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Long-Term Depression Properties in a Simple System
Abstract
Long-term depression (LTD) in pairs of cultured rodent hippocampal neurons was examined to study the molecular basis of this form of synaptic plasticity. We have previously characterized two components of transmitter release: a synchronous, fast phase that requires synaptotagmin I, and an asynchronous, slow component that persists in the absence of synaptotagmin I. Are these two release components differentially affected by the presynaptic changes of LTD, or is the mechanism of plasticity common to both? We find that LTD is expressed as parallel changes in the fast and slow components of release, and that this form of synaptic plasticity is still seen in the absence of functional synaptotagmin I. Any alterations in the presynaptic release machinery observed during LTD thus involve mechanisms shared by both modes of release.
... One of the first forms of LTD to be intensively studied was found in autaptic excitatory hippocampal cultures (Bekkers and Stevens 1991; Goda and Stevens 1996). While much was learned about the synaptic qualities of LTD in autaptic hippocampal neurons, the mechanisms of induction and expres-sion of autaptic LTD (autLTD) have remained elusive. ...
... Neurons exhibiting washout (30 -50% per data set) were excluded from analysis. In the case of 4-Hz stimulus-evoked autLTD, spontaneous miniature EPSCs (mEPSCs) were obtained from the latter half of long (1 s) traces following EPSC stimulation at which point there is no appreciable contamination of mEPSCs by evoked EPSCs (Goda and Stevens 1996). All other mEPSCs were obtained in the presence of tetrodotoxin (1 M). ...
... Examples of postsynaptic action might be: a change in glutamate receptor number, or change in responsiveness or coupling to downstream effectors. Consistent with previous findings in rat (Goda and Stevens 1996), we found that the frequency of minis in mouse neurons decreased following LTD (Fig. 8, A and B; mini frequency before LTD protocol: 2.5 Ϯ 0.6/s, frequency after LTD protocol: 1.3 Ϯ 0.3/s, n ϭ 6; P Ͻ 0.005 by paired t-test). The amplitude of minis did not change significantly (Fig. 8, A and B; mini amplitude control: 12.9 Ϯ 1.6 pA; after LTD: 11.3 Ϯ 1.2, n ϭ 5). ...
Long-term depression (LTD) of synaptic signaling-lasting from tens of minutes to hours or longer-is a widespread form of synaptic plasticity in the brain. Neurons express diverse forms of LTD, including autaptic LTD (autLTD) observed in cultured hippocampal neurons, the mechanism of which remains unknown. We have recently reported that autaptic neurons express both endocannabinoid-mediated depolarization-induced suppression of excitation (DSE) and metabotropic suppression of excitation (MSE). We now report that activating cannabinoid CB(1) receptors is necessary for the induction of autLTD. Most surprisingly, CB(1) does not induce autLTD via the G(i/o) proteins typically activated by this receptor nor with G(s). Rather, the requirements of presynaptic phospholipase C and filled calcium stores suggest G(q). In autLTD, a 3- to 4-min activation of the receptor by the endocannabinoid 2-arachidonoyl glycerol leads to prolonged inhibition while leaving short-term inhibition (e.g., DSE) intact. autLTD requires activation of both metabo- and ionotropic glutamate receptors. autLTD also requires MEK/ERK activation. Under certain conditions, one or more DSE stimuli will elicit autLTD. It is becoming evident that cannabinoids mediate multiple forms of plasticity at a single synapse, stretching temporally from tens of seconds (DSE/MSE) to tens of minutes (autLTD) to hours (CB(1) desensitization). Our findings imply a remarkable flexibility for the cannabinoid signaling system whereby discrete mechanisms of CB(1) activation within a single neuron yield temporally and mechanistically distinct forms of plasticity.
... Probably because only about a half of the synapses in our cultures are on spines (C. Boyer, T. Schikorski, and C.F.S., unpublished data), LTD is only partially and variably blocked by the standard pharmacological and membrane potential manipulations (24). These manipulations are therefore of limited usefulness in evaluating the contribution of ''tiring out'' to culture LTD. ...
... Because the application of hypertonic solution to synapses causes depletion of the readily releasable pool without participation of the usual calcium dependent mechanisms (14,24), this method for assaying pool size is the preferred one. The difficulty with the assay, however, is that it measures the average pool size for all of the synapses on the dendritic branches to which the hypertonic solution is applied. ...
... To estimate pool size before and after LTD, then, we must make sure all synapses have undergone the plastic changes. The only situation in which all synaptic inputs to a neuron are identified and express the same (average) amount of LTD is an autaptic circuit in which an isolated neuron has only itself as a potential target (24). For this reason, we have restricted our investigation of pool size before and after LTD induction to autapses. ...
We have estimated, for hippocampal neurons in culture, the size of the autaptic readily releasable pool before and after stimulation of the sort that produces culture long term depression (LTD). This stimulation protocol causes a decrease in the pool size that is proportional to the depression of synaptic currents. To determine if depression in this system is synapse specific rather than general, we have also monitored synaptic transmission between pairs of cultured hippocampal neurons that are autaptically and reciprocally interconnected. We find that the change in synaptic strength is restricted to the synapses on the target neuron that were active during LTD induction. When viewed from the perspective of the presynaptic neuron, however, synapse specificity is partial rather than complete: synapses active during induction that were not on the target neuron were partially depressed.
... Several groups have reported that long-term depression (LTD) in culture is resistant to the NMDA receptor antagonist 5-aminopyridine (AP-5) (Deisseroth et al., 1996;Goda and Stevens, 1996). Spines are frequently considered to be important for forming a compartment that limits calcium ions to the immediate postsynaptic region and also isolates the spine compartment from the dendritic shaft. ...
... The increase in calcium concentration necessary to induce LTD could be derived for shaft synapses from dendritic calcium channels rather than from NMDA receptor channels if the spine neck is required to exclude dendritic calcium from the spine head. Our morphological observations are in agreement with this notion because approximately half of the LTD in the Goda and Stevens (1996) experiments, performed in cultures similar to the ones studied here, were resistant to NMDA receptor antagonists, just as approximately half of the synapses were on spines and half were on the dendritic shaft. Presumably, calcium entry through dendritic calcium channels would have ready access to the postsynaptic membrane and could substitute for calcium that normally would have had to enter through NMDA receptor channels. ...
We have quantified hippocampal spine structure at the light and ultrastructural levels in cell cultures approximately 1- 3 weeks old and in the brains of rodents 5 and 21 d old. The number of spines bearing synapses increases with age in cultures and in brain, but the structures are similar in both. In culture, about half of the synapses are formed on spines and the remainder are formed on dendritic shafts. In the 5-d-old brain, about half of the synapses occur on dendritic shafts, by 3 weeks of age only approximately 20% of synapses are found on dendritic shafts, and in the adult shaft synapses are very rare.
... Instead, the result argues against long-term presynaptic depression as a cause for the decrease in fractional destaining measured with FM4-64. We note, however, that the result does not contradict previous reports of presynaptic long-term depression where induction required postsynaptic depolarization (Goda and Stevens, 1996) because postsynaptic depolarization was likely prevented in the present study by glutamate receptor antagonists. Figure 2C -ruling out selective depletion of the readily releasable pool as the cause of the decrease. ...
Vesicles within presynaptic terminals are thought to be segregated into a variety of readily releasable and reserve pools. The nature of the pools and trafficking between them is not well understood, but pools that are slow to mobilize when synapses are active are often assumed to feed pools that are mobilized more quickly, in a series. However, electrophysiological studies of synaptic transmission have suggested instead a parallel organization where vesicles within slowly and quickly mobilized reserve pools would separately feed independent reluctant- and fast-releasing subdivisions of the readily releasable pool. Here, we use FM-dyes to confirm the existence of multiple reserve pools at hippocampal synapses and a parallel organization that prevents intermixing between the pools, even when stimulation is intense enough to drive exocytosis at the maximum rate. The experiments additionally demonstrate extensive heterogeneity among synapses in the relative sizes of the slowly and quickly mobilized reserve pools, which suggests equivalent heterogeneity in the numbers of reluctant and fast-releasing readily releasable vesicles that may be relevant for understanding information processing and storage.
... It has been reported that autapses in single neuron cultures and synapses in dissociated neuron cultures share similar biophysical properties (Bekkers and Stevens, 1991;Shi and Rayport, 1994). In addition, previous studies of the synaptic vesicle cycle at autapses (Chamberland and Tóth, 2016) and synaptic plasticity (Goda and Stevens, 1996;Straiker and Mackie, 2005;Kellogg et al., 2009;Rost et al., 2010) indicate the similarity between autapses and synapses in the intact brain. ...
A morphologically present but non-functioning synapse is termed a silent synapse. Silent synapses are categorized into “postsynaptically silent synapses,” where AMPA receptors are either absent or non-functional, and “presynaptically silent synapses,” where neurotransmitters cannot be released from nerve terminals. The presence of presynaptically silent synapses remains enigmatic, and their physiological significance is highly intriguing. In this study, we examined the distribution and developmental changes of presynaptically active and silent synapses in individual neurons. Our findings show a gradual increase in the number of excitatory synapses, along with a corresponding decrease in the percentage of presynaptically silent synapses during neuronal development. To pinpoint the distribution of presynaptically active and silent synapses, i.e., their positional information, we employed Sholl analysis. Our results indicate that the distribution of presynaptically silent synapses within a single neuron does not exhibit a distinct pattern during synapse development in different distance from the cell body. However, irrespective of neuronal development, the proportion of presynaptically silent synapses tends to rise as the projection site moves farther from the cell body, suggesting that synapses near the cell body may exhibit higher synaptic transmission efficiency. This study represents the first observation of changes in the distribution of presynaptically active and silent synapses within a single neuron.
... Instead, the result argues against long-term presynaptic depression as a cause for the decrease in fractional destaining measured with FM4-64. We note, however, that the result does not contradict previous reports of presynaptic long-term depression where induction required postsynaptic depolarization (Goda and Stevens, 1996) because postsynaptic depolarization was likely prevented in the present study by glutamate receptor antagonists. ...
Vesicles within presynaptic terminals are thought to be segregated into a variety of readily releasable and reserve pools. The nature of the pools and trafficking between them is not well understood, but pools that are slow to mobilize when synapses are active are usually assumed to feed pools that are mobilized more quickly, in a series. However, results from electrophysiological studies of synaptic transmission suggested instead a parallel organization where vesicles within slowly and quickly mobilized reserve pools would separately feed independent reluctant- and fast-releasing subdivisions of the readily releasable pool, without intermixing. We now use FM-dyes to confirm the existence of multiple reserve pools at hippocampal synapses. We then confirm the prediction that slowly and quickly mobilized reserve pools do not intermix, even when mobilized by high frequency stimulation. The result provides a simplifying new constraint on the dynamics of vesicle recycling within presynaptic terminals. The experiments additionally demonstrated extensive heterogeneity among synapses in the relative sizes of slowly and quickly mobilized reserve pools. The heterogeneity suggests equivalent heterogeneity in the probability of release among readily releasable vesicles that may be relevant for understanding information processing and storage.
... For example, modulation of the AMPA receptor may be an important clinical target, because it showed greater efficacy in seizures control than the NMDA receptors (Hunt and Castillo 2012) included in the pathological neuroplasticity (Goda and Stevens 1996). ...
Epilepsy is a neurological disorder in which an imbalance between excitatory and inhibitory transmission is observed. Glutamate is the principal excitatory neurotransmitter that acts through ionic and metabotropic receptors; both types of receptors are involved in temporal lobe epilepsy (TLE). High frequency oscillations called fast ripples (FR, 250–600 Hz) have been observed, particularly in the hippocampus, and they are involved in epileptogenesis. The present study analyzed the immunoreactivity of the principal glutamate receptors associated with epilepsy in epileptic animals with FR activity. Male Swiss-Wistar rats (210–250 gr) were injected with pilocarpine (2.4 mg/2 µl) and were video monitored (24/7) until the appearance of spontaneous and recurrent seizures. Then, a deep microelectrode implantation surgery was performed in the DG, CA3 and CA1 regions, and FR activity was observed 1-, 2-, 3-, 7-, and 14-day postsurgery. The animals were sacrificed on day 15, and fluorescence immunohistochemistry was carried out in the hippocampus for the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-D-aspartate (NMDA) and mGlu-R5 glutamate receptors as well as Neuronal Nuclear Protein (NeuN) and Glial Fibrillary Acidic Protein (GFAP). An increase in the immunoreactivity for the three receptors was found. However, the AMPA receptor showed an increase in the three regions analyzed (i.e., DG, CA1 and CA3). The findings showed a decrease of NeuN in the DG and an increase of GFAP. These results suggest an important role of glutamate receptors in the hippocampus of epileptic rats with FR activity.
