Evidence for postsynaptic induction and expression of NMDA receptor independent LTP

Whole cell/patch-clamp and extracellular field potential recordings were used to study the induction and expression of N-methyl-D-aspartate (NMDA) receptor independent long-term potentiation (LTP) in area CA1 of the in vitro rat hippocampus. Induction of NMDA receptor independent LTP was prevented by manipulations that inhibited postsynaptic depolarization during tetanic stimulation: direct hyperpolarization of postsynaptic neurons and bath application of an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate receptor antagonist. NMDA receptor independent LTP also was blocked by intracellular application of the lidocaine derivative, N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide (QX-314), to CA1 pyramidal neurons. These results complement the previous findings that NMDA receptor independent LTP was inhibited by postsynaptic injections of the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and also was inhibited by a L-type voltage-dependent calcium channel antagonist (nifedipine). Collectively, these data make a strong case for the postsynaptic induction of this form of LTP. This paper also provides evidence for postsynaptic expression of NMDA receptor independent LTP. In an experiment where AMPA- and NMDA-receptor-mediated excitatory postsynaptic potentials (EPSPs) were isolated pharmacologically, LTP was found for only the AMPA-receptor-mediated EPSPs. In a separate experiment, paired-pulse facilitation (PPF) was measured during NMDA receptor independent LTP. Although there was an initial decrease in PPF, suggesting a posttetanic increase in the probability of glutamate release, the change in PPF decayed within 30-40 min of the tetanic stimulation, whereas the magnitude of the LTP was constant over this same time period. In addition, the LTP, but not the corresponding change in PPF, was blocked by the metabotropic glutamate receptor antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine. These results are accounted for most easily by a selective increase in postsynaptic AMPA receptor function, but one type of presynaptic modification-an increase in the number of release sites without an overall change in the probability of release-also could account for these results (assuming that the level of glutamate release before LTP induction fully saturated NMDA, but not AMPA, receptors). One possible presynaptic modification, an increase in axon excitability, was ruled out by analysis of the presynaptic fiber volley, which was not increased at any time after LTP induction.     

Modulation by substance P of synaptic transmission in the mouse hippocampal slice

The modulatory action of substance P on synaptic transmission of CA1 neurons was studied using intra- or extracellular recording from the mouse hippocampal slice preparation. Bath-applied substance P (2-4 microM) or the selective NK1 receptor agonist substance P methylester (SPME, 10 nM-5 microM) depressed field potentials (recorded from stratum pyramidale) evoked by focal stimulation of Schaffer collaterals. This effect was apparently mediated via NK1 receptors since it was completely blocked by the selective NK1 antagonist SR 140333. The field potential depression by SPME was significantly reduced in the presence of bicuculline. Intracellular recording from CA1 pyramidal neurons showed that evoked excitatory postsynaptic potentials (EPSPs) and evoked inhibitory postsynaptic potentials (IPSPs) were similarly depressed by SPME, which at the same time increased the frequency of spontaneous GABAergic events and reduced that of spontaneous glutamatergic events. The effects of SPME on spontaneous and evoked IPSPs were prevented by the ionotropic glutamate receptor blocker kynurenic acid. In tetrodotoxin (TTX) solution, no change in either the frequency of spontaneous GABAergic and glutamatergic events or in the amplitude of responses of pyramidal neurons to 4 microM alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or 10 microM N-methyl-D-aspartate (NMDA) was observed. On the same cells, SPME produced minimal changes in passive membrane properties unable to account for the main effects on synaptic transmission. The present data indicate that SPME exerted its action on CA1 pyramidal neurons via a complex network mechanism, which is hypothesized to involve facilitation of a subset of GABAergic neurons with widely distributed connections to excitatory and inhibitory cells in the CA1 area.     

