Primary afterdischarge in organotypic hippocampal slice cultures: effects of standard antiepileptic drugs.

PURPOSE: In the hippocampus intense high frequency electrical stimulation induces a long-lasting rhythmic synchronization (primary afterdischarge). In order to examine the suitability of primary afterdischarges (PADs) in organotypic hippocampal slice cultures (OHSCs) as an in vitro model of evoked seizures, we have worked out in detail the sensitivity of PADs to standard antiepileptic drugs (AEDs) and compared the necessary concentrations to those that were effective in animal models of partial and generalized tonic-clonic seizures. METHODS: OHSCs were prepared according to the interface culture method from 8 to 11-day-old Wistar rats. A PAD in CA1 was elicited by stimulating the stratum radiatum with an intensity of two times that required to elicit a maximal population spike. The effects of carbamazepine, phenytoin, valproic acid, phenobarbital, diazepam, and ethosuximide on the duration and on frequency properties of PADs and the tonic-like and clonic-like subdivisions of PADs were determined, and as a measure of the AEDs potency half maximal effective concentration (EC(50) ) values were calculated from concentration-response curves. KEY FINDING: Carbamazepine, phenytoin, valproic acid, phenobarbital, and diazepam reduced the durations of PADs and tonic-like and clonic-like subdivisions of PADs. The effects were concentration dependent and reversible. Ethosuximide was ineffective. The effects on subdivisions of PADs differed between AEDs. Carbamazepine and phenytoin shortened the tonic-like and clonic-like subdivisions at similar proportions, whereas phenobarbital, diazepam, and valproic acid preferentially shortened the clonic-like subdivision. Diazepam at low concentrations increased the duration of tonic-like subdivisions, an effect not seen with the other AEDs. The suppressive effects of AEDs on frequency properties of tonic-like and clonic-like subdivisions were variable and observed only at higher concentrations. SIGNIFICANCE: Carbamazepine and phenytoin were more effective in the PAD test in OHSCs than in the maximal electroshock and kindled seizures tests. The effectiveness of phenobarbital, diazepam, and valproic acid in the PAD test matched their effectiveness in the MES test and-with the exception of valproic acid and diazepam-in kindled seizures tests. Valproic acid was less effective in OHSCs than in the kindled seizure tests, and diazepam was more (generalized seizures) or less (focal seizures and afterdischarge durations) effective in this animal model than in OHSCs. We conclude that the PAD test in OHSCs is a suitable in vitro model of evoked seizures. The model could serve as an initial screen to identify the most promising leads for further evaluation and characterization in in vivo models of efficacy and toxicity.

Age- and region-specific effects of anticonvulsants and bumetanide on 4-aminopyridine-induced seizure-like events in immature rat hippocampal-entorhinal cortex slices.

PURPOSE: Seizure-like events (SLEs) induced by 4-aminopyridine in rat organotypic slices cultures, which are prepared early after birth, are resistant to standard antiepileptic drugs. In this study we tested the hypothesis that pharmacoresistance may be an intrinsic property of the immature brain. METHODS: Frequently recurring SLEs presumably representing status epilepticus were induced by 4-aminopyridine in acute rat hippocampal-entorhinal cortex slices obtained from postnatal day 3-19 (P3-P19), and the effects of carbamazepine, phenytoin, valproic acid, and phenobarbital were examined. In addition, bumetanide was tested, which blocks the Na(+) -K(+) -2Cl(-) (NKCC1) cotransporter, and also acetazolamide, which blocks the carbonic anhydrase and thereby the accumulation of bicarbonate inside neurons. RESULTS: The efficacy of all antiepileptic drugs in blocking SLEs was dependent on postnatal age, with low efficacy in P3-P5 slices. Antiepileptic drugs suppressed SLEs more readily in the medial entorhinal cortex (ECm) than in the CA3. In P3-P5 slices, valproic acid and phenobarbital increased both tonic and clonic seizure-like activities in the CA3, whereas phenytoin and carbamazepine blocked tonic-like but prolonged clonic-like activity. In P3-P5 slices, bumetanide often blocked SLEs in the CA3, but was not as effective in the ECm. Like with other antiepileptic drugs, the seizure-suppressing effects of acetazolamide increased with postnatal age. CONCLUSION: We conclude that pharmacoresistance may be inherent to very immature tissue and suggest that expression of the NKCC1 cotransporter might contribute to pharmacoresistance.

Endogenous nitric oxide is a key promoting factor for initiation of seizure-like events in hippocampal and entorhinal cortex slices.

