Procion brown: an intracellular dye for light and electron microscopy.

Procion browvn has been used to inject lamprey giant interneutros. The dye is opaque to both light and electrons, and imparts a pink color to the neuron in situ during iontophoresis. It has the advantage over cobaltolus chloride that postinjection treatment with ammonium sulfide is eliminated and the integrity of the surrounding neuropil is ensured.

Direct electrical connections between transient amacrine cells in the catfish retina.

Transient amacrine cells were identified by their intracellularly recorded response to flashes of light. These cells typically respond with a transient depolarization, often followed by a steady-state response during the stimulus. When two electrodes were placed in different transient amacrine cells, current of either polarity passed through one electrode produced a steady-state voltage change that was recorded by the electrode in the nearby cell. Following identification of the physiological type, transient amacrine cells were injected with horseradish peroxidase and the tissue was processed for light and electron microscopy. Both conventional chemical synaptic junctions and gap junctions were found to connect amacrine cells.

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.

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.

Sodium and calcium currents measured in isolated catfish horizontal cells under voltage clamp.

A low resistance suction microelectrode was used to record intracellularly from enzymatically dissociated horizontal cells obtained from catfish retina (Ictalurus punctatus). This microelectrode was connected to a voltage clamp circuit and the transmembrane currents were recorded during depolarizing and hyperpolarizing clamp pulses. Two fast transient inward currents were recorded, one sensitive to tetrodotoxin and one insensitive to this drug. A slower and persistent tetrodotoxin insensitive inward current was also recorded. The results indicate that separate membrane conductances exist for both sodium and calcium ions. We suggest that the action potential recorded from isolated horizontal cells is generated by both sodium and calcium currents.

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).

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.

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.

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.

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.

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.

Distribution of electrotonic synapses on identified lamprey neurons: a comparison of a model prediction with an electron microscopic analysis.

1. Intracellular recordings of the monosynaptic excitatory postsynaptic potential (EPSP) were made from an identified giant interneuron in the lamprey spinal cord following stimulation of a single identified presynaptic giant axon. The EPSP had both electrotonic and chemical components. 2. The giant interneuron and sometimes the giant axon were labeled with horseradish peroxidase (HRP). Using combined light and electron microscopy (EM), gap junctions formed by the axon on the HRP-labeled dendritic trees of seven interneurons were identified and their position marked on dendritic diagrams made from tracings of the cells. 3. The total number of gap junctions on a giant interneuron varied from a minimum of 9 to a maximum of 23. The number of gap junctions on a branch of a dendritic tree varied from 1 to 9. For all seven neurons the closest gap junction was located approximately 65 microns from the soma and the furthest 280 microns from the soma. 4. When gap junction position was plotted as a function of electrotonic distance from the soma, the variability in their distribution decreased significantly. Synaptic location was found to be on the average about 0.5 space constants from the soma, and this value compared favorably with that determined from an analysis of half-width and rise time of the electrotonic synaptic potential for these neurons. 5. These results suggest that the presynaptic axon tends to make, on the average, synaptic contact at the same electrical distance from the soma.

Morphological correlates of synaptic transmission in lamprey spinal cord.

The dye Procion brown was used to identify in the light and electron microscope, synaptic contacts made between monosynaptically coupled neurons in the lamprey spinal cord whose synaptic interaction had been recorded. Synaptic contacts were made on different dendrites of the postsynaptic cell at different distances from the soma. Some of the contacts were made on dentritic spines and some on the smooth shaft of the dentrites. Serial sections through synaptic contacts made on dendritic processess of the postsynaptic cells were used for three-dimensional reconstruction of the synapses using computer graphics techniques. The computer reconstructions and detailed examination of the serial EM micrographs revealed the large proliferation of membrane involved in making these en passant synapses as well as the morphological changes due to stimulation of the presynaptic axon. These changes include depletion of synaptic vesicles and formation of complex vesicles and synaptic cisternae. Besides chemical synaptic contacts, four electrotonic contacts were located, confirming the mixed electrochemical synaptic response recorded from the postsynaptic cell. The mean quantum content was estimated and compared with the estimate of the available transmitter pool, assuming the quantal release hypothesis applies at these synapses. The total transmitter pool was estimated by counting all synaptic vesicles in all synaptic contacts. It was estimated that about 6% of the total transmitter pool is available for release at these synapses. This compares with less than 1% at the neuromuscular junction and about 20% at sympathetic synapses. These results support the hypothesis that synaptic vesicles may be recycled as described by Heuser and Reese (22) at the neuromuscular junction. Ongoing studies are investigating the effect on a variety of synaptic junctions to stimulation for different periods of time of presynaptic axons. The methods described in this study can also be used to test the models of synaptic interaction on dendritic trees described by Rall (39) and Jack and Redman (24).

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.

A quantitative analysis of ultrastructural changes induced by electrical stimulation of identified spinal cord axons in the larval lamprey.

Microelectrodes filled with horseradish peroxidase (HRP) were used to label single identified giant axons in the isolated lamprey spinal cord. Subsequent to the iontophoretic injection of HRP, the spinal cord was stimulated at repetition rates of 20-30/s and the activity in labelled axons monitored. Immediately following failure of the action potential, the spinal cord was fixed by immersion and processed for light and electron microscopy. Electron micrographs were taken of synaptic contacts made by the labelled axons. Several quantitative measures were made from each synapse using a digitizing tablet interfaced with a digital computer. These measures included vesicle number (VN), vesicle area (VA), length of differentiated membrane (DM), vesicle density (VD = VN/VA), vesicle frequency (VF = VN/DM), and a relative measure of the amount of vesicle membrane added to the axolemma during the stimulation period, the curvature ratio (CR). Measures from 106 stimulated synapses were compared with 134 synapses from injected but unstimulated giant axons. The results from these experiments suggest that measurable ultrastructural changes occur during transmitter release at identified C.N.S. synapses, which are consistent with the hypothesis of synaptic vesicle recycling.