... Certain types of synaptic plasticity that have been studied in slices are also faithfully replicated in autaptic cultures. For instance, depolarizationinduced suppression of excitation (DSE; Straiker and Mackie, 2009) and inhibition (DSI; Straiker and Mackie, 2005;Kellogg et al., 2009) are present at autapses, as is long-term depression (LTD; Goda and Stevens, 1996;Tong et al., 1996;Kumura et al., 2000). Cultured dentate granule cells form autapses that resemble mossy fiber inputs to CA3 pyramidal cells, including expression of a presynaptic form of long-term potentiation (Rost et al., 2010). ...
Neurons typically form daisy chains of synaptic connections with other neurons, but they can also form synapses with themselves. Although such self-synapses, or autapses, are comparatively rare in vivo, they are surprisingly common in dissociated neuronal cultures. At first glance, autapses in culture seem like a mere curiosity. However, by providing a simple model system in which a single recording electrode gives simultaneous access to the pre- and postsynaptic compartments, autaptic cultures have proven to be invaluable in facilitating important and elegant experiments in the area of synaptic neuroscience. Here, I provide detailed protocols for preparing and recording from autaptic cultures (also called micro-island or microdot cultures). Variations on the basic procedure are presented, as well as practical tips for optimizing the outcomes. I also illustrate the utility of autaptic cultures by reviewing the types of experiments that have used them over the past three decades. These examples serve to highlight the power and elegance of this simple model system, and will hopefully inspire new experiments for the interrogation of synaptic function.
... , [54,65] . ...
... LTD, prolonged and stable depression of the postsynaptic response has been examined in an isolated pair of hippocampus culture neurons [15]. Synaptic strength decreased after depression stimulations means the coupling of two neurons are depressed [16]. ...
Amorphous indium–gallium–zinc oxide (a-IGZO) -based synaptic transistors with hafnium oxide (HfOx) insulating layer were fabricated to mimic synaptic long-term depression (LTD) characteristics. The fabrication temperature was less than 120℃. Interval time of presynaptic spikes dependent synaptic depression was first demonstrated in these IGZO-based synaptic transistors, which is important for computation system coding by time. The depression effect in our synaptic transistor is erasable, using ultraviolet (UV) light (λ = 365 nm) to erase the electrons trapped in the defects of the HfOx layer. Our device is in great significance for future brain-like artificial neuromorphic computation system since LTD has been verified as a contributor to learning and memory function in brains.
... Long-lasting alterations in synaptic strength in dissociated cultures can be induced by electrical stimulation [15,16]. These changes, termed synaptic plasticity, are thought to underlie certain forms of information storage in the brain [17]. ...
Neural circuits are responsible for the brain's ability to process and store information. Reductionist approaches to understanding the brain include isolation of individual neurons for detailed characterization. When maintained in vitro for several days or weeks, dissociated neurons self-assemble into randomly connected networks that produce synchronized activity and are capable of learning. This review focuses on efforts to control neuronal connectivity in vitro and construct living neural circuits of increasing complexity and precision. Microfabrication-based methods have been developed to guide network self-assembly, accomplishing control over in vitro circuit size and connectivity. The ability to control neural connectivity and synchronized activity led to the implementation of logic functions using living neurons. Techniques to construct and control three-dimensional circuits have also been established. Advances in multiple electrode arrays as well as genetically encoded, optical activity sensors and transducers enabled highly specific interfaces to circuits composed of thousands of neurons. Further advances in on-chip neural circuits may lead to better understanding of the brain.
... When the same stimulation paradigm was given to a cell pair in which the presynaptic neuron expressed VGLUT-pHluorin, a reporter of synaptic vesicle exo-endocyosis, the readily releasable vesicle pool that was proportional to p r (1) also showed potentiation (two of six pairs), depression (one of six pairs), or no change (three of six pairs), which further confirmed the presynaptic origin of plasticity (Fig. S2). This observation was consistent with presynaptic expression of plasticity that had been previously reported for hippocampal synapses (28,30,33,(35)(36)(37). ...
Significance
We addressed the basic mechanisms underlying synapse heterogeneity, and we identified a form of regulation that serves to increase the variations in the efficacy with which neurons communicate with each other through synapses. We demonstrate that this process requires astrocytes, which, previously, have been thought to play mostly a passive role in maintaining neuronal functions. The cellular mechanism that regulates synaptic efficacy requires astrocyte membrane depolarization, activation of astrocyte NMDA receptors, and astrocyte calcium signaling. The fundamental nature of the regulation is underscored by the preservation of the mechanism from acute brain slices down to dissociated cultures that lack the native topology of brain networks.
... Here, we tested for such a facilitation in single, cultured rat hippocampal neurons. When grown on glial microislands, the neurons form extensive synaptic connections onto themselves ("autapses") ( Van der Loos and Glaser, 1972), which have properties very similar to those of synapses between neurons (Bekkers and Stevens, 1991;Johnson and Yee, 1995;Goda and Stevens, 1996). Such autapses show large postsynaptic responses and thereby provide a convenient system for studying short-term plasticity, especially under conditions in which neurotransmission is reduced such as during G-protein-mediated inhibition and presynaptic calcium channel blockade. ...
G-protein inhibition of voltage-gated calcium channels can be transiently relieved by repetitive physiological stimuli. Here, we provide evidence that such relief of inhibition contributes to short-term synaptic plasticity in microisland-cultured hippocampal neurons. With G-protein inhibition induced by the GABA(B) receptor agonist baclofen or the adenosine A1 receptor agonist 2-chloroadenosine, short-term synaptic facilitation emerged during action potential trains. The facilitation decayed with a time constant of similar to 100 msec. However, addition of the calcium channel inhibitor Cd2+ at 2-3 mu M had no such effect and did not alter baseline synaptic depression. As expected of facilitation from relief of channel inhibition, analysis of miniature EPSCs implicated presynaptic modulation, and elevating presynaptic Ca2+ entry blunted the facilitation. Most telling was the near occlusion of synaptic facilitation after selective blockade of P/Q- but not N-type calcium channels. This was as predicted from experiments using recombinant calcium channels expressed in human embryonic kidney (HEK) 293 cells; we found significantly stronger relief of G-protein inhibition in recombinant P/Q- versus N-type channels during action potential trains. G-protein inhibition in HEK 293 cells was induced via recombinant M2 muscarinic acetylcholine receptors activated by carbachol, an acetylcholine analog. Thus, relief of G-protein inhibition appears to produce a novel form of short-term synaptic facilitation in cultured neurons. Similar short-term synaptic plasticity may be present at a wide variety of synapses, as it could occur during autoreceptor inhibition by glutamate or GABA, heterosynaptic inhibition by GABA, tonic adenosine inhibition, and in many other instances.
... Post tetanus (min) Pre 5 10 15 Tetanised pathway (%) 100 390 380 332 Control pathway (%) 100 74 67 73 Figure 9. LTD before and after stimulation. The initial synaptic strength was set to 1. Reprinted from [52], Copyright (1996), with permission from Elsevier. ...
The synapse is a crucial element in biological neural networks, but a simple electronic equivalent has been absent. This complicates the development of hardware that imitates biological architectures in the nervous system. Now, the recent progress in the experimental realization of memristive devices has renewed interest in artificial neural networks. The resistance of a memristive system depends on its past states and exactly this functionality can be used to mimic the synaptic connections in a (human) brain. After a short introduction to memristors, we present and explain the relevant mechanisms in a biological neural network, such as long-term potentiation and spike time-dependent plasticity, and determine the minimal requirements for an artificial neural network. We review the implementations of these processes using basic electric circuits and more complex mechanisms that either imitate biological systems or could act as a model system for them.
... There is also some evidence that low frequency stimulation generates long term depression (LTD) (Hansel, Linden, and D'Angelo, 2001 ). These effects of tetanic stimulation applied to granule cells parallel the effects of similar protocols in generating LTP and LTD at excitatory synapses in cells from the hippocampus (Bliss and Collingridge, 1993; Goda and Stevens, 1996) and the cerebral cortex (Glazewski et al., 1998; Yasuda and Tsumoto, 1996). Excitatory synapses in pyramidal cells of the hippocampus (Bi and Poo, 1998; Debanne, Gähwiler, and Thompson, 1998) and the cerebral cortex (Markram et al., 1997; Feldman, 2000) can also develop a spike timing-dependent plasticity (STDP). ...
A model of post-synaptic signaling cascades is used to predict spike-timing dependency of synaptic plasticity in cerebellar granule cells. The mossy fiber-cerebellar granule cell synapse has been found to express a protein kinase C-dependent long-term potentiation after a high frequency presynaptic tetanus. The model simulates the electrophysiological behavior of the granule cell in response to current injections and includes dendrites, glutamatergic synapses, and aspects of intracellular Ca 2+ handling. A minimal set of enzymes and other proteins have been modeled to test a mechanism of synaptic plasticity based on regulated protein-protein interactions. The frequency dependence of persistent enzyme accumulation near the synapse is interpreted as inducing synaptic plasticity. The model predicts the spike-timing dependency of synaptic change due to repeated pairings of pre-and postsynaptic spikes as constrained by experimental results from frequency-dependent long-term plasticity.
... Thus, in the study of Lu et al. (2001) there were similar changes in mEPSC frequency (30%) and mEPSC amplitude (20%) whereas our effect was predominantly expressed by a change in mEPSC frequency and was typically much larger. One possibility for this is that the longer stimulation period used by Lu et al. (2001) may have resulted in a mixture of LTP and LTD, at different synapses, since stimulation for 3 min at 5 Hz couple to postsyanptic depolarisation has been shown to induce LTD in culture (Deisseroth et al., 1996;Goda and Stevens, 1996). Another explanation for these differences may be our use of postnatal, rather than embryonic, cultures. ...
Long-term potentiation (LTP) of synaptic transmission is under intense investigation. It is believed that the mechanisms involved in its induction and expression are critically involved in synaptic processes that are important for learning and memory and other physiological functions. A reliable means of inducing LTP in dissociated cultured neurones would facilitate investigations into the molecular basis of LTP but has been hard to achieve. Here we report a mechanism for inducing LTP in postnatal dissociated hippocampal neurones using transient depolarisation. This form of LTP is prevented by NMDA receptor antagonists and by chelating Ca2+ in the postsynaptic neurone. It is manifest primarily as an increase in the frequency of mEPSCs.
... A number of these requirements are shared with LTP, thus the inescapable theory that their mechanisms may share, at least some, common steps (Table 1). For an LTD to be induced, a number of requirements must be met that include (1) the coactivation of multiple synapses of the same postsynaptic neuron (Holland & Wagner, 1998;Pocket, Brookes, & Bindman, 1990;Stäubli & Lynch, 1987Stäubli & Zi, 1996;Yang, Connor, & Faber, 1994), (2) the electrical uncoupling of the NMDA-R (Abraham & Bear, 1996;Malenka et al., 1998), and (3) Ca ++ must enter the postsynaptic spine (Christie, Magee, & Johnston, 1996;Cummings, Mulkey, Nicoll, & Malenka, 1996;Nevian & Sackmann, 2006;Reyes-Harde & Stanton, 1998Bashir et al., 1993;Nicoll et al., 1998), (2)protein phosphatases, such as phosphatase I, must be activated (Mulkey, Endo, Shenolikar, & Malenka, 1994;Mulkey, Herron, & Malenka, 1993;Thiels, Norman, Barrionuevo, & Klann, 1998;Zhuo et al., 1999), and (3)phospholipase C must be activated and triphosphoric inositol (IP3) must be produced (Malenka & Bear, 2004;Reyes-HardeGoda & Stevens, 1996;Mulkey & Malenka, 1992;Nicoll & Malenka, 1997Howland & Wang, 2008;Kessels & Malinow, 2009;Teyler, 1987;Teyler & Discenna, 1984). ...
Since it was first observed, synaptic plasticity has been considered as the experimental paradigm most likely to provide us with an understanding of how information is stored in the vertebrate brain. Various types have been demonstrated over these past 45 years, most notably long-term potentiation and long-term depression, and their established characteristics as well as their induction and consolidation requirements are highly indicative of this plasticity being the substrate for skills acquisition and mnemonic engraving. The molecular, biochemical, and structural models that have been proposed in the past, although most accommodate some aspect of synaptic plasticity observations, admittedly cannot offer a universally functional connection between all the phenomena that surround and result in the different modifications of synaptic efficacy. As a result, there are a number of persisting questions. In an attempt toward synthesis, we reviewed the most important studies in the field and believe that we can now propose a unifying Model for synaptic plasticity that can accommodate the experimental evidence and reconcile most of the contradictions. Moreover, from this model emerge potential answers to several unyielding questions, namely, accounting for the induction and expression of long-term depression, identifying the plasticity switch, offering a possible explanation for the sliding modification threshold, and proposing a new mechanism for synaptic tagging.
