Status epilepticus alters hippocampal PKAbeta and PKAgamma expression in mice.

OBJECTIVES: To investigate the localization and progressive changes of cyclic-AMP dependent protein kinase (cPKA) in the mouse hippocampus at acute stages during and after pilocarpine induced status epilepticus. METHODS: Pilocarpine induced status epilepticus mice were sacrificed 30 min, 2 h or 1 day after the start of a approximately 7 h lasting status as assessed by video-electroencephalography. Brains were processed for quantitative immunohistochemistry of hippocampal cPKAbeta and cPKAgamma, and immunohistochemical co-localization of cPKAbeta and cPKAgamma with calbindin (CB), calretinin (CR), and parvalbumin (PV). RESULTS: Based on anatomical and morphological assessment, cPKAbeta was primarily expressed by principal cells and cPKAgamma by interneurons. In CA1, cPKAbeta co-localized with 76% of CB, 41% of CR, and 95% of PV-immunopositive cells, while cPKAgamma co-localized with 50% of CB, 29% of CR, and 80% of PV-immunopositive cells. Upon induction of status epilepticus, cPKAbeta expression was transiently reduced in CA1, whereas cPKAgamma expression was sustainably reduced. CONCLUSION: cPKA may play an important role in neuronal hyperexcitability, death and epileptogenesis during and after pilocarpine induced status epilepticus.

Morpho-physiologic characteristics of dorsal subicular network in mice after pilocarpine-induced status epilepticus.

The goal of this study was to examine the morpho-physiologic changes in the dorsal subiculum network in the mouse model of temporal lobe epilepsy using extracellular recording, juxtacellular and immunofluorescence double labeling, and anterograde tracing methods. A significant loss of total dorsal subicular neurons, particularly calbindin, parvalbumin (PV) and immunopositive interneurons, was found at 2 months after pilocarpine-induced status epilepticus (SE). However, the sprouting of axons from lateral entorhinal cortex (LEnt) was observed to contact with surviving subicular neurons. These neurons had two predominant discharge patterns: bursting and fast irregular discharges. The bursting neurons were mainly pyramidal cells, and their dendritic spine density and bursting discharge rates were increased significantly in SE mice compared with the control group. Fast irregular discharge neurons were PV-immunopositive interneurons and had less dendritic spines in SE mice when compared with the control mice. When LEnt was stimulated, bursting and fast irregular discharge neurons had much shorter latency and stronger excitatory response in SE mice compared with the control group. Our results illustrate that morpho-physiologic changes in the dorsal subiculum could be part of a multilevel pathologic network that occurs simultaneously in many brain areas to contribute to the generation of epileptiform activity.

mGluR5-PLCbeta4-PKCbeta2/PKCgamma pathways in hippocampal CA1 pyramidal neurons in pilocarpine model of status epilepticus in mGluR5+/+ mice.

While it is generally accepted that phospholipase C (PLC) and protein kinase C (PKC) are down-stream proteins involved in metabotropic glutamate receptor 5 (mGluR5)-related signal transduction, we still do not know which subtype of PLC or PKC is specifically regulated after mGluR5 activation. In the present study in mGluR5 wild-type (mGluR5+/+) mice, we showed induced PKCbeta2 or PKCgamma expression at the border between the stratum oriens and alveus (O/A border) at 2h during pilocarpine induced status epilepticus (SE), and in the stratum pyramidale in CA1 area at 1 day after pilocarpine induced SE; at 1 day, induced expression of PLCbeta4 in the stratum pyramidale of CA1 area was observed. Furthermore, double labeling revealed the co-localization of induced PKCbeta2 or PKCgamma with mGluR5 or with induced PLCbeta4 in the stratum pyramidale of CA1 area. These induced expression, however, were not found in mGluR5 mutant (mGluR5-/-) mice. It suggests that induced PLCbeta4-PKCbeta2/PKCgamma at 1 day after pilocarpine induced SE in pyramidal neurons or PKCbeta2 or PKCgamma in interneurons at O/A border at 2h during pilocarpine induced SE may be specifically linked to the activation of mGluR5. When compared to mGluR5+/+ mice, significant shorter latency (from pilocarpine injection to the occurrence of status epilepticus) and maintenance period (from beginning to the end of status epilepticus) for status epilepticus in mGluR5-/- mice were also demonstrated. It is possible that mGluR5 may play a negative role in initiation of status epilepticus by interacting with muscarinic acetylcholine receptor in mGluR5+/+ mice.

