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Solid pieces of fetal locus coeruleus (LC) or superior cervical ganglion (SCG) were placed into a fimbria-fornix lesion cavity in 6-hydroxydopamine-treated, noradrenaline (NA)-denervated rats. Six to 8 months later, all animals were subjected to electrical kindling stimulations in the hippocampus until they had reached the fully kindled state. Nongrafted lesioned animals showed markedly increased kindling rate which was partly attenuated by LC but not SCG grafts. In both LC- and SCG-grafted animals, dopamine beta-hydroxylase immunocytochemistry demonstrated a high density of graft-derived noradrenergic fibers in the dorsal hippocampus, whereas reinnervation of the ventral hippocampus was much more sparse. Subregional distribution of these fibers within the hippocampus was different in the two grafted groups. Both grafts partly restored basal extracellular NA levels in the hippocampus and reacted to generalized seizures by a significant (two- to threefold) increase of NA release, as measured by intracerebral microdialysis. Our data indicate (i) that seizure activity can regulate transmitter release from noradrenergic neurons in both LC and SCG grafts, (ii) that only fetal LC grafts retard seizure development in kindling, and (iii) that the inability of SCG implants to influence kindling epileptogenesis could be due to a lack of synaptic contacts between the graft-derived ganglionic fibers and host hippocampal neurons.

GABAA receptor alpha 1, beta 3 and gamma 2 subunit mRNA levels have been measured in hippocampus using in situ hybridization, following 1, 10 and 40 seizures produced by rapid kindling stimulations. Major alterations of gene expression were largely confined to the dentate gyrus. One stimulus-induced seizure reduced gamma 2 mRNA levels in the dentate gyrus by 30%. In contrast, mRNA expression increased for alpha 1 in CA1 and CA3 and for beta 3 in CA1 to around 30% above control values. Ten stimulations reduced beta 3 (by 19%) and gamma 2 (by 37%) mRNA expression in the dentate gyrus. No changes were observed in other hippocampal subregions. Forty kindling-induced seizures led to biphasic alterations of subunit mRNA levels in dentate gyrus with only minor changes in CA1-CA3. Up to 4 h after the last seizure mRNA expression for alpha 1 was slightly decreased in dentate gyrus, whereas marked reductions were observed for beta 3 and gamma 2 (by 41% and 48%, respectively). Between 12 and 48 h there were major increases of alpha 1 (by 59%) and gamma 2 (by 35%) mRNA levels but no significant changes of beta 3 mRNA expression. Subunit mRNA levels had returned to control values after 5 days, which argues against a direct involvement of GABAA receptor in kindling-evoked hyperexcitability. The rapid and transient, biphasic changes of GABAA receptor subunits following recurrent seizures could play an important role in stabilizing granule cell excitability, thereby reducing seizure susceptibility. The differential regulation of subunit mRNA levels following seizures suggests a novel mechanism for changing the physiological properties of dentate granule cells through possible GABAA receptor complexes with different subunit composition.

N-methyl-D-aspartate-receptors are implicated in several neuropathological conditions including epilepsy. As a model of complex partial seizures, rapid hippocampal kindling was chosen to investigate changes in the expression of messenger RNAs encoding the N-methyl-D-aspartate-receptor subunits NR1, NR2A and NR2B both during and in the period immediately following the induction of the kindled state. The study demonstrates a cell-specific, time-dependent modulation of the N-methyl-D-aspartate-receptor subunit messenger RNAs almost entirely restricted to the granule cells of the dentate gyrus. In partially kindled animals (10 stimulations), while the NR1 subunit messenger RNA remained unaltered after a period of 2 h, the NR2A and NR2B subunit messenger RNAs were bilaterally reduced in dentate gyrus granule cells by around 50% below control values. In fully kindled animals (40 stimulations), a progressive reduction in NR1 subunit messenger RNA levels in the dentate gyrus was observed, being maximal after 4 h (-67%). At the same time point, NR2A and NR2B transcript levels were transiently increased by 102% and 46% above control values, respectively. These data point to a differential regulation of N-methyl-D-aspartate-receptor subunit messenger RNAs. No alterations were detected in pyramidal cells. Long-term maintenance of the kindled state was not associated with alterations in N-methyl-D-aspartate-receptor subunit messenger RNAs since control levels of messenger RNA were attained by 12 h and persisted for at least five days. The early changes in messenger RNAs described in this study indicate that the expression of N-methyl-D-aspartate-receptor subunits is under independent regulatory control. This phenomenon may contribute to epileptogenesis and to kindling-associated plasticity by mediating a structural reorganization of N-methyl-D-aspartate-receptors, leading to an altered excitability of dentate gyrus granule cells.