Role of temporal summation in age-related long-term potentiation-induction deficits

Hippocampal long-term potentiation (LTP) is reduced in aged relative to young F-344 rats when peri-threshold stimulation protocols (several stimulus pulses at 100-200 Hz) are used. The present study was designed to examine the possibility that this LTP-induction deficit is caused by a reduced overlap of Schaffer-collateral inputs onto CA1 pyramidal cells (input cooperativity). This reduced input cooperativity would decrease the levels of postsynaptic depolarization during LTP induction, which might account for the age-related LTP deficit. Both behavioral data (Morris Water Maze) and electrophysiological data (intracellular recordings from hippocampal slices) were collected from adult and aged F-344 rats. To counter the effects of reduced input cooperativity, stimulus intensities were adjusted to elicit baseline excitatory postsynaptic potentials (EPSPs) of equivalent amplitude in aged and young rats. Contrary to expectations, however, an age-related LTP-induction deficit was still observed. Further evaluation of the electrophysiological data revealed that temporal summation of multiple EPSPs during high-frequency stimulation was impaired in the aged rats. Thus, despite the equalization across age groups of the baseline EPSP amplitudes, the cells of aged rats were less depolarized during the LTP-inducing stimulation than were those of young rats. This reduced total depolarization was not an artifact of the higher stimulus intensity used on aged animals, nor was it caused by a failure of aged rats' CA1 afferents to follow high-frequency stimulation. The present data therefore suggest that there is a deficit in the ability of aged rats' synapses to provide the sustained depolarization necessary to active the LTP-induction cascade.     

Bidirectional associative plasticity of unitary CA3-CA1 EPSPs in the rat hippocampus in vitro

Associative long-term potentiation (LTP) and depression of compound and unitary CA3-CA excitatory postsynaptic potentials (EPSPs) were investigated in rat hippocampal slice cultures. The induction of LTP with synchronous pairing of synaptic activation and postsynaptic depolarization resulted in an increase in the amplitude of EPSPs to the same absolute level, regardless of whether the input was naive or had been previously depressed by asynchronous pairing of pre- and postsynaptic activity. Saturated LTP of compound and unitary EPSPs was reversed by asynchronous pairing and could be reinduced by synchronous pairing. The likelihood that an action potential in a presynaptic CA3 cell failed to trigger an unitary EPSP in a postsynaptic CA1 cell decreased after induction of associative potentiation and increased after induction of associative depotentiation. These changes in the rate of transmission failures were accompanied by large changes in the amplitude of nonfailure EPSPs. We conclude that the same CA3-CA1 synapses can alternatively undergo associative potentiation and depression, perhaps through opposite changes in a single expression mechanism.     

Anoxic depression of excitatory and inhibitory postsynaptic potentials in rat neocortical slices

1. The effects of brief anoxia (4-6 min replacement of O2 by N2) on synaptic potentials evoked from layer IV and/or the white matter were studied in pyramidal neurons of layers II-III from rat neocortical slices. 2. The early and late components of excitatory postsynaptic potentials (EPSPs) showed differential sensitivity to anoxia: within 2 min the late EPSP (lEPSP) disappeared, whereas the amplitude of the early EPSP (eEPSP) decreased by 70% at 5 min of anoxia. Recovery was complete within 4-11 min. 3. Both fast and slow inhibitory postsynaptic potentials (IPSPs) were extremely sensitive to lack of O2 and were abolished earlier than the lEPSP evoked by the same stimulus. As well, recovery of the IPSPs was always more delayed than that of the EPSPs. 4. A transient increase in excitability during early anoxia and/or midrecovery, manifested as enhanced probability of spiking in 25% of neurons, is attributed to the higher sensitivity of IPSPs compared with EPSPs. 5. The anoxic-induced depression of the lEPSP and IPSPs, which are generated close to the soma, is not due to depolarization-induced occlusion; however, occlusion may cause an attenuation of the eEPSP at dendritic sites. 6. The depression of the EPSPs is not a result of a decreased transmembrane Na+ gradient after inactivation of Na-K-adenosine triphosphatase (Na-K-ATPase). Although ouabain induced a depolarization similar to that of anoxia, it did not affect EPSP amplitude.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stratum radiatum giant cells: a type of principal cell in the rat hippocampus

