Biomedical subjects

B N Christensen

Publications and source records attributed to B N Christensen.

At least 19 recordsLinked to original sources

PubMed2001

Role of calcium-dependent protease(s) in globulization of isolated rat lens cortical fiber cells.

PURPOSE: To investigate the role of calcium-activated proteases in calcium-dependent disintegrative globulization of isolated rat lens cortex fiber cells. METHODS: Rat lens fiber cells were isolated and plated on coverslips at the bottom of a temperature-controlled chamber. The fiber cells were incubated with 10 microM protease substrate, (t-butoxycarbonyl-leu-met-7-amino-4-chloromethylcoumarin:BOC-Leu-M et- CMAC) and the proteolytic activity in the fiber cells was determined by observing the increase in fluorescence, using an excitation wavelength of 360 nm, and measuring emission at 410 nm. Free intracellular calcium was measured using the cell-permeable calcium indicator Fluo-3-AM, and the globulization time (T(g)) was determined using image analysis. RESULTS: T:(g) of fiber cells superfused with Ringer's solution containing 2 x 10(-)(3) M, 10(-)(6) M, and 10(-)(8) M [Ca(2+)](o) were: 24.7 +/- 1.3, 53.0 +/- 2.8, and more than 120 minutes, respectively. A significant increase in T:(g) ( approximately 95 minutes) was observed when the fibers were preincubated with acetoxymethyl ester of 1,2-bis (2-amino-phenoxy) ethane N:, N:, N:, N:-tetra-acetic acid (BAPTA-AM) to buffer changes in [Ca(2+)](i), or the protease substrate to competitively inhibit degradation of cellular proteins. In the presence of Ringer's solution containing 2 x 10(-)(3) M [Ca(2+)](o) and 0.5 mM of the cysteine protease inhibitor, leupeptin, T:(g) increased to 100 minutes, without affecting [Ca(2+)](i). The proteolytic activity of fiber cells in Ringer's solution containing 10(-)(6) M and 2 x 10(-)(3) M [Ca(2+)](o) increased by approximately 7- and 12-fold, respectively, compared with sucrose-EDTA solution or Ringer's solution containing 10(-)(8) M [Ca(2+)](o). This increase in proteolytic activity was inhibited by leupeptin. CONCLUSIONS: Elevation of calcium in the medium results in a proportionate increase in [Ca(2+)](i) and the proteolytic activity in isolated lens fiber cells. The increase in the proteolytic activity is accompanied by an increase in the rate of globulization of the fiber cells. Inhibition of the proteolytic activity by leupeptin increases T:(g) without affecting the gain in [Ca(2+)](i). These results suggest that globulization of isolated fiber cells in physiological salt solutions is mediated by Ca(2+)-activated protease(s).

Aniline Compounds↗

PubMed1999

Multiple protein kinase pathways are involved in gastrin-releasing peptide receptor-regulated secretion.

Gastrin-releasing peptide (GRP) and its amphibian homolog, bombesin, are potent secretogogues in mammals. We determined the roles of intracellular free Ca(2+) ([Ca(2+)](i)), protein kinase C (PKC), and mitogen-activated protein kinases (MAPK) in GRP receptor (GRP-R)-regulated secretion. Bombesin induced either [Ca(2+)](i) oscillations or a biphasic elevation in [Ca(2+)](i). The biphasic response was associated with peptide secretion. Receptor-activated secretion was blocked by removal of extracellular Ca(2+), by chelation of [Ca(2+)](i), and by treatment with inhibitors of phospholipase C, conventional PKC isozymes, and MAPK kinase (MEK). Agonist-induced increases in [Ca(2+)](i) were also inhibited by dominant negative MEK-1 and the MEK inhibitor, PD89059, but not by an inhibitor of PKC. Direct activation of PKC by a phorbol ester activated MAPK and stimulated peptide secretion without a concomitant increase in [Ca(2+)](i). Inhibition of MEK blocked both bombesin- and phorbol 12-myristate 13-acetate-induced secretion. GRP-R-regulated secretion is initiated by an increase in [Ca(2+)](i); however, elevated [Ca(2+)](i) is insufficient to stimulate secretion in the absence of activation of PKC and the downstream MEK/MAPK pathways. We demonstrated that the activity of MEK is important for maintaining elevated [Ca(2+)](i) levels induced by GRP-R activation, suggesting that MEK may affect receptor-regulated secretion by modulating the activity of Ca(2+)-sensitive PKC.

