Altered PLCβ-1 expression in the gerbil hippocampal complex following spontaneous seizure

Although the phospholipase C (PLC)β-1 isoform is associated with spontaneous seizure and distinctively expressed in the telencephalon, the distribution of PLCβ-1 expression in the epileptic gerbil hippocampus remains controversial. Therefore, we determined whether PLCβ-1 is associated with spontaneous seizure in an animal model of genetic epilepsy. In the present study, PLCβ-1 immunoreactivity was down-regulated in seizure-sensitive (SS) gerbils more than in seizure-resistant (SR) gerbils. The expression of PLCβ-1 within calretinin (CR)- positive neurons was rarely detected within the dentate hilar region of SS gerbils. PLCβ-1 immunoreactivity in the hippocampus was significantly elevated as compared to that in pre-seizure SS gerbil 3 h post-ictal. These findings suggest that alterations in PLCβ-1 immunoreactivity in the SS gerbil hippocampus may be closely related to the epileptic state of the gerbil brain and transiently elevated PLCβ-1 protein levels following seizure episodes. Such alterations may be compensatory responses in the SS gerbil hippocampus.     

Changes in the expression of calbindin D-28k in the gerbil hippocampus following seizure

Previous studies have reported that calbindin D-28k (CB), a calcium-binding protein, containing neurons in the hippocampus play an important role in hippocampal excitability in epilepsy, because CB modulates the free calcium ion during seizure. Hence, in the present study, we investigated changes of CB expression in the hippocampus and its association in the Mongolian gerbil to identify roles of CB in epileptogenesis. CB immunoreactivity in the hippocampus was significantly lower in the pre-seizure group of seizure sensitive (SS) gerbils as compared with those seen in the seizure resistant (SR) gerbils. The distribution of CB immunoreactivity in the hippocampus showed significant difference after seizure on-set in SS gerbils. CB immunoreactivity in the hippocampal CA1, CA2 areas, and subiculum was lowest at 3h after seizure on-set; thereafter, the immunoreactivity became to increase to 12h after seizure on-set. Mossy fibers, Schaffer collaterals and dentate granule cells showed the highest CB immunoreactivity at 3h after seizure on-set; thereafter, the immunoreactivity became to decrease. In the case of the intrinsic and output connections of the hippocampus, a rapid decrease of CB serves an inhibitory function, which regulates the seizure activity and output signals from the hippocampus.     

The altered expression of GABA shunt enzymes in the gerbil hippocampus before and after seizure generation

In the present study, the distribution of succinic semialdehyde dehydrogenase (SSADH) and succinic semialdehyde reductase (SSAR) in the hippocampus of the Mongolian gerbil and its association with various sequelae of spontaneous seizure were investigated in order to identify the roles of GABA shunt in the epileptogenesis and the recovery mechanisms in these animals. Both SSADH and SSAR immunoreactivities in the GABAergic neurons were significantly higher in the pre-seizure groups of seizure sensitive (SS) gerbil as compared to those seen in the seizure resistant (SR) gerbils. The distributions of both SSADH and SSAR immunoreactivities in the hippocampus showed significant differences after the on-set of seizure. At 3 h postictal, when compared to the pre-seizure group of SS gerbils, a decline in the immunoreactivities in the perikarya was observed. At 12 h after seizure on-set, the densities of both SSADH and SSAR immunoreactivities were begun to recover to the pre-seizure level of SS gerbils. These results suggest that the GABAergic neurons in the hippocampal complex of the SS gerbil may be highly activated. In addition, the imbalance of GABA shunt expressions in the GABAergic neurons may imply a malfunction of the metabolism of GABAergic neurons in the SS gerbils, and this defect may trigger seizure on-set. Therefore, the initiation of seizure, at least in gerbils, may be the result of a malfunction in GABA shunt in the GABAergic neurons.     

