Individual Boutons Research Articles

Pharmacological dissection of calcium channel subtype-related components of strontium inflow in large mossy fiber boutons of mouse hippocampus.

Several subtypes of voltage-dependent calcium channels (VDCCs) are present in the presynaptic terminals. In the mammalian hippocampus, P/Q-, N-, and R- but not L-type VDCCs are involved in the fast transmitter release from large mossy fiber (MF) boutons, which are associated with CA3 pyramidal cell dendrites. We investigated whether L-type VDCCs are indeed absent in these large MF boutons. With the use of Sr2+ as the Ca2+ substitute, the stimulus-evoked Sr2+ increment (delta[Sr2+]pre) was evaluated fluorometrically. Delta[Sr2+]pre appeared to be proportional to Sr2+ inflow through VDCCs and was specifically attenuated by conventional VDCC subtype-selective antagonists. The P/Q-type selective omega-agatoxin IVA (AgTx(IVA)) blocked delta[Sr2+]pre with an IC50 of 28 nM and by 30-35% at its maximum effective concentration of 0.5 microM. The N-type selective omega-conotoxin GVIA (CgTx(GVIA)) blocked delta[Sr2+]pre with an IC50 of 15 nM and by 20-25% at its maximum effective concentration of 1 microM. The R-type selective SNX-482 blocked delta[Sr2+]pre with an IC50 of 79 nM and by 20-25% at its maximum effective concentration of 1 microM. The effects of these toxins did not overlap at their maximum effective concentrations and about 70-80% of delta[Sr +]pre was blocked by the simultaneous exposure to these toxins. delta[Sr2+]pre component that is resistant to AgTx(IVA), CgTx(IVA), and SNX-482 was significantly potentiated by an L-type agonist, (S)-(-)-Bay K8644, and attenuated by an L-type antagonist, nimodipine, suggesting that L-type VDCCs are present in large MF terminals. The L-type agonist, (+/-)-Bay K8644, also potentiated Sr2+ inflow into individual boutons identified as large MF boutons under confocal microscopy. Almost similar results were observed for Ca2+ inflow-dependent fluorescence increments. L-type VDCCs appear to be present in large MF boutons and mediate a substantial Ca2+ inflow into presynaptic terminals during action potentials.

Intraterminal Ca2+ concentration and asynchronous transmitter release at single GABAergic boutons in rat collicular cultures

Neurotransmitter release in response to a single action potential has a precise time course. A significant fraction of the releasable vesicles is exocytosed synchronously, within a few milliseconds after the arrival of an action potential. If repeatedly activated, stimulus-locked phasic synchronous release declines, but synaptic transmission can be maintained through tonic asynchronous transmitter release. The desynchronisation of release during repetitive activation is generally attributed to a build-up of intraterminal Ca2+ concentration. However, the precise relationship between presynaptic Ca2+ level and asynchronous release rate at small central synapses has remained unclear. Here we characterise this relationship for single GABAergic terminals in rat collicular cultures. In the presence of tetrodotoxin, inhibitory postsynaptic currents (IPSCs) and presynaptic Ca2+ transients were recorded in response to direct presynaptic depolarisation of individual boutons. Repetitive stimulation indeed resulted in a shift from phasic to asynchronous neurotransmitter release. A clear dominance of the asynchronous release mode was observed after 10 pulses. The steady-state asynchronous release rate showed a third-power dependency on the presynaptic Ca2+ concentration, which is similar to that of evoked release. The Ca2+ sensor for asynchronous release exhibited a high affinity for Ca2+ and was far from saturation. These properties of the Ca2+ sensor should make the asynchronous release very sensitive to any modification of presynaptic Ca2+ concentration, including those resulting from changes in presynaptic activity patterns. Thus, asynchronous release represents a powerful but delicately regulated mechanism that ensures the maintenance of appropriate inhibition when the readily releasable pool of vesicles is depleted.

