Hypoglycemia-induced alterations in hippocampal intrinsic rhythms: Decreased inhibition, increased excitation, seizures and spreading depression.

UNLABELLED: Seizures are the most common clinical presentation of severe hypoglycemia, usually as a side effect of insulin treatment for juvenile onset type 1 diabetes mellitus and advanced type 2 diabetes. We used the mouse thick hippocampal slice preparation to study the pathophysiology of hypoglycemia-induced seizures and the effects of severe glucose depletion on the isolated hippocampal rhythms from the CA3 circuitry. METHODS AND RESULTS: Dropping the glucose perfusate concentration from the standard 10 mM to 1 mM produced epileptiform activity in 14/16 of the slices. Seizure-like events (SLEs) originated in the CA3 region and then spread into the CA1 region. Following the SLE, a spreading-depression (SD)-like event occurred (12/16 slices) with irreversible synaptic failure in the CA1 region (8/12 slices). CA3 SD-like events followed ~30 s after the SD-like event in the CA1 region. Less commonly, SD-like events originated in the CA3 region (4/12). Additionally, prior to the onset of the SLE in the CA3 area, there was decreased GABA correlated baseline SPW activity (bSPW), while there was increased large-amplitude sharp wave (LASW) activity, thought to originate from synchronous pyramidal cell firing. CA3 pyramidal cells displayed progressive tonic depolarization prior to the seizure which was resistant to synaptic transmission blockade. The initiation of hypoglycemic seizures and SD was prevented by AMPA/kainate or NMDA receptor blockade. CONCLUSIONS: Severe glucose depletion induces rapid changes initiated in the intrinsic CA3 rhythms of the hippocampus including depressed inhibition and enhanced excitation, which may underlie the mechanisms of seizure generation and delayed spreading depression.

In vitro recordings of human neocortical oscillations.

Electrophysiological oscillations are thought to create temporal windows of communication between brain regions. We show here that human cortical slices maintained in vitro can generate oscillations similar to those observed in vivo. We have characterized these oscillations using local field potential and whole-cell recordings obtained from neocortical slices acquired during epilepsy surgery. We confirmed that such neocortical slices maintain the necessary cellular and circuitry components, and in particular inhibitory mechanisms, to manifest oscillatory activity when exposed to glutamatergic and cholinergic agonists. The generation of oscillations was dependent on intact synaptic activity and muscarinic receptors. Such oscillations differed in electrographic and pharmacological properties from epileptiform activity. Two types of activity, theta oscillations and high gamma activity, uniquely characterized this model-activity not typically observed in animal cortical slices. We observed theta oscillations to be synchronous across cortical laminae suggesting a novel role of theta as a substrate for interlaminar communication. As well, we observed cross-frequency coupling (CFC) between theta phase and high gamma amplitude similar to that observed in vivo. The high gamma "bursts" generated by such CFC varied in their frequency content, suggesting that this variability may underlie the broadband nature of high gamma activity.

Transition to seizure: from "macro"- to "micro"-mysteries.

One of the most terrifying aspects of epilepsy is the sudden and apparently unpredictable transition of the brain into the pathological state of an epileptic seizure. The pathophysiology of the transition to seizure still remains mysterious. Herein we review some of the key concepts and relevant literatures dealing with this enigmatic transitioning of brain states. At the "MACRO" level, electroencephalographic (EEG) recordings at time display preictal phenomena followed by pathological high-frequency oscillations at the seizure onset. Numerous seizure prediction algorithms predicated on identifying changes prior to seizure onset have met with little success, underscoring our lack of understanding of the dynamics of transition to seizure, amongst other inherent limitation. We then discuss the concept of synchronized hyperexcited oscillatory networks underlying seizure generation. We consider these networks as weakly coupled oscillators, a concept which forms the basis of some relevant mathematical modeling of seizure transitions. Next, the underlying "MICRO" processes involved in seizure generation are discussed. The depolarization of the GABA(A) chloride reversal potential is a major concept, facilitating epileptogenesis, particularly in immature brain. Also the balance of inhibitory and excitatory local neuronal networks plays an important role in the process of transitioning to seizure. Gap junctional communication, including that which occurs between glia, as well as ephaptic interactions are increasingly recognized as critical for seizure generation. In brief, this review examines the evidence regarding the characterization of the transition to seizure at both the "MACRO" and "MICRO" levels, trying to characterize this mysterious yet critical problem of the brain state transitioning into a seizure.