Altered Na+-K+ ATPase immunoreactivity within GABAergic neurons in the gerbil hippocampal complex induced by spontaneous seizure and vigabatrin treatment

In the present study, the expression of Na(+)-K(+) ATPase in the gerbil hippocampus associated with various sequelae of spontaneous seizures were investigated in order to identify the roles of Na(+)-K(+) ATPase in the epileptogenesis and the recovery mechanisms in these animals. The population of Na(+)-K(+) ATPase immunoreactive neurons and Na(+)-K(+) ATPase immunodensity were significantly lower in the pre-seizure group of SS gerbils than those in SR gerbils. At 30-min postictal, the Na(+)-K(+) ATPase immunoreactivity was significantly elevated in the hippocampal complex. At 3-h postictal, the Na(+)-K(+) ATPase immunoreactivity in the hippocampus was declined, as compared to the 30-min postictal. At 12h after seizure on-set, Na(+)-K(+) ATPase expression was re-enhanced in the all regions of the hippocampal complex including the dentate hilus. Following administration of vigabatrin Na(+)-K(+) ATPase expression was also increased. The present data suggest that altered Na(+)-K(+) ATPase expression may contribute the regulation of the seizure activity in this animal.     

Altered corticotropin-releasing factor (CRF) receptor immunoreactivity in the gerbil hippocampal complex following spontaneous seizure

Considerable attention has been focused on the role of corticotropin-releasing factor (CRF) in neuropsychiatric disorders and neurodegenerative diseases including epilepsy. Therefore, in the present study, we investigated the temporal and spatial alteration of CRF receptor in the gerbil hippocampal complex in order to characterize the possible changes and associations with different sequelae of spontaneous seizure in these animals. Thirty minutes postictal, a decline in CRF receptor immunoreactivity was observed in the granule cells and hilar neurons. In the subiculum, CRF receptor immunoreactivity was also significantly decreased at this time point. Twenty-four hours after seizure onset, the immunoreactivity in these regions recovered to the pre-seizure level. Moreover, 30 min after seizure in the entorhinal cortex, the density of CRF receptor immunoreactivity began to decrease, particularly in the layers II and III, compared to pre-seizure group. Nevertheless, 24h after seizure onset, CRF receptor immunodensity had recovered to its seizure-sensitive (SS) level. These results suggest that altered CRF receptor expression in the hippocampal complex may affect tissue excitability and seizure activity in SS gerbils.     

Altered expression of adrenocorticotropic hormone in the epileptic gerbil hippocampus following spontaneous seizure

We investigated the temporal alterations of adrenocorticotropic hormone (ACTH) immunoreactivity in the hippocampus after seizure onset. Expression of ACTH was observed within interneurons in the pre-seizure group of seizure sensitive gerbils, whereas its immunoreactivities were rarely detected in seizure resistant gerbil. Three hr after the seizure, ACTH immunoreactivity was significantly increased in interneurons within all hippocampal regions. On the basis of their localization and morphology through immunofluorescence staining, these cells were identified as GABA_A α1-containing interneurons. At the 12 hr postictal period, ACTH expression in these regions was down-regulated, in a similar manner to the pre-seizure group of gerbils. These findings support the increase in ACTH synthesis that contributes to a reduction of corticotrophin-releasing factor via the negative feedback system which in turn provides an opportunity to enhance the excitability of GABAergic interneurons. Therefore, ACTH may play an important role in the reduction of excitotoxicity in all hippocampal regions. [BMB Reports 2013; 46(2): 80-85]     

The alteration of γ-aminobutyric acid-transaminase expression in the gerbil hippocampus induced by seizure

It is well established that GABA degradation may play a key role in epileptogenesis. However, whether or not the expression of GABA-transaminase (GABA-T), which catalyzes GABA degradation and participates in the neuronal metabolism via GABA shunt, changes chronologically after on-set of seizure remains to be clarified. To identify the change of GABA-T expression in seizure, GABA-T expression in the gerbil hippocampus, associated with different sequelae of spontaneous seizures, was investigated. The distribution pattern of GABA-T immunoreactive neurons in the hippocampus between the seizure-resistant and pre-seizure group of seizure sensitive gerbils was similar. Interestingly, at 30 min postictal, the enhancement of GABA-T immunoreactivity in the perikarya was apparently observed. This contrasted with the decline in GABA-T immunoreactivity in the granular and pyramidal layer. At 12–24 h postictal, GABA-T immunoreactivity in the hilar neurons had declined significantly. However, the GABA-T immunoreactivity in the granular layer increased. These findings suggest that in the gerbil, the alteration in GABA-T expressions may play an important role in the self-recovery mechanism from seizure attack via both GABA degradation and regulation of neuronal metabolism.     