Neurons of a distinct type in CA1 area stratum radiatum of the rat hippocampus have been found to express a direct cellular form of long-term potentiation (LTP, Maccaferri & McBain, 1996, J. Neurosci. 16, 5334), but their functional identity, i.e. whether interneuron or principal cell, remained unknown. Whole cell recording from hippocampal slices in vitro was combined with light and electron microscopy to answer this question. LTP was robustly induced by a pairing protocol and physiological properties were measured in radiatum giant cells (RGCs) using biocytin containing pipettes. Reconstruction of the cells' dendritic and axonal arbor revealed morphological properties similar to CA1 pyramidal cells with some characteristic differences. They typically had two large diameter apical dendrites, or when only one dendrite arose, it soon bifurcated. Apical dendrites formed a dendritic tuft in stratum lacunosum-moleculare and the dendrites, but not the somata, were densely covered with conventional spines. The axon arose from the basal pole of the soma, descended to stratum oriens and emitted several axon terminals bearing collaterals that travelled horizontally, remaining in stratum oriens. The main, myelinated axon trunks turned towards the fimbria. In the electron microscope axon terminals were found to form asymmetrical synapses on postsynaptic dendritic shafts and dendritic spines in stratum oriens. The dendrites received asymmetrical synapses, mostly on their spines. The axon initial segments also received several synapses, a feature never observed on interneurons. All the above characteristics support the conclusion that RGCs are excitatory principal neurons.     

Calcium dynamics in single spines during coincident pre- and postsynaptic activity depend on relative timing of back-propagating action potentials and subthreshold excitatory postsynaptic potentials

We compared the transient increase of Ca2+ in single spines on basal dendrites of rat neocortical layer 5 pyramidal neurons evoked by subthreshold excitatory postsynaptic potentials (EPSPs) and back-propagating action potentials (APs) by using calcium fluorescence imaging. AP-evoked Ca2+ transients were detected in both the spines and in the adjacent dendritic shaft, whereas Ca2+ transients evoked by single EPSPs were largely restricted to a single active spine head. Calcium transients elicited in the active spines by a single AP or EPSP, in spines up to 80 micro(m) for the soma, were of comparable amplitude. The Ca2+ transient in an active spine evoked by pairing an EPSP and a back-propagating AP separated by a time interval of 50 ms was larger if the AP followed the EPSP than if it preceded it. This difference reflected supra- and sublinear summation of Ca2+ transients, respectively. A comparable dependence of spinous Ca2+ transients on relative timing was observed also when short bursts of APs and EPSPs were paired. These results indicate that the amplitude of the spinous Ca2+ transients during coincident pre- and postsynaptic activity depended critically on the relative order of subthreshold EPSPs and back-propagating APs. Thus, in neocortical neurons the amplitude of spinous Ca2+ transients could encode small time differences between pre- and postsynaptic activity.     

Evidence from simultaneous intracellular recordings in rat hippocampal slices that area CA3 pyramidal cells innervate dentate hilar mossy cells