Common time-frequency analysis of local field potential and pyramidal cell activity in seizure-like events of the rat hippocampus.

To study cell-field dynamics, physiologists simultaneously record local field potentials and the activity of individual cells from animals performing cognitive tasks, during various brain states or under pathological conditions. However, apart from spike shape and spike timing analyses, few studies have focused on elucidating the common time-frequency structure of local field activity relative to surrounding cells across different periods of phenomena. We have used two algorithms, multi-window time frequency analysis and wavelet phase coherence (WPC), to study common intracellular-extracellular (I-E) spectral features in spontaneous seizure-like events (SLEs) from rat hippocampal slices in a low magnesium epilepsy model. Both algorithms were applied to 'pairs' of simultaneously observed I-E signals from slices in the CA1 hippocampal region. Analyses were performed over a frequency range of 1-100 Hz. I-E spectral commonality varied in frequency and time. Higher commonality was observed from 1 to 15 Hz, and lower commonality was observed in the 15-100 Hz frequency range. WPC was lower in the non-SLE region compared to SLE activity; however, there was no statistical difference in the 30-45 Hz band between SLE and non-SLE modes. This work provides evidence of strong commonality in various frequency bands of I-E SLEs in the rat hippocampus, not only during SLEs but also immediately before and after.

The role of altered tissue osmolality on the characteristics and propagation of seizure activity in the intact isolated mouse hippocampus.

OBJECTIVES: To study the role of altered tissue osmolality on the characteristics and propagation dynamics of seizure activity and on interictal activity, in a low-Mg(+2) artificial cerebrospinal fluid (ACSF) model of recurrent seizures, using the immature (P8-P25) intact isolated mouse hippocampus. METHODS: Recordings were obtained extracellularly from a single site in the CA1 region and from multiple sites along the septotemporal axis measuring spontaneous epileptiform field activity in ACSFs of different osmolalities. RESULTS: In normal osmolar ACSF (310 mOsmol), the average duration of recorded seizures was 90+/-10 s and the average peak amplitude was 0.9+/-0.1 mV. In a hypoosmolar ACSF (270 mOsmol), the seizures were significantly prolonged at 165+/-20 s (p<0.05) with a peak amplitude of 1.2+/-0.3 mV, whereas interictal activity was suppressed. Hyperosmolar ACSF (340 mOsmol) reduced the duration (65+/-15 s) and peak amplitude (0.6+/-0.1 mV, p<0.05) from control, but interictal activity was not affected. No differences in seizure recurrence rate were noted in all three osmolar states. CONCLUSION: The present study, the first to assess of the role of altered tissue osmolality in an intact in vitro preparation, demonstrates that changes in perfusate osmolality play a significant role on the amplitude, duration, and propagation velocity of seizure-like events, and the characteristics of interictal activity, without affecting seizure recurrence rate. SIGNIFICANCE: Increasing tissue osmolality should be considered as a valid target for anticonvulsant treatment.

Impaired presynaptic cytosolic and mitochondrial calcium dynamics in aged compared to young adult hippocampal CA1 synapses ameliorated by calcium chelation.

