Properties of short-term synaptic depression at larval neuromuscular synapses in wild-type and temperature-sensitive paralytic mutants of Drosophila

The larval neuromuscular synapse of Drosophila serves as an important model for genetic and molecular analysis of synaptic development and function. Further functional characterization of this synapse, as well as adult neuromuscular synapses, will greatly enhance the impact of this model system on our understanding of synaptic transmission. Here we describe a form of short-term synaptic depression observed at larval, but not adult, neuromuscular synapses and explore the underlying mechanisms. Larval neuromuscular synapses exhibited a form of short-term depression that was strongly dependent on stimulation frequency over a narrow range of low frequencies (0.1-1 Hz). This form of synaptic depression, referred to here as low-frequency short-term depression (LF-STD), results from an activity-dependent reduction in neurotransmitter release. However, in contrast to the predictions of depletion models, the degree of depression was independent of the initial level of neurotransmitter release over a range of extracellular calcium concentrations. This conclusion was confirmed in two temperature-sensitive (TS) paralytic mutants, cacophony and shibire, which exhibit reduced neurotransmitter release resulting from conditional disruption of presynaptic calcium channels and dynamin, respectively. Higher stimulation frequencies (40 or 60 Hz) produced two components of depression that appeared to include LF-STD as well as a more conventional component of short-term depression. These findings reveal novel properties of short-term synaptic depression and suggest that complementary genetic analysis of larval and adult neuromuscular synapses will further define the in vivo mechanisms of neurotransmitter release and short-term synaptic plasticity.     

Activity-dependent depression of local excitatory connections in the CA1 region of mouse hippocampus

The existence of recurrent excitatory synapses between pyramidal cells in the hippocampal CA1 region has been known for some time yet little is known about activity-dependent forms of plasticity at these synapses. Here we demonstrate that under certain experimental conditions, Schaffer collateral/commissural fiber stimulation can elicit robust polysynaptic excitatory postsynaptic potentials due to recurrent synaptic inputs onto CA1 pyramidal cells. In contrast to CA3 pyramidal cell inputs, recurrent synapses onto CA1 pyramidal cells exhibited robust paired-pulse depression and a sustained, but rapidly reversible, depression in response to low-frequency trains of Schaffer collateral fiber stimulation. Blocking GABA(B) receptors abolished paired-pulse depression but had little effect on low-frequency stimulation (LFS)-induced depression. Instead, LFS-induced depression was significantly attenuated by an inhibitor of A1 type adenosine receptors. Blocking the postsynaptic effects of GABA(B) and A1 receptor activation on CA1 pyramidal cell excitability with an inhibitor of G-protein-activated inwardly rectifying potassium channels had no effect on either paired-pulse depression or LFS-induced depression. Thus activation of presynaptic GABA(B) and adenosine receptors appears to have an important role in activity-dependent depression at recurrent synapses. Together, our results indicate that CA3-CA1 and CA1-CA1 synapses exhibit strikingly different forms of short-term synaptic plasticity and suggest that activity-dependent changes in recurrent synaptic transmission can transform the CA1 region from a sparsely connected recurrent network into a predominantly feedforward circuit.     

Presynaptic depolarization facilitates neurotrophin-induced synaptic potentiation

Neurotrophins have been proposed to participate in activity-dependent modifications of neuronal connectivity and synaptic efficacy. Preferential strengthening of active inputs requires restriction of putative neurotrophin-mediated synaptic potentiation to active synapses. Here we report that potentiation of synaptic efficacy by brain-derived neurotrophic factor (BDNF) is greatly facilitated by presynaptic depolarization at developing neuromuscular synapses. Brief depolarization in the presence of low-level BDNF results in a marked potentiation of both evoked and spontaneous synaptic transmission, whereas exposure to either BDNF or depolarization alone is without effect. This potentiation depends on the relative timing of depolarization and reflects an enhancement of transmitter secretion from the presynaptic neuron. Thus synapses made by active inputs may be selectively strengthened by secreted neurotrophins as part of activity-dependent refinement of developing connections or of mature synapses.     

