Effects of cholinergic denervation on seizure development and neurotrophin messenger RNA regulation in rapid hippocampal kindling.

Intraventricular 192 IgG-saporin was used to induce a selective lesion of basal forebrain cholinergic neurons in rats. When subjected to 40 rapid hippocampal kindling stimulations with 5-min intervals, these animals exhibited increased number of generalized seizures and a higher mean seizure grade in response to the first five stimulations, and required fewer stimuli to develop focal behavioural seizures, as compared to non-lesioned rats. In contrast, both groups showed similarly enhanced responsiveness when test stimulated four weeks later. Using in situ hybridization, cholinergic denervation was found to cause a significant decrease of basal brain-derived neurotrophic factor messenger RNA levels in the hippocampal formation and piriform cortex, whereas gene expression for nerve growth factor, neurotrophin-3, and TrkB and TrkC was unchanged. Four weeks after rapid kindling stimulations, basal levels of brain-derived neurotrophic factor messenger RNA in the dentate granule cells were restored to normal in the lesioned rats, whereas neurotrophin-3 messenger RNA levels were decreased. No differences in the seizure-evoked levels of neurotrophin and Trk messenger RNAs were detected, except in the dentate granule cell layer, which had significantly higher brain-derived neurotrophic factor messenger RNA expression in the lesioned animals at 2 h. In conclusion, the basal forebrain cholinergic system (i) dampens the severity of recurring seizures induced by rapid hippocampal kindling stimulations, but has no effect on the subsequent delayed phase of epileptogenesis; and (ii) exerts a tonic stimulation of basal brain-derived neurotrophic factor messenger RNA levels in the hippocampal formation and piriform cortex. The findings also indicate that the cholinergic lesion does not affect neurotrophin and Trk gene expression after recurring seizures, and that the kindling process leads to long-term changes in basal brain-derived neurotrophic factor and neurotrophin-3 messenger RNA levels in the denervated animals.

Septal cholinergic neurons suppress seizure development in hippocampal kindling in rats: comparison with noradrenergic neurons.

Widespread lesions of forebrain cholinergic or noradrenergic projections by intraventricular administration of 192 IgG-saporin or 6-hydroxydopamine, respectively, accelerate kindling epileptogenesis. Here we demonstrate both quantitative and qualitative differences between the two lesions in their effects on hippocampal kindling in rats. Epileptogenesis was significantly faster after noradrenergic as compared to cholinergic denervation, and when both lesions were combined, kindling development resembled that in animals with 6-hydroxydopamine lesion alone. Furthermore, whereas the 192 IgG-saporin lesion promoted the development only of the early stages of kindling, administration of 6-hydroxydopamine or both neurotoxins accelerated the late stages also. To investigate the contribution of different subparts of the basal forebrain cholinergic system to its seizure-suppressant action in hippocampal kindling, 192 IgG-saporin was injected into medial septum/vertical limb of the diagonal band of Broca or nucleus basalis magnocellularis, leading to selective hippocampal or cortical cholinergic deafferentation, respectively. The denervation of the hippocampus facilitated kindling similar to the extensive lesion caused by intraventricular 192 IgG-saporin, whereas the cortical lesion had no effect. These results indicate that although both noradrenergic and cholinergic projections to the forebrain exert powerful inhibitory effects on hippocampal kindling epileptogenesis, the action of the cholinergic system is less pronounced and occurs specifically prior to seizure generalization. In contrast, noradrenergic neurons inhibit the development of both focal and generalized seizures. The septo-hippocampal neurons are responsible for the antiepileptogenic effect of the cholinergic system in hippocampal kindling, whereas the cortical projection is not significantly involved. Conversely, we have previously shown [Ferencz I. et al. (2000) Eur. J. Neurosci., 12, 2107-2116] that seizure-suppression in amygdala kindling is exerted through the cortical and not the hippocampal cholinergic projection. This shows that, depending on the location of the primary epileptic focus, i.e. the site of stimulation, basal forebrain cholinergic neurons operate through different subsystems to counteract seizure development in kindling.

Immunolesioning of basal forebrain cholinergic neurons facilitates hippocampal kindling and perturbs neurotrophin messenger RNA regulation.