1. Simultaneous intracellular recordings of area CA3 pyramidal cells and dentate hilar "mossy" cells were made in rat hippocampal slices to test the hypothesis that area CA3 pyramidal cells excite mossy cells monosynaptically. Mossy cells and pyramidal cells were differentiated by location and electrophysiological characteristics. When cells were impaled near the border of area CA3 and the hilus, their identity was confirmed morphologically after injection of the marker Neurobiotin. 2. Evidence for monosynaptic excitation of a mossy cell by a pyramidal cell was obtained in 7 of 481 (1.4%) paired recordings. In these cases, a pyramidal cell action potential was followed immediately by a 0.40 to 6.75 (mean, 2.26) mV depolarization in the simultaneously recorded mossy cell (mossy cell membrane potentials, -60 to -70 mV). Given that pyramidal cells used an excitatory amino acid as a neurotransmitter (Cotman and Nadler 1987; Ottersen and Storm-Mathisen 1987) and recordings were made in the presence of the GABAA receptor antagonist bicuculline (25 microM), it is likely that the depolarizations were unitary excitatory postsynaptic potentials (EPSPs). 3. Unitary EPSPs of mossy cells were prone to apparent "failure." The probability of failure was extremely high (up to 0.72; mean = 0.48) if the effects of all presynaptic action potentials were examined, including action potentials triggered inadvertently during other spontaneous EPSPs of the mossy cell. Probability of failure was relatively low (as low as 0; mean = 0.24) if action potentials that occurred during spontaneous activity of the mossy cell were excluded. These data suggest that unitary EPSPs produced by pyramidal cells are strongly affected by concurrent synaptic inputs to the mossy cell. 4. Unitary EPSPs were not clearly affected by manipulation of the mossy cell's membrane potential. This is consistent with the recent report that area CA3 pyramidal cells innervate distal dendrites of mossy cells (Kunkel et al. 1993). Such a distal location also may contribute to the high incidence of apparent failures. 5. Characteristics of unitary EPSPs generated by pyramidal cells were compared with the properties of the unitary EPSPs produced by granule cells. In two slices, pyramidal cell and granule cell inputs to the same mossy cell were compared. In other slices, inputs to different mossy cells were compared. In all experiments, unitary EPSPs produced by granule cells were larger in amplitude but similar in time course to unitary EPSPs produced by pyramidal cells. Probability of failure was lower and paired-pulse facilitation more common among EPSPs triggered by granule cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytochrome-oxidase blobs and intrinsic horizontal connections of layer 2/3 pyramidal neurons in primate V1

Pyramidal neurons in superficial layers of cerebral cortex have extensive horizontal axons that provide a substrate for lateral interactions across cortical columns. These connections are believed to link functionally similar regions, as suggested by the observation that cytochrome-oxidase blobs in the monkey primary visual cortex (V1) are preferentially connected to blobs and interblobs to interblobs. To better understand the precise relationship between horizontal connections and blobs, we intracellularly labeled 20 layer 2/3 pyramidal neurons in tangential living brain slices from V1 of macaque monkeys. The locations of each cell body and the cell's synaptic boutons relative to blobs were quantitatively analyzed. We found evidence for two cell types located at characteristic distances from blob centers: (1) neurons lacking long-distance, clustered axons (somata 130-200 microm from blob centers) and (2) cells with clustered, long-distance axon collaterals (somata < 130 microm or >200 microm from blob centers). For all cells, synaptic boutons close to the cell body were located at similar distances from blob centers as the cell body. The majority of boutons from cells lacking distal axon clusters were close to their cell bodies. Cells located more than 200 microm from blob centers were in interblobs and had long-distance clustered axon collaterals selectively targeting distant interblob regions. Cells located less than 130 microm from blob centers were found within both blobs and interblobs, but many were close to traditionally defined borders. The distant synaptic boutons from these cells were generally located relatively near to blob centers, but the neurons closest to blob centers had synaptic boutons closer to blob centers than those farther away. There was not a sharp transition that would suggest specificity for blobs and interblobs as discrete, binary entities. Instead they appear to be extremes along a continuum. These observations have important implications for the function of lateral interactions within V1.     

Metabotropic glutamate receptors are involved in long-term potentiation in isolated slices of rat medial frontal cortex

