Effects of carbonic anhydrase activity on the excitability of hippocampal axons during high-frequency firing.

Epileptic activity is known to cause a lowering of intraneuronal pH, which has been suggested to serve as a feedback signal to terminate seizures. The mechanism of such signaling is unclear, but likely involves an altered function of several types of ligand- and voltage-gated channels in postsynaptic membranes caused by increasing cytosolic and extracellular [H+]. In addition, axonal conduction properties may be altered by endogenous pH signals, but this has not been investigated. In the present study, we have recorded the axonal compound action potential (fiber volley) in hippocampal slices in the presence of glutamatergic and GABAergic antagonists. During high-frequency stimulation (HFS) of the Schaffer collaterals, the fiber volley was depressed and its latency from stimulus to peak increased. In the CA1 stratum radiatum these changes were enhanced when the carbonic anhydrase inhibitor acetazolamide (1 mM) was co-perfused. The enhancing effect of acetazolamide was absent after lowering of [Ca2+] in the perfusion medium. Acetazolamide had no detectable effect on HFS-evoked fiber volleys recorded from a more proximal site along the Schaffer collaterals (at the CA2-CA3 border) or from axons in the alveus of CA1. Intracellular acidification imposed by washout of NH4Cl (5 mM) had qualitatively similar effects on fiber volleys evoked at low frequency as those observed with acetazolamide during HFS in CA1 stratum radiatum. The results suggest that carbonic anhydrase-dependent pH regulation counteracts activity-induced reduction of the excitability of Schaffer collateral axons in CA1. A possible influence from local synaptic terminals on this effect is discussed.

Differential role of oxidative stress in synaptic and nonsynaptic in vitro ictogenesis.

It is well established that dysfunctional glucose metabolism and in particular hypoglycemia can lead to hyperexcitability and exacerbate epileptic seizures. The precise mechanisms behind this form of hyperexcitability are still unresolved. The present study investigates to what extent oxidative stress can account for the acute proconvulsant effect of hypoglycemia. We used the glucose derivative 2-deoxy-d-glucose (2-DG) to mimic glucose deprivation in hippocampal slices during the extracellular recording of interictal-like (IED) and seizure-like (SLE) epileptic discharge in areas CA3 and CA1. After induction of IED in area CA3 by perfusion of Cs+ (3 mM), MK801 (10 µM), and bicuculline (10 µM), subsequent application of 2-DG (10 mM) resulted in the appearance of SLE in 78.3% of experiments. This effect was only observed in area CA3 and was reversibly blocked by tempol (2 mM), a scavenger of reactive oxygen species, in 60% of experiments. Preincubation with tempol reduced the incidence of 2-DG-induced SLE to 40%. Low-Mg2+-induced SLE in area CA3 and in the entorhinal cortex (EC) was also reduced by tempol. In contrast, to the above models, which depend on synaptic transmission, nonsynaptic epileptiform field bursts induced in area CA3 by a combination of Cs+ (5 mM) and Cd2+ (200 µM), or in area CA1 using the "low-Ca2+ model," was unaffected or even enhanced by tempol. These results indicate that oxidative stress significantly contributes to 2-DG-induced seizures in area CA3 and that the impact of oxidative stress differs between synaptic and nonsynaptic ictogenesis.NEW & NOTEWORTHY The main findings of the current study are that area CA3 but not area CA1 can support 2-DG-induced seizure activity, that oxidative stress significantly contributes to 2-DG-induced seizure activity in area CA3, and that the impact of oxidative stress differs between synaptic and nonsynaptic epileptiform activity. In in vitro models where ictogenesis depends on synaptic interactions, oxidative stress lowers the seizure threshold, whereas in nonsynaptic models seizure threshold is unchanged or even increased.

Effect of acute mitochondrial dysfunction on hyperexcitable network activity in rat hippocampus in vitro.

