Feng Ru Tang

Metabotropic glutamate receptor 2/3 in the hippocampus of patients with mesial temporal lobe epilepsy, and of rats and mice after pilocarpine-induced status epilepticus

A comparative study of the expression of metabotropic glutamate receptor 2/3 (mGluR2/3) was done in the hippocampus of rats and mice after pilocarpine-induced status epilepticus (APISE), and of patients with mesial temporal lobe epilepsy. At 1 day APISE, there was a marked increase in mGluR2/3 immunoreactivity in the stratum lacunosum moleculare (SLM) of CA1 area and in the middle one-third of the molecular layer (MM) of the dentate gyrus. Immuno-electron microscopic study showed degenerating mGluR2/3 positive axons in the SLM of CA1 area at 1 day APISE. From 7 days, mGluR2/3 immunopositive product decreased, and by 31 days APISE, it almost disappeared in two-thirds of the SLM near CA2. In the mouse model at 2 months APISE, mGluR2/3 immunopositive product in two-thirds of the SLM near the stratum radiatum disappeared, and so did in the whole SLM of CA1 area in patients with mesial temporal lobe epilepsy. Neuropharmacological study by intravenous injection of mGluR2/3 agonist 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate [(2R,4R)-APDC] at different doses at 1h during pilocarpine induced status epilepticus showed that (2R,4R)-APDC could not stop seizures and neuronal death in the hilus of the dentate gyrus. The present study, therefore, suggests that the reduction of mGluR2/3 immunopositive product in the SLM of CA1 is a consequence of neuronal loss in either the entorhinal cortex or CA1 area of the hippocampus, and at the dosage range from 12.5 to 600 mg/kg, (2R,4R)-APDC may not be effective in the prevention of seizures or neuronal death in the hilus of the dentate gyrus.

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.

Expression of metabotropic glutamate receptor 1α in the hippocampus of rat pilocarpine model of status epilepticus

The expression of metabotropic glutamate receptor 1alpha was studied in the rat hippocampus after pilocarpine-induced status epilepticus by Western blot and immunocytochemistry at both light and electron microscopic levels. At 1 day after pilocarpine-induced status epilepticus, there was marked decrease in metabotropic glutamate receptor 1alpha immunoreactivity at the border between stratum oriens and alveus in CA1 and CA3, and in the hilus of dentate gyrus. Between 3 and 31 days after pilocarpine-induced status epilepticus, metabotropic glutamate receptor 1alpha-immunoreactive dendrites and cell bodies in the border between stratum oriens and alveus gradually reappeared. Upregulation of metabotropic glutamate receptor 1alpha, however, was observed in the stratum oriens of CA1 at day 1, but returned to baseline by day 7. By electron microscopy, the metabotropic glutamate receptor 1alpha-immunoreactive product was demonstrated only in the post-synaptic elements in the border between the stratum oriens and alveus of CA1 and the hilus of the dentate gyrus in both control and experimental rats. At 1 day after pilocarpine-induced status epilepticus, metabotropic glutamate receptor 1alpha-immunoreactive degenerating neurons were identified in the border between stratum oriens and alveus of CA1 and the hilus of the dentate gyrus. At 7 and 31 days, many degenerating axons were also found. Present results suggest that excitoneurotoxicity mediated through post-synaptic metabotropic glutamate receptor 1alpha may be involved in degeneration and death of interneurons in the hilus of dentate gyrus, and the border between stratum oriens and alveus of CA1 in the early stage after pilocarpine-induced status epilepticus.

mGluR5–PLCβ4–PKCβ2/PKCγ 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.

Cyto-, axo- and dendro-architectonic changes of neurons in the limbic system in the mouse pilocarpine model of temporal lobe epilepsy

While different hypotheses have been proposed to explain the mechanism of onset of temporal lobe epilepsy (TLE), most of them are based on structural, electrophysiological, cellular or molecular changes in one particular area. Extensive neuronal loss, axon reorganization, dendrite and dendritic spine growth make it impossible to apply one hypothesis to explain epileptogenesis for patients or animal models with different pathophysiological changes in the brain. It is therefore hypothesized that cyto-, axo- and dendro-architectonic changes at multiple brain regions may be involved in epileptogenesis in TLE. In the review, structural changes of the limbic system, in particular, hippocampus, entorhinal cortex, subiculum and amygdale, in the mouse pilocarpine model of TLE will be summarized. Their functional significance will be discussed. The final conclusion and future research directions will then be made.

