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PubMed1984

A miniature molecular-sieving column assay for cytoplasmic vitamin A-binding proteins.

Cytoplasmic vitamin A-binding proteins were measured by a method using centrifugation of gel-exclusion columns and compared to the sucrose gradient method. The gel-exclusion method analyzed 18 samples simultaneously on one table-top centrifuge, while the sucrose gradient method required use of three ultracentrifuges to process 18 samples simultaneously. Multiple 2-min low-speed centrifugations of test cytosol applied to miniature molecular-sieving columns was a faster (1/2 the working time for 18 samples), more convenient, and more accurate method for measuring cytoplasmic vitamin A-binding proteins than was the sucrose gradient method. Using the same cytosol and [3H]retinoid preparations, the rapid gel-exclusion method results were only slightly lower than those obtained by radioimmunoassay but 50% higher than the values obtained by the sucrose gradient assay. The methodology described may be useful not only for cytoplasmic vitamin A-binding proteins but also for other similar binding protein assays.

Animals↗

PubMed1989

Polyacrylamide gel miniaturization improves protein visualization and autoradiographic detection.

Polyacrylamide gels shrink to one-quarter of their original area when soaked in a 50% (w/v) solution of polyethylene glycol. Gel miniaturization improves the contrast of protein bands, with four valuable consequences. (i) A 5- to 10-fold increase in sensitivity for Coomassie blue is observed. (ii) Gels are more durable; i.e., they resist tearing when wet and they do not crack during drying under vacuum. (iii) Shrunken gels give sharper photographic images and provide better interlane protein band comparisons. (iv) Condensed protein bands lead to an increased sensitivity for detecting low-abundance, radioactively-labeled proteins by fluorography.

Affinity Labels↗

PubMed1978

Small mode miniature end plate potentials are increased and evoked in fatigued preparations and in high Mg2+ saline.

Amplitude histograms of miniature end-plate potentials (MEPPs) from small sartorius fibers (6-20 micron diam.) of very small frogs were found to have multiple peaks. All MEPPs had the same time characteristics which demonstrates that they arose at the same junction. The amplitude of the smallest mode (s-MEPPs) was one-seventh that of the major mode (m-MEPPs), and in the unstressed preparation s-MEPPs composed only a few per cent of the total. After repetitive nerve stimulation (1 min at 20 Hz) the percentage of s-MEPPs was initially increased. Successive periods of stimulation gradually reduced the end-plate potential (EPP) (above 90% failures) to the same size as s-MEPPs. A 12.5 MM Mg2+ and half normal Ca2+ saline was also found to increase the percentage of s-MEPPs and reduce EPPs so that profiles of EPP amplitude histograms showed a class of small EPPs the same size as s-MEPPs. The data demonstrate that quanta the size of s-MEPPs were released with nerve stimulation in both the Mg2+ blocked and fatigued preparation.

Action Potentials↗

PubMed1980

Effects of temperature on the decline in miniature end-plate potential frequency following a tetanus.

When frog nerve-muscle preparations are stimulated tetanically in saline solutions containing Ca2+, Mn2+, Co2+, or Ni2+ there is a substantial rise in miniature end-plate potential (MEPP) frequency. After stimulation is ended, there is a decline back toward pre-stimulus MEPP frequencies. If we assume that MEPP frequencies are an index of the concentration of the divalent cations at some critical site in the nerve terminal, then the fall in MEPP frequency reflects the clearing of the divalent metal cation from the critical region. As a first step in investigating the clearing mechanism, we have studied the effects of temperature on the decline in MEPP frequencies following a tetanus. The Q10s for the fall in MEPP frequencies following the tetanus in Ca2+ or Mn2+ range between 1.2 and 1.6. The results can be interpreted in terms of a two-stage model for the fall-off in release probability following stimulation. In Co2+ or Ni2+ containing solutions at 7 degrees C or lower the MEPP frequency is sustained at an elevated level following the tetanus; at higher temperatures the decline does not reach the initial control level.

Animals↗

PubMed1981

Tetanic stimulation increases the frequency of miniature end-plate potentials at the frog neuromuscular junction in Mn2+-, CO2+-, and Ni2+-saline solutions.

