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The voltage-dependent conductances of Limulus ventral photoreceptors have been investigated using a voltage-clamp technique. Depolarization in the dark induces inward and outward currents. The inward current is reduced by removing Na+ or Ca2+ and is abolished by removing both ions. These results suggest that both Na+ and Ca2+ carry voltage-dependent inward current. Inward current is insensitive to tetrodotoxin but is blocked by external Ni2+. The outward current has a large transient component that is followed by a smaller maintained component. Intracellular tetraethylammonium preferentially reduces the maintained component, and extracellular 4-amino pyridine preferentially reduces the transient component. Neither component is strongly affected by removal of extracellular Ca2+ or by intracellular injection of EGTA. It is concluded that the photoreceptors contain at least three separate voltage-dependent conductances: 1) a conductance giving rise to inward currents; 2) a delayed rectifier giving rise to maintained outward K+ current; and 3) a rapidly inactivating K+ conductance similar to the A current of molluscan neurons.

The Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is a central regulator of learning and memory, which poses a problem for targeting it therapeutically. Indeed, our study supports prior conclusions that long-term interference with CaMKII signaling can erase pre-formed memories. By contrast, short-term pharmacological CaMKII inhibition with the neuroprotective peptide tatCN19o interfered with learning in mice only mildly and transiently (for less than 1 h) and did not at all reverse pre-formed memories. These results were obtained with ≥500-fold of the dose that protected hippocampal neurons from cell death after a highly clinically relevant pig model of transient global cerebral ischemia: ventricular fibrillation followed by advanced life support and electrical defibrillation to induce the return of spontaneous circulation. Of additional importance for therapy development, our preliminary cardiovascular safety studies in mice and pig did not indicate any concerns with acute tatCN19o injection. Taken together, although prolonged interference with CaMKII signaling can erase memory, acute short-term CaMKII inhibition with tatCN19o did not cause such retrograde amnesia that would pose a contraindication for therapy., Competing Interests: Conflict of interest K. U. B. is co-founder and board member of Neurexis Therapeutics, a company that seeks to develop the tatCN19o inhibitor of CaMKII into a therapeutic drug for cerebral ischemia. O. R. B. is director of research and development at the same company., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is a central regulator of learning and memory, which poses a problem for targeting it therapeutically. Indeed, our study supports prior conclusions that long-term interference with CaMKII signaling can erase pre-formed memories. By contrast, short-term pharmacological CaMKII inhibition with tatCN19o interfered with learning in mice only mildly and transiently (for less than 1 h) and did not at all reverse pre-formed memories. This was at ≥500fold of the dose that protected hippocampal neurons from cell death after a highly clinically relevant pig model of transient global cerebral ischemia: ventricular fibrillation followed by advanced life support and electrical defibrillation to induce return of spontaneous circulation. Of additional importance for therapeutic development, cardiovascular safety studies in mice and pig did not indicate any concerns with acute tatCN19o injection. Taken together, even though prolonged interference with CaMKII signaling can erase memory, acute short-term CaMKII inhibition with tatCN19o did not cause such retrograde amnesia that would pose a contraindication for therapy.

Hippocampal place cells represent nonspatial information through a process called rate remapping, which involves a change in the firing rate of a place cell without changes in its spatial specificity. However, many hippocampal phenomena occur on very short time scales over which long-term average firing rates are not an appropriate description of activity. To understand how rate remapping relates to fine-scale temporal firing phenomena, we asked how rate remapping affected burst firing and trial-to-trial spike count variability. In addition, we looked at how rate remapping relates to the theta-frequency oscillations of the hippocampus, which are thought to temporally organize firing on time scales faster than 100 ms. We found that theta phase coding was preserved through changes in firing rate due to rate remapping. Interestingly, rate remapping in CA1 in response to task demands preferentially occurred during the first half of the theta cycle. The other half of the theta cycle contained preferential expression of phase precession, a phenomenon associated with place cell sequences, in agreement with previous results. This difference of place cell coding during different halves of the theta cycle supports recent theoretical suggestions that different processes occur during the two halves of the theta cycle. The differentiation between the halves of the theta cycle was not clear in recordings from CA3 during rate remapping induced by task-irrelevant sensory changes. These findings provide new insight into the way that temporal coding is utilized in the hippocampus and how rate remapping is expressed through that temporal code., (© 2018 Wiley Periodicals, Inc.)

High speed imaging of ion concentration changes in neurons is an important and growing tool for neuroscientists. We previously developed a system for simultaneously measuring sodium and calcium changes in small compartments in neurons (Miyazaki and Ross, 2015). We used this technique to analyze the dynamics of these ions in individual pyramidal neuron dendritic spines (Miyazaki and Ross, 2017). This system is based on high speed multiplexing of light emitting diodes (LEDs) and classic organic indicators. To improve this system we made additional changes, primarily incorporating lasers in addition to the LEDs, more sophisticated imaging protocols, and the use of newer sodium and calcium indicators. This new system generates signals with higher signal to noise ratio (S/N), less background fluorescence, and less photodynamic damage. In addition, by using longer wavelength indicators instead of indicators sensitive in the UV range, it allows for the incorporation of focal uncaging along with simultaneous imaging, which should extend the range of experiments.

Complex spatial working memory tasks have been shown to require both hippocampal sharp-wave ripple (SWR) activity and dentate gyrus (DG) neuronal activity. We therefore asked whether DG inputs to CA3 contribute to spatial working memory by promoting SWR generation. Recordings from DG and CA3 while rats performed a dentate-dependent working memory task on an eight-arm radial maze revealed that the activity of dentate neurons and the incidence rate of SWRs both increased during reward consumption. We then found reduced reward-related CA3 SWR generation without direct input from dentate granule neurons. Furthermore, CA3 cells with place fields in not-yet-visited arms preferentially fired during SWRs at reward locations, and these prospective CA3 firing patterns were more pronounced for correct trials and were dentate-dependent. These results indicate that coordination of CA3 neuronal activity patterns by DG is necessary for the generation of neuronal firing patterns that support goal-directed behavior and memory.