Unitary Assembly of Presynaptic Active Zones from Piccolo-Bassoon Transport Vesicles

Recent studies indicate that active zones (AZs)—sites of neurotransmitter release—may be assembled from preassembled AZ precursor vesicles inserted into the presynaptic plasma membrane. Here we report that one putative AZ precursor vesicle of CNS synapses—the Piccolo-Bassoon transport vesicle (PTV)—carries a comprehensive set of AZ proteins genetically and functionally coupled to synaptic vesicle exocytosis. Time-lapse imaging reveals that PTVs are highly mobile, consistent with a role in intracellular transport. Quantitative analysis reveals that the Bassoon, Piccolo, and RIM content of individual PTVs is, on average, half of that of individual presynaptic boutons and shows that the synaptic content of these molecules can be quantitatively accounted for by incorporation of integer numbers (typically two to three) of PTVs into presynaptic membranes. These findings suggest that AZs are assembled from unitary amounts of AZ material carried on PTVs.

Intraterminal Ca2+ concentration and asynchronous transmitter release at single GABAergic boutons in rat collicular cultures.

Neurotransmitter release in response to a single action potential has a precise time course. A significant fraction of the releasable vesicles is exocytosed synchronously, within a few milliseconds after the arrival of an action potential. If repeatedly activated, stimulus-locked phasic synchronous release declines, but synaptic transmission can be maintained through tonic asynchronous transmitter release. The desynchronisation of release during repetitive activation is generally attributed to a build-up of intraterminal Ca2+ concentration. However, the precise relationship between presynaptic Ca2+ level and asynchronous release rate at small central synapses has remained unclear. Here we characterise this relationship for single GABAergic terminals in rat collicular cultures. In the presence of tetrodotoxin, inhibitory postsynaptic currents (IPSCs) and presynaptic Ca2+ transients were recorded in response to direct presynaptic depolarisation of individual boutons. Repetitive stimulation indeed resulted in a shift from phasic to asynchronous neurotransmitter release. A clear dominance of the asynchronous release mode was observed after 10 pulses. The steady-state asynchronous release rate showed a third-power dependency on the presynaptic Ca2+ concentration, which is similar to that of evoked release. The Ca2+ sensor for asynchronous release exhibited a high affinity for Ca2+ and was far from saturation. These properties of the Ca2+ sensor should make the asynchronous release very sensitive to any modification of presynaptic Ca2+ concentration, including those resulting from changes in presynaptic activity patterns. Thus, asynchronous release represents a powerful but delicately regulated mechanism that ensures the maintenance of appropriate inhibition when the readily releasable pool of vesicles is depleted.

Extracellular Ca 2+ Depletion Contributes to Fast Activity-Dependent Modulation of Synaptic Transmission in the Brain

Synaptic activation is associated with rapid changes in intracellular Ca 2+, while the extracellular Ca 2+ level is generally assumed to be constant. Here, using a novel optical method to measure changes in extracellular Ca 2+ at high spatial and temporal resolution, we find that brief trains of synaptic transmission in hippocampal area CA1 induce transient depletion of extracellular Ca 2+. We show that this depletion, which depends on postsynaptic NMDA receptor activation, decreases the Ca 2+ available to enter individual presynaptic boutons of CA3 pyramidal cells. This in turn reduces the probability of consecutive synaptic releases at CA3−CA1 synapses and therefore contributes to short-term paired-pulse depression of minimal responses. This activity-dependent depletion of extracellular Ca 2+ represents a novel form of fast retrograde synaptic signaling that can modulate glutamatergic information transfer in the brain.

Differential Regulation of Active Zone Density during Long-Term Strengthening ofDrosophilaNeuromuscular Junctions

In this study we established a transgenic Ca2+ imaging technique in Drosophila that enabled us to target the Ca2+ sensor protein yellow Cameleon-2 specifically to larval neurons. This noninvasive method allowed us to measure evoked Ca2+ signals in presynaptic terminals of larval neuromuscular junctions (NMJs). We combined transgenic Ca2+ imaging with electrophysiological recordings and morphological examinations of larval NMJs to analyze the mechanisms underlying persistently enhanced evoked vesicle release in two independent mutants. We show that persistent strengthening of junctional vesicle release relies on the recruitment of additional active zones, the spacing of which correlated with the evoked presynaptic Ca2+ dynamics of individual presynaptic terminals. Knock-out mutants of the postsynaptic glutamate receptor (GluR) subunit DGluR-IIA, which showed a reduced quantal size, developed NMJs with a smaller number of presynaptic boutons but a strong compensatory increase in the density of active zones. This resulted in an increased evoked vesicle release on single action potentials and larger evoked Ca2+ signals within individual boutons; however, the transmission of higher frequency stimuli was strongly depressed. A second mutant (pabp(P970)/+), which showed enhanced evoked vesicle release triggered by elevated subsynaptic protein synthesis, developed NMJs with an increased number of presynaptic boutons and active zones; however, the density of active zones was maintained at a value typical for wild-type animals. This resulted in wild-type evoked Ca2+ signals but persistently strengthened junctional signal transmission. These data suggest that the consolidation of strengthened signal transmission relies not only on the recruitment of active zones but also on their equal distribution in newly grown boutons.