Nitric oxide (NO) modulates synaptic transmission, and its level is elevated during epileptic activity in animal models of epilepsy. However, the role of NO for development and maintenance of epileptic activity is controversial. We studied this aspect in rat organotypic hippocampal slice cultures and acute hippocampal-entorhinal cortex slices from wild-type and neuronal NO synthase (nNOS) knock-out mice combining electrophysiological and fluorescence imaging techniques. Slice cultures contained nNOS-positive neurons and an elaborated network of nNOS-positive fibers. Lowering of extracellular Mg(2+) concentration led to development of epileptiform activity and increased NO formation as revealed by NO-selective probes, 4-amino-5-methylamino-2',7'-difluorofluorescein and 1,2-diaminoanthraquinone sulfate. NO deprivation by NOS inhibitors and NO scavengers caused depression of both EPSCs and IPSCs and prevented initiation of seizure-like events (SLEs) in 75% of slice cultures and 100% of hippocampal-entorhinal cortex slices. This effect was independent of the guanylyl cyclase/cGMP pathway. Suppression of SLE initiation in acute slices from mice was achieved by both the broad-spectrum NOS inhibitor N-methyl-L-arginine acetate and the nNOS-selective inhibitor 7-nitroindazole, whereas inhibition of inducible NOS by aminoguanidine was ineffective, suggesting that nNOS activity was crucial for SLE initiation. Additional evidence was obtained from knock-out animals because SLEs developed in a significantly lower percentage of slices from nNOS(-/-) mice and showed different characteristics, such as prolongation of onset latency and higher variability of SLE intervals. We conclude that enhancement of synaptic transmission by NO under epileptic conditions represents a positive feedback mechanism for the initiation of seizure-like events.

In search of models of pharmacoresistant epilepsy.

Clinically available anticonvulsant drugs fail to control seizures in approximately 30% of epileptic patients. If hippocampal sclerosis is combined with focal dysplasia or similar developmental alterations, the likelihood of incomplete seizure control may reach >90%. Because only a minority of epilepsy patients benefit from epilepsy surgery, we need more research into the mechanisms of drug refractoriness. In this review we analyze different approaches to study pharmacoresistance and underlying mechanisms using in vitro models. Epileptiform discharges after prolonged application of low Mg(2+) artificial cerebrospinal fluid (ACSF) or 4-aminopyridine (4-AP), or combined application of these convulsants with bicuculline in acute hippocampal-entorhinal cortex slices reveal pharmacoresistance and point to loss of gamma-aminobutyric acid (GABA)ergic function, in part due to reduced delivery of GABA from presynaptic terminals. Interestingly, epileptiform activity in immature tissue (organotypic hippocampal slice cultures and acute intact hippocampus) is immediately resistant to available antiepileptic drugs, and preliminary evidence points to a role of alterations in Cl(-) homeostasis. Seizure-like events can also be induced in dissected tissues from human epileptic patients. Future research on human tissue may provide useful information for understanding the mechanisms underlying pharmacoresistance.

Gamma oscillations and spontaneous network activity in the hippocampus are highly sensitive to decreases in pO2 and concomitant changes in mitochondrial redox state.

Gamma oscillations have been implicated in higher cognitive processes and might critically depend on proper mitochondrial function. Using electrophysiology, oxygen sensor microelectrode, and imaging techniques, we investigated the interactions of neuronal activity, interstitial pO2, and mitochondrial redox state [NAD(P)H and FAD (flavin adenine dinucleotide) fluorescence] in the CA3 subfield of organotypic hippocampal slice cultures. We find that gamma oscillations and spontaneous network activity decrease significantly at pO2 levels that do not affect neuronal population responses as elicited by moderate electrical stimuli. Moreover, pO2 and mitochondrial redox states are tightly coupled, and electrical stimuli reveal transient alterations of redox responses when pO2 decreases within the normoxic range. Finally, evoked redox responses are distinct in somatic and synaptic neuronal compartments and show different sensitivity to changes in pO2. We conclude that the threshold of interstitial pO2 for robust CA3 network activities and required mitochondrial function is clearly above the "critical" value, which causes spreading depression as a result of generalized energy failure. Our study highlights the importance of a functional understanding of mitochondria and their implications on activities of individual neurons and neuronal networks.

Independent positioning of microelectrodes for multisite recordings in vitro.

A robust and easy to handle, inexpensive multisite recording system is described which allows independent positioning of several microelectrodes with high precision axial movement of each electrode. The basic units of the system are a manually operated manipulator for left-right and up-down movement and a micrometer for positioning in the direction of the micrometer axis. The micrometer is actuated with a DC-motor operated by a stand-alone controller module allowing for remote control of the motor in either step mode or continuous mode. The multisite recording system has been proven to allow stable simultaneous recordings of single unit and population activities, extracellular ion concentrations and intracellular potentials in organotypic hippocampal slice cultures (OHSCs) of rat.

Lack of Exuberance in Clustered Intrinsic Connections in the Striate Cortex of One-month-old Kitten.

