Excitatory transmitter release induced by high concentrations of γ-aminobutyric acid (GABA) in crayfish neuromuscular junctions

At the neuromuscular junction of very small crayfish (0.4–2 g) addition of -aminobutyric acid (GABA) to the superfusing solution at concentrations exceeding 100 mmol/l elicited high frequency release of excitatory transmitter quanta. In seven experiments single application of 500 mmol/l GABA gave rise to instantaneous release of 70,000 to 130,000 quanta. These stores of transmitter were released by GABA in a first order process with time constants, _q, of between 9 s and 20 s, the maximum rate of release,ñ ₀, reaching 10,000 quanta/s in some cases. After release had ceased in the presence of GABA, the preparation was allowed to recover for five minutes in normal solution. Subsequently, a second trial evoked about 50% of the release induced during the first application of GABA. Pretreatment of the preparation with 2 mol/l serotonin (5-HT) facilitated GABA-induced transmitter release resulting in larger rates of release and consequently in a larger output of transmitter by a factor of about 3. The largest amount of transmitter released on a single application of GABA in the presence of serotonin comprised about 220,000 quanta with a maximum rate of releaseñ ₀ 25,000 quanta/s. The release evoked by high GABA-concentrations did not depend markedly on extracellular Ca²⁺ or Mg²⁺, but required extracellular Na⁺. The effects induced by high concentrations of GABA on release of excitatory transmitter quanta were quantitatively similar to the effects of high glycine-concentrations on release of quanta from the inhibitory terminals (Finger 1983a, b).This investigation was supported by the Deutsche Forschungs-gemeinschaft, Project Fi 305/1-3     

Differential effect of intraterminal sodium on spontaneous quantal release of transmitter in two neuromuscular junctions of crayfish

Nerve terminals on the superficial abdominal extensor muscle and the claw opener muscle of small crayfish were loaded with sodium by bath application of 100 μmol/l veratridine in superfusates where normal Ca²⁺ was removed (low-Ca²⁺ superfusate). In both preparations this caused an increase in spontaneous quantal release of excitatory and inhibitory transmitter which was evaluated by means of the noise analysis technique. About 2.5 min after application of veratridine, when spontaneous quantal release had largely ceased, the normal Ca²⁺ concentration was reestablished. This increased transiently the quantal release rate a second time. However, release activated by Ca²⁺ application was much more vigorous at the superficial abdominal extensor muscle than at the claw opener. At the superficial abdominal extensor muscle on average about 8% of the total number of quanta could be released in low Ca²⁺ and 92% in normal Ca²⁺ superfusate, while at the claw opener 75% of the quanta were released in low Ca²⁺ and 25% in normal Ca²⁺ superfusate. The experiments suggest that intraterminal sodium has a differential effect in the terminals of the two preparations. Possibly, the intraterminal source from which Na⁺ may liberate Ca²⁺ is more restricted in the superficial abdominal extensor muscle than in the opener muscle of the claw.     

Quantal stores of excitatory transmitter in nerve-muscle synapses of crayfish evaluated from high-frequency asynchronous quantal release induced by veratridine or high concentrations of potassium

At single voltage-clamped opener muscle fibres of crayfish claw, 10–100 mol/l veratridine increased within a few seconds the rate of asynchronous quantal release, ñ, of excitatory transmitter from ñ<1 quantum/s to ñ10,000 quanta/s. Thereafter ñ declined exponentially either with a single, (2)50 s, or with two time constants (1)19 s, (2)50 s. In total (t), about 0.3 million quanta were released by veratridine in a single short fibre of about 1 mm length. These values were estimated by means of the noise analysis technique and they agreed with equivalent parameters of release when 100 mmol/l K⁺ were used as release stimulus. Strong quantal release could be elicited only once in a single muscle by veratridine. Furthermore, the effect of veratridine on quantal release could be completely prevented by pretreatment with tetrodotoxin. In another nerve-muscle preparation of crayfish, the abdominal superficial extensor muscle, up to 3 million excitatory quanta could be released by veratridine in a single fibre. In the latter muscle veratridine-induced asynchronous quantal release was strongly dependent on the extracellular concentration of Ca²⁺ whereas in the claw opener dependence of quantal release on extracellular Ca²⁺ was negligible.This investigation was supported by the Deutsche Forschungsgemeinschaft, SFB 220     