Bombesin↗

PubMed1998

Na+/Ca2+ exchange in catfish retina horizontal cells: regulation of intracellular Ca2+ store function.

The role of the Na+/Ca2+ exchanger in intracellular Ca2+ regulation was investigated in freshly dissociated catfish retinal horizontal cells (HC). Ca2+-permeable glutamate receptors and L-type Ca2+ channels as well as inositol 1,4,5-trisphosphate-sensitive and caffeine-sensitive intracellular Ca2+ stores regulate intracellular Ca2+ in these cells. We used the Ca2+-sensitive dye fluo 3 to measure changes in intracellular Ca2+ concentration ([Ca2+]i) under conditions in which Na+/Ca2+ exchange was altered. In addition, the role of the Na+/Ca2+ exchanger in the refilling of the caffeine-sensitive Ca2+ store following caffeine-stimulated Ca2+ release was assessed. Brief applications of caffeine (1-10 s) produced rapid and transient changes in [Ca2+]i. Repeated applications of caffeine produced smaller Ca2+ transients until no further Ca2+ was released. Store refilling occurred within 1-2 min and required extracellular Ca2+. Ouabain-induced increases in intracellular Na+ concentration ([Na+]i) increased both basal free [Ca2+]i and caffeine-stimulated Ca2+ release. Reduction of external Na+ concentration ([Na+]o) further and reversibly increased [Ca2+]i in ouabain-treated HC. This effect was not abolished by the Ca2+ channel blocker nifedipine, suggesting that increases in [Na+]i promote net extracellular Ca2+ influx through a Na+/Ca2+ exchanger. Moreover, when [Na+]o was replaced by Li+, caffeine did not stimulate release of Ca2+ from the caffeine-sensitive store after Ca2+ depletion. The Na+/Ca2+ exchanger inhibitor 2',4'-dimethylbenzamil significantly reduced the caffeine-evoked Ca2+ response 1 and 2 min after store depletion.

Amiloride↗

PubMed1997

Connexin43 immunoreactivity in the catfish retina.

We used antibodies directed against rat heart connexin43 (Cx43) to perform immunoblot and immunohistochemical (IHC) analyses of the catfish retina. The antibodies recognized a retinal protein which co-migrated with mouse brain Cx43. IHC staining resulted in punctate labeling of the external limiting membrane and the outer nuclear layer. Although infrequent, labeling was also observed in the inner nuclear layer. These results suggest that a Cx43 isoform may be present in Muller glial cells and neurons of the distal catfish retina.

Animals↗

PubMed1996

Distribution of the inositol trisphosphate receptor in the catfish retina.

Inositol 1,4,5-trisphosphate (InsP3) mobilizes intracellular stored Ca2+ by binding to specific receptors that are similar to the ryanodine receptor of skeletal and cardiac muscle. We have immunolocalized the InsP3 receptor to the inner nuclear layer and limiting membranes of the catfish retina. Immunocytochemistry on dissociated retinal cells further localized the receptor in the horizontal, bipolar and Müller glial cells. Immunostaining of the rat retina localized the InsP3 receptor to the plexiform layers. These data show a different distribution of InsP3 receptor in the catfish retina compared to that of other vertebrates, that may be suggestive of a different functional role for this receptor in different species.

Animals↗

PubMed1996

Proton inhibition of the NMDA-gated channel in isolated catfish cone horizontal cells.

The effect of H+ on the N-methyl-D-aspartate-induced (NMDA) membrane current in enzymatically isolated catfish cone horizontal cells was investigated. Extracellular acidification to pH 5.5 blocked nearly completely the NMDA-induced current and reduced desensitization. The pK for the H+ effect was 6.5, near that for the free amino acid histidine. Protons did not alter the receptor affinity for NMDA and the inhibition was insensitive to the membrane potential and surface charge screening. However, extracellular H+ increased the IC50 for Zn2+. These results indicate that protons can modulate the NMDA-induced membrane current by a mechanism that may include interaction with histidine residues.