... As mentioned above, activation of L-type channels has been demonstrated to play a role in the induction of heterosynaptic LTD in area CA1 , as well as the dentate gyrus (Christie and Abraham, 1994). While most studies indicate that NMDAR antagonists block homosynaptic LTD in neonates, other researchers report synaptic depression in the presence of NMDAR blockade, or impaired induction during bath application of VDCC antagonists (Velisek et al., 1993;Bolshakov and Siegelbaum, 1994;Christie et al., 1996Christie et al., , 1997Cummings et al., 1996;Goda and Stevens, 1996;however, see Selig et al., 1995). Together, the results indicate the involvement of VDCCs in LTD-induction under certain conditions and suggest that an NMDAR-independent form of LTD may exist. ...
Altered calcium (Ca2+) homeostasis is thought to play a key role in aging and neuropathology resulting in memory deficits. Several forms of hippocampal synaptic plasticity are dependent on Ca2+, providing a potential link between altered Ca2+ homeostasis and memory deficits associated with aging. The current study reviews evidence for Ca2+ dysregulation during aging which could interact with Ca(2+)-dependent synaptic plasticity. The authors suggest that changes in Ca2+ regulation could adjust the thresholds for synaptic modification, favoring processes for depression of synaptic strength during aging.
... Although the induction of LTD in area CA1 often depends on the activation of NMDARs, it is becoming increasingly clear that, under certain conditions, L-channels also contribute to the induction process. For instance, although LTD induction in immature animals usually is blocked by NMDAR antagonists (Bear and Abraham, 1996), other investigators have observed substantial LTD during NMDAR blockade or the inhibition of LTD in the presence of L-channel antagonists (Velisek et al., 1993;Bolshakov and Siegelbaum, 1994;Christie et al., 1996Christie et al., , 1997Cummings et al., 1996;Goda and Stevens, 1996) (but see Selig et al., 1995). Because LTD induction depends on a modest rise of cytosolic Ca 2ϩ (Bear, 1995), elevated Ca 2ϩ influx through L-channels may lower the rate of synaptic activity that is necessary to increase cytosolic Ca 2ϩ beyond the LTD threshold (Debanne et al., 1994;. ...
The role of L-type Ca2+ channels in the induction of synaptic plasticity in hippocampal slices of aged (22-24 months) and young adult (4-6 months) male Fischer 344 rats was investigated. Prolonged 1 Hz stimulation (900 pulses) of Schaffer collaterals, which normally depresses CA3/CA1 synaptic strength in aged rat slices, failed to induce long-term depression (LTD) during bath application of the L-channel antagonist nifedipine (10 microM). When 5 Hz stimulation (900 pulses) was used to modify synaptic strength, nifedipine facilitated synaptic enhancement in slices from aged, but not young, adult rats. This enhancement was pathway-specific, reversible, and impaired by the NMDA receptor (NMDAR) antagonist DL-2-amino-5-phosphonopentanoic acid (AP5). Induction of long-term potentiation (LTP) in aged rats, using 100 Hz stimulation, occluded subsequent synaptic enhancement by 5 Hz stimulation, suggesting that nifedipine-facilitated enhancement shares mechanisms in common with conventional LTP. Facilitation of synaptic enhancement by nifedipine likely was attributable to a reduction ( approximately 30%) in the Ca2+-dependent K+-mediated afterhyperpolarization (AHP), because the K+ channel blocker apamin (1 microM) similarly reduced the AHP and promoted synaptic enhancement by 5 Hz stimulation. In contrast, apamin did not block LTD induction using 1 Hz stimulation, suggesting that, in aged rats, the AHP does not influence LTD and LTP induction in a similar way. The results indicate that, during aging, L-channels can (1) facilitate LTD induction during low rates of synaptic activity and (2) impair LTP induction during higher levels of synaptic activation via an increase in the Ca2+-dependent AHP.
... Correspondingly, the mean reduction in EPSC amplitude fol-the stimulation rate, a protocol termed "pairing" and one which has been used to elicit LTD in hippocampal lowing pairing declined significantly with age ( Figure 2B; ANOVA, p Ͻ 0.02). The greatest mean LTD was observed CA1 neurons (Selig et al., 1995;Goda and Stevens, 1996). Figure 1 shows an example of a cell in which at P4-P5 (Ϫ28.2% ...
Sensory experience during an early critical period guides the development of thalamocortical circuits in many cortical areas. This process has been hypothesized to involve long-term potentiation (LTP) and long-term depression (LTD) at thalamocortical synapses. Here, we show that thalamocortical synapses in rat barrel cortex can express LTD, and that LTD is most readily induced during a developmental period that is similar to the critical period for thalamocortical plasticity in vivo. Thalamocortical LTD is homosynaptic and dependent on activation of N-methyl-D-aspartate (NMDA) receptors. The age-related decline of LTD is not due to changes in inhibition nor to changes in NMDA receptor voltage dependence. Minimal stimulation experiments indicate that, unlike thalamocortical LTP, thalamocortical LTD is not associated with a significant change in failure rate. The existence of LTD and its developmental time course suggest that LTD, like LTP, may contribute to the refinement of thalamocortical inputs in vivo.
... In particular, associative LTP is induced when a strong postsynaptic calcium entry is temporally associated with a synaptic activation whereas a moderate and persistent elevation in calcium concentration decreases synaptic strength at the active synapse. For example, associative LTD is induced in hippocampal (Selig et al. 1995;Goda and Stevens 1996) and cortical ( Feldman et al. 1998) ...
Several forms of synaptic plasticity in the neocortex and hippocampus depend on the temporal coincidence of presynaptic activity and postsynaptic trains of action potentials (APs). This requirement is consistent with the Hebbian, or correlational, type of cellular learning rule used in many studies of associative synaptic plasticity. Recent experimental evidence suggests that APs initiated in the axosomatic area are actively back-propagated to the dendritic arborization of neocortical and pyramidal cells. High-frequency trains of postsynaptic APs that are used as conditioning stimuli for the induction of Hebbian-like plasticity in both neocortical and hippocampal pyramidal cells display attenuation of the dendritic AP amplitude during the train. This attenuation has been shown to be modulated by neurotransmitters and by electrical activity. We suggest here that both spike train attenuation in the dendrite and its modulation by neurotransmitters and electrical activity may have important functional consequences on the magnitude and/or the sign of the synaptic plasticity induced by a Hebbian pairing procedure.
... Here, we tested for such a facilitation in single, cultured rat hippocampal neurons. When grown on glial microislands, the neurons form extensive synaptic connections onto themselves ("autapses") ( Van der Loos and Glaser, 1972), which have properties very similar to those of synapses between neurons (Bekkers and Stevens, 1991;Johnson and Yee, 1995;Goda and Stevens, 1996). Such autapses show large postsynaptic responses and thereby provide a convenient system for studying short-term plasticity, especially under conditions in which neurotransmission is reduced such as during G-protein-mediated inhibition and presynaptic calcium channel blockade. ...
G-protein inhibition of voltage-gated calcium channels can be transiently relieved by repetitive physiological stimuli. Here, we provide evidence that such relief of inhibition contributes to short-term synaptic plasticity in microisland-cultured hippocampal neurons. With G-protein inhibition induced by the GABA(B) receptor agonist baclofen or the adenosine A1 receptor agonist 2-chloroadenosine, short-term synaptic facilitation emerged during action potential trains. The facilitation decayed with a time constant of approximately 100 msec. However, addition of the calcium channel inhibitor Cd(2+) at 2-3 microM had no such effect and did not alter baseline synaptic depression. As expected of facilitation from relief of channel inhibition, analysis of miniature EPSCs implicated presynaptic modulation, and elevating presynaptic Ca(2+) entry blunted the facilitation. Most telling was the near occlusion of synaptic facilitation after selective blockade of P/Q- but not N-type calcium channels. This was as predicted from experiments using recombinant calcium channels expressed in human embryonic kidney (HEK) 293 cells; we found significantly stronger relief of G-protein inhibition in recombinant P/Q- versus N-type channels during action potential trains. G-protein inhibition in HEK 293 cells was induced via recombinant M2 muscarinic acetylcholine receptors activated by carbachol, an acetylcholine analog. Thus, relief of G-protein inhibition appears to produce a novel form of short-term synaptic facilitation in cultured neurons. Similar short-term synaptic plasticity may be present at a wide variety of synapses, as it could occur during autoreceptor inhibition by glutamate or GABA, heterosynaptic inhibition by GABA, tonic adenosine inhibition, and in many other instances.
Vesicles within presynaptic terminals are thought to be segregated into a variety of readily releasable and reserve pools. The nature of the pools and trafficking between them is not well understood, but pools that are slow to mobilize when synapses are active are usually assumed to feed pools that are mobilized more quickly, in a series. However, results from electrophysiological studies of synaptic transmission suggested instead a parallel organization where vesicles within slowly and quickly mobilized reserve pools would separately feed independent reluctant- and fast-releasing subdivisions of the readily releasable pool, without intermixing. We now use FM-dyes to confirm the existence of multiple reserve pools at hippocampal synapses. We then confirm the prediction that slowly and quickly mobilized reserve pools do not intermix, even when mobilized by high frequency stimulation. The result provides a simplifying new constraint on the dynamics of vesicle recycling within presynaptic terminals. The experiments additionally demonstrated extensive heterogeneity among synapses in the relative sizes of slowly and quickly mobilized reserve pools. The heterogeneity suggests equivalent heterogeneity in the probability of release among readily releasable vesicles that may be relevant for understanding information processing and storage.
We describe a biological network and the principal mechanisms that are responsible for learning and memory. We start with a description of the morphology of these networks and their components, such as neurons and synapses. Then, we will identify crucial components of the information processing, such as ion flux and the induced mechanisms, e.g., long-term potentiation and depression. Next, we will compare the behaviour of a memristive system with the mechanisms identified in biological systems and present corresponding experiments and a few simulations. Finally, we will present more abstract ways of using memristors to solve complex problems.
The release of neurotransmitter molecules, which are stored in synaptic vesicles, is the first step in the process of communication between neurons through chemical synaptic transmission.17 This is achieved by coalescence of synaptic vesicles membrane with the plasma membrane of the synaptic terminal, i.e. the process of exocytosis and it is reversed or compensated for by endocytosis, the process of surface membrane withdrawal.10,14–15,58 Exocytosis and endocytosis are supposedly coupled events, and their balance maintains a stable number of functional synaptic vesicles and prevent the nerve terminal from enlarging and losing its specific features due to dilution with synaptic vesicles membrane components. The usual way to look at exo-endocytosis in neuronal cells is to record from the cell body of postsynaptic elements the electrical responses produced by synaptic release.17 In the central nervous system, these responses typically reflect the activity of several thousands synaptic contacts and most of our knowledge about synaptic properties comes from studies where large populations of synapses have been simultaneously activated.34 Although this approach has been essential to unravel many aspects of quantal transmission, individual differences between synapses which could be very important for the physiology of the brain are evidently obscured.34,44,38,53–54,60
In neural networks, information can be stored by altering the efficacy of synaptic transmission. Synaptic plasticity therefore provides a candidate mechanism underlying learning and memory. Since its initial discovery in 1973, long-term potentiation (LTP) has been in the spotlight as a cellular correlate of various forms of learning, particularly those mediated by the hippocampus. Later, it was discovered that synapses can also undergo long-term depression (LTD), often in an activity-dependent way. In this article, we describe the molecular and cellular mechanisms of LTD induction and expression at (1) hippocampal Schaffer collateral-CA1 pyramidal cell synapses and (2) parallel fiber and climbing fiber synapses, respectively, onto cerebellar Purkinje cells. Using these types of synapses as examples, we characterize the different roles of LTD and compare bidirectional plasticity mechanisms at hippocampal and cerebellar synapses.
For many years the importance of internal calcium stores (ICSs) in excitation–contraction coupling and endocrine function has been well recognized. With the discovery of ICSs in the CNS, evidence has accumulated regarding their role in neuronal function, and in particular, synaptic plasticity. In this review we focus on the involvement of ICSs in synaptic plasticity in the hippocampus.