Localization of synaptic input on dendrites of a lamprey spinal cord neurone from physiological measurements of membrane properties.

1. Composite excitatory post-synaptic potentials (e.p.s.p.s) resulting from electrotonic and chemical synaptic junctions were recorded from eighteen interneurones following stimulation of the I2 burster axon in the isolated lamprey spinal cord. 2. In each cell, the half-width of the electrotonic e.p.s.p. was measured and used, together with the cable parameters estimated for the same neurone, to locate the position of synaptic contact made by the I2 axon on the dendrites of the interneurone. The synaptic location ranged from 0.05 to 1.35 space constants with a mean of 0.46. 3. The synaptic potential was simulated using the Rall model of the neurone. When compared with the experimentally recorded e.p.s.p. with the same half-width, the rise-time of the simulated synaptic potential was found to be faster. By changing the value of synaptic distance and/or synaptic current duration the half-width, rise-time, and decay of the simulated synaptic potential fit closely the experimental e.p.s.p. The range of synaptic distance estimated from the simulation decreased considerably (0.2--0.7 space constants; mean 0.52). 4. Direct comparison of synaptic location estimated from histological tracings of dendritic trees from these same cells injected with horseradish peroxidase compared favourably with synaptic location estimated from the simulations. 5. These results support the hypothesis that functionally similar presynaptic axons make synaptic connexions at the same electrotonic distance from the soma on functionally similar post-synaptic cells. This occurs in the face of large variations in physical distance for these same synaptic contacts.

Estimates of cable parameters in lamprey spinal cord neurones.

1. Two micro-electrodes were used to penetrate giant interneurones in the isolated lamprey spinal cord. A brief (50--100 microsec) current pulse was applied to one electrode while the other recorded the voltage transient response. 2. A formal analysis of the voltage transient was achieved by the simplifying reduction of each neurone. Somas were treated as a parallel combination of resistance and capacitance. Dendrite trees were reduced to an equivalent cylinder (Rall, 1959). 3. The voltage transients were analysed according to the procedure suggested by Jack & Redman (1971b) to estimate the cable parameters governing the passive propagation of transmembrane potentials. Membrane time constant (tau m), dendritic to soma conductance ratio (rho 00), and electrotonic length (L) of the equivalent cylinder were estimated from these data. 4. In thirty-two interneurones it was possible to determine the membrane time constant, but rho 00 and L were determined in only twenty-two. 5. For the twenty-two neurones in which all cable parameters were estimated, the electrotonic length of the equivalent cylinder was similar to that found for cat spinal motoneurones (1--2 space constants). 6. Simulations of the voltage transient using the Rall model of the motoneurone as developed by Jack & Redman (1971b) resulted in a voltage response which closely ditted the experimental data. 7. These results suggest that the Rall model of the motoneurone accurately describes the propagation of passive transmembrane potentials in lamprey spinal cord neurones. It is further concluded that the time constant for soma and dendritic membrane is similar in these neurones.

The relationship between some measures of synaptic ultrastructure as a function of distance from the soma on lamprey reticulospinal neurons.

Synaptic junctions located on the dendrites of lamprey (Petromyzon marinus) reticulospinal neurons labelled with intracellularly-injected horseradish peroxidase were studied. The normal ultrastructure of the synaptic junctions was defined and several quantitative measures made from each junction in order to test the hypothesis that distally-located synapses are ultrastructurally different from those located at proximal dendritic sites. A total of 820 contacts from one neuron and 279 from a second neuron ranging from 20 to 340 microns from the soma were quantified. The vast majority of the presynaptic endings contained round, clear-cored vesicles and formed an asymmetrical membrane differentiation with the postsynaptic dendrite. A small fraction of the population contained flattened or pleomorphic vesicles and these synapses were equally distributed with respect to distance from the soma. Many of the terminals contained a few large dark- and clear-cored vesicles. Four quantitative measures of each synaptic contact were made. These included vesicle number, length of differentiated membrane, vesicle area and terminal area. Four ratios relating the different quantitative measures were also calculated. Each ratio or measurement from the synaptic junctions was plotted as a function of distance from the soma to determine if differences existed at any distance. It was found that synaptic junctions are uniformly similar and that distal junctions did not differ significantly (P greater than 0.05) from those at proximal dendritic sites. It is concluded that if distal synapses do compensate for their remote location they do this is some other way, possibly by increasing the number of synaptic contacts made by each presynaptic axon.

Estimates of probability of transmitter release at the mammalian neuromuscular junction.

1. Intracellular records of end-plate potentials (e.p.p.s) were obtained from curarized neuromuscular junctions in the rat diaphragm.2. Statistical estimates of the quantal release probability (p) were made at individual junctions from measurements of the means and variances of e.p.p. amplitudes at two different levels of Ca concentration. It was assumed that the release process was binomial and that the number of quanta available for release (n) was independent of external Ca.3. These estimates of p were compared with those obtained by measuring depression of e.p.p. amplitudes after single conditioning shocks and assuming that the depression was due to depletion of n. The statistical estimates were consistently smaller.4. This disparity, plus the additional observation that depression was not linearly related to the number of quanta released by one or more conditioning shocks, suggested that depression was not due entirely to depletion and that the depletion assumption led to an over-estimate of p.5. A third method of estimating p from measurements of the decline of e.p.p. amplitudes during rapid stimulation also appeared to result in an over-estimate.

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.