Animals↗

PubMed1995

Intracellular Ca2+ stores in chick cerebellum Purkinje neurons: ontogenetic and functional studies.

The molecular composition of intracellular Ca2+ stores in developing chicken cerebellum Purkinje neurons from embryonic day 11 (E11) to posthatching day 2 (P2) was studied by immunocytochemistry using specific antibodies for three molecular constituents, the receptor (R) and/or channel sensitive to inositol 1,4,5-trisphosphate (IP3), Ca(2+)-adenosinetriphosphatase (ATPase), and calsequestrin (CS). CS, IP3R, and Ca(2+)-ATPase were first detected by light-microscopic immunofluorescence in migrating Purkinje cells at E11-E12 and throughout late phases of embryonic development. Ontogenesis of CS, IP3R, and Ca(2+)-ATPase accompanied well-defined stages of cerebellum histogenesis and cytogenesis and was accomplished before hatching. High-resolution immunogold electronmicroscopy revealed that, at E18-P1, CS was still largely distributed to the endoplasmic reticulum (ER) lumen and began to be segregated to ER subcompartments (calciosomes) only by P2, whereas the IP3R was concentrated into ER cisternal stacks as early as E18. Both ionotropic and metabotropic plasma membrane receptors were present in dissociated single chicken Purkinje cells from E16 onward, as indicated by measurements of membrane currents (whole cell recording mode) and of cytoplasmic Ca2+ transients monitored with the cell-trappable fluorescent indicator fura 2-acetoxymethyl ester, respectively. Cytoplasmic Ca2+ transients were detected after either activation of glutamate metabotropic receptors, i.e., evidence of IP3-sensitive Ca2+ channels, or application of caffeine, i.e., evidence of ryanodine-sensitive Ca2+ channels. Intracellular Ca2+ stores appear to be functional during embryonic development.

Aging↗

PubMed1992

Excitatory amino acid regulation of intracellular Ca2+ in isolated catfish cone horizontal cells measured under voltage- and concentration-clamp conditions.

[Ca2+]i was measured using fura-2-loaded isolated catfish horizontal cells in the presence of L-glutamate and the glutamate analogs kainate (KA), quisqualate (QA), and NMDA. Caffeine was used to release Ca2+ from intracellular stores. Cell membrane potential was controlled with a voltage clamp to prevent activation of voltage-dependent Ca2+ channels in the presence of agonist. All excitatory amino acid agonists produced a rapid and sustained rise in [Ca2+]i with the order of potency being QA greater than Glu greater than KA greater than NMDA. The agonist-induced [Ca2+]i increase was blocked in reduced [Ca2+]o and by 6-cyano-7-nitroquinoxaline-2,3-dione and 2-amino-5-phosphonopentanoate, which are specific blockers for QA/KA and NMDA receptors, respectively. The metabotropic receptor agonist trans-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD; 10-200 microM) had no effect on [Ca2+]i. Hill coefficients from curves fitted to concentration-response data suggested an amplification of the Ca2+ signal that was interpreted as calcium-induced calcium release (CICR) from intracellular Ca2+ stores. Caffeine (10 mM) produced a rapid transient rise in [Ca2+]i, confirming the existence of a Ca(2+)-sensitive store. Following caffeine-induced depletion of Ca2+ from intracellular stores, agonists were still able to produce increases in [Ca2+]i, confirming Ca2+ influx through the agonist-gated channel. The agonist-induced increase in [Ca2+]i was decreased following caffeine-induced depletion, confirming a process of CICR. These results are consistent with the hypothesis that excitatory amino acids can produce direct modulation of [Ca2+]i by influx through the agonist-gated channel and by CICR from intracellular stores.

Amino Acids↗

PubMed1990

Protonation of histidine groups inhibits gating of the quisqualate/kainate channel protein in isolated catfish cone horizontal cells.

