Long-lasting Enhancement Of Synaptic Transmission Research Articles

Modulation of Long-Term Potentiation by Gamma Frequency Transcranial Alternating Current Stimulation in Transgenic Mouse Models of Alzheimer's Disease.

Transcranial alternating current stimulation (tACS) is a neuromodulation procedure that is currently studied for the purpose of improving cognitive function in various diseases. A few studies have shown positive effects of tACS in Alzheimer’s disease (AD). However, the mechanism underlying tACS has not been established. The purpose of this study was to investigate the mechanism of tACS in five familial AD mutation (5xFAD) mouse models. We prepared twenty 4-month-old mice and divided them into four groups: wild-type mice without stimulation (WT-NT group), wild-type mice with tACS (WT-T group), 5xFAD mice without stimulation (AD-NT group), and 5xFAD mice with tACS (AD-T group). The protocol implemented was as follows: gamma frequency 200 μA over the bilateral frontal lobe for 20 min over 2 weeks. The following tests were conducted: excitatory postsynaptic potential (EPSP) recording, Western blot analysis (cyclic AMP response element-binding (CREB) proteins, phosphorylated CREB proteins, brain-derived neurotrophic factor, and parvalbumin) to examine the synaptic plasticity. The EPSP was remarkably increased in the AD-T group compared with in the AD-NT group. In the Western blot analysis, the differences among the groups were not significant. Hence, tACS can affect the long-lasting enhancement of synaptic transmission in mice models of AD.

BDNF induces in vivo long-lasting enhancement of synaptic transmission and structural reorganization at the hippocampal mossy fibers in a transcription and translation-independent manner.

Brain-derived neurotrophic factor (BDNF) is an essential product of protein synthesis with a prominent impact on brain signaling and synaptic plasticity. Exogenous application of this neurotrophin is able to induce long-term potentiation (LTP) in several brain structures such as the hippocampus along with increases in gene transcription and translation of proteins involved in functional and structural plasticity. In this regard, our previous studies have demonstrated that acute intrahippocampal administration of BDNF induces long-lasting enhancement of synaptic transmission at the mossy fibers projection (MF) accompanied by a structural reorganization at the CA3 hippocampus area. Thus, considering the non-canonical molecular mechanisms underlying MF-CA3-LTP and the high expression of this neurotrophin in the CA3 area, we wonder whether transcriptional and translational inhibition interferes with the persistence of the MF functional and structural synaptic plasticity elicited by BDNF in adult rats in vivo. Our results show that BDNF is able to induce a lasting potentiation of synaptic efficacy at the MF projection accompanied by a structural reorganization at the CA3 area in an mRNA synthesis and protein translation-independent manner. The present findings support the idea that BDNF is an essential plasticity related product, which is necessary and sufficient to induce and maintain functional and structural synaptic plasticity at the MF-CA3 pathway.

Paradoxical sleep: A vigilance state to gate long-term brain plasticity?

Memory consolidation is the process for long-term storage of information and protection against interferences. It has been proposed that long-term potentiation (LTP), the long-lasting enhancementof synaptic transmission, is a cellular model for memory consolidation. Since consolidation of several forms of memory is facilitated by paradoxical sleep (PS) we ask whether PS modulates the cellular and molecular pathways underlying LTP. The long-lasting form of LTP (L-LTP) is dependent on the activation of transcription factors, enzymatic cascades and the secreted neurotrophin BDNF. By using PS deprivation, immunohistochemistry and quantitative real-time polymerase chain reaction (qPCR), we showed that an increase in PS amount (produced by rebound in PS deprived rats) is able to up-regulate the expression level of transcription factors Zif268 and c-Fos as well as Arc and BDNF in the CA1 and CA3 areas of the hippocampus. Several studies involved these factors in dendritic protein synthesis and in long-term structural changes of synapses underlying L-LTP. The present study together with the work of others (Ribeiro et al., 2002) suggest that by this mechanism, a post-learning increase in PS quantity (post-learning PS window) could convert a transient form of LTP to L-LTP.

