CA3 Burst Research Articles

Sex-specific neurobiological actions of prophylactic (R,S)-ketamine, (2R,6R)-hydroxynorketamine, and (2S,6S)-hydroxynorketamine.

Enhancing stress resilience in at-risk populations could significantly reduce the incidence of stress-related psychiatric disorders. We have previously reported that the administration of (R,S)-ketamine prevents stress-induced depressive-like behavior in male mice, perhaps by altering α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated transmission in hippocampal CA3. However, it is still unknown whether metabolites of (R,S)-ketamine can be prophylactic in both sexes. We administered (R,S)-ketamine or its metabolites (2R,6R)-hydroxynorketamine ((2R,6R)-HNK) and (2S,6S)-hydroxynorketamine ((2S,6S)-HNK) at various doses 1 week before one of a number of stressors in male and female 129S6/SvEv mice. Patch clamp electrophysiology was used to determine the effect of prophylactic drug administration on glutamatergic activity in CA3. To examine the interaction between ovarian hormones and stress resilience, female mice also underwent ovariectomy (OVX) surgery and a hormone replacement protocol prior to drug administration. (2S,6S)-HNK and (2R,6R)-HNK protected against distinct stress-induced behaviors in both sexes, with (2S,6S)-HNK attenuating learned fear in male mice, and (2R,6R)-HNK preventing stress-induced depressive-like behavior in both sexes. (R,S)-ketamine and (2R,6R)-HNK, but not (2S,6S)-HNK, attenuated large-amplitude AMPAR-mediated bursts in hippocampal CA3. All three compounds reduced N-methyl-D-aspartate receptor (NMDAR)-mediated currents 1 week after administration. Furthermore, ovarian-derived hormones were necessary for and sufficient to restore (R,S)-ketamine- and (2R,6R)-HNK-mediated prophylaxis in female mice. Our data provide further evidence that resilience-enhancing prophylactics may alter AMPAR-mediated glutamatergic transmission in CA3. Moreover, we show that prophylactics against stress-induced depressive-like behavior can be developed in a sex-specific manner and demonstrate that ovarian hormones are necessary for the prophylactic efficacy of (R,S)-ketamine and (2R,6R)-HNK in female mice.

The Glycolytic Metabolite, Fructose-1,6-bisphosphate, Blocks Epileptiform Bursts by Attenuating Voltage-Activated Calcium Currents in Hippocampal Slices.

Manipulation of metabolic pathways (e.g., ketogenic diet (KD), glycolytic inhibition) alters neural excitability and represents a novel strategy for treatment of drug-refractory seizures. We have previously shown that inhibition of glycolysis suppresses epileptiform activity in hippocampal slices. In the present study, we aimed to examine the role of a “branching” metabolic pathway stemming off glycolysis (i.e., the pentose-phosphate pathway, PPP) in regulating seizure activity, by using a potent PPP stimulator and glycolytic intermediate, fructose-1,6-bisphosphate (F1,6BP). Employing electrophysiological approaches, we investigated the action of F1,6BP on epileptiform population bursts, intrinsic neuronal firing, glutamatergic and GABAergic synaptic transmission and voltage-activated calcium currents (ICa) in the CA3 area of hippocampal slices. Bath application of F1,6BP (2.5–5 mM) blocked epileptiform population bursts induced in Mg2+-free medium containing 4-aminopyridine, in ~2/3 of the slices. The blockade occurred relatively rapidly (~4 min), suggesting an extracellular mechanism. However, F1,6BP did not block spontaneous intrinsic firing of the CA3 neurons (when synaptic transmission was eliminated with DNQX, AP-5 and SR95531), nor did it significantly reduce AMPA or NMDA receptor-mediated excitatory postsynaptic currents (EPSCAMPA and EPSCNMDA). In contrast, F1,6BP caused moderate reduction (~50%) in GABAA receptor-mediated current, suggesting it affects excitatory and inhibitory synapses differently. Finally and unexpectedly, F1,6BP consistently attenuated ICa by ~40% without altering channel activation or inactivation kinetics, which may explain its anticonvulsant action, at least in this in vitro seizure model. Consistent with these results, epileptiform population bursts in CA3 were readily blocked by the nonspecific Ca2+ channel blocker, CdCl2 (20 μM), suggesting that these bursts are calcium dependent. Altogether, these data demonstrate that the glycolytic metabolite, F1,6BP, blocks epileptiform activity via a previously unrecognized extracellular effect on ICa, which provides new insight into the metabolic control of neural excitability.