Increases in the extracellular hydrogen ion concentration ([H+]o) but not the intracellular concentration ([H+]i) antagonized the inward going membrane currents recorded from isolated cone horizontal cells during application of quisqualate, alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid, and kainate. The pK determined from a titration curve was 6.5 with a slope greater than 1, indicating protonation of several histidines. The reduction in membrane current was voltage-independent. The affinity of the agonist for the receptor, the single-channel conductance, and the open time were unaffected by [H+]o. [H+]o antagonism was not the result of charge neutralization such as screening surface charge. Diethylpyrocarbonate, a histidine-modifying reagent, reduced the agonist-induced current, but disulfide- and sulfhydryl-modifying reagents were ineffective. These results suggest that histidine groups on the external face of the channel protein provide a functional site regulating channel gating.

Animals↗

PubMed1989

A voltage-clamp study of isolated stingray horizontal cell non-NMDA excitatory amino acid receptors.

1. Horizontal cells enzymatically isolated from retinas of the Atlantic stingray (Dasyatis sabina) were voltage-clamped using the patch electrode in the whole-cell mode. A rapid microsuperfusion system was used to apply excitatory amino acid agonists and antagonists. 2. The isolated cells responded to glutamate (GLU), kainate (KA), quisqualate (QA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Responses elicited by GLU, QA, and AMPA but not KA exhibited a concentration-dependent and concanavalin A- (Con-A) sensitive desensitization. No responses were elicited by aspartate, N-methyl-D-aspartate, or quinolinate at concentrations as high as 1.0 mM. 3. Judging from the concentration producing one-half of the maximal current response (EC50), the rank order affinities of the agonists was QA greater than or equal to GLU greater than AMPA greater than KA. Whereas KA had the lowest affinity of the agonists tested it was the most efficacious, producing the largest currents. Hill coefficients of the concentration-response data were near two for KA and GLU and near one for QA and AMPA. 4. The agonists differed in their sensitivity to various excitatory amino acid receptor antagonists. Kynurenate (KYN) produced a nearly complete block of horizontal cell responses to GLU and KA at concentrations that had little effect on QA and AMPA. Piperidine-2,3-dicarboxylic acid (cis-PDA), 1-(4-chlorobenzoyl)-piperazine-2,3-dicarboxylic acid (pCB-PzDA), and folic acid were less potent antagonists than KYN but were also better blockers of KA and GLU responses than of QA- and AMPA-elicited responses. 5. When QA, AMPA, or GLU were applied in combination with 55.0 microM KA the current was less than that produced by KA alone. The rank order potency for the inhibition of KA-elicited responses was QA greater than AMPA greater than GLU. In the presence of low concentrations of KA (1.0-20.0 microM), QA- and AMPA-elicited responses were potentiated. This potentiation was prevented by KYN. 6. Single-channel conductance and mean open time were estimated from the current noise fluctuations in the presence of agonist. The mean single-channel conductance for QA was 9 pS that was almost twice as large as the conductance for KA (5.9 pS) and GLU (5.7 pS). The mean open time in the presence of QA or GLU was approximately 1 ms, which was about one-half of that for KA (2.0 ms). 7. These results are best explained by the existence of a single receptor protein with multiple but not identical ligand-binding sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗

PubMed1989

Horizontal cells isolated from catfish retina contain two types of excitatory amino acid receptors.