Impaired regulation of presynaptic intracellular calcium is thought to adversely affect synaptic plasticity and cognition in the aged brain. We studied presynaptic cytosolic and mitochondrial calcium (Ca) dynamics using axonally loaded Calcium Green-AM and Rhod-2 AM fluorescence respectively in young (2-3 months) and aged (23-26 months) CA3 to CA1 Schaffer collateral excitatory synapses in hippocampal brain slices from Fisher 344 rats. After a tetanus (100 Hz, 200 ms), the presynaptic cytosolic Ca peaked at approximately 10 s in the young and approximately 12 s in the aged synapses. Administration of the membrane permeant Ca chelator, bis (O-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid (BAPTA-AM), significantly attenuated the Ca response in the aged slices, but not in the young slices. The presynaptic mitochondrial Ca signal was much slower, peaking at approximately 90 s in both young and aged synapses, returning to baseline by 300 s. BAPTA-AM significantly attenuated the mitochondrial calcium signal only in the young synapses. Uncoupling mitochondrial respiration by carbonyl cyanide m-chlorophenylhydrazone (CCCP) application evoked a massive intracellular cytosolic Ca increase and a significant drop of mitochondrial Ca, especially in aged slices wherein the cytosolic Ca signal disappeared after approximately 150 s of washout and the mitochondrial Ca signal disappeared after 25 s of washout. These signals were preserved in aged slices by BAPTA-AM. Five minutes of oxygen glucose deprivation (OGD) was associated with a significant increase in cytosolic Ca in both young and aged synapses, which was irreversible in the aged synapses. These responses were significantly attenuated by BAPTA-AM in both the young and aged synapses. These results support the hypothesis that increasing intracellular calcium neuronal buffering in aged rats ameliorates age-related impaired presynaptic Ca regulation.

Enhanced Ih depresses rat entopeduncular nucleus neuronal activity from high-frequency stimulation or raised Ke+.

High-frequency stimulation (HFS) is used to treat a variety of neurological diseases, yet its underlying therapeutic action is not fully elucidated. Previously, we reported that HFS-induced elevation in [K(+)](e) or bath perfusion of raised K(e)(+) depressed rat entopeduncular nucleus (EP) neuronal activity via an enhancement of an ionic conductance leading to marked depolarization. Herein, we show that the hyperpolarization-activated (I(h)) channel mediates the HFS- or K(+)-induced depression of EP neuronal activity. The perfusion of an I(h) channel inhibitor, 50 microM ZD7288 or 2 mM CsCl, increased input resistance by 23.5 +/- 7% (ZD7288) or 35 +/- 10% (CsCl), hyperpolarized cells by 3.4 +/- 1.7 mV (ZD7288) or 2.3 +/- 0.9 mV (CsCl), and decreased spontaneous action potential (AP) frequency by 51.5 +/- 12.5% (ZD7288) or 80 +/- 13.5% (CsCl). The I(h) sag was absent with either treatment, suggesting a block of I(h) channel activity. Inhibition of the I(h) channel prior to HFS or 6 mM K(+) perfusion not only prevented the previously observed decrease in AP frequency, but increased neuronal activity. Under voltage-clamp conditions, I(h) currents were enhanced in the presence of 6 mM K(+). Calcium is also involved in the depression of EP neuronal activity, since its removal during raised K(e)(+) application prevented this attenuation and blocked the I(h) sag. We conclude that the enhancement of I(h) channel activity initiates the HFS- and K(+)-induced depression of EP neuronal activity. This mechanism could underlie the inhibitory effects of HFS used in deep brain stimulation in output basal ganglia nuclei.

Transition to seizures in the isolated immature mouse hippocampus: a switch from dominant phasic inhibition to dominant phasic excitation.