Membrane ultrastructure of the giant synapse of the squidLoligo pealei

The ultrastructure of the ‘giant synapse’ of the stellate ganglion of the squid was studied with freeze-fracture and thin-sectioning techniques. A sheath of glial cells separates the pre- and post-synaptic axons. At intervals, round-topped processes of the postsynaptic axon pierce the sheath to contact the presynaptic axon. This area of synaptic contact is marked by a widened intercellular cleft containing electron-dense material and by a cluster of synaptic vesicles within the presynaptic cytoplasm. The number of synaptic vesicles in such clusters was greatly reduced by electrical stimulation of the synapse during fixation. Freeze-fracture reveals a roughly circular patch (0.3 μm diameter) of 10 nm particles on the cytoplasmic leaflet of the presynaptic membrane. A similar patch of particles lies on the external leaflet of the apposed postsynaptic membrane.The squid giant synapse thus consists of multiple small pre- and postsynaptic active zones where neurotransmitter is released from the presynaptic terminal and sensed by postsynaptic receptors. Comparison of the structure of these postsynaptic active zones with those at synapses where the transmitter or transmitter action is known suggests that the excitatory transmitter at this synapse is an amino acid.Presumptive gap junctions, marked by particles in the cytoplasmic leaflet, are found between small-diameter axons in the stellate ganglion but not at the giant synapse. Glial-cell membranes contain aggregates of particles and pits suggestive of gap junctions. The aggregates of pits are embedded within linear arrays of particles which somewhat resemble tight junctions.     

Identification of a cholinergic synapse in the giant fiber pathway of Drosophila using conditional mutations of acetylcholine synthesis

Temperature-sensitive mutations of the choline acetyltransferase (Cha) gene, which lead to reduced choline acetyltransferase (ChAT) activity and acetylcholine (ACh) levels, have been used in an attempt to identify the neurotransmitter at a chemical synapse in the giant fiber pathway (GFP) of Drosophila melanogaster. Prolonged incubation of adult mutant flies at non-permissive temperatures blocked the response of this pathway to brain stimulation, whereas shorter incubation times disturbed various parameters of the normal response. Even at permissive temperature subnormal responses were still evident. These defects in the giant fiber pathway's function suggest a specific cholinergic synapse within the pathway, the first synapse of this type implicated in Drosophila. When the function of this synapse was experimentally stressed, disruptions of GFP function paralleling decreased ChAT activity began to appear at enzyme levels estimated to be approximately 80% of wild-type.     

Drosophila CAKI/CMG protein, a homolog of human CASK, is essential for regulation of neurotransmitter vesicle release

Vertebrate CASK is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins. CASK is present in the nervous system where it binds to neurexin, a transmembrane protein localized in the presynaptic membrane. The Drosophila homologue of CASK is CAKI or CAMGUK. CAKI is expressed in the nervous system of larvae and adult flies. In adult flies, the expression of caki is particularly evident in the visual brain regions. To elucidate the functional role of CASK, we employed a caki null mutant in the model organism Drosophila melanogaster. By means of electrophysiological methods, we analyzed, in adult flies, the spontaneous and evoked neurotransmitter release at the neuromuscular junction (NMJ) as well as the functional status of the giant fiber pathway and of the visual system. We found that in caki mutants, when synaptic activity is modified, the spontaneous neurotransmitter release of the indirect flight muscle NMJ was increased, the response of the giant fiber pathway to continuous stimulation was impaired, and electroretinographic responses to single and continuous repetitive stimuli were altered and optomotor behavior was abnormal. These results support the involvement of CAKI in neurotransmitter release and nervous system function.     