Inter-bouton variability of synaptic strength correlates with heterogeneity of presynaptic Ca(2+) signals.

The elevation of presynaptic calcium concentration is a crucial step in excitation-secretion coupling. However, the amplitudes of action-potential-induced presynaptic calcium transients can display high variability among different terminals. The aim of this study was to clarify whether, at individual boutons, synaptic strength correlates with the average amplitude of presynaptic calcium transients. Low-density collicular cultures were loaded with the calcium indicator Oregon Green bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) 1. Action potentials were blocked with tetrodotoxin. Presynaptic terminals were identified with FM4-64, a use-dependent vesicle marker. Presynaptic calcium influx was elicited by a focal electrical stimulation of single boutons. Whole cell patch-clamp and calcium imaging techniques were used to record GABAergic evoked inhibitory postsynaptic currents (eIPSCs) and presynaptic fluorescence changes in the stimulated terminal. To make the eIPSCs from different boutons comparable, they were normalized to the mean value of miniature IPSCs (mIPSCs) of the postsynaptic cell. Records from 47 boutons showed that eIPSCs varied between 0.5 and 3.0 and presynaptic calcium transients varied between 0.1 and 1.3. However, there was a strong correlation between the mean amplitudes of eIPSCs and presynaptic calcium responses. The eIPSC-[Ca(2+)](pre) relationship allows to use the amplitudes of presynaptic calcium transients as an indicator of release efficacy and, in a set of contacts made by one axon, to predict the relative impact of individual terminals.

Presynaptic and postsynaptic mechanisms underlie paired pulse depression at single GABAergic boutons in rat collicular cultures.

Paired pulse depression (PPD) is a common form of short-term synaptic plasticity. The aim of this study was to characterise PPD at the level of a single inhibitory bouton. Low-density collicular cultures were loaded with the Ca2+ indicator Oregon Green-1, active boutons were stained with RH414, and action potentials were blocked with TTX. Evoked IPSCs (eIPSCs) and presynaptic Ca2+ transients were recorded in response to direct presynaptic depolarisation of an individual bouton. The single bouton eIPSCs had a low failure rate (< 0.1), large average quantal content (3-6) and slow decay (tau(1) = 15 ms, tau(2) = 81 ms). The PPD of eIPSCs had two distinct components: PPD(fast) and PPD(slow) (tau = 86 ms and 2 s). PPD(slow) showed no dependence on extracellular Ca2+ concentration, or on the first eIPSC's failure rate or amplitude. Most probably, it reflects a release-independent inhibition of exocytosis. PPD(fast) was only observed in normal or elevated Ca2+. It decreased with the failure rate and increased with the amplitude of the first eIPSC. It coincided with paired pulse depression of the presynaptic Ca2+ transients (tau = 120 ms). The decay of the latter was accelerated by EGTA, which also reduced PPD(fast). Therefore, a suppressive effect of residual presynaptic Ca2+ on subsequent Ca2+ influx is considered the most likely cause of PPD(fast). PPD(fast) may also have a postsynaptic component, because exposure to a low-affinity GABA(A) receptor antagonist (TPMPA; 300 microM) counteracted PPD(fast), and asynchronous IPSC amplitudes were depressed for a short interval following an eIPSC. Thus, at these synapses, PPD is produced by at least two release-independent presynaptic mechanisms and one release-dependent postsynaptic mechanism.