Cytoarchitectonics and afferent/efferent reorganization of neurons in layers II and III of the lateral entorhinal cortex in the mouse pilocarpine model of temporal lobe epilepsy.

With the mouse pilocarpine model of temporal lobe epilepsy (TLE), we showed a progressive loss of both principal cells and calbindin (CB)-, calretinin (CR)-, and parvalbumin (PV)-immunopositive interneurons in layers II-III of lateral entorhinal cortex (LEnt) from 2 months to 1 year after pilocarpine-induced status epilepticus (PISE). In the efferent pathway of LEnt, more Phaseolus vulgaris leucoagglutinin (PHA-L)-labelled en passant and terminal boutons with larger diameters were shown in the hippocampus and subiculum; in the prefrontal, piriform, and perirhinal cortices; and in the amygdaloid complex in experimental mice at the two time points compared with the control after iontophoretical injection of an anterograde tracer PHA-L into the LEnt. Furthermore, the numbers of CB- or CR-immunopositive neurons contacted by PHA-L-labelled en passant and terminal boutons decreased in most of these areas at 2 months or 1 year after PISE. In the afferent pathway of LEnt, the numbers of retrogradely labelled neurons were reduced significantly in the ipsilateral piriform cortex and endopiriform nucleus at 2 months and 1 year and in the reuniens thalamic nucleus only at 1 year after injection of a retrograde tracer cholera toxin B subunit (CTB) into the LEnt. The percentages of the number of CTB and CB or CR double-labelled neurons of all the retrogradely labelled neurons were also decreased in the reunions thalamic nucleus at 1 year after PISE. It is concluded that both cytoarchitectonic change and reorganization of afferent and efferent pathways in LEnt may be involved in the occurrence of TLE.

Two-methyl-6-phenylethynyl-pyridine (MPEP), a metabotropic glutamate receptor 5 antagonist, with low doses of MK801 and diazepam: a novel approach for controlling status epilepticus.

By intravenous administration of group I metabotropic glutamate receptor antagonists at 1 or 2h during pilocarpine induced status epilepticus (PISE), we showed that mGluR1 antagonists AIDA or LY367385 (at dosages ranging from 25 to 200mg/kg), mGluR5 antagonists SIB1757 (at dosages ranging from 25 to 200mg/kg), SIB1893 (from 25 to 100mg/kg), MPEP (from 25 to 100mg/kg) injected at 1 or 2h during PISE were ineffective in controlling status epilepticus (SE). However, when administered at 1h during PISE, MPEP at 200mg/kg, combination of MPEP (200mg/kg) with MK801 (0.1mg/kg) or with MK801 (0.1mg/kg) and diazepam (0.5mg/kg), combination of SIB1893 (200mg/kg) with MK801 (0.1mg/kg) could effectively control behavioral SE, and were neuroprotective. In particular, the combination of MPEP with MK801 and diazepam could stop both behavioral SE and electrical SE (under EEG monitoring) within a few minutes after the administration. HPLC study showed that a high level of MPEP was maintained in the blood and its metabolism rate was slow in experimental mice with PISE. We therefore concluded that the combination of MPEP (200mg/kg) with MK801 (0.1mg/kg) and diazepam (0.5mg/kg) could effectively stop SE and its subsequent neuronal loss in the hippocampus when administered 1h during PISE. It may provide a new approach to effectively control intractable SE.

Anticonvulsive effect of a selective mGluR8 agonist (S)-3,4-dicarboxyphenylglycine (S-3,4-DCPG) in the mouse pilocarpine model of status epilepticus.