Excitability and depolarization-release characteristics of excitatory nerve terminals in a tail muscle of spiny lobster

In the deep abdominal L₁-extensor muscle of the spiny lobster (Panulirus penicillatus) quantal excitatory postsynaptic currents (EPSCs) were recorded through macropatch-clamp electrodes. Release of transmitter quanta from terminals was also elicited by depolarizing current pulses given through the recording electrode. The majoritiy of terminals were excitable: on increasing the depolarization pulses, release was triggered at a threshold in an all-ornothing manner. If excitation was blocked by tetrodotoxin (TTX), release was graded with depolarization reaching the amplitude of the all-or-nothing response at pulse amplitudes several times higher than the former threshold level. Some inexcitable terminals were also found: in these, release was graded for increasing depolarization pulses, and TTX did not alter the depolarization-release relation. Among the other types of terminals studied with the same technique, the proportion of excitable terminals in this lobster tail muscle is higher than in the crayfish opener and lower than in the frog's cutaneous pectoris muscle. The contribution of the increase in intraterminal Ca concentration to the control of release was estimated using facilitation of a test EPSC as an indicator of Ca inflow during a preceding depolarization pulse. This facilitation was found to have a maximum at a certain pulse amplitude, , and to decline for larger depolarizations. Release, however, rose considerably for depolarizations larger than those effected at .It is concluded that, like in crayfish and frog motor terminals, release is controlled directly by depolarization in addition to the control by Ca-inflow. Supported by a grant of the Deutsche Forschungsgemeinschaft to J. Dudel and I. Parnas     

Calcium dependence of quantal release triggered by graded depolarization pulses to nerve terminals on crayfish and frog muscle

Quantal transmitter release was measured in small portions of neuromuscular junctions by means of a perfused macro-patch-clamp electrode. Release was elicited by graded current pulses through the recording electrode (excitation blocked by TTX). On increasing the stimulation current from a threshold amplitude, release rose steeply for several orders of magnitude and finally approached a saturation level of about 10 quanta/pulse. Reduction of the Ca concentration in the perfusate of the electrode, Ca_e, depressed the saturation level of release relatively little and had practically no effect on the threshold current amplitude, as long as the Ca concentration in the superfusion of the bath, Ca_b, remained high. When Ca_b was reduced too, the depression of release was more severe. The dependence of release on Ca_e was determined for a large range of Ca_e for saturating depolarization pulses. In crayfish, at 0 Ca_b, in double-logarithmic release-Ca_e plots the maximum slope was on average 3.9, and this slope dropped to on average 2.1 in 13.5 mM Ca_b. In frog, at 0 Ca_b, the respective double-logarithmic slope was 3.5, while in 1.8 mM Ca_b this slope declined dramatically, the rate of release decreasing on average only by a factor of 3.8 from 10 mM to 0.02 mM Ca_e. These results are interpreted by the assumption that the resting Ca concentration in the terminal, Ca_ir, has strong influence on the rate of release due to depolarization pulses in low Ca_e, and that Ca_b has control on Ca_ir in the terminal.Supported by the Deutsche Forschungsgemeinschaft     

Calcium and depolarization dependence of twin-pulse facilitation of synaptic release at nerve terminals of crayfish and frog muscle

