Mice deficient for the extracellular matrix glycoprotein tenascin-r show physiological and structural hallmarks of increased hippocampal excitability, but no increased susceptibility to seizures in the pilocarpine model of epilepsy.

Recognition molecules provide important cues for neuronal survival, axonal fasciculation, axonal pathfinding, synaptogenesis, synaptic plasticity, and regeneration. Our previous studies revealed a link between perisomatic inhibition and the extracellular matrix glycoprotein tenascin-R (TN-R). Therefore, we here studied neuronal excitability and epileptic susceptibility in mice constitutively deficient in TN-R. In vitro analysis of populational spikes in hippocampal slices of TN-R-deficient mice revealed a significant increase in multiple spikes in the CA1 region, as compared with wild-type mice. This difference between genotypes was only partially reduced after blockade of GABA(A) receptors with picrotoxin, indicating a deficit in GABAergic inhibition and an increase in intrinsic excitability of CA1 pyramidal cells in TN-R-deficient mice. Using a battery of immunohistochemical markers and histological stainings, we were able to identify two abnormalities in the hippocampus of TN-R-deficient mice possibly related to increased excitability: the high number of glial fibrillary acidic protein-positive astrocytes and low number of calretinin-positive interneurons in the CA1 and CA3 regions. In order to test whether the revealed abnormalities give rise to increased susceptibility to seizures in TN-R-deficient mice, we used the pilocarpine model of epilepsy. No genotype-specific differences were found with regard to the time-course of pilocarpine-induced and spontaneous seizures, neuronal cell loss, aberrant sprouting and distribution of synaptic and inhibitory interneuron markers. However, pilocarpine-induced astrogliosis and reduction in calretinin-positive interneurons were less pronounced in TN-R mutants, thereby resulting in an occlusion of effects induced by TN-R deficiency and pilocarpine. Thus, TN-R-deficient mutants show several electrophysiological and morphological hallmarks of increased neuronal excitability, which, however, do not give rise to more accelerated or severe epileptogenesis in the pilocarpine model of epilepsy.

Distribution of alpha and beta integrin subunits in the adult rat hippocampus after pilocarpine-induced neuronal cell loss, axonal reorganization and reactive astrogliosis.

Integrins are alphabeta-heterodimers that act as cell-extracellular matrix (ECM) and cell-cell adhesion molecules. During development, they are involved in axonal guidance, synaptogenesis, and in astrocytic maturation and migration. Here, we have examined the potential role of the integrin subunits alpha1-alpha5 and beta1-beta5 in axonal sprouting, synaptogenesis and reactive astrogliosis in the adult rat brain caused by pilocarpine-induced status epilepticus (SE). Strong hippocampal immunoreactivity of alpha1-alpha5, beta1, beta3, beta4, and beta5 was observed in the pia mater, in vascular endothelia, and in astrocytes at the pial surface. beta2 immunoreactivity was found exclusively in vascular endothelia. Pyramidal cells and interneurons of CA3-CA1, as well as hilar neurons revealed moderate alpha5 labeling in their cell bodies. Mossy fibers were immunoreactive for alpha2, beta4, and beta5. After pilocarpine-induced SE, strong immunoreactivity for alpha1, alpha2, alpha4, alpha5, beta1, beta3, and beta4 was observed in reactive astrocytes. Our results show that members of the integrin family are differently distributed in cellular and subcellular compartments of the hippocampus and undergo specific patterns of regulation, which may be important for lesion-induced reactive changes in the adult brain.

Annexin VII: an astroglial protein exhibiting a Ca2+-dependent subcellular distribution.

A fundamental issue in neuronal and glial cells is how intracellular rises in Ca2+ are coupled to signaling cascades and changes in subcellular morphology. We studied the expression and localization of annexin VII (synexin), a Ca(2+)-/GTP-dependent membrane fusion protein, in the human CNS. Here, we demonstrate the presence of two annexin VII isoforms (47 and 51 kDa) in human brain tissue as well as its exclusive expression in astroglial cells. An in vitro study of astrocyte-derived C6 rat glioblastoma cells expressing a GFP tagged annexin VII fusion protein demonstrates a sequential redistribution of the fusion protein in response to rising intracellular Ca2+ concentrations. Our findings indicate a role of annexin VII in the regulation of intracellular Ca(2+)-dependent processes in astroglial cells.

Temporal lobe epilepsy associated up-regulation of metabotropic glutamate receptors: correlated changes in mGluR1 mRNA and protein expression in experimental animals and human patients.