The prelimbic region of medial frontal cortex in the rat receives a direct input from the hippocampus and this functional connection is essential for aspects of spatial memory. Activity-dependent changes in the effectiveness of synaptic transmission in the medial frontal cortex, namely long-term potentiation (LTP) and long-term depression (LTD) can persist for tens of minutes or hours and may be the basis of learning and memory storage. Glutamatergic activation of ionotropic receptors is required to induce both LTP and LTD. We now present evidence of the involvement of metabotropic glutamate receptors in LTP in isolated slices of frontal cortex. Repetitive bursts of stimulation at theta frequencies (TBS) were applied to layer II, and monosynaptic EPSPs were monitored in layer V neurons of the prelimbic area. TBS was found to be more effective at inducing LTP than tetanic stimulation at 100 Hz and produced LTP that lasted >30 min in 8 out of 14 neurons. Tetanic stimulation at 100 Hz in the presence of the N-methyl--aspartate (NMDA)-antagonist 2-amino-5-phosphonopentanoate (AP5) was reported to be a reliable method of inducing LTD in prelimbic cortex (). However we found that this protocol did not facilitate the induction of LTD. The role of metabotropic glutamate receptors (mGluR) in LTP was assessed by using the selective, broad-spectrum antagonist (R, S)-alpha-methyl-4- carboxyphenylglycine (MCPG). This drug significantly reduced the incidence of LTP after TBS to only 1 of 14 neurons (P < 0.02, chi2 test). The pooled responses to TBS in MCPG showed significantly reduced potentiation [(P < 0.02, analysis of variance (ANOVA)]. The broad-spectrum mGluR agonist (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) and the selective group I agonist S-3 hydroxyphenylglycine(S-3HPG) both produced membrane depolarization, an increase in number of spikes evoked by depolarizing current pulses, and a reduction in the afterhyperpolarization. Similar effects were produced by these agonists even when synaptic transmission was blocked by use of the gamma-aminobutyric acid-B (GABAB) receptor agonist, 200 microM baclofen, which suggests that group I mGluRs are present on layer V neurons. We conclude that mGluRs participate in the production of LTP in prelimbic cortex, and that this excitatory effect could be mediated by the postsynaptic group I mGluRs.     

Developmental influence of glycinergic transmission: regulation of NMDA receptor-mediated EPSPs

The influence of excitatory transmission on postsynaptic structure is well established in developing animals, but little is known about the role of synaptic inhibition. We addressed this issue in developing gerbils with two manipulations designed to decrease glycinergic transmission in an auditory nucleus, the lateral superior olive (LSO), before the onset of sound-evoked activity. First, contralateral cochlear ablation functionally denervated the glycinergic pathway from the medial nucleus of the trapezoid body (MNTB) to the LSO, while leaving the excitatory pathway intact. Second, continuous release of a glycine receptor antagonist, strychnine (SN), was used to decrease transmission. The strength of excitatory and inhibitory synapses was examined with whole-cell recordings from LSO neurons in a brain-slice preparation. The percentage of LSO neurons exhibiting MNTB-evoked IPSPs was reduced in both ablated and SN-treated animals. In those neurons displaying IPSPs, the amplitude was significantly reduced. This decrease was accompanied by an 8 mV depolarization in the IPSP equilibrium potential. In contrast, the ipsilaterally evoked EPSPs were of unusually long duration in experimental animals. These long-duration EPSPs were significantly shortened by hyperpolarizing the neuron to -90 mV or exposing them to aminophosphonopentanoic acid (AP-5), an NMDA receptor antagonist. Membrane hyperpolarization and AP-5 had little effect in control neurons. In addition, LSO neurons from ablated or SN-treated animals displayed broad rebound depolarizations after membrane hyperpolarization, and these were abolished in the presence of Ni2+. Because both cochlear ablation and SN-rearing were initiated before the onset of sound-evoked activity, the results suggest that spontaneous glycinergic transmission influences the development of postsynaptic properties, including the IPSP reversal potential, NMDA receptor function, and a Ca2+ conductance.     

Silent synapses in the developing rat visual cortex: evidence for postsynaptic expression of synaptic plasticity

