Characteristics of local excitatory circuits studied with glutamate microapplication in the CA3 area of rat hippocampal slices

1. Local neuronal circuits in CA3 of hippocampal slices were studied by recording excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) intracellularly during glutamate microapplication in CA3. Control experiments validated this approach by providing evidence that glutamate microdrops stimulated neurons but not axons-of-passage or axon terminals in CA3. 2. Glutamate microdrops (10-20 mM, 10-20 microns diam) increased the firing frequency of extracellularly recorded dentate granule cells for 5-10 s when applied to their somata but not when applied to their mossy fiber axons and terminals in the hilus and in CA3. 3. Glutamate microapplications to granule cell somata, but not to mossy fiber axons, also increased the frequency of intracellularly recorded EPSPs in CA3 pyramidal cells for 5-10 s. This provided a second line of evidence that glutamate did not cause firing in mossy fiber axons synapsing in CA3. 4. In slices where the CA3 region was surgically separated from the dentate gyrus and CA2, glutamate microdrops placed in the CA3 stratum pyramidale within 400 microns of intracellularly recorded pyramidal cells increased the frequency of EPSPs and IPSPs. Tetrodotoxin (1 microgram/ml) blocked these increases in PSP frequency, indicating that they did not result from glutamate-induced depolarization and associated transmitter release from presynaptic terminals. Increases in PSP frequency were interpreted to reflect glutamate activations of CA3 neurons with local synaptic connections to recorded cells. 5. Low concentrations of picrotoxin (PTX, 5-10 microM) blocked glutamate-induced increases in IPSP frequency and often revealed increases in EPSP frequency where they were not previously observed. This suggests that recurrent inhibitory circuits normally mask or block transmission through recurrent excitatory pathways in CA3. 6. In five experiments following PTX treatment (7.5-10 microM), large and prolonged (up to 2 min) increases in EPSP frequency were observed in CA3 pyramidal cells to glutamate microapplications in CA3. Rhythmic epileptiform bursts eventually occurred in two of these cases, suggesting that the protracted increases in EPSP frequency represent a form of reverberating excitation during a transition from normal to epileptic states. 7. Sixteen CA3 pyramidal cells were recorded in PTX (5-10 microM) during glutamate microapplications at 200 and 400 microns on each side of the recording site. The most consistent glutamate-induced increases in EPSP frequency occurred to microapplications 200 microns from recording sites on the hilar side.(ABSTRACT TRUNCATED AT 400 WORDS)     

Electrophysiological evidence from glutamate microapplications for local excitatory circuits in the CA1 area of rat hippocampal slices

1. Evidence for local excitatory synaptic connections in CA1 of the rat hippocampus was obtained by recording excitatory postsynaptic potentials (EPSPs) intracellularly from pyramidal cells during local microapplications of glutamate. 2. Experiments were performed in hippocampal slices cut parallel to (transverse slice) or perpendicular to (longitudinal slice) alvear fibers. In normal solutions, glutamate microdrops (10-20 mM, 10-20 micron diam) applied in CA1 within 400 micron of recorded cells sometimes increased the frequency of inhibitory postsynaptic potentials for 5-10 s in both transverse and longitudinal slices. Increases in EPSP frequency were also occasionally observed, but only in transverse slices. Tetrodotoxin (1 microgram/ml) blocked glutamate-induced increases in PSP frequency, thus indicating that they were not caused by subthreshold effects on presynaptic terminals. Increases in PSP frequency were interpreted to result from glutamate activation of hippocampal neurons with inhibitory and excitatory connections to recorded neurons. 3. In both slice orientations, local excitatory circuits were studied in more isolated conditions by surgically separating CA1 from CA3 (transverse slices) and by blocking GABAergic inhibitory synapses with picrotoxin (5-10 microM). Microdrops were systematically applied at 200 and 400 micron on each side of the recording site. Significant glutamate-induced increases in EPSP frequency were observed in neurons from both slice orientations to microdrops in at least one of the locations. This provided evidence that excitatory synapses are present in both transverse and longitudinal slices. 4. Substantial increases in EPSP frequency only occurred in neurons from longitudinal slices when glutamate was microapplied 200 micron or less from the recording site. In transverse slices, however, large increases in EPSP frequency were observed to glutamate microapplications at 200 or 400 micron. These data suggest that CA1 local excitatory connections project for longer distances in the transverse than in the longitudinal plane of section. 5. Increases in EPSP frequency, averaged across cells, did not differ significantly in the four microapplication sites in either transverse or longitudinal slices. Thus local excitation in CA1 does not appear to be asymmetrically arranged in the way suggested for CA3. 6. The densities of local excitatory circuits in CA1 versus CA3 were studied by quantitatively comparing glutamate-induced increases in EPSP frequency.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rat hippocampal neurons in culture: responses to electrical and chemical stimuli

