Impaired reelin processing and secretion by Cajal–Retzius cells contributes to granule cell dispersion in a mouse model of temporal lobe epilepsy

Cajal–Retzius cells play a crucial role during ontogeny in regulating cortical lamination via release of reelin. In adult brain, they comprise small calretinin‐positive interneurons located in the marginal zone of the cerebral cortex and in the hippocampal fissure. Alterations of reelin signaling or expression have been involved in major neurological disorders, and they underlie granule cell dispersion (GCD) in mesial temporal lobe epilepsy (TLE). Here, we investigated in a mouse model of TLE the contribution of Cajal–Retzius cells to reelin production in epileptic hippocampus and the molecular mechanisms underlying GCD. Following unilateral intrahippocampal Kainic acid injection in adult mice to induce an epileptic focus, we observed that Cajal–Retzius cells gradually became strongly immunopositive for reelin, due to intracellular accumulation. This phenotype resembled the morphology of Cajal–Retzius cells in reeler Orleans (reln^orl/orl) mice, which express a secretion‐deficient 310‐kDa reelin fragment. The possibility that GCD might result from abnormal reelin processing in Cajal–Retzius cells, leading to a lack of reelin secretion, was confirmed by KA injection in reln^orl/+ mice, which induced severe GCD. Furthermore, Western blot analysis in KA‐treated wildtype mice revealed increased production of ～300‐kDa reelin fragments, confirming abnormal proteolytic processing. This effect was not seen upon treatment with Botulinum neurotoxin E (BoNT/E), which prevents GCD in KA‐lesioned hippocampus by chronic blockade of synaptic transmission. Furthermore, BoNT/E blocked upregulation of TrkB in Cajal–Retzius cells, suggesting that production of truncated reelin in KA‐treated hippocampus is activity‐dependent and regulated by BDNF. Altogether, these data reveal that GCD results from abnormal reelin processing in Cajal–Retzius cells under the control of BDNF. Our findings highlight the critical role played by Cajal–Retzius cells for hippocampal neuronal reorganization in TLE. © 2010 Wiley‐Liss, Inc.     

Expression of neuropeptide Y1 receptors in the amygdala and hippocampus and anxiety-like behavior associated with Ammon's horn sclerosis following intrahippocampal kainate injection in C57BL/6J mice

Damage to the amygdala is often linked to Ammon's horn sclerosis (AHS) in surgical specimens of patients suffering from temporal lobe epilepsy (TLE). Moreover, amygdalar pathology is thought to contribute to the development of anxiety symptoms frequently found in TLE. The neuropeptide Y (NPY) Y1 receptor is critical in the regulation of anxiety-related behavior and epileptiform activity in TLE. Therefore, intrahippocampal kainate (KA) injection was performed to induce AHS-associated TLE and to investigate behavioral and cytoarchitectural changes that occur in the amygdala related to Y1 receptor expression. Status epilepticus was induced by intrahippocampal KA injection in C57BL/6J mice. Anxiety-like behavior was assessed using the elevated plus maze (EPM). Pathology of hippocampus and amygdala (volume loss and gliosis) was examined in KA-injected and saline-injected controls. Y1 receptor expression was measured using immunohistochemistry and ELISA. Animal injected with KA showed increased anxiety-like behaviors and reduced risk assessment in the EPM test compared with saline-injected controls. In the ipsilateral hippocampus of KA-injected animals, CA1 ablation, granule cell dispersion, and volume reduction were accompanied by astrogliosis indicating the development of AHS. In the amygdala, a significant decrease in the volume of nuclei and numbers of neurons was observed in the ipsilateral lateral, basolateral, and central amygdalar nuclei, which was accompanied by astrogliosis. In addition, a decrease in Y1 receptor-expressing cells in the ipsilateral CA1 and CA3 sectors of the hippocampus, ipsilateral and contralateral granule cell layer of the dentate gyrus, and ipsilateral central nucleus of the amygdala was found, consistent with a reduction in Y1 receptor protein levels. Our results suggest that plastic changes in hippocampal and/or amygdalar Y1 receptor expression may negatively impact anxiety levels. Moreover, intrahippocampal KA injection can induce amygdalar damage suggesting that AHS-associated amygdala damage may contribute to behavioral alterations seen in patients with TLE.     