Glycolytic inhibition by 2-deoxy-d-glucose abolishes both neuronal and network bursts in an in vitro seizure model

Neuronal activity is energy demanding and coupled to cellular metabolism. In this study, we investigated the effects of glycolytic inhibition with 2-deoxy-d-glucose (2-DG) on basal membrane properties, spontaneous neuronal firing, and epileptiform network bursts in hippocampal slices. The effect of glycolytic inhibition on basal membrane properties was examined in hippocampal CA1 neurons, which are not ordinarily active spontaneously. Intracellular application of 2-DG did not significantly alter the membrane input resistance, action-potential threshold, firing pattern, or input-output relationship of these neurons compared with simultaneously recorded neighboring neurons without intracellular 2-DG. The effect of glycolytic inhibition on neuronal firing was tested in spontaneously active hippocampal neurons (CA3) when synaptic transmission was left intact or blocked with AMPA, NMDA, and GABAA receptor antagonists (DNQX, APV, and bicuculline, respectively). Under both conditions (synaptic activity intact or blocked), bath application of 2-DG (2 mM) blocked spontaneous firing in ~2/3 (67 and 71%, respectively) of CA3 pyramidal neurons. In contrast, neuronal firing of CA3 neurons persisted when 2-DG was applied intracellularly, suggesting that glycolytic inhibition of individual neurons is not sufficient to stop neuronal firing. The effects of 2-DG on epileptiform network bursts in area CA3 were tested in Mg2+-free medium containing 50 µM 4-aminopyridine. Bath application of 2-DG abolished these epileptiform bursts in a dose-dependent and all-or-none manner. Taken together, these data suggest that altered glucose metabolism profoundly affects cellular and network hyperexcitability and that glycolytic inhibition by 2-DG can effectively abrogate epileptiform activity.NEW & NOTEWORTHY Neuronal activity is highly energy demanding and coupled to cellular metabolism. In this study, we demonstrate that glycolytic inhibition with 2-deoxy-d-glucose (2-DG) effectively suppresses spontaneous neuronal firing and epileptiform bursts in hippocampal slices. These data suggest that an altered metabolic state can profoundly affect cellular and network excitability, and that the glycolytic inhibitor 2-DG may hold promise as a novel treatment of drug-resistant epilepsy.

Repeating Spatial-Temporal Motifs of CA3 Activity Dependent on Engineered Inputs from Dentate Gyrus Neurons in Live Hippocampal Networks.

Anatomical and behavioral studies, and in vivo and slice electrophysiology of the hippocampus suggest specific functions of the dentate gyrus (DG) and the CA3 subregions, but the underlying activity dynamics and repeatability of information processing remains poorly understood. To approach this problem, we engineered separate living networks of the DG and CA3 neurons that develop connections through 51 tunnels for axonal communication. Growing these networks on top of an electrode array enabled us to determine whether the subregion dynamics were separable and repeatable. We found spontaneous development of polarized propagation of 80% of the activity in the native direction from DG to CA3 and different spike and burst dynamics for these subregions. Spatial-temporal differences emerged when the relationships of target CA3 activity were categorized with to the number and timing of inputs from the apposing network. Compared to times of CA3 activity when there was no recorded tunnel input, DG input led to CA3 activity bursts that were 7× more frequent, increased in amplitude and extended in temporal envelope. Logistic regression indicated that a high number of tunnel inputs predict CA3 activity with 90% sensitivity and 70% specificity. Compared to no tunnel input, patterns of >80% tunnel inputs from DG specified different patterns of first-to-fire neurons in the CA3 target well. Clustering dendrograms revealed repeating motifs of three or more patterns at up to 17 sites in CA3 that were importantly associated with specific spatial-temporal patterns of tunnel activity. The number of these motifs recorded in 3 min was significantly higher than shuffled spike activity and not seen above chance in control networks in which CA3 was apposed to CA3 or DG to DG. Together, these results demonstrate spontaneous input-dependent repeatable coding of distributed activity in CA3 networks driven by engineered inputs from DG networks. These functional configurations at measured times of activation (motifs) emerge from anatomically accurate feed-forward connections from DG through tunnels to CA3.