Quantal Current Fields around Individual Boutons in Sympathetic Ganglia

The release of a quantum of neurotransmitter from an active zone of a bouton is accompanied by the flow of extracellular current that creates a potential field about the site of transmitter action beneath the bouton. It is shown theoretically that the density of the field at the peak of the quantal current gives rise to an extracellular potential that declines to values of less than 5 μV at 1.3 μm distance in the circumferential direction around the neuron and equally rapidly in the radial direction away from the neuron. A loose-patch electrode placed over a bouton distorts the quantal field about the bouton and calculations show that under current-clamp conditions, potentials of over 40 μV can be recorded with an electrode of tip diameter 2 μm, provided the separation between the tip and the neuron's surface is about 0.1 μm. Quantal release recorded from visualized boutons on rat monopolar pelvic ganglion cells with loose-patch electrodes is in agreement with the properties of the quantal potential field given in the theoretical analysis.

Depression of transmitter release at synapses in the rat superior cervical ganglion: the role of transmitter depletion

The characteristics of depression of the excitatory postsynaptic potential (EPSP) during a short train of impulses to the rat superior cervical ganglion (SCG) have been ascertained with the object of determining the relative contributions of transmitter depletion and autoreceptors to depression. Successive EPSPs in a short train were depressed after the first ( V o) up to about the fourth impulse when a steady-state depressed EPSP level ( V ss) was reached. V ss increased with the stimulation frequency between 1 and 30 Hz. V o recovered after a short train with a time constant of about 2.8 s in the frequency range from 5 to 30 Hz. In order to determine if depression was related to changes in calcium influx with successive impulses in the train, preganglionic boutons were loaded with the calcium indicator Oregon Green™ 488 BAPTA-1 and line scans taken through individual boutons with a confocal laser microscope. Successive calcium transients were of about the same amplitude in boutons during short trains of impulses at 5 Hz. The contribution of autoreceptors activated by the action of endogenously derived adenosine on the extent of depression of the EPSP during short trains was ascertained by blocking these receptors with 8-phenyltheophylline (10 μM). There was no change in the extent or time course of development of depression. Similar results were obtained with the opioid receptor antagonist naloxone (10 μM) and the adrenergic receptor antagonist yohimbine (10 μM). Factors, which increased the extent of transmitter release during a train, such as increasing the external calcium concentration from 0.8 to 2.5 mM, increased depression. Factors, which decreased the extent of transmitter release such as increasing the exogenous adenosine concentration between 1 and 200 μM decreased depression. These results are interpreted in terms of a model in which vesicles are mobilised by a calcium-dependent process from a store into an available pool of docked vesicles. Depletion of the docked vesicles during exocytosis then leads to depression of transmitter release during a train of impulses.

Estrogen increases synaptic connectivity between single presynaptic inputs and multiple postsynaptic CA1 pyramidal cells: a serial electron-microscopic study.

Dendritic spines are sites of the vast majority of excitatory synaptic input to hippocampal CA1 pyramidal cells. Estrogen has been shown to increase the density of dendritic spines on CA1 pyramidal cell dendrites in adult female rats. In parallel with increased spine density, estrogen has been shown also to increase the number of spine synapses formed with multiple synapse boutons (MSBs). These findings suggest that estrogen-induced dendritic spines form synaptic contacts with preexisting presynaptic boutons, transforming some previously single synapse boutons (SSBs) into MSBs. The goal of the current study was to determine whether estrogen-induced MSBs form multiple synapses with the same or different postsynaptic cells. To quantify same-cell vs. different-cell MSBs, we filled individual CA1 pyramidal cells with biocytin and serially reconstructed dendrites and dendritic spines of the labeled cells, as well as presynaptic boutons in synaptic contact with labeled and unlabeled (i.e., different-cell) spines. We found that the overwhelming majority of MSBs in estrogen-treated animals form synapses with more than one postsynaptic cell. Thus, in addition to increasing the density of excitatory synaptic input to individual CA1 pyramidal cells, estrogen also increases the divergence of input from individual presynaptic boutons to multiple postsynaptic CA1 pyramidal cells. These findings suggest the formation of new synaptic connections between previously unconnected hippocampal neurons.