Granule cell dispersion develops without neurogenesis and does not fully depend on astroglial cell generation in a mouse model of temporal lobe epilepsy

Purpose: Granule cell dispersion (GCD) appears as a characteristic morphological feature of the mesial temporal lobe epilepsy (MTLE). It has been suggested that this phenomenon could be due to an increased neurogenesis in the dentate gyrus. However, this hypothesis is still debated and recent clinical and experimental studies have shown that neurogenesis is rather decreased in MTLE. To further determine the role of neural and astroglial cell generation in GCD we examined the consequences of aging and irradiation, which are known to reduce progenitor cells, in a mouse model of MTLE induced by intrahippocampal kainate (KA) injection. Methods: We injected KA in hippocampus of three different types of mice; (1) young adult, (2) aged, and (3) irradiated mice. Newly generated cells were labeled by Bromodeoxyuridine (BrdU) and were characterized by immunohistochemistry. The extent of GCD was compared among the three animal groups. Results: In young adult mice, BrdU‐labeled neurons as well as doublecortin‐ and NeuroD‐positive cells decreased progressively after KA injection whereas BrdU‐labeled astrocytes and microglias increased. In aged and irradiated mice, where basal neurogenesis was already strongly reduced, GCD developed after KA injection to the same extent as in young adult mice. However, augmentation of the BrdU‐labeled astrocytes after KA was less than 40% in irradiated mice in comparison to young and aged mice. Conclusions: Our data show that GCD occurs without neurogenesis. Furthermore GCD developed regardless of the degree of astroglial cell proliferation, suggesting that neural stem cell generation is not crucial for GCD.     

Dentate gyrus and hilus transection blocks seizure propagation and granule cell dispersion in a mouse model for mesial temporal lobe epilepsy

Epilepsy‐associated changes of the anatomical organization of the dentate gyrus and hilus may play a critical role in the initiation and propagation of seizures in mesial temporal lobe epilepsy (MTLE). This study evaluated the role of longitudinal projections in the propagation of hippocampal paroxysmal discharges (HPD) in dorsal hippocampus by performing a selective transection in a mouse model for MTLE obtained by a single unilateral intrahippocampal injection of kainic acid (KA). Full transections of the dentate gyrus and hilus were performed in the transverse axis at 22 days after KA injection when spontaneous HPD were fully developed. They: (i) significantly reduced the occurrence of HPD; (ii) increased their duration at the KA injection site; (iii) abolished their spread along the longitudinal axis of the hippocampal formation and; (iv) limited granule cell dispersion (GCD) of the dentate gyrus posterior to the transection. These data suggest that: (i) longitudinal projections through the dentate gyrus and hilus are involved in HPD spread; (ii) distant hippocampal circuits participate in the generation and cessation of HPD and; (iii) GCD requires continuous HPD to develop, even when seizures are established. Our data reveal a critical role for longitudinal projections in the generation and spread of hippocampal seizures. © 2010 Wiley‐Liss, Inc.     

Cystatin C expression is associated with granule cell dispersion in epilepsy

Human temporal lobe epilepsy (TLE) is associated with cellular alterations (eg, hilar cell death, neurogenesis, and granule cell dispersion) in the dentate gyrus but their underlying molecular mechanism are not known. We previously demonstrated increased expression of cystatin C, a protease inhibitor linked to both neurodegeneration and neurogenesis, during epileptogenesis in the rat hippocampus. Here, we investigated cystatin C expression in the dentate gyrus in chronic epilepsy and its association with neuronal loss and neurogenesis. In both rats with epilepsy and human patients with TLE, cystatin C expression was increased in glial cells in the molecular layer of the dentate gyrus, being most prominent in cases with granule cell dispersion. In patients with TLE, high cystatin C expression associated with greater numbers of polysialylated neural cell adhesion molecule-positive newborn cells in the molecular layer, although the overall number was decreased, indicating that the newborn cells migrate to abnormal locations in the epileptic dentate gyrus. These data thus demonstrate that cystatin C expression is altered during the chronic phase of epilepsy and suggest that cystatin C plays a role in network reorganization in the epileptic dentate gyrus, especially in granule cell dispersion and guidance of migrating newborn granule cells.     