Intracellular activity was recorded from dissociated rat hippocampal neurons maintained in tissue culture conditions for 4-6 wk. The cells developed dense interconnections and had typical morphological characteristics similar to hippocampal neurons in situ. The recorded neurons possessed similar electrophysiological properties to those observed in situ or in a slice preparation. Their input resistance (42 M omega), resting membrane potential (-60 mV), membrane time constant (16.2 ms), total electrotonic length (0.92), and spike size (68.3 mV) were similar to values obtained in hippocampal cells in a slice. The connections among adjacent neurons were largely inhibitory. The inhibitory postsynaptic potentials (IPSPs) had longer durations than excitatory postsynaptic potentials (EPSPs) when these were detected. Synaptic delay varied between 0.3 and 3.0 ms. There were no electrotonic connections among neurons. Reciprocal connections were common. Most neurons reacted to acetylcholine (ACh) by an increase in frequency of spontaneous EPSPs, action-potential discharges, and IPSPs. Concurrently, there was a marked reduction in the magnitude of the evoked PSPs tested in pairs of cells. This effect is probably presynaptic to the recorded neurons. A statistical analysis of quantal properties of the synaptic interactions among neurons revealed that ACh causes a reduction of magnitude of PSPs by reducing the number of releasing elements (m). This effect is different from the reduction of evoked PSPs caused by postsynaptic depolarization.     

Physiology and pharmacology of epileptiform activity induced by 4-aminopyridine in rat hippocampal slices.

1. Conventional intracellular and extracellular recording techniques were used to investigate the physiology and pharmacology of epileptiform bursts induced by 4-aminopyridine (4-AP, 50 microM) in the CA3 area of rat hippocampal slices maintained in vitro. 2. 4-AP-induced epileptiform bursts, consisting of a 25-to 80-ms depolarizing shift of the neuronal membrane associated with three to six fast action potentials, occurred at the frequency of 0.61 +/- 0.29 (SD)/s. The bursts were generated synchronously by CA3 neurons and were triggered by giant excitatory postsynaptic potentials (EPSPs). A second type of spontaneous activity consisting of a slow depolarization also occurred but at a lower rate (0.04 +/- 0.2/s). 3. The effects of 4-AP on EPSPs and inhibitory postsynaptic potentials (IPSPs) evoked by mossy fiber stimulation were studied on neurons impaled with a mixture of K acetate and 2(triethyl-amino)-N-(2,6-dimethylphenyl) acetamide (QX-314)-filled microelectrodes. After the addition of 4-AP, the EPSP became potentiated and was followed by the appearance of a giant EPSP. This giant EPSP completely obscured the early IPSP recorded under control conditions and inverted at -32 +/- 3.9 mV (n = 4), suggesting that both inhibitory and excitatory conductances were involved in its generation. IPSPs evoked by Schaffer collateral stimulation increased in amplitude and duration after 4-AP application. 4. The spontaneous field bursts and the stimulus-induced giant EPSP induced by 4-AP were not affected by N-methyl-D-aspartate (NMDA) receptor antagonists 3-3 (2-carboxy piperazine-4-yl) propyl-1-phosphonate (CPP) and DL-2-amino-5-phosphonovalerate (APV) but were blocked by quisqualate/kainate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX). CNQX also abolished the presence of small spontaneously occurring EPSPs, thereby disclosing the presence of bicuculline-sensitive (BMI, 20 microM) IPSPs. 5. Small, nonsynchronous EPSPs played an important role in the generation of 4-AP-induced epileptiform activity. 1) After the addition of 4-AP, small EPSPs appeared randomly on the baseline and then became clustered to produce a depolarizing envelope of irregular shape that progressively formed an epileptiform burst, 2) These small EPSPs were more numerous in the 100 ms period that preceded burst onset. 3) The frequency of occurrence of small EPSPs was positively correlated with the frequency of occurrence of synchronous bursts. 4) Small EPSPs and bursts were similarly decreased after the addition of different concentrations of CNQX (IC50 in both cases of approximately 1.2 microM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of cellular and synaptic variability in the lamprey spinal cord