Metabolic stress imposed by epileptic seizures can result in mitochondrial dysfunction, believed to act as positive feedback on epileptogenesis and seizure susceptibility. As the mechanism behind this positive feedback is unclear, the aim of the present study was to investigate the causal link between acute mitochondrial dysfunction and increased seizure susceptibility in hyperexcitable hippocampal networks. Following the induction of spontaneous interictal-like discharges, acute selective pharmacological blockade of either of the mitochondrial respiratory complexes (MRC) I-IV induced seizure-like events (SLE) in 78-100% of experiments. A similar result was obtained by uncoupling the oxidative phosphorylation (OXPHOS) but not by selective blockade of MRCV (ATP synthase) which did not induce SLE. The reactive oxygen species (ROS) scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (tempol, 2 mM) significantly reduced the proconvulsant effect of blocking MRCI but did not reduce the proconvulsant effect of OXPHOS uncoupling. These findings indicate that acute mitochondrial dysfunction can lead to a convulsive state within a short timeframe, and that increased ROS production makes substantial contribution to such induction in addition to other mitochondrial related factors, which appears to be independent of changes in ROS and ATP production.

Role of GABAB receptors in proepileptic and antiepileptic effects of an applied electric field in rat hippocampus in vitro.

The mechanisms underlying antiepileptic effects of deep brain stimulation (DBS) are complex and poorly understood. Studies on the effects of applied electric fields on epileptic nervous tissue could enable future advances in DBS treatments. Applied electric fields are known to inhibit or enhance epileptic activity in vitro through direct effects on local neurons, but it is unclear whether trans-synaptic effects participate in such actions. The present study investigates, in an epileptic brain slice model, the influence of GABAB receptor activation on excitatory and suppressive effects of a short-duration (10 ms) electric field in rat hippocampus. The results show that perfusion of the GABAB receptor antagonist, CGP 55845 (2 µM), could abolish applied-field induced suppression of orthodromic-stimulus evoked epileptiform afterdischarge activity in the CA1 region. GABAB receptor blockade was associated with an enhanced excitatory (proepileptic) effect of the applied field. However, the suppressive effect, observed in isolation using weak field stimuli, was left unchanged. The G-protein-activated inwardly rectifying K+ channel (GIRK) antagonist, tertiapin (30-50 nM), mimicked the effects of CGP 55845. The results suggest that the applied field activate (elements of) local interneurons to release GABA onto GABAB receptors. The resulting activation of postsynaptic GIRK channels inhibits neuronal activity thereby dampening the direct stimulatory effect of the applied field. The study indicates that local-stimulus induced GABAB receptor activation can serve a protective role under antiepileptic paradigms by preventing electrical stimulation from causing hyperexcitation.

Opposing effects of 2-deoxy-d-glucose on interictal- and ictal-like activity when K+ currents and GABAA receptors are blocked in rat hippocampus in vitro.

The ketogenic diet (KD), a high-fat, carbohydrate-restricted diet, is used as an alternative treatment for drug-resistant epileptic patients. Evidence suggests that compromised glucose metabolism has a significant role in the anticonvulsant action of the KD; however, it is unclear what part of the glucose metabolism that is important. The present study investigates how selective alterations in glycolysis and oxidative phosphorylation influence epileptiform activity induced by blocking K+ currents and GABAA and NMDA receptors in the hippocampal slice preparation. Blocking glycolysis with the glucose derivative 2-deoxy-d-glucose (2-DG; 10 mM) gave a fast reduction of the frequency of interictal discharge (IED) consistent with findings in other in vitro models. However, this was followed by the induction of seizure-like discharges in area CA1 and CA3. Substituting glucose with sucrose (glucopenia) had effects similar to those of 2-DG, whereas substitution with l-lactate or pyruvate reduced the IED but had a less proconvulsant effect. Blockade of ATP-sensitive K+ channels, glycine or adenosine 1 receptors, or depletion of the endogenous anticonvulsant compound glutathione did not prevent the actions of 2-DG. Baclofen (2 µM) reproduced the effect of 2-DG on IED activity. The proconvulsant effect of 2-DG could be reproduced by blocking the oxidative phosphorylation with the complex I toxin rotenone (4 µM). The data suggest that inhibition of IED, induced by 2-DG and glucopenia, is a direct consequence of impairment of glycolysis, likely exerted via a decreased recurrent excitatory synaptic transmission in area CA3. The accompanying proconvulsant effect is caused by an excitatory mechanism, depending on impairment of oxidative phosphorylation. NEW & NOTEWORTHY This study reveals two opposing effects of 2-deoxy-d-glucose (2-DG) and glucopenia on in vitro epileptiform discharge observed during combined blockade of K+ currents and GABAA receptors. Interictal-like activity is inhibited by a mechanism that selectively depends on impairment of glycolysis and that results from a decrease in the strength of excitatory recurrent synaptic transmission in area CA3. In contrast, 2-DG and glucopenia facilitate ictal-like activity by an excitatory mechanism, depending on impairment of mitochondrial oxidative phosphorylation.