In the developing visual cortex activity-dependent refinement of synaptic connectivity is thought to involve synaptic plasticity processes analogous to long-term potentiation (LTP). The recently described conversion of so-called silent synapses to functional ones might underlie some forms of LTP. Using whole-cell recording and minimal stimulation procedures in immature pyramidal neurons, we demonstrate here the existence of functionally silent synapses, i.e., glutamatergic synapses that show only NMDA receptor-mediated transmission, in the neonatal rat visual cortex. The incidence of silent synapses strongly decreased during early postnatal development. After pairing presynaptic stimulation with postsynaptic depolarization, silent synapses were converted to functional ones in an LTP-like manner, as indicated by the long-lasting induction of AMPA receptor-mediated synaptic transmission. This conversion was dependent on the activation of NMDA receptors during the pairing protocol. The selective activation of NMDA receptors at silent synapses could be explained presynaptically by assuming a lower glutamate concentration compared with functional ones. However, we found no differences in glutamate concentration-dependent properties of NMDA receptor-mediated PSCs, suggesting that synaptic glutamate concentration is similar in silent and functional synapses. Our results thus support a postsynaptic mechanism underlying silent synapses, i.e., that they do not contain functional AMPA receptors. Synaptic plasticity at silent synapses might be expressed postsynaptically by modification of nonfunctional AMPA receptors or rapid membrane insertion of AMPA receptors. This conversion of silent synapses to functional ones might play a major role in activity-dependent synaptic refinement during development of the visual cortex.     

Double Resonance Sub-Doppler Study of the Allowed and DeltaK = -3 Forbidden Q(3, 3) Transitions to the nu2 Vibrational State of 14NH3

Whereas much is now known about the behavioral importance of the lateral nucleus of the amygdala for the storage of implicit memories of fear, little is known in molecular terms about the signal transduction pathways required for long-term potentiation (LTP) in this nucleus. Using brain slices containing the amygdala, we have studied LTP in the pathway from external capsule to the lateral nucleus, a pathway that mediates information from the auditory cortex important for fear conditioning. We found the induction of LTP is postsynaptic; it is dependent on postsynaptic depolarization, on the influx of Ca2+ into the postsynaptic cell and, at least in part, on the activation of N-methyl-D-aspartate (NMDA) receptors. The LTP is associated with a decrease of paired-pulse facilitation (PPF) and is blocked by bath application but not blocked by postsynaptic injection of inhibitors of the cyclic adenosine monophosphate-dependent (cAMP-dependent) protein kinase (PKA). Consistent with the possibility that the expression might involve PKA presynaptically, the adenylyl cyclase activator forskolin induced synaptic potentiation of this pathway that also was associated with a decrease of PPF, and this potentiation occluded the tetanus-induced LTP.     

Conditions for the induction of long-term potentiation in layer II/III horizontal connections of the rat motor cortex

1. The present studies investigated conditions for the induction of long-term potentiation (LTP) in the local horizontal pathways of layers II/III in the primary motor cortex (MI) of the adult rat. Field potential and intracellular recordings demonstrated synaptic interactions across the superficial layers within MI that could be enhanced transiently by focal application of the gamma-aminobuturic acid-A receptor antagonist bicuculline methiodide (Bic) at the recording site. 2. Field potentials evoked in the superficial MI horizontal pathways increased in amplitude after tetanizing, theta burst stimulation (TBS), but only when Bic was applied transiently at the recording site immediately before TBS. In the absence of Bic, TBS failed to produce long-lasting increases in horizontally evoked field responses. By contrast, TBS delivery during focal Bic application increased field potential amplitudes by 25-35% when measured 25-30 min after stimulation. The amount of potentiation was greater when two converging horizontal inputs were stimulated together but was not increased with higher intensity stimulation. Persistent effects of Bic application alone were evident. However, these effects were small unless Bic application continued until evoked field potential amplitude increase exceeded 200% of baseline. 3. The synaptic nature of field potential increases were confirmed using intracellular recordings of layer II/III neurons located near field potential electrodes. 4. LTP also could be induced without Bic application by cotetanization of vertical pathways simultaneously with horizontal activation. Vertical conditioning alone at 2 Hz, which affects inhibitory efficacy, was shown to transiently relieve depression of successive responses that ordinarily occurs during a burst of three horizontal stimuli. These results suggest that LTP of horizontal pathways may be regulated by spatiotemporal interactions between horizontal and vertical pathways. 5. Horizontal LTP was blocked reversibly by bath application of the N-methyl-D-aspartate (NMDA) antagonist 2-amino-5-phosphonovaleric acid, thereby implicating NMDA-receptor activation in LTP induction for these pathways. 6. The results confirm and extend our previous finding that the potential for activity-dependent modification of synaptic connections exists within the intrinsic horizontal connections of the superficial cortical layers. Synaptic modification across horizontally connected neurons appears to be regulated both by the arrangement of intrinsic circuitry and by the availability of mechanisms for modification at individual synapses. The properties of horizontal connections indicate that they form a spatial substrate and provide an activity-dependent mechanism for plasticity of adult cortical representations.     