Topological organization of CA3‐to‐CA1 excitation

The CA1-projecting axons of CA3 pyramidal cells, called Schaffer collaterals, constitute one of the major information flow routes in the hippocampal formation. Recent anatomical studies have revealed the non-random structural connectivity between CA3 and CA1, but little is known regarding the functional connectivity (i.e. how CA3 network activity is functionally transmitted downstream to the CA1 network). Using functional multi-neuron calcium imaging of rat hippocampal slices, we monitored the spatiotemporal patterns of spontaneous CA3 and CA1 burst activity under pharmacological GABAergic blockade. We found that spatially clustered CA3 activity patterns were transformed into layered CA1 activity sequences. Specifically, synchronized bursts initiated from multiple hot spots in CA3 ensembles, and CA1 neurons located deeper in the pyramidal cell layer were recruited during earlier phases of the burst events. The order of these sequential activations was maintained across the bursts, but the sequence velocity varied depending on the inter-burst intervals. Thus, CA3 axons innervate CA1 neurons in a highly topographical fashion.

Bidirectional NMDA receptor plasticity controls CA3 output and heterosynaptic metaplasticity

N–methyl–d–aspartate glutamate receptors (NMDARs) are classically known as coincidence detectors for the induction of long–term synaptic plasticity, and have been implicated in hippocampal CA3–dependent spatial memory functions that likely rely on dynamic cellular ensemble encoding of space. The unique functional properties of both NMDARs and mossy fiber (MF) projections to CA3 pyramidal cells place MF–NMDARs in a prime position to influence CA3 ensemble dynamics. By mimicking pre and postsynaptic activity patterns observed in–vivo, we report a burst timing–dependent paradigm for bidirectional long–term NMDAR plasticity at MF–CA3 synapses in rat hippocampal slices. This form of plasticity imparts bimodal control of MF–driven CA3 burst–firing and spike temporal fidelity. Moreover, we show that MF–NMDARs mediate heterosynaptic metaplasticity between MF and associational/commissural synapses. Thus, bidirectional NMDAR plasticity at MF–CA3 synapses could significantly contribute to the formation, storage, and recall of CA3 cell assembly patterns.

Extracellular field signatures of CA1 spiking cell assemblies during sharp wave-ripple complexes

Although postsynaptic and transmembrane currents over local neuronal populations are considered the main factors for shaping local field potential (LFP) and current source density (CSD) fluctuations [1], high-frequency oscillatory LFPs can also be shaped by extracellular action potentials of pyramidal cell populations [2]. Sharp wave-ripple complexes (SWRs) are typical examples of such high-frequency oscillatory events, observed in hippocampal LFPs during deep sleep and awake immobility. They consist of an extensive depolarization in the CA1 dendritic layer (sharp wave) arising from population bursts in CA3, accompanied by a ~150-200 Hz LFP oscillation in the CA1 pyramidal layer (ripple). During SWRs, temporal firing patterns of correlated place cells, acquired during wakeful exploration, are replayed in fast-scale, providing a strong indication for the participation of SWRs in memory consolidation. Yet the particular effects of these pattern replays on the hippocampal extracellular field are largely unknown. How are the different ensembles of spiking cells encoded in the emerging ripple-LFPs? Here, we study this association through both a modeling and an experimental approach.

Diphenytoin, riluzole and lidocaine: Three sodium channel blockers, with different mechanisms of action, decrease hippocampal epileptiform activity

Epilepsy is a condition affecting 1–2% of the population, characterized by the presence of spontaneous, recurrent seizures. The most common type of acquired epilepsy is temporal lobe epilepsy (TLE). Up to 30% of patients with TLE are refractory to currently available compounds, and there is an urgent need to identify novel targets for therapy. Here, we utilized the in-vitro CA3 burst preparation to examine alterations in network excitability, characterized by changes in interburst interval. Specifically, we show that bath application of three different sodium channel blockers—diphenytoin, riluzole, and lidocaine—slow spontaneous CA3 bursts. This in turn, decreased the epileptiform activity. These compounds work at different sites on voltage-gated sodium channels, but produce a similar network phenotype of decreased excitability. In the case of diphenytoin and riluzole, the change in network activity (i.e., increased interburst intervals) was persistent following drug washout. Lidocaine application, however, only increased the CA3 interburst interval when it was in the bath solution. Thus, its action was not permanent and resulted in returning CA3 bursting to baseline levels. These data demonstrate that the CA3 burst preparation provides a relatively easy and quick platform for identifying compounds that can decrease network excitability, providing the initial screen for further and more complex in-vivo, freely-behaving animal studies.