Furosemide depresses the presynaptic fiber volley and modifies frequency-dependent axonal excitability in rat hippocampus.

The loop diuretic furosemide is known to have anticonvulsant effects, believed to be exerted through blockade of glial Na+-K+-2Cl- cotransport causing altered volume regulation in brain tissue. The possibility that direct effects of furosemide on neuronal properties could also be involved is supported by previous observations, but such effects have not been thoroughly investigated. In the present study we show that furosemide has two opposing effects on stimulus-induced postsynaptic excitation in the nonepileptic rat hippocampal slice: 1) an enhancement of e-s coupling, which depended on intact GABAA transmission and was partially mimicked by selective blockade of K+-2Cl- cotransport, and 2) a decrement of field excitatory postsynaptic potentials. The balance between these effects varied, depending on the amount of synaptic drive. In addition, the compound action potential (fiber volley) recorded from the stimulated Schaffer collateral axons in stratum radiatum showed a progressive decrease during perfusion of furosemide. This effect was activity-independent, was mimicked by the stilbene derivative DIDS, and could be reproduced on fiber volleys in the alveus. Furosemide also reduced the initial enhancement of the fiber volley observed during trains of high-frequency stimulation (HFS). Results of hyperosmotic expansion of the extracellular volume, with 30 mM sucrose, indicated that both the induction and antagonism of the HFS-induced enhancement were independent of signaling via the extracellular space. Furosemide caused an increased decay of paired-pulse-induced supranormal axonal excitability, which was antagonized by ZD7288. We conclude that furosemide decreases axonal excitability and prevents HFS-induced hyperexcitability via mechanisms downstream of blockage of anion transport, which could include hyperpolarization of axonal membranes.NEW & NOTEWORTHY This study shows that the anion transporter antagonists furosemide and DIDS cause a marked decrease of axonal excitability in rat hippocampal CA1 region and prevent the induction of activity-dependent hyperexcitability in Schaffer collateral axons. The data are consistent with direct effects on axonal membrane properties. We also find that activity-dependent enhancement and depression of axonal excitability can be modified independently, suggesting that these events are governed by different underlying processes.

Cognitive deficits caused by a disease-mutation in the α3 Na(+)/K(+)-ATPase isoform.

The Na(+)/K(+)-ATPases maintain Na(+) and K(+) electrochemical gradients across the plasma membrane, a prerequisite for electrical excitability and secondary transport in neurons. Autosomal dominant mutations in the human ATP1A3 gene encoding the neuron-specific Na(+)/K(+)-ATPase α3 isoform cause different neurological diseases, including rapid-onset dystonia-parkinsonism (RDP) and alternating hemiplegia of childhood (AHC) with overlapping symptoms, including hemiplegia, dystonia, ataxia, hyperactivity, epileptic seizures, and cognitive deficits. Position D801 in the α3 isoform is a mutational hotspot, with the D801N, D801E and D801V mutations causing AHC and the D801Y mutation causing RDP or mild AHC. Despite intensive research, mechanisms underlying these disorders remain largely unknown. To study the genotype-to-phenotype relationship, a heterozygous knock-in mouse harboring the D801Y mutation (α3(+/D801Y)) was generated. The α3(+/D801Y) mice displayed hyperactivity, increased sensitivity to chemically induced epileptic seizures and cognitive deficits. Interestingly, no change in the excitability of CA1 pyramidal neurons in the α3(+/D801Y) mice was observed. The cognitive deficits were rescued by administration of the benzodiazepine, clonazepam, a GABA positive allosteric modulator. Our findings reveal the functional significance of the Na(+)/K(+)-ATPase α3 isoform in the control of spatial learning and memory and suggest a link to GABA transmission.

A novel mechanism for the anticonvulsant effect of furosemide in rat hippocampus in vitro.