Single-Cell Analysis of Drosophila Larval Neuromuscular Synapses

The neuromuscular system of Drosophila has been widely used in studies on synaptic development. In the embryo, the cellular components of this model system are well established, with uniquely identified motoneurons displaying specific connectivity with distinct muscles. Such knowledge is essential to analyzing axon guidance and synaptic matching mechanisms with single-cell resolution. In contrast, to date the cellular identities of the larval neuromuscular synapses are hardly established. It is not known whether synaptic connections seen in the embryo persist, nor is it known how individual motor endings may differentiate through the larval stages. In this study, we combine single-cell dye labeling of individual synaptic boutons and counterstaining of the entire nervous system to characterize the synaptic partners and bouton differentiation of the 30 motoneuron axons from four nerve branches (ISN, SNa, SNb, and SNd). We also show the cell body locations of 4 larval motoneurons (RP3, RP5, V, and MN13-Ib) and the types of innervation they develop. Our observations support the following: (1) Only 1 motoneuron axon of a given bouton type innervates a single muscle, while up to 4 motoneuron axons of different bouton types can innervate the same muscle. (2) The type of boutons which each motoneuron axon forms is likely influenced by cell-autonomous factors. The data offer a basis for studying the properties of synaptic differentiation, maintenance, and plasticity with a high cellular resolution.

Do synaptic rearrangements underlie compensatory reflex enhancement in spinal motoneurons after partial cell loss?

In adult cats, avulsion of a spinal ventral root induces retrograde cell death among the corresponding motoneurons and, also, enhanced monosynaptic reflexes ipsilaterally in the adjacent uninjured spinal cord segments. The present study investigates possible mechanisms behind this reflex potentiation. At 1–12 weeks after unilateral L7 ventral root avulsion, the L7 dorsal root ganglia were bilaterally injected with choleragenoid-HRP to light microscopically quantify the amount of HRP-labeled terminals in the motor nuclei of the lesioned L7 segment and adjacent intact L6+S1 segments. In addition, motoneuron synaptology and individual HRP-labeled boutons were analyzed electron microscopically. In the L7 segment, the loss of motoneurons at 12 weeks after ventral root avulsion was accompanied by a marked loss of HRP-labeled boutons in the corresponding ventral horn. In the L6/S1 segments, the monosynaptic reflex enhancement found ipsilaterally at 12 weeks postoperatively (mean 212%) was not accompanied by an increased HRP-labeling in the ventral horn (mean 109%), indicating that no sprouting or enlargement of the monosynaptic boutons had occurred. Ultrastructurally, the values for apposition length, total active site length, cross-sectional area, and mitochondrial density of the labeled boutons were also similar between the two sides. However, ipsilaterally the L6/S1 motoneurons exhibited an increased membrane covering by presumably excitatory boutons. The present results indicate that after partial cell death in a motoneuron pool the remaining motoneurons may undergo compensatory synaptic rearrangements leading to increased excitability and enhanced reflexes. Synapse 38:384–391, 2000. © 2000 Wiley-Liss, Inc.

Developmental increase in asynchronous GABA release in cultured hippocampal neurons

Developmental changes in GABAergic synaptic transmission were examined in cultured hippocampal neurons using patch-clamp recordings and Ca 2+ imaging. In paired recordings, tetanization of the presynaptic GABAergic neuron with 80 pulses at either 40 or 80 Hz was accompanied by tetanic depression of inhibitory postsynaptic responses. In neurons that had been cultured for more than two weeks, asynchronous inhibitory postsynaptic currents often appeared during the tetanus and continued for several seconds following stimulation. There was little asynchronous activity in neurons that had been cultured for shorter times. However, no age-related changes were observed in the amplitude of single synchronous inhibitory postsynaptic currents, paired-pulse depression or post-tetanic potentiation of inhibitory postsynaptic currents. Following equimolar replacement of extracellular Ca 2+ with strontium ions (Sr 2+), single autaptic inhibitory postsynaptic currents were depressed in amplitude and asynchronous inhibitory postsynaptic currents were present on the decaying phase. Sr 2+-induced asynchronous inhibitory postsynaptic currents showed no dependence on age in culture. Imaging of Ca 2+ in single GABAergic boutons was performed by including Fluo-3 in the patch pipette. During action potential firing induced by stimulating at 80 Hz for 1 s, intracellular calcium [Ca 2+] i increased rapidly in individual boutons. Following the stimulus, [Ca 2+] i decayed back to baseline within 10–15 s. The half-time of decay increased from 1.7±0.2 s at 15 days in vitro to 4.0±0.2 s at 30 days in vitro ( P<0.05), with a developmental profile that closely matched the increase in asynchronous inhibitory postsynaptic currents. We propose that the increase in tetanus-induced asynchronous GABA-release during the first month of synapse maturation in vitro is caused by a slowing of the Ca 2+-clearing mechanisms in the GABAergic boutons. This results in larger and more prolonged elevations of [Ca 2+] i during tetanic stimulation, which leads to enhanced asynchronous transmitter release. We propose that the results of this study demonstrate a potentially important aspect of synapse maturation during development, and also imply that GABA release is up-regulated in conditions of decreased Ca 2+ buffering and clearing.