The effects of Mn2+, Co2+, and of Ni2+ on quantal acetylcholine (ACh) release have been studied with conventional microelectrode techniques. Increasing the [Co2+]0 or [Ni2+]0 (in the absence of extracellular Ca2+) caused an increase in miniature endplate potential (MEPP) frequency. [Mn2+]0 caused some increase in frequency at low levels, but then there was no rise as the concentration was increased further. In preparations depolarized with 20 mM K+, the MEPP frequency was a monotonically increasing function of [Co2+]0 or [Ni2+]0. In increasing concentrations of [Mn2+]0 there was an increase followed by a levelling off or a depression at higher concentrations. Tetanic stimulation of the motor nerve in solutions containing no added divalent cations or containing MgEGTA produced slight or no increases in MEPP frequency. In Mn2+-, Co2+- or Ni2+- saline solution stimulation of the motor nerve led to substantial increases in MEPP frequencies. The maximum frequency attained in Mn2+, Co2+, or Ni2+ was a power function of: (a) the duration of the tetanus; (b) the frequency of stimulation during the tetanus; or (c) the extracellular concentration of the divalent cation. During stimulation in Mn2+-saline solution the MEPP frequency reached a maximum; further stimulation led to a fall in frequency. We conclude that Mn2+, Co2+, and Ni2+ can enter the nerve terminal through a voltage-gated channel. Once within the terminal, they can stimulate quantal release by releasing Ca2+ or by causing the liberation of an activator, like H+, within the terminal.

Acetylcholine↗

PubMed1981

Bursts of miniature end-plate potentials can be released from localized regions of the frog motor nerve terminal.

We examined spontaneous transmitter release by simultaneously recording from end-plates with both an intracellular electrode and a focal extracellular electrode. In normal Ringer's solution the focal releases appear to be a near random portion of the pooled output. But in elevated [Sr2+]out bursts of miniature end-plate potentials (m.e.p.p.s.) occur, and these can be localized at a portion of the presynaptic terminal. The bursts are not artifacts caused by the focal recording technique. In some conditions, sections of the nerve terminal can exhibit an enhanced probability of quantal release that persists for seconds. We occasionally observe extracellular events that must be generated by m.e.p.p.s. in adjacent fibers.

Animals↗

PubMed1981

The timing of channel opening during miniature end-plate currents.

Many chemical transmitters act by opening channels with exponentially distributed life-times. We present a way to analyze a synaptic current in terms of its component channels. We can estimate the numbers and times of channel opening within the synaptic current. This approach is used to study miniature end-plate currents (m.e.p.c.s) at the frog neuromuscular junction. The results support the idea that some transmitter rebinds after dissociation from post-synaptic receptors, and suggest that the time of channel closing is related to the time at which acetylcholine leaves the receptor.

Acetylcholine↗

PubMed1982

Miniature endplate potentials related to neuronal injury.

Intracellular recordings were made in vivo from muscle fibers of anesthetized or spinalized frogs. Characteristic 'spontaneous' miniature endplate potentials were rarely recorded in intact muscles by comparison with muscles acutely denervated in the same animals. It is concluded that little or no spontaneous quantal release of transmitter occurs in vivo, in contrast with isolated preparations. The presence effectively 'silent' endplates was confirmed by evoking quantal release with hypertonic saline.

Animals↗

PubMed1982

The interaction of extracellular H+, Na+, Ca2+ and Sr2+ on the decay of miniature end-plate currents.

We suggested previously that increases in the extracellular [Ca2+] or [Mg2+] lengthen miniature end-plate currents (m.e.p.c.s.) because the divalent cations decrease the negative surface potential on the end-plate, and thereby increase the voltage drop within the membrane. The increased voltage drop would lengthen the open time of the end-plate channels. Increased extracellular [H+] is also known to lengthen m.e.p.c.s.15; perhaps also acting by changing the surface potential. There has been dispute about the observations and their interpretation. Reducing the [Na+]0 at pH = 7.4 from 120 to 12 mM decreased the half-time of m.e.p.c. decay by nearly a factor of two. When the same changes in [Na+]0 were performed at pH = 5.2, no change in m.e.p.c. decay was detected. This suggests that the H+ titrates the negative surface charges, so they no longer can be affected by [Na+]0. The data do not fit the hypothesis that lifetime is determined by the presence of permeant ions in the channel 4,16. At pH = 7.4 replacing the 2.5 mM CaCl2 by SrCl2 decreased the m.e.p.c. decay rate by 60%17. This effect is not seen at pH = 5.2. We suggest that at pH = 7.4, Sr2+ binds to fixed negative charges on the end-plate and thereby slows the decay. H+ titrates the fixed charges and decreases the Sr2+ effect. In low [Na+] saline, raising [Ca2+]0 shortened m.e.p.c. decay--an effect exactly opposite to that expected from the change in surface potential. Under these conditions, increasing the [Ca2+] decreases the m.e.p.c. decay rate by some other mechanism; this second effect of Ca2+ on m.e.p.c. decay may account for some of the different results reported.

Animals↗

PubMed1983

Elevated tonicity increases miniature end-plate potential frequency during tetanic stimulation at frog neuromuscular junction in low calcium and in manganese saline solutions.

