Multiphoton absorption and nonlinear refraction of GaAs in the mid-infrared.

We report the wavelength dependencies of the two- and three-photon absorption coefficients of undoped GaAs in the spectral range 1.3-3.5 microm, as well as nonlinear refractive index n2 in the range 1.7-3.25 microm. The data were obtained by using the single-beam Z-scan method with 100-fs-long optical pulses. Anisotropy of the three-photon absorption coefficient was observed and found to be consistent with the crystal symmetry of GaAs.

Terahertz wave propagation in one-dimensional periodic dielectrics.

We demonstrate the temporal evolution of terahertz (THz) wave propagation in one-dimensional periodic dielectrics. Distributed Bragg reflectors and a resonant cavity are investigated: The structures involve air gaps interleaved between polymer films. Transmitted and reflected broadband THz waves are measured by means of THz time-domain spectroscopy. The experimental results agree well with transfer matrix calculations.

Alpha-latrotoxin channels in neuroblastoma cells.

The changes in ionic permeability induced by the application of alpha-latrotoxin to NG108-15 neuroblastoma x glioma cells were examined using the nystatin perforated-patch technique for whole-cell recording. Complex single channel activity appeared in the plasmalemmas after delays that ranged from 1-20 min in Krebs' solution. The conductance of a channel fluctuated among at least three broad, approximately equispaced bands, the maximum conductance being about 300 pS, and the reversal potential approximately 0 mV. The channels were permeable to Na+, K+, Ca2+ and Mg2+, poorly permeable to glucosamineH+ and Cl-, and were blocked by La3+. The channels stayed fully open in Ca(2+)-free solutions with 4 mM Mg2+, in solutions with no divalent cations and in solutions with 2 mM Ca2+ and 96 mM Mg2+. They opened infrequently if both internal and external Cl- were replaced by glutamate-. If alpha-latrotoxin opened similar channels in nerve terminals, the flux of ions through them could account for the massive release of neurotransmitter induced by the toxin.

Correlation between quantal secretion and vesicle loss at the frog neuromuscular junction.

1. We measured the rate of occurrence of miniature endplate potentials (MEPPs) at identified endplates in frog cutaneous pectoris muscles treated with crude black widow spider venom (BWSV) or purified alpha-latrotoxin (alpha-LTX) in calcium-free solutions, and we examined the relationship between the length of the nerve terminal and the total number of quanta secreted, and the relationship between the number of quanta secreted and the number of vesicles remaining at different times. 2. The venom, or toxin, was applied in a modified Ringer solution with tetrodotoxin, 1 mM-EGTA and no divalent cations, and quantal secretion was started by applying Ca2(+)-free solutions with Mg2+. This was done to synchronize the quantal discharge at the various junctions in a muscle. Ringer solution was applied after the MEPP rate had declined to low levels, and then the muscle fibre was injected with Lucifer Yellow, the endplate stained for acetylcholinesterase and the length of the nerve terminal and the length of a sarcomere were measured on the fluorescent fibre. 3. The total number of quanta secreted by a terminal was measured under a wide variety of experimental conditions: the weights of the frogs ranged from 13 to 68 g, the temperature from 9 to 28 degrees C, and the concentration of Mg2+ from 2 to 10 mM. In one series of experiments the Mg2+ was withdrawn after 3-4 min and reapplied 35-40 min later in order to divide the total output of quanta into two approximately equal bouts of secretion that were well separated in time. 4. The total number of MEPPs recorded at a junction was loosely correlated with the length of its nerve terminal, but it was not affected by the temperature, the concentration of Mg2+ or the division of secretion into well-separated bouts of quantal release. The average total secretion per unit length was about 3700 quanta/sarcomere or about 1200 quanta/microns. 5. The average time course of quantal secretion per micrometre of terminal was determined at single junctions in muscles held at 22-23 degrees C or at 9-10 degrees C. Other muscles were fixed at various times during the course of secretion at each temperature and the number of synaptic vesicles remaining in cross-sections of the terminals were counted on electron micrographs. The number of vesicles remaining per micrometre of terminal was determined from the number per cross-section and the section thickness.(ABSTRACT TRUNCATED AT 400 WORDS)

A re-examination of the effects of lanthanum on the frog neuromuscular junction.