Windup leads to characteristics of central sensitization

To elucidate the physiological role of Fyn, we analysed the properties of synaptic transmission and synaptic plasticity in hippocampal slices of mice overexpressing either wild-type Fyn (w-Fyn) or its constitutively active mutant (m-Fyn). These fyn-transgenes were driven by the calcium/calmodulin-dependent protein kinase II alpha promoter which turned on in the forebrain neurons including hippocampal pyramidal cells and in late neural development. In the hippocampal slices expressing m-Fyn the paired-pulse facilitation was reduced and the basal synaptic transmission was enhanced. A weak theta-burst stimulation, which was subthreshold for the induction of long-term potentiation (LTP) in control slices, elicited LTP in CA1 region of the slices expressing m-Fyn. When a relatively strong stimulation was applied, the magnitude of LTP in m-Fyn slices was similar to that in control slices. By contrast, the basal synaptic transmission and the threshold for the induction of LTP were not altered in the slices overexpressing wild-type Fyn. To examine the effect of expression of m-Fyn on GABAergic inhibitory system, we applied bicuculline, a GABAA receptor blocker, to the hippocampal slices. The ability of bicuculline to enhance excitatory postsynaptic potentials was attenuated in slices expressing m-Fyn, suggesting that the overexpression of m-Fyn reduced the GABAergic inhibition. The enhancement of synaptic transmission and the reduction of GABAergic inhibition may contribute to the enhanced seizure susceptibility in the mice expressing m-Fyn. Thus, these results suggest that regulation of Fyn tyrosine kinase activity is important for both synaptic transmission and plasticity.     

Hebbian induction of LTP in visual cortex: perforated patch-clamp study in cultured neurons

1. To see whether presynaptic activation paired with postsynaptic depolarization is necessary for the induction of long-term potentiation (LTP) in visual cortex or whether an activation of postsynaptic receptors in conjunction with depolarization is sufficient, we carried out perforated patch-clamp recordings with nystatin from cultured cortical neurons of rats. 2. Recorded neurons were monosynaptically activated either by electrical stimulation of an adjacent neuron or by direct activation of glutamate on "hot spots" of dendrites through iontophoresis or pressure ejection. In experiments in which cultured neurons were stained immunocytochemically with antibody against synaptophysin after electrophysiological recordings, hot spots were found to correspond to probable synaptic sites. 3. Excitatory postsynaptic currents (EPSCs) evoked by test stimulation applied to the adjacent neuron at 0.1 Hz were recorded at a holding potential of -60 or -70 mV for 5-10 min after an establishment of the whole cell recording configuration. Then, stimulation was paired with postsynaptic depolarization (0 mV for 200 ms) at 1 Hz for 30 or 60 s. LTP of EPSCs was induced in 7 of the 15 cells from which stable recordings were obtained for 18-30 min after pairing. 4. When postsynaptic depolarization was paired with direct glutamate application in the absence of presynaptic stimulation in 12 cells, only 1 showed LTP. Postsynaptic depolarization alone did not induce LTP in any of the six cells tested. Also, presynaptic stimulation alone did not induce LTP in any of the five cells tested. 5. These results suggest that the concurrent activation of presynaptic elements with postsynaptic depolarization is necessary for the induction of LTP in visual cortex.     