PURPOSE: We sought to investigate the anticonvulsive and neuroprotective effect of a selective metabotropic glutamate receptor 8 (mGluR8) agonist (S)-3,4-dicarboxyphenylglycines (S-3,4-DCPG) on pilocarpine-induced status epilepticus (PISE) and subsequent loss of hilar neurons in the dentate gyrus after systemic (intravenous) or local (intracerebroventricular) administration. We compared the difference in granular cell responses after paired-pulse stimulation of the perforant pathway and the sensitivity to local injection of S-3,4-DCPG into the stratum granulosum in the control and mice at 2 months after PISE. METHODS: We used intravenous, intracerebroventricular, or intrahippocampal administration of S-3,4-DCPG to mice with status epilepticus or temporal lobe epilepsy and neurophysiologic recording of somatic field excitatory postsynaptic potential (sfEPSP) and population spike (PS) of granular cells in response to perforant-pathway stimulation or S-3,4-DCPG treatment. RESULTS: Intracerebroventricular (1.91 micromol) but not systemic administration of S-3,4-DCPG (at doses of 12.5, 50, 100, 200, 400, 800, and 1,200 mg/kg) could control PISE with no neuroprotective effect. In epileptic mice, mGluR8-mediated inhibition of fEPSPs was reduced significantly in granular cell bodies. CONCLUSIONS: At doses ranging from 12.5 to 1,200 mg/kg, intravenous administration of S-3,4-DCPG may not be effective in controlling status epilepticus. Down-regulation of mGluR8 may be related to reduced S-3,4-DCPG-mediated inhibition and the subsequent occurrence of spontaneously recurrent seizures.

Ca(v)1.2, Ca(v)1.3, and Ca(v)2.1 in the mouse hippocampus during and after pilocarpine-induced status epilepticus.

Calcium binding proteins are well known to be expressed by different groups of hippocampal interneurons; however, whether voltage-dependent calcium channels (Ca(v)) are also localized in these neurons, changed during and after status epilepticus (SE), and involved in epileptic activity have not been reported. In the present study, we showed the colocalization of three subtypes of voltage-gated calcium channels (Ca(v)1.2, Ca(v)1.3, or Ca(v)2.1) with different calcium binding proteins such as calbindin (CB), calretinin (CR), and parvalbumin (PV). At early stages during and after pilocarpine-induced status epilepticus (PISE), significant changes of expression of Ca(v)1.2, Ca(v)1.3 (L-type), and Ca(v)2.1 (P/Q-type) were found in different groups of hippocampal neurons. Induced expression of Ca(v)1.3 or Ca(v)2.1 in reactive astrocytes was shown at 1 week and 2 months after PISE. At the latter time point, higher percentages of colocalization of PV and Ca(v)1.2, CB, or PV and Ca(v)1.3 or Ca(v)2.1, lower percentages of CR and Ca(v)1.3 or Ca(v)2.1 immunoposivie neurons were observed in gliotic CA1 area. We therefore conclude that voltage-gated calcium channels are expressed by different groups of hippocampal interneurons in the mouse. At acute stages during and after PISE, up- or down-regulation of Ca(v)1.2, Ca(v)1.3, or Ca(v)2.1 in functionally different groups of interneurons in CA1 area may be related to the changes of their plasticity. Up-regulation of Ca(v)1.2, Ca(v)1.3, or Ca(v)2.1 in granule cells may be directly related to the occurrence of SE. The induced expression of Ca(v)1.3 or Ca(v)2.1 in reactive astrocytes at 1 week and 2 months after PISE suggests that Ca(v)1.3 or Ca(v)2.1-related calcium signaling in reactive astrocytes may be involved in initiation, maintenance or spread of seizure activity. In gliotic CA1 area at chronic stage (i.e., 2 months after PISE), the occurrence of higher percentages of colocalization of PV and Ca(v)1.2, CB, or PV and Ca(v)1.3 or Ca(v)2.1, lower percentages of CR and Ca(v)1.3 or Ca(v)2.1 immunopositive neurons may suggest that such colocalizations may be linked to the survival or loss of particular group of hippocampal neurons.

CCR7, CCR8, CCR9 and CCR10 in the mouse hippocampal CA1 area and the dentate gyrus during and after pilocarpine-induced status epilepticus.