Clathrin-Mediated Endocytosis near Active Zones in Snake Motor Boutons

We have used the activity-dependent probe FM1-43 with electron microscopy (EM) to examine endocytosis at the vertebrate nerve-muscle synapse. Preparations were fixed after very brief neural stimulation at reduced temperature, and internalized FM1-43 was photoconverted into an electron-dense reaction product. To locate the reaction product, we reconstructed computer renderings of individual terminal boutons from serial EM sections. Most of the reaction product was seen in 40-60 nm vesicles. All of the labeled vesicles were clathrin-coated, and 92% of them were located within 300 nm of the plasma membrane, suggesting that they had undergone little processing after retrieval from their endocytic sites. The vesicles (and by inference the sites) were not dispersed randomly near the plane of the membrane but instead were clustered significantly near active zones. Additional reaction product was found within putative macropinosomes; these appeared to form from deep membrane invaginations near active zones. Thus two mechanisms of endocytosis were evident after brief stimulation. Endocytosis near active zones is consistent with the existence of local exo/endocytic cycling pools. This mechanism also might serve to maintain alignment of active zones with postsynaptic folds during periods of activity when vesicular and plasma membranes are interchanged.

Role of cAMP Cascade in Synaptic Stability and Plasticity: Ultrastructural and Physiological Analyses of Individual Synaptic Boutons inDrosophilaMemory Mutants

Mutations of the genes rutabaga (rut) and dunce (dnc) affect the synthesis and degradation of cAMP, respectively, and disrupt learning in Drosophila. Combined ultrastructural analysis and focal electrophysiological recording in the larval neuromuscular junction revealed a loss of stability and fine tuning of synaptic structure and function in both mutants. Increased ratios of docked/undocked vesicles and poorly defined synaptic specializations characterized dnc synapses. In contrast, rut boutons possessed fewer, although larger, synapses with lower proportions of docked vesicles. At reduced Ca(2+) levels, decreased quantal content coupled with an increase in failure rate was seen in rut boutons and reduced pair-pulse facilitation were found in both rut and dnc mutants. At physiological Ca(2+) levels, strong enhancement, instead of depression, in evoked release was observed in some dnc and rut boutons during 10 Hz tetanus. Furthermore, increased variability of synaptic transmission, including fluctuation and asynchronicity of evoked release, paralleled an increase in synapse size variation in both dnc and rut boutons, which might impose problems for effective signal processing in the nervous system. Pharmacological and genetic studies indicated broader ranges of physiological alteration by dnc and rut mutations than either the acute effects of cAMP analogs or the available mutations that affect cAMP-dependent protein kinase (PKA) activity. This is consistent with previous reports of more severe learning defects in dnc and rut mutations than these PKA mutants and allows identification of the phenotypes involving long-term developmental regulation and those conferred by PKA.

Synaptic Transmission at Single Boutons in Sympathetic Ganglia.

Synaptic transmission has traditionally been studied at the level of the entire nerve terminal rather than at one of its constituent boutons. Autonomic ganglia provide preparations for recording from individual boutons, as well as for determining the calcium transients necessary for transmitter release at these boutons. The results suggest a new paradigm for synaptic transmission.

Do synaptic rearrangements underlie compensatory reflex enhancement in spinal motoneurons after partial cell loss?