Grafts of fetal noradrenaline (NA)-rich locus coeruleus (LC) tissue implanted bilaterally into the hippocampus in 6-hydroxydopamine (6-OHDA) treated, hyperexcitable rats retard seizure development in hippocampal kindling. We report that bilateral intrahippocampal LC grafts failed to affect the severity of seizures when implanted in 6-OHDA-treated, already kindled rats. Furthermore, a noradrenergic hyperinnervation of the hippocampus, produced by grafting fetal LC tissue into the intact brain, did not influence hippocampal kindling development. Kindling leads to long-term reduction in basal hippocampal NA release from the intrinsic locus coeruleus system as monitored using intracerebral microdialysis. This deficit could be reversed by intrahippocampal LC grafts implanted into previously kindled rats. However, these unilateral grafts had no effect on the severity of the kindled convulsions despite a noradrenergic hyperinnervation of the hippocampus around the grafts. In conclusion, these data show a distinct functional specificity of intrahippocampal LC grafts in the kindling model of epilepsy. Similar to the intrinsic noradrenergic system, the grafted neurons have a major inhibitory influence on seizure development but are ineffective against established convulsions.

Levels of messenger RNA for nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and the tyrosine kinase receptors trkA, trkB and trkC have been studied using in situ hybridization in the rat brain 2 h and four weeks after kindling-induced seizures. Epileptiform activity evoked by hippocampal stimulation and exceeding 70 s lead to a concomitant and transient increase of brain- derived neurotrophic factor, nerve growth factor, trkB and trkC messenger RNA expression in dentate granule cells after both focal and generalized seizures. Brain-derived neurotrophic factor messenger RNA levels were also increased bilaterally in the CA1-CA3 regions, amygdala and the piriform, entorhinal, perirhinal, retrosplenial and temporal cortices after generalized seizures. The magnitude of the increases was similar throughout the development of kindling and in the fully kindled brain. No changes of trkA messenger RNA were observed. In amygdalar kindling, elevated brain-derived neurotrophic factor messenger RNA levels developed more rapidly in the amygdala-piriform cortex than after stimulation in the hippocampus but changes in the hippocampal formation were only seen in few animals. Intraventricular 6-hydroxydopamine or a bilateral fimbria-fornix lesion did not alter basal expression or seizure-evoked changes in messenger RNA levels for neurotrophins or trk receptors but increased the number of animals exhibiting elevated levels after the first stimulation, probably due to a prolongation of seizure activity. Both in sham-operated and fimbria-fornix-lesioned rats seizure activity caused a marked reduction of neurotrophin-3 messenger RNA levels in dentate granule cells. The results indicate that activation of the brain-derived neurotrophic factor gene, at least in dentate granule cells, is an "all-or-none" type of response and dependent on the duration but not the severity of seizures or the stage of kindling epileptogenesis. Changes in brain-derived neurotrophic factor, nerve growth factor, neurotrophin-3 and trkB and trkC were observed concomitantly in the dentate gyrus, which suggests that seizure activity sets in motion a cascade of genomic events possibly mediated via a common mechanism. Since altered messenger RNA levels outside hippocampus were detected only for brain-derived neurotrophic factor, neurotrophin and trk gene expression in these regions seems to be regulated differently.

Nerve growth factor (NGF) protein levels were determined in various forebrain regions using a two-site immunoassay following kindling-induced seizures. In the dentate gyrus the NGF content was significantly elevated 7 days after the last seizure (to 152% of control). In the piriform and parietal cortices, maximal increases were seen at 12 h (to 261% of control) and at 24 h (to 169% of control), respectively, and the NGF content was then normalized at 7 days. The increased production of NGF might be a protective response or could be involved in plastic changes underlying kindling epileptogenesis.

Kindling, induced by repeated subconvulsive electrical or chemical stimulations leads to progressive and permanent amplification of seizure activity, culminating in generalized seizures. We report that kindling induced by electrical stimulation in the ventral hippocampus leads to a marked and transient increase in mRNA for NGF and BDNF in the dentate gyrus, the parietal cortex, and the piriform cortex. BDNF mRNA increased also in the pyramidal layer of hippocampus and in the amygdaloid complex. No change was seen in the level of HDNF/NT-3 mRNA. The increased expression of NGF and BDNF mRNAs was not influenced by pretreatment with the NMDA receptor antagonist MK801, but was partially blocked by the quisqualate, AMPA receptor antagonist NBQX. The presumed subsequent increase of the trophic factors themselves may be important for kindling-associated plasticity in specific neuronal systems in the hippocampus, which could promote hyperexcitability and contribute to the development of epileptic syndromes.