The neural dynamics and mechanisms responsible for the transition from the interictal to the ictal state (seizures) are unresolved questions in epilepsy. It has been suggested that a shift from inhibitory to excitatory GABAergic drive can promote seizure generation. In this study, we utilized an experimental model of temporal lobe epilepsy which produces recurrent seizure-like events in the isolated immature mouse hippocampus (P8-16), perfused with low magnesium ACSF, to investigate the cellular dynamics of seizure transition. Whole-cell and perforated patch recordings from CA1 pyramidal cells and from fast- and non-fast-spiking interneurons in the CA1 stratum oriens hippocampal region showed a change in intracellular signal integration during the transition period, starting with dominant phasic inhibitory synaptic input, followed by dominant phasic excitation prior to a seizure. Efflux of bicarbonate ions through the GABA A receptor did not fully account for this excitation and GABAergic excitation via reversed IPSPs was also excluded as the prime mechanism generating the dominant excitation, since somatic and dendritic GABA A responses to externally applied muscimol remained hyperpolarizing throughout the transition period. In addition, abolishing EPSPs in a single neuron by intracellularly injected QX222, revealed that inhibitory synaptic drive was maintained throughout the entire transition period. We suggest that rather than a major shift from inhibitory to excitatory GABAergic drive prior to seizure onset, there is a change in the interaction between afferent synaptic inhibition, and afferent and intrinsic excitatory processes in pyramidal neurons and interneurons, with maintained inhibition and increasing, entrained 'overpowering' excitation during the transition to seizure.

High frequency stimulation or elevated K+ depresses neuronal activity in the rat entopeduncular nucleus.

High frequency stimulation (HFS) is applied to many brain regions to treat a variety of neurological disorders/diseases, yet the mechanism(s) underlying its effects remains unclear. While some studies showed that HFS inhibits the stimulated nucleus, others report excitation. In this in vitro study, we stimulated the rat globus pallidus interna (entopeduncular nucleus, EP), a commonly stimulated area for Parkinson's disease, to investigate the effect of HFS-induced elevation of extracellular potassium (K(+)(e)) on rat EP neuronal activity. Whole-cell patch-clamp recordings and [K(+)](e) measurements were obtained in rat EP brain slices before, during and after HFS. After HFS (150 Hz, 10 s), [K(+)](e) increased from 2.5-9.6+/-1.4 mM, the resting membrane potential of EP neurons depolarized by 11.1+/-2.5 mV, spiking activity was significantly depressed, and input resistance decreased by 25+/-6%. The GABA(A) receptor blocker, gabazine, did not prevent these effects. The bath perfusion of 6 or 10 mM K(+), with or without synaptic blockers, mimicked the HFS-mediated effects: inhibition of spike activity, a 20+/-9% decrease in input resistance and a 17.4+/-3.0 mV depolarization. This depolarization exceeded predicted values of elevated [K(+)](e) on the resting membrane potential. A depolarization block did not fully account for the K(+)-induced inhibition of EP neuronal activity. Taken together, our results show that HFS-induced elevation of [K(+)](e) decreased EP neuronal activity by the activation of an ion conductance resulting in membrane depolarization, independent of synaptic involvement. These findings could explain the inhibitory effects of HFS on neurons of the stimulated nucleus.

L-type calcium channel blockade modifies anesthetic actions on aged hippocampal neurons.

