RESUMEN
Neurotransmitter release from different parts of frog motor nerve terminals is often non-uniform. There is a decrease in release efficacy from the distal regions of frog motor nerve terminal branches. Since release is thought to occur near the double arrays of large intramembranous particles that constitute the pre-synaptic active zones (AZs), we have examined quantitatively the proximal-distal distribution of AZ structure, using a novel freeze-fracture technique that produces replicas of large fractions of terminals, including the region of nerve entry. This enables us to know the proximal distal orientation of each branch. From 23 end-plates we have obtained fractures of 72 branches. For 27 of these branches we have obtained continuous fractures both greater than 25 microm in length and with sufficient information to determine their proximal distal polarity. Only a few of these branches showed a marked distal decrease in AZ length/unit length of terminal, while several junctions had short regions (5-10 microm), either proximally or distally, that exhibited amounts of AZ that were substantially greater or smaller than the mean value for that terminal branch. The terminal area, post-synaptic gutter width and nerve terminal width all exhibit some distal decline concomitant with the distal tapering of nerve terminal branches. AZ length tends to have the least decline compared to the other parameters. Thus, the vast majority of frog motor nerve terminal branches do not display a significant proximal-distal gradient in the amount of AZ structure / microm terminal length. The present data do not provide an obvious ultrastructural correlate for the distal decline in transmitter release that some authors have observed.
Asunto(s)
Técnica de Fractura por Congelación , Unión Neuromuscular/ultraestructura , Rana pipiens/anatomía & histología , Sinapsis/ultraestructura , Animales , Microscopía Electrónica , Músculo Esquelético/inervación , Músculo Esquelético/ultraestructuraRESUMEN
The orderly arrays of intramembranous particles (IMPs) found in the p-face of freeze-fracture replicas of the frog neuromuscular junction ('active zones') are believed to be involved in transmitter release. Some or all of the particles represent voltage-dependent Ca2+ channels. Since there is a great heterogeneity in the amount of transmitter released by different frog motor nerve terminals we sought to determine whether active zone (AZ) structure displayed a similar heterogeneity by using a novel freeze-fracture procedure providing large, intact replicas containing significant portions of motor nerve terminals from the cutaneous pectoris muscle of the frog, Rana pipiens. Using only junctions in which more than 50 AZs or more than 50 microm of nerve terminal were included in the fractures, we measured AZ length, AZ intramembranous particle density, terminal width at each AZ, space between AZs, the angle of AZ orientation with respect to the longitudinal axis of the nerve terminal, exposed pre-synaptic nerve terminal surface area and a calculated value for mean AZ length per unit terminal length. The analysis led to the following conclusions. There is an approximate 5-fold range in mean AZ length/micrometre terminal length. Terminal width is a good predictor of AZ length. Particle density does not vary significantly within a given AZ, nor between AZs from the same or different junctions. The distance between AZs is not related to AZ length, i.e. shorter AZs are no more or less likely to be closer to the adjacent AZ. The probability of release from any AZ on action potential invasion is small. If most of the IMPs are Ca2+ channels, either the probability of channel opening or the efficacy of triggering release is very low or both. That the variability in release efficacy in different terminals is much greater than ultrastructural variability in terminals suggests that regulation of release is dominated by physiological processes that do not have obvious ultrastructural correlates. On the other hand, the apparent excess of AZ relative to the number of vesicles released indicates that the amount and variability in amount of AZ is important in ways that need to be elucidated.
Asunto(s)
Técnica de Fractura por Congelación , Unión Neuromuscular/ultraestructura , Rana pipiens/anatomía & histología , Sinapsis/ultraestructura , Potenciales de Acción , Animales , Canales de Calcio/ultraestructura , Microscopía Electrónica , Placa Motora/ultraestructura , Músculo Esquelético/inervación , Músculo Esquelético/ultraestructuraRESUMEN
The neurotransmitter L-glutamate has been associated with a number of developmental events within the central nervous system including synaptogenesis and the refinement of topographically ordered neural maps. As a model for studying such events at the molecular level, we have examined the expression of glutamate and glutamate receptors in neurons that develop from P19 cells in response to retinoids. We report here that many P19-derived neurons do contain glutamate in secretory vesicles and that this glutamate appears to function as a neurotransmitter. The neurotransmitter GABA is also present in these cultures and both glutamate and GABA appeared to co-localize in some neuronal processes. Both neurotransmitters were released from the neurons in response to membrane depolarization. These neurons also express various glutamate receptor subunits including GluR1, GluR4 and NMDAR1 as detected by immunological methods. Using whole-cell patch-clamping, we have recorded spontaneous postsynaptic potentials which increase in both amplitude and frequency with time in culture and which are sensitive to the glutamate antagonist kynurenic acid Thus, P19-derived neurons mature in culture and form electrically active neural networks involving glutamate and glutamate receptors.