GABA(B) receptors couple to potassium and calcium channels on identified lateral perforant pathway projection neurons

Activation of presynaptic GABA(B) receptors inhibits neurotransmitter release at most cortical synapses, at least in part because of inhibition of voltage-gated calcium channels. One synapse where this is not the case is the lateral perforant pathway synapse onto dentate granule cells in the hippocampus. The current study was conducted to determine whether the neurons that make these synapses express GABA(B) receptors that can couple to ion channels. Perforant pathway projection neurons were labeled by injecting retrograde tracer into the dorsal hippocampus. The GABA(B) receptor agonist baclofen (10 microM) activated inwardly rectifying potassium channels and inhibited currents mediated by voltage-gated calcium channels in retrogradely labeled neurons in layer II of the lateral entorhinal cortex. These effects were reversed by coapplication of the selective GABA(B) receptor antagonist CGP 55845A (1 microM). Equivalent effects were produced by 100 microM adenosine, which inhibits neurotransmitter release at lateral perforant pathway synapses. The effects of baclofen and adenosine on inward currents were largely occlusive. These results suggest that the absence of GABA(B) receptor-mediated presynaptic inhibition at lateral perforant pathway synapses is not simply due to a failure to express these receptors and imply that GABA(B) receptors can either be selectively localized or regulated at terminal versus somatodendritic domains.     

Activity-dependent elimination of neuromuscular synapses

At developing neuromuscular synapses in vertebrates, different motor axon inputs to muscle fibers compete for maintenance of their synapses. Competition results in progressive changes in synaptic structure and strength that lead to the weakening and loss of some inputs, a process that has been called synapse elimination. At the same time, a single input is strengthened and maintained throughout adult life, consistently recruiting muscle fibers to contract even at rapid firing rates. Work over the last decade has led to an understanding of some of the cell biological mechanisms that underlie competition and how these culminate in synapse elimination. We discuss current ideas about how activity modulates neuromuscular synaptic competition, how competition leads to synapse loss, and how these processes are modulated by cell-cell signaling. A common feature of competition at neuromuscular as well as CNS synapses is that temporally correlated activity seems to slow or prevent competition, while uncorrelated activity seems to trigger or enhance competition. Important questions that remain to be addressed include how patterns of motor neuron activity affect synaptic strength, what is the temporal relationship between changes in synaptic strength and structure, and what cellular signals mediate synapse loss. Answers to these questions will expand our understanding of the mechanisms by which activity edits synaptic structure and function, writing permanent changes in neural circuitry.     

Kainate receptor-dependent presynaptic modulation and plasticity

Kainate-type ionotropic glutamate receptors (KARs) distribute widely and heterogenously throughout the central nervous system (CNS). There is now increasing evidence showing that, in addition to conventional action to mediate postsynaptic excitation, KAR also exerts presynaptic action modulating the amount of transmitter release at certain synapses in the CNS. The mechanism and physiological function of presynaptic KARs have been studied most extensively at the hippocampal mossy fiber (MF)-CA3 synapse, one of the CNS regions where the highest density of KAR subunits is expressed. One unique feature of presynaptic KARs is that their activation modulates transmitter release bi-directionally; weak activation enhances glutamate release, while strong activation leads to inhibition. These findings may be explained by their possible ionotropic action leading to axonal depolarization, which in turn regulates several voltage-dependent channels involved in action potential-dependent Ca2+ entry processes. Furthermore, physiological activation of presynaptic KAR involves an activity-dependent process. Large frequency facilitation, a form of short-term plasticity characteristic of the MF-CA3 synapse, is mediated, at least partly, by presynaptic KAR. Bi-directional and activity-dependent regulation of transmitter release by kainate autoreceptors might have physiological significance in information processing in the hippocampus and other CNS regions, as well as its well-known pathological action contributing to epileptogenesis.     