Variability is increasingly recognized as a characteristic feature of cellular, synaptic, and network properties. While studies have traditionally focused on mean values, significant effects can result from changes in variance. This study has examined cellular and synaptic variability in the lamprey spinal cord and its modulation by the neuropeptide substance P. Cellular and synaptic variability differed in different types of cell and synapse. Substance P reduced the variability of subthreshold locomotor-related depolarizations and spiking in motor neurons during network activity. These effects were associated with a reduction in the variability of spiking in glutamatergic excitatory network interneurons and with a reduction in the variance of excitatory interneuron-evoked excitatory postsynaptic potentials (EPSPs). Substance P also reduced the variance of postsynpatic potentials (PSPs) from crossing inhibitory and excitatory interneurons, but it increased the variance of inhibitory postsynpatic potentials (IPSPs) from ipsilateral inhibitory interneurons. The effects on the variance of different PSPs could occur with or without changes in the PSP amplitude. The reduction in the variance of excitatory interneuron-evoked EPSPs was protein kinase A, calcium, and N-methyl-d-aspartate (NMDA) dependent. The NMDA dependence suggested that substance P was acting postsynaptically. This was supported by the reduced variability of postsynaptic responses to glutamate by substance P. However, ultrastructural analyses suggested that there may also be a presynaptic component to the modulation, because substance P reduced the variability of synaptic vesicle diameters in putative glutamatergic terminals. These results suggest that cellular and synaptic variability can be targeted for modulation, making it an additional source of spinal cord plasticity.     

Neuropeptide Y reduces epileptiform discharges and excitatory synaptic transmission in rat frontal cortex in vitro

Neuropeptide Y reduced spontaneous and stimulation-evoked epileptiform discharges in rat frontal cortex slices perfused with a magnesium-free solution and with the GABA(A) receptor antagonist picrotoxin. To investigate the mechanism of that action, effects of neuropeptide Y on intrinsic membrane properties and synaptic responses of layer II/III cortical neurons were studied using intracellular recording. Neuropeptide Y (1 microM) had no detectable effect on the membrane properties of neurons. The evoked synaptic potentials were attenuated by neuropeptide Y. Moreover, the pharmacologically isolated excitatory postsynaptic potentials, mediated by N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors, were reversibly depressed by neuropeptide Y. The most pronounced inhibitory effect of neuropeptide Y was observed on late polysynaptic excitatory postsynaptic potentials. To assess a putative postsynaptic action of neuropeptide Y, N-methyl-D-aspartate was locally applied in the presence of tetrodotoxin. The N-methyl-D-aspartate-evoked depolarizations were unaffected by neuropeptide Y, which suggests that the depression of excitatory postsynaptic potentials was due to an action at sites presynaptic to the recorded neurons.These data show that neuropeptide Y attenuates epileptiform discharges and the glutamate receptor-mediated synaptic transmission in the rat frontal cortex. The above results indicate that neuropeptide Y may regulate neuronal excitability within the cortex, and that neuropeptide Y receptors are potential targets for an anticonvulsant therapy.     

Target-cell-specific facilitation and depression in neocortical circuits

In neocortical circuits, repetitively active neurons evoke unitary postsynaptic potentials (PSPs) whose peak amplitudes either increase (facilitate) or decrease (depress) progressively. To examine the basis for these different synaptic responses, we made simultaneous recordings from three classes of neurons in cortical layer 2/3. We induced repetitive action potentials in pyramidal cells and recorded the evoked unitary excitatory (E)PSPs in two classes of GABAergic neurons. We observed facilitation of EPSPs in bitufted GABAergic interneurons, many of which expressed somatostatin immunoreactivity. EPSPs recorded from multipolar interneurons, however, showed depression. Some of these neurons were immunopositive for parvalbumin. Unitary inhibitory (I)PSPs evoked by repetitive stimulation of a bitufted neuron also showed a less pronounced but significant difference between the two target neurons. Facilitation and depression involve presynaptic mechanisms, and because a single neuron can express both behaviors simultaneously, we infer that local differences in the molecular structure of presynaptic nerve terminals are induced by retrograde signals from different classes of target neurons. Because bitufted and multipolar neurons both formed reciprocal inhibitory connections with pyramidal cells, the results imply that the balance of activation between two recurrent inhibitory pathways in the neocortex depends on the frequency of action potentials in pyramidal cells.     