Asunto(s)
Ácido Glutámico/fisiología , Neuronas/fisiología , Animales , Northern Blotting , Diferenciación Celular , Línea Celular , Electrofisiología , Técnica del Anticuerpo Fluorescente Directa , Inmunohistoquímica , Neuronas/ultraestructura , Técnicas de Placa-Clamp , Ratas , Receptores de Glutamato/metabolismo , Receptores de Glutamato/fisiología , Sinapsis/fisiología , Sinapsis/ultraestructura , Ácido gamma-Aminobutírico/fisiologíaRESUMEN
Previous anatomical studies suggest that androgen regulates synapse elimination in the androgen-sensitive levator ani(LA) muscle of the rat. Androgen treatment beginning on postnatal day 7 (P7) prevents some of the normal loss of multiaxonal innervation in this muscle. The present study used physiological techniques to measure the number and size of LA motor units during the synapse elimination period in muscles from normals, and castrates treated with either testosterone propionate or oil. The number of increments in LA twitch tension as nerve stimulation intensity increased, a measure of the number of motor units, was the same at the end (P28) of synapse elimination as near the beginning (P7) of this process. This result indicates that motoneuronal cell death does not contribute to synapse elimination in the LA. Moreover, androgen during this period did not influence the number of LA motor units. In contrast, between P7 and P28, there was a dramatic decline in the size of LA motor units, as indicated by a decrease in the percentage of twitch or tetanus tension of individual motor units relative to the maximal twitch or tetanus tension of the whole muscle. In addition, androgen treatment of castrated males during this period prevented some of the normal decline in the size of LA motor units. Estimates of the number of inputs per LA muscle fiber derived from the number of LA motor units and their average size indicate that androgen maintains polyneuronal innervation in the LA muscle. This finding supports previous anatomical studies suggesting that androgen can prevent synapse elimination in this muscle. The strength of LA synapses was also examined by measuring the tetanus: twitch ratio of individual motor units and by measuring the safety margin of LA synapses. Both measurements indicated that the average strength of LA synapses increases during synapse elimination. Moreover, androgen appeared to spare synapses from elimination without increasing their strength, since androgen-treated muscles generally had larger motor units but the same mean tetanus:twitch ratio and safety margins as untreated LA muscles except at P28, when synapses in androgen-treated LA muscles had appreciably lower safety margins than normal. These results suggest that androgen regulates synapse elimination through a mechanism(s) independent of synaptic strength.
Asunto(s)
Andrógenos/fisiología , Neuronas Motoras/citología , Músculos/inervación , Sinapsis/fisiología , Animales , Femenino , Masculino , Neuronas Motoras/fisiología , Contracción Muscular , Fibras Nerviosas/fisiología , Pelvis , Ratas , Ratas Sprague-Dawley , Tiempo de ReacciónRESUMEN
This paper describes the extent of release and terminal variability among normal frog sartorius neuromuscular junctions and seeks physiological correlates for these differences. Terminal length varied over approximately a 10-fold range, quantal content and release per unit terminal length ("release efficacy") over much larger ranges. For purposes of comparison of different junctions, release efficacy in a Ringer's containing 0.25 mM Ca2+ was determined in all cases. In a Ringer's containing 0.1 mM Ca2+, tetanic stimulation causes a buildup of evoked release and of miniature endplate potential (mEPP) frequency. The mEPP frequency at the end of the tetanus is proportional to the evoked release level. Following the tetanus, the mEPP frequency declines in a multiexponential fashion, with the 2 longest decay phases, representing augmentation and posttetanic potentiation (PTP), both having time constants that are positively linearly correlated with the synaptic release efficacy. Longer or higher-frequency tetanic stimulation resulted in a longer time course of decay of mEPP frequency. In a Ca2(+)-free/EGTA Ringer's, tetanic stimulation causes no evoked release, but does lead to an increased mEPP frequency, presumably due to a buildup of free Ca2+ displaced from internal stores by the Na+ influx. Following the tetanus, the mEPP frequency declines to resting level with a time constant that is essentially the same for all junctions, regardless of their release efficacy in Ca2(+)-containing Ringer's. These findings indicate that stronger terminals have a greater influx of Ca2+ per unit length during action potential invasion, but that in the absence of external Ca2+, tetanic stimulation results in comparable release of Ca2+ from internal stores in all terminals and comparable accumulation of Ca2+ in some large compartment, the subsequent emptying of which determines the time course of PTP.