The topographies of the intrinsic tangential connections in layers II - IVab of one-month-old and adult cat's striate cortex were compared by plotting the distribution of retrogradely labelled neurons following injection of small amounts of wheat-germ agglutinin - horseradish peroxidase. In both cortices, the tracer reveals the characteristic cluster-like arrangement of intrinsic tangential connections. No differences were found between the two age groups in terms of either the numbers of cell clusters, their periodicities or their profiles, nor was there any difference in the lateral extent of the tangential connections. We conclude that one month postnatally, tangentially projecting intrinsic networks in the upper layers of the cat striate cortex lack exuberance and have an adult-like topography.

Network activity in hippocampal slice cultures revealed by long-term in vitro recordings.

Organotypic hippocampal slice cultures (OHSCs) are widely used for anatomical, molecular and electrophysiological studies of the development of neuronal networks. Electrophysiological recordings are usually limited to a single time point during development, and recording conditions differ greatly based on culture conditions. Consequently, little is known about the maturation of neuronal network activity in vitro. Here, we describe a simple method that allows long-term electrophysiological recordings during culture maintenance in a CO2 incubator. We compared the occurrence of spontaneous network activity, including epileptiform activity, in OHSCs (maintained in Neurobasal/B27 serum-free medium) prepared at different postnatal days and investigated the effects of changes in osmolality and pH. Recordings over 48 h revealed spontaneous network activity culminating in seizure-like events (SLEs) in 65.4% of the OHSCs (n=78). SLE incidence peaked during the first 6h following implantation of the microelectrodes and a secondary increase in SLE-incidence began after 9h of recording and averaged 2.65SLEs/h. The initial peak was likely initiated by transient alkalosis induced by the low pCO2 during the positioning of the electrodes, whereas successive changes in the composition of the culture medium might explain the secondary increase in SLE incidence. Notably, changes in osmolality had no effect on SLE induction. In conclusion, long-term recordings in OHSCs will help to reveal changes in spontaneous network activity during maturation. The extent to which the axonal reorganization known to occur in OHSCs contributes to the susceptibility to epileptogenesis remains to be determined.

Drug refractoriness of epileptiform activity in organotypic hippocampal slice cultures depends on the mode of provocation.

We previously reported that low Mg(2+) or 4-aminopyridine induced seizure-like events in organotypic hippocampal slice cultures (OHSCs) were resistant to standard antiepileptic drugs (Albus et al., 2008). We now show that high-frequency electrical stimulation induced ictal-like afterdischarges in OHSCs are effectively suppressed by the standard antiepileptic drugs phenytoin and carbamazepine. Thus, a single OHSC can express both pharmacoresistant and pharmacosensitive epileptiform activities.

Effects of gamma-aminobutyric acid (GABA) agonists and a GABA uptake inhibitor on pharmacoresistant seizure like events in organotypic hippocampal slice cultures.

PURPOSE: Seizure like events (SLEs) induced by low magnesium or 4-aminopyridine in organotypic hippocampal slice cultures (OHSCs) are resistant to standard antiepileptic drugs including phenobarbital, and 1,4-benzodiazepines [Albus, K., Wahab, A., Heinemann, U., 2008. Standard antiepileptic drugs fail to block epileptiform activity in rat organotypic hippocampal slice cultures. Br. J. Pharmacol. 154, 709-724]. The present study was undertaken in order to test the effects of other compounds on SLEs in OHSCs that enhance GABA-mediated actions. METHODS: Six to 12 days old Wistar rats were used to cultivate OHSCs according to the interface method [Stoppini, L., Buchs, P.A., Muller, D., 1991. A simple method for organotypic cultures of nervous tissue. J. Neurosci. Methods 37, 173-182]. Neuronal activity and extracellular potassium concentration were recorded under submerged conditions. SLEs were induced by lowering the magnesium concentration. The effects of GABA(A) agonists muscimol and isoguvacine, the GABA(B) agonist baclofen, the GABA uptake blocker nipecotic acid and the neurosteroid alfaxalone on induction and ongoing SLEs were analyzed. RESULTS: Low magnesium induced SLEs were dose dependently suppressed by the GABA(A) receptor agonists muscimol, isoguvacine and alfaxalone and by the GABA uptake inhibitor nipecotic acid whereas the GABA(B) receptor agonist baclofen attenuated but did not suppress SLE. DISCUSSION: Our findings demonstrate that in OHSCs GABA has an inhibitory effect on SLEs. Proconvulsant effects of GABA agonists on spontaneous neuronal activity and seizure like activity were never observed. Our findings exclude a possible contribution of impaired/altered GABA-ergic mechanisms based on immaturity of receptors and/or low receptor density to seizure susceptibility and pharmacoresistance in OHSCs.