Serotonin induces excitatory postsynaptic potentials in apical dendrites of neocortical pyramidal cells

By intracellular and whole cell recording in rat brain slices, it was found that bath-applied serotonin (5-HT) produces an increase in the frequency and amplitude of spontaneous excitatory postsynaptic potentials/currents (EPSPs/EPSCs) in layer V pyramidal cells of neocortex and transitional cortex (e.g. medial prefrontal, cigulate and frontoparietal). The EPSCs were suppressed by LY293558, an antagonist selective for the AMPA subtype of excitatory amino acid receptor, and by two selective 5-HT2A receptor antagonists, MDL 100907 and SR 46349B. In addition, the EPSCs were suppressed by the fast sodium channel blocker tetrodotoxin (TTX) and were dependent upon external calcium. However, despite being TTX-sensitive and calcium dependent, there was no evidence that the EPSPs resulted from an increase in impulse flow in excitatory neuronal afferents to layer V pyramidal cells. The EPSCs could be induced rapidly by the microiontophoresis of 5-HT directly to "hot spots" within the apical (but not basilar) dendritic field of recorded neurons, indicating that excitatory amino acids may be released by a TTX-sensitive focal action of 5-HT on a subset of glutamatergic terminals in this region. Consistent with such a presynaptic action, the inhibitory metabotropic glutamate receptor agonist (1S,3S)-aminocyclopentane-1,3-dicarboxylate markedly reduced the induction of EPSPs by 5-HT. Postsynaptically, 5-HT enhanced a subthreshold TTX-sensitive sodium current, potentially contributing to an amplification of EPSC amplitudes. These data suggest 5-HT. via 5-HT2A receptors, enhances spontaneous EPSPs/EPSCs in neocortical layer V pyramidal cells through a TTX-sensitive focal action in the apical dendritic field which may involve both pre- and postsynaptic mechanisms.     

Transplanted embryonic entorhinal neurons make functional synapses in adult host hippocampus

Grafts of embryonic entorhinal cortex (EC) or non-entorhinal cortex (NEC) were placed into the hippocampus of adult rats with transection of the perforant paths. Graft-host connectivity was investigated at 4-6 months post-transplantation by recording extracellular evoked responses in hippocampal slice preparations. Electrical stimulation of the grafts evoked excitatory postsynaptic potentials (EPSPs) in the outer molecular layer of the dentate gyrus, and the stratum lacunosum moleculare of CA1, CA3, and elicited population spikes in the granule cell layer and the pyramidal cell layer of CA1, but not CA3. While the latencies and the forms of these evoked response were similar to those in matched control slices from the normal animals, the amplitudes were smaller than normal controls. However, in the slices with NEC grafts, no such responses were recorded when stimulus was applied in similar position in the grafts. The findings suggest that grafted entorhinal neurons make viable synaptic connections with the host hippocampus.     

N-methyl-D-aspartate receptors are indispensable for the formation of long-term potentiation in the rat suprachiasmatic nucleus in vitro

Optic nerve (ON) stimulation caused a postsynaptic field potential in the suprachiasmatic nucleus (SCN) of rat hypothalamic slices. The postsynaptic field potential was suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist, in a concentration-dependent manner, but not affected by D-amino-5-phosphonovaleric acid (APV), a competitive NMDA receptor antagonist. Tetanic stimulation to the ON induced long-term potentiation (LTP) in the SCN. Application of APV at 50 microM inhibited the induction of LTP by tetanic stimulation but CNQX at lower dose (5 microM) didn't inhibit it. These results suggest that NMDA receptors are indispensable for the induction of LTP after tetanic stimulation.     

Src activation in the induction of long-term potentiation in CA1 hippocampal neurons

Long-term potentiation (LTP) is an activity-dependent strengthening of synaptic efficacy that is considered to be a model of learning and memory. Protein tyrosine phosphorylation is necessary to induce LTP. Here, induction of LTP in CA1 pyramidal cells of rats was prevented by blocking the tyrosine kinase Src, and Src activity was increased by stimulation producing LTP. Directly activating Src in the postsynaptic neuron enhanced excitatory synaptic responses, occluding LTP. Src-induced enhancement of alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) receptor-mediated synaptic responses required raised intracellular Ca2+ and N-methyl-D-aspartate (NMDA) receptors. Thus, Src activation is necessary and sufficient for inducing LTP and may function by up-regulating NMDA receptors.