GABA-A Receptors Mediate Tonic Inhibition and Neurosteroid Sensitivity in the Brain.

Neurosteroids like allopregnanolone (AP) are positive allosteric modulators of synaptic and extrasynaptic GABA-A receptors. AP and related neurosteroids exhibit a greater potency for δ-containing extrasynaptic receptors. The δGABA-A receptors, which are expressed extrasynaptically in the dentate gyrus and other regions, contribute to tonic inhibition, promoting network shunting as well as reducing seizure susceptibility. Levels of endogenous neurosteroids fluctuate with ovarian cycle. Natural and synthetic neurosteroids maximally potentiate tonic inhibition in the hippocampus and provide robust protection against a variety of limbic seizures and status epilepticus. Recently, a consensus neurosteroid pharmacophore model has been proposed at extrasynaptic δGABA-A receptors based on structure-activity relationship for functional activation of tonic currents and seizure protection. Aside from anticonvulsant actions, neurosteroids have been found to be powerful anxiolytic and anesthetic agents. Neurosteroids and Zn2+ have preferential affinity for δ-containing receptors. Thus, Zn2+ can prevent neurosteroid activation of extrasynaptic δGABA-A receptor-mediated tonic inhibition. Recently, we demonstrated that Zn2+ selectively inhibits extrasynaptic δGABA-A receptors and thereby fully prevents AP activation of tonic inhibition and seizure protection. We confirmed that neurosteroids exhibit greater sensitivity at extrasynaptic δGABA-A receptors. Overall, extrasynaptic GABA-A receptors are primary mediators of tonic inhibition in the brain and play a key role in the pathophysiology of epilepsy and other neurological disorders.

Exclusive Activation of Caspase-3 in Mossy Fibers and Altered Dynamics of Autophagy Markers in the Mice Hippocampus upon Status Epilepticus Induced by Kainic Acid.

Epileptic seizures are generally associated with pathological changes in the hippocampus such as astrogliosis, mossy fiber sprouting, and neuronal damage. However, more than 30% of temporal lobe epilepsy in humans shows neither neuronal damage nor mossy fiber sprouting despite chronic epileptic seizures. A similar situation exists in certain commonly used strains of mice, specifically C57BL/6 and BALB/c, which exhibit epileptic seizures, but no neuronal damage upon kainic acid administration. This suggests that intrinsic factors may influence the pathological manifestations of epilepsy. Mechanisms which are behind the resistance of hippocampal cells to KA-induced neuronal death are unknown. Autophagy seems to be involved in the pathogenesis of many brain insults and to have a dual nature in neuroprotection and cell death. This study addresses the role of autophagy upon status epilepticus (SE) that has been induced by kainic acid (KA) in the C57BL/6 strain which is classified as seizure resistant. We analyzed the dynamics in the expression of autophagic and cell death markers in the hippocampus upon SE. Immunofluorescence data show that KA did not induce neuronal death in the hippocampal CA1-CA3 subfields; however, it leads to an exclusive activation of caspase-3 in the mossy fibers. We also found alterations in the expression of core proteins of the autophagic machinery. Levels of MAP1LC3, phospho-mTOR/mTOR, and Beclin 1 were significantly increased after induction of seizures. However, levels of Atg3, Atg14, Atg5-Atg12, Atg7, BAG3, Hsp70, and LAMP1 showed no significant alterations compared to controls. Although KA did not induce neuronal death, this study provides morphological and biochemical evidence that status epilepticus induced by KA activates caspase-3 in mossy fibers and induces autophagy in the C57BL/6 hippocampus. These data indicate that autophagic factors may modulate the sensitivity of pyramidal cells to KA and that autophagy may constitute a part of an endogenous neuroprotective arsenal which might be behind the resistance of C57BL/6-hippocampal cells to KA-induced neuronal death.

Roles of Rho guanine nucleotide triphosphatases in hippocampal mossy fiber sprouting in the pentylenetetrazole kindling model.