Previous studies in our laboratory demonstrated a reversal of anesthetic actions on aged neurons by decreasing extracellular [Ca(2+)] in hippocampal slices. Such maneuver indirectly attenuated Ca(2+) influx, hence decreased exogenous intraneuronal Ca(2+) loads during neuronal activity and consequently improved intracellular Ca(2+) concentration homeostasis. Therefore, in the present study we hypothesized that decreasing exogenous Ca(2+) loads by blocking voltage-gated calcium influx in aged neurons would oppose isoflurane actions. Conversely, increasing endogenous Ca(2+) loads by suppressing calcium efflux during forced reversal of Na(+)/Ca(2+) exchanger function would enhance anesthetic effects. Hippocampal slices were prepared from young (2-4 months) and old (24-26 months) Fischer 344 rats. Isoflurane depressed the evoked dendritic field excitatory postsynaptic potentials by approximately 45% in slices taken from old animals. However, application of isoflurane in addition with CoCl(2) or nifedipine opposed the anesthetic actions, which then depressed the evoked dendritic field postsynaptic potentials by only 15%. Selective blockade of the N-type and P/Q-type calcium channels with omega-conotoxin GVIA and omega-conotoxin MVIIC respectively caused rapid but partial depression of synaptic transmission in slices taken from old Fischer 344 rats. However, isoflurane actions in these aged slices were not affected compared with slices perfused only with normal artificial cerebrospinal fluid. Young and aged slices were then exposed to a low sodium perfusate that forces the Na(+)/Ca(2+) exchanger protein into a reverse mode, thus increasing intracellular Ca(2+) concentration. Isoflurane actions under such conditions were profoundly potentiated in aged slices but were not altered in young hippocampi. The current results show that in aged central neurons, selectively blocking L-type calcium channels opposes anesthetic-induced depression of excitatory synaptic transmission. On the contrary, increasing calcium loads in aged neurons potentiates these actions.

Brain-Silicon Interface for High-Resolution in vitro Neural Recording.

A 256-channel integrated interface for simultaneous recording of distributed neural activity from acute brain slices is presented. An array of 16 times 16 Au recording electrodes are fabricated directly on the die. Each channel implements differential voltage acquisition, amplification and band-pass filtering. In-channel analog memory stores an electronic image of neural activity. A 3 mm times 4.5 mm integrated prototype fabricated in a 0.35-mum CMOS technology is experimentally validated in single-channel extracellular in vitro recordings from the hippocampus of mice and in multichannel simultaneous recordings in a controlled environment.

Bidirectional multisite seizure propagation in the intact isolated hippocampus: the multifocality of the seizure "focus".

Localizing the seizure focus is difficult and frequently, multiple sites are found. This reflects our poor understanding of the fundamental mechanisms of seizure generation and propagation. We used multisite electrophysiological recordings in two seizure models and voltage-sensitive dye imaging, to spatiotemporally characterize the initiation and propagation of seizures in an intact epileptogenic brain region, the isolated hippocampus. In low-magnesium perfusate, seizures always originated in the temporal region, and propagated along the septotemporal axis to the septal region. After the seizure spread across the hippocampus, the bursts within a seizure became bidirectional, with different propagation patterns at different frequencies. When the intact hippocampus was separated along the septotemporal axis, independent bidirectional activity was observed in the two halves, and region-specific cuts to the tissue reveal that the CA3 region is critical for seizure generation and propagation. In a second seizure model, using focal tetanic stimulation of the septal and temporal CA3 region, seizures always originated at the stimulated site with bidirectionality later developing at different frequencies, as noted in the low magnesium model, behavior compatible with coupled neuronal network oscillators. These data provide novel insights into the dynamic multifocality of seizure onset and propagation, revealing that the current concept of a single seizure "focus" is complex.

Postnatal development of intrinsic GABAergic rhythms in mouse hippocampus.

The local circuitry of the mammalian limbic cortices, including the hippocampus, is capable of generating spontaneous rhythmic activities of 0.5-4 Hz when isolated in vitro. These rhythmic activities are mediated by synchronous inhibitory postsynaptic potentials in pyramidal neurons as the result of repeated discharges of inhibitory interneurons. As such, they are thought to represent an intrinsic inhibitory rhythm. It is unknown at present whether such a rhythm occurs in the immature rodent hippocampus and, if so, the postnatal time window in which it develops. We explored these issues using our recently developed whole mouse hippocampal isolate preparation in vitro. We found that spontaneous rhythmic field potentials started to emerge in mouse hippocampal isolates around postnatal day 10, stabilized after postnatal day 15 and persisted into adulthood. In postnatal days 11-14 mouse hippocampi, the properties of these rhythmic potentials were in keeping with a CA3-driven, IPSP-based intrinsic network activity. The lack of spontaneous field rhythm in neonatal (postnatal days 2-7) hippocampi cannot be attributed to the excitatory activities mediated by gamma-aminobutyric acid type A (GABA-A) receptors, as chloride-dependent hyperpolarizing inhibitory postsynaptic potentials were detectable in neonatal pyramidal neurons at voltages near resting potentials and pharmacological antagonisms of GABA-A receptors produced robust epileptiform discharges in neonatal hippocampi. High frequency afferent stimulation or applications of 4-aminopyridine at low micromolar concentrations failed to induce persistent field rhythm in neonatal hippocampi, suggesting that an overall weak glutamatergic drive is not the sole causing factor. We suggest that the inhibitory postsynaptic potential-based spontaneous rhythmic field potentials develop in a discrete time window during the second postnatal week in the mouse hippocampus due to a fine-tuning in the structure and function of CA3 recurrent circuitry and associated GABAergic inhibitory interneurons.