Granule cell dispersion is not accompanied by enhanced neurogenesis in temporal lobe epilepsy patients

Granule cell dispersion (GCD) in the dentate gyrus is a frequent feature of Ammon's horn sclerosis (AHS) which is often associated with temporal lobe epilepsy (TLE). It has been hypothesized that GCD may be caused by an abnormal migration of newly born granule cells. To test this hypothesis, we used markers of proliferation and neurogenesis and immunocytochemical methods as well as quantitative Western blot and real-time RT-PCR analyses in surgically resected hippocampi from TLE patients and controls. Below the age of 1 year, Ki-67-immunopositive nuclei were detected in the subgranular zone of the dentate gyrus, but not in the dentate of TLE patients independent of age. The expression of the proliferation marker minichromosome maintenance protein 2 (mcm2) and of doublecortin (DCX) decreased significantly with age in controls and in TLE patients, but the expression of both proteins was independent of the degree of AHS and GCD. Quantitative real-time RT-PCR confirmed these findings at the level of gene expression. In contrast, immunocytochemistry for glial fibrillary acidic protein (GFAP) and vimentin as well as Golgi staining revealed a radially aligned glial network in the region of GCD. GFAP-positive fiber length significantly increased with the severity of GCD. These results indicate that epileptic activity does not stimulate neurogenesis in the human dentate gyrus and that GCD probably does not result from a malpositioning of newly generated granule cells, but rather from an abnormal migration of mature granule cells along a radial glial scaffold.     

Disruption of the neurogenic potential of the dentate gyrus in a mouse model of temporal lobe epilepsy with focal seizures

Adult hippocampal neurogenesis is enhanced in response to multiple stimuli including seizures. However, the relationship between neurogenesis and the development of temporal lobe epilepsy (TLE) remains unclear. Unilateral intrahippocampal injection of kainate in adult mice models the morphological characteristics (e.g. neuronal loss, gliosis, granule cell dispersion and hypertrophy) and occurrence of chronic, spontaneous recurrent partial seizures observed in human TLE. We investigated the influence of a kainate-induced epileptogenic focus on hippocampal neurogenesis, comparing neural stem cell proliferation following status epilepticus and spontaneous recurrent partial seizures. Cell proliferation in the subgranular zone was transiently increased bilaterally after kainate treatment. As a result, neurogenesis was stimulated in the contralateral dentate gyrus. In contrast, the epileptic hippocampus exhibited a strongly reduced neurogenic potential, even after onset of spontaneous recurrent partial seizures, possibly due to an alteration of the neurogenic niche in the subgranular zone. These results show that neurogenesis does not contribute to the formation of the epileptic focus and may be affected when dispersion of dentate gyrus granule cells occurs. Therefore, in patients with TLE, hippocampal sclerosis and granule cell dispersion may play a significant role in disrupting the potential for hippocampal neurogenesis.     

Glutamate receptor antagonists and benzodiazepine inhibit the progression of granule cell dispersion in a mouse model of mesial temporal lobe epilepsy

PURPOSE: Unilateral intrahippocampal injection of kainic acid (KA) in adult mice induces the progressive dispersion of dentate granule cells, one of the characteristic pathologic changes of mesial temporal lobe epilepsy. However, little is known about the mechanisms that trigger this dispersion. In this study, the possible involvement of glutamatergic and gamma-aminobutyric acid (GABA)ergic neurotransmissions in the development of granule cell dispersion (GCD) was examined in this model. METHODS: Antagonists of N-methyl-d-aspartate (NMDA) receptor (MK-801) and non-NMDA receptor (GYKI52466), and an agonist of benzodiazepine-GABA(A) receptor (midazolam) were injected before and after KA in various ways, and the morphologic changes of the hippocampus, especially GCD, were examined. RESULTS: MK-801 (5 mg/kg, i.p.) did not reduce GCD when injected 2 h before KA injection but inhibited GCD almost completely for 相似文献   

Chronic temporal lobe epilepsy is associated with severely declined dentate neurogenesis in the adult hippocampus