Progressive, potassium-sensitive epileptiform activity in hippocampal area CA3 of pilocarpine-treated rats with recurrent seizures

Rat hippocampal area CA3 pyramidal cells synchronously discharge in rhythmic bursts of action potentials after acute disinhibition or convulsant treatment in vitro. These burst discharges resemble epileptiform activity, and are of interest because they may shed light on mechanisms underlying limbic seizures. However, few studies have examined CA3 burst discharges in an animal model of epilepsy, because a period of prolonged, severe seizures (status epilepticus) is often used to induce the epileptic state, which can lead to extensive neuronal loss in CA3. Therefore, the severity of pilocarpine-induced status epilepticus was decreased with anticonvulsant treatment to reduce damage. Rhythmic burst discharges were recorded in the majority of slices from these animals, between two weeks and nine months after status epilepticus. The incidence and amplitude of bursts progressively increased with time after status, even after spontaneous behavioral seizures had begun. The results suggest that modifying the pilocarpine models of temporal lobe epilepsy to reduce neuronal loss leads to robust network synchronization in area CA3. The finding that these bursts increase long after spontaneous behavioral seizures begin supports previous arguments that temporal lobe epilepsy exhibits progressive pathophysiology.

Modeling sharp wave - ripple complexes and their interactions with cortical slow oscillations through a cortico-CA3-CA1 model

The hippocampus, and particularly the CA3 and CA1 areas, exhibit a variety of oscillatory rhythms that span frequencies from the slow theta range (4-10 Hz) up to fast ripples (~200 Hz). Various computational models of different complexities have been developed in an effort to simulate such population oscillations. Nevertheless the mechanism that underlies the so called sharp wave-ripple complex, observed in extracellular recordings in CA1, still remains elusive. We present a combination of two computational models of the rat CA3 and CA1 hippocampal areas respectively. Both models are simple one-dimensional arrays of pyramidal and interneuronal populations, interacting only via fast AMPA and GABAA synapses. Connectivity schemes and postsynaptic potentials are based on biological data, rendering the network topology as realistic as possible. Both models are first validated individually by showing that they reproduce a number of established anatomical and/or functional characteristic properties of the corresponding areas, including population bursts in CA3 and gamma oscillations in CA1. The two networks are coupled in a full CA3-CA1 model through a feedforward scheme mimicking Schaffer collaterals. Quasi-synchronous population bursts in CA3 result in transient responses in CA1, consisting of deep depolarizations in the dendritic layer accompanied by transient ~150-200 Hz field oscillations in the somatic layer. These responses are shown to accurately reproduce a number of basic characteristic features of sharp wave-ripple complexes, reported previously in a variety of neurophysiological studies. By examining these features through our model, we are led to the formulation of a novel mechanism for the generation of ripples, based purely on chemical interactions and avoiding any use of gap junctions. The depolarizing input from CA3, produces intense interneuronal firing in CA1 which is regulated and synchronized by strong, fast-decaying, recurrent inhibition, resulting in a field oscillation within the ripple frequency range. Pyramidal cells, trapped within this inhibitory barrage, have a more passive role, with a small subset of them producing most of the excitatory spikes in a ripple. Through our proposed mechanism, we are able to interpret neurophysiological observations, such as the ripple disruption by halothane and the selective firing of pyramidal cells during ripples, which has various implications on memory consolidation. Finally, the CA3-CA1 model is coupled with a cortical network exhibiting slow oscillations, in a feedback loop involving the mossy fiber and temporoammonic pathway inputs to the hippocampus and monosynaptic projections from CA1 to prefrontal cortex. We study the resulting correlations between hippocampal and cortical activity in an effort to uncover important parameters and mechanisms on which they depend. We show how the spiking activities of CA3 and CA1 depend on the inhibition-excitation ratio, induced by the two hippocampal inputs, and how this ratio can affect established correlations between cortical UP states and ripples. Such correlations have been suggested to be important for the transferring of memories to the cortex for long-term storage.