Though both in vivo and in vitro studies have demonstrated an anticonvulsant effect of the loop diuretic furosemide, the precise mechanism behind this effect is still debated. The current study investigates the effect of furosemide on Cs-induced epileptiform activity (Cs-FP) evoked in area CA1 of rat hippocampal slices in the presence of Cs(+) (5mM) and ionotropic glutamatergic and GABAergic receptor antagonists. As this model diverges in several respects from other epilepsy models it can offer new insight into the mechanism behind the anticonvulsive effect of furosemide. The present study shows that furosemide suppresses the Cs-FP in a dose-dependent manner with a near complete block at concentrations ≥ 1.25 mM. Because furosemide targets several types of ion transporters we examined the effect of more selective antagonists. Bumetanide (20 µM), which selectively inhibits the Na-K-2Cl co-transporter (NKCC1), had no significant effect on the Cs-FP. VU0240551 (10 µM), a selective antagonist of the K-Cl co-transporter (KCC2), reduced the ictal-like phase by 51.73 ± 8.5% without affecting the interictal-like phase of the Cs-FP. DIDS (50 µM), a nonselective antagonist of Cl(-)/HCO3(-)-exchangers, Na(+)-HCO3(-)-cotransporters, chloride channels and KCC2, suppressed the ictal-like phase by 60.8 ± 8.1% without affecting the interictal-like phase. At 500 µM, DIDS completely suppressed the Cs-FP. Based on these results we propose that the anticonvulsant action of furosemide in the Cs(+)-model is exerted through blockade of the neuronal KCC2 and Na(+)-independent Cl(-)/HCO3(-)-exchanger (AE3) leading to stabilization of the activity-induced intracellular acidification in CA1 pyramidal neurons.

Intrinsic Ca2+-dependent theta oscillations in apical dendrites of hippocampal CA1 pyramidal cells in vitro.

Behavior-associated theta-frequency oscillation in the hippocampal network involves a patterned activation of place cells in the CA1, which can be accounted for by a somatic-dendritic interference model predicting the existence of an intrinsic dendritic oscillator. Here we describe an intrinsic oscillatory mechanism in apical dendrites of in vitro CA1 pyramidal cells, which is induced by suprathreshold depolarization and consists of rhythmic firing of slow spikes in the theta-frequency band. The incidence of slow spiking (29%) increased to 78% and 100% in the presence of the ß-adrenergic agonist isoproterenol (2 µM) or 4-aminopyridine (2 mM), respectively. Prior depolarization facilitated the induction of slow spiking. Applied electrical field polarization revealed a distal dendritic origin of slow spikes. The oscillations were largely insensitive to tetrodotoxin, but blocked by nimodipine (10 µM), indicating that they depend on activation of L-type Ca2+ channels. Antagonists of T-, R-, N-, and P/Q-type Ca2+ channels had no detectable effect. The slow spike dimension and frequency was sensitive to 4-aminopyridine (0.1-2 mM) and TEA (10 mM), suggesting the contribution from voltage-dependent K+ channels to the oscillation mechanism. α-Dendrotoxin (10 µM), stromatoxin (2 µM), iberiotoxin (0.2 µM), apamin (0.5 µM), linorpidine (30 µM), and ZD7288 (20 µM) were without effect. Oscillations induced by sine-wave current injection or theta-burst synaptic stimulation were voltage-dependently attenuated by nimodipine, indicating an amplifying function of L-type Ca2+ channels on imposed signals. These results show that the apical dendrites have intrinsic oscillatory properties capable of generating rhythmic voltage fluctuations in the theta-frequency band.

Suppression of epileptiform activity by a single short-duration electric field in rat hippocampus in vitro.

The mechanisms behind the therapeutic effects of electrical stimulation of the brain in epilepsy and other disorders are poorly understood. Previous studies in vitro have shown that uniform electric fields can suppress epileptiform activity through a direct polarizing effect on neuronal membranes. Such an effect depends on continuous DC stimulation with unbalanced charge. Here we describe a suppressive effect of a brief (10 ms) DC field on stimulus-evoked epileptiform activity in rat hippocampal brain slices exposed to Cs(+) (3.5 mM). This effect was independent of field polarity, was uncorrelated to changes in synchronized population activity, and persisted during blockade of synaptic transmission with Cd(2+) (500 µM). Antagonists of A(1), P(2X), or P(2Y) receptors were without effect. The suppressive effect depended on the alignment of the external field with the somato-dendritic axis of CA1 pyramidal cells; however, temporal coincidence with the epileptiform activity was not essential, as suppression was detectable for up to 1 s after the field. Pyramidal cells, recorded during epileptiform activity, showed decreased discharge duration and truncation of depolarizing plateau potentials in response to field application. In the absence of hyperactivity, the applied field was followed by slow membrane potential changes, accompanied by decreased input resistance and attenuation of the depolarizing afterpotential following action potentials. These effects recovered over a 1-s period. The study suggests that a brief electric field induces a prolonged suppression of epileptiform activity, which can be related to changes in neuronal membrane properties, including attenuation of signals depending on the persisting Na(+) current.