Epileptiform activity in hippocampal slice cultures with normal inhibitory synaptic drive

The synaptic events responsible for epileptiform burst discharge are often difficult to define. Blockade of inhibition has been used to produce epileptiform events, but it is unclear whether increased excitatory activity in the presence of normal inhibition can also result in burst discharge. In the hippocampal slice culture preparation, a small percentage of cultures exhibit spontaneous bursts. To determine whether the absence of inhibitory postsynaptic potentials (IPSPs) is responsible for these spontaneous bursts, we applied the glutamate antagonist, kynurenic acid (KYN) to block burst activity, and unmask any underlying IPSPs. KYN (10 mM) quickly reduced synaptic activity with concomitant loss of burst discharge. Washout of KYN resulted in a gradual return of synaptic activity, during which time both fast and slow IPSPs were clearly observed. As burst activity returned to control levels, excitatory postsynaptic potentials (EPSPs) were increasingly superimposed within the inhibitory events, obscuring (but not eliminating) the IPSPs. In these hippocampal slice cultures, therefore, epileptiform bursts appear to be the result of an abnormally high level of excitatory synaptic drive, not a reduction in inhibition.     

Presynaptic GABA(B) receptors inhibit synaptic inputs to rat subthalamic neurons.

Effects of baclofen on synaptic transmission were studied in rat subthalamic neurons using whole-cell patch clamp recording from brain slices. Focal electrical stimulation of the brain slice evoked GABAergic inhibitory postsynaptic currents and glutamatergic excitatory postsynaptic currents. Baclofen reduced the amplitude of evoked inhibitory postsynaptic currents in a concentration-dependent manner with an IC(50) of 0.6+/-0.2 microM. Evoked excitatory postsynaptic currents were also reduced by baclofen concentration-dependently (IC(50) of 1.6+/-0.2 microM), but baclofen was more potent at reducing the GABA(A) receptor inhibitory postsynaptic currents. The GABA(B) receptor antagonist CGP 35348 blocked these inhibitory effects of baclofen on evoked inhibitory and excitatory postsynaptic currents. Baclofen increased the paired-pulse ratios of evoked inhibitory and excitatory postsynaptic currents. Furthermore, baclofen reduced the frequency of spontaneous miniature excitatory postsynaptic currents, but had no effect on their amplitude.These results provide evidence for presence of presynaptic GABA(B) receptors that modulate both GABA and glutamate release from afferent terminals in the subthalamus.     

Transient depression of excitatory synapses on interneurons contributes to epileptiform bursts during gamma oscillations in the mouse hippocampal slice

Persistent gamma frequency (30-70 Hz) network oscillations occur in hippocampal slices under conditions of metabotropic glutamate receptor (mGluR) activation. Excessive mGluR activation generated a bistable pattern of network activity during which epochs of gamma oscillations of increasing amplitude were terminated by synchronized bursts and very fast oscillations (>70 Hz). We provide experimental evidence that, during this behavior, pyramidal cell-to-interneuron synaptic depression takes place, occurring spontaneously during the gamma rhythm and associated with the onset of epileptiform bursts. We further provide evidence that excitatory postsynaptic potentials (EPSPs) in pyramidal cells are potentiated during the interburst gamma oscillation. When these two types of synaptic plasticity are incorporated, phenomenologically, into a network model previously shown to account for many features of persistent gamma oscillations, we find that epochs of gamma do indeed alternate with epochs of very fast oscillations and epileptiform bursts. Thus the same neuronal network can generate either gamma oscillations or epileptiform bursts, in a manner depending on the degree of network drive and network-induced fluctuations in synaptic efficacies.     