Vagus nerve stimulation induced long-lasting enhancement of synaptic transmission and decreased granule cell discharge in the hippocampal dentate gyrus of urethane-anesthetized rats

Vagus nerve stimulation (VNS) ameliorates deficits of hippocampal functions, such as contextual learning and memory, probably through direct modulation of neuronal activity. Previous studies showed that VNS enhanced excitatory synaptic transmission in the hippocampal CA3 area via activation of β-adrenergic receptors. However, effects of VNS on excitatory synaptic transmission and action potential (AP) discharge of granule cells (GCs) in the dentate gyrus have not been studied. Urethane-anesthetized rats were used to investigate whether VNS influences synaptic transmission efficacy at perforant path-GC synapses and population spike discharge in the dentate gyrus. Intermittent burst stimulation of the left vagus nerve (30Hz for 30s at an inter-train interval of 5min for 1h) significantly enhanced the perforant path-GC synaptic transmission efficacy in the dentate gyrus for at least 2h, indicating that VNS can induce a long-lasting enhancement of synaptic transmission in this area, similar to the situation observed in the CA3 area. In contrast, a 60-min period of VNS significantly reduced population spike amplitude (a parameter reflecting synchronized AP discharge of GCs) for a given excitatory postsynaptic potential. These findings suggest that acute VNS enhances the excitatory synaptic transmission and reduces synchronized AP discharge of GCs in the dentate gyrus. It is likely that enhancement of excitatory synaptic transmission and reduction of GC excitability contribute VNS treatment efficacy for learning deficits and intractable epilepsy, respectively.

Activity-Induced Long-Term Potentiation of Excitatory Synapses in Developing Zebrafish Retina In Vivo

Neural activity-induced long-term potentiation (LTP) of synaptic transmission is believed to be one of the cellular mechanisms underlying experience-dependent developmental refinement of neural circuits. Although it is well established that visual experience and neural activity are critical for the refinement of retinal circuits, whether and how LTP occurs in the retina remain unknown. Using in vivo perforated whole-cell recording and two-photon calcium imaging, we find that both repeated electrical and visual stimulations can induce LTP at excitatory synapses formed by bipolar cells on retinal ganglion cells in larval but not juvenile zebrafish. LTP induction requires the activation of postsynaptic N-methyl-D-aspartate receptors, and its expression involves arachidonic acid-dependent presynaptic changes in calcium dynamics and neurotransmitter release. Physiologically, both electrical and visual stimulation-induced LTP can enhance visual responses of retinal ganglion cells. Thus, LTP exists in developing retinae with a presynaptic locus and may serve for visual experience-dependent refinement of retinal circuits.

Facilitation of AMPA Receptor Synaptic Delivery as a Molecular Mechanism for Cognitive Enhancement

Author SummaryThe human brain contains trillions of neuronal connections, called synapses, whose pattern of activity controls all our cognitive functions. These synaptic connections are dynamic and constantly changing in their strength and properties, and this process of synaptic plasticity is essential for learning and memory. Alterations in synaptic plasticity mechanisms are thought to be responsible for multiple cognitive deficits, such as autism, Alzheimer's disease, and several forms of mental retardation. In this study, we show that synapses can be made more plastic using a small protein fragment (peptide) derived from a neuronal protein involved in cell-to-cell communication. This peptide (FGL) initiates a cascade of events inside the neuron that results in the facilitation of synaptic plasticity. Specifically, we find that FGL triggers delivery of a specific type of glutamate receptor (AMPA receptors) to synapses in a region of the brain called the hippocampus, which is known to be involved in multiple forms of learning and memory. Importantly, when this peptide was administered to rats, their ability to learn and retain spatial information was enhanced. Therefore, this work demonstrates that cognitive function can be improved pharmacologically in adult animals by enhancing the plasticity of synaptic connections in the brain.

Cellular Signalling Pathways of Spinal Pain Neuroplasticity as Targets for Analgesic Development

Nociceptive inputs from primary afferents are primarily mediated at fast glutamatergic synapses onto second order neurons in the dorsal horn of the spinal cord through activation of AMPA/kainate and NMDA receptor subtypes of ionotropic glutamate receptors. At these glutamatergic synapses several forms of short-lasting and long-lasting enhancement of synaptic transmission are known. Enhancement of excitatory synaptic transmission in nociceptive pathways is thought to be a key neural substrate underlying chronic pain, and thus the cellular and molecular mechanisms producing this enhancement represent potential targets for developing novel forms of therapeutics. Central to the mechanisms for pain hypersensitivity is the NMDA receptor, the activity of which is facilitated by convergent intracellular biochemical cascades in dorsal horn neurons. Cellular changes are not restricted to neurons in the dorsal horn, however, and there is growing evidence for involvement of glia, and of glia-neuronal signaling, in initiating and sustaining enhancement of nociceptive transmission. In particular, a role has emerged for microglia in pain hypersensitivity following nerve injury. This expanded understanding of cellular and molecular signalling mechanisms in the dorsal horn, that includes both neurons and glia, provides a basis of creating new types of strategies for management, and also for diagnosis, of chronic pain.