Heterogeneous firing behavior during ictal-like epileptiform activity in vitro.

Seizure activity in vivo is caused by populations of neurons displaying a high degree of variability in activity pattern during the attack. The reason for this variability is not well understood. Here we show in an in vitro preparation that hippocampal CA1 pyramidal cells display four types of afterdischarge behavior during stimulus-induced ictal-like events in the presence of Cs(+) (5 mM): type I (43.7%) consisting of high-frequency firing riding on a plateau potential; type II (28.2%) consisting of low-frequency firing with no plateau potential; type III (18.3%) consisting of high-frequency firing with each action potential preceded by a transient hyperpolarization and time-locked to population activity, no plateau potential; "passive" (9.9%) typified by no afterdischarge. Type I behavior was blocked by TTX (0.2 µM) and intracellular injection of QX314 (12.5-25 mM). TTX (0.2 µM) or phenytoin (50 µM) terminated ictal-like events, suggesting that the persistent Na(+) current (I(NaP)) is pivotal for type I behavior. Type I behavior was not correlated to intrinsic bursting capability. Blockade of the M current (I(M)) with linopirdine (10 µM) increased the ratio of type I neurons to 100%, whereas enhancing I(M) with retigabine (50-100 µM) greatly reduced the epileptiform activity. These results suggest an important role of I(M) in determining afterdischarge behavior through control of I(NaP) expression. We propose that type I neurons act as pacemakers, which, through synchronization, leads to recruitment of type III neurons. Together, they provide the "critical mass" necessary for ictogenesis to become regenerative.

Baclofen and adenosine inhibition of synaptic transmission at CA3-CA1 synapses display differential sensitivity to K+ channel blockade.

The metabotropic GABA(B) and adenosine A(1) receptors mediate presynaptic inhibition through regulation of voltage-dependent Ca(2+) channels, whereas K(+) channel regulation is believed to have no role at the CA3-CA1 synapse. We show here that the inhibitory effect of baclofen (20 µM) and adenosine (300 µM) on field EPSPs are differentially sensitive to Cs(+) (3.5 mM) and Ba(2+) (200 µM), but not 4-aminopyridine (100 µM). Barium had no effect on paired-pulse facilitation (PPF) in itself, but gave significant reduction (14 ± 5%) when applied in the presence of baclofen, but not adenosine, suggesting that the effect is presynaptic and selective on the GABA(B) receptor-mediated response. The effect of Ba(2+) on PPF was not mimicked by tertiapin (30 nM), indicating that the underlying mechanism does not involve GIRK channels. Barium did not affect PPF in slices from young rats (P7-P8), suggesting developmental regulation. The above effects of Ba(2+) on adult tissue were reproduced when measuring evoked whole-cell EPSCs from CA1 pyramidal neurons: PPF was reduced by 22 ± 3% in the presence of baclofen and unaltered in adenosine. In contrast, Ba(2+) caused no significant change in frequency or amplitude of miniature EPSCs. The Ba(2+)-induced reduction of PPF was antagonized by LY341495, suggesting metabotropic glutamate receptor involvement. We propose that these novel effects of Ba(2+) and Cs(+) are exerted through blockade of inwardly rectifying K(+) channels in glial cells, which are functionally interacting with the GABA(B) receptor-dependent glutamate release that generates heterosynaptic depression.

Differential influence of non-synaptic mechanisms in two in vitro models of epileptic field bursts.

Non-synaptic interactions are known to promote epileptiform activity through mechanisms that have primarily been studied in one particular in vitro model (low Ca(2+) model). Here we characterize another non-synaptic model, where ictal-like field bursts are induced in the CA1 area of rat hippocampal slices by exposure to Cs(+) (4-5mM) together with blockers of fast chemical synaptic transmission, and compare it with the low Ca(2+) model. The Cs-induced field bursts were blocked by 1 microM tetrodotoxin, but persisted in the presence of 200 microM Cd(2+) or 300 microM Ni(2+). Hyperosmotic condition (addition of 30 mM sucrose), reduced burst amplitude, but, unlike field bursts induced by 0mM Ca(2+)/5.25 mM K(+), sucrose had no effect on frequency or duration. Intracellular alkalinization-acidification sequence induced by NH(4)Cl potentiated and blocked, respectively, the field bursts. Octanol (100-250 microM) blocked all activity in most experiments. A quantitative comparison of three gap junction antagonists (carbenoxolone (100 microM), quinidine (100-250 microM), and endothelin-3 (1-2 microM)) indicated that gap junction communication is implicated in both models. However, endothelin-3 had selective effect on the low Ca(2+)-induced field burst. The data suggest that extracellular space-dependent processes, including field effects, significantly contribute to ongoing field burst activity, whereas initiation of a field burst can occur with or without the aid of such interactions, depending on the level of neuronal excitability. Gap junctions seem to have a general role in initiating field bursts. However, the contribution to this effect from neuronal versus glial connexin types differs in the two epileptic models studied.