Aberrant axonal reorganization and altered distribution of neurotransmitter receptor subtypes have been proposed as major pathogenic mechanisms for hippocampal hyperexcitability in chronic temporal lobe epilepsies (TLE). Recent data point to excitatory class I metabotropic glutamate receptors (mGluR1 and mGluR5) as interesting candidates. Here, we have analyzed the hippocampal distribution and mRNA expression of mGluR1 and mGluR5 in two rat models of limbic seizures, i.e. electrical kindling and intraperitoneal kainate injections, as well as in human TLE. Quantitative RT-PCR analysis detected a significant increase of hippocampal mGluR1 gene transcript levels in kainate treated and kindled rats. In addition, microdissected hippocampal tissue samples localized this increase to the dentate gyrus. Using immunohistochemistry with mGluR1alpha subtype specific antibodies, increased labeling was observed within the dentate gyrus molecular layer (DG-ML). A similar pattern of increased mGluR1alpha neuropil staining was found within the DG-ML of epilepsy patients (n = 42) compared with peritumoral hippocampus specimens obtained from nonepileptic patients (biopsy controls, n = 3). This increase was detected in TLE patients with segmental hippocampal cell loss, as well as in TLE patients with focal lesions but no histopathological alterations of the hippocampus. In contrast, mGluR5 immunoreactivity and mRNA expression were not significantly altered in the DG-ML. Our data demonstrate a striking regional induction of mGluR1alpha in the hippocampal dentate gyrus of experimental animals with limbic seizures as well as in human patients with chronic, intractable TLE. This increase corresponds to functional alterations of class I mGluRs observed in seizure models and may significantly contribute to hippocampal hyperexcitability in focal human epilepsies.

Surviving granule cells of the sclerotic human hippocampus have reduced Ca(2+) influx because of a loss of calbindin-D(28k) in temporal lobe epilepsy.

In mesial temporal lobe epilepsy (mTLE), the predominant form of epilepsy in adults, and in animal models of the disease, there is a conspicuous loss of the intracellular Ca(2+)-binding protein calbindin-D(28k) (CB) from granule cells (GCs) of the dentate gyrus. The role of this protein in nerve cell function is controversial, but here we provide evidence for its role in controlling Ca(2+) influx into human neurons. In patients with Ammon's horn sclerosis (AHS), the loss of CB from GCs markedly increased the Ca(2+)-dependent inactivation of voltage-dependent Ca(2+) currents (I(Ca)), thereby diminishing Ca(2+) influx during repetitive neuronal firing. Introducing purified CB into GCs restored Ca(2+) current inactivation to levels observed in cells with normal CB content harvested from mTLE patients without AHS. Our data are consistent with the possibility of neuroprotection secondary to the CB loss. By limiting Ca(2+) influx through an enhanced Ca(2+)-dependent inactivation of voltage-dependent Ca(2+) channels during prolonged neuronal discharges, the loss of CB may contribute to the resistance of surviving human granule cells in AHS.

Up-regulation of the metabotropic glutamate receptor mGluR4 in hippocampal neurons with reduced seizure vulnerability.

Selective hippocampal cell loss and altered neurotransmitter receptor expression have been proposed as pathogenic mechanisms in the development of chronic mesial temporal lobe epilepsy (TLE). Studies in animal models point to metabotropic glutamate receptors (mGluRs) as modulators of hippocampal epileptogenesis. In addition, mGluRs may constitute specific targets for the development of novel anticonvulsive drugs. As mGluR4 represents an inhibitory class III mGluR associated with the reduction of intracellular cyclic AMP levels and calcium influx, we have analyzed the regional and cellular expression of mGluR4 in surgical hippocampal specimens obtained from patients with TLE by using immunohistochemistry and in situ hybridization. Although the hippocampi of control specimens (n = 11) were almost devoid of mGluR4 immunolabeling, all TLE specimens (n = 35) showed a striking up-regulation of mGluR4 immunoreactivity, in particular within the dentate gyrus. Immunoelectron microscopy localized the receptor protein to the periphery of presynaptic and postsynaptic membranes. In situ hybridization revealed increased transcript levels of mGluR4 in dentate granule cells and residual CA4 neurons of TLE specimens compared with controls. Our results suggest a potential role of mGluR4 in counteracting excitatory hippocampal activity and in modulating seizure-associated vulnerability of hippocampal neurons. These data may also provide a basis for pharmacological studies of mGluR4 agonists as potential novel drugs in the treatment of TLE.

Cellular pathology of hilar neurons in Ammon's horn sclerosis.