α-Synuclein produces a long-lasting increase in neurotransmitter release

Wild-type alpha-synuclein, a protein of unknown function, has received much attention because of its involvement in a series of diseases that are known as synucleinopathies. We find that long-lasting potentiation of synaptic transmission between cultured hippocampal neurons is accompanied by an increase in the number of alpha-synuclein clusters. Conversely, suppression of alpha-synuclein expression through antisense nucleotide and knockout techniques blocks the potentiation, as well as the glutamate-induced increase in presynaptic functional bouton number. Consistent with these findings, alpha-synuclein introduction into the presynaptic neuron of a pair of monosynaptically connected cells causes a rapid and long-lasting enhancement of synaptic transmission, and rescues the block of potentiation in alpha-synuclein null mouse cultures. Also, we report that the application of nitric oxide (NO) increases the number of alpha-synuclein clusters, and inhibitors of NO-synthase block this increase, supporting the hypothesis that NO is involved in the enhancement of the number of alpha-synuclein clusters. Thus, alpha-synuclein is involved in synaptic plasticity by augmenting transmitter release from the presynaptic terminal.

Synaptic plasticity of local connections in rat motor cortex

This paper reviews studies that investigated mechanisms of the induction of long-term synaptic efficacy increase in local horizontal connections in slices of adult rat motor cortex. Long-term potentiation (LTP) could be induced by electrical stimulation of afferents using theta burst stimulation (TBS) conditionally, when synaptic inhibition was transiently blocked by focal application of GABAA receptor antagonist. Robust, long-lasting enhancement of synaptic transmission in horizontal connections was induced by brief application of the potassium channel blocker, tetraethylammonium (TEA, 25 mM), to the incubation medium. This TEA-LTP could be blocked by nifedipine, a voltage-dependent calcium channel blocker. A transient exposure of slices to elevated extracellular calcium (5 mM) resulted in a long-lasting enhancement of responses, termed Ca-LTP, which could be blocked by the antagonist of NMDA receptors, APV. The induction of both TEA-LTP and Ca-LTP, could be prevented by inhibitors of the extracellular signal regulated kinase (ERK) cascade U0126 and PD 98059. A transient activation of the ERK, 15 min after application of TEA or elevated [Ca2+], was demonstrated using immunofluorescence. Both forms of plasticity could also be prevented by the inhibitor of cAMP-dependent protein kinases (PKA), Rp-cAMPS. These studies indicate the involvement of the ERK and PKA signaling mechanisms in synaptic plasticity of the motor cortex in vitro. Since LTP in horizontal connections of the motor cortex has previously been shown to be related to the acquisition of a motor skill, it is suggested that the ERK and PKA signaling pathways may be involved in motor learning.

Protein kinase C is required for long-lasting synaptic enhancement by the neuropeptide DRNFLRFamide in crayfish.

The FMRFamide-related neuropeptide AspArgAsnPheLeuArgPhe-NH2 (DRNFLRFamide, DF2) induces a long-lasting enhancement of synaptic transmission at neuromuscular junctions on the crayfish deep abdominal extensor muscles. Here we investigated the function of protein kinase C (PKC) in this effect because PKC has been implied in the control of long-term synaptic modulation in other systems. The general kinase antagonist staurosporine reduced both the initial increase in excitatory postsynaptic potential (EPSP) amplitude and the duration of synaptic enhancement. Unlike staurosporine, the selective PKC inhibitors, chelerythrine and bisindolylmaleimide, augmented the initial EPSP increase. However, like staurosporine, they also reduced the duration of synaptic enhancement. The PKC activator, phorbol-12-myristate 13-acetate, induced a long-lasting synaptic enhancement that was blocked by chelerythrine. These results show that synaptic enhancement by DF2 is mediated by different intracellular signaling systems that act in temporal sequence. The initial increase in EPSP amplitudes is negatively regulated by PKC and involves another, staurosporine-sensitive, kinase; whereas, the maintenance of synaptic enhancement requires PKC.