Transmitter quanta were elicited from nerve terminals of crayfish and frog muscle by depolarization pulses through a macro-patch-clamp electrode. The rates of quantal release for twin pulses and their ratio, twin pulse facilitation, F_d, were determined. When the electrode was perfused with normal Ca_e (13.5 mM for crayfish, 1.8 mM for frog), F_d was low for threshold depolarizations, increased to a maximum at medium depolarizations, decreased when the rate of release due to the first pulse approached saturation, and increased again for larger depolarizations. If under these conditions the superfusion of the muscle outside the electrode was changed from normal to 0 Ca_b and high Mg_b solution, F_d increased. When the Ca concentration around the terminal, Ca_e, was reduced to levels at which release did not reach the saturation level for large depolarizations, F_d in dependence on depolarization did not show the minimum at higher depolarizations. The amplitude of F_d measured for large, constant depolarization pulses showed a maximum at a Ca_e below that of the normal solution. The maximum of F_d was much higher if the superfusion of the bath contained 0 Ca_b and high Mg_b than when normal bathing solution was superfused. The maxima of F_d at a low value of Ca inflow are predicted by the residual Ca theory of facilitation, if release is influenced by a resting low internal Ca concentration, Ca_ir, and reaches a saturation level for large Ca-inflow. It is also predicted that decreasing Ca_ir (as in low Ca_b) will increase F_d.Supported by the Deutsche Forschungsgemeinschaft (SFB 220)     

Reciprocal axo-axonal synapses between the common inhibitor and excitor motoneurons in crustacean limb muscles

Summary Nerve terminals of the common inhibitor motoneuron in a crab (Eriphia spinifrons) limb closer muscle and in a crayfish (Procambarus clarkii) limb accessory flexor muscle make neuromuscular synapses with the muscle membrane (postsynaptic inhibition) as well as axo-axonal synapses with the terminals of the excitatory axon (presynaptic inhibition). That transmission is from the inhibitor to the excitor terminals at these axo-axonal synapses is indicated by the occurrence on the inhibitor membrane of presynaptic dense bars denoting sites of transmitter release. Axo-axonal synapses with the opposite polarity, in which transmission is from an excitatory onto an inhibitory terminal, were occasionally seen either adjacent to or separate from the inhibitory axo-axonal synapse. Nerve terminals of the specific inhibitor in the crayfish opener muscle were seen to make numerous axo-axonal output synapses upon excitatory nerve terminals but excitor nerve terminals were not seen to make output synapses onto inhibitor terminals. Thus reciprocal axo-axonal synapses appear to be a feature of the common inhibitor but not of the specific inhibitor. The excitor-to-inhibitor component of these reciprocal synapses may serve to limit transmitter output in the common inhibitor axon by activating glutamate_B receptors which facilitate efflux of K⁺ and hyperpolarization of the membrane.     

Gustatory reaction time under variable stimulus parameters in human adults

Reaction times and perceived intensities for NaCl solutions were measured in sixteen human adults. Stimulus delivery was by means of a circular piece of solution-soaked filter paper held with forceps. The reaction time (T) decreased and the perceived intensity (S) increased with increasing the concentration (C) of NaCl solution applied to a fixed (78.5 mm²) tongue area. The comprehensive relations among T, S and C can be expressed by the following formulae; $\text{T = a +}\text{b}\text{log(}\text{C}\text{C}{}_0\text{)}$, $\text{T = p +}\text{q}\text{log(}\text{S}\text{S}{}_0\text{)}$, and logS = mlogC + logn, where a, b, p, q, m, n = constants, and S₀ indicates the perceived intensity at the threshold concentration (C₀). Meanwhile, the reaction time decreased and the perceived intensity increased with increasing the stimulated area (A) under a fixed (1.0 M) concentration of NaCl solution. The relations among T, S and A can also be expressed by the following equations; $\text{T = a′ +}\text{b′}\text{log(}\text{A}\text{A}{}_0\text{)}$, $\text{T = p′ +}\text{q′}\text{log(}\text{S}\text{S}{}_0\text{)}$, and logS = m′logA + logn′, where a′, b′, p′, q′, m′, n′ = constants, and A₀ indicates the threshold size of stimulated area.     