Asunto(s)
Contracción Muscular , Unión Neuromuscular/fisiología , Animales , Calcio/farmacología , Ácido Egtácico/farmacología , Estimulación Eléctrica , Soluciones Isotónicas/farmacología , Rana pipiens , Solución de Ringer , Sinapsis/fisiologíaRESUMEN
1. The calcium dependence of spontaneous transmitter release from nerve terminals of different lengths was examined at neuromuscular junctions in frog muscle. Miniature endplate potential (MEPP) frequency was positively correlated with the endplate potential (EPP) quantal content and was dependent on external Ca2+. The higher the resting MEPP frequency in a 0.25 mM-Ca2+ Ringer solution, the greater the dependence on external Ca2+. MEPP frequency in all terminals dropped to approximately the same low level in a Ca2(+)-free Ringer solution containing EGTA. This suggests that terminals with higher release levels have a larger Ca2+ influx at rest. 2. Several tests were done to try to characterize the mode of Ca2+ entry into resting terminals. omega-Conotoxin (omega-CgTx) blocked evoked release and reduced MEPP frequency, but not as effectively as zero Ca2(+)-EGTA Ringer solution. Some component of Ca2+ influx thus appears to enter through channels insensitive to omega-CgTx. Tetrodotoxin (TTX) did not affect MEPP frequency, indicating that the Ca2+ did not enter through TTX-sensitive Na+ channels that might be opening spontaneously at rest. Hyperpolarization of the terminal by reducing the K+ in the Ringer solution caused no consistent differences in MEPP frequency, suggesting that the Ca2+ influx is relatively insensitive to small changes in membrane potential around the resting level. Strong buffering of the Ringer solution with citrate, to overwhelm any differences in Ca2+ buffering within different junctional clefts, had no significant effect on the MEPP frequency. 3. Evidence that the Na(+)-Ca2+ exchanger helps set the internal Ca2+ level was obtained. Reduction of the Na+ concentration in the Ringer solution caused increases in MEPP frequency ranging from 6 to 440%. However, these changes were not correlated with resting MEPP frequency, hence differences in MEPP frequency probably are not the result of differences in Na(+)-Ca2+ exchanger function in terminals having a uniform Ca2+ leak. 4. Although MEPP frequency was generally correlated with quantal content, in subsets of junctions grouped according to their similar quantal contents, there was a positive correlation between MEPP frequency and terminal length. 5. In zero Ca2(+)-EGTA Ringer solution, the low residual MEPP frequency is independent of terminal length, even when MPP frequency is sharply increased by tetanic stimulation.
Asunto(s)
Calcio/farmacología , Unión Neuromuscular/metabolismo , Neurotransmisores/metabolismo , Animales , Calcio/metabolismo , Canales de Calcio/metabolismo , Proteínas Portadoras/metabolismo , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/ultraestructura , Rana pipiens , Canales de Sodio/metabolismo , Intercambiador de Sodio-Calcio , Sinapsis/fisiologíaRESUMEN
Periodic oscillations in miniature endplate potential (MEPP) frequency have been described at the frog neuromuscular junction. It is assumed that the periodic oscillations in MEPP frequency reflect cytosolic oscillations in intracellular Ca2+ concentration. In the course of a study related to describing the differences between weak and strong neuromuscular junctions by using the post-tetanic potentiation of MEPP frequency, we noted periodic oscillations in MEPP frequency in the first few minutes after a tetanus. The period of this oscillation (i.e. the time interval of one complete oscillation cycle) was inversely related to synaptic release efficacy, as measured by quantal content released per 100 microns of nerve terminal length. Junctions of high release efficacy have an oscillation period of 20 s or less whereas the oscillations in weaker junctions have periods of up to 60 s or longer. This relation is very similar during post-tetanic recovery in either a calcium containing Ringer solution or in a zero calcium-EGTA Ringer solution, indicating that external calcium is not necessary to express the phenomenon. We also found that the oscillations are apparent in resting junctions preceding a tetanus and that they are similar in period and show the same inverse relation to synaptic strength.
Asunto(s)
Calcio/fisiología , Placa Motora/fisiología , Unión Neuromuscular/fisiología , Animales , Relojes Biológicos , Ácido Egtácico , Contracción Muscular , Relajación Muscular , Periodicidad , Rana pipiens , Factores de TiempoRESUMEN
During a 2 1/2 year period, we studied 298 identified endplates from 40 sartorius muscles, correlating their morphology with their synaptic release properties. There is a remarkably large range in evoked and spontaneous quantal release levels when junctions are studied in a low-Ca2+ Ringer. This diversity in release efficacy persists when release is normalized to identified nerve terminal length. We also found that release was significantly larger in the winter (Dec-Feb) than in the spring and summer (Mar-Aug), suggesting seasonal regulation of release properties.