This thesis is a collection of manuscripts addressing connectivity of neural circuits in cultured hippocampal neurons. These studies begin with an investigation of dopaminergic modulation of excitatory synapses in small circuits of neurons grown on glial micro islands. We found that dopamine transiently depressed excitatory synaptic transmission. Scaling up to larger circuits of neurons proved more challenging, since finding connected pairs became combinatorially more improbable. The discovery and use of light-activatable ion channel channel rhodopsin-2 (ChR2) promised to revolutionize the way in which we could map connectivity in vitro. We successfully delivered the gene for ChR2 in hippocampal cultures using recombinant adeno-associated virus and characterized the spatial resolution, as well as the reliability of stimulating action potentials. However, there were limitations to this technique that would render circuit maps ambiguous and incomplete. More recently, the engineering of rabies virus (RV) as a neural circuit tracer has produced an exciting method whereby viral infection can be targeted to a population of neurons and spread of the virus restricted to monosynaptically connected neurons. We further investigated potential mechanisms for previous observations which claim that RV spread is restricted to synaptically connected neurons by manipulating neural activity and synaptic vesicle release. We found that RV spread increased for blockade of synaptic vesicle exocytosis and for blockade of neural activity. The underlying premise for pursuing these methods to elucidate connectivity is that the computational power of the brain comes from changeable, malleable connectivity and that to test network models of computation in a biological brain, we must map the connectivity between individual neurons. This thesis builds a framework for experiments designed to bridge the gap between computational learning theories and networks of live neurons.
It has been proposed that during cognitive processes, “online” memory traces in the brain are carried by reverberatory activity
in neuronal circuits. However, the nature of such reverberation has remained elusive from experimental studies, largely due
to the enormous complexity of intact circuits. Recent works have attempted to address this issue using cultured neuronal network
and have revealed new dynamic properties of network reverberation as well as the underlying cellular mechanisms. These results
demonstrate the effectiveness of in vitro networks as a useful tool for mechanistic dissection of neuronal circuit dynamics.
The aims of this paper are to provide a comprehensive and up to date review of the mechanisms of induction and expression of long-term depression (LTD) of synaptic transmission. The review will focus largely on homosynaptic LTD and other forms of LTD will be considered only where appropriate for a fuller understanding of LTD mechanisms. We shall concentrate on what are felt to be some of the most interesting recent findings concerning LTD in the central nervous system. Wherever possible we shall try to consider some of the disparities in results and possible reasons for these. Finally, we shall briefly consider some of the possible functional consequences of LTD for normal physiological function.
Memristors cover a gap in the capabilities of basic electronic
components by remembering the history of the applied electric
potentials, and are considered to bring neuromorphic computers closer by
imitating the performance of synapses. We used memristive magnetic
tunnel junctions based on MgO to demonstrate that the synaptic
functionality is complemented by neuron-like behavior in these
nanoscopic devices. The synaptic functionality originates in a
resistance change caused by a voltage-driven oxygen vacancy motion
within the MgO layer. The additional functionality provided by magnetic
electrodes enabled a current-driven resistance modulation due to
spin-transfer torque. We report on memristive magnetic tunnel junctions
characterized by the simultaneous occurrence of resistive switching and
tunnel magnetoresistance. Since resistivity provides a natural measure
of the synaptic strength, and because of the bipolar nature of the
resistance change, long term potentiation and long term depression were
emulated. Furthermore, we show that the flux is a good variable for
describing voltage-induced resistance variation, which provides the
scope for the emulation of spike timing dependend plasticity as well.
Goda investigates how neurons fine-tune their signals.
Long-term potentiation (LTP) has received attention because of its proposed role in learning and memory. Despite substantial effort the pre- or postsynaptic expression site of LTP remains unsettled. It has been proposed that LTP is expressed postsynaptically through the functional conversion of "silent synapses." We had shown that Schaffer collateral (SC) silent and "functional synapses," which lack and express AMPA receptors, respectively exhibit distinct transmitter release properties. Therefore the functional conversion of silent synapses with LTP should be associated with presynaptic modifications. We now show that the pairing-induced LTP at SC synapses is mediated by combined pre- and postsynaptic modifications involving the postsynaptic emergence of an AMPA response coupled with an enhanced glutamate release. BDNF replicates the changes associated with this LTP by activating TrkBRs, suggesting that the neurotrophin is required for the coordinated changes on both sides of the synaptic cleft.
We present a method for studying synaptic transmission in mass cultures of dissociated hippocampal neurons based on patch clamp recording combined with laser stimulation of neurons expressing channelrhodopsin-2 (ChR2). Our goal was to use the high spatial resolution of laser illumination to come as close as possible to the ideal of identifying monosynaptically coupled pairs of neurons, which is conventionally done using microisland rather than mass cultures. Using recombinant adeno-associated virus (rAAV) to deliver the ChR2 gene, we focused on the time period between 14 and 20 days in vitro, during which expression levels are high, and spontaneous bursting activity has not yet started. Stimulation by wide-field illumination is sufficient to make the majority of ChR2-expressing neurons spike. Stimulation with a laser spot at least 10 microm in diameter also produces action potentials, but in a reduced fraction of neurons. We studied synaptic transmission by voltage-clamping a neuron with low expression of ChR2 and scanning a 40 microm laser spot at surrounding locations. Responses were observed to stimulation at a subset of locations in the culture, indicating spatial localization of stimulation. Pharmacological means were used to identify responses that were synaptic. Many responses were of smaller amplitude than those typically found in microisland cultures. We were unable to find an entirely reliable criterion for distinguishing between monosynaptic and polysynaptic responses. However, we propose that postsynaptic currents with small amplitudes, simple shapes, and latencies not much greater than 8 ms are reasonable candidates for monosynaptic interactions.
Phosphorylation of the transcription factor CREB is thought to be important in processes underlying long-term memory. It is unclear whether CREB phosphorylation can carry information about the sign of changes in synaptic strength, whether CREB pathways are equally activated in neurons receiving or providing synaptic input, or how synapse-to-nucleus communication is mediated. We found that Ca(2+)-dependent nuclear CREB phosphorylation was rapidly evoked by synaptic stimuli including, but not limited to, those that induced potentiation and depression of synaptic strength. In striking contrast, high frequency action potential firing alone failed to trigger CREB phosphorylation. Activation of a submembranous Ca2+ sensor, just beneath sites of Ca2+ entry, appears critical for triggering nuclear CREB phosphorylation via calmodulin and a Ca2+/calmodulin-dependent protein kinase.
It has been hypothesized that the direction of synaptic weight change elicited by synaptic activity depends on the magnitude of the activity-dependent rise in intracellular Ca2+ concentration. Several aspects of this hypothesis were examined at the Schaffer collateral CA1 synapse, where both long-term depression (LTD) and long-term potentiation (LTP) can be elicited and are Ca2+ dependent. Brief tetanic stimulation, which normally generated LTP, could induce LTD when Ca2+ entry via NMDA receptors was limited either by moderate concentrations of D-APV or by voltage clamping cells at negative membrane potentials. Repetitive activation of voltage-dependent Ca2+ channels in the absence of afferent stimulation could also elicit an LTD that was Ca2+ dependent and was occluded by prior generation of homosynaptic LTD using prolonged low evidence that the minimal requirements for inducing LTD involve simply a transient influx of Ca2+ into the postsynaptic cell, via either NMDA receptors or voltage-dependent Ca2+ channels.
The requirement for cooperative interactions between multiple synaptic inputs in the induction of long-term potentiation (LTP) and long-term depression (LTD) has been tested at Schaffer collateral synapses with paired recordings from monosynaptically coupled CA3-CA1 cell pairs in rat hippocampal slice cultures. Tetanization of single presynaptic neurons at 50 Hz (repeated 5-7 times for 300-500 ms each) induced only a transient potentiation (< 3 min) of excitatory postsynaptic potentials (EPSPs). Persistent potentiation (> 15 min) was induced only when single presynaptic action potentials were synchronously paired with directly induced postsynaptic depolarizing pulses (repeated 50-100 times). Tetanus-induced potentiation of extracellularly evoked EPSPs lasting > 4 min could only be obtained if the EPSP was > 4 mV. Because unitary EPSP amplitudes average approximately 1 mV, we conclude that high-frequency discharge must occur synchronously] in 4-5 CA3 cells for LTP to be induced in a common postsynaptic CA1 cell. Asynchronous pairing of presynaptic action potentials with postsynaptic depolarizing current pulses (preceding each EPSP by 800 ms) depressed both naive and previously potentiated unitary EPSPs. Likewise, homosynaptic LTD of unitary EPSPs was induced when the presynaptic cell was tetanized at 3 Hz for 3 min, regardless of their amplitude (0.3-3.2 mV). Homosynaptic LTD of extracellularly evoked Schaffer collateral EPSPs < 4 mV could be induced if no inhibitory postsynaptic potential was apparent, but was prevented by eliciting a large inhibitory postsynaptic potential or by injection of hyperpolarizing current in the postsynaptic cell. We conclude that cooperative interactions among multiple excitatory inputs are not required for induction of homosynaptic LTD of unitary EPSPs.
Nitric oxide (NO.) does not react significantly with thiol groups under physiological conditions, whereas a variety of endogenous NO donor molecules facilitate rapid transfer to thiol of nitrosonium ion (NO+, with one less electron than NO.). Here, nitrosonium donors are shown to decrease the efficacy of evoked neurotransmission while increasing the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs). In contrast, pure NO donors have little effect (displaying at most only a slight increase) on the amplitude of evoked EPSCs and frequency of spontaneous mEPSCs in our preparations. These findings may help explain heretofore paradoxical observations that the NO moiety can either increase, decrease, or have no net effect on synaptic activity in various preparations.
We describe how endogenous adenosine can prevent the induction of homosynaptic long-term depression (LTD) in the CA1 region of slices of adult rat hippocampus. Neither of two consecutive periods of prolonged low frequency stimulation (LFS; 1 Hz, 900 stimuli) of the Schaffer collateral-commissural fibres resulted in the induction of LTD in the CA1 region of hippocampal slices from adult (8-30 week) animals. However, in the presence of adenosine deaminase or the selective adenosine A1 receptor antagonist, 1,3-dipropyl-8-cyclopentyl-xanthine (DPCPX), LTD was induced by each of the first and second of two periods of LFS. The first period of LFS did not, but the second period of LFS did, induce LTD in the presence of DPCPX and the NMDA receptor antagonist, D-2-amino-5-phosphonopentanoate (AP5). The present results show that A1 receptor activation by endogenous adenosine can prevent the induction of LTD in the adult hippocampus.
The distinction between pre- or postsynaptic expression of synaptic plasticity is difficult to make, unless the postsynaptic receptors can be investigated in isolation. We have studied single synaptic contacts in dissociated cultures of rat hippocampus. The reaction of postsynaptic receptor assemblies to the induction of synaptic plasticity was measured and compared with changes in the rate of spontaneous miniature excitatory postsynaptic currents (mEPSCs), which can reflect changes in the transmitter release mechanism. The response of a receptor assembly to locally applied exogenous glutamate was measured before and after synchronized application of glutamate and a train of postsynaptic depolarizations ('pairing'). Pairing induced a variety of changes: (i) the majority of the receptor assemblies showed no change in their response to glutamate before and after pairing; (ii) the postsynaptic current due to exogenous glutamate showed a rapid increase in five out of 26 cases. This was not due to changes in the single channel conductance; (iii) the rate of mEPSCs increased, if it had previously been below 25 Hz; (iv) the rate of mEPSCs decreased, if it had previously been above 25 Hz. Effects 2 and 3 were blocked by antagonists of NMDA receptors. These findings provide direct evidence for an increase of the number of glutamate receptors at a subset of the investigated postsynaptic sites during synaptic potentiation.
Triple whole-cell recordings from simple networks of cultured hippocampal neurons show that Induction of long-term depression at glutamatergic synapses is accompanied by a back propagation of depression to Input synapses on the dendrite of the presynaptic neuron. The depression also propagates laterally to divergent outputs of the presynaptic neuron and to convergent inputs on the postsynaptic neuron. There is no forward propagation of depression to the output of the postsynaptic neuron and no presynaptic propagation accompanying long-term depression at GABAergic synapses. Activity-induced synaptic modification is therefore not restricted to the activated synapse, but selectively propagates throughout the neural network.
The time course of development of autaptic and synaptic connections and the contribution of endogenously activated cAMP signaling to the regulation of AMPA/kainate receptor-mediated synaptic transmission were studied in microcultures of isolated single hippocampal neurons or of pairs of neurons grown on astrocytic islands in serum-free culture medium. Standard whole cell patch clamp techniques were employed to monitor evoked and spontaneous autaptic and synaptic currents. Glutamatergic synaptic transmission became detectable after 4 days in vitro (DIV). After 9-10 DIV more than 80% of the neurons had developed glutamatergic autaptic and synaptic connections. Elevation of intracellular cAMP levels by application of forskolin (20 microM) or IBMX (200 microM) to autaptic neurons resulted in enhanced autaptic current amplitudes (forskolin: 146 +/- 9%, IBMX: 177 +/- 21% of control) and impaired paired pulse facilitation (PPF). Likewise, intracellular application of cAMP via the patch pipette into autaptic neurons or into the presynaptic neuron of a synaptically connected pair also resulted in enhanced autaptic/synaptic current amplitudes (170 +/- 16% of control). In contrast, injection of cAMP into the postsynaptic neuron of a synaptic pair failed to significantly enhance the synaptic responses. The magnitude of the cAMP-mediated enhancement depended on the initial autaptic/synaptic strength observed in an individual cell, with small autapses/synapses being enhanced more effectively. Application of an inhibitor of cAMP-mediated processes (Rp-cAMPS) reversibly reduced autaptic/synaptic current amplitudes (to 75 +/- 5% of control). Taken together, these results suggest that cAMP-mediated processes endogenously enhance the efficacy of developing glutamatergic autaptic and synaptic connections in serum-free microcultures of isolated hippocampal neurons.