The present study showed CCR7, CCR8, CCR9 and CCR10 in the normal Swiss mouse hippocampus at both protein and mRNA levels. CCR7, CCR9 and CCR10 were mainly localized in hippocampal principal cells and some interneurons. CCR9 was also found in the mossy fibres and/or terminals, suggesting an axonal or presynaptic localization, and CCR10 in apical dendrites of pyramidal neurons in the CA1 area. CCR8 was observed in interneurons. Double-labelling immunocytochemistry revealed that most of calbindin (CB)-, calretinin (CR)- and parvalbumin (PV)-immunopositive neurons expressed CCR7-10, except CR-immunopositive cells in which only 10 to 12% expressed CCR8. During and after pilocarpine-induced status epilepticus, progressive changes of each of CCR7, CCR8, CCR9 and CCR10 proteins occurred in different patterns at various time points. Sensitive real-time PCR showed similar change patterns at mRNA level. At the chronic stage, i.e. at 2 months after pilocarpine-induced status epilepticus, significant reduction of CCR7-10 expression in CB-, CR- and PV-immunpositive interneurons may suggest the phenotype change of surviving interneurons. Double labelling of CCR7, CCR8 and CCR9 with glial fibrillary acidic protein (GFAP) at the chronic stage may suggest an induced expression in reactive astrocytes. The present study may, therefore, for the first time, provide evidence that CCR7-10 may be involved in normal hippocampal activity. The demonstration of the progressive changes of CCR7-10 during and after status epilepticus may open a new area to reveal the mechanism of neuronal loss after status epilepticus and of epileptogenesis.

Spastin in the human and mouse central nervous system with special reference to its expression in the hippocampus of mouse pilocarpine model of status epilepticus and temporal lobe epilepsy.

In the present in situ hybridization and immunocytochemical studies in the mouse central nervous system (CNS), a strong expression of spastin mRNA and protein was found in Purkinje cells and dentate nucleus in the cerebellum, in hippocampal principal cells and hilar neurons, in amygdala, substantia nigra, striatum, in the motor nuclei of the cranial nerves and in different layers of the cerebral cortex except piriform and entorhinal cortices where only neurons in layer II were strongly stained. Spastin protein and mRNA were weakly expressed in most of the thalamic nuclei. In selected human brain regions such as the cerebral cortex, cerebellum, hippocampus, amygdala, substania nigra and striatum, similar results were obtained. Electron microscopy showed spastin immunopositive staining in the cytoplasma, dendrites, axon terminals and nucleus. In the mouse pilocarpine model of status epilepticus and subsequent temporal lobe epilepsy, spastin expression disappeared in hilar neurons as early as at 2h during pilocarpine induced status epilepticus, and never recovered. At 7 days and 2 months after pilocarpine induced status epilepticus, spastin expression was down-regulated in granule cells in the dentate gyrus, but induced expression was found in reactive astrocytes. The demonstration of widespread distribution of spastin in functionally different brain regions in the present study may provide neuroanatomical basis to explain why different neurological, psychological disorders and cognitive impairment occur in patients with spastin mutation. Down-regulation or loss of spastin expression in hilar neurons may be related to their degeneration and may therefore initiate epileptogenetic events, leading to temporal lobe epilepsy.

Reorganization of CA3 area of the mouse hippocampus after pilocarpine induced temporal lobe epilepsy with special reference to the CA3-septum pathway.

We showed that when CA3 pyramidal neurons in the caudal 80% of the dorsal hippocampus had almost disappeared completely, the efferent pathway of CA3 was rarely detectable. We used the mouse pilocarpine model of temporal lobe epilepsy (TLE), and injected iontophoretically the anterograde tracer phaseolus vulgaris leucoagglutinin (PHA-L) into gliotic CA3, medial septum and the nucleus of diagonal band of Broca, median raphe, and lateral supramammillary nuclei, or the retrograde tracer cholera toxin B subunit (CTB) into gliotic CA3 area of hippocampus. In the afferent pathway, the number of neurons projecting to CA3 from medial septum and the nucleus of diagonal band of Broca, median raphe, and lateral supramammillary nuclei increased significantly. In the hippocampus, where CA3 pyramidal neurons were partially lost, calbindin, calretinin, parvalbumin immunopositive back-projection neurons from CA1-CA3 area were observed. Sprouting of Schaffer collaterals with increased number of large boutons in both sides of CA1 area, particularly in the stratum pyramidale, was found. When CA3 pyramidal neurons in caudal 80% of the dorsal hippocampus have almost disappeared completely, surviving CA3 neurons in the rostral 20% of the dorsal hippocampus may play an important role in transmitting hyperactivity of granule cells to surviving CA1 neurons or to dorsal part of the lateral septum. We concluded that reorganization of CA3 area with its downstream or upstream nuclei may be involved in the occurrence of epilepsy.