BACKGROUND: One unique feature of chronic human and experimental epilepsy is hippocampal dentate granule cell axon (mossy fiber) sprouting which creates an aberrant positive-feedback circuit that may be epileptogenic. However, the mechanism underlying this process remains unclear. Rho guanine nucleotide triphosphatases (RhoGTP ases) Rac1 and RhoA are important regulators of axon growth and synaptic plasticity and can be blocked by treatment with fasudil. We hypothesized that Rac1 and RhoA are involved in aberrant mossy fiber sprouting (MFS). METHODS: A temporal lobe epilepsy model was established by intraperitoneal pentylenetetrazole (PTZ) injection for animals in PTZ group, and fasudil was injected 30 minutes prior to PTZ injection for animals in PTZ + Fas group. The expression of Rac1 and RhoA in the rat hippocampus was tested at different time points by immunohistochemistry, Western blot and quantitative real-time PCR. Mossy fiber sprouting in the hippocampus was evaluated by Timm staining. RESULTS: Rac1 and RhoA were significantly up-regulated in the PTZ group, and as predicted, the degree of aberrant MFS was correspondingly increased. However, the expression of Rac1 and RhoA was not inhibited in the PTZ + Fas group, and the epileptiform activity, EEG and aberrant MFS were not suppressed following PTZ + Fas treatment. CONCLUSIONS: RhoGTPases play a role in MFS but fasudil is not sufficient to inhibit RhoGTPases and MFS in the PTZ kindling model.

Maintenance of long-term potentiation in hippocampal mossy fiber-CA3 pathway requires fine-tuned MMP-9 proteolytic activity.

Mechanisms of synaptic plasticity involve proteolytic activity mediated by a complex system of proteases, including members of metalloproteinase (MMP) family. In particular, MMP-9 is critical in LTP maintenance in the Schaffer collateral-CA1 pathway and in the acquisition of hippocampus-dependent memory. Recent studies from this laboratory revealed that in the mossy fiber-CA3 (MF-CA3) projection, where LTP induction and expression are largely presynaptic, MMPs blockade disrupts LTP maintenance and that LTP induction is associated with increased MMP-9 expression. Here we used acute brain slices from MMP-9 knock-out mice and transgenic rats overexpressing MMP-9 to determine how manipulations in endogenous MMP-9 affect LTP in the MF-CA3 projection. Both types of transgenic models showed a normal basal synaptic transmission and short-term plasticity. Interestingly, the maintenance of LTP induced in slices from knock-out mice and overexpressing rats was nearly abolished. However, in the presence of active MMP-9, a gradual fEPSP autopotentiation was observed and tetanization evoked a marked LTP in knock-out mice. Additionally, in MMP-9-treated slices from wild-type mice, fEPSP autopotentiation also occurred and partially occluded LTP. This indicates that exogenous protease can restore LTP in null mice whereas in the wild-type, MMP-9 excess impairs LTP. We expected that LTP maintenance in transgenic rats could be re-established by a partial MMP blockade but non-saturating concentrations of MMP inhibitor were ineffective. In conclusion, we demonstrate that LTP maintenance in MF-CA3 pathway requires fine-tuned MMP-9 activity and raises the possibility that altered MMP-9 level might be detrimental for cognitive processes as observed in some neuropathologies.

Dab2IP GTPase activating protein regulates dendrite development and synapse number in cerebellum.

DOC-2/DAB-2 interacting protein (Dab2IP) is a GTPase activating protein that binds to Disabled-1, a cytosolic adapter protein involved in Reelin signaling and brain development. Dab2IP regulates PI3K-AKT signaling and is associated with metastatic prostate cancer, abdominal aortic aneurysms and coronary heart disease. To date, the physiological function of Dab2IP in the nervous system, where it is highly expressed, is relatively unknown. In this study, we generated a mouse model with a targeted disruption of Dab2IP using a retrovirus gene trap strategy. Unlike reeler mice, Dab2IP knock-down mice did not exhibit severe ataxia or cerebellar hypoplasia. However, Dab2IP deficiency produced a number of cerebellar abnormalities such as a delay in the development of Purkinje cell (PC) dendrites, a decrease in the parallel fiber synaptic marker VGluT1, and an increase in the climbing fiber synaptic marker VGluT2. These findings demonstrate for the first time that Dab2IP plays an important role in dendrite development and regulates the number of synapses in the cerebellum.

Lovastatin modulates glycogen synthase kinase-3ß pathway and inhibits mossy fiber sprouting after pilocarpine-induced status epilepticus.