We have investigated the site of expression of striatal long-term synaptic depression (LTD) using analysis of Sr2+-induced asynchronous release of quanta from stimulated synapses. The cumulative amplitude distribution of Sr2+-induced asynchronous synaptic responses overlaps with that of miniature EPSCs (mEPSCs), suggesting that Sr2+-induced asynchronous responses are quantal. Quantal amplitude at stimulated synapses is not significantly altered after LTD induction, whereas quantal frequency decreases after LTD induction. The decrease in quantal frequency is prevented when LTD expression is blocked by dialyzing 10 mM EGTA into the postsynaptic neuron. Our findings are most consistent with the idea that expression of striatal LTD involves decreased neurotransmitter release with no change in quantal amplitude, despite the fact that induction of striatal LTD involves postsynaptic mechanisms.
Retrograde signaling from the postsynaptic cell to the presynaptic neuron is essential for the development, maintenance, and activity-dependent modification of synaptic connections. This review covers various forms of retrograde interactions at developing and mature synapses. First, we discuss evidence for early retrograde inductive events during synaptogenesis and how maturation of presynaptic structure and function is affected by signals from the postsynaptic cell. Second, we review the evidence that retrograde interactions are involved in activity-dependent synapse competition and elimination in developing nervous systems and in long-term potentiation and depression at mature synapses. Third, we review evidence for various forms of retrograde signaling via membrane-permeant factors, secreted factors, and membrane-bound factors. Finally, we discuss the evidence and physiological implications of the long-range propagation of retrograde signals to the cell body and other parts of the presynaptic neuron.
Glutamate is the primary excitatory transmitter in axons innervating the hypothalamic suprachiasmatic nucleus (SCN) and is responsible for light-induced phase shifts of circadian rhythms generated by the SCN. By using self-innervating single neuron cultures and patch-clamp electrophysiology, we studied metabotropic glutamate receptors (mGluRs) expressed by SCN neurons. The selective agonists for group I (3,5-dihydroxy-phenylglycine), group II ((S)-4-carboxy-3-hydroxyphenylglycine), and group III ((+)-2-amino-4-phosphonobutyric acid) mGluRs all depressed the evoked IPSC in a subset (33%) of single autaptic neurons, suggesting a coexpression of all three groups of mGluRs in the same axon terminals of a single neuron. Other neurons showed a variety of combinations of mGluRs, including an expression of only one group of mGluR (18%) or coexpression of two groups of mGluRs (27%). Some neurons had no response to any of the three agonists (22%). The three mGluR agonists had no effect on postsynaptic gamma-aminobutyric acid (GABA) receptor responses, indicating a presynaptic modulation of GABA release by mGluRs. We conclude that multiple mGluRs that act through different second messenger pathways are coexpressed in single axon terminals of SCN neurons where they modulate the release of GABA presynaptically, usually inhibiting release.
In cultures of dissociated rat hippocampal neurons, persistent potentiation and depression of glutamatergic synapses were induced by correlated spiking of presynaptic and postsynaptic neurons. The relative timing between the presynaptic and postsynaptic spiking determined the direction and the extent of synaptic changes. Repetitive postsynaptic spiking within a time window of 20 msec after presynaptic activation resulted in long-term potentiation (LTP), whereas postsynaptic spiking within a window of 20 msec before the repetitive presynaptic activation led to long-term depression (LTD). Significant LTP occurred only at synapses with relatively low initial strength, whereas the extent of LTD did not show obvious dependence on the initial synaptic strength. Both LTP and LTD depended on the activation of NMDA receptors and were absent in cases in which the postsynaptic neurons were GABAergic in nature. Blockade of L-type calcium channels with nimodipine abolished the induction of LTD and reduced the extent of LTP. These results underscore the importance of precise spike timing, synaptic strength, and postsynaptic cell type in the activity-induced modification of central synapses and suggest that Hebb's rule may need to incorporate a quantitative consideration of spike timing that reflects the narrow and asymmetric window for the induction of synaptic modification.
Synaptic strength can be altered by a variety of pre- or postsynaptic modifications. Here we test the hypothesis that long-term depression (LTD) involves a decrease in the number of glutamate receptors that are clustered at individual synapses in primary cultures of hippocampal neurons. Similar to a prominent form of LTD observed in hippocampal slices, LTD in hippocampal cultures required NMDA receptor activation and was accompanied by a decrease in the amplitude and frequency of miniature excitatory postsynaptic currents. Immunocytochemical analysis revealed that induction of LTD caused a concurrent decrease in the number of AMPA receptors clustered at synapses but had no effect on synaptic NMDA receptor clusters. These results suggest that a subtype-specific redistribution of synaptic glutamate receptors contributes to NMDA receptor-dependent LTD.
Minimal excitatory postsynaptic potentials were evoked in CA3 pyramidal neurons by activation of the mossy fibres in hippocampal slices from seven- to 16-day-old rats. Conditioning intracellular depolarizing pulses were delivered as 50- or 100-Hz bursts. A statistically significant depression and potentiation was induced in four and five of 13 cases, respectively. The initial state of the synapses influenced the effect: the amplitude changes correlated with the pretetanic paired-pulse facilitation ratio. Afferent (mossy fibre) tetanization produced a significant depression in four of six inputs, and no significant changes in two inputs. Quantal content decreased or increased following induction of the depression or potentiation, respectively, whereas no significant changes in quantal size were observed. Compatible with presynaptic maintenance mechanisms of both depression and potentiation, changes in the mean quantal content were associated with modifications in the paired-pulse facilitation ratios, coefficient of variation of response amplitudes and number of response failures. Cases were encountered when apparently "presynaptically silent" synapses were converted into functional synapses during potentiation or when effective synapses became "presynaptically silent" when depression was induced, suggesting respective changes in the probability of transmitter release. It is concluded that, in juvenile rats, it is possible to induce lasting potentiation at the mossy fibre-CA3 synapses by purely postsynaptic stimulation, while afferent tetanization is accompanied by long-lasting depression. The data support the existence not only of a presynaptically induced, but also a postsynaptically induced form of long-term potentiation in the mossy fibre-CA3 synapse. Despite a postsynaptic induction mechanism, maintenance of both potentiation and depression is likely to occur presynaptically.
During intense presynaptic activity, the readily releasable pool (RRP) of synaptic vesicles empties more quickly than it can be refilled, and short-term depression results. Ordinarily, the pool refills within 20 s, but long, high-frequency trains of action potentials often induce a form of short-term depression that persists for a much longer time. Here, we report that replenishment of the RRP is governed by two simple processes: the previously identified mechanism termed refilling, and another process that appears after extensive exocytosis and produces a transient decrease in the capacity of the pool, lasting for several minutes. The data presented here place stringent constraints on the types of kinetic models that can be used to describe synaptic vesicular cycling and are inconsistent with the traditional multipool models of vesicular mobilization.
The proenzyme of a Ca2+-dependent protease-activated protein kinase previously obtained from mammalian tissues (Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616) was enzymatically fully active without limited proteolysis when Ca2+ and a membrane-associated factor were simultaneously present in the reaction mixture. The activation process was reversed by removing Ca2+ with ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid. An apparent Ka value for Ca2+ was less than 5 x 10(-5) M. Other divalent cations were inactive except for Sr2+, which was 5% as active as Ca2+. The factor was almost exclusively localized in membrane fractions of various tissues including brain, liver, kidney, skeletal muscle, blood cells, and adipose tissue. It was easily extractable with chloroform/methanol (2:1), and was recovered in the phospholipid fraction. In fact, this membrane factor could be replaced by chromatographically pure phosphatidylinositol, phosphatidylserine, phosphatidic acid, or diphosphatidylglycerol. Phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin were far less effective under the comparable conditions. Ca2+-dependent modulator protein was unable to support enzymatic activity. The enzyme thus activated showed an ability to phosphorylate five histone fractions and muscle phosphorylase kinase, and appeared to possess multifunctional catalytic activities.
We tested a theoretical prediction that patterns of excitatory input activity that consistently fail to activate target neurons sufficiently to induce synaptic potentiation will instead cause a specific synaptic depression. To realize this situation experimentally, the Schaffer collateral projection to area CA1 in rat hippocampal slices was stimulated electrically at frequencies ranging from 0.5 to 50 Hz. Nine hundred pulses at 1-3 Hz consistently yielded a depression of the CA1 population excitatory postsynaptic potential that persisted without signs of recovery for greater than 1 hr after cessation of the conditioning stimulation. This long-term depression was specific to the conditioned input, ruling out generalized changes in postsynaptic responsiveness or excitability. Three lines of evidence suggest that this effect is accounted for by a modification of synaptic effectiveness rather than damage to or fatigue of the stimulated inputs. First, the effect was dependent on the stimulation frequency; 900 pulses at 10 Hz caused no lasting change, and at 50 Hz a synaptic potentiation was usually observed. Second, the depressed synapses continued to support long-term potentiation in response to a high-frequency tetanus. Third, the effects of conditioning stimulation could be prevented by application of NMDA receptor antagonists. Thus, our data suggest that synaptic depression can be triggered by prolonged NMDA receptor activation that is below the threshold for inducing synaptic potentiation. We propose that this mechanism is important for the modifications of hippocampal response properties that underlie some forms of learning and memory.
Individual rat hippocampal neurons, grown in isolation from other neurons on small spots of permissive substrate, were studied in order to characterize the electrical properties of the synapses that such cells formed with themselves (autapses). Excitatory (probably glutamatergic) or inhibitory (probably type A gamma-aminobutyratergic) autapses were frequently found. Excitatory autaptic currents reversed near the potential expected for monovalent cations were blocked by the glutamatergic antagonist kynurenic acid, and possessed a slow component with the pharmacological profile of N-methyl-D-aspartate-type channels. These currents also exhibited trial-to-trial statistical fluctuations in their amplitudes, this being well-described by quantal analysis. Inhibitory autaptic currents reversed at hyperpolarized potentials, as expected for chloride-permeable pores and were blocked by picrotoxin, a type A gamma-aminobutyric receptor antagonist. It is concluded that autaptic currents in culture are identical to those found at synapses.
The size of synaptic quanta has been found to display considerable variation in cultured hippocampal neurons, but the source of this variability was previously unknown. We have now compared the properties of locally evoked miniature excitatory postsynaptic currents in cultured hippocampal neurons and in thin hippocampal slices using whole-cell patch-clamp recordings. The variability in miniature excitatory postsynaptic current size was similar in both preparations and occurred in cultured neurons when only one or a few synaptic boutons were stimulated. Thus, the variability in miniature excitatory postsynaptic current amplitude is not an artifact of cultured neurons and arises predominantly from variability within a single bouton. Possible origins of this variability are discussed.
Depolarization-induced (potassium-stimulated) influx of 45Ca, 85Sr, and 133Ba was measured in synaptosomes prepared from rat brain. There are two phases of divalent cation entry, "fast" and "slow;" each phase is mediated by channels with distinctive characteristics. The fast channels inactivate (within 1 s) and are blocked by low concentrations (less than 1 micro M) of La. The slow channels do not inactivate (within 10 s), and are blocked by high concentrations (greater than 50 micro M) of La. Divalent cation influx through both channels saturates with increasing concentrations of permeant divalent cation; in addition, each permeant divalent cation species competitively blocks the influx of other permeant species. These results are consistent with the presence of "binding sites" for divalent cations in the fast and slow channels. The Ca:Sr:Ba permeability ratio, determined by measuring the influx of all three species in triple-label experiments, was 6:3:2 for the fast channel and 6:3:1 for the slow channel. A simple model for ion selectivity, based on the presence of a binding site in the channel, could account well for slow and, to some extent, for fast, channel selectivity data.