1. Inward going membrane currents elicited by N-methyl-D-aspartate (NMDA), glutamate (GLU), and glutamate analogues were recorded from isolated catfish (Ictalurus punctatus) cone horizontal cells using the patch-clamp technique in the whole-cell mode. 2. Currents elicited by the N-methyl-D-aspartate receptor agonists NMDA, L-aspartate (ASP) or L-homocysteate (L-HCA) in nominally Mg-free saline were completely blocked by 100 microM 2-amino-5-phosphonovalerate (AP-5) but responses to non-NMDA receptor agonists kainate (KA), quisqualate (QA), or alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) in normal Mg2+ saline were unaffected. Responses to GLU were partially blocked. Kynurenic acid (1 mM) effectively blocked responses to all agonists. 3. Concentration-response curves obtained from measured responses in the presence of different NMDA receptor agonists gave Hill coefficients of near 1 indicating a single binding site for channel activation. The rank order of agonist affinity at the NMDA receptor is L-HCA greater than NMDA greater than ASP. Glycine potentiates responses to NMDA in horizontal cells. 4. The NMDA-activated channel is blocked in a voltage-dependent manner by Mg2+, Ni2+, and Co2+ and in a voltage-independent manner by Zn2+. Both the NMDA- and KA-activated channel were permeable to monovalent cations but the NMDA-activated channel appeared to have a greater permeability to Ca2+ than the KA-activated channel. 5. Concentration-response curves measured from responses to the non-NMDA receptor agonists QA, KA, and AMPA gave Hill coefficients of approximately 1.5 suggesting multiple binding sites for channel activation and cooperativity. The rank-order affinity was QA greater than AMPA greater than GLU greater than KA. KA was the most efficacious of the agonists resulting in the largest Imax. Rapid desensitization was observed only in the presence of QA, AMPA, or GLU and this desensitization could be removed by pretreatment with conconavalin A (Con A). 6. Single-channel conductance and mean open time were measured from the fluctuations in current noise in the presence of the agonists. The single-channel conductance estimated from the slope of a linear regression obtained from a plot of the variance of the conductance versus the whole-cell conductance measured in NMDA and ASP was 4.7 pS. The mean channel open time was 1.3 ms. These same parameters measured for KA and QA were 5.7 and 5.9 pS and 1.1 to 1.3 ms, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

2-Amino-5-phosphonovalerate↗

PubMed1989

Studies of solitary semicircular canal hair cells in the adult pigeon. I. Frequency- and time-domain analysis of active and passive membrane properties.

1. Hair cells were enzymatically dissociated from the neuroepithelium (cristae ampullares) of the semicircular canals of white king pigeons (Columba livia). Those hair cells determined to be type II by an anatomic criterion, the ratio of the minimum width of the neck to the width of the cuticular plate, were studied with the use of the whole cell patch-clamp technique. 2. The mean +/- SD zero-current membrane potential, Vz, was found to be -54 +/- 12 mV for anterior crista hair cells (n = 71), -62 +/- 14 mV for posterior crista hair cells (n = 14), and -55 +/- 12 mV for lateral (horizontal) crista hair cells (n = 18). The mean +/- SD value of Vz for hair cells from all cristae (n = 103) was -56 +/- 13 mV. 3. Active and passive membrane properties were calculated in the time domain, in voltage- or current-clamp mode, from responses to voltage or current pulses and, in the frequency domain, by fitting a membrane model to admittance magnitude and phase data resulting from current responses to sum-of-sines voltages at different d.c. levels of voltage-clamp membrane potential. 4. The average value +/- SE of input resistance (Rin), over the range from -100 to -60 mV, was found to 1.5 +/- 0.3 G omega from a mean-voltage-as-a-function-of-current plot, V-I, (n = 7) and a mean of 1.4 +/- 0.3 G omega from individual (n = 15) current-as-a-function-of-voltage plots, I-V. A lower mean value 0.8 +/- 0.4 G omega was obtained for the input resistance from frequency-domain calculations for a different set of cells (n = 21). Also, in two different sets of cells, average input capacitance (Cin) was determined to be 12 +/- 3 pF (n = 7) from time-domain estimates and 14 +/- 3 pF (n = 21) from frequency-domain estimates. The (Rin)(Cin) product was 11 ms based on frequency-domain estimates and 17 ms from time-domain estimates. 5. I-V curves for hair cells voltage clamped at -60 mV showed some anomalous rectification for hyperpolarizations between -60 and -120 mV but no detectable N-shape for depolarizations between -50 and 90 mV. The I-V relation showed increasing slope with depolarization through the resting potential (Vz) and increased linearly between -40 and 80 mV; the best-fit straight-line maximum slope conductance for six cells over this range was 17.4 +/- 0.3 nS.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1988

Mecamylamine is a selective non-competitive antagonist of N-methyl-D-aspartate- and aspartate-induced currents in horizontal cells dissociated from the catfish retina.