A requirement for local protein synthesis in neurotrophin-induced hippocampal synaptic plasticity.

Two neurotrophic factors, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), are able to produce a long-lasting enhancement of synaptic transmission in the hippocampus. Unlike other forms of plasticity, neurotrophin-induced plasticity exhibited an immediate requirement for protein synthesis. Plasticity in rat hippocampal slices in which the synaptic neuropil was isolated from the principal cell bodies also required early protein synthesis. Thus, the neurotrophins may stimulate the synthesis of proteins in either axonal or dendritic compartments, allowing synapses to exert local control over the complement of proteins expressed at individual synaptic sites.

Long-lasting forms of synaptic potentiation in the mammalian hippocampus.

I n t r o d u c t i o n Memory storage in the mammalian brain is commonly divided into at least two distinct temporal phases: short-term memory, lasting minutes to hours, and long-term memory, which can persist for days, weeks, or even years (for review, see Polster et al. 1991). Whereas short-term memory requires only covalent modifications of pre existing proteins, long-term memory requires the synthesis of new mRNA and protein (Davis and Squire 1984; Castellucci et al. 1989; Tully et al. 1994). The idea that memory storage in the brain results from activity-dependent changes in synaptic strength was elaborated by Hebb (1949), who proposed that synapses linking two cells could be strengthened if both cells were active simultaneously. The first such Hebbian synapses to be found in the mammalian brain were the connections made by entorhinal perforant path fibers onto dentate granule cells in the hippocampus (Bliss and Lomo 1973). Brief, high-frequency stimulation of this excitatory pathway elicited a long-lasting enhancement of synaptic transmission. This effect, now known as long-term potentiation (LTP), can last for 8 -10 hr in hippocampal slices, and for several days and even weeks in intact animals. LTP has now been shown in all three major monosynaptic excitatory pathways within the hippocampus ( the perforant pathway, mossy fiber pathway, and Schaffer collateral pathway) (Bliss and Collingridge 1993). Hippocampal LTP has a number of properties that make it an attractive model for memory storage. These include associativity, synapse specificity, cooperativity, and persistence (see Bliss and Collingridge 1993 for a more complete description). In this review, we will focus on the long-term maintenance (late phase) of LTP and its underlying mechanisms. We will try to illustrate how pharmacological and molecular biological approaches, some with genetically modified animals, have elucidated some of the mechanisms that contribute to long-term changes in synaptic strength. Most of the experiments that we will discuss use the hippocampal slice preparation, and they focus on synaptic changes occurring over hours. Thus, these changes pertain primarily to the initial phase of the cellular changes that contribute to long-term memory storage. Other, still longer term changes in synaptic strength that may occur over days or weeks can be studied in the intact animal, but these will not be discussed here.

Interactions between arachidonic acid and metabotropic glutamate receptors in the induction of synaptic potentiation in the rat hippocampal slice

Perfusion of neither the metabotropic glutamate receptor agonist (1 S,3 R)-1-aminocyclopentane-1,3-dicar☐ylic acid (ACPD), nor arachidonic acid caused any long-term enhancement of synaptic transmission in the CA1 region of the rat hippocampal slice. However, co-perfusion of ACPD (50 μM) and arachidonic acid (10 μM) for 5 min induced a rapidly evoked and long-lasting enhancement of synaptic transmission. This enhancement persisted in the presence of d(−)-2-amino-5-phosphonopentanoic acid (40 μM) and is therefore independent of NMDA receptor activation. The potentiation was mimicked by perfusion of the phospholipase A 2 activator melittin (10 μ/ml) for 5 or 10 min, or exogenous phospholipase A 2 (1 μg/ml) for 5 min, immediately before ACPD application. We propose a role for arachidonic acid in the induction of synaptic potentiation, possibly as a retrograde transmitter substance.

Medial amygdala enhances synaptic transmission and synaptic plasticity in the dentate gyrus of rats in vivo.