The slow Ca2+ -dependent K+ -current facilitates synchronization of hyperexcitable pyramidal neurons.

Studies on in vivo and in vitro epilepsy models have shown that progression and maintenance of epileptiform activity can be affected by the slow Ca(2+)-dependent K(+) current (I(sAHP)). This study aimed to investigate the influence of the I(sAHP) on population activity and single cell activity during the transition from the interictal- to the ictal-like phase of an epileptiform field potential induced by Cs(+). Extracellular and intracellular recordings were performed in area CA1 on 400 microm thick hippocampal slices from adult male Wistar rats. During maintained exposure to Cs(+), the transition between the two phases underwent a slow, time-dependent change, where synchronized population activity gradually disappeared and a plateau-like prolongation of the interictal-like phase emerged. In parallel, the size of the ictal-like phase increased. These effects could be ascribed to a gradual block of the I(sAHP) and were mimicked by the I(sAHP) antagonists carbacholine (2 microM), isoproterenol (4 microM) and Ba(2+) (0.2 mM). Cessation of population activity generally occurred without a concomitant decrease in firing rate of single CA1 pyramidal neurons, but was accompanied by the disappearance of hyperpolarizing prepotentials, indicating a shift in the mechanism of action potential initiation. These findings suggest that the presence of the I(sAHP) increases the tendency of hyperexcitable neurons to fire in synchrony, but at the same time serves to dampen the ictal-like activity that follows the hyperexcitable state. Our data indicate that both effects can be attributed to the influence of this current on the steady-state membrane potential in the period of the transition from interictal- to ictal-like activity.

Inwardly rectifying K(+) (Kir) channels antagonize ictal-like epileptiform activity in area CA1 of the rat hippocampus.

Reactive glial cells, for example, from patients with temporal lope epilepsy have a reduced density of inward rectifying K(+) (Kir) channels and thus a reduced K(+) buffering capacity. Evidence is accumulating that this downregulation of Kir channels could be implicated in epileptogenesis. In rat hippocampal brain slices, prolonged exposure to the nonselective Kir channel antagonist, Cs(+) (5 mM), gives rise to an epileptiform field potential (Cs-FP) in area CA1 composed of an initial positive (interictal-like) phase followed by a prolonged negative (ictal-like) phase. We have previously shown that the interictal-like phase depends on synaptic activation. The present study extends these findings by showing that the ictal-like phase of the Cs-FP is (i) sensitive to osmotic expansion of the extracellular space, (ii) reversed very quickly during wash out of Cs(+), and (iii) re-established in the presence of Ba(2+) (30-200 microM) or isosmotic low extracellular concentration of Na(+) ([Na(+)](o), 51.25 mM). The interictal-like phase showed less or no sensitivity to these treatments. In the complete absence of Cs(+), the Cs-FP could be fully reconstructed by the combined application of 4-aminopyridine (0.5 mM), an isosmotic high extracellular concentration of K(+) ([K(+)](o), 7 mM), and low [Na(+)](o) (51.25 mM). These results suggest that the interictal-like phase is initiated through synaptic activation and results from an unspecific increase in neuronal excitability, whereas the ictal-like phase is entirely dependent on blockade of Kir channels in CA1. We propose that glial dysfunction-related loss of Kir channels may not alone be sufficient for starting the induction process, but will likely increase the tendency of an epileptogenic process to proceed into seizure activity.

Postnatal development of a new type of epileptiform activity in the rat hippocampus.