Intramembrane organization of synapses in the lobster stretch receptor organ

Summary The intramembrane organization of axodendritic and neuromuscular synapses in the lobster stretch receptor organ was investigated by freeze-fracturing. Based on ultrastructural criteria which are known to be correlated with physiological properties, we identified three types of synapse: the inhibitory axodendritic, the inhibitory neuromuscular, and the excitatory neuromuscular synapse. Although these synapses have some features in common, each has a characteristic arrangement of intramembrane particles in both the presynaptic and postsynaptic membranes. All three have, in their presynaptic membranes, aggregates of P-face particles and associated depressions representing sites of synaptic vesicle exocytosis, features which together define active zones. However, in the inhibitory axodendritic synapse the P-face contains short ridges in this region. These ridges may occur singly or in pairs oriented in V-shaped configurations. The ridges are decorated with particles along their entire length. In the inhibitory neuromuscular synapse, no ridges are present. Clusters of particles are present, but they are scattered randomly over a large expanse of presynaptic membrane. In the excitatory neuromuscular synapse, isolated clusters of particles are associated with the P-face and are occasionally located on circular elevations of the membrane. The postsynaptic membrane also shows structural diversity in the three types of synapse. In the inhibitory axodendritic synapse, there is no apparent specialization. However, in the inhibitory neuromuscular synapse, P-face particles are arranged in double rows which are separated by particle-free strips of membrane. In the excitatory neuromuscular synapse, particles are confined to a narrow band that borders the synaptic cleft. This band is demarcated by a single intermittent strand of particles arranged in the direction of the long axis of the muscle fibre. Therefore, intramembrane specializations of both the presynaptic and postsynaptic membranes are sufficiently distinctive that three different types of synapse can be recognized.     

Excitatory potentials induced by stimulation of the inhibitory axon at the crustacean neuromuscular junction.

Synaptic events in a chloride-deficient condition were studied to elucidate functional aspects of presynaptic inhibitory synapses. The extracellular junctional potentials and nerve terminal potentials were concurrently recorded from a synaptic region. Inhibitory stimulation produced repetitive spikes on the inhibitory nerve terminal and then the excitatory nerve terminal, which resulted in the extracellular excitatory junctional potentials. Excitatory stimulation did not produce repetitive spikes on the inhibitory nerve terminal, indicating one-way signal transmission in this axo-axonal synapse from inhibitory to excitatory axon. The interval required for an inhibitory stimulation to produce the first response in the postsynaptic muscle membrane ranged widely from 10 to 800 msec. When gamma-aminobutyric acid (GABA, 1 times 10-minus 4 M) was added in these experimental conditions, the muscle membrane was transiently depolarized by about 10 mV. The action of GABA mimics that of the neurotransmitter at presynaptic inhibitory synapses. The experimental observations may be well explained by the concept of synapses on synapses, i.e., presynaptic inhibition, where the neurotransmitter may be GABA and chloride ions may be playing essential roles as in the case of postsynaptic inhibition.     

Presynaptic CB1 receptors regulate synaptic plasticity at cerebellar parallel fiber synapses

Endocannabinoids are potent regulators of synaptic strength. They are generally thought to modify neurotransmitter release through retrograde activation of presynaptic type 1 cannabinoid receptors (CB1Rs). In the cerebellar cortex, CB1Rs regulate several forms of synaptic plasticity at synapses onto Purkinje cells, including presynaptically expressed short-term plasticity and, somewhat paradoxically, a postsynaptic form of long-term depression (LTD). Here we have generated mice in which CB1Rs were selectively eliminated from cerebellar granule cells, whose axons form parallel fibers. We find that in these mice, endocannabinoid-dependent short-term plasticity is eliminated at parallel fiber, but not inhibitory interneuron, synapses onto Purkinje cells. Further, parallel fiber LTD is not observed in these mice, indicating that presynaptic CB1Rs regulate long-term plasticity at this synapse.     

Mechanisms of tonic, graded release: lessons from the vertebrate photoreceptor

The release of neurotransmitter via exocytosis is a highly conserved, fundamental feature of nervous system function. At conventional synapses, neurotransmitter release occurs as a brief burst of exocytosis triggered by an action potential. By contrast, at the first synapse of the vertebrate visual pathway, not only is the calcium-dependent release of neurotransmitter typically graded with respect to the presynaptic membrane potential, but release can be maintained throughout the duration of a sustained stimulus. The specializations that provide for graded and sustained release are not well-defined. However, recent advances in our understanding of basic synaptic vesicle dynamics and the calcium sensitivity of the release process at these and other central, glutamatergic neurons have shed some light on the photoreceptor's extraordinary abilities.     