While it is clear that acute hippocampal injury or status epilepticus increases the production of new neurons in the adult dentate gyrus (DG), the effects of chronic epilepsy on dentate neurogenesis are unknown. We hypothesize that epileptogenic changes and spontaneous recurrent motor seizures (SRMS) that ensue after hippocampal injury or status epilepticus considerably decrease dentate neurogenesis. We addressed this issue by quantifying the number of cells that are positive for doublecortin (DCX, a marker of new neurons) in the DG of adult F344 rats at 16 days and 5 months after an intracerebroventricular kainic acid (ICV KA) administration or after graded intraperitoneal KA (IP KA) injections, models of temporal lobe epilepsy (TLE). At early post-KA administration, the injured hippocampus exhibited increased dentate neurogenesis in both models. Conversely, at 5 months post-KA administration, the chronically epileptic hippocampus demonstrated severely declined neurogenesis, which was associated with considerable SRMS in both KA models. Additionally, stem/progenitor cell proliferation factors, FGF-2 and IGF-1, were decreased in the chronically epileptic hippocampus. Interestingly, the overall decrease in neurogenesis and the extent of SRMS were greater in rats receiving IP KA than rats receiving ICV KA, suggesting that the extent of neurogenesis during chronic TLE exhibits an inverse relationship with SRMS. These results provide novel evidence that chronic TLE is associated with extremely declined dentate neurogenesis. As fraction of newly born neurons become GABA-ergic interneurons, declined neurogenesis may contribute to the increased seizure-susceptibility of the DG in chronic TLE. Likewise, the hippocampal-dependent learning and memory deficits observed in chronic TLE could be linked at least partially to the declined neurogenesis.     

Increased leak conductance in dentate gyrus granule cells of temporal lobe epilepsy patients with Ammon's horn sclerosis

Purpose:   Temporal lobe epilepsy (TLE) is often associated with Ammon's horn sclerosis (AHS) characterized by hippocampal cell death and dentate gyrus granule cell dispersion (GCD). Granule cells survive AHS and have been proposed to be hyperexcitable in TLE. Here we studied whether the passive excitability of granule cells correlates with the severity of AHS.
Methods:   We analyzed the passive membrane properties of identified granule cells using patch-clamp recordings in acute tissue slices obtained from TLE surgery. Independent Wyler grading and GCD measurements were used to assess the severity of AHS.
Results:   The input resistances and membrane time constants of granule cells were reduced in high-grade versus low-grade AHS samples and negatively correlated with the degree of GCD. Granule cells possessed large Ba²⁺-sensitive, inwardly rectifying K⁺ conductances.
Discussion:   The increased leak conductance, likely mediated by K⁺ channels, does not argue for an increased excitability of granule cells but rather points to a neuroprotective mechanism in the sclerotic focus in TLE.     

PSA‐NCAM‐dependent GDNF signaling limits neurodegeneration and epileptogenesis in temporal lobe epilepsy

Polysialylated neuronal cell adhesion molecule (PSA‐NCAM), a polysialylated protein constitutively expressed in the hippocampus, is involved in neuronal growth, synaptic plasticity and neurotrophin signaling. In particular, PSA‐NCAM mediates Ret‐independent glial‐derived neurotrophic factor (GDNF) signaling, leading to downstream FAK activation. GDNF has potent seizure‐suppressant action, whereas PSA‐NCAM is upregulated by seizure activity. However, the involvement of Ret‐independent GDNF signaling in temporal lobe epilepsy (TLE) is not established. We tested the effects of PSA‐NCAM inactivation on neurodegeneration and epileptogenesis in a mouse model of TLE. In this model, unilateral intrahippocampal kainic acid (KA) injection induced degeneration of CA1, CA3c and hilar neurons, followed by spontaneous recurrent focal seizures. In the contralateral, morphologically preserved hippocampus, a long‐lasting increase of PSA‐NCAM immunoreactivity was observed. Inactivation of PSA‐NCAM by endoneuraminidase (EndoN) administration into the contralateral ventricle of KA‐treated mice caused severe degeneration of CA3a,b neurons and dentate gyrus granule cells in the epileptic focus, and led to early onset of focal seizures. This striking trans‐hemispheric alteration suggested that PSA‐NCAM mediates GDNF signaling, leading to transport of neuroprotective signals into the lesioned hippocampus. This hypothesis was confirmed by injecting GDNF antibodies into the contralateral hippocampus of KA‐treated mice, thereby reproducing the enhanced neurodegeneration seen after PSA‐NCAM inactivation. Furthermore, contralateral EndoN and anti‐GDNF treatment decreased GDNF family receptor α1 immunoreactivity and FAK phosphorylation in the epileptic focus. Thus, Ret‐independent GDNF signaling across the commissural projection might protect CA3a,b neurons and delay seizure onset. These findings implicate GDNF in the control of epileptogenesis and offer a possible mechanism explaining lesion asymmetry in mesial TLE.     