Both synaptic and intrinsic mechanisms underlie the different properties of population bursts in the hippocampal CA3 area of immature versus adult rats

Pharmacological blockade of GABA(A) receptors on CA3 pyramidal cells in hippocampal slices from immature rats (i.e. second to third postnatal weeks), compared to CA3 slices from adult rats, is known to cause prolonged burst discharges (i.e. several seconds vs. tens of milliseconds). Synaptic and intrinsic mechanisms responsible for this developmental difference in burst duration were analysed in isolated minislices of the CA3 area. The frequency and amplitude of spontaneous EPSCs in CA3 pyramidal cells were greater in slices from immature than mature rats. In the presence of GABA(A)- and GABA(B)-receptor antagonists, the burst discharges of immature CA3 pyramidal cells were still prolonged in thinner slices (350 microm, vs. 450 microm in adults, to compensate for developmental differences in neuronal density) and in NMDA- and mGlu1-receptor antagonists. The AMPA receptor antagonist DNQX blocked the remaining burst discharges, suggesting that differences in recurrent excitatory circuits contributed to the prolonged bursts of immature CA3 pyramidal cells. In slices from immature versus adult rats, the CA3 recurrent synaptic responses showed potentiation to repetitive stimulation, suggestive of a lower transmitter release probability. The intrinsic firing ability was greater in CA3 pyramidal neurons from immature than adult rats, and the medium-duration afterhyperpolarization was smaller. These data suggest that, compared to adults, the CA3 area of immature rats contains a more robust recurrent excitatory synaptic network, greater intrinsic membrane excitability, and an increased capacity for sustained transmitter release, which together may account for the more prolonged network bursts in immature versus adult CA3.

Excitotoxic-mediated transcriptional decreases in HCN2 channel function increase network excitability in CA1

Changes in the conductance of the hyperpolarization-activated, cyclic nucleotide-gated (HCN) channel that mediates I h are proposed to contribute to increased network excitability. Synchronous neuronal burst activity is a good reflection of network excitability and can be generated in isolated hippocampal slice cultures by removing Mg 2+ from the extracellular fluid. We demonstrate that I h contributes to this activity by increasing both the frequency and duration of bursting events. Changes in HCN channel function are also implicated in altered seizure susceptibility. Short-term application of kainic acid (KA) is known to initiate long lasting changes in neuronal networks that result in seizures, and in slice cultures was found to alter HCN mRNA levels in an isoform and hippocampal sub-region specific manner. These changes correlate with the ability of each sub-region to develop synchronous burst activity following KA that we have previously reported. Specifically, a loss of synchronous activity in the CA3 correlated with an increase in HCN2 mRNA levels that normalized concomitantly with the restoration of CA3 burst activity 7 days post insult. In contrast, in CA1 an increase in synchronous burst duration correlated with a reduction in HCN2 mRNA levels and both changes were still evident for 7 days post insult. Lamotrigine, known to increase I h, reversed the impact of KA on burst duration in CA1 at both time-points linking a transcriptional reduction in HCN2 function to increased burst duration.

α 2 Adrenergic Modulation of Hippocampal CA3 Network Activity

Norepinephrine (NE) has been shown to have profound effects on epileptogenesis. The CA3 region of the hippocampus is believed to be a common focus in temporal lobe epilepsy, generating a type of epileptiform activity which propagates throughout the brain. Our previous work suggests that NE attenuates CA3 burst discharge activity by activating an α2 adrenergic receptor (AR) expressed on hippocampal CA3 pyramidal cells. To characterize the α2AR responsible for decreasing hippocampal epileptiform activity; a pharmacological profile for various α2AR agonists was generated. EC50 values obtained were consistent with an α2AR-mediated response. To confirm the involvement of α2ARs, Schild regressions were constructed from concentration-response curves using the AR agonist epinephrine in the presence of the selective α2AR antagonist RS79948. The pA2 values obtained correlated with affinity values for α2ARs. Similar experiments were performed in the presence of subtype-selective α2AR antagonists. pA2 values obtained from rauwolscine, BRL44408 and ARC 239, correlated with affinities for the α2AAR. Surgical and pharmacological isolation of CA3 pyramidal neurons suggest that NE is acting locally on these cells. These results suggest that NE is directly inhibiting hippocampal CA3 burst activity through the α2AAR. Insight into this effect may lead to a greater understanding of how NE modulates hippocampal networks and its role in epilepsy. This study was funded by NSF CAREER Award 0347259 (VAD), ND EPSCoR through NSF EPS-0447679 (VAD) and NIH 5P20RR017699 from the COBRE program (JEP, VAD).