The alteration of gamma-aminobutyric acid-transaminase expression in the gerbil hippocampus induced by seizure

It is well established that GABA degradation may play a key role in epileptogenesis. However, whether or not the expression of GABA-transaminase (GABA-T), which catalyzes GABA degradation and participates in the neuronal metabolism via GABA shunt, changes chronologically after on-set of seizure remains to be clarified. To identify the change of GABA-T expression in seizure, GABA-T expression in the gerbil hippocampus, associated with different sequelae of spontaneous seizures, was investigated. The distribution pattern of GABA-T immunoreactive neurons in the hippocampus between the seizure-resistant and pre-seizure group of seizure sensitive gerbils was similar. Interestingly, at 30 min postictal, the enhancement of GABA-T immunoreactivity in the perikarya was apparently observed. This contrasted with the decline in GABA-T immunoreactivity in the granular and pyramidal layer. At 12-24 h postictal, GABA-T immunoreactivity in the hilar neurons had declined significantly. However, the GABA-T immunoreactivity in the granular layer increased. These findings suggest that in the gerbil, the alteration in GABA-T expressions may play an important role in the self-recovery mechanism from seizure attack via both GABA degradation and regulation of neuronal metabolism.     

Elevated P/Q type (alpha1A) and L2 type (alpha1D) Purkinje cell voltage-gated calcium channels in the cerebella of seizure prone gerbils

Differences in expression of N-methyl-D-aspartate (NMDA) receptor and voltage gated Ca2+ channels (VGCC) in the gerbil cerebellum were investigated to identify routes of Ca2+ influx that may be involved in Purkinje cell damage. Immunodensities of NR1 and NR2A/B were the same in seizure resistant (SR) and seizure sensitive (SS) gerbils. However, both P/Q type (alpha1A) and L2 type (alpha1D) VGCC levels were higher in the Purkinje cells of SS gerbils than in those of SR gerbils, whereas N type (alpha1B) and L1 type (alpha1C) VGCC levels were similar in the two groups. Our findings suggest that increases in P/Q type (alpha1A) and L2 type (alpha1D) VGCC are implicated in the degeneration of Purkinje cells in SS gerbils.     

Accumulation of microtubule-associated proteins in the hippocampal neurons of seizure-sensitive gerbils

We have carried out a chronological and comparative analysis of microtubule-associated protein 1A (MAP1A) and microtubule-associated protein 2 (MAP2) immunoreactivities in the hippocampi of seizure-resistant (SR) and seizure-sensitive (SS) gerbils. These animals represent excellent genetic models of epilepsy associated with different sequelae of spontaneous seizures. Both MAP1A and MAP2 immunoreactivities were detected in the granule cell layer and in the hilar neurons of SR gerbils. In contrast with the SR gerbils, some neurons containing MAPs immunoreactivities were scattered in the molecular layer of the dentate gyrus as well as being concentrated in the hilar neurons in the SS gerbils. An increase in MAP1A immunoreactivity was evident in the perikarya of the dentate gyrus at 30 min postictal, whereas MAP2 immunoreactivity decreased. MAP1A immunoreactivity in the hilar neurons declined significantly by 3 h postictal, whereas MAP2 immunoreactivity increased. These results suggest that the immunoreactivity of MAPs in the hippocampal complex differs between SR and SS gerbils, and that this difference may be the results of seizure activity.     