Lanthanum (La3+, 0.1-2mM) was applied to frog cutaneous pectoris muscles at 20-25 degrees C, or at 3-5 degrees C, and the mean amplitude and rate of occurrence, mean value of r, of the miniature endplate potentials (mepps) were measured as functions of time at single neuromuscular junctions. Some muscles were fixed at 3-5 degrees C and their nerve terminals examined in the electron microscope. When 1 or 2 mM La3+ was applied at room temperature, mean value of r rose to peak values of 0.8-3.4 x 10(3) and then declined to less than 100/s after 30-60 min. When the results are corrected for the dispersion in mepp amplitudes, we estimate that approximately 1.8 x 10(6) mepps occurred in this time. If 0.1 mM La3+ was applied, or if 1 mM La3+ was removed when mean value of r was near its peak, then mean value of r remained high for at least 1 h and approximately 4 x 10(6) mepps occurred. All these mepp counts exceed the 0.7 x 10(6) quanta stored in resting nerve terminals. When 1 or 2 mM La3+ was applied at 3-5 degrees C, mean value of r rose to peak values of 50-700/s and then fell to 20-200/s after 20-30 min. If the La3+ was then removed, mean value of r declined approximately 50% over the next hour; approximately 0.7 x 10(6) mepps occurred at the junctions treated with 1 mM La3+, and their terminals still contained about 69% of their vesicles. Thus, vesicles can be recycled at 3-5 degrees C. Millimolar concentrations of La3+ reduced the mepp amplitude by 70-80% at both temperatures and abolished almost completely the depolarization produced by bath applied acetylcholine or carbachol. These effects were reversible.

Effect of alpha-latrotoxin on the frog neuromuscular junction at low temperature.

1. alpha-Latrotoxin (alpha-LTx) was applied to frog cutaneous pectoris muscles bathed at 1-3 degrees C in either Ringer solution, Ca2+-free Ringer solution with 1 mM-EGTA and 4 mM-Mg2+ or Ringer solution plus 4 mM-Mg2+, and its effects on miniature end-plate potential (MEPP) frequency, nerve terminal ultrastructure and uptake of horseradish peroxidase (HRP) were studied. 2. Large concentrations (2 micrograms/ml) of alpha-LTx increased MEPP rates to levels above 100/s at all junctions, but the time course of the increases depended upon the divalent cation content of the bathing solution. However, similar numbers of MEPPs (0.3-0.7 x 10(6] were recorded at all junctions during 2 h of secretion. 3. Nerve terminals exposed to alpha-LTx for 2 h lost 60-75% of their synaptic vesicles and were swollen; their presynaptic membranes were deeply infolded and they often contained many large vesicular structures. Terminals in Ringer solution retained the largest number of synaptic vesicles; terminals in Ringer solution plus Mg2+ swelled the least and contained the largest number of coated vesicles. The average number of synaptic vesicles lost was approximately equal to the average number of MEPPs recorded. 4. Few vesicles became loaded with HRP when this extracellular tracer was present in the bathing solution and the muscles were fixed near the peak of secretion. 5. When the terminals were warmed to 20 degrees C, those in the Ca2+-free solution with Mg2+ secreted additional quanta and lost almost all their residual vesicles; those in Ringer solution without Mg2+ secreted few additional quanta and retained most of their residual vesicles. 6. These results suggest that recycling was blocked at these terminals and that for each quantum secreted a vesicle became permanently incorporated into the axolemma.

Effects of black widow spider venom and Ca2+ on quantal secretion at the frog neuromuscular junction.