1. We examined the effects of the metabotropic glutamate receptor (mGluR) antagonist alpha-methyl-4-carboxyphenylglycine (MCPG) on the induction of long-term potentiation (LTP) long-term depression (LTD), and depotentiation in CA1 hippocampal neurons using extracellular recording techniques. 2. MCPG (500 microM) strongly antagonized the presynaptic inhibitory action of the mGluR agonist 1-aminocyclopentane-(1S,3R)-dicarboxylic acid yet failed to block LTP induced with either tetanic stimulation (100 Hz, 1 s) or theta-burst stimulation. 3. To test the possibility that our failure to block LTP was due to prior activation of a "molecular switch" that in its "on" state obviates the need for mGluR activation to generate LTP, we gave repeated periods of prolonged low-frequency stimulation (LFS; 1 Hz, 10 min), a manipulation reported to turn the switch "off." Although this stimulation saturated LTD, subsequent application of MCPG still failed to block LTP. 4. MCPG did not block LFS-induced depotentiation in older slices (4-6 wk) or LFS-induced LTD in older, young (11-18 days), or neonatal (3-7 days) slices. 5. These results demonstrate that MCPG-sensitive mGluRs are not necessary for the induction of LTP, LTD, or depotentiation in hippocampal CA1 pyramidal cells. The possibility remains, however, that their activation may modify the threshold for the induction of these long-term plastic changes.
Long-term potentiation (LTP) is a synaptic mechanism thought to be involved in learning and memory. Long-term depression (LTD), an activity-dependent decrease in synaptic efficacy, may be an equally important mechanism which permits neural networks to store information more effectively. One form of LTD that has been observed in the hippocampus requires activation of postsynaptic NMDA (N-methyl-D-aspartate) receptors, a change in postsynaptic calcium concentration, and activation of postsynaptic serine/threonine protein phosphatase 1 (PP1) or 2A (PP2A). The mechanism by which PP1 or PP2A is regulated by synaptic activity is unclear because these protein phosphatases are not directly influenced by calcium concentration. LTD induction may require activation of a more complex protein phosphatase cascade consisting of the Ca2+/calmodulin-dependent protein phosphatase, calcineurin, its phosphoprotein substrate, inhibitor-1, and PP1. We tested this hypothesis using calcineurin inhibitors as well as different forms of inhibitor-1 loaded into postsynaptic cells. Our results suggest a signalling pathway in which calcineurin dephosphorylates and inactivates inhibitor-1. This in turn increases PP1 activity and contributes to the generation of LTD.
Presynaptic injection of inositol 1,3,4,5-tetraphosphate, inositol 1,3,4,5,6-pentakisphosphate, or inositol 1,2,3,4,5,6-hexakisphosphate--which we denote here the inositol high-polyphosphate series (IHPS)--is shown to block synaptic transmission when injected into the preterminal of the squid giant synapse. This effect is not produced by injection of inositol 1,4,5-trisphosphate. The synaptic block is characterized by a time course in the order of 15-45 min, depending on the injection site in the preterminal fiber; the fastest block occurs when the injection is made at the terminal release site. Presynaptic voltage clamp during transmitter release demonstrates that IHPS block did not modify the presynaptic inward, calcium current. Analysis of synaptic noise at the postsynaptic axon shows that both the evoked and spontaneous transmitter release are blocked by the IHPS. Tetanic stimulation of the presynaptic fiber at frequencies of 100 Hz indicates that block is accompanied by gradual reduction of the postsynaptic response, demonstrating that the block interferes with vesicular fusion rather than with vesicular docking. These results, in combination with the recently demonstrated observation that the IHPS bind the C2B domain in synaptotagmin [Fukada, M., Aruga, J., Niinobe, M., Aimoto, S. & Mikoshiba, K. (1994) J. Biol. Chem. 269, 29206-29211], suggest that IHPS elements are involved in vesicle fusion and exocytosis. In addition, a scheme is proposed in which synaptotagmin triggers transmitter release directly by promoting the fusion of synaptic vesicles with the presynaptic plasmalemma, in agreement with the very rapid nature of transmitter release in chemical synapses.
After the arrival of a presynaptic nerve impulse at an excitatory synapse in hippocampal neurons, the rate of neurotransmitter release increases rapidly and then returns to low levels with a biphasic decay. The two kinetically distinct components are differentially affected when Sr2+ is substituted for Ca2+ ions. Our findings are comparable to those of the classical studies for the frog neuromuscular junction, and thus the basic aspects of Ca(2+)-activated transmitter release machinery appear to be conserved in central synapses. The method we have used, in addition, permits us to estimate the average neurotransmitter release rate for a single bouton. The observation of differential Ca2+/Sr2+ sensitivity is consistent with a release mechanism mediated by two Ca2+ sensors with distinct Ca2+ affinities: the low-affinity Ca2+ sensor facilitates the fast synchronous phase of release, whereas the high-affinity sensor sustains the slow asynchronous phase of release.
Synaptic transmission in the hippocampus is rather unreliable, with many presynaptic action potentials failing to release neurotransmitter. How is this unreliability affected by the alterations in synaptic strength seen in long-term potentiation (LTP) and long-term depression (LTD)? We find that LTP increases synaptic reliability, and LTD decreases it, both without a change in the size of those postsynaptic currents that do occur. Thus LTD is a functional inverse of LTP.
Synaptotagmin is an integral synaptic vesicle protein proposed to be involved in Ca(2+)-dependent exocytosis during synaptic transmission. Null mutations in synaptotagmin have been made in Drosophila, and the protein's in vivo function has been assayed at the neuromuscular synapse. In the absence of synaptotagmin, synaptic transmission is dramatically impaired but is not abolished. In null mutants, evoked vesicle release is decreased by a factor of 10. Moreover, the fidelity of excitation-secretion coupling is impaired so that a given stimulus generates a more variable amount of secretion. However, this residual evoked release shows Ca(2+)-dependence similar to normal release, suggesting either that synaptotagmin is not the Ca2+ sensor or that a second, independent Ca2+ sensor exists. While evoked transmission is suppressed, the rate of spontaneous vesicle fusion is increased by a factor of 5. We conclude that synaptotagmin is not an absolutely essential component of the Ca(2+)-dependent secretion pathway in synaptic transmission but is necessary for normal levels of transmission. Our data support a model in which synaptotagmin functions as a negative regulator of spontaneous vesicle fusion and acts to increase the efficiency of excitation-secretion coupling during synaptic transmission.
Transmission at individual synaptic contacts on CA1 hippocampal pyramidal neurons has been found to be very unreliable, with greater than half of the arriving presynaptic nerve impulses failing to evoke a postsynaptic response. This conclusion has been reached using the method of minimal stimulation of Schaffer collaterals and whole cell recording in hippocampal slices; with minimal stimulation only one or a few synapses are activated on the target neuron and the behavior of individual synapses can be examined. Four sources for the unreliability of synaptic transmission have been investigated: (i) the fluctuation of axon thresholds at the site of stimulation causing the failure to generate a nerve impulse in the appropriate Schaffer collaterals, (ii) the failure of nerve impulses generated at the site of stimulation to arrive at the synapse because of conduction failures at axon branch points, (iii) an artifactual synaptic unreliability due to performing experiments in vitro at temperatures well below the normal mammalian body temperature, and (iv) transmission failures due to probabilistic release mechanisms at synapses with a very low capacity to release transmitter. We eliminate the first three causes as significant contributions and conclude that probabilistic release mechanisms at low capacity synapses are the main cause of unreliability of synaptic transmission.
IP4BP/Synaptotagmin II is an inositol-1,3,4,5-tetrakisphosphate (IP4) or inositol polyphosphate-binding protein, which is accumulated at nerve terminals. Here we report a novel function of the C2B domain, which was originally thought to be responsible for Ca(2+)-dependent binding to phospholipid membranes. A study of deletion mutants showed that about 30 amino acids of the central region of the C2B domain of mouse IP4BP/synaptotagmin II (315 IHLMQNGKRLKKKKTTVKKKTLNPYFNESFSF 346) are essential for inositol polyphosphate binding. This binding domain includes a sequence corresponding to the squid Pep20 peptide, which is also known to be essential for neurotransmitter release (Bommert, K., Charlton, M. P., DeBello, W. M., Chin, G. J., Betz, H., and Augustine, G. J. (1993) Nature 363, 163-165), suggesting that inositol polyphosphate has some effect on neurotransmitter release. Rabphilin 3A, another neuronal protein containing C2 domains, cannot bind IP4, indicating that the IP4 binding property is specific to the C2B domain of synaptotagmin. Phospholipid and IP4 binding experiments clearly indicated that the C2A and C2B domains have different functions. The C2A domain binds phospholipid in a Ca(2+)-dependent manner, but the C2B domain binds inositol polyphosphate and phospholipid irrespective of the presence of Ca2+. Our data suggest that the C2B domain of synaptotogamin is the inositol polyphosphate sensor at the synaptic vesicle and may be involved in synaptic function.
Since the demonstration that Ca2+ influx into the presynaptic terminal is essential for neurotransmitter release, there has been much speculation about the Ca2+ receptor responsible for initiating exocytosis. Numerous experiments have shown that the protein, or protein complex, binds multiple Ca2+ ions, resides near the site of Ca2+ influx, and has a relatively low affinity for Ca2+. Synaptotagmin is an integral membrane protein of synaptic vesicles that contains two copies of a domain known to be involved in Ca(2+)-dependent membrane interactions. Synaptotagmin has been shown to bind Ca2+ in vitro with a relatively low affinity. In addition, synaptotagmin has been shown to bind indirectly to Ca2+ channels, positioning the protein close to the site of Ca2+ influx. Recently, a negative regulatory role for synaptotagmin has been proposed, in which it functions as a clamp to prevent fusion of synaptic vesicles with the presynaptic membrane. Release of the clamp would allow exocytosis. Here we present genetic and electrophysiological evidence that synaptotagmin forms a multimeric complex that can function as a clamp in vivo. However, upon nerve stimulation and Ca2+ influx, all synaptotagmin mutations dramatically decrease the ability of Ca2+ to promote release, suggesting that synaptotagmin probably plays a key role in activation of synaptic vesicle fusion. This activity cannot simply be attributed to the removal of a barrier to secretion, as we can electrophysiologically separate the increase in rate of spontaneous vesicle fusion from the decrease in evoked response. We also find that some syt mutations, including those that lack the second Ca(2+)-binding domain, decrease the fourth-order dependence of release on Ca2+ by approximately half, consistent with the hypothesis that a synaptotagmin complex functions as a Ca2+ receptor for initiating exocytosis.
Synaptotagmin I is a Ca(2+)- and phospholipid-binding protein of synaptic vesicles with an essential function in neurotransmission. Ca2+/phospholipid binding by synaptotagmin I may be mediated by its C2 domains, sequence motifs that have been implicated in the Ca2+ regulation of a variety of proteins. However, it is currently unknown if C2 domains are sufficient for Ca2+/phospholipid binding or if they even directly participate in Ca2+/phospholipid binding. In order to address this question, we have studied the Ca2+/phospholipid-binding properties of the first C2 domain of synaptotagmin I. Our results show that this C2 domain by itself binds Ca2+ and phospholipids with high affinity (half-maximal binding at 4-6 microM free Ca2+) and exhibits strong positive cooperativity. The C2 domain is specific for negatively charged phospholipids and for those divalent cations that are known to stimulate synaptic vesicle exocytosis (Ca2+ > Sr2+, Ba2+ > Mg2+). These studies establish that C2 domains can serve as independently folding Ca2+/phospholipid-binding domains. Furthermore, the cation specificity and the cooperativity of Ca2+ binding by the C2 domain from synaptotagmin I support a role for this protein in mediating the Ca2+ signal in neurotransmitter release.
The N-ethylmaleimide-sensitive fusion protein (NSF) and the soluble NSF attachment proteins (SNAPs) appear to be essential components of the intracellular membrane fusion apparatus. An affinity purification procedure based on the natural binding of these proteins to their targets was used to isolate SNAP receptors (SNAREs) from bovine brain. Remarkably, the four principal proteins isolated were all proteins associated with the synapse, with one type located in the synaptic vesicle and another in the plasma membrane, suggesting a simple mechanism for vesicle docking. The existence of numerous SNARE-related proteins, each apparently specific for a single kind of vesicle or target membrane, indicates that NSF and SNAPs may be universal components of a vesicle fusion apparatus common to both constitutive and regulated fusion (including neurotransmitter release), in which the SNAREs may help to ensure vesicle-to-target specificity.
p65 (synaptotagmin), an abundant synaptic vesicle protein, has been implicated in the processes of vesicle docking and fusion. To characterize further the properties of this important neuronal protein, we have investigated its phosphorylation in vitro. Immunoprecipitation of p65 results in coprecipitation of a protein kinase that phosphorylates p65 as well as syntaxin, a plasma membrane protein that interacts with p65. p65 is phosphorylated on a threonine residue (Thr-128) within the cytoplasmic domain near the transmembrane region. The coprecipitating protein kinase was identified as casein kinase II based on its catalytic properties, the sequence surrounding Thr-128, and Western blot analysis of the anti-p65 immunoprecipitates. Affinity chromatography utilizing bacterially expressed fragments of p65 demonstrated that casein kinase II interacts with a domain of p65 distinct from the phosphorylation site. In a synaptic vesicle fraction, the phosphorylation of p65 is stimulated by sphingosine and by detergent solubilization, suggesting that p65 phosphorylation may be subject to regulatory processes.