Time Course of Changes in Immunoreactivities of GABA Degradation Enzymes in the Hippocampal CA1 Region after Adrenalectomy in Gerbils

Adrenalectomy (ADX) has been useful for a good in vivo model for apoptosis in the hippocampus by the absence of corticosteroids following ADX. In some neurodegenerative diseases, GABAergic neurons are more resistant to neuronal damage as compared with glutamatergic neurons. In the present study, we observed chronological changes in three GABA degradation enzymes, e.g., GABA transaminase (GABA-T), succinic semialdehyde dehydrogenase (SSADH) and succinic semialdehyde reductase (SSAR) immunoreactivity and protein levels in the gerbil hippocampal CA1 region after ADX. Changes in their immunoreactivities were distinct in the stratum pyramidale of the CA1 region. GABA-T immunoreactivity and protein level were significantly increased in the CA1 region 3 h after ADX, in contrast, SSAR and SSADH immunoreactivity and protein level were increased 12 h and 3–12 h, respectively, after ADX. These results suggest that the increases of GABA-T, SSADH and SSAR immunoreactivity and protein levels in the hippocampal CA1 region in ADX gerbils may be associated with the control of GABA levels in this region.     

Identification of ryanodine receptor isoforms in prostate DU-145, LNCaP, and PWR-1E cells

We previously reported that left ventricular (LV) slices from isoproterenol (ISO)-induced hypertrophied rat hearts showed an increase of energy expenditure due to remodeling of Ca(2+) handling in excitation-contraction coupling, i.e., suppressed SERCA2a activity and enhanced Na(+)/Ca(2+)exchanger-1 (NCX-1) activity. Na(+)/H(+) exchanger-1 (NHE-1) inhibitor (NHEI) has been demonstrated to exert beneficial effects in the development of cardiac remodeling. We hypothesized that a novel NHE-1 selective inhibitor, BIIB723 prevents remodeling of Ca(2+) handling in LV slices of ISO-induced hypertrophied rat hearts mediated by inhibiting NCX-1 activity. The significant shortening in duration of multi-cellular Ca(2+) transient in ISO group was normalized in ISO+BIIB723 group. The significant increase in amplitude of multi-cellular Ca(2+) waves (CaW) generated at high [Ca(2+)](o) of LV slices in ISO group was also normalized in ISO+BIIB723 group. However, the enhanced NCX-1 activity was not antagonized by BIIB723. We recently reported that ISO-induced down-regulation of a Ca(2+) handling protein, SERCA2a, was normalized by BIIB723. Therefore, it seems likely that BIIB723 normalized shortened multi-cellular Ca(2+) transient duration and increased CaW amplitude in LV slices mediated via normalization of SERCA2a activity. Furthermore, the results presented here suggest the multi-cellular Ca(2+) transient duration and CaW amplitude in LV slices might be better indices reflecting SERCA2a activity than SERCA2a protein expression level.     

The decreases in calcium binding proteins and neurofilament immunoreactivities in the Purkinje cell of the seizure sensitive gerbils.

In previous studies, it has been reported that Purkinje cell degeneration during seizure is evoked by excitotoxicity due to an increase in the intracellular Ca(2+) level, though calbindin D-28k (CB) and parvalbumin (PV), intracellular free calcium buffers, are abundantly colocalized in these cells. In the present study, we investigated the expressions of CB, PV, neurofilament (NF) 68, 150, 200, and polyphosphorylated epitope in NF (RT 97), in the cerebellum of gerbils to identify the mechanism of Purkinje cell damages induced by seizure. In seizure resistant gerbils, nearly all the Purkinje cells showed CB, PA, NF 150, NF 200 and RT 97 immunoreactivity. In SS gerbils, however, a clear decrease in the number of CB(+) and PV(+) Purkinje cells was observed. The NF and RT 97 immunoreactivities, in the Purkinje cells was also lower (except NF 68), but not absent. These results suggest several points. First, the decrease in the concentrations of CB and PV may render the Purkinje cells more susceptible to intermittent Ca(2+) fluctuations and more prone to accumulating intolerable quantities of Ca(2+). Second, during the Ca(2+)-PV interaction PV plays an important role in facilitating donations of Mg(2+), which is a potent enzyme activator in phosphorylation. Thus the decline in PV concentration also implicated the defects of phosphorylation in the NF. Third, increases in both the intracellular Ca(2+) level and dephosphorylation trigger the degradation of the NF, particularly NF 200. Finally, these degradations in the NF induce the functional defects in Purkinje cell, which then cause Purkinje cell degeneration.     

