. As in our previous work, the b-wave, but not the a-wave, showed a reliable circadian rhythm (CR) in amplitude, with an acrophase near projected noon. Both the a- and b-waves showed a CR in peak time (implicit time, or IT), with the a-wave IT being longest near midnight, and the b-wave IT at midday. Acrophases were shifted when animals were housed on a phase-shifted light-dark cycle. The ERG CR was unaffected by removal of the parietal organ, but it was virtually abolished by removal of the pineal gland, thus suggesting that pineal output (probably melatonin) modulates retinal responses. In addition to the ERG, the tectal light-evoked potential exhibited a CR--a finding compatible with a circadian variation in retinal output. Lastly, the amplitude of the ERG component waveforms showed a seasonal variation, but the ERG CR was constant across the year.

Animals↗
PubMed1992

Light-evoked expansion of subretinal space volume in the retina of the frog.

The retina of the frog was superfused with a Ringer solution containing impermeant "probe" cations and anions. Light-evoked concentration changes in these probe ions were measured in the subretinal space (SRS) with ion-selective microelectrodes. A decrease in probe ion concentration was found, and several observations suggest that this is caused by a light-evoked expansion of the SRS. The probe ion decrease was not seen in the isolated retina; thus, the pigment epithelial (PE) cells are important for its generation. Pharmacological studies suggest that K+ channels in the PE cells are important--perhaps the PE cells shrink in response to the light-evoked decrease in SRS [K+]. The light-evoked decrease of SRS volume may be important in the understanding of SRS solute concentrations, retina-PE adhesivity, photoreceptor-PE cell interactions, and the interphotoreceptor matrix.

Animals↗
PubMed1991

Comparison of pharmacological agents (aspartate vs. aminophosphonobutyric plus kynurenic acids) to block synaptic transmission from retinal photoreceptors in frog.

The combination of aminophosphonobutyric plus kynurenic acids (APB/Kyn) was compared to aspartate with respect to its ability to block synaptic transmission from photoreceptors. Like aspartate, APB/Kyn blocks photoreceptor synaptic transmission, as monitored by the b- and d-waves of the electroretinogram, by the proximal negative response and M-wave of the proximal retina, and by the light-evoked increase in extracellular K+ concentration in the inner plexiform layer. Unlike aspartate, APB/Kyn has relatively minor effects on retinal resistance, light-evoked changes in K+ and Ca2+ concentrations in the subretinal space, light-evoked changes in subretinal space volume, resting extracellular concentrations of K+ and Ca2+ in the proximal and distal retina, and the c-wave. Effects of APB/Kyn are generally more reversible than effects of Asp. A disadvantage of APB/Kyn is that the a-wave usually becomes smaller and slower. Overall, APB/Kyn disrupts the retina less than aspartate. Therefore, in some situations in which blockade of photoreceptor synaptic transmission is desired, the use of APB/Kyn may be preferable to that of aspartate.

Aminobutyrates↗
PubMed1990

Light-evoked changes in extracellular calcium concentration in frog retina.

Light-evoked changes in extracellular Ca2+ concentration were recorded with Ca2(+)-selective microelectrodes in the retina of the frog eyecup. A Ca-decrease at light onset and offset was found in the inner plexiform layer, and its properties are consistent with it resulting from Ca2+ influx into activated neuronal terminals. In the subretinal space, a Ca-increase at light onset and a Ca-decrease at offset were observed, and these likely arise directly from photoreceptors. A slower ON Ca-decrease was also seen here. Because it survives pharmacological isolation of the photoreceptors from post-synaptic interactions, but not physical isolation of the retina from the pigment epithelium, this component probably depends on pigment epithelial activity.

Animals↗
PubMed1989

Spatial buffering of light-evoked potassium increases by retinal Müller (glial) cells.

Activity-dependent variations in extracellular potassium concentration in the central nervous system may be regulated, in part, by potassium spatial buffering currents in glial cells. The role of spatial buffering in the retina was assessed by measuring light-evoked potassium changes in amphibian eyecups. The amplitude of potassium increases in the vitreous humor was reduced to approximately 10 percent by 50 micromolar barium, while potassium increases in the inner plexiform layer were largely unchanged. The decrease in the vitreal potassium response was accurately simulated with a numerical model of potassium current flow through Müller cells, the principal glial cells of the retina. Barium also substantially increased the input resistance of Müller cells and blocked the Müller cell-generated M-wave, indicating that barium blocks the potassium channels of Müller cells. Thus, after a light-evoked potassium increase within the retina, there is a substantial transfer of potassium from the retina to the vitreous humor by potassium current flow through Müller cells.

Ambystoma↗
PubMed1989

Light-evoked changes in extracellular pH in frog retina.

Light-induced changes in extracellular H+ concentration (delta pH0) were studied with intraretinal H(+)-sensitive double-barreled microelectrodes in frog eyecup and isolated retina preparations. The most prominent delta pH0 were found in the inner plexiform layer, as pH increases (alkalinizations) at light onset and offset. With a small-spot stimulus (0.3 mm dia.), 30 sec in duration, the delta pH0 were relatively small (0.03 pH units), and long lasting (peak at 25-30 sec). They were enhanced by flicker (0.3 Hz). Depth profiles paralleled those of the field potentials (PNR/M-wave), the ON delta pH0 peaking 40 microns more proximal than the OFF response. The delta pH0 exhibited surround antagonism, which was blocked by tetrodotoxin (TTX), indicating an independence from action potentials. The mechanism for these pH increases in proximal retina is not yet understood. In the subretinal space diffuse retinal illumination produced a small pH increase, consistent with a presumed decrease in photoreceptor lactate production. Inhibition of carbonic anhydrase (CA) with acetazolamide or methazolamide increased both the proximal and distal retinal delta pH0, suggesting that CA is involved in buffering retinal pH.