This study was undertaken to assay the effect of lovastatin on the glycogen synthase kinase-3 beta (GSK-3ß) and collapsin responsive mediator protein-2 (CRMP-2) signaling pathway and mossy fiber sprouting (MFS) in epileptic rats. MFS in the dentate gyrus (DG) is an important feature of temporal lobe epilepsy (TLE) and is highly related to the severity and the frequency of spontaneous recurrent seizures. However, the molecular mechanism of MFS is mostly unknown. GSK-3ß and CRMP-2 are the genes responsible for axonal growth and neuronal polarity in the hippocampus, therefore this pathway is a potential target to investigate MFS. Pilocarpine-induced status epilepticus animal model was taken as our researching material. Western blot, histological and electrophysiological techniques were used as the studying tools. The results showed that the expression level of GSK-3ß and CRMP-2 were elevated after seizure induction, and the administration of lovastatin reversed this effect and significantly reduced the extent of MFS in both DG and CA3 region in the hippocampus. The alteration of expression level of GSK-3ß and CRMP-2 after seizure induction proposes that GSK-3ß and CRMP-2 are crucial for MFS and epiletogenesis. The fact that lovastatin reversed the expression level of GSK-3ß and CRMP-2 indicated that GSK-3ß and CRMP-2 are possible to be a novel mechanism of lovatstain to suppress MFS and revealed a new therapeutic target and researching direction for studying the mechanism of MFS and epileptogenesis.

Long term potentiation affects intracellular metalloproteinases activity in the mossy fiber-CA3 pathway.

Matrix Metalloproteinases (MMPs) are a family of endopeptidases known to process extracellular proteins. In the last decade, studies carried out mainly on the Schaffer collateral-CA1 hippocampal projection have provided solid evidence that MMPs regulate synaptic plasticity and learning. Recently, our group has shown that MMP blockade disrupts LTP maintenance also in the mossy fiber-CA3 (mf-CA3) projection (Wojtowicz and Mozrzymas, 2010), where LTP mechanisms are profoundly different (NMDAR-independent and presynaptic expression site). However, how plasticity of this pathway correlates with activity and expression of MMPs remains unknown. Interestingly, several potential MMP substrates (especially of gelatinases) are localized intracellularly but little is known about MMP activity in this compartment. In the present study we have asked whether LTP is associated with the expression and activity of gelatinases in apparent intra- and extracellular compartments along mf-CA3 projection. In situ zymography showed that LTP induction was associated with increased gelatinases activity in the cytoplasm of the hilar and CA3 neurons. Using gelatin zymography, immunohistochemistry and immunofluorescent staining we found that this effect was due to de novo synthesis and activation of MMP-9 which, 2-3h after LTP induction was particularly evident in the cytoplasm. In contrast, MMP-2 was localized preferentially in the nuclei and was not affected by LTP induction. In conclusion, we demonstrate that LTP induction in the mf-CA3 pathway correlates with increased expression and activity of MMP-9 and provide the first evidence that this increase is particularly evident in the neuronal cytoplasm and nucleus.

Glutamate decarboxylase 67 is expressed in hippocampal mossy fibers of temporal lobe epilepsy patients.

Recently, expression of glutamate decarboxylase-67 (GAD67), a key enzyme of GABA synthesis, was detected in the otherwise glutamatergic mossy fibers of the rat hippocampus. Synthesis of the enzyme was markedly enhanced after experimentally induced status epilepticus. Here, we investigated the expression of GAD67 protein and mRNA in 44 hippocampal specimens from patients with mesial temporal lobe epilepsy (TLE) using double immunofluorescence histochemistry, immunoblotting, and in situ hybridization. Both in specimens with (n = 37) and without (n = 7) hippocampal sclerosis, GAD67 was highly coexpressed with dynorphin in terminal areas of mossy fibers, including the dentate hilus and the stratum lucidum of sector CA3. In the cases with Ammon's horn sclerosis, also the inner molecular layer of the dentate gyrus contained strong staining for GAD67 immunoreactivity, indicating labeling of mossy fiber terminals that specifically sprout into this area. Double immunofluorescence revealed the colocalization of GAD67 immunoreactivity with the mossy fiber marker dynorphin. The extent of colabeling correlated with the number of seizures experienced by the patients. Furthermore, GAD67 mRNA was found in granule cells of the dentate gyrus. Levels, both of GAD67 mRNA and of GAD67 immunoreactivity were similar in sclerotic and nonsclerotic specimens and appeared to be increased compared to post mortem controls. Provided that the strong expression of GAD67 results in synthesis of GABA in hippocampal mossy fibers this may represent a self-protecting mechanism in TLE. In addition GAD67 expression also may result in conversion of excessive intracellular glutamate to nontoxic GABA within mossy fiber terminals.