Memory storage involves activity-dependent strengthening of synaptic transmission, a process termed long-term potentiation (LTP). The late phase of LTP is thought to encode long-term memory and involves structural processes that enlarge the synapse. Hence, understanding how synapse size is graded provides fundamental information about the information storage capability of synapses. Recent work using electron microscopy (EM) to quantify synapse dimensions has suggested that synapses may structurally encode as many as 26 functionally distinct states, which correspond to a series of proportionally spaced synapse sizes. Other recent evidence using super-resolution microscopy has revealed that synapses are composed of stereotyped nanoclusters of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and scaffolding proteins; furthermore, synapse size varies linearly with the number of nanoclusters. Here we have sought to develop a model of synapse structure and growth that is consistent with both the EM and super-resolution data. We argue that synapses are composed of modules consisting of matrix material and potentially one nanocluster. LTP induction can add a trans-synaptic nanocluster to a module, thereby converting a silent module to an AMPA functional module. LTP can also add modules by a linear process, thereby producing an approximately 10-fold gradation in synapse size and strength.This article is part of the themed issue 'Integrating Hebbian and homeostatic plasticity'., (© 2017 The Author(s).)

A goal of current schizophrenia (SZ) research is to understand how multiple risk genes work together with environmental factors to produce the disease. In schizophrenia, there is elevated delta frequency EEG power in the awake state, an elevation that can be mimicked in rodents by N-methyl-d-aspartate receptor (NMDAR) antagonist action in the thalamus. This thalamic delta can be blocked by dopamine D2 receptor antagonists, agents known to be therapeutic in SZ. Experiments suggest that these oscillations can interfere with brain function and may thus be causal in producing psychosis. Here we evaluate the question of whether well-established schizophrenia risk genes may interact to affect the delta generation process. We identify 19 risk genes that can plausibly work in a synergistic fashion to generate delta oscillations., Competing Interests: John Lisman received speaker’s honoraria from Pfizer Pharmaceuticals and Roche Pharmaceuticals. Edwin A. Richard, Elizaveta Khlestova and Roshan Nanu report no potential conflicts of interest., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Because CaMKII is the critical Ca(2+) sensor that triggers long-term potentiation (LTP), understanding its activation and deactivation is important. A major advance has been the development of a FRET indicator of the conformational state of CaMKII called Camui. Experiments using Camui have demonstrated that the open (active) conformation increases during LTP induction and then decays in tens of seconds, with the major fast component decaying with a time-constant of ~ 6 sec (tau1). Because this decay is faster if autophosphorylation of T286 is prevented (the autophosphorylation prolongs activity by making the enzyme active even after Ca(2+) falls), it seemed likely that the fast decay is due to the T286 dephosphorylation. To test this interpretation, we studied the effect of phosphatase inhibitors on the single-spine Camui signal evoked by two-photon glutamate uncaging. We applied inhibitors of PP1 and PP2A, two phosphatases that are present at synapses and that have been shown to dephosphorylate CaMKII in vitro. The inhibitors increased the basal Camui activation state, indicating their effectiveness in cells. However, in no case did we find that tau1 was prolonged, contrary to what would be expected if the decay was phosphatase-dependent. This could either mean that decay was due to some unknown phosphatase or that the decay was not due to dephosphorylation. To distinguish between these possibilities, we expressed pseudo-phosphorylated Camui (T286D) (plus additional mutations [T/A] that prevented inhibitory 305/306 phosphorylation). This form had an elevated basal activation state, but was further activated during glutamate uncaging; importantly the activation state decayed with tau1 nearly the same as that of WT Camui. Therefore, the data strongly indicate that tau1 is not due to T286 dephosphorylation. We conclude that, although Camui is an excellent tool for observing CaMKII signaling, further experimentation is needed to determine how CaMKII is turned off by its dephosphorylation.

During long-term potentiation (LTP) of excitatory synapses, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is activated by Ca(2+) influx through NMDA receptors that potentiate AMPA receptor currents by insertion of additional GluR1-containing receptors at the synapse and by increasing AMPA channel conductance, as well as by stimulating structural changes. CaMKII is also involved in the maintenance of LTP and contributes to maintenance of behavioral sensitization by cocaine or amphetamine. Recent studies show that transient expression of catalytically dead αCaMKII K42M mutant after exposure to amphetamine persistently reverses the behavioral effects of the addiction. A suggested interpretation is that this mutant acts as a dominant negative in the control of synaptic strength, but this interpretation has not been physiologically tested. Here we investigate the effect of αCaMKII K42M mutant expressed in single CA1 pyramidal neurons on basal excitatory neurotransmission in cultured rat hippocampal organotypic slices. The mutant caused nearly 50% reduction in the basal CA3-CA1 transmission, while overexpression of the wild-type αCaMKII had no effect. This result is consistent with the dominant negative hypothesis, but there are complexities. We found that the decrease in basal transmission did not occur when activity in the slices was suppressed after transfection by TTX or when NMDA receptors were blocked by APV. Thus, the dominant negative effect requires neural activity for its expression.

The notion of attractor networks is the leading hypothesis for how associative memories are stored and recalled. A defining anatomical feature of such networks is excitatory recurrent connections. These "attract" the firing pattern of the network to a stored pattern, even when the external input is incomplete (pattern completion). The CA3 region of the hippocampus has been postulated to be such an attractor network; however, the experimental evidence has been ambiguous, leading to the suggestion that CA3 is not an attractor network. In order to resolve this controversy and to better understand how CA3 functions, we simulated CA3 and its input structures. In our simulation, we could reproduce critical experimental results and establish the criteria for identifying attractor properties. Notably, under conditions in which there is continuous input, the output should be "attracted" to a stored pattern. However, contrary to previous expectations, as a pattern is gradually "morphed" from one stored pattern to another, a sharp transition between output patterns is not expected. The observed firing patterns of CA3 meet these criteria and can be quantitatively accounted for by our model. Notably, as morphing proceeds, the activity pattern in the dentate gyrus changes; in contrast, the activity pattern in the downstream CA3 network is attracted to a stored pattern and thus undergoes little change. We furthermore show that other aspects of the observed firing patterns can be explained by learning that occurs during behavioral testing. The CA3 thus displays both the learning and recall signatures of an attractor network. These observations, taken together with existing anatomical and behavioral evidence, make the strong case that CA3 constructs associative memories based on attractor dynamics.

The exchange of CaMKII enzymes between larger structures called holoenzymes may provide the molecular mechanism underlying the long-term stability of memories.