A modification of the classical procedure of fluctuation analysis is used to measure the waveform, w(t), mean amplitude, (h), and mean rate of occurrence, (r), of miniature endplate potentials (MEPPs) at frog cutaneous pectoris neuromuscular junctions treated with black widow spider venom (BWSV). MEPP parameters are determined from the power spectrum of the fluctuating potential and the second (variance), third (skew), and fourth semi-invariants (cumulants) of high-pass-filtered records of the potential. The method gives valid results even when the mean potential undergoes slow changes unrelated to MEPPs and when the MEPP rate is not stationary; it detects changes in the distribution of MEPP amplitudes and corrects for the nonlinear summation of MEPPs. The effects of Ca2+ on BWSV-induced secretion are studied in detail. When Ca2+ is absent, the power spectrum of the fluctuations is shaped like the spectrum of w(t) and secretion is quasi-stationary; (r) rises smoothly to peak values of approximately 1,500/s and then quickly subsides to levels near 10/s. Many relatively small and some "giant" MEPPs occur at the ends of the experiments, and the distribution of MEPP amplitudes broadens. When the effects of this broadening are corrected for, we find that approximately 0.7 X 10(6) MEPPs occurred during the 30 min of intense secretion. Since BWSV depletes nerve terminals of their quanta of transmitter and their synaptic vesicles, this figure is an upper limit for the quantal store in a resting terminal. When Ca2+ is present, the noise spectrum deviates from the spectrum of w(t) and secretion is nonstationary; (r) rises to similar peak values but is sustained at levels near 400/s for up to an hour and at least 1.5 X 10(6) quanta are secreted within this period. Thus, the quantal store must have turned over at least twice under this condition. Data previously obtained at junctions treated with La3+ are corrected for nonlinear summation and for the distribution of MEPP amplitudes. The two corrections roughly compensate each other, and the corrected results confirm the previous conclusion that the number of quanta secreted from La3+-treated terminals during 1 h is not strongly dependent upon the extracellular concentration of Ca2+; approximately 2 X 10(6) quanta are released even when Ca2+ is absent.

Fluctuation analysis of nonideal shot noise. Application to the neuromuscular junction.

Procedures are described for analyzing shot noise and determining the waveform, w(t), mean amplitude, (h), and mean rate of occurrence, (r), of the shots under a variety of nonideal conditions that include: (a) slow, spurious changes in the mean, (b) nonstationary shot rates, (c) nonuniform distribution of shot amplitudes, and (d) nonlinear summation of the shots. The procedures are based upon Rice's (1944. Bell Telephone System Journal. 23: 282-332) extension of Campbell's theorem to the second (variance), lambda 2, third (skew), lambda 3, and fourth, lambda 4, semi-invariants (cumulants) of the noise. It is shown that the spectra of lambda 2 and lambda 3 of nonstationary shot noise contain a set of components that are proportional to (r) and arise from w(t), and a set of components that are independent of (r) and arise from the temporal variations in r(t). Since the latter components are additive and are limited by the bandwidth of r(t), they can be removed by appropriate filters; then (r) and (h) can be determined from the lambda 2 and lambda 3 of the filtered noise. We also show that a factor related to the ratio (lambda 3)2/(lambda 2)(lambda 4) monitors the spread in the distribution of shot amplitudes and can be used to correct the estimates of (r) and (h) for the effects of that spread, if the shape of the distribution is known and if r(t) is stationary. The accuracy of the measurements of lambda 4 is assessed and corrections for the effects of nonlinear summation of lambda 2, lambda 3, and lambda 4 are derived. The procedures give valid results when they are used to analyze shot noise produced by the (linear) summation of simulated miniature endplate potentials, which are generated either at nonstationary rates or with a distribution of amplitudes.

Miniature endplate potential frequency and amplitude determined by an extension of Campbell's theorem.

A method based upon an extension of Campbell's theorem is used to measure the amplitude, waveform, and frequency of occurrence of miniature endplate potentials (mepps) at rapidly secreting neuromuscular junctions of frog cutaneous pectoris muscles. Measurements of the variance, skew, and power spectrum of the fluctuations in membrane potential are used to deduce the mepp parameters. These estimates of mepp amplitude and frequency are insensitive to slow drifts in membrane potential that preclude the conventional application of Campbell's theorem, which uses the mean and variance. The new method becomes unreliable at high mepp frequencies because the distribution of the values of membrane potential approaches a Gaussian thereby reducing the accuracy of skew measurements. Frequencies approaching 10(4) s-1 can be measured, however, if the data are high-pass filtered. The method has been tested with computer simulated data and applied to junctions exposed to La3+; the effects of Ca2+ on the La3+-induced secretion have been explored. Some muscles were fixed after treatment with La3+, and changes in nerve terminal ultrastructure were assessed by morphometric analysis of electron micrographs. Horseradish peroxidase was used to obtain information about vesicle recycling.

Post-tetanic potentiation of acetylcholine release at the frog neuromuscular junction develops after stimulation in Ca2+-free solutions.

At many synapses, previous activity increases the amount of transmitter released by a single action potential. This potentiation of transmitter release is usually attributed to the local accumulation of the calcium ions that cross the axolemma during an action potential. We found that potentiated transmitter release can be observed at frog neuromuscular junctions after periods of repetitive stimulation in Ca2+-free solutions, if Ca2+ is restored after the tetanus. Potentiation is greater and more prolonged, the lower the level of extracellular K+. This component of potentiation may be due to Ca2+ that accumulates within the terminal in exchange for intracellular Na+.