1. The present experiment was designed to test whether synaptic transmission and synaptic plasticity in the dentate gyrus were modulated by the medial amygdala (MeA). Field potentials in the dentate gyrus (DG) evoked by stimulations of the medial perforant path (PP) were extracellularly recorded in anesthetized rats. 2. Although single-pulse stimulation of the MeA augmented PP stimulation-evoked population spike amplitude in the DG transiently, high-frequency stimulation (100 Hz for 1 s) of the MeA induced long-lasting enhancement of synaptic transmission that was not occluded by PP tetanus-induced long-term potentiation (LTP). 3. When high-frequency stimulation of the MeA was applied concurrently with weak tetanus of the PP, which alone induced only marginal LTP, the magnitude of LTP increased considerably. 4. These results demonstrate that neuron activities in the MeA induce short- and long-lasting changes in the excitability of the PP-DG synapses and thereby enhance their synaptic plasticity.

Long-term potentiation at low temperature is stronger in hippocampal slices from hibernating Turkish hamsters compared to warm-acclimated hamsters and rats

Tetanus-induced long-term potentiation (LTP) of population action potentials at 22°C was investigated in area CA1 of hippocampal slices prepared from hibernating (HH) and warm-acclimated Turkish hamsters (WH) and rats. UP elicited at this temperature was significantly stronger in HH slices compared to WH and rat slices. Hibernation-related improvement of the ability to develop long-lasting enhancement of synaptic transmission at low temperatures is interpreted as supporting hippocampal function during arousal from hibernation.

Activity-dependent enhancement of synaptic transmission in hippocampal slices treated with the phosphatase inhibitor calyculin A

The role of protein phosphatases in regulating synaptic transmission in the CA1 region of the hippocampus was examined using slices pretreated with calyculin A, a specific inhibitor of protein phosphatases 1 and 2A. Stimulation of afferents at 1 Hz (but not 0.1 Hz) for periods of 5-10 min caused a long-lasting enhancement of synaptic transmission. The increase in synaptic responses was not due to a change in fiber excitability, as there was a shift to the left in the input-output curve following the synaptic enhancement. The enhancement was observed only in the input that received the 1 Hz stimulation and not in an independent control pathway, indicating that the increase in synaptic strength is input specific and limited to repetitively activated synapses. Applying 1 Hz stimulation when synaptic transmission was blocked by replacing extracellular Ca2+ with Mg2+ prevented or significantly reduced any change in synaptic efficacy after reperfusion with normal Ca(2+)-containing medium. In contrast, 1 Hz stimulation given when synaptic transmission was blocked by non-NMDA and NMDA glutamate receptor antagonists still caused a synaptic enhancement following washout of the antagonists. The enhancement of synaptic transmission also was not blocked by loading CA1 cells with the calcium chelator BAPTA. Thus, influx of Ca2+ into presynaptic elements is required for the synaptic enhancement elicited by 1 Hz stimulation in calyculin A-treated hippocampal slices. Consistent with the activation of processes that cause an increase in transmitter release, the magnitude of paired-pulse facilitation decreased following the synaptic enhancement, and the NMDA receptor-mediated component of the synaptic response was increased by 1 Hz stimulation. These results suggest that when protein phosphatases are inhibited by calyculin A, prolonged periods of 1 Hz stimulation lead to activation of presynaptic Ca(2+)-dependent protein kinases, resulting in a persistent increase in evoked transmitter release. They also indicate that the activity of presynaptic protein phosphatases is critically important for limiting increases in synaptic strength following repetitive afferent activity.

Prolonged enhancement of synaptic transmission in area CA1 of rat hippocampal slices induced by NaF/AlCl 3 does not require NMDA receptor activation but is suppressed by inhibitors of phosphoinositide-mediated signalling pathways

The processes underlying the action of AlF 4 − (10 mM NaF/10 μM AlCl 3) in inducing long-lasting enhancement of synaptic transmission in area CA1 of rat hippocampal slices have been investigated. Exposure of hippocampal slices to AlF 4 − for 10 min caused population EPSP slope to rise by approximately 50% within 60 min of washing off the NaF/AlCl 3 saline. This effect was not inhibited either by APV (50 μM), or by temporary interruption of the delivery of test stimuli during and for up to 20 min after application of the AlF 4 −-containing medium. However, pretreatment of preparations with either thapsigargin (1 μM) or staurosporine (1 μM), or ommission of Ca 2+ from the AlF 4 −-containing saline (no addition of EGTA) prevented the potentiating action of NaF/AlCl 3. We conclude that the potentiating effect of AlF 4 − is via a G-protein linked to phosphoinositide turnover, and that both arms of this signalling pathway are necessary for potentiation to occur. Ca 2+ influx is also a requirement, but does not occur through the NMDA receptor.