The mechanisms responsible for long-lasting, activity-dependent decreases in synaptic efficacy are not well understood. We have examined the initial steps required for the induction of long-term depression (LTD) in CA1 pyramidal cells by repetitive low frequency (1 Hz) synaptic stimulation. This form of LTD was synapse specific, was saturable, and required activation of post-synaptic NMDA receptors. Loading CA1 cells with the Ca2+ chelator BAPTA prevented LTD, whereas lowering extracellular Ca2+ resulted in the induction of LTD by stimulation that previously elicited long-term potentiation. Following LTD, synaptic strength could be increased to its original maximal level, indicating that LTD is reversible and not due to deterioration of individual synapses. Induction of homosynaptic LTD therefore requires an NMDA receptor-dependent change in postsynaptic Ca2+ which may be distinct from that required for long-term potentiation.
Single channel and whole cell recordings were used to study ion permeation through Ca channels in isolated ventricular heart cells of guinea pigs. We evaluated the permeability to various divalent and monovalent cations in two ways, by measuring either unitary current amplitude or reversal potential (Erev). According to whole cell measurements of Erev, the relative permeability sequence is Ca2+ greater than Sr2+ greater than Ba2+ for divalent ions; Mg2+ is not measurably permeant. Monovalent ions follow the sequence Li+ greater than Na+ greater than K+ greater than Cs+, and are much less permeant than the divalents. These whole cell measurements were supported by single channel recordings, which showed clear outward currents through single Ca channels at strong depolarizations, similar values of Erev, and similar inflections in the current-voltage relation near Erev. Information from Erev measurements stands in contrast to estimates of open channel flux or single channel conductance, which give the sequence Na+ (85 pS) greater than Li+ (45 pS) greater than Ba2+ (20 pS) greater than Ca2+ (9 pS) near 0 mV with 110-150 mM charge carrier. Thus, ions with a higher permeability, judged by Erev, have lower ion transfer rates. In another comparison, whole cell Na currents through Ca channels are halved by less than 2 microM [Ca]o, but greater than 10 mM [Ca]o is required to produce half-maximal unitary Ca current. All of these observations seem consistent with a recent hypothesis for the mechanism of Ca channel permeation, which proposes that: ions pass through the pore in single file, interacting with multiple binding sites along the way; selectivity is largely determined by ion affinity to the binding sites rather than by exclusion by a selectivity filter; occupancy by only one Ca ion is sufficient to block the pore's high conductance for monovalent ions like Na+; rapid permeation by Ca ions depends upon double occupancy, which only becomes significant at millimolar [Ca]o, because of electrostatic repulsion or some other interaction between ions; and once double occupancy occurs, the ion-ion interaction helps promote a quick exit of Ca ions from the pore into the cell.
AT the neuromuscular junction1, as well as at neuronal synapses2, calcium plays an essential and direct part in the process whereby depolarization of the presynaptic nerve terminal leads to release of the transmitter substance. It is also known that a nerve impulse releases the transmitter in quantal form, that is, in discrete multi-molecular amounts of a fairly standard size, and that calcium acts by increasing the probability of such transmitter release. The question is raised whether other ions can replace calcium in this process, and if so, is the transmitter still released in quantal form ? Or is the quantal nature dependent on the specific combination of calcium with a receptor in the nerve membrane ?
We present experimental procedures describing the creation of perforated patches by use of amphotericin B. In 13 different cellular preparations, access resistances below 10 M omega were achieved and with blunt electrode tips, access resistances of 3-4 M omega were possible. In addition to using the techniques to measure whole cell currents, we have used them to measure single channel currents in a new "outside-out patch" preparation and we have utilized them to measure the resting voltage of epithelial monolayers. We conclude that these new approaches can provide a substantial increase in versatility and quality for many kinds of electrophysiological measurements.
Neurotransmitters are released at synapses by the Ca2(+)-regulated exocytosis of synaptic vesicles, which are specialized secretory organelles that store high concentrations of neurotransmitters. The rapid Ca2(+)-triggered fusion of synaptic vesicles is presumably mediated by specific proteins that must interact with Ca2+ and the phospholipid bilayer. We now report that the cytoplasmic domain of p65, a synaptic vesicle-specific protein that binds calmodulin contains an internally repeated sequence that is homologous to the regulatory C2-region of protein kinase C (PKC). The cytoplasmic domain of recombinant p65 binds acidic phospholipids with a specificity indicating an interaction of p65 with the hydrophobic core as well as the headgroups of the phospholipids. The binding specificity resembles PKC, except that p65 also binds calmodulin, placing the C2-regions in a context of potential Ca2(+)-regulation that is different from PKC. This is a novel homology between a cellular protein and the regulatory domain of protein kinase C. The structure and properties of p65 suggest that it may have a role in mediating membrane interactions during synaptic vesicle exocytosis.
1. Fluctuations in the latency of focally recorded end‐plate currents were analysed to determine the time course of the probabilistic presynaptic process underlying quantal release evoked after single nerve stimuli at the frog neuromuscular junction.
2. The early falling phase of the presynaptic probability function can be fitted by a single exponential over two orders of magnitude of quantal release rate. The time constant of the early falling phase is about 0·5 msec at 11° C, and increases with decreasing temperature with a Q 10 of at least 4 over the range 1–12° C.
3. After this early exponential fall, quantal release probability returns to control levels with a much slower time course.
4. Conditioning nerve stimuli increase the magnitude and slightly prolong the early time course of release evoked by a test stimulus. When facilitation is calculated for matched time intervals following the conditioning and testing stimuli, it is found that the magnitude of the small, late residual tail of release is facilitated by a greater percentage than the magnitude of larger, early portions of release.
5. These results are discussed in terms of the hypothesis (Katz & Miledi, 1968) that evoked release and facilitation are mediated by a common presynaptic factor which activates release in a non‐linear manner.
1. The quantitative dependence of transmitter release on external calcium concentration has been studied at the frog neuromuscular junction, using intracellular recording and taking the amplitude of the end‐plate potential (e.p.p.) as an index of the number of packets released.
2. The relation between [Ca] and the e.p.p. is highly non‐linear. The initial part of this relation on double logarithmic co‐ordinates gives a straight line with a slope of nearly four (mean 3·78 ± 0·2 S.D. in 28 experiments). Addition of a constant amount of Mg reduces the e.p.p. without altering the slope of the log e.p.p./log Ca relation.
3. The slope of this logarithmic relation diminishes as [Ca] is raised towards the normal level.
4. The results are explained quantitatively on the hypothesis that Ca ions combine with a specific site X on the nerve terminal forming CaX, and that the number of packets of acetylcholine released is proportional to the fourth power of [CaX].
5. The analysis suggests that a co‐operative action of about four calcium ions is necessary for the release of each quantal packet of transmitter by the nerve impulse.
A soluble Ca2+/calmodulin-dependent protein kinase has been purified from rat brain to near homogeneity by using casein as substrate. The enzyme was purified by using hydroxylapatite adsorption chromatography, phosphocellulose ion-exchange chromatography, Sepharose 6B gel filtration, affinity chromatography using calmodulin-Sepharose 4B, and ammonium sulfate precipitation. On sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels, the purified enzyme consists of three protein bands: a single polypeptide of 51 000 daltons and a doublet of 60 000 daltons. Measurements of the Stokes radius by gel filtration (81.3 +/- 3.7 A) and the sedimentation coefficient by sucrose density sedimentation (13.7 +/- 0.7 S) were used to calculate a native molecular mass of 460 000 +/- 29 000 daltons. The kinase autophosphorylated both the 51 000-dalton polypeptide and the 60 000-dalton doublet, resulting in a decreased mobility in NaDodSO4 gels. Comparison of the phosphopeptides produced by partial proteolysis of autophosphorylated enzyme reveals substantial similarities between subunits. These patterns, however, suggest that the 51 000-dalton subunit is not a proteolytic fragment of the 60 000-dalton doublet. Purified Ca2+/calmodulin-dependent casein kinase activity was dependent upon Ca2+, calmodulin, and ATP X Mg2+ or ATP X Mn2+ when measured under saturating casein concentrations. Co2+, Mn2+, and La3+ could substitute for Ca2+ in the presence of Mg2+ and saturating calmodulin concentrations. In addition to casein, the purified enzyme displayed a broad substrate specificity which suggests that it may be a "general" protein kinase with the potential for mediating numerous processes in brain and possibly other tissues.
Calmodulin is the major intracellular, and intramembrane, Ca2+ receptor. Its function is to detect free Ca2+ and translate changes in Ca2+ concentrations into altered states of metabolism. Liganded calmodulin interacts with a large number of enzymes, particularly those of nucleotide phosphate transfer and hydrolysis. In so doing, the protein provides a simple mechanism by which systems under Ca2+ control are regulated. Specificity of response in the calmodulin system is almost certainly dictated through a combination of compartmentalization and genetic regulation. Parallel genetic regulation of specific sets of calmodulin-sensitive enzymes required to produce specific internal biochemical responses in a particular cell type is also important in providing for differentiated cellular responsiveness. Calmodulin serves as a coupling link between the two major second-messenger systems, Ca2+ and CAMP. It does so because of its interactions with some, but not all, species of each of the enzymes related to cAMP function: cyclic nucleotide phosphodiesterase, adenylate cyclase, and cAMP-regulated protein kinase. Both Ca2+-calmodulin and cAMP are fundamental stimulators of enzyme action.
While the mechanisms responsible for LTP and LTD of excitatory synaptic responses mediated by AMPA receptors (AMPARs) have been extensively characterized, much less is known about the regulation of NMDA receptors (NMDARs) by synaptic activity. In hippocampal CA1 cells, prolonged low frequency afferent stimulation depresses synaptic responses mediated by either NMDARs or AMPARs. However, this apparently similar LTD is accompanied by a change in the coefficient of variation (CV) of only the AMPAR-mediated synaptic responses; the CV of the NMDAR-mediated synaptic responses is unaffected. Moreover, by varying the pattern of synaptic stimulation, the responses mediated by one receptor subtype can be modified without affecting the responses mediated by the other. These results indicate that the mechanisms underlying activity-dependent plasticity of NMDAR-mediated synaptic responses are different from those responsible for plasticity of AMPAR-mediated synaptic responses.
Developmental changes in rat hippocampal transmitter release and synaptic plasticity were investigated. Recordings from pairs
of pyramidal neurons in slices showed that an action potential in a CA3 neuron released only a single quantum of transmitter
onto a CA1 neuron. Failures of synaptic transmission reflected probabilistic transmitter release. The probability of release
(Pr) was 0.9 in 4- to 8-day-old rats and decreased to less than 0.5 at 2 to 3 weeks. Long-term potentiation (LTP) in 2- to
3-week-old rats was associated with an increase in Pr from a single synaptic site. The high initial Pr in 4- to 8-day-old
rats normally occludes the expression of LTP at this stage.
Recent work has suggested that some proportion of excitatory synapses on hippocampal CA1 pyramidal cells that express NMDA receptors (NMDARs) may not express functional AMPA receptors (AMPARs), thus making these synapses silent at the resting membrane potential. In agreement with this hypothesis, we demonstrate here that it is possible to stimulate synapses that yield no detectable excitatory postsynaptic currents (EPSCs) when the cell is held at -60 mV; yet at positive holding potentials (+30 to +60 mV), EPSCs can be elicited that are completely blocked by the NMDAR antagonist, D-APV. When these functionally silent synapses are subjected to an LTP induction protocol, EPSCs mediated by AMPARs appear and remain for the duration of the experiment. This conversion of silent synapses to functional synapses is blocked by D-APV. These results suggest that LTP may involve modification of AMPARs that, prior to LTP, were either not present in the postsynaptic membrane or electrophysiologically silent. This mechanism may account for several experimental results previously attributed to presynaptic changes in quantal content.
Long-term potentiation (LTP) is an enhancement of synaptic strength that can be produced by pairing of presynaptic activity with postsynaptic depolarization. LTP in the hippocampus has been extensively studied as a cellular model of learning and memory, but the nature of the underlying synaptic modification remains elusive, partly because our knowledge of central synapses is still limited. One proposal is that the modification is postsynaptic, and that synapses expressing only NMDA (N-methyl-D-aspartate) receptors before potentiation are induced by LTP to express functional AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate) receptors. Here we report that a high proportion of synapses in hippocampal area CA1 transmit with NMDA receptors but not AMPA receptors, making these synapses effectively non-functional at normal resting potentials. These silent synapses acquire AMPA-type responses following LTP induction. Our findings challenge the view that LTP in CA1 involves a presynaptic modification, and suggest instead a simple postsynaptic mechanism for both induction and expression of LTP.