We have followed the increase in miniature end-plate potential (MEPP) frequency that occurs during tetanic stimulation of the motor nerve in low Ca2+ and in Mn2+ saline solutions. During stimulation in either solution the frequency rises over the first minutes to reach an asymptote. In low Ca2+ solutions, hypertonicity increases the rate at which the MEPP frequency rises. Raising the [Mn2+] also increases the rate at which the MEPP frequency rises and elevates the level of the asymptote. The data are interpreted by using a model in which quantal release frequency is proportional to the [( Ca2+])n in the nerve terminal; the model specifically includes the [Ca2+] in the terminal before stimulation. There is evidence that hypertonic solutions elevate the [Ca2+]i. We suggest that the effects of tonicity increases can be accounted for solely by the rise in [Ca2+]i before the beginning of the tetanus. If the number of Ca2+ cooperating in release, n, is 4, then with each stimulus there must be an increase in Ca2+ influx. If the value of n is higher, for example 50, the amount of Ca2+ entering with each stimulus is roughly constant. The data from the Mn2+ experiments also suggest than n may be greater than 4. It is pointed out that for exocytotic transmitter release, many Ca2+ ions may cooperate in the release process, so high values of n are not unreasonable.

Acetylcholine↗

PubMed1984

Miniature synaptic potentials recorded intracellularly from Purkinje cell dendrites in guinea pig cerebellar slices.

Intracellular recordings from Purkinje cell dendrites in guinea pig cerebellar slices revealed the existence of miniature spikes with various amplitudes, which were firing apparently without any externally applied stimulations. These spikes were in a hyperpolarizing direction at a resting membrane potential, and their apparent reversal potential was at about -60 mV, indicating their inhibitory nature. Based on the blocking actions of high-Mg2+, tetrodotoxin and amino acid antagonists such as bicuculline etc., these spikes were suggested to be inhibitory synaptic potentials generated by cerebellar interneurons.

Amino Acids↗

PubMed1985

Time course of miniature postsynaptic potentials at the Mauthner fiber giant synapse of the hatchetfish.

The hatchetfish Mauthner fiber is presynaptic to 8-14 large myelinated axons in the medulla; the large ('giant') synapses formed by these fibers appear to be nicotinic cholinergic. Miniature postsynaptic potentials (mPSPs) were recorded from single identified synapses. The mPSPs were averaged to more accurately determine their shape; the rise time was approximately 70 microseconds, and the fall usually was biphasic with time constants of decay for the two phases of 280 and 800 microseconds. In 25% of the records analyzed a third, slow tail of decay was seen which had an average decay constant of 4.2 ms. The biphasic decay of mPSPs largely accounts for the similar shape of the postsynaptic current following a presynaptic impulse, which is described in the accompanying paper.

Animals↗

PubMed1985

The aminoglycoside antibiotic, gentamicin, fails to block increases in miniature endplate potential frequency induced by the sulfhydryl reagent, N-ethylmaleimide, in low calcium solutions.

N-ethylmaleimide (NEM) increases the frequency of miniature endplate potentials (MEPPs) at the adult rat hemidiaphragm. This sulfhydryl-alkylating agent produces comparable effects in the absence of added calcium (2 mM EGTA), suggesting that the drug releases calcium from internal stores, or promotes calcium-independent release by depolarizing the nerve terminal or interacting more directly with the release mechanism. These increases in frequency are not blocked by the aminoglycoside antibiotic, gentamicin; although the latter agent reduces quantal content and the elevations in MEPP frequency induced by high potassium solutions. The results suggest that gentamicin and NEM act at different sites at the presynaptic terminal, and that the aminoglycosides block voltage-dependent presynaptic calcium influx.

Action Potentials↗

PubMed1985

Distribution of exponentiality in miniature endplate current decay.

Exponential functions are commonly used to describe miniature endplate current (MEPC) decay; under control conditions a monoexponential is usually regarded as sufficient, whereas in the presence of some drugs a biexponential may be necessary. Using an automated fitting procedure which estimated exponential parameters and the period of decay from peak to baseline, a unimodal distribution of curvature was found for control MEPCs recorded in frog sartorius muscle. The majority of MEPCs were of monoexponential form, while the remainder were biexponential with either less or greater curvature than expected for a simple exponential (hypoexponential or hyperexponential, respectively). The proportion of MEPCs in each of the 3 groups was constant for a given endplate but varied between endplates. A possible explanation for this phenomenon could be differences in synaptic geometry within and between endplates. The increased curvature of individual MEPCs in the hyperexponential group was analyzed by assuming a sequential model for agonist blockade or desensitization, and calculating closing and reopening rate constants. These rate constants were altered by procaine and verapamil (100 microM) in a manner consistent with blockade of the acetylcholine receptor by enhancement of agonist-induced desensitization.