Acetazolamide↗
PubMed1989

Current-evoked transcellular K+ flux in frog retina.

1. Changes in extracellular K+ concentration (delta[K+]o) evoked by electrical current were measured with K+-selective microelectrodes (K-ISMs) in the retina of the frog eyecup. 2. In the superfusate at 20 microns above the inner limiting membrane (ILM), current-evoked delta[K+] was a function of current polarity and strength; its amplitude decreased as the K-ISM was moved higher above the ILM. Responses were similar whether measured with K-ISMs containing the Corning exchanger or a valinomycin-based liquid membrane. No current-evoked delta[Ca2+] could be detected with Ca-selective microelectrodes (Ca-ISMs). 3. Within the retina, a complex spatiotemporal profile of current-evoked delta[K+]o was observed. Strophanthidin abolished responses in the proximal retina, but had little effect on the response in the superfusate. A blocker of K+ channels (Ba2+) depressed responses in the superfusate, but not in the proximal retina. 4. Quantitative analysis of these responses indicates a transport number for K+ of 0.18 at onset of current, and that decreases over a few seconds. In contrast, a transport number of approximately 0.01 is predicted from the expected ionic concentrations within extracellular space. 5. These findings are compatible with the delta[K+] above the ILM being due to transcellular movement of K+ through Müller cells. The results suggest that K+ spatial buffering may be particularly potent in the retina. Furthermore, determinations of tissue characteristics by passage of electrical current must take into account that at least 17% of the current does not travel through extracellular space.

Action Potentials↗
PubMed1988

Generation of the e-wave of the electroretinogram in the frog retina.

The e-wave and a delayed-OFF increase in extracellular K+ concentration are both maximum in the distal half of the inner plexiform layer. These responses also have similar latency, time-course, intensity-dependence, surround properties, and sensitivity to tetrodotoxin. Current source-density analysis of the e-wave reveals a current sink through the proximal retina, a source at the retinal surface, and, in some cases, a weaker source in the mid-retina. These results suggest a model for e-wave generation: delayed-OFF activity in proximal neurons releases K+, which enters Muller cells in the inner plexiform layer; a current exists Muller cells primarily via their endfeet, and the return flow through extracellular space produces the e-wave.

Action Potentials↗
PubMed1988

Changes in [K+]0 induced by transretinal currents in frog retina.

Current-induced changes in extracellular potassium concentration (delta [K+]0) were measured with K+-selective microelectrodes in frog retina. A characteristic depth profile of delta[K+]0 was detected, which included significant changes in the superfusate just above the inner limiting membrane. This response was almost eliminated by Ba2+, a blocker of K+ channels. The delta[K+]0 in the superfusate is likely due to transcellular flux of K+, probably through Muller cell endfeet.

Animals↗
PubMed1987

Sources and sinks of light-evoked delta [K+]o in the vertebrate retina.

In the vertebrate retina, recordings of light-evoked changes in extracellular K+ concentration delta [K+]o are of particular interest because this tissue is complex and multilayered, yet can be activated routinely with its "natural" stimulus (i.e., light). This review identifies the components of the spatiotemporal profile of retinal light-evoked delta [K+]o and then presents evidence concerning the specific neural origins of these components as well as the mechanisms by which these delta [K+]o are dispersed from extracellular space. Finally, to gain improved resolution of K+ sources and sinks, the technique of ion source density is introduced and applied to both model and real spatiotemporal distributions of delta [K+]o.

Animals↗
PubMed1985

Light-evoked increases in extracellular K+ in the plexiform layers of amphibian retinas.

Recordings of light-evoked changes in extracellular K+ concentration (delta[K+]o) were obtained in the retinas of frog and mudpuppy. In eyecup preparations, various recording approaches were used and provided evidence for a K increase near the outer plexiform layer (distal K increase). This distal K increase could be pharmacologically dissociated from the well-known, large K increase in the proximal retina by the application of ethanol and gamma-aminobutyric acid. The distal K increase also often showed surround antagonism. A retinal slice preparation was used to permit electrode placement into the desired retinal layers under direct visual control and without the risk of electrode damage to adjacent layers. In the slice, a distinct distal K increase was found in the outer plexiform layer, in addition to the prominent K increase in the inner plexiform layer. Compared with eyecups, only weak K increases were found in the nuclear layers of the slice. This suggests that the K responses observed in the nuclear layers of eyecups may be generated by K+ diffusing along the electrode track from the plexiform layers. In the context of current models of ERG b-wave generation, the magnitude of the recorded distal K increase, compared with the proximal K increase, seems too small to give rise to the b-wave. However, the distal K increase may be differentially depressed by electrode dead space. It is also possible that if certain aspects of the models of b-wave generation were modified, then the observed distal K increase could give rise to the b-wave.

Animals↗