Previous studies have shown that appetitive motivation enhances episodic memory formation via a network including the substantia nigra/ventral tegmental area (SN/VTA), striatum and hippocampus. This functional magnetic resonance imaging (fMRI) study now contrasted the impact of aversive and appetitive motivation on episodic long-term memory. Cue pictures predicted monetary reward or punishment in alternating experimental blocks. One day later, episodic memory for the cue pictures was tested. We also investigated how the neural processing of appetitive and aversive motivation and episodic memory were modulated by dopaminergic mechanisms. To that end, participants were selected on the basis of their genotype for a variable number of tandem repeat polymorphism of the dopamine transporter (DAT) gene. The resulting groups were carefully matched for the 5-HTTLPR polymorphism of the serotonin transporter gene. Recognition memory for cues from both motivational categories was enhanced in participants homozygous for the 10-repeat allele of the DAT, the functional effects of which are not known yet, but not in heterozygous subjects. In comparison with heterozygous participants, 10-repeat homozygous participants also showed increased striatal activity for anticipation of motivational outcomes compared to neutral outcomes. In a subsequent memory analysis, encoding activity in striatum and hippocampus was found to be higher for later recognized items in 10-repeat homozygotes compared to 9/10-repeat heterozygotes. These findings suggest that processing of appetitive and aversive motivation in the human striatum involve the dopaminergic system and that dopamine plays a role in memory for both types of motivational information. In accordance with animal studies, these data support the idea that encoding of motivational events depends on dopaminergic processes in the hippocampus., (© 2013 The Authors. Published by Elsevier Ltd. All rights reserved.)

Previous studies have shown that appetitive motivation enhances episodic memory formation via a network including the substantia nigra/ventral tegmental area (SN/VTA), striatum and hippocampus. This functional magnetic resonance imaging (fMRI) study now contrasted the impact of aversive and appetitive motivation on episodic long-term memory. Cue pictures predicted monetary reward or punishment in alternating experimental blocks. One day later, episodic memory for the cue pictures was tested. We also investigated how the neural processing of appetitive and aversive motivation and episodic memory were modulated by dopaminergic mechanisms. To that end, participants were selected on the basis of their genotype for a variable number of tandem repeat polymorphism of the dopamine transporter (DAT) gene. The resulting groups were carefully matched for the 5-HTTLPR polymorphism of the serotonin transporter gene. Recognition memory for cues from both motivational categories was enhanced in participants homozygous for the 10-repeat allele of the DAT, the functional effects of which are not known yet, but not in heterozygous subjects. In comparison with heterozygous participants, 10-repeat homozygous participants also showed increased striatal activity for anticipation of motivational outcomes compared to neutral outcomes. In a subsequent memory analysis, encoding activity in striatum and hippocampus was found to be higher for later recognized items in 10-repeat homozygotes compared to 9/10-repeat heterozygotes. These findings suggest that processing of appetitive and aversive motivation in the human striatum involve the dopaminergic system and that dopamine plays a role in memory for both types of motivational information. In accordance with animal studies, these data support the idea that encoding of motivational events depends on dopaminergic processes in the hippocampus., (© 2013 The Authors. Published by Elsevier Ltd. All rights reserved.)

Theta and gamma frequency oscillations occur in the same brain regions and interact with each other, a process called cross-frequency coupling. Here, we review evidence for the following hypothesis: that the dual oscillations form a code for representing multiple items in an ordered way. This form of coding has been most clearly demonstrated in the hippocampus, where different spatial information is represented in different gamma subcycles of a theta cycle. Other experiments have tested the functional importance of oscillations and their coupling. These involve correlation of oscillatory properties with memory states, correlation with memory performance, and effects of disrupting oscillations on memory. Recent work suggests that this coding scheme coordinates communication between brain regions and is involved in sensory as well as memory processes., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Networks that produce persistent firing in response to novel input patterns are thought to be important in working memory and other information storage functions. One possible mechanism for maintaining persistent firing is dendritic voltage bistability in which the depolarized state depends on the voltage dependence of the NMDA conductance at recurrent synapses. In previous models, the hyperpolarized state is dependent on voltage-independent conductances, including GABA(A). The interplay of these conductances leads to bistability, but its robustness is limited by the fact that the conductance ratio must be within a narrow range. The GABA(B) component of inhibitory transmission was not considered in previous analyses. Here, we show that the voltage dependence of the inwardly rectifying potassium (KIR) conductance activated by GABA(B) receptors adds substantial robustness to network simulations of bistability and the persistent firing that it underlies. The hyperpolarized state is robust because, at hyperpolarized potentials, the GABA(B)/KIR conductance is high and the NMDA conductance is low; the depolarized state is robust because, at depolarized potentials, the NMDA conductance is high and the GABA(B)/KIR conductance is low. Our results suggest that this complementary voltage dependence of GABA(B)/KIR and NMDA conductances makes them a "perfect couple" for producing voltage bistability.

Work on schizophrenia demonstrates the involvement of the hippocampus in the disease and points specifically to hyperactivity of CA1. Many symptoms of schizophrenia can be mimicked by N-methyl-d-aspartate receptor (NMDAR) antagonist; notably, delta frequency oscillations in the awake state are enhanced in schizophrenia, an abnormality that can be mimicked by NMDAR antagonist action in the thalamus. Given that CA1 receives input from the nucleus reuniens of the thalamus, we sought to determine whether an NMDAR antagonist in the thalamus can affect hippocampal processes. We found that a systemic NMDAR antagonist (ketamine; 50 mg/kg) increased the firing rate of cells in the reuniens and CA1 in awake rats. Furthermore, ketamine increased the power of delta oscillations in both structures. The thalamic origin of the change in hippocampal properties was demonstrated in three ways: 1) oscillations in the two structures were coherent; 2) the hippocampal changes induced by systematic ketamine were reduced by thalamic injection of muscimol; and 3) the hippocampal changes could be induced by local injection of ketamine into the thalamus. Lower doses of ketamine (20 mg/kg) did not evoke delta oscillations but did increase hippocampal gamma power, an effect not dependent on the thalamus. There are thus at least two mechanisms for ketamine action on the hippocampus: a low-dose mechanism that affects gamma through a nonthalamic mechanism and a high-dose mechanism that increases CA1 activity and delta oscillations as a result of input from the thalamus. Both mechanisms may be important in producing symptoms of schizophrenia.