In adult cats, avulsion of a spinal ventral root induces retrograde cell death among the corresponding motoneurons and, also, enhanced monosynaptic reflexes ipsilaterally in the adjacent uninjured spinal cord segments. The present study investigates possible mechanisms behind this reflex potentiation. At 1-12 weeks after unilateral L7 ventral root avulsion, the L7 dorsal root ganglia were bilaterally injected with choleragenoid-HRP to light microscopically quantify the amount of HRP-labeled terminals in the motor nuclei of the lesioned L7 segment and adjacent intact L6+S1 segments. In addition, motoneuron synaptology and individual HRP-labeled boutons were analyzed electron microscopically. In the L7 segment, the loss of motoneurons at 12 weeks after ventral root avulsion was accompanied by a marked loss of HRP-labeled boutons in the corresponding ventral horn. In the L6/S1 segments, the monosynaptic reflex enhancement found ipsilaterally at 12 weeks postoperatively (mean 212%) was not accompanied by an increased HRP-labeling in the ventral horn (mean 109%), indicating that no sprouting or enlargement of the monosynaptic boutons had occurred. Ultrastructurally, the values for apposition length, total active site length, cross-sectional area, and mitochondrial density of the labeled boutons were also similar between the two sides. However, ipsilaterally the L6/S1 motoneurons exhibited an increased membrane covering by presumably excitatory boutons. The present results indicate that after partial cell death in a motoneuron pool the remaining motoneurons may undergo compensatory synaptic rearrangements leading to increased excitability and enhanced reflexes.

Quantal unit populations at the Drosophila larval neuromuscular junction.

Focal extracellular recording at visualized boutons of the Drosophila larval neuromuscular junction was used to determine frequency and time course of the spontaneously occurring quantal events. When simultaneous intracellular recordings from the innervated muscle cell were made, more than one class of quantal event occurred at some of the individual boutons. "True" signals (arising at the bouton within the focal macropatch electrode) were often contaminated by additional signals generated outside the lumen of the focal electrode. Inclusion of these contaminating signals gave spuriously low values for relative amplitude, and spuriously high values for spontaneous quantal emission, for the synapses within the focal electrode. The contaminating signals, which appeared to be conducted along the subsynaptic reticulum surrounding the nerve terminals, generally were characterized by relatively small extracellular signals associated with normal intracellular events in the muscle fiber. From plots of simultaneous extracellular and intracellular recordings, the individual data points were classified according to the angles they subtended with the x axis (extracellular signal axis). Statistical procedures were developed to separate the true signals and contaminants with a high level of confidence. Populations of quantal events were found to be well described by Gaussian mixtures of two or three components, one of which could be characterized as the true signal population. Separation of signals from contaminants provides a basis for improving the estimates of quantal size and spontaneous frequency for the synapses sampled by the focal extracellular electrode.

Allatostatin modulates skeletal muscle performance in crustaceans through pre- and postsynaptic effects.

Allatostatins, originally identified in insects as peptide inhibitors of juvenile hormone biosynthesis, are regarded as potent inhibitory regulators of intestinal muscles in insects and crustaceans. However, accumulating data indicate that allatostatins might also be involved in modulation of skeletal neuromuscular events. We show that most ganglia of two isopod crustaceans (Idotea baltica and I. emarginata) contain pairs of large, allatostatin-immunoreactive motor neurons which supply several segmental muscles. Among them are the dorsal extensor muscles, of which some fibres receive immunoreactive, varicose innervation. We demonstrate, on identified muscle fibres, that allatostatin exerts a twofold inhibitory effect: it reduces contractions of single voltage-clamped fibres, and it decreases the amplitude of evoked excitatory junctional currents recorded from individual release boutons. No change in excitation-contraction threshold or in passive membrane parameters was observed. As the amplitude of miniature currents generated by spontaneously released single transmitter quanta was not changed, the inhibitory effect of the peptide on junctional currents must be of presynaptic origin. Supportive results were obtained on leg muscles of the crab Eriphia spinifrons, where allatostatin decreased evoked synaptic currents by reducing the mean number of transmitter quanta released by presynaptic depolarization without affecting the amplitudes of currents generated by single quanta. This effect of allatostatin was similar for two functionally different neurons, the slow and the fast closer excitor. The data show that allatostatin occurs in identified motor neurons of Idotea and exerts complementary pre- and postsynaptic modulatory effects which reduce muscle responses. Thus, allatostatin counteracts the effects of another neuropeptide, proctolin, which is also present in Idotea and causes potentiating effects on the same muscle fibres.