The in vivo microdialysis technique was used to monitor steady-state noradrenaline release in the rat hippocampus after hippocampal kindling. At 8 weeks after the last seizure, the noradrenaline release was reduced by 62% in the stimulated hippocampus in kindled animals as compared to non-kindled rats. The reduction was not due to the repeated handling of the animals as assessed in a separate experiment. The results suggest a decrease of inhibitory noradrenergic influence at the primary kindling site, which could play a role in kindling epileptogenesis.

Intrahippocampal cell suspension grafts, prepared from the locus coeruleus region of rat fetuses, have previously been shown to retard seizure development in rats made hypersensitive to hippocampal kindling by a lesion of the forebrain noradrenergic system. The objective of the present study was to provide evidence that the seizure-suppressant effect elicited by the grafts is mediated via noradrenergic mechanisms. Two groups of rats received 6-hydroxydopamine in the lateral ventricle and then bilateral intrahippocampal locus coeruleus grafts. After 3 months, the grafted animals and a group of normal rats were subjected to hippocampal kindling. One group of grafted animals and the normal rats were injected intraperitoneally with the alpha-2 adrenergic receptor blocker idazoxan before each kindling stimulation. The other grafted rats received vehicle injections. The development of seizures was significantly faster in the grafted and normal rats that had been given idazoxan than in the grafted rats that had not been subjected to alpha-2 receptor blockade. Our data suggest that the seizure-suppressant action exerted by grafts of fetal locus coeruleus in hippocampal kindling is mediated via noradrenergic mechanisms, most likely via activation of postsynaptic alpha-2 adrenoreceptors.

The intracerebral microdialysis technique has been used to monitor extracellular levels of noradrenaline, 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in the rat hippocampus in vivo in response to focal and generalized seizures induced by hippocampal kindling stimulation. In fully kindled animals a stimulus-induced generalized seizure gave rise to a three-fold increase of noradrenaline levels in the stimulated hippocampus as compared to baseline levels (15-min samples). The maximal increase of noradrenaline levels occurred within the first minutes after onset of seizure activity, as assessed in 2-min sample fractions with the noradrenaline uptake blocker desipramine added to the perfusion medium. After the peak increase, the noradrenaline levels tapered off, reaching baseline after 8-10 min. In 6-hydroxydopamine-treated animals, baseline noradrenaline levels were markedly reduced and there was no significant increase in noradrenaline release in response to a generalized seizure. These data support the hypothesis that the high extracellular levels of noradrenaline measured in seizures are of neuronal origin. There were no significant changes in extracellular 5-hydroxytryptamine or 5-hydroxyindoleacetic acid levels after a generalized seizure. In non-kindled animals the steady state noradrenaline levels during uptake blockade were two-three times higher than in the kindled rats. However, the peak noradrenaline levels measured in both hippocampi after the first two electrical kindling stimulations giving rise to focal epileptiform activity (afterdischarge) were similar to those observed in the kindled animals in response to generalized seizures. The increase of noradrenaline release in the non-kindled animals was significantly correlated to the duration of afterdischarge. In conclusion, the present study demonstrates the usefulness of the intracerebral dialysis technique for monitoring noradrenaline, 5-hydroxytryptamine and 5-hydroxyindoleacetic acid release during seizures. The results indicate that both focal and generalized hippocampal seizures evoked by electrical kindling stimulation lead to a marked increase of transmitter release from noradrenergic but not from serotonergic neurons in the hippocampus. The ability of the noradrenergic system to respond by increased transmitter release to epileptic seizures is thus retained also in the kindled state.

Rats were subjected to hippocampal kindling with short interstimulus intervals (5 min), so-called rapid kindling. Severe depletion of forebrain noradrenaline (NA) with intraventricular 6-hydroxydopamine or blockade of alpha 2 adrenergic receptors with idazoxan markedly reduced the number of stimulations needed to induce the first severe limbic seizure and increased the total number of such seizures during 40 stimulations. Intracerebral microdialysis demonstrated a 3-fold increase of extracellular NA levels in response to the first kindling stimulations in both the stimulated and the non-stimulated hippocampus. The NA levels then gradually tapered off reaching baseline levels after 14 stimulations. We conclude that central noradrenergic neurons exert a powerful suppressant action, most probably mediated via an alpha 2 adrenergic receptor, on seizure development in rapid hippocampal kindling.