Granule cells of the dentate gyrus (DG) generally have multiple place fields, whereas CA3 cells, which are second order, have only a single place field. Here, we explore the mechanisms by which the high selectivity of CA3 cells is achieved. Previous work showed that the multiple place fields of DG neurons could be quantitatively accounted for by a model based on the number and strength of grid cell inputs and a competitive network interaction in the DG that is mediated by gamma frequency feedback inhibition. We have now built a model of CA3 based on similar principles. CA3 cells receive input from an average of one active DG cell and from 1,400 cortical grid cells. Based on experimental findings, we have assumed a linear interaction of the two pathways. The results show that simulated CA3 cells generally have a single place field, as observed experimentally. Thus, a two-step process based on simple rules (and that can occur without learning) is able to explain how grid cell inputs to the hippocampus give rise to cells having ultimate spatial selectivity. The CA3 processes that produce a single place depend critically on the competitive network processes and do not require the direct cortical inputs to CA3, which are therefore likely to perform some other unknown function., (Copyright © 2010 Wiley Periodicals, Inc.)

CaMKII and PSD-95 are the two most abundant postsynaptic proteins in the postsynaptic density (PSD). Overexpression of either can dramatically increase synaptic strength and saturate long-term potentiation (LTP). To do so, CaMKII must be activated, but the same is not true for PSD-95; expressing wild-type PSD-95 is sufficient. This raises the question of whether PSD-95's effects are simply an equilibrium process [increasing the number of AMPA receptor (AMPAR) slots] or whether activity is somehow involved. To examine this question, we blocked activity in cultured hippocampal slices with TTX and found that the effects of PSD-95 overexpression were greatly reduced. We next studied the type of receptors involved. The effects of PSD-95 were prevented by antagonists of group I metabotropic glutamate receptors (mGluRs) but not by antagonists of ionotropic glutamate receptors. The inhibition of PSD-95-induced strengthening was not simply a result of inhibition of PSD-95 synthesis. To understand the mechanisms involved, we tested the role of CaMKII. Overexpression of a CaMKII inhibitor, CN19, greatly reduced the effect of PSD-95. We conclude that PSD-95 cannot itself increase synaptic strength simply by increasing the number of AMPAR slots; rather, PSD-95's effects on synaptic strength require an activity-dependent process involving mGluR and CaMKII.

NMDAR antagonists can evoke delta frequency bursting in the nucleus reticularis of the thalamus (nRT). The mechanism of this oscillation was determined; antagonist blocks an NR2C-like conductance that has low Mg block at resting potential and thus can contribute a resting inward current in response to ambient glutamate. Block of this current hyperpolarizes the cell, deinactivating T-type Ca channels and thus triggering delta frequency bursting. The basis for assuming a NR2C-like conductance was that (1) transcripts for NR2C are abundant in the thalamus and (2) the current-voltage curve of the synaptically evoked NMDAR current has the low rectification characteristic of NR2C. In the current study, we have sought to determine whether the channels that generate the NMDAR current are NR2C-like or are actually comprised of receptors containing NR2C. We studied the current-voltage curve of synaptically evoked NMDAR current in the nRT of NR2C knockout mice. In wild-type mice, the current was weakly voltage dependent, as previously observed in rats. This weak rectification was absent in NR2C KO mice. In contrast, NR2C KO had no effect on the strongly rectifying NMDAR current in pyramidal cells of the prefrontal cortex. These results demonstrate that the low rectification normally observed in the nRT is due to NR2C.

Visual object recognition occurs easily despite differences in position, size, and rotation of the object, but the neural mechanisms responsible for this invariance are not known. We have found a set of transforms that achieve invariance in a neurally plausible way. We find that a transform based on local spatial frequency analysis of oriented segments and on logarithmic mapping, when applied twice in an iterative fashion, produces an output image that is unique to the object and that remains constant as the input image is shifted, scaled, or rotated.

Many forms of signal transduction occur when Ca(2+) enters the cytoplasm of a cell. It has been generally thought that there is a fast buffer that rapidly reduces the free Ca(2+) level and that it is this buffered level of Ca(2+) that triggers downstream biochemical processes, notably the activation of calmodulin (CaM) and the resulting activation of CaM-dependent enzymes. Given the importance of these transduction processes, it is crucial to understand exactly how Ca(2+) activates CaM. We have determined the rate at which Ca(2+) binds to CaM and found that Ca(2+) binds more rapidly to CaM than to other Ca(2+)-binding proteins. This property of CaM and its high concentration support a new view of signal transduction: CaM directly intercepts incoming Ca(2+) and sets the free Ca(2+) level (that is, it strongly contributes to fast Ca(2+) buffering) rather than responding to the lower Ca(2+) level set by other buffers. This property is crucial for making CaM an efficient transducer. Our results also suggest that other Ca(2+) binding proteins have a previously undescribed role in regulating the lifetime of Ca(2+) bound to CaM and thereby setting the gain of signal transduction.