Convulsant and Anticonvulsant Effects on Spontaneous CA3 Population Bursts

This paper analyzes the effects of a convulsant and an anticonvulsant manipulation on spontaneous bursts in CA3 pyramidal cells in the in vitro slice preparation under conditions of low (3.3 mM [K(+)](o)) and high (8.5 mM [K(+)](o)) burst probability. When burst probability was low, the anticonvulsant, pentobarbital, produced the anticipated effects: the burst duration decreased and interburst interval increased. However, when burst probability was high, both anticonvulsant and convulsant manipulations decreased the interburst interval and the burst duration. To reconcile these findings, we utilized a model in which CA3 burst duration is limited by activity-dependent depression of CA3 excitatory recurrent collateral synapses and the interburst interval is determined by the time required to recover from this depression. We defined the burst end threshold as the level of synaptic depression at which bursts terminate, and the burst start threshold as the level of synaptic depression at which burst initiation is possible. Synapses were considered to oscillate between these thresholds. When average burst duration and interburst interval data were fit using this model, the paradoxically similar effects of the convulsant and anticonvulsant manipulations could be quantitatively interpreted. The convulsant maneuver decreased both the burst start and end thresholds. The start threshold decreased more than the end threshold, so that the thresholds were closer together. This decreased the time needed to transition from one threshold to the other, i.e., the interburst interval and burst duration. The anticonvulsant manipulation primarily increased the burst end threshold. This also decreased the difference between thresholds, decreasing both interburst interval and burst duration. This model resolves the paradoxical proconvulsant effects of pentobarbital in the CA3 preparation and provides insights into the effects of anticonvulsants on epileptiform discharges in the human EEG.

Statistical model relating CA3 burst probability to recovery from burst-induced depression at recurrent collateral synapses.

When neuronal excitability is increased in area CA3 of the hippocampus in vitro, the pyramidal cells generate periodic bursts of action potentials that are synchronized across the network. We have previously provided evidence that synaptic depression at the excitatory recurrent collateral synapses in the CA3 network terminates each population burst so that the next burst cannot begin until these synapses have recovered. These findings raise the possibility that burst timing can be described in terms of the probability of recovery of this population of synapses. Here we demonstrate that when neuronal excitability is changed in the CA3 network, the mean and variance of the interburst interval change in a manner that is consistent with a timing mechanism comprised of a pool of exponentially relaxing pacemakers. The relaxation time constant of these pacemakers is the same as the time constant describing the recovery from activity-dependent depression of recurrent collateral synapses. Recovery was estimated from the rate of spontaneous transmitter release versus time elapsed since the last CA3 burst. Pharmacological and long-term alterations of synaptic strength and network excitability affected CA3 burst timing as predicted by the cumulative binomial distribution if the burst pace-maker consists of a pool of recovering recurrent synapses. These findings indicate that the recovery of a pool of synapses from burst-induced depression is a sufficient explanation for burst timing in the in vitro CA3 neuronal network. These findings also demonstrate how information regarding the nature of a pacemaker can be derived from the temporal pattern of synchronous network activity. This information could also be extracted from less accessible networks such as those generating interictal epileptiform discharges in vivo.

Long-term change in synaptic transmission in CA3 circuits followed by spontaneous rhythmic activity in rat hippocampal slices

The relevance of long-term potentiation (LTP) at excitatory synapses in CA3 circuits to generation of spontaneous epileptiform bursts in CA3 was investigated using rat hippocampal slices. CA3 pyramidal cells were antidromically stimulated through Schaffer collaterals. Evoked field potentials were extracellularly recorded from the stratum pyramidale and the stratum radiatum in CA3. Therefore, field potentials reflecting recurrent excitatory post-synaptic potentials (EPSPs) and inhibitory post-synaptic potentials (IPSPs) were positive at the stratum pyramidale and negative at the stratum radiatum. First, we tested how the amplitude of the evoked field potentials depends on a gamma-aminobutyric acid (GABA(A)) antagonist. Both of the positive and negative field potential peaks reduced in the medium containing penicillin (2 mM) or bicuculline (20 microM). This suggests that unmasked EPSPs due to suppression of IPSPs do not result in an increase in the evoked potentials. Second, CA3 pyramidal cells were antidromically stimulated by tetanic stimulation of Schaffer collaterals in order to induce LTP at synapses in CA3 circuits. Both of the positive and negative field potentials increased, suggesting that recurrent EPSPs were enhanced by tetanic stimulation. Induction of LTP at recurrent excitatory synapses was followed by spontaneous epileptiform bursts which persisted throughout experiments (approximately 1.5 h), while LTP of afferent synaptic potential evoked by hilar test stimulation was not induced. These results suggest that LTP at the afferent synapses is not necessary to spontaneous epileptiform bursts in CA3, but LTP at excitatory synapses between CA3 pyramidal cells contribute to spontaneous epileptiform bursts.