Impairment of dentate gyrus neuronal progenitor cell differentiation in a mouse model of temporal lobe epilepsy

Unilateral intrahippocampal injection of kainic acid (KA) in adult mice induces an epileptic focus replicating major histopathological features of temporal lobe epilepsy (TLE). In this model, neurogenesis is impaired in the lesioned dentate gyrus, although cell proliferation transiently is increased bilaterally in the subgranular zone (SGZ). To investigate further the relationship between epileptogenesis and neurogenesis, we compared the differentiation of cells born shortly before and after KA injection. Immunohistochemical staining for doublecortin and PSA-NCAM, two markers of young neurons, revealed a rapid downregulation of both markers ipsilaterally, whereas they were increased transiently on the contralateral side. To determine whether KA treatment directly affects neural progenitors in the SGZ, dividing cells were prelabeled with 5'-bromo-2'deoxyuridine (BrdU) treatment before unilateral injection of KA. Double staining with the proliferation marker PCNA showed that prelabeled BrdU cells survived KA exposure and proliferated bilaterally. Unexpectedly, the neuronal differentiation of these cells, as assessed after 2 weeks with doublecortin and NeuN triple-staining, occurred to the same extent as on the contralateral side. Only 5% of pre-labeled BrdU cells were GFAP-positive within the lesion. Therefore, SGZ progenitor cells committed to a neuronal phenotype before KA treatment complete their differentiation despite the rapid down-regulation of doublecortin and PSA-NCAM. These findings suggest impaired fate commitment and/or early differentiation of proliferating cells in the lesioned dentate gyrus. Loss of neurogenesis in this TLE model likely reflects an irreversible alteration of the SGZ germinal niche during development of the epileptic focus and may therefore be relevant for human TLE.     

Proportional loss of parvalbumin-immunoreactive synaptic boutons and granule cells from the hippocampus of sea lions with temporal lobe epilepsy

One in 26 people develop epilepsy and in these temporal lobe epilepsy (TLE) is common. Many patients display a pattern of neuron loss called hippocampal sclerosis. Seizures usually start in the hippocampus but underlying mechanisms remain unclear. One possibility is insufficient inhibition of dentate granule cells. Normally parvalbumin-immunoreactive (PV) interneurons strongly inhibit granule cells. Humans with TLE display loss of PV interneurons in the dentate gyrus but questions persist. To address this, we evaluated PV interneuron and bouton numbers in California sea lions (Zalophus californianus) that naturally develop TLE after exposure to domoic acid, a neurotoxin that enters the marine food chain during harmful algal blooms. Sclerotic hippocampi were identified by the loss of Nissl-stained hilar neurons. Stereological methods were used to estimate the number of granule cells and PV interneurons per dentate gyrus. Sclerotic hippocampi contained fewer granule cells, fewer PV interneurons, and fewer PV synaptic boutons, and the ratio of granule cells to PV interneurons was higher than in controls. To test whether fewer boutons was attributable to loss versus reduced immunoreactivity, expression of synaptotagmin-2 (syt2) was evaluated. Syt2 is also expressed in boutons of PV interneurons. Sclerotic hippocampi displayed proportional losses of syt2-immunoreactive boutons, PV boutons, and granule cells. There was no significant difference in the average numbers of PV- or syt2-positive boutons per granule cell between control and sclerotic hippocampi. These findings do not address functionality of surviving synapses but suggest reduced granule cell inhibition in TLE is not attributable to anatomical loss of PV boutons.     