APV-sensitive dorsal root afferent transmission to neonate rat sympathetic preganglionic neurons in vitro

1. Intracellular recordings were made from antidromically identified sympathetic preganglionic neurons (SPNs) in transverse thoracolumbar spinal cord slices from neonate (12- to 22-day-old) rats. 2. Electrical stimulation of dorsal roots or dorsal root entry zone elicited in SPNs an excitatory postsynaptic potential (EPSP) or multiple EPSPs of varying latencies. The EPSP could be graded by varying the stimulus intensity and, on reaching the threshold, discharged an action potential. 3. The dorsal root-evoked EPSPs had a mean synaptic latency of 2.6 ms (range: 1.2-11 ms), suggesting a polysynaptic pathway. The EPSPs were characteristically slow in onset with a mean rise time and half-decay time of 8.3 and 23 ms, respectively. 4. At the resting membrane potential of -50 to -60 mV, the amplitude of EPSPs recorded in normal (1.3 mM Mg2+) Krebs solution was reduced by membrane hyperpolarization or depolarization. In Mg2(+)-free solution, EPSPs were potentiated and reached threshold for spike discharge. 5. The EPSPs were suppressed by the nonselective glutamate receptor antagonist kynurenic acid (0.1-0.5 mM) and by the N-methyl-D-aspartate (NMDA) receptor antagonists D-2-amino-5-phosphonovaleric acid (APV; 1-10 microM) and ketamine (5-10 microM), but not by the quisqualate (QA)/kainate (KA) receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX, 1-10 microM). The latter depressed the EPSPs elicited by stimulation of lateral funiculus in the same SPNs. 6. NMDA applied by pressure elicited a depolarization in the SPNs. In normal Krebs solution the response was voltage dependent with the peak amplitude occurring around -60 mV; conditioning depolarization or hyperpolarization diminished the response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presynaptic dopamine D1 receptors attenuate excitatory and inhibitory limbic inputs to the shell region of the rat nucleus accumbens studied in vitro.

1. Intracellular recordings were made from the shell region of the nucleus accumbens in an in vitro slice preparation. The mean resting membrane potential, input resistance, and action potential amplitude of these neurons were -76 +/- 1 mV, 87 +/- 5 M omega and 94 +/- 2 mV (N = 108), respectively. A sample of these neurons (N = 18) was identified as medium spiny neurons with the use of the biocytin-avidin labeling technique. 2. Electrical stimulation of the fornix, subcortical fibers, or neuropil within the nucleus accumbens shell itself elicited a depolarizing postsynaptic potential (PSP). Dopamine (10-100 microM) attenuated PSPs elicited by stimulation of all of these sites. In a paired-pulse stimulation protocol, dopamine was observed to enhance the facilitation of the test response with respect to the conditioning response. 3. The suppressive effect of dopamine was mimicked by the D1 receptor agonist SKF 82958 (10-30 microM), whereas the D2 receptor agonist quinpirole (10-30 microM) was ineffective. The action of dopamine was antagonized by the D1 receptor antagonist Sch 23390 (10-30 microM), but not by the D2 receptor antagonist sulpiride (10-50 microM) or various adrenergic receptor antagonists. 4. The PSP was usually composed of an excitatory postsynaptic potential (EPSP)-inhibitory postsynaptic potential (IPSP) sequence. Dopamine equally attenuated the excitatory and inhibitory component of the synaptic response. The attenuation of both EPSP and IPSP did not depend on membrane potential. 5. Dopamine effects on the resting membrane potential and input resistance were variable and did not correlate with changes in the PSP. Two further indications were found in favor of a presynaptic locus of dopaminergic modulation. First, the time course of the PSP was not altered during dopamine application. Second, dopamine did not attenuate depolarizations induced by bath-applied L-glutamate. In extracellular recordings, it was found that dopamine reduced the population spike but not the presynaptic fiber volley. 6. These findings strongly indicate that dopaminergic modulation of synaptic responses in neurons located in the accumbens shell region is mediated by presynaptic D1 receptors. Notably, dopamine does not exert a purely inhibitory effect on synaptic excitability in the nucleus accumbens, because it suppresses both the excitatory and inhibitory component of the synaptic response.     

Kindling-induced long-lasting changes in synaptic transmission in the basolateral amygdala.