Rate remapping is a recently revealed neural code in which sensory information modulates the firing rate of hippocampal place cells. The mechanism underlying rate remapping is unknown. Its characteristic modulation, however, must arise from the interaction of the two major inputs to the hippocampus, the medial entorhinal cortex (MEC), in which grid cells represent the spatial position of the rat, and the lateral entorhinal cortex (LEC), in which cells represent the sensory properties of the environment. We have used computational methods to elucidate the mechanism by which this interaction produces rate remapping. We show that the convergence of LEC and MEC inputs, in conjunction with a competitive network process mediated by feedback inhibition, can account quantitatively for this phenomenon. The same principle accounts for why different place fields of the same cell vary independently as sensory information is altered. Our results show that rate remapping can be explained in terms of known mechanisms., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The N-methyl-D-aspartate receptor (NMDAR) hypofunction model of schizophrenia is based on the ability of NMDAR antagonists to produce many symptoms of the disease. Recent work in rats shows that NMDAR antagonist works synergistically with dopamine to produce delta frequency bursting in the thalamus. This finding, together with other results in the literature, suggests a mechanism for the sudden onset of schizophrenia. Among the thalamic nuclei most activated by NMDAR antagonist is the nucleus reuniens. This nucleus excites the cornu ammonis area 1 (CA1) region of the hippocampus. Experiments indicate that such activation can lead to excitation of dopaminergic cells of the ventral tegmental area by a polysynaptic pathway. The resulting elevation of dopamine in the thalamus will enhance thalamic bursting, thereby creating a loop with the potential for positive feedback. We show through computer simulations that in individuals with susceptibility to schizophrenia (e.g., because of partially compromised NMDAR function), an event that stimulates the dopamine system, such as stress, can cause the system to reach the threshold for thalamic bursting. When this occurs, positive feedback in the loop will cause all components to become highly active and to remain active after the triggering stimulus is removed. This is a physiologically specific hypothesis for the sudden and lasting transition that underlies the psychotic break in schizophrenia. Furthermore, the model provides an explanation for the observed selective activation of the CA1 hippocampal region in schizophrenia. The model also predicts an increase of basal activity in the dopamine system and thalamus; the relevant evidence is reviewed., (Copyright 2010 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Injection of NMDAR antagonist into the thalamus can produce delta frequency EEG oscillations in the thalamocortical system. It is surprising that an antagonist of an excitatory neurotransmitter should trigger such activity, and the mechanism is unknown. One hypothesis is that the antagonist blocks excitation of GABAergic cells, thus producing disinhibition. To test this hypothesis, we investigated the effect of NMDAR antagonist (APV) on cells of the nucleus reticularis (nRT) in rat brain slices, a thalamic nucleus that can serve as a pacemaker for thalamocortical delta oscillations and that is composed entirely of GABAergic neurons. We found, unexpectedly, that nRT cells are hyperpolarized by APV. This occurs because these cells have an unusual form of NMDAR (probably NR2C) that contributes inward current at resting potential in response to ambient glutamate. The hyperpolarization produced by APV is sufficient to deinactivate T-type calcium channels, and these trigger rhythmic bursting at delta frequency. The APV-induced delta frequency bursting is abolished by dopamine D2 receptor antagonist, indicating that dopamine and NMDAR antagonist work synergistically to stimulate delta frequency bursting. Our results have significant implications concerning the electrophysiological basis of schizophrenia and bring together the NMDAR hypofunction, dopamine, and GABA theories of the disease. Our results suggest that NMDAR hypofunction and dopamine work synergistically on the GABAergic cells of the nRT to generate the delta frequency EEG oscillations, a thalamocortical dysrhythmia (TCD) in the awake state that is an established abnormality in schizophrenia.

The pre/post debate involves the question of whether long-term potentiation (LTP) is mediated by enhancement of release, enhancement of postsynaptic receptors, or both. Recent papers have presented evidence for purely postsynaptic or purely presynaptic changes, and a paper by Ahmed and Siegelbam (in this issue of Neuron) suggests a mechanism by which release is enhanced. This debate is increasingly constrained by technical advances that allow central synapses to be studied with increasing precision. A possible of way of reconciling conflicting evidence is suggested.

Grid cells in the rat medial entorhinal cortex fire (periodically) over the entire environment. These cells provide input to hippocampal granule cells whose output is characterized by one or more small place fields. We sought to understand how this input-output transformation occurs. Available information allows simulation of this process with no freely adjustable parameters. We first examined the spatial distribution of excitation in granule cells produced by the convergence of excitatory inputs from randomly chosen grid cells. Because the resulting summation depends on the number of inputs, it is necessary to use a realistic number (approximately 1200) and to take into consideration their 20-fold variation in strength. The resulting excitation maps have only modest peaks and valleys. To analyze how this excitation interacts with inhibition, we used an E%-max (percentage of maximal suprathreshold excitation) winner-take-all rule that describes how gamma-frequency inhibition affects firing. We found that simulated granule cells have firing maps that have one or more place fields whose size and number approximates those observed experimentally. A substantial fraction of granule cells have no place fields, as observed experimentally. Because the input firing rates and synaptic properties are known, the excitatory charge into granule cells could be calculated (2-3 pC) and was found to be only somewhat larger than required to fire granule cells (1 pC). We conclude that the input-output transformation of dentate granule does not depend strongly on synaptic modification; place field formation can be understood in terms of simple summation of randomly chosen excitatory inputs, in conjunction with a winner-take-all network mechanism.

The role of gamma oscillations in producing synchronized firing of groups of principal cells is well known. Here, we argue that gamma oscillations have a second function: they select which principal cells fire. This selection process occurs through the interaction of excitation with gamma frequency feedback inhibition. We sought to understand the rules that govern this process. One possibility is that a constant fraction of cells fire. Our analysis shows, however, that the fraction is not robust because it depends on the distribution of excitation to different cells. A robust description is termed E%-max: cells fire if they have suprathreshold excitation (E) within E% of the cell that has maximum excitation. The value of E%-max is approximated by the ratio of the delay of feedback inhibition to the membrane time constant. From measured values, we estimate that E%-max is 5-15%. Thus, an E%-max winner-take-all process can discriminate between groups of cells that have only small differences in excitation. To test the utility of this framework, we analyzed the role of oscillations in V1, one of the few systems in which both spiking and intracellular excitation have been directly measured. We show that an E%-max winner-take-all process provides a simple explanation for why the orientation tuning of firing is narrower than that of the excitatory input and why this difference is not affected by increasing excitation. Because gamma oscillations occur in many brain regions, the framework we have developed for understanding the second function of gamma is likely to have wide applicability.

Studies of long-term potentiation (LTP) and long-term depression (LTD) strongly suggest that individual synapses can be bidirectionally modified. A central question is the biochemical mechanisms that make LTP and LTD persistent. Previous theoretical models have proposed that the autophosphorylation properties of CaMKII could underlie a bistable molecular switch that maintains LTP, and there is experimental support for this mechanism. In contrast, there has been comparatively little theoretical or experimental work regarding the mechanisms that maintain LTD. Several lines of evidence indicate that LTD is not simply a reversal of previous LTP but rather involves separate biochemical reactions. These findings indicate that a minimal model of the synapse must involve a tristable system. Here, we describe a phosphatase (PP2A) switch, which together with a kinase switch form a tristable system. PP2A can be activated by a Ca(2+)-dependent process but can also be phosphorylated and inactivated by CaMKII. When dephosphorylated, PP2A can dephosphorylate itself. We show that these properties can lead to a persistent increase in PP2A during LTD (as reported experimentally), thus forming a phosphatase switch. We show that the coupled PP2A and CaMKII switches lead to a tristable system in which the kinase activity is high in the LTP state; the PP2A activity is high in the LTD state, and neither activity is high in the basal state. Our results provide an explanation for the recent finding that inhibition of PP2A prevents LTD induction.