Ca2+-dependent recycling of synaptic vesicles at the frog neuromuscular junction.

Frog cutaneous pectoris muscles were treated with low doses of crude black widow spider venom (BWSV) or purified alpha-latrotoxin, and neuromuscular transmission, quantal secretion, changes in ultrastructure and uptake of horseradish peroxidase (HRP) were studied. When these agents were applied to muscles bathed in a Ca2+-free solution with 1 mM EGTA and 4 mM Mg2+, the rate of quantal secretion rose to high levels but quickly subsided; neuromuscular transmission was totally and irreversibly blocked within 1 h. The terminals became swollen and were depleted of vesicles; HRP was not taken up. When BWSV was applied to other muscles bathed in a solution with 1.8 mM Ca2+ and 4 mM Mg2+, the rate of secretion rose to high levels and then declined to intermediate levels that were sustained throughot the hour of exposure. Neuromuscular transmission was blocked in fewer than 50% of these fibers. The ultrastructure of these terminals was normal and they contained large numbers of synaptic vesicles. If HRP had been present, most of the synaptic vesicles were labeled with reaction product. These observations suggest that Ca2+ plays an important role in endocytosis at the frog neuromuscular junction.

Tests of an electrostatic screening hypothesis of the inhibition of neurotransmitter release by cations at the frog neuromuscular junction.

We have investigated an electrostatic screening hypothesis of cationic inhibition of quantal release at the neuromuscular junction of the frog (Rana pipiens). According to this hypothesis, increasing the extracellular concentration of an inhibitory cation reduces the quantal content (m) of the end-plate potential by reducing the ability of negative surface charge to attract Ca2+ to the external surface of the presynaptic membrane. The inhibitory power of various cations should depend only on their net ionic charge and should increase strongly with increasing charge. We have demonstrated, in Ringer's solutions containing modified concentrations of Na+, Ca+, and Mg2+, that at fixed concentrations of Ca2+ and Na+ (a) the dependence of m on [Mg2+]0 is satisfactorily accounted for by electrostatic theory and (b) the dependence of m on the univalent cation concentration of the modified Ringer's solution is satisfactorily predicted from the Mg2+ inhibition of m. (Glucosamine or arginine was used to replace a fraction of the Na+ content of Ringer's solution in the latter experiments.) These results are consistent with electrostatic screening actions of Mg2+ and univalent cations in the inhibition of m. We have also re-examined the inhibition of m caused by the addition to Ringer's solution of two trace concentration divalent cations, Mn2+ and Sr2+. Our data suggest that the inhibition of m by Sr2+ at high quantal contents may also be due to surface charge screening, while the potent inhibitory actions of Mn2+ may be due to its ability to bind negative surface charge.

Freeze-fracture studies of frog neuromuscular junctions during intense release of neurotransmitter. III. A morphometric analysis of the number and diameter of intramembrane particles.

The intramembrane particles on the presynaptic membrane and on the membrane of synaptic vesicles were studied at freeze-fractured neuromuscular junctions of the frog. The particles on the P face of the presynaptic membrane belong to two major classes: small particles with diameters less than 9 nm and large particles with diameters between 9 and 13 nm. In addition, there were a few extralarge particles with diameters greater than 13 nm. Indirect stimulation of the muscle, or the application of black widow spider venom, decreased the concentration of small particles on the presynaptic membrane but did not change the concentration of large particles. Three similar classes of particles were found on the P face of the membrane of the synaptic vesicles. The concentrations of large and extralarge particles on the vesicle membrane were comparable to the concentrations of these particles on the presynaptic membrane, whereas the concentration of small particles on the vesicle membrane was less than than the concentration of small particles on the presynaptic membrane. These results are compatible with the idea that synaptic vesicles fuse with the presynaptic membrane when quanta of transmitter are released. However, neither the large nor the extralarge particles on the P face of the presynaptic membrane can be used to trace the movement of vesicle membrane that has been incorporated into the axolemma.

Freeze-fracture studies of frog neuromuscular junctions during intense release of neurotransmitter. I. Effects of black widow spider venom and Ca2+-free solutions on the structure of the active zone.