The immunotoxin 192 IgG-saporin induces an efficient and specific lesion of low-affinity nerve growth factor receptor-bearing cholinergic neurons in the basal forebrain. Intraventricular injection of 192 IgG-saporin, which caused a complete loss of cholinergic afferents to the hippocampus and neocortex and a partial denervation of amygdala and piriform cortex, was found to markedly facilitate the initial stages of seizure development in hippocampal kindling. In contrast, the progression of kindling process from focal to generalized seizures was not affected. In situ hybridization demonstrated that basal levels of brain-derived neutrotrophic factor messenger RNA in the hippocampal formation and piriform cortex were significantly decreased by the lesion, which also attenuated the seizure-induced increase of brain-derived neurotrophic factor messenger RNA expression in the hippocampus and frontal cortex. In the dentate gyrus, the 192 IgG-saporin lesion selectively reduced the upregulation of messenger RNAs for brain-derived neurotrophic factor exons I and III after a generalized seizure, whereas the increase of exon II messenger RNA was unchanged. The lesion abolished the seizure-evoked increase of nerve growth factor and TrkC messenger RNA levels and decrease of neutrophin-3 messenger RNA expression in dentate granule cells, while TrkB messenger RNA levels were not affected. We conclude that the basal forebrain cholinergic system (1) suppresses kindling epileptogenesis in the hippocampus, and (2) enhances both basal and seizure-evoked brain-derived neurotrophic factor synthesis in the hippocampal formation and some cortical areas through a specific pattern of activation of promoters within the brain-derived neurotrophic factor gene.

Delayed kindling development after rapidly recurring seizures: relation to mossy fiber sprouting and neurotrophin, GAP-43 and dynorphin gene expression.

Development of kindling and mossy fiber sprouting, and changes of gene expression were studied after 40 seizures produced during about 3 h by electrical stimulation every 5 min in the ventral hippocampus. As assessed by 5 test stimulations, enhanced responsiveness was present already after 6-24 h but from 1 week post-seizure increased gradually up to 4 weeks without additional stimuli. Sprouting of mossy fibers in the dentate gyrus was demonstrated only at 4 weeks with Timm's staining. In situ hybridization showed a transient increase (maximum at 2 h) of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), TrkB and TrkC mRNA levels and reduction (maximum at 12-24 h) of neurotrophin-3 (NT-3) mRNA expression in dentate granule cells after the seizures. In addition, BDNF mRNA levels were elevated in CA1 and CA3 regions, amygdala and piriform cortex. Marked increases of mRNA for growth-associated protein (GAP-43), with maximum expression at 12-24 h, were observed in dentate granule cells and in amygdala-piriform cortex. Dynorphin mRNA levels showed biphasic changes in dentate granule cells with an increase at 2 h followed by a decrease at 24 h. No long-term alterations of gene expression were observed. These findings indicate that increased responsiveness develops rapidly after recurring seizures but that the kindled state is reached gradually in about 4 weeks. Mossy fiber sprouting occurs in parallel to epileptogenesis and may play a causative role. Short-term changes of neurotrophin and Trk, GAP-43 and dynorphin mRNA levels and the assumed alterations of the corresponding proteins could trigger structural rearrangements underlying kindling but might also contribute to the initial increase of seizure susceptibility.

Ethanol stimulates basal and serotonin-induced formation of [32P]phosphatidic acid in human platelets.

The addition of serotonin to preparations of 32P-labelled human platelets resulted in a time- and dose-dependent hydrolysis of [32P]phosphatidylinositol 4,5-bisphosphate (PIP2) and formation of [32P]phosphatidic acid (PA). This response was inhibited by the serotonin2 receptor antagonist ritanserin, indicating that the stimulation was mediated via the serotonin2 receptor. The addition of 50-150 mM of ethanol prior to stimulation with 10(-5) M serotonin resulted in an increased accumulation of [32P]PA, but had no effect on [32P]PIP2. Ethanol stimulated [32P]PA formation at all serotonin concentrations studied (10(-7)-10(-5) M). Furthermore, in the absence of serotonin, ethanol increased basal [32P]PA formation.

Suppressed kindling epileptogenesis and perturbed BDNF and TrkB gene regulation in NT-3 mutant mice.

In the kindling model of epilepsy, repeated electrical stimulations lead to progressive and permanent intensification of seizure activity. We find that the development of amygdala kindling is markedly retarded in mice heterozygous for a deletion of the neurotrophin-3 (NT-3) gene (NT-3+/- mice). These mice did not reach the fully kindled state (3rd grade 5 seizure) until after 28 +/- 4 days of stimulation compared to 17 +/- 2 days in the wild-type animals. The deficit in the NT-3+/- mice reflected dampening of the progression from focal to generalized seizures. The number of stimulations required to evoke focal (grade 1 and 2) seizures did not differ between the groups, but the NT-3 mutants spent a considerably longer period of time (13 +/- 3 days) than wild-type mice (2 +/- 1 days) in grade 2 seizures. As assessed by test stimulation 4-12 weeks after the 10th grade 5 seizure, kindling was maintained in the NT-3 mutants. In situ hybridization showed 30% reduction of basal NT-3 mRNA levels and lack of upregulation of TrkC mRNA expression at 2 h after a generalized seizure in dentate granule cells of the NT-3+/- mice, whereas the seizure-evoked increase in brain-derived neurotrophic factor (BDNF) and TrkB mRNA levels was enhanced. These results indicate that endogenous NT-3 levels can influence the rate of epileptogenesis, and suggest a link between NT-3 and BDNF gene regulation in dentate granule cells.