Deficits in Behavioral and Neuronal Pattern Separation in Temporal Lobe Epilepsy

In temporal lobe epilepsy, the ability of the dentate gyrus to limit excitatory cortical input to the hippocampus breaks down, leading to seizures. The dentate gyrus is also thought to help discriminate between similar memories by performing pattern separation, but whether epilepsy leads to a breakdown in this neural computation, and thus to mnemonic discrimination impairments, remains unknown. Here we show that temporal lobe epilepsy is characterized by behavioral deficits in mnemonic discrimination tasks, in both humans (females and males) and mice (C57Bl6 males, systemic low-dose kainate model). Using a recently developed assay in brain slices of the same epileptic mice, we reveal a decreased ability of the dentate gyrus to perform certain forms of pattern separation. This is because of a subset of granule cells with abnormal bursting that can develop independently of early EEG abnormalities. Overall, our results linking physiology, computation, and cognition in the same mice advance our understanding of episodic memory mechanisms and their dysfunction in epilepsy.SIGNIFICANCE STATEMENT People with temporal lobe epilepsy (TLE) often have learning and memory impairments, sometimes occurring earlier than the first seizure, but those symptoms and their biological underpinnings are poorly understood. We focused on the dentate gyrus, a brain region that is critical to avoid confusion between similar memories and is anatomically disorganized in TLE. We show that both humans and mice with TLE experience confusion between similar situations. This impairment coincides with a failure of the dentate gyrus to disambiguate similar input signals because of pathologic bursting in a subset of neurons. Our work bridges seizure-oriented and memory-oriented views of the dentate gyrus function, suggests a mechanism for cognitive symptoms in TLE, and supports a long-standing hypothesis of episodic memory theories.     

Neurogenesis in the human hippocampus and its relevance to temporal lobe epilepsies

Ample evidence points to the dentate gyrus as anatomical region for persistent neurogenesis in the adult mammalian brain. This has been confirmed in a variety of animal models under physiological as well as pathophysiological conditions. Notwithstanding, similar experiments are difficult to perform in humans. Postmortem studies demonstrated persisting neurogenesis in the elderly human brain. In addition, neural precursor cells can be isolated from surgical specimens obtained from patients with intractable temporal lobe epilepsy (TLE) and propagated or differentiated into neuronal and glial lineages. It remains a controversial issue, whether epileptic seizures have an effect on or even increase hippocampal neurogenesis in humans. Recent data support the notion that seizures induce neurogenesis in young patients, whereas the capacity of neuronal recruitment and proliferation decreases with age. Animal models of TLE further indicate that these newly generated neurons integrate into epileptogenic networks and contribute to increased seizure susceptibility. However, pathomorphological disturbances within the epileptic hippocampus, such as granule cell dispersion (GCD), may not directly result from compromised neurogenesis. Still, the majority of adult TLE patients present with significant dentate granule cell loss at an end stage of the disease, which relates to severe memory and learning disabilities. In conclusion, surgical specimens obtained from TLE patients represent an important tool to study mechanisms of stem cell recruitment, proliferation and differentiation in the human brain. In addition, increasing availability of surgical specimens opens new avenues to systematically explore disease pathomechanisms in chronic epilepsies.     

Granule cell dispersion and aberrant neurogenesis in the adult hippocampus of an LIS1 mutant mouse

Human type 1 lissencephaly is a severe brain malformation associated with cognitive dysfunction and intractable epilepsy. Mutant mice with a heterozygous deletion of LIS1 show varying degrees of hippocampal abnormality and enhanced excitability. Whether a reduction of LIS1 function affects adult hippocampal neurogenesis, and if so, whether aberrant neurogenesis contributes to the generation of a disorganized hippocampus remain unknown. Previous reports indicate the presence of multiple pyramidal cell layers and granule cell dispersion in LIS1 mutant mice. Here we observed disruption of the subgranular zone and glial fibrillary acidic protein-immunoreactive radial astrocytes in the dentate gyrus of adult LIS1 mice. Using pulse-chase bromodeoxyuridine (BrdU) labeling combined with neuronal and glial antibody staining we provide evidence for ectopic adult neurogenesis in LIS1 mice. A gradually decreased survival rate for these newborn granule cells was also demonstrated in LIS1 mice 7 days after BrdU injection. This reduced survival rate was associated with impaired neuronal differentiation 28 days after BrdU administration. Thus, LIS1 haploinsufficiency can lead to abnormal cell proliferation, migration and differentiation in the adult dentate gyrus.     