Postmortem studies have shown that schizophrenia produces a reduction in the 67-kilodalton isoform of glutamic acid decarboxylase (GAD67), a key enzyme for gamma-aminobutyric acid (GABA) synthesis. N-methyl-d-aspartate receptor (NMDAR) antagonists have been extensively used to study schizophrenia because they can induce many aspects of the disease, including the decrease in GAD67. It is generally thought that this reduction in GAD implies a reduction in functional inhibition, but direct evidence had been lacking. We have therefore performed physiological studies in slices of prefrontal cortex taken from rats treated with the NMDAR antagonist ketamine. Both frequency and amplitude of miniature inhibitory postsynaptic currents were reduced. Consistent with a reduction of inhibition, we observed an increase in postsynaptic excitability. The increased excitability is likely to result from disinhibition because miniature excitatory postsynaptic current properties and intrinsic excitability were not changed. Ketamine did not affect inhibition or GAD levels in young rats, indicating a developmental regulation that may be related to the developmental increase in ketamine sensitivity that occurs in humans. Our results show that NMDAR antagonist produces biochemical changes in the GABA system that lead to a functional disinhibition. Such disinhibition would be expected to decrease gamma oscillations, which are reduced in schizophrenia.

Many risk genes interact synergistically to produce schizophrenia and many neurotransmitter interactions have been implicated. We have developed a circuit-based framework for understanding gene and neurotransmitter interactions. NMDAR hypofunction has been implicated in schizophrenia because NMDAR antagonists reproduce symptoms of the disease. One action of antagonists is to reduce the excitation of fast-spiking interneurons, resulting in disinhibition of pyramidal cells. Overactive pyramidal cells, notably those in the hippocampus, can drive a hyperdopaminergic state that produces psychosis. Additional aspects of interneuron function can be understood in this framework, as follows. (i) In animal models, NMDAR antagonists reduce parvalbumin and GAD67, as found in schizophrenia. These changes produce further disinhibition and can be viewed as the aberrant response of a homeostatic system having a faulty activity sensor (the NMDAR). (ii) Disinhibition decreases the power of gamma oscillation and might thereby produce negative and cognitive symptoms. (iii) Nicotine enhances the output of interneurons, and might thereby contribute to its therapeutic effect in schizophrenia.

The existence of recurrent synaptic connections in CA3 led to the hypothesis that CA3 is an autoassociative network similar to the Hopfield networks studied by theorists. CA3 undergoes gamma frequency periodic inhibition that prevents a persistent attractor state. This argues against the analogy to Hopfield nets, in which an attractor state can be used for working memory. However, we show that such periodic inhibition allows one cycle of recurrent excitatory activity and that this is sufficient for memory retrieval (within milliseconds). Thus, gamma oscillations are compatible with a long-term autoassociative memory function for CA3. A second goal of our work was to evaluate previous methods for estimating the memory capacity (P) of CA3. We confirm the equation, P = c/a(2), where c is the probability that any two cells are recurrently connected and a is the fraction of cells representing a memory item. In applying this to CA3, we focus on CA3a, the subregion where recurrent connections are most numerous (c = 0.2) and approximate randomness. We estimate that a memory item is represented by approximately 225 of the 70,000 neurons in CA3a (a = 0.003) and that approximately 20,000 memory items can be stored. Our general conclusion is that the physiological and anatomical findings of CA3a are consistent with an autoassociative function. The nature of the information that is associated in CA3a is discussed. We also discuss how the autoassociative properties of CA3 and the heteroassociative properties of dentate synapses (linking sequential memories) form an integrated system for the storage and recall of item sequences. The recall process generates the phase precession in dentate, CA3, and entorhinal cortex.

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is activated by Ca(2+) entry into neurons. Autophosphorylation of T286 is of special importance because it makes the enzyme active in the absence of Ca(2+), providing a biochemical memory that is critical for plasticity. To understand the factors controlling the duration of this state of CaMKII, we studied dephosphorylation of CaMKII in the post-synaptic density (PSD), a structure that defines a neuronal subcompartment critical for plasticity. We found that PSD-resident PP1 can dephosphorylate many sites on CaMKII, but not the T286 site that produces Ca(2+)-independent activity. This, together with previous work showing that soluble PP2A cannot dephosphorylate PSD CaMKII, provides a novel explanation for the in vivo persistence of T286 phosphorylation: after activated CaMKII translocates from the cytoplasm to the PSD, structural constraints prevent phosphatases from dephosphorylating T286. These results also suggest that the PSD is more than a simple scaffold for synaptic proteins; it may act to regulate the activity of proteins by positioning them in orientations that either prevent or favor specific biochemical reactions.

The properties of synaptic transmission were first elucidated at the neuromuscular junction. More recent work has examined transmission at synapses within the brain. Here we review the remarkable progress in understanding the biophysical and molecular basis of the sequential steps in this process. These steps include the elevation of Ca2+ in microdomains of the presynaptic terminal, the diffusion of transmitter through the fusion pore into the synaptic cleft and the activation of postsynaptic receptors. The results give insight into the factors that control the precision of quantal transmission and provide a framework for understanding synaptic plasticity.

Long-term potentiation (LTP) is an activity-dependent strengthening of synapses that is thought to underlie memory storage. Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been a leading candidate as a memory molecule because it is persistently activated after LTP induction and can enhance transmission. Furthermore, a mutation that blocks persistent activation blocks LTP and forms of learning. However, direct evidence for a role of the kinase in maintaining synaptic strength has been lacking. Here, we show that a newly developed noncompetitive inhibitor of CaMKII strongly reduces synaptic transmission in the CA1 region of the hippocampal slice. This occurs through both presynaptic and postsynaptic action. To study the role of CaMKII in the maintenance of LTP, inhibitor was applied after LTP induction and then removed. Inhibition occurred in both LTP and control pathways but only partially recovered. The nonrecovering component was attributable primarily to a postsynaptic change. To test whether nonrecovery was attributable to a persistent reversal of LTP, we first saturated LTP and then transiently applied inhibitor. This procedure allowed additional LTP to be induced, indicating a reversal of an LTP maintenance mechanism. This is the first procedure that can reverse LTP by chemical means and suggests that a component of synaptic memory is attributable to CaMKII. The procedure also enhanced the LTP that could be induced in the control pathway, consistent with the idea that CaMKII is involved in controlling basal synaptic strength, perhaps as a result of LTP that occurred in vivo.