The nicotinic acetylcholine channel blockers mecamylamine (MECA) and pempidine (PEMP) blocked voltage-clamped isolated catfish retina cone horizontal responses to aspartate (Asp) and N-methyl-D-aspartate (NMDA) but had little effect on currents induced by kainate and quisqualate. Concentration response curves for NMDA and Asp in the presence of MECA suggested that MECA was a non-competitive inhibitor of NMDA and Asp responses. Further, the MECA and PEMP block of NMDA and Asp-induced currents was highly voltage-sensitive. The non-competitive and voltage-sensitive block of NMDA and Asp-induced currents by MECA suggest that MECA (and PEMP) block the NMDA receptor ion channel.

Animals↗

PubMed1988

Transfer impedances between different regions of branched excitable cells.

1. The excitable properties of branched cells were measured using a combination of voltage-clamp and frequency-domain techniques. Point impedance functions from either the soma or growth cone of NG-108 cells were curve fitted with a reduced cable model at different membrane potentials to establish kinetic parameters. 2. Transfer impedance functions between the soma and growth cone were measured and simulated with a morphologically determined model. In these experiments the membrane potential was controlled by a single-electrode voltage clamp thus allowing an estimate of transfer functions for any arbitrary input, such as a single synaptic current for differing degrees of tonic synaptic drive. Furthermore, the integration of different regional inputs was evaluated based on the transfer functions between different locations on an individual cell. 3. The activation of an outward steady-state current leads to resonating impedance functions that were used to evaluate the kinetic properties of ionic channels in different regions of branched excitable cells. For simple branching patterns the point and transfer impedances show lower resonant frequencies for active growth cones compared with active somas. 4. More complex branching patterns showed the unexpected result that the voltage-dependent resonant frequency was higher for the growth cone recording than the soma. The presence of a higher resonant frequency when the growth cone is activated does not require more rapid kinetics of the active potassium conductance, since the time constant of the active conductance can be the same in the growth cone and the soma membrane. 5. In conclusion, the resonant frequencies, as well as all other aspects of the impedance functions, are complicated interactions of the detailed branching patterns and active conductances. In general, these interactions are not predictable from a passive electrotonic analysis, especially when the voltage-dependent conductances are distributed throughout the dendritic tree.

Animals↗

PubMed1988

Effect of subthreshold voltage-dependent conductances on the transfer function of branched excitable cells and the conduction of synaptic potentials.

1. Impulse response functions were determined from complex point impedance and transfer functions from cultured NG-108 cells to simulate the propagation of a synaptic potential in response to the release of transmitter. In general, the flow of synaptic current has a much shorter duration than the normal membrane time constant, thereby making the use of impulse response functions useful approximations to synaptic events. 2. The resonance observed during the activation of the potassium conductance was reflected in the impulse response function as a pronounced damped oscillation. A comparison of the impulse response functions calculated from point impedance and transfer functions showed similar results for current injections in the growth cone. 3. In addition to the resonance effects of the voltage-dependent conductances on transfer and impulse response functions due principally to the activation of conductances for outward currents, transfer functions were measured during the activation of a steady-state negative conductance. Under these conditions the phase function approaches 180 degrees, indicating that the voltage response is out of phase with the current. 4. In the steady state, the effect of a negative conductance is to algebraically add to the positive conductances and generally decrease the absolute conductance unless there is a net negative current. The decreased conductance enhances the impulse response and the DC space constant, thus leading to a better propagation of slow potentials. This effect can be seen as a decrease in the electrotonic length, L, with intermediate depolarizations. At large depolarizations the steady-state activation of the K conductance generally dominates and leads to a greatly increased electrotonic length. 5. Both the net conductances and the associated kinetics play a role in shaping the potential changes during a synaptic current. This is especially critical if there is a net negative steady-state conductance. Under these conditions there is a surprising reduction in the impulse response function. 6. Thus, during a subthreshold activation of the voltage-dependent negative conductances, the observable synaptic potentials would be either large potential responses due to an apparent increase in the impedance (algebraic summation of positive and negative conductances with a net positive conductance) or a minimal response because of the phasic cancellation due to a net negative conductance. The latter condition could exist near the synaptic reversal potential due to a large synaptic drive and would appear experimentally as a form of inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1988

Ionic currents in neurones cultured from embryonic cockroach (Periplaneta americana) brains.