In addition to functionally affected neuronal signaling pathways, altered axonal, dendritic, and synaptic morphology may contribute to hippocampal hyperexcitability in chronic mesial temporal lobe epilepsies (MTLE). The sclerotic hippocampus in Ammon's horn sclerosis (AHS)-associated MTLE, which shows segmental neuronal cell loss, axonal reorganization, and astrogliosis, would appear particularly susceptible to such changes. To characterize the cellular hippocampal pathology in MTLE, we have analyzed hilar neurons in surgical hippocampus specimens from patients with MTLE. Anatomically well-preserved hippocampal specimens from patients with AHS (n = 44) and from patients with focal temporal lesions (non-AHS; n = 20) were studied using confocal laser scanning microscopy (CFLSM) and electron microscopy (EM). Hippocampal samples from three tumor patients without chronic epilepsies and autopsy samples were used as controls. Using intracellular Lucifer Yellow injection and CFLSM, spiny pyramidal, multipolar, and mossy cells as well as non-spiny multipolar neurons have been identified as major hilar cell types in controls and lesion-associated MTLE specimens. In contrast, none of the hilar neurons from AHS specimens displayed a morphology reminiscent of mossy cells. In AHS, a major portion of the pyramidal and multipolar neurons showed extensive dendritic ramification and periodic nodular swellings of dendritic shafts. EM analysis confirmed the altered cellular morphology, with an accumulation of cytoskeletal filaments and increased numbers of mitochondria as the most prominent findings. To characterize cytoskeletal alterations in hilar neurons further, immunohistochemical reactions for neurofilament proteins (NFP), microtubule-associated proteins, and tau were performed. This analysis specifically identified large and atypical hilar neurons with an accumulation of low weight NFP. Our data demonstrate striking structural alterations in hilar neurons of patients with AHS compared with controls and non-sclerotic MTLE specimens. Such changes may develop during cellular reorganization in the epileptogenic hippocampus and are likely to contribute to the pathogenesis or maintenance of temporal lobe epilepsy.

Molecular neuropathology of human mesial temporal lobe epilepsy.

With the recent progress in surgical treatment modalities, human brain tissue from patients with intractable focal epilepsies will increasingly become available for studies on the molecular pathology, electrophysiological changes and pathogenesis of human focal epilepsies. An inherent problem for studies on human temporal lobe epilepsy (TLE) is the lack of suitable controls. Strategies to alleviate this obstacle include the use of human post mortem samples, hippocampus from experimental animals and, in particular, the comparative analysis of surgical specimens from patients with Ammon's horn sclerosis (AHS) and with focal temporal lesions but anatomically preserved hippocampal structures. In this review we focus on selected aspects of the molecular neuropathology of TLE: (1) the potential impact of persisting calretinin-immunoreactive neurons with Cajal-Retzius cell morphology, (2) astrocytic tenascin-C induction and redistribution as potential regulator of aberrant axonal sprouting and (3) alterations of Ca2+ -mediated hippocampal signalling pathways. The diverse and complex changes described so far in human TLE specimens require a systematic interdisciplinary approach to distinguish primary, epileptogenic alterations and secondary, compensatory mechanisms in the pathogenesis of human temporal lobe epilepsies.

Distribution of voltage-dependent calcium channel beta subunits in the hippocampus of patients with temporal lobe epilepsy.

Voltage-dependent Ca2+ channels constitute a major class of plasma membrane channels through which a significant amount of extracellular Ca2+ enters neuronal cells. Their pore-forming alpha1 subunits are associated with cytoplasmic regulatory beta subunits, which modify the distinct biophysical and pharmacological properties of the alpha1 subunits. Studies in animal models indicate altered expression of alpha1 and/or beta subunits in epilepsy. We have focused on the regulatory beta subunits and have analysed the immunoreactivity patterns of the beta1, beta2, beta3 and beta4 subunits in the hippocampus of patients with temporal lobe epilepsy (n = 18) compared to control specimens (n = 2). Temporal lobe epilepsy specimens were classified as Ammon's horn sclerosis (n = 9) or focal lesions without alteration of hippocampal cytoarchitecture (n = 9). Immunoreactivity for the beta subunits was observed in neuronal cell bodies, dendrites and neuropil. The beta1, beta2 and beta3 subunits were found mainly in cell bodies while the beta4 subunit was primarily localized to dendrites. Compared to the control specimens, epilepsy specimens of the Ammon's horn sclerosis and of the lesion group showed a similar beta subunit distribution, except for beta1 and beta2 staining in the Ammon's horn sclerosis group: in the severely sclerotic hippocampal subfields of these specimens, beta1 and beta2 immunoreactivity was enhanced in some of the remaining neuronal cell bodies and, in addition, strongly marked dendrites. Thus, hippocampal neurons apparently express multiple classes of beta subunits which segregate into particular subcellular domains. In addition, the enhancement of beta1 and beta2 immunoreactivity in neuronal cell bodies and the additional shift of the beta1 and beta2 subunits into the dendritic compartment in severely sclerotic hippocampal regions indicate specific changes in Ammon's horn sclerosis. Altered expression of these beta subunits may lead to increased currents carried by voltage-dependent calcium channels and to enhanced synaptic excitability.