Asunto(s)
Neuronas Motoras/fisiología , Músculos/inervación , Unión Neuromuscular/fisiología , Rana pipiens/fisiología , Estaciones del Año , Animales , Neuronas Motoras/metabolismo , Rana pipiens/metabolismoRESUMEN
1. A monosynaptic, chemical synapse exists between two identified neurons in the subesophageal ganglia of the pulmonate mollusc, Achatina fulica. The snail undergoes a direct development, i.e., there is no intervening metamorphic period. The presynaptic (V2) and postsynaptic (RPr1) cells are two of the largest neurons found in the ganglia. The development of transmission at this synapse was studied from the last one-third of embryonic life to adulthood. 2. Synaptic transmission was studied by eliciting an action potential in V2 and recording the resultant excitatory postsynaptic potential (EPSP) in RPr1. In a train of repetitive stimuli, the ratio of the mean amplitude of the second EPSP to that of the first EPSP (EPSP2/EPSP1) is always greater than 1, indicating that short-term facilitation is present at all developmental ages studied. Following the initial short-term facilitation, embryonic synapses undergo a profound synaptic depression. Postembryonically there is a progressive increase in the amount of frequency facilitation with age, suggesting that the synapse shows a developmental trend towards an increased capacity for transmitter release. 3. In contrast to the progressive growth of frequency facilitation, the amplitude of the first EPSP in a series of responses (EPSP1) is not significantly related to age. 4. When transmitter release is reduced to approximately 25% of normal levels by a low-Ca2+/high-Mg2+ saline, the synaptic depression that is observed in the younger synapses disappears and is replaced by an adult-like frequency facilitation. 5. The adult synapse displays a phenomenon similar to posttetanic potentiation, which we refer to as the "retention of frequency facilitation." If an initial train of 150 stimuli at 0.2 Hz is followed by a second, identical train after an interval of 1 h, the postsynaptic response is greater during the second train than during the first. This phenomenon only becomes apparent in the second month after hatching, indicating that this separate synaptic plasticity develops at a different rate than does frequency facilitation.
Asunto(s)
Plasticidad Neuronal , Sinapsis/fisiología , Transmisión Sináptica , Animales , Electrólitos/análisis , Potenciales Evocados , Hemolinfa/análisis , Técnicas In Vitro , CaracolesRESUMEN
1. A quantal analysis of transmission at the identified molluscan synapse, V2-RPr1, was performed during development. The study was intended to determine the pre- and postsynaptic contributions to the marked changes in transmitter release described in the previous report. 2. The success of the quantal analysis was predicated on overcoming the problems associated with extending the quantal analysis technique to central synapses. This involved adopting the following strategies: 1) using a low-noise recording system coupled with electrical filtering; 2) establishing objective criteria for failures recognition; and 3) using three methods to determine the quantal content: amplitude histograms, failures analysis, and the coefficient of variation. 3. The correlation of the results obtained from an analysis of amplitude histograms and from failures analysis were highly significant (P less than 0.01) at all times studied. A similar significant correlation was observed between the failures method and the coefficient of variation methods. 4. The amplitude of the quantal unit declined progressively during development (range: 131-25 microV), in parallel with the decrease in the postsynaptic input resistance (range: 103-5 M omega). 5. At both frequencies of stimulation (0.02 and 0.2 Hz), there is an approximately 20-fold increase in quantal content over the period of the study. Frequency facilitation at the synapse is due to an increase in quantal content. 6. Possible structural correlates for the developmental increase in quantal content were discussed.
Asunto(s)
Sinapsis/fisiología , Transmisión Sináptica , Envejecimiento , Animales , Calcio/farmacología , Potenciales Evocados , Magnesio/farmacología , Modelos Neurológicos , Especificidad de Órganos , Teoría Cuántica , Caracoles/crecimiento & desarrollo , Especificidad de la Especie , Sinapsis/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacosRESUMEN
We have studied developmentally the electrotonic fate of synaptic potentials in an identified giant neuron of the Pulmonate mollusc Achatina fulica. Our experimental preparation enabled us to sample spontaneously occurring excitatory postsynaptic potentials (EPSPs) from embryogenesis onward, while concomitantly analyzing the postsynaptic membrane properties. We observe non-monotonic changes in the electrotonic attenuation of synaptic inputs which invade the somata. Our data suggest that non-monotonic changes in the specific membrane resistance can account for these observations.
Asunto(s)
Ganglios/fisiología , Potenciales de la Membrana , Transmisión Sináptica , Animales , Ganglios/citología , Ganglios/crecimiento & desarrollo , Moluscos , Neuronas/fisiología , Sinapsis/fisiologíaRESUMEN
We have sought physiological explanations for large differences in synaptic strength of different frog sartorius neuromuscular junctions, using the time course of posttetanic potentiation (PTP) of mEPP frequency as an indicator of the kinetics of Ca2+-metabolism in junctions of varying strengths. Results obtained in Ca2+ vs Ca2+-free/EGTA Ringers suggest that differences in the Ca2+-influx contribute to the physiological differences between strong and weak junctions.