Shifts in the voltage dependence of synaptic release due to changes in the extracellular calcium concentration at nerve terminals on muscle of crayfish and frogs

The rate of release of transmitter quanta, elicited by variable depolarization pulses applied to a nerve terminal by means of a macro-patch-clamp electrode, was measured in muscles of crayfish and frogs. The electrode was perfused with solutions containing different Ca concentrations, Ca_e. The bath was superfused separately, usually with solutions containing nominally no Ca_b and elevated Mg_b. A fixed depolarization pulse followed the variable test pulse within 7–10 ms, and facilitation, F_c, of release after the fixed pulse was determined as a measure of Ca-inflow during the test pulse. As described before, F_c always showed a peak, F_c, at depolarization amplitudes of the test pulse below the saturation level of release. When Ca_e was changed, the depolarization levels generating F_c shifted in a negative direction if Ca_e was lowered, and in a positive direction if Ca_e was increased. These shifts agreed with the known dependence of the effective membrane potential (controlling e.g. Ca inward current) on Ca_e which is due to shielding of surface changes by Ca²⁺ (cf. Hille 1984). Changes of Ca_b, at constant Ca_e, did not affect the depolarization dependence of F_c. It is concluded that Ca inflow is not the only factor controlling quantal release, and that at least in depolarizations beyond those eliciting F_c another potential dependent factor increases release while Ca inflow presumably falls.Supported by the Deutsche Forschungsgemeinschaft (SFB 220)     

Tonic depolarization of excitatory nerve terminals in crayfish muscle by high concentrations of extracellular potassium

At voltage-clamped fibres of the claw opener muscle of small crayfish, spontaneous quantal release of excitatory transmitter elicited by raising extracellular K+ to 100 mM was investigated. On application of the high K+ concentration, the rates of quantal release increased to n = 10,000-25,000 quanta/s within 10 s, and thereafter declined exponentially, either with a single (tau congruent to 15-40 s) or with two (tau 1 congruent to 15-40 s, tau 2 greater than 70 s) time constants. The total number of quanta released per trial ranged from s = 200,000 to 800,000 quanta. The results were derived by means of the fluctuation analysis technique.     

Presynaptic effect of gamma-aminobutyric acid on the inhibitory nerve and nerve terminals in the crayfish neuromuscular junction

Experiments were carried out in voltage-clamped fibres of the opener muscle of the first walking leg or claw of small crayfish. Repetitive discharges in the inhibitory nerve innervating the muscle were induced by adding serotonin (10(-6) mol/l) and forskolin (10(-4) mol/l) to the superfusate. Rates of nerve discharge were determined by recording nerve evoked inhibitory postsynaptic currents (IPSCs) in the voltage-clamped muscle fibre. Subsequently, the effect of gamma-aminobutyric acid (GABA) on the rate of IPSCs in normal and Cl- -deficient superfusate was investigated. In normal superfusate GABA (10(-5) mol/l) abolished the IPSCs whereas in Cl- -deficient superfusate GABA (10(-4) mol/l) enhanced the rate of IPSCs. Moreover, in Cl- -deficient superfusate the rate of asynchronous quantal release of inhibitory transmitter could be enhanced by GABA. The results indicate that in the crayfish neuromuscular junction the inhibitory axon is supplied with GABA receptors which may affect (a) axonal excitation and (b) quantal output at the inhibitory axon terminals.     

A pocket calculator program for the evaluation of the oxygen diffusion to perfusion ratio

A pocket calculator program has been written for the evaluation of the oxygen difusion to perfusion ratio (). It allows one to avoid the computational errors involved by approximating the oxygen dissociation curve through a straight line.     

Effects of exogenous nitric oxide on neurotransmitter secretion and ionic currents in motor terminals

Extracellular recording was used to study the effect of sodium nitroprusside, a donor of NO, on endplate transmitter release and ionic currents in frog cutaneous pectoris muscle. Exogenous NO inhibits induced transmiter secretion, and this effect is antagonized by extracellular Ca²⁺. Exogenous NO increases potential-dependent outward potassium current and inhibits Ca²⁺-activated potassium current in the motor nerve terminals. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 8, pp. 144–147, August, 1999     

Effect of lithium on veratridine-induced quantal and non-quantal release from inhibitory nerve terminals in crayfish muscle