Presynaptic plasticity at two giant auditory synapses in normal and deaf mice

Large calyceal synapses are often regarded as simple relay points, built for high-fidelity and high-frequency synaptic transmission and a minimal requirement for synaptic plasticity, but this view is oversimplified. Calyceal synapses can exhibit surprising activity-dependent developmental plasticity. Here we compare basal synaptic transmission and activity-dependent plasticity at two stereotypical calyceal synapses in the auditory pathway, the endbulb and the calyx of Held. Basal synaptic transmission was more powerful at the calyx than the endbulb synapse: the amplitude of evoked AMPA receptor-mediated excitatory postsynaptic currents (eEPSCs) was significantly greater at the calyx, as were the release probability, and the number of release sites. The quantal amplitude was smaller at the calyx, consistent with the smaller amplitude of spontaneous miniature EPSCs at this synapse. High-frequency trains of stimuli revealed that the calyx had a larger readily releasable pool of vesicles (RRP), less tetanic depression and less asynchronous transmitter release. Activity-dependent synaptic plasticity was assessed in congenitally deaf mutant mice ( dn/dn ). Previously we showed that a lack of synaptic activity in deaf mice increases synaptic strength at the endbulb of Held via presynaptic mechanisms. In contrast, we have now found that deafness does not affect synaptic transmission at the calyx synapse, as eEPSC and mEPSC amplitude, release probability, number of release sites, size of RRP, tetanic depression and asynchronous release were unchanged compared to normal mice. Synaptic transmission at the calyx synapse is more powerful and has less capacity for developmental plasticity compared to the endbulb synapse.     

Extracellular potassium and trasmitter release at the giant synapse of squid.

1. The effects of changes in extracellular K concentration, [K]0, on synaptic transmission were studied at the squid giant synapse with intracellular recording from the presynaptic terminal and post-synaptic axon. 2. The amplitudes of both the presynaptic spike and the e.p.s.p. varied inversely with [K]0. On the average, a 10 mV change in spike height was accompanied by a 3-1 mV change in e.p.s.p. amplitude. 3. The amplitude of the presynaptic spike after-hyperpolarization (AH) varied inversely with [K]0. On the average, increasing [K]0 resulted in a 20% change in e.p.s.p. amplitude per mV change in presynaptic spike AH. 4. Repetitive antidromic stimulation of the post-synaptic giant axon resulted in an exponential decline in the post-synaptic spike AH, a depolarization of the presynaptic membrane potential and a reduction in the AHs of presynaptic spikes. This suggests that the K which accumulates in the extracellular spaces around the post-synaptic axon also affects the presynaptic terminal. 5. Repetitive antidromic stimulation of the post-synaptic axon resulted in a reduction in the amplitude of e.p.s.p.s. elicted by stimulation of the presynaptic axon. The reduction in e.p.s.p. amplitude relative to the change in presynaptic spike AH was quantitatively close to the change produced by increasing [K]0, suggesting that the reduction in e.p.s.p. amplitude is due to the accumulation of extracellular K at the presynaptic terminal. 6. Repetitive stimulation of the presynaptic axon reduced the amplitudes of the e.p.s.p. and the presynaptic spike AH. On the average, a 1 mV change in presynaptic spike AH was accompanied by a 204% change in e.p.s.p. amplitude, suggesting that K accumulation may only contribute to a small extent, under these conditions, to the depression of transmitter release.     

Competitive Hebbian learning through spike-timing-dependent synaptic plasticity

Hebbian models of development and learning require both activity-dependent synaptic plasticity and a mechanism that induces competition between different synapses. One form of experimentally observed long-term synaptic plasticity, which we call spike-timing-dependent plasticity (STDP), depends on the relative timing of pre- and postsynaptic action potentials. In modeling studies, we find that this form of synaptic modification can automatically balance synaptic strengths to make postsynaptic firing irregular but more sensitive to presynaptic spike timing. It has been argued that neurons in vivo operate in such a balanced regime. Synapses modifiable by STDP compete for control of the timing of postsynaptic action potentials. Inputs that fire the postsynaptic neuron with short latency or that act in correlated groups are able to compete most successfully and develop strong synapses, while synapses of longer-latency or less-effective inputs are weakened.     