Long-term application of Cs(+) (5 mM) induces an epileptiform field potential (Cs-FP) in area CA1 of the rat hippocampus, which is independent of N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors and gamma-aminobutyric acid (GABA)(A) receptors. To gain insight into possible mechanisms for the induction of the Cs-FP, we investigated the postnatal development of the response. In brain slices prepared from rats at different ages, the Cs-FP was evoked by stimulation of the Schaffer-collateral-commisural pathway. We found that expression of this potential was clearly dependent on the postnatal age. Thus, the Cs-FP was completely absent at 1 week of age. By 2 weeks, a reduced form of the response was observed, whereas slices taken from 3-week-old rats, displayed full Cs-FP, which were indistinguishable in size and shape from the adult form. In the presence of 4-aminopyridine, potentials resembling the Cs-FP were evoked. These potentials showed a similar age-dependency as the Cs-FP. The Na(+)/K(+) pump inhibitor dihydroouabain (DHO), when present during wash-in of Cs(+), gave a partial block of the Cs-FP in adult slices. This effect was not seen when DHO was applied after development of the Cs-FP. The data indicate that the processes necessary for expression of the Cs-PF are absent at birth and develop during the second postnatal week. We propose that the Cs-FP depends on Cs(+) entry into presynaptic neurons, and that the Na(+)/K(+) pump contributes to this transport of Cs(+). The observed age-dependency could therefore, in part, reflect the delayed development of the Na(+)/K(+) pump.

New type of synaptically mediated epileptiform activity independent of known glutamate and GABA receptors.

It is well known that excitatory synaptic transmission at the hippocampal CA3-CA1 synapse depends on the binding of released glutamate to ionotropic receptors. Here we report that during long-term application of Cs+ (5 mM), stimulation of the Schaffer collateral-commisural pathway evokes an epileptic field potential (Cs-FP) in area CA1 of the rat hippocampal slice, which is resistant to antagonists of ionotropic glutamate and GABA(A) receptors. The Cs-FP was blocked by N-type but not L-type Ca2+ channel antagonists and was attenuated by adenosine (0.5 mM), as expected for a synaptically mediated response. These properties make the Cs-FP fundamentally different from other types of Cs(+)-induced epileptiform activity. Replacement of Cs+ with antagonists of the hyperpolarization-activated nonselective cation current I(h) and inwardly rectifying potassium channels (K(IR)) or partial inhibition of the Na(+)/K+ pump did not cause Cs-FP-like potentials, which indicates that such actions of Cs+ were not responsible for the Cs-FP. The effect of Cs+ was partly mimicked by 4-aminopyridine (4-AP; 2 mM), suggesting that an increase in transmitter release is involved. The group I metabotropic glutamate receptor (mGluR) agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) attenuated the Cs-FP. This effect was not, however, antagonized by group I mGluR antagonists. Selective and nonselective mGluR antagonists did not attenuate the Cs-FP. We conclude that long-term exposure to Cs+ induces a state where excitatory synaptic transmission can exist between area CA3 and CA1 in the hippocampus, independent of ionotropic and metabotropic glutamate receptors and GABA(A) receptors.

Influence of the hyperpolarization-activated cation current, I(h), on the electrotonic properties of the distal apical dendrites of hippocampal CA1 pyramidal neurones.

The electrical field application technique has revealed that the electrotonic length of the distal apical dendrites of hippocampal CA1 pyramidal neurones is long compared to the rest of the cell. This difference may be due to an asymmetrical distribution of channels responsible for the leak conductance in distal and proximal membrane segments. One such conductance, the hyperpolarization-activated cation current, I(h), is reported to display an increasing density with distance from the soma along the apical dendrite. Such asymmetry of I(h) could be a major cause of the increased electrotonic length of the distal apical dendrite. In the present study we found that blockade of I(h), by bath application of Cs(+) (3 mM) or ZD7288 (20 microM), reduced the electrical field-induced transmembrane polarization (TMP) in the distal apical dendrites. In some neurones the polarization reversed polarity, reflecting a movement of the indifference point (site of zero polarization) from the distal dendrites, across the recording site to a more proximal position. These effects were more pronounced when Cs(+) and ZD7288 were applied locally to the distal apical dendrites. Bath application of another antagonist of leak conductance, Ba(2+) (1 mM), also decreased the average field-induced polarization. This latter effect, however, did not reach statistical significance. These data suggest that I(h) is partly responsible for the distal location of the indifference point, and indicate that an elevated activity of I(h) contributes to the relatively increased electrotonic length of the most distal part of the apical dendrites.