Muscle fibres of small crayfish were voltage clamped and superfused for about 10 min with Li⁺ saline (Na⁺ replaced by Li⁺) which contained 5 mmol/l glutamate to desensitize excitatory postsynaptic receptors. Then 100 mol/l veratridine were added to the superfusate which caused strong asynchronous quantal release of inhibitory transmitter. However, in the presence of Li⁺ strong inhibitory quantal release was only transient. It could be activated a second time by removal of Li⁺ and readministration of Na⁺. From the total of 0.7 to 1.1 million quanta released by veratridine only about 30–35% could be released in Li⁺ saline. The voltage clamp DC-currents recorded during veratridine-induced quantal release suggested that a nonquantal release component is additionally involved. This non-quantal release component was most prominent during the period of quantal release in Li⁺ superfusate while it was less obvious during the second enhancement of quantal release in normal saline. Together with previous results (Martin and Finger 1988) it may be concluded that quantal release, but not non-quantal release, is decreased by Li⁺ in the nerve terminals.This investigation was supported by the Deutsche Forschungsgemeinschaft, SFB 220     

Neurotransmitter release and its facilitation in crayfish

Excitatory postsynaptic currents (EPSCs) were recorded extracellularly from synaptic spots on crayfish opener muscles. Release of transmitter was determined by counting the average number of quanta which appear after a stimulus. When [Mg]₀ was increased from 2.5 to 12.5 mM, release was inhibited. Quantitatively the effect of [Mg]₀ could be described by a competitive inhibition of the entry (not of the release) of Ca²⁺ after an impulse, with apparent dissociation constants K_Mg between 1.4 and 18 mM [Mg]₀, assuming saturation kinetics for entry of Ca²⁺ and release. At constant [Ca]₀, twin pulse facilitation (F _s ) for short intervals (about 10 ms) increased when [Mg]₀ was raised from low values, reached a maximum at a certain {ie237-1} and unexpectedly decreased again at higher [Mg]₀. At higher [Ca]₀, {ie237-2} shifted to higher values. This maximum of facilitation is predicted qualitatively by our theoretical model. However, the amplitude of facilitation was larger than predicted theoretically, and the {ie237-3} were smaller than predicted. The theoretical possibilities to correct these discrepancies within the framework of residual calcium based facilitation and saturation kinetics of entry and release were analyzed, but all were in conflict with experimental findings. It is concluded that an essential element is missing in the present theory of facilitation.Supported by the Deutsche Forschungsgemeinschaft and by the USA-Israel Binational Foundation     

Pre- and postsynaptic actions of nifedipine at an identified cholinergic central synapse of Aplysia

The effects of the dihydropyridine (DHP) Ca²⁺ channel antagonist, nifedipine, were studied on the cholinergic synapse between the presynaptic neurones B4/B5 and the postsynaptic neurones B3/B6 located in the buccal ganglion of Aplysia californica. Nifedipine (10 M) decreased the presynaptic Ca²⁺ current by 30%–40%. Blockade of DHP-sensitive Ca²⁺ channels, however, did not affect quantal transmitter release from the presynaptic neurones. Thus, at this synapse, DHP-sensitive Ca²⁺ channels appear not to be involved in acetylcholine (ACh) release. The postsynaptic response to an ionophoretic application of ACh was decreased by nifedipine, pointing to a blocking action of the drug on the postsynaptic receptor/channel complex. Nifedipine was also found to activate protein kinase C, which in turn induces an increase in the nifedipine-resistant presynaptic Ca²⁺ influx and in the number of released ACh quanta. These effects of nifedipine could be prevented by a previous application of 1, 5-(isoquinolinylsulfonyl)-2-methyl-piperazine (H-7), a protein kinase blocker.     

Purification and some properties of beet yellow virus

Beet yellows virus was purified by a method, based on ultracentrifuging at 35,000 g, that preserved the normal length of the virus particle and eliminated a uv-absorbing contaminant retained by previously described methods. The purified particles had a modal length in shadowed preparations of 1250 nm and sedimented in the analytical centrifuge usually as one component at 130 S. Purified preparations had an $\text{A}{}_{260}\text{A}{}_{280}$ ratio of 1.44 and an extinction coefficient at 260 nm of 2.9. In Cs₂SO₄ isopycnic banding, the virus had a density of 1.285 g/cm³, while in CsCl two bands were produced at 1.307 and 1.312 g/cm³.     