Neurones isolated from embryonic cockroach brains were maintained in culture for up to 8 weeks. A single patch electrode was used to record voltage changes in response to injected current, membrane ionic currents under whole-cell voltage-clamp conditions or single-channel currents from isolated membrane patches. The voltage changes in response to injected current that depolarized the cell indicated increases in membrane permeability to calcium and potassium. These observations were confirmed using a voltage clamp. The potassium current observed in the youngest cultures turned on with a delay and was blocked by tetraethylammonium (TEA) and 4-aminopyridine (4-AP). Two kinds of decrease in the outward potassium current were observed. One may be associated with extracellular potassium accumulation, inactivation of the potassium channel or inactivation of a calcium channel. The other appears to be a voltage-dependent inactivation. The magnitude of the calcium permeability appeared to increase as the cultures developed, being most prominent in cultures more than 2 weeks old. Single-channel conductance measured from an analysis of records from six isolated membrane patches ranged from 15 to 110 pS. Except for one channel, the probability of the channels being open did not change appreciably with membrane potential. Our results suggest that much of the increase in potassium permeability may be due an increase in intracellular calcium level.

Animals↗

PubMed1986

N-methyl-D-aspartate receptors coexist with kainate and quisqualate receptors on single isolated catfish horizontal cells.

Horizontal cells enzymatically isolated from catfish retina were exposed to the putative neurotransmitters aspartate (Asp) or N-methyl-D-aspartate (NMDA). Under voltage clamp conditions, inward currents were recorded when the holding potential was more negative than zero and outward currents were recorded when the membrane potential was more positive than zero. The current voltage curve was highly non-linear in the range of membrane potential between -30 and -100 mV. This non-linearity was largely removed in zero magnesium solution. 2-Amino-phosphonovaleric acid selectively blocked Asp and NMDA responses. These response characteristics are consistent with the presence of NMDA receptors in these cells.

Animals↗

PubMed1986

The synaptic ultrastructure in the outer plexiform layer of the catfish retina: a three-dimensional study with HVEM and conventional EM of Golgi-impregnated bipolar and horizontal cells.

Synaptic structures between receptors and horizontal and bipolar cells in the outer plexiform layer (OPL) of Golgi-impregnated catfish retina were examined by conventional electron microscopy of serial ultrathin sections and by high-voltage electron microscopy (HVEM) of thick sections. Cone terminals contained multiple synaptic ribbons and rod terminals contained single synaptic ribbons. This observation was used to identify these two types of photoreceptors. The cone horizontal cell, located in the most distal part of the inner nuclear layer (INL), invaginated only cone terminals, whereas the rod horizontal cell, located in the proximal part of the INL, invaginated only rod terminals. Both lateral elements of the triad in the rod terminal originated from a single rod horizontal cell whereas the same structures in the cone terminal were often derived from several cone horizontal cells. Golgi-impregnated catfish bipolar cells were classified into two types based on the differences in their axonal arborization as described by Famiglietti et al. ('77). Axonal endings of type a bipolar cells were located in the distal part, sublamina a, of the inner plexiform layer (IPL), and axonal endings of type b cells were located in the proximal part, sublamina b, of the IPL. Dendrites from type a bipolar cells made direct contact with the synaptic ribbons in both rod and cone terminals whereas those from type b cells made indirect contact with the ribbons in both rod and cone terminals, but rare direct contact with the ribbon in rod terminals were also seen. In addition, bipolar cells made basal junctions or superficial contacts in both rod and cone terminals. The "lateral" processes of bipolar cells invaginating rods penetrated between the rod terminal and rod horizontal cell processes, and made basal junctions with both rod terminals and rod horizontal cells. There was no definitive morphological feature that could be associated with sign-conserving and sign-inverting signal transmission.

Animals↗