Synaptotagmins (Syts) are brain-specific Ca2+/phospholipid-binding proteins. In hippocampal synapses, Syt I is essential for fast Ca(2+)-dependent synaptic vesicle exocytosis but not for Ca(2+)-independent exocytosis. In vertebrates and invertebrates, Syt may therefore participate in Ca(2+)-dependent synaptic membrane fusion, either by serving as the Ca2+ sensor in the last step of fast Ca(2+)-triggered neurotransmitter release, or by collaborating with an additional Ca2+ sensor. While Syt I binds Ca2+ (refs 10, 11), its phospholipid binding is triggered at lower calcium concentrations (EC50 = 3-6 microM) than those required for exocytosis. Furthermore, Syts bind clathrin-AP2 with high affinity, indicating that they may play a general role in endocytosis rather than being confined to a specialized function in regulated exocytosis. Here we resolve this apparent contradiction by describing four Syts, three of which (Syt VI, VII and VIII) are widely expressed in non-neural tissues. All Syts tested share a common domain structure, with a cytoplasmic region composed of two C2 domains that interacts with clathrin-AP2 (Kd = 0.1-1.0 nM) and with neural and non-neural syntaxins. The first C2 domains of Syt I, II, III, V and VII, but not of IV, VI or VIII, bind phospholipids with a similar Ca(2+)-concentration dependence (EC50 = 3-6 microM). The same C2 domains also bind syntaxin as a function of Ca2+ but the Ca(2+)-concentration dependence of Syt I, II and V (> 200 microM) differs from that of Syt III and VII (< 10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)
A change in the probability of neurotransmitter release (Pr) is an important mechanism underlying synaptic plasticity. Although Pr is often assumed to be the same for all terminals at a single synapse, this assumption is difficult to reconcile with the nonuniform size and structure of synaptic terminals in the central nervous system. Release probability was measured at excitatory synapses on cultured hippocampal neurons by analysis of the progressive block of N-methyl-D-aspartate receptor-mediated synaptic currents by the irreversible open channel blocker MK-801. Release probability was nonuniform (range of 0.09 to 0.54) for terminals arising from a single axon, the majority of which had a low Pr. However, terminals with high Pr are more likely to be affected by the activity-dependent modulation that occurs in long-term potentiation.
When an action potential reaches a synaptic terminal, fusion of a transmitter-containing vesicle with the presynaptic membrane occurs with a probability (pr) of less than one. Despite the fundamental importance of this parameter, pr has not been directly measured in the central nervous system. Here we describe a novel approach to determine pr, monitoring the decrement of NMDA (N-methyl-D-aspartate)-receptor mediated synaptic currents in the presence of the use-dependent channel blocker MK-801 (ref. 2). On a single postsynaptic CA1 hippocampal slice neuron, two classes of synapses with a sixfold difference in pr are resolved. Synapses with low pr contribute to over half of transmission and are more sensitive to drugs enhancing transmitter release. Switching between these two classes of synapses provides the potential for large changes in synaptic efficacy and could underlie forms of activity-dependent plasticity.
Long-term depression (LTD) is an activity-dependent decrease in synaptic efficacy that together with its counterpart, long-term
potentiation, is thought to be an important cellular mechanism for learning and memory in the mammalian brain. The induction
of LTD in hippocampal CA1 pyramidal neurons in neonatal rats is shown to depend on postsynaptic calcium ion entry through
L-type voltage-gated calcium channels paired with the activation of metabotropic glutamate receptors. Although induced postsynaptically,
LTD is due to a long-term decrease in transmitter release from presynaptic terminals. This suggests that LTD is likely to
require the production of a retrograde messenger.
Rapid calcium-dependent exocytosis underlies neurotransmitter release from nerve terminals. Despite the fundamental importance of this process, neither the relationship between presynaptic intracellular calcium ion concentration ([Ca2+]i) and rate of exocytosis, nor the maximal rate of secretion is known quantitatively. To provide this information, we have used flash photolysis of caged Ca2+ to elevate [Ca2+]i rapidly and uniformly in synaptic terminals, while measuring membrane capacitance as an index of exocytosis and monitoring [Ca2+]i with a Ca(2+)-indicator dye. When [Ca2+]i was abruptly increased to > 10 microM, capacitance rose at a rate that increased steeply with [Ca2+]i. The steepness suggested that at least four calcium ions must bind to activate synaptic vesicle fusion. Half-saturation was at 194 microM, and the maximal rate constant was 2,000-3,000 s-1. A given synaptic vesicle can exocytose with high probability within a few hundred microseconds, if [Ca2+]i rises above 100 microM. These properties provide for the extremely rapid signalling required for neuronal communication.
In the CA1 hippocampal region low-frequency (1-2 Hz) afferent activation leads to a long-term depression of excitatory synaptic potentials that is induced by calcium influx through postsynaptic N-methyl-D-aspartate receptor channels. In the present experiments using 2- to 3-week-old rats, long-term depressions of field excitatory postsynaptic potentials mediated by amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and by N-methyl-D-aspartate receptor channels were examined in parallel, using a perfusion solution containing low concentrations of an AMPA receptor antagonist and of magnesium (0.1 mM). These experiments revealed that long-term depression was associated with equal relative changes in the two components of the field potential, compatible with a presynaptic location of the expression mechanism for the long-term depression.
Mice carrying a mutation in the synaptotagmin I gene were generated by homologous recombination. Mutant mice are phenotypically normal as heterozygotes, but die within 48 hr after birth as homozygotes. Studies of hippocampal neurons cultured from homozygous mutant mice reveal that synaptic transmission is severely impaired. The synchronous, fast component of Ca(2+)-dependent neurotransmitter release is decreased, whereas asynchronous release processes, including spontaneous synaptic activity (miniature excitatory postsynaptic current frequency) and release triggered by hypertonic solution or alpha-latrotoxin, are unaffected. Our findings demonstrate that synaptotagmin I function is required for Ca2+ triggering of synchronous neurotransmitter release, but is not essential for asynchronous or Ca(2+)-independent release. We propose that synaptotagmin I is the major low affinity Ca2+ sensor mediating Ca2+ regulation of synchronous neurotransmitter release in hippocampal neurons.
Perhaps the most fundamental and remarkable feature of the mammalian central nervous system is its ability to process and store large amounts of information. For many decades it has been postulated that the brain uses Ionglasting modifications of synaptic strength in critical neural circuits to accomplish this feat. One such activitydependent modification is long-term potentiation (LTP) in the hippocampus, a sustained increase in synaptic strength that is elicited by brief high frequency stimulation of excitatory afferents. Recent excitement about the phenomenon of LTP has arisen from three major sources. First, compelling evidence from lesion studies in higher primates, including humans, shows that the hippocampus is a critical component of a neural system that is required for the initial storage of certain forms of long-term memory (Squire and Zola-Morgan, 1991). Second, several properties of LTP make it an attractive cellular mechanism for information storage or memory (Bliss and Collingridge, 1993). Like memories, LTP can be generated rapidly and is strengthened with repetition. It exhibits input specificity; that is, LTP occurs only at synapses stimulated by afferent activity but not at adjacent synapses on the same postsynaptic cell. Input specificity presumbably dramatically increases the storage capacity within a neural circuit. Most importantly, LTP is associative; temporally pairing activity in a “weak” input (incapable of generating LTP by itself) with activation of a strong input (capable of eliciting LTP at adjacent synapses on the same postsynaptic cell) results in LTP of the weak input. This associative property, which is reminiscent of classical conditioning, can be considered a cellular analog of associative learning. Third, LTP is readily elicited in in vitro preparations of the hippocampus, and this makes it amenable to rigorous experimental manipulations. There are several different forms of LTP, but the majority of experimental work has focused on the LTP observed in hippocampal CA1 pyramidal cells. This minireview will provide an update on the cellular mechanisms of LTP and distinguish those mechanisms that are firmly established from those that remain contentious. The evidence connecting LTP to real learning and memory also will be reviewed briefly. The Induction of LTP: NMDA Receptors and Cap+ It is well accepted that the induction of LTP requires activation of postsynaptic N-methyl-o-aspartic acid (NMDA) receptors (a subtype of glutamate receptor) during postsynaptic depolarization, which is normally generated by high frequency afferent activity. This results in a rise in CaH concentration ([Ca%],), a necessary trigger for LTP. Figure 1 shows that during normal low frequency synaptic transmission, the excitatory neurotransmitter glutamate is
The effectiveness of long-term potentiation (LTP) as a mechanism for information storage would be severely limited if processes
that decrease synaptic strength did not also exist. In area CA1 of the rat hippocampus, prolonged periods of low-frequency
afferent stimulation elicit a long-term depression (LTD) that is specific to the stimulated input. The induction of LTD was
blocked by the extracellular application of okadaic acid or calyculin A, two inhibitors of protein phosphatases 1 and 2A.
The loading of CA1 cells with microcystin LR, a membrane-impermeable protein phosphatase inhibitor, or calmodulin antagonists
also blocked or attenuated LTD. The application of calyculin A after the induction of LTD reversed the synaptic depression,
suggesting that phosphatase activity is required for the maintenance of LTD. These findings indicate that the synaptic activation
of protein phosphatases plays an important role in the regulation of synaptic transmission.
Long-term potentiation of synaptic transmission in the hippocampus is the primary experimental model for investigating the synaptic basis of learning and memory in vertebrates. The best understood form of long-term potentiation is induced by the activation of the N-methyl-D-aspartate receptor complex. This subtype of glutamate receptor endows long-term potentiation with Hebbian characteristics, and allows electrical events at the postsynaptic membrane to be transduced into chemical signals which, in turn, are thought to activate both pre- and postsynaptic mechanisms to generate a persistent increase in synaptic strength.
Transmission at excitatory synapses in the mammalian brain is thought to depend on the release of transmitter quanta through exocytosis of presynaptic vesicles (Katz, 1969). The number of vesicles released by a single presynaptic action potential is important for understanding the impact of a single synapse, and the variability in transmission from one impulse to the next. In addition, the number of vesicles released may be an important factor for synaptic regulation and plasticity, such as facilitation, post-tetanic potentiation and long-term potentiation (LTP). Three recent studies suggest that an increase in the number of transmitter quanta underlies hippocampal LTP (Malinow and Tsien, 1990; Bekkers and Stevens 1990; Malinow, 1991), whereas other reports suggest a postsynaptic mechanism (Kauer et al., 1988; Muller et al., 1988; Foster and McNaughton, 1989). We have used the whole-cell recording technique to compare putative quantal and single fibre responses at excitatory synapses in rat hippocampal slices, and find (i) a surprisingly large variability in single fibre excitatory postsynaptic currents (sfEPSCs); (ii) an equally large variability of putative quantal (pq) EPSCs elicited by hyperosmolar media or ruthenium red; (iii) the observed amplitude ranges for the sfEPSCs and the pqEPSCs overlap almost completely; and (iv) in neither case can the variability be attributed to a scatter in electrotonic distance from the soma of the engaged synapses. Thus, the data are compatible with the hypothesis that a presynaptic action potential usually releases only a single quantum. Other possibilities are also discussed.
Focal recording from active spots of a neuromuscular junction was used to measure the 'synaptic delay' between terminal axon spike and end-plate current (e.p.c.). Synaptic delay is defined as the time interval between peak of inward current through the presynaptic membrane and commencement of inward current through the postsynaptic membrane. By substituting magnesium for calcium in the medium, and by adjustable electrophoretic application of calcium from the recording electrode, the e.p.c. can be restricted to the small portion of a single junction which is in contact with the microelectrode, and the statistical average amplitude of the e.p.c. can be reduced to less than quantal unit size. Under these conditions, the latency of the unit components of the e.p.c. can be determined and its statistical fluctuations studied. The synaptic delay at a single end-plate spot has a minimum value, at 20 degrees C, of 0\cdot 4 to 0\cdot 5 ms and a modal value of about 0\cdot 75 ms. There is considerable fluctuation of the measured intervals during a series of nerve impulses; over 50% occur within a range of 0.5 ms, the rest being spread out in declining fashion over a further 1 to 4 ms. These latency fluctuations are shown to be a statistical consequence of the quantal process of transmitter release. The contribution of various factors to the minimum synaptic delay are discussed. Terminal conduction time has been effectively eliminated by the method of focal recording. Diffusion of acetylcholine towards the receptors, and its reaction with them must cause delays whose exact values are uncertain, but whose extreme upper limits can be shown to make up only a small part of the observed minimum delay. It is concluded that the synaptic interval arises chiefly from a delay in the release of transmitter after the arrival of the nerve impulse.
The probability depotentiation
- Ltp
- Ltd
- N A Hessler
- A M Shirke
- R Malinow
amination of the effects of MCPG on hippocampal LTP, LTD, and Hessler, N.A., Shirke, A.M., and Malinow, R. (1993). The probability depotentiation. J. Neurophysiol. 74, 1075–1082.