1. Intracellular current-clamp recordings were obtained from neurons of the basolateral amygdala (BLA) in an in vitro slice preparation from control and kindled animals. Postsynaptic potentials, elicited by stimulation of the stria terminalis (ST) or lateral amygdaloid nucleus (LA), were used to investigate the role of excitatory and inhibitory amino acid transmission in kindling-induced epileptiform activity. The contributions of glutamatergic and GABAergic receptor subtypes were analyzed by use of the non-N-methyl-D-aspartate (non-NMDA) antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), the NMDA antagonist DL-2-amino-5-phosphonovaleric acid (APV), and the GABAA antagonist bicuculline methiodide (BMI). 2. The synaptic waveform evoked in control neurons consisted of an excitatory postsynaptic potential (EPSP), a fast inhibitory postsynaptic potential (f-IPSP), and a slow inhibitory postsynaptic potential (s-IPSP). Stimulation of the ST or LA pathways evoked a burst-firing response in BLA neurons contralateral from the site of stimulation of kindled animals. 3. APV (50 microM) reduced, but CNQX (10 microM) completely blocked, the burst-firing response in BLA neurons from kindled animals and bicuculline-induced bursting in control neurons. 4. Kindling significantly increased the amplitude of both the slow NMDA- and the fast non-NMDA-receptor-mediated components of synaptic transmission (s- and f-EPSPs, respectively). Furthermore, the stimulus intensities required to evoke EPSPs just subthreshold for action potential generation were significantly lower in slices from kindled animals. 5. In kindled neurons no significant change was observed in the membrane input resistance and resting membrane potential or in the number of action potentials elicited in response to depolarizating current injection. 6. Kindling resulted in a pathway-specific loss of ST- and LA-evoked feedforward GABAergic synaptic transmission and of spontaneous IPSPs. In the same BLA neurons, direct GABAergic inhibition via stimulation of the LA was not affected by kindling. 7. The enhanced glutamatergic transmission was not due to disinhibition, because, in the presence of BMI (and CNQX to prevent BMI-induced bursting), the s-EPSP amplitude was still greater in kindled than in control neurons. 8. These results provide evidence that the epileptiform activity observed in BLA neurons after kindling results from an increase in excitatory NMDA- and non-NMDA-receptor-mediated glutamatergic transmission and a decrease in inhibitory gamma-aminobutyric acid (GABA)-receptor-mediated transmission; the enhanced excitatory transmission cannot be accounted for by reduced inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of dopamine D1 and NMDA receptors mediates acute clozapine potentiation of glutamate EPSPs in rat prefrontal cortex

The atypical antipsychotic drug clozapine effectively alleviates both negative and positive symptoms of schizophrenia via unclear cellular mechanisms. Clozapine may modulate both glutamatergic and dopaminergic transmission in the prefrontal cortex (PFC) to achieve part of its therapeutic actions. Using whole cell patch-clamp techniques, current-clamp recordings in layers V-VI pyramidal neurons from rat PFC slices showed that stimulation of local afferents (in 2 microM bicuculline) evoked mixed [AMPA/kainate and N-methyl-D-aspartate (NMDA) receptors] glutamate receptor-mediated excitatory postsynaptic potentials (EPSPs). Clozapine (1 microM) potentiated polysynaptically mediated evoked EPSPs (V(Hold) = -65 mV), or reversed EPSPs (rEPSP, V(Hold) = +20 mV) for >30 min. The potentiated EPSPs or rEPSPs were attenuated by elevating [Ca(2+)](O) (7 mM), by application of NMDA receptor antagonist 2-amino5-phosphonovaleric acid (50 microM), or by pretreatment with dopamine D1/D5 receptor antagonist SCH23390 (1 microM) but could be further enhanced by a dopamine reuptake inhibitor bupropion (1 microM). Clozapine had no significant effect on pharmacologically isolated evoked NMDA-rEPSP or AMPA-rEPSPs but increased spontaneous EPSPs without changing the steady-state resting membrane potential. Under voltage clamp, clozapine (1 microM) enhanced the frequency, and the number of low-amplitude (5-10 pA) AMPA receptor-mediated spontaneous EPSCs, while there was no such changes with the mini-EPSCs (in 1 microM TTX). Taken together these data suggest that acute clozapine can increase spike-dependent presynaptic release of glutamate and dopamine. The glutamate stimulates distal dendritic AMPA receptors to increase spontaneous EPSCs and enabled a voltage-dependent activation of neuronal NMDA receptors. The dopamine released stimulates postsynaptic D1 receptor to modulate a lasting potentiation of the NMDA receptor component of the glutamatergic synaptic responses in the PFC neuronal network. This sequence of early synaptic events induced by acute clozapine may comprise part of the activity that leads to later cognitive improvement in schizophrenia.     