Rapid and long-term alterations of hippocampal GABAB receptors in a mouse model of temporal lobe epilepsy

Alterations of gamma-aminobutyric acid (GABA)B receptor expression have been reported in human temporal lobe epilepsy (TLE). Here, changes in regional and cellular expression of the GABAB receptor subunits R1 (GBR1) and R2 (GBR2) were investigated in a mouse model that replicates major functional and histopathological features of TLE. Adult mice received a single, unilateral injection of kainic acid (KA) into the dorsal hippocampus, and GABAB receptor immunoreactivity was analysed between 1 day and 3 months thereafter. In control mice, GBR1 and GBR2 were distributed uniformly across the dendritic layers of CA1-CA3 and dentate gyrus. In addition, some interneurons were labelled selectively for GBR1. At 1 day post-KA, staining for both GBR1 and GBR2 was profoundly reduced in CA1, CA3c and the hilus, and no interneurons were visible anymore. At later stages, the loss of GABAB receptors persisted in CA1 and CA3, whereas staining increased gradually in dentate gyrus granule cells, which become dispersed in this model. Most strikingly, a subpopulation of strongly labelled interneurons reappeared, mainly in the hilus and CA3 starting at 1 week post-KA. In double-staining experiments, these cells were selectively labelled for neuropeptide Y. The number of GBR1-positive interneurons also increased contralaterally in the hilus. The rapid KA-induced loss of GABAB receptors might contribute to epileptogenesis because of a reduction in both presynaptic control of transmitter release and postsynaptic inhibition. In turn, the long-term increase in GABAB receptors in granule cells and specific subtypes of interneurons may represent a compensatory response to recurrent seizures.     

Adoptive transfer of T lymphocytes in immunodeficient mice influences epileptogenesis and neurodegeneration in a model of temporal lobe epilepsy

Intra-hippocampal injection of kainic acid (KA) in adult mice causes a focal lesion in the CA1 area and hilus of the dentate gyrus, as well as pronounced granule cell hypertrophy and dispersion. The lesion results in chronic focal seizures, with a two-week delay following KA-induced status epilepticus. Furthermore, seizures are preceded by infiltration of T lymphocytes into the lesioned tissue and of macrophage-like cells, strongly immunopositive for the monocyte marker F4/80, into the dentate gyrus, where they regulate granule cell dispersion and survival. Unexpectedly, depletion of CD4(+) and/or CD8(+) T lymphocytes by targeted gene deletion results in a marked shortening of the delay prior to seizure onset, suggesting a role of adaptive immunity in epileptogenesis (Zattoni et al. 2011, J. Neurosci. 31, 4037). Here, we investigated the specific role of adaptive immunity in this TLE model by adoptive i.v. transfer of immunopurified T cells in mutant mice lacking either CD4(+) T cells (MHCII-knockout), CD8(+) T cells (β2-microglobulin-knockout), or both populations (RAG1-knockout mice). EEG analysis in mutants mice injected with KA two days after the T cell transfer revealed that grafting of the missing T cell population had no influence on seizure onset, but strongly influenced F4/80(+) macrophage-like cell infiltration in the dentate gyrus. Specifically, CD8(+) T cells in β2-microgloblin-knockout mice enhanced macrophage recruitment, whereas CD4(+) T cells transferred in MHCII-knockout and in RAG1-knockout mice blocked macrophage infiltration, leading to reduced granule cell dispersion and survival, thereby worsening the KA-induced lesion. These results suggest that intact adaptive immunity is required for delayed seizure onset in this mouse model of TLE and unravel complex interactions between T cells and mononuclear phagocytes for the control of neuronal integrity and survival in the lesioned brain.     