Glutamic acid decarboxylase immunoreactivity in the mossy fiber terminals of the hippocampus of genetic absence epileptic rats.

AIM: Genetic absence epilepsy rats from Strasbourg (GAERS) provide a model of absence epilepsy. Although excessive GABA mediation within the thalamo-cortico-thalamic circuit has been shown to play a role in absence epilepsy, neuronal networks of hippocampus have recently received attention. Glutamic acid decarboxylase (GAD) was previously shown to be increased after convulsive seizures in the mossy fiber terminals (MFTs) of hippocampus. The aim of the present study was to investigate whether the change in the level of this enzyme in convulsive seizures is also observed in rats having genetic absence epilepsy. MATERIAL AND METHODS: Hippocampal CA3 and dentate regions were processed for transmission electron microscopic evaluations. Thin sections were incubated with anti-GAD65/67 antibody. The NIH Image Analysis program was used for the quantitative analysis. RESULTS: It was observed that GAD65/67 immunoreactivity was positive in CA3 and dentate gyrus MFTs of both groups and the difference in the density of immunolabeling between the groups was not statistically significant. CONCLUSION: The present study demonstrated that GABA synthesizing enzyme, GAD, is found in MFTs of Wistar and GAERS hippocampus and this enzyme does not show an increase in these terminals in absence epilepsy, in contrast to convulsive seizures.

Rac1 and Rac3 GTPases regulate the development of hilar mossy cells by affecting the migration of their precursors to the hilus.

We have previously shown that double deletion of the genes for Rac1 and Rac3 GTPases during neuronal development affects late developmental events that perturb the circuitry of the hippocampus, with ensuing epileptic phenotype. These effects include a defect in mossy cells, the major class of excitatory neurons of the hilus. Here, we have addressed the mechanisms that affect the loss of hilar mossy cells in the dorsal hippocampus of mice depleted of the two Rac GTPases. Quantification showed that the loss of mossy cells was evident already at postnatal day 8, soon after these cells become identifiable by a specific marker in the dorsal hilus. Comparative analysis of the hilar region from control and double mutant mice revealed that synaptogenesis was affected in the double mutants, with strongly reduced presynaptic input from dentate granule cells. We found that apoptosis was equally low in the hippocampus of both control and double knockout mice. Labelling with bromodeoxyuridine at embryonic day 12.5 showed no evident difference in the proliferation of neuronal precursors in the hippocampal primordium, while differences in the number of bromodeoxyuridine-labelled cells in the developing hilus revealed a defect in the migration of immature, developing mossy cells in the brain of double knockout mice. Overall, our data show that Rac1 and Rac3 GTPases participate in the normal development of hilar mossy cells, and indicate that they are involved in the regulation of the migration of the mossy cell precursor by preventing their arrival to the dorsal hilus.

Imaging of sialidase activity in rat brain sections by a highly sensitive fluorescent histochemical method.

Sialidase (EC 3.2.1.18) removes sialic acid from sialoglycoconjugates. Since sialidase extracellularly applied to the rat hippocampus influences many neural functions, including synaptic plasticity and innervations of glutamatergic neurons, endogenous sialidase activities on the extracellular membrane surface could also affect neural functions. However, the distribution of sialidase activity in the brain remains unknown. To visualize extracellular sialidase activity on the membrane surface in the rat brain, acute brain slices were incubated with 5-bromo-4-chloroindol-3-yl-α-d-N-acetylneuraminic acid (X-Neu5Ac) and Fast Red Violet LB (FRV LB) at pH 7.3. After 1h, myelin-abundant regions showed intense fluorescence in the rat brain. Although the hippocampus showed weak fluorescence in the brain, mossy fiber terminals in the hippocampus showed relatively intense fluorescence. These fluorescence intensities were attenuated with a sialidase-specific inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA, 1mM). Additionally, the fluorescence intensities caused by X-Neu5Ac and FRV LB were correlated with the sialidase activity measured with 4-methylumbelliferyl-α-d-N-acetylneuraminic acid (4MU-Neu5Ac), a classical substrate for quantitative measurement of sialidase activity, in each brain region. Therefore, staining with X-Neu5Ac and FRV LB is specific for sialidase and useful for quantitative analysis of sialidase activities. The results suggest that white matter of the rat brain has intense sialidase activity.