MT2 Melatonin Receptor Immunoreactivity in Neurons is Very High in the Aged Hippocampal Formation in Gerbils

Melatonin exerts many physiological functions via its G protein-coupled receptors. In the present study, we investigated age-related changes in MT2 melatonin receptor immunoreactivity and its levels in the gerbil hippocampus during normal aging. In the postnatal month 1 (PM 1) group, MT2 immunoreaction was well observed in neurons in all subregions of the gerbil hippocampus. In the PM 3 and 6 groups, MT2 immunoreactivity in neurons was decreased compared to that in the PM 1 group. Thereafter, MT2 immunoreactivity in neurons was increased. In the PM 18 and 24 groups, MT2 immunoreactivity in neurons was strong in all subregions of the gerbil hippocampus. In addition, the number of MT2 immunoreactive cells was lowest at PM 3 and highest at PM 24. From western blot analysis, age-dependent change pattern in MT2 level in the gerbil hippocampus was similar to the immunohistochemical result. These results indicate that MT2 immunoreactivity and levels are altered in the gerbil hippocampus during normal aging; lowest at young adult stage and highest at aged stage.     

Appearance of parvalbumin-specific immunoreactivity in the cerebral cortex and hippocampus of the developing rat and gerbil brain

Summary Developmental changes in the distribution of parvalbumin-specific immunoreactivity in the brain, in particular in the cerebral cortex and hippocampus, were followed immunohistochemically in two different species, the rat and the Mongolian gerbil (Meriones unguiculatus) using an antibody raised against for rat parvalbumin. The gerbil is known to develop its auditory and visual capacity later than rat. In both the rat and gerbil, parvalbumin-specific immunoreactivity appeared after birth in both the cerebral cortex and hippocampus. The timing of the development of expression of parvalbumin varied among different parts of the cerebral cortex. The parietal cortex showed evidence of the earliest expression of parvalbumin whilst the occipital and temporal cortices expressed parvalbumin at a later stage of a development. This feature was common to both the rat and gerbil but occurred at a relatively later stage in the gerbil. The profile of the distribution of parvalbumin in the brain of the developing and adult gerbil was similar to that of the rat, but there were some differences. The frequency of bead-like structures on the dendrites of the parvalbumin-positive cells in the CA1 region of the hippocampus was markedly lower in the gerbil; instead, straight non-beaded fibers which ran vertically into the pyramidal layer were stained. Parvalbumin-positive fibers were also found in the cerebral cortex of the gerbil.     

Cyclooxygenase-2 Immunoreactivity and Protein Level in the Gerbil Hippocampus During Normal Aging

Cyclooxygenases-2 (COX-2) is not only related to inflammation but also plays critical roles in brain development and synaptic signaling. In the present study, we investigated age-related changes in COX-2 immunoreactivity and protein levels in the gerbil hippocampus. In the hippocampal CA1 region (CA1) and dentate gyrus (DG), weak COX-2 immunoreactivity was observed at postnatal month 1 (PM 1), and COX-2 immunoreactivity was markedly increased at PM 18 and 24. In the CA2/3, COX-2 immunoreactivity was strong at PM 1. COX-2 immunoreactivities in the PM 3, 6 and 12 groups were decreased compared to that in the PM 1 group, and it was increased at PM 18 and 24. In addition, age-related changes in COX-2 levels were similar with immunohistochemical results in the CA2/3. These results suggest that COX-2 immunoreactivity and levels were high in the hippocampus of aged gerbils.     