Black widow spider venom (BWSV) was applied to frog nerve-muscle preparations bathed in Ca2+-containing, or Ca2+-free, solutions and the neuromuscular junctions were studied by the freeze-fracture technique. When BWSV was applied for short periods (10-15 min) in the presence of Ca2+, numerous dimples (P face) or protuberances (E face) appeared on the presynaptive membrane and approximately 86% were located immediately adjacent to the double rows of large intramembrane particles that line the active zones. When BWSV was applied for 1 h in the presence of Ca2+, the nerve terminals were depleted of vesicles, few dimples or protuberances were seen, and the active zones were almost completely disorganized. The P face of the presynaptic membrane still contained large intramembrane particles. When muscles were soaked for 2-3 h in Ca2+-free solutions, the active zones became disorganized, and isolated remnants of the double rows of particles were found scattered over the P face of the presynaptic membrane. When BWSV was applied to these preparations, dimples or protuberances occurred almost exclusively alongside disorganized active zones or alongside dispersed fragments of the active zones. The loss of synaptic vesicles from terminals treated with BWSV probably occurs because BWSV interferes with the endocytosis of vesicle membrane. Therefore, we assume that the dimples or protuberances seen on these terminals identify the sites of exocytosis, and we conclude that exocytosis can occur mostly in the immediate vicinity of the large intramembrane particles. Extracellular Ca2+ seems to be required to maintain the grouping of the large particles into double rows at the active zones, but is not required for these particles to specify the sites of exocytosis.

Freeze-fracture studies of frog neuromuscular junctions during intense release of neurotransmitter. II. Effects of electrical stimulation and high potassium.

Frog cutaneous pectoris nerve muscle preparations were studied by the freeze-fracture technique under the following conditions: (a) during repetitive indirect stimulation for 20 min, 10/s; (b) during recovery from this stimulation; and (c) during treatment with 20 mM K+. Indirect stimulation causes numerous dimples or protuberances to appear on the presynaptic membrane of nerve terminal, and most are located near the active zones. Deep infoldings of the axolemma often develop between the active zones. Neither the number nor the distribution of dimples, protuberances, of infoldings changes markedly during the first minute of recovery. The number of dimples, protuberances, and infoldings is greatly reduced after 10 min of recovery. Since endocytosis proceeds vigorously during the recovery periods, we conclude that endocytosis occurs mostly at the active zones, close to the sites of exocytosis. 20 mM K+ also causes many dimples or protuberances to appear on the axolemma of the nerve terminal but they are distributed almost uniformly along the presynaptic membrane. Experiments with horseradish peroxidase (HRP) show that recycling of synaptic vesicles occurs in 20 mM K+. This recycling is not accompanied by changes in the number of coated vesicles. Since both exocytosis and endocytosis occur in 20 mM K+, it is difficult to account for this unique distribution. However, we suggest that K+ causes dimples or protuberances to appear between the active zones because it activates latent sites of exocytosis specified by small numbers of large intramembrane particles located between active zones. The activation of latent release sites may be related to the complex effects that K+ has on the quantal release of neurotransmitter.

Action of black widow spider venom on quantized release of acetylcholine at the frog neuromuscular junction: dependence upon external Mg2+.

Black widow spider (Latrodectus tredecimguttatus) venom (BWSV) increases several hundredfold the frequency of occurrance of minature end-plate potentials (Fmepp) at frog neuromuscular junctions bathed in Ringer's solutions containing either Ca2+ of Mg2+, but it has little effect on Fmepp at junctions bathed in modified Ringer's solution containing 1--2 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) but no Ca2+ or Mg2+. When Mg2+ is added to preparations that have been treated with BWSV in the modified solution, Fmepp increases exponentially with time. Fmepp falls again to low values when the Mg2+ is removed. The rate constant of the exponential rise is proportional to [Mg2+]o in the range 1--4 mM, and the threshold [Mg2+]o is 0.1--0.5 mM. Increasing the K+ concentration of the bathing solution decreases the ability of Mg2+ to increase Fmepp. Addition of Ca2+, Co2+, Mn2+, or Zn2+ also leads to a large increase in Fmepp. These results are consistent with the possibility that BWSV increases the permeability of the nerve terminal to divalent cations. BWSV can, however, increase Fmepp in hypertonic solutions in the absence of external divalent cations. This result suggests that the effects of BWSV on the nerve terminal may not be confined to increasing the permeability of the plasma-lemma.