Model of frequent, recurrent, and spontaneous seizures in the intact mouse hippocampus.

This study presents a model of chronic, recurrent, spontaneous seizures in the intact isolated hippocampal preparation from mice aged P8-P25. Field activity from the CA1 pyramidal cell layer was recorded and recurrent, spontaneous seizure-like events (SLEs) were observed in the presence of low Mg2+ (0.25 mM) artificial cerebrospinal fluid (ACSF). Hippocampi also showed interictal epileptiform discharges (IEDs) of 0.9-4.2 Hz occurring between seizures. No age-specific differences were found in SLE occurrence (2 SLEs per 10 min, on average), duration, and corresponding frequencies. After long exposure to low Mg2+ ACSF (>3 h), SLEs were completely reversible within minutes with the application of normal (2 mM Mg2+) ACSF. The AMPA antagonist, CNQX, blocked all epileptiform activity, whereas the NMDA antagonist, APV, did not. The gamma-aminobutyric acid (GABA)A antagonist, bicuculline, attenuated and fragmented SLEs, implicating interneurons in SLE generation. The L-type Ca2+ blocker, nifedipine, enhanced epileptiform activity. Analysis of dual site recordings along the septotemporal hippocampus demonstrated that epileptiform activity began first in the temporal pole of the hippocampus, as illustrated by disconnection experiments. Once an SLE had been established, however, the septal hippocampus was sometimes seen to lead the epileptiform activity. The whole hippocampus with intact local circuitry, treated with low Mg2+, provides a realistic model of recurrent spontaneous seizures, which may be used, in normal and genetically modified mice, to study the dynamics of seizures and seizure evolution, as well as the mechanisms of action of anti-epileptic drugs and other therapeutic modalities.

Dynamics of intracellular calcium and free radical production during ischemia in pyramidal neurons.

Biochemical cascades initiated by oxidative stress and excitotoxic intracellular calcium rises are thought to converge on mitochondrial dysfunction. We investigated the contribution of mitochondrial dysfunction to free radical (FR) overproduction in rat CA1 pyramidal neurons of organotypic slices subjected to a hypoxic-hypoglycemic insult. Ischemia-induced FR generation was decreased by the mitochondrial complex I blocker, rotenone, indicating that mitochondria are the principal source of ischemic FR production. Measurements of mitochondrial calcium with the mitochondrial calcium probe dihydroRhod-2, revealed that FR production during and after the anoxic episode correlates with the accumulation of mitochondrial calcium. However, the mitochondrial calcium uptake inhibitor Ru360 did not prevent FR generation during ischemia and attenuated it to some degree during reoxygenation. On the other hand, the mitochondrial permeability transition blocker cyclosporinA (CsA) completely arrested both ischemic FR generation and mitochondrial calcium overload, and prevented deterioration of neuronal intrinsic membrane properties. CsA had no effect on the accumulation of intracellular calcium during ischemia-reperfusion. Nicotinamide, a blocker of NAD+ hydrolysis, reproduced the CsA effects on FR generation, mitochondrial calcium accumulation and cytoplasmic calcium increases. These observations suggest that a major determinant of ischemic FR generation in pyramidal neurons is the uncoupling of the mitochondrial respiratory chain, which may be associated with the mitochondrial permeability transition.