Nicotinic and muscarinic reduction of unitary excitatory postsynaptic potentials in sensory cortex; dual intracellular recording in vitro

We studied the cholinergic modulation of glutamatergic transmission between neighboring layer 5 regular-spiking pyramidal neurons in somatosensory cortical slices from young rats (P10-P26). Brief bath application of 5-10 microM carbachol, a nonspecific cholinergic agonist, decreased the amplitude of evoked unitary excitatory postsynaptic potentials (EPSPs). This effect was blocked by 1 microM atropine, a muscarinic receptor antagonist. Nicotine (10 microM), in contrast to carbachol, reduced EPSPs in nominally magnesium-free solution but not in the presence of 1 mM Mg+2, indicating the involvement of NMDA receptors. Likewise, when the postsynaptic cell was depolarized under voltage clamp to allow NMDA receptor activation in the presence of 1 mM Mg+2, synaptic currents were reduced by nicotine. Nicotinic EPSP reduction was prevented by the NMDA receptor antagonist D-AP5 (50 microM) and by the nicotinic receptor antagonist mecamylamine (10 microM). Both carbachol and nicotine reduced short-term depression of EPSPs evoked by 10 Hz stimulation, indicating that EPSP reduction happens via reduction of presynaptic glutamate release. In the case of nicotine, several possible mechanisms for NMDAR-dependent EPSP reduction are discussed. As a result of NMDA receptor dependence, nicotinic EPSP reduction may serve to reduce the local spread of cortical excitation during heightened sensory activity.     

Local excitatory circuits in the intermediate gray layer of the superior colliculus

We have used photostimulation and whole cell patch-clamp recording techniques to examine local synaptic interactions in slices from the superior colliculus of the tree shrew. Uncaging glutamate 10-75 microm from the somata of neurons in the intermediate gray layer elicited a long-lasting inward current, due to direct activation of glutamate receptors on these neurons, and brief inward currents caused by activation of presynaptic neurons. The synaptic responses occurred as individual currents or as clusters that lasted up to several hundred milliseconds. Excitatory synaptic responses, which reversed at membrane potentials near 0 mV, could be evoked by uncaging glutamate anywhere within 75 microm of an intermediate layer neuron. Our results indicate the presence of extensive local excitatory circuits in the intermediate layer of the superior colliculus and support the hypothesis that such intrinsic circuitry contributes to the development of presaccadic command bursts.     

Cell-specific, spike timing-dependent plasticities in the dorsal cochlear nucleus

In the dorsal cochlear nucleus, long-term synaptic plasticity can be induced at the parallel fiber inputs that synapse onto both fusiform principal neurons and cartwheel feedforward inhibitory interneurons. Here we report that in mouse fusiform cells, spikes evoked 5 ms after parallel-fiber excitatory postsynaptic potentials (EPSPs) led to long-term potentiation (LTP), whereas spikes evoked 5 ms before EPSPs led to long-term depression (LTD) of the synapse. The EPSP-spike protocol led to LTD in cartwheel cells, but no synaptic changes resulted from the reverse sequence (spike-EPSP). Plasticity in fusiform and cartwheel cells therefore followed Hebbian and anti-Hebbian learning rules, respectively. Similarly, spikes generated by summing EPSPs from different groups of parallel fibers produced LTP in fusiform cells, and LTD in cartwheel cells. LTD could also be induced in glutamatergic inputs of cartwheel cells by pairing parallel-fiber EPSPs with depolarizing glycinergic PSPs from neighboring cartwheel cells. Thus, synaptic learning rules vary with the postsynaptic cell, and may require the interaction of different transmitter systems.     