Synaptic GABAA Activation Inhibits AMPA-Kainate Receptor–Mediated Bursting in the Newborn (P0–P2) Rat Hippocampus

The mechanisms of synaptic transmission in the rat hippocampus at birth are assumed to be fundamentally different from those found in the adult. It has been reported that in the CA3-CA1 pyramidal cells a conversion of "silent" glutamatergic synapses to conductive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) synapses starts gradually after P2. Further, GABA via its depolarizing action seems to give rise to grossly synchronous yet slow calcium oscillations. Therefore, GABA is generally thought to have a purely excitatory rather than an inhibitory role during the first postnatal week. In the present study field potential recordings and gramicidin perforated and whole cell clamp techniques as well as K(+)-selective microelectrodes were used to examine the relative contributions of AMPA and GABA(A) receptors to network activity of CA3-CA1 pyramidal cells in the newborn rat hippocampus. As early as postnatal day (P0-P2), highly coherent spontaneous firing of CA3 pyramidal cells was seen in vitro. Negative-going extracellular spikes confined to periodic bursts (interval 16 +/- 3 s) consisting of 2.9 +/- 0.1 spikes were observed in stratum pyramidale. The spikes were accompanied by AMPA-R-mediated postsynaptic currents (PSCs) in simultaneously recorded pyramidal neurons (7.6 +/- 3.0 unitary currents per burst). In CA1 pyramidal cells synchronous discharging of CA3 circuitry produced a barrage of AMPA currents at >20 Hz frequencies, thus demonstrating a transfer of the fast CA3 network activity to CA1 area. Despite its depolarizing action, GABA(A)-R-mediated transmission appeared to exert inhibition in the CA3 pyramidal cell population. The GABA(A)-R antagonist bicuculline hypersynchronized the output of glutamatergic CA3 circuitry and increased the network-driven excitatory input to the pyramidal neurons, whereas the GABA(A)-R agonist muscimol (100 nM) did the opposite. However, the occurrence of unitary GABA(A)-R currents was increased after muscimol application from 0.66 +/- 0.16 s(-1) to 1.43 +/- 0.29 s(-1). It was concluded that AMPA synapses are critical in the generation of spontaneous high-frequency bursts in CA3 as well as in CA3-CA1 transmission as early as P0-P2 in rat hippocampus. Concurrently, although GABA(A)-R-mediated depolarization may excite hippocampal interneurons, in CA3 pyramidal neurons it can restrain excitatory inputs and limit the size of the activated neuronal population.

Chloride-cotransport blockade desynchronizes neuronal discharge in the "epileptic" hippocampal slice.

Antagonism of the chloride-cotransport system in hippocampal slices has been shown to block spontaneous epileptiform (i.e., hypersynchronized) discharges without diminishing excitatory synaptic transmission. Here we test the hypotheses that chloride-cotransport blockade, with furosemide or low-chloride (low-[Cl(-)](o)) medium, desynchronizes the firing activity of neuronal populations and that this desynchronization is mediated through nonsynaptic mechanisms. Spontaneous epileptiform discharges were recorded from the CA1 and CA3 cell body layers of hippocampal slices. Treatment with low-[Cl(-)](o) medium led to cessation of spontaneous synchronized bursting in CA1 >/=5-10 min before its disappearance from CA3. During the time that CA3 continued to burst spontaneously but CA1 was silent, electrical stimulation of the Schaffer collaterals showed that hyperexcited CA1 synaptic responses were maintained. Paired intracellular recordings from CA1 pyramidal cells showed that during low-[Cl(-)](o) treatment, the timing of action potential discharges became desynchronized; desynchronization was identified with phase lags in firing times of action potentials between pairs of neurons as well as a with a broadening and diminution of the CA1 field amplitude. Continued exposure to low-[Cl(-)](o) medium increased the degree of the firing-time phase shifts between pairs of CA1 pyramidal cells until the epileptiform CA1 field potential was abolished completely. Intracellular recordings during 4-aminopyridine (4-AP) treatment showed that prolonged low-[Cl(-)](o) exposure did not diminish the frequency or amplitude of spontaneous postsynaptic potentials. CA3 antidromic responses to Schaffer collateral stimulation were not significantly affected by prolonged low-[Cl(-)](o) exposure. In contrast to CA1, paired intracellular recordings from CA3 pyramidal cells showed that chloride-cotransport blockade did not cause a significant desynchronization of action potential firing times in the CA3 subregion at the time that CA1 synchronous discharge was blocked but did reduce the number of action potentials associated with CA3 burst discharges. These data support our hypothesis that the anti-epileptic effects of chloride-cotransport antagonism in CA1 are mediated through the desynchronization of population activity. We hypothesize that interference with Na(+),K(+),2Cl(-) cotransport results in an increase in extracellular potassium ([K(+)](o)) that reduces the number of action potentials that are able to invade axonal arborizations and varicosities in all hippocampal subregions. This reduced efficacy of presynaptic action potential propagation ultimately leads to a reduction of synaptic drive and a desynchronization of the firing of CA1 pyramidal cells.