Upregulation of gap junction connexin 32 with epileptiform activity in the isolated mouse hippocampus.

Gap junctions, which serve as intercellular channels providing direct cytoplasmic continuity and ionic current flow between adjacent cells, are constituted by connexin proteins. Using an in vitro model of bicuculline-induced epileptiform activity, we asked whether increased connexin levels occur during epileptiform activity in the intact whole hippocampus, freshly isolated from young (15-day-old) mouse brain. Exposure to bicuculline (10 microM), for 2-10 h, induced persistent changes in electrical activities that included enhanced spontaneous field activity (4 h), an epileptiform response to single electrical stimulation (6 h), and spontaneous epileptiform activity (6 h). These electrophysiological changes were not reversed by up to 60 min perfusion with normal artificial cerebrospinal fluid, but were greatly depressed by the gap junction uncoupler, carbenoxolone (120 microM, 10 min). Data from RNase protection assay and immunoblotting showed that among several detected gap junctions, only connexin 32 was affected. After 2-6 h exposure to bicuculline, the connexin 32 mRNA expression was upregulated to 2-3-fold control (P < 0.01), and its protein level was significantly elevated the following 6 h (P < 0.01), at which time electrophysiologically measured evidence of clearly epileptiform activity was apparent. In addition, the transcription factor, c-fos protein, but not the cAMP response element-binding protein, was also found to be increased at the early stage of bicuculline exposure (2 h) compared to control (P < 0.05).Thus, we have found that exposing the acutely isolated hippocampus to bicuculline, induced increased c-fos protein, followed by increased connexin 32 transcript and protein, and concurrently, persistent epileptiform activity that was depressed by carbenoxolone.

Artificial electrotonic coupling affects neuronal firing patterns depending upon cellular characteristics.

While there have been numerous theoretical studies indicating that electrotonic coupling via gap junctions interacts with the intrinsic characteristics of the coupled neurons to modify their electrical behaviour, little experimental evidence has been provided in coupled mammalian neurons. Using an artificial electrotonic junction, two distant uncoupled neurons were coupled through the computer, and the coupling conductance was varied. Tonically firing CA1 hippocampal pyramidal neurons reduced their spike firing frequency when coupled to thalamic or pyramidal cells, showing that the electrical coupling can be considered as a low-pass filter. The strength of coupling needed to entrain spike bursts of pyramidal neurons was considerably lower than the coupling needed to synchronize two neurons with different cellular characteristics (thalamic and pyramidal cells). Coupling promoted burst firing in a non-bursting cell if it was coupled to a spontaneously bursting neuron. These results support modelling studies that indicate a role for gap-junctional coupling in the synchronization of neuronal firing and the expression of low-frequency bursting.

Progestin receptors mediate progesterone suppression of epileptiform activity in tetanized hippocampal slices in vitro.