Recurrent excitatory connectivity in the dentate gyrus of kindled and kainic acid-treated rats

Repeated seizures induce mossy fiber axon sprouting, which reorganizes synaptic connectivity in the dentate gyrus. To examine the possibility that sprouted mossy fiber axons may form recurrent excitatory circuits, connectivity between granule cells in the dentate gyrus was examined in transverse hippocampal slices from normal rats and epileptic rats that experienced seizures induced by kindling and kainic acid. The experiments were designed to functionally assess seizure-induced development of recurrent circuitry by exploiting information available about the time course of seizure-induced synaptic reorganization in the kindling model and detailed anatomic characterization of sprouted fibers in the kainic acid model. When recurrent inhibitory circuits were blocked by the GABA(A) receptor antagonist bicuculline, focal application of glutamate microdrops at locations in the granule cell layer remote from the recorded granule cell evoked trains of excitatory postsynaptic potentials (EPSPs) and population burst discharges in epileptic rats, which were never observed in slices from normal rats. The EPSPs and burst discharges were blocked by bath application of 1 microM tetrodotoxin and were therefore dependent on network-driven synaptic events. Excitatory connections were detected between blades of the dentate gyrus in hippocampal slices from rats that experienced kainic acid-induced status epilepticus. Trains of EPSPs and burst discharges were also evoked in granule cells from kindled rats obtained after > or = 1 wk of kindled seizures, but were not evoked in slices examined 24 h after a single afterdischarge, before the development of sprouting. Excitatory connectivity between blades of the dentate gyrus was also assessed in slices deafferented by transection of the perforant path, and bathed in artificial cerebrospinal fluid (ACSF) containing bicuculline to block GABA(A) receptor-dependent recurrent inhibitory circuits and 10 mM [Ca(2+)](o) to suppress polysynaptic activity. Low-intensity electrical stimulation of the infrapyramidal blade under these conditions failed to evoke a response in suprapyramidal granule cells from normal rats (n = 15), but in slices from epileptic rats evoked an EPSP at a short latency (2.59 +/- 0.36 ms) in 5 of 18 suprapyramidal granule cells. The results are consistent with formation of monosynaptic excitatory connections between blades of the dentate gyrus. Recurrent excitatory circuits developed in the dentate gyrus of epileptic rats in a time course that corresponded to the development of mossy fiber sprouting and demonstrated patterns of functional connectivity corresponding to anatomic features of the sprouted mossy fiber pathway.     

The actions of 2-hydroxy-saclofen at presynaptic GABAB receptors in the rat hippocampus

The actions of 2-hydroxy-saclofen (2-OH-S), a recently developed analog of baclofen, were studied at presynaptic GABAB receptors in the rat hippocampal slice. Baclofen (0.5-20 microM) reduces the amplitude of excitatory postsynaptic potentials (EPSPs) recorded from hippocampal CA1 pyramidal neurons. In the presence of 200-500 microM 2-OH-S, the synaptic depressant action of baclofen is significantly reduced. These data show that 2-OH-S is an effective antagonist at presynaptic GABAB receptors on excitatory terminals in the hippocampus.     

Regulation of synaptic input to hypothalamic presympathetic neurons by GABA(B) receptors

The hypothalamic paraventricular (PVN) neurons projecting to the spinal cord and brainstem play an important role in the control of homeostasis and the sympathetic nervous system. Although GABA(B) receptors are present in the PVN, their function in the control of synaptic inputs to PVN presympathetic neurons is not clear. Using retrograde tracing and whole-cell patch-clamp recordings in rat brain slices, we determined the role of presynaptic GABA(B) receptors in regulation of glutamatergic and GABAergic inputs to spinally projecting PVN neurons. The GABA(B) receptor agonist baclofen (1-50 microM) dose-dependently decreased the frequency but not the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and inhibitory postsynaptic currents (sIPSCs). The effect of baclofen on sEPSCs and sIPSCs was completely blocked by 10 microM CGP52432, a selective GABA(B) receptor antagonist. Baclofen also significantly reduced the frequency of both miniature excitatory and miniature inhibitory postsynaptic currents (mEPSCs and mIPSCs). Furthermore, uncoupling pertussis toxin-sensitive G(i/o) proteins with N-ethylmaleimide abolished baclofen-induced inhibition of mEPSCs and mIPSCs. However, the inhibitory effect of baclofen on the frequency of mIPSCs and mEPSCs persisted in the presence of either Cd2+, a voltage-gated Ca2+ channel blocker, or 4-aminopyridine, a blocker of voltage-gated K+ channels. Our results suggest that activation of presynaptic GABA(B) receptors inhibits synaptic GABA and glutamate release to PVN presympathetic neurons. This presynaptic action of GABA(B) receptors is mediated by the N-ethylmaleimide-sensitive G(i/o) proteins, but independent of voltage-gated Ca2+ and K+ channels.