Suppression of kindling epileptogenesis in rats by intrahippocampal cholinergic grafts.

Selective immunolesioning of the basal forebrain cholinergic system by 192 IgG-saporin, which leads to a dramatic loss of the cholinergic innervation in cortical and hippocampal regions, facilitates the development of hippocampal kindling in rats. The aim of the present study was to explore whether grafted cholinergic neurones are able to reverse the lesion-induced increase of seizure susceptibility. Intraventricular 192 IgG-saporin was administered to rats which 3 weeks later were implanted with rat embryonic, acetylcholine-rich septal-diagonal band tissue ('cholinergic grafts') or cortical tissue/vehicle ('sham grafts') bilaterally into the hippocampal formation. After 3 months, the grafted animals as well as non-lesioned control rats were subjected to daily hippocampal kindling stimulations. In the animals with cholinergic grafts, which had reinnervated the hippocampus and dentate gyrus bilaterally, there was a marked suppression of the development of seizures as compared with the hyperexcitable, sham-grafted rats. This effect was significantly correlated to the density of the graft-derived cholinergic innervation of the host hippocampal formation. The kindling rate in the rats with cholinergic grafts was similar to that in non-lesioned controls. These results provide further evidence that the intrinsic basal forebrain cholinergic system dampens kindling epileptogenesis and demonstrate that this function can be exerted also by grafted cholinergic neurones.

Basal forebrain neurons suppress amygdala kindling via cortical but not hippocampal cholinergic projections in rats.

Intraventricular administration of the immunotoxin 192 IgG-saporin in rats has been shown to cause a selective loss of cholinergic afferents to the hippocampus and cortical areas, and to facilitate seizure development in hippocampal kindling. Here we demonstrate that this lesion also accelerates seizure progression when kindling is induced by electrical stimulations in the amygdala. However, whereas intraventricular 192 IgG-saporin facilitated the development of the initial stages of hippocampal kindling, the same lesion promoted the late stages of amygdala kindling. To explore the role of various parts of the basal forebrain cholinergic system in amygdala kindling, selective lesions of the cholinergic projections to either hippocampus or cortex were produced by intraparenchymal injections of 192 IgG-saporin into medial septum/vertical limb of the diagonal band or nucleus basalis, respectively. Cholinergic denervation of the cortical regions caused acceleration of amygdala kindling closely resembling that observed after the more widespread lesion induced by intraventricular 192 IgG-saporin. In contrast, removal of the cholinergic input to the hippocampus had no effect on the development of amygdala kindling. These data indicate that basal forebrain cholinergic neurons suppress kindling elicited from amygdala, and that this dampening effect is mediated via cortical but not hippocampal projections.

Cerebral complications after cardiac surgery assessed by S-100 and NSE levels in blood.

OBJECTIVE: Assessment of the value of blood analysis of the astroglia protein, S-100, and neuron-specific enolase for the detection of nervous system dysfunction after cardiac surgery. DESIGN: Prospective study. Neurologists blinded from laboratory results. SETTING: University hospital. PARTICIPANTS: 38 patients undergoing cardiac surgery. INTERVENTIONS: 21 patients were operated for coronary artery disease; seven patients with replacement of the aortic valve of whom 2 also had coronary bypass. Four patients had mitral valve replacement of whom 2 also had coronary bypass. One patient had both aortic and mitral valve replacement and coronary bypass. Two patients were operated on because of aortic arch aneurysm. MEASUREMENTS AND MAIN RESULTS: Neurologic examinations were performed before and after surgery. General behavior of the patients was repeatedly assessed. Blood samples for analysis were collected before operation and on the second day after surgery. In 8/38 patients (21%), a neurologic complication, one of which was lethal, occurred. In 27 patients (71%), the neurologic outcome was uncomplicated, and in 3 (8%), it could not be classified. Elevated S-100 and neuron-specific enolase levels were found in 7/8 patients who endured a neurologic complication and in 4/27 free of complication. (Fisher's exact test p < 0.001). Positive and negative predictive values were 64% and 96%, respectively. S-100 (range 0.5 to 1.3 micrograms/L) and neuron-specific enolase levels (range 8.6 to 16.7 micrograms/L) were lower for the 7 patients with nonlethal complications than for the patient who died (9.5 micrograms/L and 31.3 micrograms/L, respectively). CONCLUSIONS: S-100 and neuron-specific enolase levels after cardiac surgery are associated with neurologic complications. The results have implications on patient-related treatment and prognosis as well as for the development of safer perfusion techniques.