Clinical and laboratory studies suggest that progesterone reduces epileptic seizure activity. The mechanisms underlying this effect are not known. The present study determined the effects of progesterone on extracellular evoked responses recorded in the CA1 field of hippocampal slices, as well as epileptiform responses recorded from tetanized slices. Slices were prepared from ovariectomized rats, with or without estrogen replacement. Hippocampal slices were superfused in vitro with one of the following treatments: progesterone with or without RU486 (a progesterone receptor antagonist); allopregnanolone (a progesterone metabolite that potentiates GABA action at GABA(A) receptors); RU5020 (a high-affinity progesterone receptor agonist); or cholesterol (control). In non-tetanized slices, a twofold increase in the excitatory postsynaptic field potential and population spike amplitude occurred during both cholesterol and progesterone superfusion. In contrast, under the same conditions, exposure to allopreganolone caused a 25% reduction in both field potential and population spike amplitude of evoked responses within 30min of treatment. In tetanized slices, progesterone and RU5020, but not allopregnanolone or cholesterol, caused significant reductions in the field potential and population spike amplitude of evoked responses. Progesterone and RU5020 also significantly reduced the duration of tetanic stimulus-induced afterdischarges and the frequency of spontaneous interictal discharges. The effects of allopregnanolone were restricted to a reduction in the primary afterdischarge duration. Estrogen replacement slightly attenuated progesterone's suppression of spontaneous discharges and depression of evoked responses. All responses to progesterone were blocked by prior or concurrent exposure to RU486. These data indicate that allopregnanolone suppresses evoked potentials in non-tetanized hippocampal slices, consistent with previous reports that this neurosteroid has marked anxiolytic and anticonvulsant effects. After tetanization, however, progesterone receptor-mediated responses become quantitatively more important as a mechanism for suppressing hippocampal electrical activity.

Analysis of single K(ATP) channels in mammalian dentate gyrus granule cells.

ATP-sensitive potassium (K(ATP)) channels are heteromultimer complexes of subunits from members of the inwardly rectifying K(+) channel and the ATP-binding cassette protein superfamilies. K(ATP) channels couple metabolic state to membrane excitability, are distributed widely, and participate in a variety of physiological functions. Understood best in pancreatic beta cells, where their activation inhibits insulin release, K(ATP) channels have been implicated also in postischemia cardio- and neuroprotection. The dentate gyrus (DG) is a brain region with a high density of K(ATP) channels and is relatively resistant to ischemia/reperfusion-induced cell death. Therefore we were interested in describing the characteristics of single K(ATP) channels in DG granule cells. We recorded single K(ATP) channels in 59/105 cell-attached patches from DG granule cells in acutely prepared hippocampal slices. Single-channel openings had an E(K) close to 0 mV (symmetrical K(+)) and were organized in bursts with a duration of 19.3 +/- 1.6 (SE) ms and a frequency of 3.5 +/- 0.8 Hz, a unitary slope conductance of 27 pS, and a low, voltage-independent, probability of opening (P(open), 0.04 +/- 0.01). Open and closed dwell-time histograms were fitted best with one (tau(open) = 1.3 +/- 0.2 ms) and the sum of two (tau(closed,fast) = 2.6 +/- 0.9 ms, tau(closed,slow) = 302.7 +/- 67. 7 ms) exponentials, respectively, consistent with a kinetic model having at least a single open and two closed states. The P(open) was reduced ostensibly to zero by the sulfonylureas, glybenclamide (500 nM, 2/6; 10 microM,11/14 patches) and tolbutamide (20 microM, 4/6; 100 microM, 4/4 patches). The blocking dynamics for glybenclamide included transition to a subconductance state (43.3 +/- 2.6% of control I(open channel)). Unlike glybenclamide, the blockade produced by tolbutamide was reversible. In 5/5 patches, application of diazoxide (100 microM) increased significantly P(open) (0.12 +/- 0.02), which was attributable to a twofold increase in the frequency of bursts (8.3 +/- 2.0 Hz). Diazoxide was without effect on tau(open) and tau(closed,fast) but decreased significantly tau(closed,slow) (24.4 +/- 2.6 ms). We observed similar effects in 6/7 patches after exposure to hypoxia/hypoglycemia, which increased significantly P(open) (0.09 +/- 0.03) and the frequency of bursts (7.1 +/- 1.7 Hz) and decreased significantly tau(closed,slow) (29.5 +/- 1.8 ms). We have presented convergent evidence consistent with single K(ATP) channel activity in DG granule cells. The subunit composition of K(ATP) channels native to DG granule cells is not known; however, the characteristics of the channel activity we recorded are representative of Kir6.1/SUR1, SUR2B-based channels.