CCL28 in the mouse hippocampal CA1 area and the dentate gyrus during and after pilocarpine-induced status epilepticus

The present study showed a wide presence of CCL28 in mouse CNS, including cerebral, cerebellum, brain stem and spinal cord. In hippocampus, the expression of CCL28 at both mRNA and protein level was clarified. The CCL28 expression was mainly localized in pyramidal cells of CA area, granular cells of dentate gyrus and some interneurons in CA area and hilus. Double-labelling immunocytochemistry revealed that most of calbindin, calretinin and parvalbumin immunopositive neurons expressed CCL28. During and after pilocarpine induced status epilepticus (SE), a down-regulated expression of CCL28 in hippocampal interneurons in the CA1 area and in the hilus of the dentate gyrus was demonstrated. The present study may, therefore provide evidence that CCL28 may have a novel role in CNS and may be involved in the loss of hippocampal interneurons, and subsequent disinhibition of pyramidal neurons.

Status epilepticus alters hippocampal PKAβ and PKAγ 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.

Alterations of L-type voltage dependent calcium channel alpha 1 subunit in the hippocampal CA3 region during and after pilocarpine-induced epilepsy

&NA; Voltage‐dependent calcium channels (VDCC) have been shown to regulate neuronal excitability and their antagonists have been used clinically for the control of seizures. While functional studies of VDCC in epileptogenesis in the CA1 area of hippocampus or the dentate gyrus have been done, few studies were carried out in the CA3 area. Given the bursting characteristics of the CA3 neurons, we speculated that VDCC in the CA3 area might play an important role in the epileptogenesis. In the present study in the mouse pilocarpine model of temporal lobe epilepsy, we investigated the alterations of alpha 1 subunits of L‐type VDCC in the CA3 area of the hippocampus at different stages of epileptogenesis, i.e., acute stage from 10 min to 1 day during and after pilocapine‐induced status epilepticus (SE), latent period at 1 week, and chronic stage with spontaneous recurrent seizures at 2 months after SE. We found that an immediate redistribution of alpha 1 subunits in the CA3 area occurred during SE which might be involved in the seizure occurrence indicated by the Racine score record. Alterations of alpha 1 subunits were also demonstrated in the latent period and chronic stage, which might be related to the epileptogenesis and occurrence of epilepsy. Cav1.3, but not Cav1.2, was expressed in reactive astrocytes of the CA3 area, indicating the involvement of Cav1.3 in the modulation of astrocytic Ca2+ homeostasis during epileptogenesis.

Cx36 in the mouse hippocampus during and after pilocarpine-induced status epilepticus.

Gap junctions play an important role in the synchronization activity of coupled cells. Hippocampal inhibitory interneurons are involved in epileptogenesis and seizure activity, and express gap junction protein connexin (Cx) 36. Cx36 is also localized in the axons (mossy fibers) of granule cells in the dentate gyrus. While it has been documented that Cx36 is involved in epileptogenesis, there are still controversies regarding the expression levels of Cx36 at different developmental stages of human and animal models of epileptogenesis. In this study, the expression of Cx36 was investigated in the mouse hippocampus at 1 h, 4 h during pilocarpine-induced status epilepticus (PISE) and 1 week, 2 months after PISE. We found that Cx36 was down-regulated in neurons at different time points during and after PISE, whereas it was increased significantly in the stratum lucidum of CA3 area at 2 months after PISE. Double immunofluorescence indicated that Cx36 was localized in parvalbumin (PV) immunopositive interneuron in CA1 area and in mossy fibers and their terminals in the stratum lucidum of CA3 area. It suggests that decreased expression of Cx36 in interneurons may be related to less effective inhibitory control of excitatory activity of hippocampal principal neurons. However, the increased Cx36 immunopositive product in mossy fibers at the chronic stage after PISE may enhance the contacts between granule cells in the dentate gyrus and pyramidal neurons in CA3 area. The two different changes of Cx36 may be implicated in the epileptogenesis.