Synergistic interactions between kainate and mGlu receptors regulate bouton Ca signalling and mossy fibre LTP.

It is currently unknown why glutamatergic presynaptic terminals express multiple types of glutamate receptors. We have addressed this question by studying both acute and long-term regulation of mossy fibre function in the hippocampus. We find that inhibition of both mGlu1 and mGlu5 receptors together can block the induction of mossy fibre LTP. Furthermore, mossy fibre LTP can be induced by the pharmacological activation of either mGlu1 or mGlu5 receptors, provided that kainate receptors are also stimulated. Like conventional mossy fibre LTP, chemically-induced mossy fibre LTP (chem-LTPm) depends on Ca²âº release from intracellular stores and the activation of PKA. Similar synergistic interactions between mGlu receptors and kainate receptors were observed at the level of Ca²âº signalling in individual giant mossy fibre boutons. Thus three distinct glutamate receptors interact, in both an AND and OR gate fashion, to regulate both immediate and long-term presynaptic function in the brain.

Critical involvement of postsynaptic protein kinase activation in long-term potentiation at hippocampal mossy fiber synapses on CA3 interneurons.

Hippocampal mossy fiber (MF) synapses on area CA3 lacunosum-moleculare (L-M) interneurons are capable of undergoing a Hebbian form of NMDA receptor (NMDAR)-independent long-term potentiation (LTP) induced by the same type of high-frequency stimulation (HFS) that induces LTP at MF synapses on pyramidal cells. LTP of MF input to L-M interneurons occurs only at synapses containing mostly calcium-impermeable (CI)-AMPA receptors (AMPARs). Here, we demonstrate that HFS-induced LTP at these MF-interneuron synapses requires postsynaptic activation of protein kinase A (PKA) and protein kinase C (PKC). Brief extracellular stimulation of PKA with forskolin (FSK) alone or in combination with 1-Methyl-3-isobutylxanthine (IBMX) induced a long-lasting synaptic enhancement at MF synapses predominantly containing CI-AMPARs. However, the FSK/IBMX-induced potentiation in cells loaded with the specific PKA inhibitor peptide PKI(6-22) failed to be maintained. Consistent with these data, delivery of HFS to MFs synapsing onto L-M interneurons loaded with PKI(6-22) induced posttetanic potentiation (PTP) but not LTP. Hippocampal sections stained for the catalytic subunit of PKA revealed abundant immunoreactivity in interneurons located in strata radiatum and L-M of area CA3. We also found that extracellular activation of PKC with phorbol 12,13-diacetate induced a pharmacological potentiation of the isolated CI-AMPAR component of the MF EPSP. However, HFS delivered to MF synapses on cells loaded with the PKC inhibitor chelerythrine exhibited PTP followed by a significant depression. Together, our data indicate that MF LTP in L-M interneurons at synapses containing primarily CI-AMPARs requires some of the same signaling cascades as does LTP of glutamatergic input to CA3 or CA1 pyramidal cells.

ZnT-1, ZnT-3, CaMK II, PRG-1 expressions in hippocampus following neonatal seizure-induced cognitive deficit in rats.

Epilepsy in children is associated with a broad spectrum of cognitive deficits, which is associated with hippocampal mossy fiber sprouting. The underlying molecular mechanisms involved in mossy fiber sprouting in hippocampus following developmental seizures are not completely known. We studied the timing of cognitive dysfunction following neonatal seizures and the relation of this cognitive impairment to zinc transporter 1 (ZnT-1), 3 (ZnT-3), calcium/calmodulin-dependent protein kinase II (CaMK II), plasticity-related gene 1 (PRG-1) expression in hippocampus. A seizure was induced by inhalant flurothyl daily in neonatal Sprague-Dawley rats from postnatal day 6 (P6). Rats were assigned into the single-seizure group (SS), the recurrent-seizure group (RS, seizures induced in six consecutive days), and the control group. During P41-P46 and P85-P90, the rats were tested for spatial learning and memory abilities with automatic Morris water maze task. At P90, mossy fiber sprouting and gene expression in hippocampus were determined subsequently by Timm staining and RT-PCR methods. The escape latencies from the water maze were significantly longer in rats of RS group than those of the control and SS groups at d4 of the first maze test and at d3, d4 of the second maze test. As far as Spatial Probe Test was concerned, the frequency of passing through the platform quadrant was significantly decreased in RS group than that in control and SS groups in the entire two probe tests. In rats with recurrent seizures (RS group), there was an increased distribution of Timm granules in both the supragranular region of the dentate gyrus and the stratum pyramidale of CA3 subfield in RS group, while remaining barely visible in control and SS groups; the Timm scores in CA3 and dentate gyrus in the RS animals were significantly higher than that in the control and SS groups. RT-PCR densitometry analysis showed that the ratios of hippocampal ZnT-1 to beta-actin of SS and RS group were decreased significantly compared with that of control group. Meanwhile, CaMK II to beta-actin of RS group was markedly lower compared with those of SS and control groups. Our results suggest that the long-term adverse effects of recurrent neonatal seizures on cognition and mossy fiber sprouting may be associated with the down-regulated expression of ZnT-1 and CaMK II in hippocampus.