Animals↗

PubMed1985

Miniature endplate potential amplitudes corrected for spatial decay are not normally distributed.

Miniature endplate potentials (MEPPs) were recorded simultaneously with two intracellular electrodes placed in the muscle fiber near the ends of the frog neuromuscular junction (nmj). The MEPP amplitudes are different in each electrode and are not normally distributed. A method is proposed to correct MEPP amplitudes for spatial decay. The MEPP amplitudes corrected for spatial decay are also not normally distributed. When the class interval of the distribution is small, multiple peaks are observed.

Action Potentials↗

PubMed1987

Neurotransmission parameters estimated from miniature endplate current growth phase.

A numerical model of miniature endplate current (mepc) generation was fitted to the rising phase of individual mepcs recorded at the frog neuromuscular junction, and estimates of 6 transmission parameters were obtained. Model fitting was enabled by assuming literature values for geometric parameters and determining single channel current by noise analysis, the channel closing rate constant from the mepc decay, and acetylcholine hydrolysis parameters from mepcs recorded in esterase-blocked endplates. Under control conditions, mean estimates were: number of molecules in a quantum = 29,000, diffusion coefficient = 2.8 X 10(-6) cm2s-1, endplate receptor density = 8500 micron-2, forward binding rate constant = 7.6 X 10(8) M-1s-1, equilibrium dissociation constant = 58 microM and channel opening rate constant = 8100 s-1.

Animals↗

PubMed1988

Emetine increases giant miniature endplate potential population at the frog neuromuscular junction.

Intracellular recordings from 90 endplates in untreated cutaneous pectoris muscles of the frog showed that giant miniature endplate potentials (gMEPPs) occurred at a low frequency of 0.017 +/- 0.002 s-1. These spontaneous potentials are several times larger in amplitude and have time to peak twice longer than the normal MEPPs (nMEPPs). The frequency of gMEPPs was not significantly influenced by the presence or absence of tetrodotoxin, calcium or high Mg2+. The frequency of gMEPPs was not dependent on the frequency of nMEPPs nor was it influenced by prior prolonged motor nerve stimulation. After a delay of 10-20 min, the antiamebic drug, emetine, (10 microM) increased the frequency of gMEPPs by about 10-fold without affecting nMEPP frequency. The emetine-induced gMEPPs were very similar in characteristics to gMEPPs occurring in untreated muscles. The ability of emetine to increase the gMEPP frequency was not affected by the absence of extracellular calcium. The results indicate that emetine enhances the frequency of the calcium-independent gMEPPs that are normally seen in untreated muscles.

Animals↗

PubMed1990

Miniature excitatory synaptic currents in cultured hippocampal neurons.

We performed patch clamp recordings in the whole cell mode from cultured embryonic mouse hippocampal neurons. In bathing solutions containing tetrodotoxin (TTX), the cells showed spontaneous inward currents (SICs) ranging in size from 1 to 100 pA. Several observations indicated that the SICs were miniature excitatory synaptic currents mediated primarily by non-NMDA (N-methyl-D-aspartate) excitatory amino acid receptors: the rising phase of SICs was fast (1 ms to half amplitude at room temperature) and smooth, suggesting unitary events. The SICs were blocked by the broad-spectrum glutamate receptor antagonist gamma-D-glutamylglycine (DGG), but not by the selective NMDA-receptor antagonist D-2-amino-5-phosphonovaleric acid (5-APV). SICs were also blocked by desensitizing concentrations of quisqualate. Incubating cells in tetanus toxin, which blocks exocytotic transmitter release, eliminated SICs. The presence of SICs was consistent with the morphological arrangement of glutamatergic innervation in the cell cultures demonstrated immunohistochemically. Spontaneous outward currents (SOCs) were blocked by bicuculline and presumed to be mediated by GABAA receptors. This is consistent with immunohistochemical demonstration of GABAergic synapses. SIC frequency was increased in a calcium dependent manner by bathing the cells in a solution high in K+, and application of the dihydropyridine L-type calcium channel agonist BAY K 8644 increased the frequency of SICs. Increases in SIC frequency produced by high K+ solutions were reversed by Cd2+ and omega-conotoxin GVIA, but not by the selective L-type channel antagonist nimodipine. This suggested that presynaptic L-type channels were in a gating mode that was not blocked by nimodipine, and/or that another class of calcium channel makes a dominant contribution to excitatory transmitter release.

2-Amino-5-phosphonovalerate↗