Somatostatin acts in CA1 and CA3 to reduce hippocampal epileptiform activity.

Although the peptide somatostatin (SST) has been speculated to function in temporal lobe epilepsy, its exact role is unclear, as in vivo studies have suggested both pro- and anticonvulsant properties. We have shown previously that SST has multiple inhibitory cellular actions in the CA1 region of the hippocampus, suggesting that in this region SST should have antiepileptic actions. To directly assess the effect of SST on epileptiform activity, we studied two in vitro models of epilepsy in the rat hippocampal slice preparation using extracellular and intracellular recording techniques. In one, GABA-mediated neurotransmission was inhibited by superfusion of the GABAA receptor antagonist bicuculline. In the second, we superfused Mg2+-free artificial cerebrospinal fluid to remove the Mg2+ block of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor. We show here that SST markedly reduces the intensity of evoked epileptiform afterdischarges and the frequency of spontaneous bursts in both CA1 and CA3. SST appears to act additively in the two regions to suppress the transmission of epileptiform events through the hippocampus. We further examined SST's actions in CA3 and found that SST dramatically reduced the frequency of paroxysmal depolarizing shifts (PDSs) recorded intracellularly in current clamp, as well as increasing the threshold for evoking "giant" excitatory postsynaptic currents (EPSCs), large polysynaptically mediated EPSCs that are the voltage-clamp correlate of PDSs. We also examined the actions of SST on pharmacologically isolated EPSCs generated at both mossy fiber (MF) and associational/commissural (A/C) synapses. SST appears to act specifically to reduce recurrent excitation between CA3 neurons because it depresses A/C- but not MF-evoked EPSCs. SST also increased paired-pulse facilitation of A/C EPSCs, suggesting a presynaptic site of action. Reciprocal activation of CA3 neurons through A/C fibers is critical for generation of epileptiform activity in hippocampus. Thus SST reduces feedforward excitation in rat hippocampus, acting to "brake" hyperexcitation. This is a function unique from that described for other hippocampal neuropeptides, which affect more standard neurotransmission. Our results suggest that SST receptors could be a unique, selective clinical target for treatment of limbic seizures.

The influence of electric fields on the epileptiform bursts induced by high potassium in CA3 region of rat hippocampal slice

The effects of pulsed direct current (de) electric fields on the frequency of spontaneous bursting in a model epileptic focus were studied. The high potassium hippocampal slice model was used to generate spontaneous burst firing activity similar to interictal spikes in the pyramidal cell layer of CA3. Electric fields were generated from platinum subdural electrodes placed in the perfusion bath. Three hundred and seventy-eight experimental trials were performed on 10 hippocampal slices from 10 rats and the effects of field polarity, field strength and duration of stimuli on firing frequency was examined. Hippocampal slices were oriented horizontally with the CA3 layer towards the positive electrodel the average interburst interval did not correlate significantly with polarity of the delivering pulses (one-way ANOVA, p = 0.96). Average interburst interval showed a significant correlation with pulse duration of200 and 400msec (p = 0.030 and p = 0.004, respectively). As a function of field strength, there were significant average interval changes for fields of 33, 46, and 73 mV/mm (p = 0.024, P = 0.001 and p = 0.001, respectively). In conclusion, CA3 burst firing activity in high potassium concentration can therefore be altered by electric fields. [Neural Res 1998; 20: 542-548]