Phosphorylation reactions in activity-dependent synapse modification at the neuromuscular junction during development

We have studied developmental activity-dependent synapse diminution in both an in vitro tissue culture chamber system and at the intact rodent neuromuscular junction (nmj). In both types of preparations, pre- and postsynaptic alterations in synapse structure and function are produced by manipulations of thrombin (Thr) and protein kinase C (PKC) activity. An opposing postsynaptic effect of PKC and protein kinase A (PKA) action on the acetycholine receptor (AChR) can be shown in vitro with PKA stabilizing and PKC destabilizing the nmj synapses. In vivo studies of normal junctional maturation show that changes in axonal inputs and postsynaptic receptor cluster morphology occur, to a substantial degree, independently of one another. Presynaptic actions of PKA are involved in the activity dependent synapse modulation that can be demonstrated in vitro. Late in the elimination process, (>12 days in vivo ) the process becomes independent of PKC, implying that diverse, redundant mechanisms are involved in this important developmental process.     

Hippocampal CA1 synaptic plasticity as a gamma transfer function

The capacity of activity-dependent synaptic modification is essential in processing and storing information, yet little is known about how synaptic plasticity alters the input-output conversion efficiency at the synapses. In the adult mouse hippocampus in vivo, we carefully compared the input-output relationship, in terms of presynaptic activity levels versus postsynaptic potentials, before and after the induction of synaptic plasticity and found that synaptic plasticity led synapses to respond more robustly to inputs, that is, synaptic gain was increased as a function of synaptic activity with an expansive, power-law nonlinearity, i.e. conforming to the so-called gamma curve. In extreme cases, long-term potentiation and depression coexist in the same synaptic pathway with long-term potentiation dominating over long-term depression at higher levels of presynaptic activity. These findings predict a novel function of synaptic plasticity, i.e. a contrast-enhancing filtering of neural information through a gamma correction-like process.     

Activity-dependent regulation of inhibitory synaptic transmission in hippocampal neurons

Neural activity regulates the number and properties of GABAergic synapses in the brain, but the mechanisms underlying these changes are unclear. We found that blocking spike activity globally in developing hippocampal neurons from rats reduced the density of GABAergic terminals as well as the frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs). Chronic inactivity later in development led to a reduction in the mIPSC amplitude, without any change in GABAergic synapse density. By contrast, hyperpolarizing or abolishing spike activity in single neurons did not alter GABAergic synaptic inputs. Suppressing activity in individual presynaptic GABAergic neurons also failed to decrease synaptic output. Our results indicate that GABAergic synapses are regulated by the level of activity in surrounding neurons. Notably, we found that the expression of GABAergic plasticity involves changes in the amount of neurotransmitter in individual vesicles.     

Synaptic dynamics on different time scales in a parallel fiber feedback pathway of the weakly electric fish

Synaptic dynamics comprise a variety of interacting processes acting on a wide range of time scales. This enables a synapse to perform a large array of computations, from temporal and spatial filtering to associative learning. In this study, we describe how changing synaptic gain via long-term plasticity can act to shape the temporal filtering of a synapse through modulation of short-term plasticity. In the weakly electric fish, parallel fibers from cerebellar granule cells provide massive feedback inputs to the pyramidal neurons of the electrosensory lateral line lobe. We demonstrate a long-term synaptic enhancement (LTE) of these synapses that is biochemically similar to the presynaptic long-term potentiation expressed by parallel fibers in the mammalian cerebellum. Using a novel stimulation protocol and a simple modeling paradigm, we then quantify the changes in short-term plasticity during the induction of LTE and show that these changes can be explained by gradual changes in only one model parameter, that which is associated with the baseline probability of transmitter release. These changes lead to a shift in the spike frequency preference of the synapse, suggesting that long-term plasticity is not only involved in controlling the gain of the parallel fiber synapse, but also provides a means of controlling synaptic filtering over multiple time scales.