The hippocampus sits at the highest level of memory processing circuits and receives two major inputs, one coming from the lateral entorhinal cortex and one coming from the medial entorhinal cortex. This duality must be of fundamental importance, but its functional meaning remains unclear. A computational model used for robot navigation (Verschure, P.F., et al. (2003). Nature, 425: 620-624) has a dual information structure that may provide insight. In this model, information is stored as couplets consisting of information about the current sensory cues and information about the current action of the robot. Sequences of such couplets are stored in a short-term memory buffer and transferred to a long-term memory store whenever a goal is found. The overall system enhances the ability of the robot to find reward sites because stored sequences enable the robot to retrace the path to a goal site whenever any of the cues along the path to a goal is subsequently encountered. A review of the literature suggests that the idea of cue/action couplets can be usefully mapped onto the function of the entorhinal cortex. Cue information may be supplied by the lateral entorhinal cortex whereas action (motor) information may be supplied by the medial entorhinal cortex. However, given that self-position information is prominent in the medial pathway and that this is not directly related to action, a modified formulation of the duality is proposed in which the fundamental distinction is between information about non-self vs. information about self. According to this view, the lateral entorhinal pathway carries information about external (non-self) cues and their positions (in egocentric coordinates) whereas the medial entorhinal pathway carries information about the organism itself, including its position (in allocentric coordinates), motor actions and goals.

The phototransduction cascade in Limulus ventral photoreceptors involves multiple second messengers, including Ca(2+) and cGMP. Light-induced Ca(2+) release from intracellular stores is an intermediate step, but the subsequent Ca(2+)-activated reaction remains to be determined. The possibility that Ca(2+)/calmodulin (Ca(2+)/CaM) might be involved is suggested by the high calmodulin content of the transducing lobe. To test whether CaM can excite the transduction cascade we injected a 25 microM Ca(2+)/CaM solution. This produced a rapid, brief depolarization similar to that produced by light, suggesting a role for CaM in the cascade. However, an important caveat is that Ca(2+) dissociating from the Ca(2+)/CaM complex might excite this process. Several control experiments argue against, but do not entirely eliminate this possibility. To test whether endogenous CaM has a function in excitation, trifluoperazine was pressure injected into the rhabdomeric region. The response to brief flashes was not affected, but the response to steady illumination was transiently attenuated by each injection. We conclude that calmodulin should be considered a candidate to couple intermediate and late stages of the transduction cascade.

Synaptic plasticity in CA1 hippocampal neurons depends on Ca2+ elevation and the resulting activation of calmodulin-dependent enzymes. Induction of long-term depression (LTD) depends on calcineurin, whereas long-term potentiation (LTP) depends on Ca2+/calmodulin-dependent protein kinase II (CaMKII). The concentration of calmodulin in neurons is considerably less than the total concentration of the apocalmodulin-binding proteins neurogranin and GAP-43, resulting in a low level of free calmodulin in the resting state. Neurogranin is highly concentrated in dendritic spines. To elucidate the role of neurogranin in synaptic plasticity, we constructed a computational model with emphasis on the interaction of calmodulin with neurogranin, calcineurin, and CaMKII. The model shows how the Ca2+ transients that occur during LTD or LTP induction affect calmodulin and how the resulting activation of calcineurin and CaMKII affects AMPA receptor-mediated transmission. In the model, knockout of neurogranin strongly diminishes the LTP induced by a single 100 Hz, 1 s tetanus and slightly enhances LTD, in accord with experimental data. Our simulations show that exchange of calmodulin between a spine and its parent dendrite is limited. Therefore, inducing LTP with a short tetanus requires calmodulin stored in spines in the form of rapidly dissociating calmodulin-neurogranin complexes.

Cortical theta appears important in sensory processing and memory. Intracanial electrode recordings provide a high spatial resolution method for studying such oscillations during cognitive tasks. Recent work revealed sites at which oscillations in the theta range (4-12 Hz) could be gated by a working-memory task: theta power was increased at task onset and continued until task offset. Using a large data set that has now been collected (10 participants/619 recording sites), we have sufficient sampling to determine how these gated sites are distributed in the cortex and how they are synchronized. A substantial fraction of sites in occipital/parietal (45/157) and temporal (23/280) cortices were gated by the task. Surprisingly, this aspect of working-memory function was virtually absent in frontal cortex (2/182). Coherence measures were used to analyze the synchronization of oscillations. We suspected that because of their coordinate regulation by the working-memory task, gated sites would have synchronized theta oscillations. We found that, whereas nearby gated sites (<20 mm) were often but not always coherent, distant gated sites were almost never coherent. Our results imply that there are local mechanisms for the generation of cortical theta.

Behavioral and electrophysiological evidence indicates that the hippocampus has a special role in the encoding and recall of memory sequences. Importantly, the hippocampal phase precession, a phenomenon recorded as a rat moves through place fields, can be interpreted as cued recall of the sequence of upcoming places. The phase precession can be recorded in all hippocampal regions, but the role of each region has been unclear. Here, we suggest how the dentate and CA3 regions can work together to learn sequences, recall sequences, and generate the phase precession. Our proposal is constrained by information regarding synaptic plasticity rules, network connectivity, timing delays and theta/gamma oscillations.

Bath-applied monoamines-dopamine (DA), serotonin (5-HT), and noradrenaline (NE)-strongly suppress the perforant path (PP) input to CA1 hippocampal region with very little effect on the Schaffer collaterals (SC) input. The effect of DA action on PP field excitatory postsynaptic potential (fEPSP) has been characterized in detail, but relatively little is known about the NE and 5-HT effects. Here we show that the maximal inhibition of the PP fEPSP by NE is approximately 55%, whereas 5-HT inhibition is weaker ( approximately 35%). The half-maximal inhibitory concentration of both 5-HT and NE is approximately 1 muM. Neither NE nor 5-HT affected paired-pulse facilitation, suggesting that the effect is not presynaptic. This is in contrast to DA, which does have a presynaptic effect. The NE effect was blocked by alpha2 antagonists, whereas the alpha1 antagonist corynanthine and beta-antagonist propranolol were ineffective. The effect of 5-HT was mimicked by the agonist, 5-carboxamidotryptamine maleate (5-CT), and not affected by adrenergic and dopaminergic antagonists. To determine the 5-HT receptors involved, we tested a number of 5-HT antagonists, but none produced a complete suppression of the 5-HT effect. Of these, only the 5-HT7 and 5-HT2 antagonists produced weak but significant inhibition of 5-HT effect. We conclude that NE inhibits the PP fEPSP through postsynaptic action on alpha2-adrenoceptors and that 5-HT7, 5-HT2, and some other receptor may be involved in 5-HT action in PP.