Restoration of calbindin after fetal hippocampal CA3 cell grafting into the injured hippocampus in a rat model of temporal lobe epilepsy

Degeneration of the CA3 pyramidal and dentate hilar neurons in the adult rat hippocampus after an intracerebroventricular kainic acid (KA) administration, a model of temporal lobe epilepsy, leads to permanent loss of the calcium binding protein calbindin in major fractions of dentate granule cells and CA1 pyramidal neurons. We hypothesize that the enduring loss of calbindin in the dentate gyrus and the CA1 subfield after CA3-lesion is due to disruption of the hippocampal circuitry leading to hyperexcitability in these regions; therefore, specific cell grafts that are capable of both reconstructing the disrupted circuitry and suppressing hyperexcitability in the injured hippocampus can restore calbindin. We compared the effects of fetal CA3 or CA1 cell grafting into the injured CA3 region of adult rats at 45 days after KA-induced injury on the hippocampal calbindin. The calbindin immunoreactivity in the dentate granule cells and the CA1 pyramidal neurons of grafted animals was evaluated at 6 months after injury (i.e. at 4.5 months post-grafting). Compared with the intact hippocampus, the calbindin in "lesion-only" hippocampus was dramatically reduced at 6 months post-lesion. However, calbindin expression was restored in the lesioned hippocampus receiving CA3 cell grafts. In contrast, in the lesioned hippocampus receiving CA1 cell grafts, calbindin expression remained less than the intact hippocampus. Thus, specific cell grafting restores the injury-induced loss of calbindin in the adult hippocampus, likely via restitution of the disrupted circuitry. Since loss of calbindin after hippocampal injury is linked to hyperexcitability, re-expression of calbindin in both dentate gyrus and CA1 subfield following CA3 cell grafting may suggest that specific cell grafting is efficacious for ameliorating injury-induced hyperexcitability in the adult hippocampus. However, electrophysiological studies of KA-lesioned hippocampus receiving CA3 cell grafts are required in future to validate this possibility.     

Differential NPY mRNA expression in granule cells and interneurons of the rat dentate gyrus after kainic acid injection

Using in situ hybridization histochemistry neuropeptide Y (NPY) mRNA expression was investigated after intraperitoneal injection of kainic acid (KA) and after local application of KA or quinolinic acid into the dentate gyrus of the rat. Enhanced concentrations of NPY mRNA were observed in interneurons of the hilus, including presumptive fusiform neurons and pyramidal-shaped basket cells already 4 hours after initiation of limbic seizures by KA (10 mg/kg, i.p.). IncreaseD NPY expression persisted in neurons resistant to seizure-induced cell death (6–48 h after i.p. KA). Exceptionally high hybridization signals were found in interneurons of the hilus and the CA1 and CA3 sectors 8 months after KA-induced limbic seizures. In the granule cell layer only a transient but pronounced increase in NPY mRNA was observed 12–24 h after injection. Only moderate changes were observed in this cell layer at later intervals. Anticonvulsant treatment with thiopental, after a brief period of generalized seizures, prevented the increase in NPY mRNA in granule cells but not in interneurons. No change in NPY message was found also in granule cells of rats which responded with mild “wet dog shake” behvior but not with motor seizures to KA injection. Local injections of low doses of KA (0.05–0.2 nmol) or quinolinic acid (6.5–100 nmol) into the dentate gyrus of the hippocampus under deep thiopental anesthesia, after 24 h, resulted in increased concentrations of NPY message in interneurons of the ipsilateral, but not of the contralateral hilus and not in granule cells. Higher doses of the excitatory amino acid analogs caused partial neurodegeneration at the injection site, but enhanced NPY expression in interneurons of the contralateral dentate. Only the highest dose of quinolinic acid (100 nmol), resulting in general neuronal cell loss at the injection area, induced enhanced NPY mRNA expression also in granule cells of the contralateral dentate gyrus. The experiments suggest different mechanisms for NPY mRNA expression in interneurons and in granule cells of the dentate gyrus. Whereas in the stratum granulosum NPY mRNA expression was only observed after generalized limbic seizures, in hilar interneurons it was augmented by only moderate neuronal stimulation or directly by KA. © 1994 Wiley-Liss, Inc.