W. Kamphuis

Kindling induced changes in parvalbumin immunoreactivity in rat hippocampus and its relation to long-term decrease in GABA-immunoreactivity

The immunoreactivity of parvalbumin (PV), a Ca2+-binding protein present in a subpopulation of interneurons, was studied in the hippocampal CA1 region during kindling epileptogenesis, induced by tetanic stimulation of the Schaffer collateral/commissural fibers. PV-immunoreactivity was increased in comparison to controls after 13 afterdischarges and after the induction of generalized seizures. A quantification of the number of PV-immunoreactive somata showed an increase of 20% in both stages of kindling. This level had returned to baseline level 31 days after the last seizure. These results imply that changes in PV-immunoreactivity are related to seizure activity rather than to the long-term increase in seizure sensitivity in kindled animals. Co-localization study in controls showed that 32% of PV-immunoreactive somata were also immunopositive for GABA. A colocalization study in stratum oriens and pyramidale on the stimulated side of kindled animals 31 days after the last generalized seizure showed neither a reduction in the number of PV-immunoreactive somata nor in the number of GABA-immunopositive cell bodies that co-localized with PV. In contrast, the number of GABA-immunoreactive somata that did not co-localize with PV was reduced by 50%. It has been shown that a large influx of Ca2+ plays a crucial role in epileptogenesis. Here we demonstrate that the presence of the calcium-binding protein parvalbumin seems to exert a protective effect against the process that leads to a decrease in GABA content.

Changes in local evoked potentials in the rat hippocampus (CA1) during kindling epileptogenesis.

Electrophysiological changes occurring during the development of a kindled focus in the CA1 region of the rat hippocampus were studied in vivo. The most conspicuous changes of the field potentials (EPs) recorded from the stratum radiatum to stimulation of the Schaffer collaterals were the following: (1) a progressive decrease of the slope of the decaying phase of the EPs which was significantly different (P less than 0.02) from controls from sessions 8-12 onwards; (2) in the EPs recorded from the stratum pyramidale/oriens a population spike emerged from sessions 7-10 onwards; the ratio amplitude of the population spike/slope of the local EP increased progressively from session 8 onwards until a saturation level was reached; and (3) a progressive attenuation of paired-pulse depression; this decreased linearly with kindling session from session 1 up to session 11 (r = 0.97, P less than 0.01) and thereafter stabilized. These results are interpreted as due to a progressive imbalance between excitatory and inhibitory processes resulting in a decrease of inhibitory control in CA1 accompanied by a decrease in threshold of pyramidal neurons.

Hippocampal kindling increases the expression of glutamate receptor-A flip and -B flip mRNA in dentate granule cells

The level of the mRNAs encoding the AMPA-selective glutamate receptors-A and -B, alternatively splice variants, Flip and Flop, was studied by in situ hybridization in the brains of rats kindled by Schaffer collateral/commissural-fiber stimulation. In comparison to control animals, the expression level of the Flip variant of both GluR-A and GluR-B mRNAs was bilaterally enhanced in the dentate granule neurons of kindled animals 24 h after last-generalized seizure, whereas no obvious alterations were observed in the GluR-A Flop and GluR-B Flop mRNA variants. In kindled animals, studied 1 month after the last seizure, GluR-A Flip and GluR-B Flip mRNA had returned to control levels. We suggest that these changes may result in an enhanced glutamate receptor sensitivity in the fascia dentata during kindling.

A long-lasting decrease in the inhibitory effect of GABA on glutamate responses of hippocampal pyramidal neurons induced by kindling epileptogenesis