Immunocytochemical evidence for in vitro release of glutamate and GABA from separate nerve terminal populations in the rat pontine nuclei

Summary A quantitative electron microscopic immunocytochemical method was used to study the synaptic handling of glutamate and GABA in slice preparations from the rat pontine nuclei. Slices were subjected to a depolarizing stimulus (55 mM K⁺, 20 min) in the presence of a physiological or low Ca²⁺concentration. Depolarization at physiological [Ca²⁺] evoked a depletion of glutamate-like immunoreactivity from nerve terminals that contain round vesicles and establish asymmetric synaptic contacts. When depolarization was induced in the presence of only 0.1 mM Ca²⁺ (10 mM Mg²⁺ added), the loss of glutamate was significantly reduced or abolished, indicative of a Ca²⁺dependent component of glutamate release. By means of a double labeling immunocytochemical method we could identify a population of nerve terminals that displayed strong GABA-like immunoreactivity, and a level of glutamate like immunoreactivity that was low but yet clearly above background level. This type of terminal contains elongated or pleomorphic vesicles and establishes symmetric synaptic contacts. In these terminals, depolarization evoked a Ca²⁺-dependent depletion of GABA like immunoreactivity, but failed to change the level of glutamate like immunoreactivity. The present study demonstrates that two different types of nerve terminal in the rat pontine nuclei contain releasable pools of glutamate and GABA, respectively, and that the GABA releasing terminals also contain a non releasable pool of glutamate. The glutamate of the latter pool could act as precursor of GABA.     

Response characteristics of a multicompartment frog skin epidermis model.

The main goal of this study was to analyze by computer simulation the responses of a multicompartment frog skin epidermis model to systematic changes in the rate coefficients (k's) which characterize the pathways of flow of Na⁺ between compartments. Responses were expressed as deviations from the behavior of a reference model (10Es) which closely simulates known laboratory data on skin epidermis. This includes transepidermal flux rates, Na⁺ pool sizes, isotope labeling fraction, efficiency of the transepidermal Na⁺ pump ($\text{ΔNa}{}^{\mathrm{+}}\text{ΔO}{}_2$). The response patterns of 10Es with its k-variants may be useful in laboratory studies aimed at localizing the effects of an applied physical or chemical stimulus on maintenance Na⁺ metabolism, or on Na⁺ fluxes in frog skin.     

A calcium regulated adenosine triphosphatase in Entamoeba histolytica

Axenically grown trophozoites of Entamoeba histolytica (NIH-200 strain) contain an active ATPase (170 nmol PO₄/min per mg protein) with maximal activity at pH 8.8, a high affinity for ATP ($\text{K}{}_{\mathrm{m}}\text{? 40 μM}$) and an absolute and specific requirement for Ca²⁺. The activation by Ca²⁺ shows positive cooperativity (n_H = 2.48) at calcium concentrations below 8 μM and no cooperativity between 8 and 25 μM. The latter concentration fully saturates the enzyme. The observed activity is insensitive to oligomycin, ouabain and ruthenium red and is unaffected by a range of inhibitors of electron transport and uncouplers of oxidative phosphorylation. The enzyme exhibits structure bound latency and is tightly bound to cellular membranes. It is sedimentable (>80%) by high speed centrifugation of cell homogenates which are either protected osmotically or in which subcellular structures are damaged by sonication or treatment with Triton X-100. Arrhenius plots of V in the temperature range of 0–38°C are linear without breaks, similar to other pyrophosphatases of E. histolytica. The calculated activation energy is 14.8 kcal/mol. This finding as well as the failure of phospholipase treatment to affect activity indicate that interactions with membrane lipids play no role in the catalytic function of this tightly membrane-bound ATPase.