In this article we develop the concept that the hippocampus and the midbrain dopaminergic neurons of the ventral tegmental area (VTA) form a functional loop. Activation of the loop begins when the hippocampus detects newly arrived information that is not already stored in its long-term memory. The resulting novelty signal is conveyed through the subiculum, accumbens, and ventral pallidum to the VTA where it contributes (along with salience and goal information) to the novelty-dependent firing of these cells. In the upward arm of the loop, dopamine (DA) is released within the hippocampus; this produces an enhancement of LTP and learning. These findings support a model whereby the hippocampal-VTA loop regulates the entry of information into long-term memory.

Molecular switches have been implicated in the storage of information in biological systems. For small structures such as synapses, these switches are composed of only a few molecules and stochastic fluctuations are therefore of importance. Such fluctuations could potentially lead to spontaneous switch reset that would limit the lifetime of information storage. We have analyzed a model of the calcium/calmodulin-dependent protein kinase II (CaMKII) switch implicated in long-term memory in the nervous system. The bistability of this switch arises from autocatalytic autophosphorylation of CaMKII, a reaction that is countered by a saturable phosphatase-1-mediated dephosphorylation. We sought to understand the factors that control switch stability and to determine the functional relationship between stability and the number of molecules involved. Using Monte Carlo simulations, we found that the lifetime of states of the switch increase exponentially with the number of CaMKII holoenzymes. Switch stability requires a balance between the kinase and phosphatase rates, and the kinase rate must remain high relative to the rate of protein turnover. Thus, a critical limit on switch stability is set by the observed turnover rate (one per 30 h on average). Our computational results show that, depending on the timescale of fluctuations in enzyme numbers, for a switch composed of about 15 CaMKII holoenzymes, the stable persistent activation can span from a few years to a human lifetime.

Encoding and recall of memory sequences is an important process. Memory encoding is thought to occur by long-term potentiation (LTP) in the hippocampus; however, it remains unclear how LTP, which has a time window for induction of approximately 100 ms, could encode the linkage between sequential items that arrive with a temporal separation >100 ms. Here, we argue that LTP can underlie the learning of such memory sequences, provided the input to the hippocampus is from a cortical multi-item working memory buffer in which theta and gamma oscillations have an important role. In such a buffer, memory items that occurred seconds apart are represented with a temporal separation of 20-30 ms, thereby bringing them within the LTP window. The physiological and behavioral evidence for such a buffer will be reviewed.

CA1 pyramidal cells receive two major excitatory inputs: the perforant path (PP) terminates in the most distal dendrites, whereas the Schaffer collaterals (SC) terminate more proximally. We have examined the mechanism of the afterhyperpolarization (AHP) that follows single subthreshold excitatory postsynaptic potentials (EPSPs) in these inputs. The AHPs were not reduced by a GABAA antagonist or by agents that block Ca2+ entry. Application of the Ih blocker, ZD7288, partially blocked the AHP in the PP; the substantial remaining component was blocked by 2-hydroxysaclofen, a GABAB antagonist. In contrast, the AHP in the SC depends nearly completely on Ih, with almost no GABAB component. Thus postsynaptic GABAB receptors appear to be preferentially involved at distal synapses, consistent with the spatial distribution of GABAB receptors and g protein-coupled inward rectifying potassium (GIRK) channels. GABAB does, however, play a role at proximal synapses through presynaptic suppression of glutamate release, a mechanism that is much weaker at distal synapses. Experiments were conducted to explore the functional role of the AHP in the PP, which has a higher N-methyl-D-aspartate (NMDA)/AMPA ratio than the SC. Blockade of the AHP converted a response that had a small NMDA component to one that had a large component. These results indicate that the Ih and postsynaptic GABAB conductances act as a brake on distally generated NMDA responses.

We have used Monte Carlo simulations to understand the generation of quantal responses at the single active zones of CA1 synapses. We constructed a model of AMPA channel activation that accounts for the responses to controlled glutamate application and a model of glutamate diffusion in the synaptic cleft. With no further adjustments to these models, we simulated the response to the release of glutamate from a single vesicle. The predicted response closely matches the rise time of observed responses, which recent measurements show is much faster (<100 micros) than previously thought. The simulations show that initial channel opening is driven by a brief (<100 micros) glutamate spike near the site of vesicle fusion, producing a hotspot of channel activation (diameter: approximately 250 nm) smaller than many synapses. Quantal size therefore depends more strongly on the density of channels than their number, a finding that has important implications for measuring synaptic strength. Recent measurements allow estimation of AMPA receptor density at CA1 synapses. Using this value, our simulations correctly predicts a quantal amplitude of approximately 10 pA. We have also analyzed the properties of excitatory postsynaptic currents (EPSCs) generated by the multivesicular release that can occur during evoked responses. We find that summation is nearly linear and that the existence of multiple narrow peaks in amplitude histograms can be accounted for. It has been unclear how to reconcile the existence of these narrow peaks, which indicate that the variation of quantal amplitude is small (CV < 0.2) with the highly variable amplitude of miniature EPSCs (mEPSCs; CV approximately 0.6). According to one theory, mEPSC variability arises from variation in vesicle glutamate content. However, both our modeling results and recent experimental results indicate that this view cannot account for the observed rise time/amplitude correlation of mEPSCs. In contrast, this correlation and the high mEPSC variability can be accounted for if some mEPSCs are generated by two or more vesicles released with small temporal jitter. We conclude that a broad range of results can be accounted for by simple principles: quantal amplitude (approximately 10 pA) is stereotyped, some mEPSCs are multivesicular at moderate and large synapses, and evoked responses are generated by quasi-linear summation of multiple quanta.