Experiments were carried out to test whether changes in the sensitivity of hippocampal pyramidal neurons to the neurotransmitters glutamate, GABA and noradrenaline may be associated with the establishment of an epileptogenic focus induced by kindling. The effects of iontophoretically applied neurotransmitters on the firing rate of single units were quantified in the rat hippocampal CA1 area in kindled and control animals. Kindling was induced by electrical tetanic stimulation of the Schaffer collateral/commissural fibers. Firing was evoked by local glutamate iontophoresis while simultaneous GABA or noradrenaline application suppressed this response. A significant reduction of the GABAergic inhibitory action on the firing rate in kindled animals studied around four or around 42 days after the last convulsion was found. In the same neurons, the suppressive effect of noradrenaline was not different from controls. The neurons of kindled animals, investigated around four days after the last seizure, had a reduced sensitivity for glutamate; more glutamate ejection current was needed to evoke firing or to evoke the maximum firing rate. In contrast, the responsiveness for glutamate was significantly increased long-term after the last convulsion. These findings demonstrate that hippocampal Schaffer collateral kindling is associated with a long-lasting reduced effectiveness of the GABA-mediated response on glutamate-evoked firing in CA1.

Decrease in number of hippocampal gamma-aminobutyric acid (GABA) immunoreactive cells in the rat kindling model of epilepsy

SummaryDaily repeated tetanic electrical stimulation (kindling) of hippocampus or other brain structures leads to progressive increase in epileptiform activity. Since kindling may involve changes in the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), the distribution of this amino acid was studied. A significant decrease in number of GABA immunoreactive positive cell bodies in stimulated CA1 region of the rat hippocampus compared to the contralateral side was found.

Current source density of sustained potential shifts associated with electrographic seizures and with spreading depression in rat hippocampus

The membrane currents responsible for the sustained potential shifts associated with electrographic seizures and with spreading depression in hippocampus were studied in the anesthetized rat. Probes incorporating 16 sensors in a straight line, spaced at 150-microns distances, were recording the potential changes with DC-coupled amplifiers in CA1 and dentate gyrus (DG) of one hemisphere. Seizures and spreading depression were provoked by repetitive stimulation of different afferent pathways. Seizures always began in DG before CA1, regardless of the pathway stimulated. Tonic seizures were associated with a sustained negative potential shift that was largest in the cell body layers. Current source density was computed from these recordings and confirmed the presence of a current sink limited to the cell body layer throughout the duration of electrographic seizures. Spreading depression was associated with a very large sink located in the layer of apical dendrites, maximal among the proximal segment of dendrites, to which the cell body layer served as a source. We conclude that seizures are associated with an inward current in neuron cell bodies, probably flowing through membrane channels of as yet no know physiological function.

Hippocampal kindling leads to different changes in paired-pulse depression of local evoked field potentials in CA1 area and in fascia dentata

Monosynaptic evoked field potentials (EPs) in response to paired-pulse stimulation (20 ms interval) were recorded in area CA1 and fascia dentata of the same animal in the course of development of a kindled focus in the CA1 region. A significant reduction of paired pulse depression in response to medium and high stimulation intensity was found in CA1. A similar change was found in the fascia dentata in response to medium intensity stimulation of the angular bundle. In contrast, at high intensity, paired pulse depression was enhanced in the fascia dentata in the course of kindling. These results indicate that kindling epileptogenesis is accompanied by regionally different changes in recurrent inhibition: a reduction in CA1 and intensity dependent changes in fascia dentata.

Changes in voltage-dependent calcium channel α1-subunit mRNA levels in the kindling model of epileptogenesis