Long-term potentiation induced by patterned stimulation of the commissural pathway to hippocampal CA1 region in freely moving rats

In urethane-anesthetized rats, stimulation of the contralateral hippocampal CA1 region resulted in activation of the homotopic CA1 region. Current-source-density analysis revealed that both basal and apical dendrites were activated. However, alvear and stratum oriens stimulation in CA1 gave about equal peak excitation of the basal and apical dendrites while CA1 stratum radiatum/moleculare and CA3c stimulation gave stronger apical than basal dendritic excitation. In chronically implanted and freely moving rats, tetanic patterned stimulation of the contralateral CA1, irrespective of depth, resulted in a robust long-term potentiation of the ipsilateral CA1 basal dendritic synapse. The population basal dendritic excitatory postsynaptic potential was initially potentiated to >200% of the baseline and decayed with a 3 h time constant; it lasted at least two days. Patterned stimulation of the commissural inputs at 2 × threshold stimulus intensity seldom potentiated the apical dendritic synapse in CA1; rather, long-term depression was sometimes observed. After tetanic stimulations at 3 × threshold, a small potentiation of the apical dendritic excitation was seen in about half of the experiments. The average apical dendritic potentiation peaked at about 25% and persisted to at least one day. This study provides original evidence that the properties of long-term potentiation are different at the commissural basal dendritic and apical dendritic synapses in CA1 of the behaving rat. Basal dendritic potentiation is low-threshold, high-amplitude and decayed rapidly in the first 3 h. Apical dendritic potentiation is high-threshold, low-amplitude and not rapidly decaying. A long-lasting enhancement of synaptic transmission has been postulated as a physiological correlate of memory. This paper reports properties of this synaptic enhancement for two different types of synapses on the same cells in the behaving animal. The basal dendritic synapse on hippocampal pyramidal cells readily increased their efficacy, up to at least two days, after a brief, patterned stimulation. In the same preparation, it was difficult to obtain a long-lasting increase in the apical dendritic excitation, in contrast to studies on isolated hippocampal slices in vitro.

Enhancement of postsynaptic responsiveness during long-term potentiation of mossy fiber synapses in guinea pig hippocampus

The primary site responsible for the long-lasting enhancement of synaptic transmission during long-term potentiation (LTP) was examined by quantal analysis of excitatory postsynaptic potentials in thin sections of the guinea pig hippocampus. With induction of LTP in mossy fiber synapses, estimated values of quantal amplitude ( q) and Pascal parameters p and r were increased significantly. No increases in quantal content ( m) were detected. The magnitude of increases in q was almost equal to that of LTP. These results indicate that LTP in mossy fiber synapses results from increases in responsiveness of postsynaptic neurons.

Activity-dependent recruitment of silent synapses.

At the crayfish neuromuscular junction, a long-lasting enhancement of synaptic transmission can be induced by tetanic stimulation of 10-20 Hz for several minutes. The long-lasting enhancement is presynaptic in origin, because quantal content increases but not quantal size, and is not dependent upon broadening or enlargement of the presynaptic action potential. The enhancement can be selectively blocked by presynaptic injection of agents that inhibit adenylate cyclase or the cyclic AMP-dependent protein kinase. Entry of calcium may not be sufficient in itself to produce the enhancement. Analyses of quantal events using both a simple binomial statistical method, and a more refined method that takes into account the possibility of unequal probabilities of responding units, have shown that the number of responding units increases during the long-lasting enhancement. In addition, there is an increase in the probability of transmitter release at preexisting units. In contrast, during short-term facilitation accompanying repetitive stimulation, response probability increases greatly whereas the number of responding units increases only moderately with frequencies of activation up to 20 Hz, which increase quantal output severalfold. These results indicate that responding units, hypothesized to be transmitting synapses, can be recruited to active transmission from an unresponsive pool by tetanic activity, and that protein phosphorylation is required for long-lasting changes to occur. The existence of an excess of synapses on crustacean nerve terminals is indicated by ultrastructural studies, which invariably show many synapses on the terminals. The number of morphologically defined synapses is always greater than the number of responding units seen in statistical analyses of quantal release for the same recording location.