Appearance of parvalbumin-specific immunoreactivity in the cerebral cortex and hippocampus of the developing rat and gerbil brain

Developmental changes in the distribution of parvalbumin-specific immunoreactivity in the brain, in particular in the cerebral cortex and hippocampus, were followed immunohistochemically in two different species, the rat and the Mongolian gerbil (Meriones unguiculatus) using an antibody raised against for rat parvalbumin. The gerbil is known to develop its auditory and visual capacity later than rat. In both the rat and gerbil, parvalbumin-specific immunoreactivity appeared after birth in both the cerebral cortex and hippocampus. The timing of the development of expression of parvalbumin varied among different parts of the cerebral cortex. The parietal cortex showed evidence of the earliest expression of parvalbumin whilst the occipital and temporal cortices expressed parvalbumin at a later stage of a development. This feature was common to both the rat and gerbil but occurred at a relatively later stage in the gerbil. The profile of the distribution of parvalbumin in the brain of the developing and adult gerbil was similar to that of the rat, but there were some differences. The frequency of bead-like structures on the dendrites of the parvalbumin-positive cells in the CA1 region of the hippocampus was markedly lower in the gerbil; instead, straight non-beaded fibers which ran vertically into the pyramidal layer were stained. Parvalbumin-positive fibers were also found in the cerebral cortex of the gerbil.     

Ionized Calcium-binding Adapter Molecule 1 Immunoreactive Cells Change in the Gerbil Hippocampal CA1 Region after Ischemia/Reperfusion

Ionized calcium-binding adapter molecule 1 (iba-1) is specifically expressed in microglia and plays an important role in the regulation of the function of microglia. We observed chronological changes of iba-1-immunoreactive cells and iba-1 level in the gerbil hippocampal CA1 region after transient ischemia. Transient forebrain ischemia in gerbils was induced by the occlusion of bilateral common carotid arteries for 5 min. Immunohistochemical and Western blot analysis of iba-1 were performed in the gerbil ischemic hippocampus. In the sham-operated group, iba-1-immunoreactive cells were detected in the CA1 region. Thirty minutes after ischemia/reperfusion, iba-1 immunoreactivity significantly increased, and its immunoreactive cells were well ramified. Three hours after ischemia/reperfusion, iba-1 immunoreactivity and level decreased, and thereafter they increased again with time after ischemia/reperfusion. Three days after ischemia/reperfusion, iba-1-immunoreactive cells had well-ramified processes, which projected to the stratum pyramidale of the CA1 region. Seven days after ischemia/reperfusion, iba-1 immunoreactivity and level were highest in the CA1 region, whereas they significantly decreased in the CA1 region 10 days after ischemia/reperfusion. Iba-1-immunoreactive cells in the ischemic CA1 region were co-localized with OX-42, a microglia marker. In brief, iba-1-immunoreactive cells change morphologically and iba-1 immunoreactivity alters in the CA1 region with time after ischemia/reperfusion. These may be associated with the delayed neuronal death of CA1 pyramidal cells in the gerbil ischemic hippocampus.     

Clioquinol Inhibits Zinc-Triggered Caspase Activation in the Hippocampal CA1 Region of a Global Ischemic Gerbil Model

Background

Excessive release of chelatable zinc from excitatory synaptic vesicles is involved in the pathogenesis of selective neuronal cell death following transient forebrain ischemia. The present study was designed to examine the neuroprotective effect of a membrane-permeable zinc chelator, clioquinol (CQ), in the CA1 region of the gerbil hippocampus after transient global ischemia.

Methodology/Principal Findings

The common carotid arteries were occluded bilaterally, and CQ (10 mg/kg, i.p.) was injected into gerbils once a day. The zinc chelating effect of CQ was examined with TSQ fluorescence and autometallography. Neuronal death, the expression levels of caspases and apoptosis inducing factor (AIF) were evaluated using TUNEL, in situ hybridization and Western blotting, respectively. We were able to show for the first time that CQ treatment attenuates the ischemia-induced zinc accumulation in the CA1 pyramidal neurons, accompanied by less neuronal loss in the CA1 field of the hippocampus after ischemia. Furthermore, the expression levels of caspase-3, -9, and AIF were significantly decreased in the hippocampus of CQ-treated gerbils.

Conclusions/Significance

The present study indicates that the neuroprotective effect of CQ is related to downregulation of zinc-triggered caspase activation in the hippocampal CA1 region of gerbils with global ischemia.