5'-Nucleotidase activity indicates sites of synaptic plasticity and reactive synaptogenesis in the human brain.

The localization and morphological assessment of plastic or newly formed synapses in the human brain remains difficult due to the lack of specific markers. The ectoenzyme 5'-nucleotidase may represent a useful marker of these structures, since in adult rodents synaptic 5'-nucleotidase activity is restricted to sites of spontaneous synaptic turnover and induced reactive synaptogenesis. However, it is unclear to what extent synaptic 5'-nucleotidase activity occurs in the normal human brain, and whether reactive synaptogenesis, as seen e.g. in temporal lobe epilepsy (TLE), is associated with this ectoenzyme. Therefore, we have investigated the histochemical distribution of 5'-nucleotidase in hippocampal control specimens (n = 3) and in the hippocampus of TLE patients (n = 13). In controls, 5'-nucleotidase activity was present in the dentate gyrus molecular layer (DG-ML) and the mossy fiber termination field within the CA4 and CA3 subfields. Compared with controls, TLE specimens revealed markedly increased 5'-nucleotidase labeling in the DG-ML, implying TLE-associated reactive synaptogenesis in this hippocampal region. In contrast to GAP-43, synaptophysin, and dynorphin A, synaptic 5'-nucleotidase activity may serve as a potential specific indicator of plastic synapses or newly formed terminals in the human brain and prove useful for the study of diseases involving aberrant sprouting or altered synaptic plasticity.

Altered patterns of Ca2+/calmodulin-dependent protein kinase II and calcineurin immunoreactivity in the hippocampus of patients with temporal lobe epilepsy.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) and calcineurin represent neuronal Ca2+-dependent enzymes which dynamically modify several common substrates in the mammalian brain via phosphorylation/dephosphorylation cycles. Studies in animal models indicate that altered expression and activity of these enzymes may be involved in epilepsy. We have analyzed their immunohistochemical distribution in hippocampi of 28 temporal lobe epilepsy (TLE) patients and 13 controls. TLE specimens were classified as Ammon's horn sclerosis (AHS) or focal lesions without alteration of hippocampal cytoarchitecture. Compared to control and lesion-associated TLE specimens, striking changes in the distribution pattern of both enzymes were found in the dentate gyrus (DG) of AHS specimens: Whereas CaMKII labeling was significantly increased in the granule cell somata and their proximal dendrites, calcineurin immunoreactivity was significantly reduced in the granule cell somata. Furthermore, calcineurin staining in controls showed high levels in the inner molecular layer with a sharp demarcation towards the outer molecular layer. In AHS, calcineurin staining was reduced in the inner molecular layer, with partial loss of this demarcation. These findings raise the possibility, that an up-regulation of CaMKII with a concomitant down-regulation of calcineurin in the DG of AHS specimens may cause a pathogenetically relevant imbalance of neuronal Ca2+/calmodulin-dependent phosphorylation/dephosphorylation systems.

Plectin in the human central nervous system: predominant expression at pia/glia and endothelia/glia interfaces.

Plectin is a high molecular weight protein that serves as a versatile cytoskeletal cross-linker molecule. Mutations of the human plectin gene have recently been identified to cause the autosomal recessive disorder epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). A subgroup of EBS-MD patients display signs of a neurodegenerative disorder suggesting that the expression of defective plectin may also interfere with the structural and functional integrity of the human central nervous system. However, the expression pattern of plectin in the human brain is still unknown. We therefore analyzed the immunohistochemical distribution of plectin in normal hippocampal specimens obtained at autopsy and in neocortical and hippocampal tissue of patients who had undergone epilepsy surgery. In general, plectin-immunoreactive cells were identified as capillary endothelia and astrocytes. A striking feature seen in all specimens was the accentuated plectin immunoreactivity of astrocytic end feet abutting on blood vessels and on the pial surface. Furthermore, the analysis of hippocampal tissue of epilepsy patients with Ammon's horn sclerosis (AHS) revealed a strong plectin labeling of reactive astrocytes. The latter finding suggests that the up-regulation of plectin, which parallels the increase of glial fibrillary acidic protein, may be a general feature of reactive astroglia. The predominant expression of plectin at pia/glia and endothelia/glia interfaces in the human brain indicates that plectin may have an integral role in the structural organization of the blood-brain barrier and the leptomeninges.