The establishment of a focus of epileptiform activity in the hippocampus of the rat, using the kindling paradigm, leads to enhanced voltage-dependent calcium conductance of CA1 pyramidal neurones (G.C. Faas, M. Vreugdenhil, W.J. Wadman, Calcium currents in pyramidal CA1 neurones in vitro after kindling epileptogenesis in the hippocampus of the rat, Neuroscience 75 (1996) 57-67; M. Vreugdenhil, W.J. Wadman, Kindling-induced long-lasting enhancement of calcium in hippocampal CA1 area of the rat: relation to calcium-dependent inactivation, Neuroscience 59 (1994) 105-114). Using semi-quantitative in situ hybridization techniques, we investigated whether these changes were associated with an altered expression of the genes that encode for the alpha1A-E-subunits of the voltage-dependent calcium channels (VDCC). Kindling epileptogenesis was induced in rats that received an electrical tetanic stimulation of the Schaffer collateral/commissural fibre pathway in the hippocampus twice daily. Two groups of rats were studied before the appearance of generalized seizures, one group after at least 5 generalized seizures (fully kindled) and one group was investigated at long-term (28 days) after the last seizure. During the initial stages of epileptogenesis, the alpha1A-, alpha1D- and alpha1E-subunit mRNA levels were significantly increased in the different hippocampal subareas in comparison to the levels in control animals. In contrast, alpha1B-subunit gene expression decreased in the CA area and dentate gyrus. No significant change was observed in the alpha1C-I and alpha1C-II expression. At the fully kindled stage, the only significant change was an up-regulation of the alpha1B-subunit mRNA levels in the CA3 area, 24 h after the last seizure. No change in VDCC alpha1-subunit gene expression was found in animals investigated long-term after the establishment of the fully kindled state. Thus, the VDCC alpha1-subunit gene expression is altered in a subclass-specific manner during the early stages of kindling and may play a role in the establishment of a kindled focus, possibly caused by an alteration of the population of VDCCs involved in neurotransmitter release. The absence of long-lasting changes suggests that the maintenance of a kindled focus is not due to persisting alterations in VDCC alpha1 mRNA levels.

Kindling increases the K(+)-evoked Ca2(+)-dependent release of endogenous GABA in area CA1 of rat hippocampus.

The release of endogenous amino acids from hippocampal CA1 subslices under basal conditions and the release evoked by high potassium (50 mM K+) depolarization was studied during kindling epileptogenesis. Emphasis was put on the release of the amino acid neurotransmitters gamma-aminobutyric acid (GABA) and glutamate. Kindling was induced by tetanic stimulation of the Schaffer-collaterals/commissural fibers of the dorsal hippocampus of the rat. The calcium-dependent GABA release in the presence of high K+ was significantly increased (40-46%) in fully kindled animals, 24 h after the last seizure, in comparison to controls. At long-term, 28 days after the last seizure, the calcium-dependent GABA release was still significantly increased (45-49%). An increased release of GABA in kindled animals was still found when GABA uptake was blocked by nipecotic acid. In contrast, no significant alterations were encountered in the basal or high potassium induced release of the excitatory amino acids aspartate and glutamate. These results suggest that kindling epileptogenesis is accompanied by a specific and long-lasting enhancement of GABA exocytosis which may lead to a desensitization of the GABA receptor, and thus determine the increase of seizure sensitivity.

Decrease in GABA immunoreactivity and alteration of GABA metabolism after kindling in the rat hippocampus

SummaryThe kindling model of epilepsy, induced by tetanic stimulation of Schaffer collateral/commissural fibers, was studied in the rat hippocampus. Gamma-aminobutyric acid immunoreactivity was used to quantify the number of GABA-immunoreactive somata per mm2 in CA1 region, 28 days after the last generalized seizure. Comparison of the numbers obtained from kindled animals with those from controls, showed a significant decrease (18%) on the ipsilateral stimulated side but none on the contralateral side. In control rats injection of the GABA-transaminase inhibitor, amino oxyacetic acid (AOAA), led to a 46% increase in the number of cell somata immunoreactive for GABA. This probably results from an accumulation of GABA, reflecting GABA synthesis by glutamate decarboxylase (GAD) activity, in somata of interneurons that had initially a GABA content below the immunocytochemical detection threshold. In kindled rats, 31 days after the last seizure, the number of GABA-immunoreactive cells that could be observed after AOAA-treatment was significantly lower (35% ipsilateral and 25% contralateral) when compared to AOAA-treated controls. This suggests that in kindled animals a GAD dependent increase in GABA content did not take place in a subpopulation of interneurons. The observations for kindled rats are interpreted as a long-term decrease in GABA content and as an alteration in GABA turnover in a subpopulation of interneuron somata, the latter possibly due to a decrease in GAD activity. The long-term enhanced seizure sensitivity, characteristic for kindled animals, may be due to a decreased GABAergic inhibitory control of the neuronal circuitry in the CA1 region of the hippocampus.