Localization of N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) expression in mouse brain: A new perspective on N-acylethanolamines as neural signaling molecules.

N-acylethanolamines (NAEs) are membrane-derived lipids that are utilized as signaling molecules in the nervous system (e.g., the endocannabinoid anandamide). An N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) that catalyzes formation of NAEs was recently identified as a member of the zinc metallohydrolase family of enzymes. NAPE-PLD(-/-) mice have greatly reduced brain levels of long-chain saturated NAEs but wild-type levels of polyunsaturated NAEs (e.g., anandamide), suggesting an important role for NAPE-PLD in the biosynthesis of at least a subset of endogenous NAEs in the mammalian nervous system. To provide a neuroanatomical basis for investigation of NAPE-PLD function, here we have analyzed expression of NAPE-PLD in the mouse brain using mRNA in situ hybridization and immunocytochemistry. NAPE-PLD(-/-) mice were utilized to establish the specificity of probes/antibodies used. The most striking feature of NAPE-PLD expression in the brain was in the dentate gyrus, where a strong mRNA signal was detected in granule cells. Accordingly, immunocytochemical analysis revealed intense NAPE-PLD immunoreactivity in the axons of granule cells (mossy fibers). Intense NAPE-PLD immunoreactivity was also detected in axons of the vomeronasal nerve that project to the accessory olfactory bulb. NAPE-PLD expression was detected in other brain regions (e.g., hippocampus, cortex, thalamus, hypothalamus), but the intensity of immunostaining was weaker than in mossy fibers. Collectively, the data obtained indicate that NAPE-PLD is expressed by specific populations of neurons in the brain and targeted to axonal processes. We suggest that NAEs generated by NAPE-PLD in axons may act as anterograde synaptic signaling molecules that regulate the activity of postsynaptic neurons.

Acute and chronic changes in glycogen phosphorylase in hippocampus and entorhinal cortex after status epilepticus in the adult male rat.

Glial cells provide energy substrates to neurons, in part from glycogen metabolism, which is influenced by glycogen phosphorylase (GP). To gain insight into the potential subfield and laminar-specific expression of GP, histochemistry can be used to evaluate active GP (GPa) or totalGP (GPa + GPb). Using this approach, we tested the hypothesis that changes in GP would occur under pathological conditions that are associated with increased energy demand, i.e. severe seizures (status epilepticus or 'status'). We also hypothesized that GP histochemistry would provide insight into changes in the days and weeks after status, particularly in the hippocampus and entorhinal cortex, where there are robust changes in structure and function. One hour after the onset of pilocarpine-induced status, GPa staining was reduced in most regions of the hippocampus and entorhinal cortex relative to saline-injected controls. One week after status, there was increased GPa and totalGP, especially in the inner molecular layer, where synaptic reorganization of granule cell mossy fibre axons occurs (mossy fibre sprouting). In addition, patches of dense GP reactivity were evident in many areas. One month after status, levels of GPa and totalGP remained elevated in some areas, suggesting an ongoing role of GP or other aspects of glycogen metabolism, possibly due to the evolution of intermittent, recurrent seizures at approximately 3-4 weeks after status. Taken together, the results suggest that GP is dynamically regulated during and after status in the adult rat, and may have an important role in the pilocarpine model of epilepsy.

[3H]-L-685,458 as a radiotracer that maps gamma-secretase complex in the rat brain: relevance to Abeta genesis and presence of active presenilin-1 components.

Gamma-secretase is a multimeric enzyme important for normal cell/neuronal proliferation, differentiation and plasticity. Determining in vivo gamma-secretase expression and activity remains a challenge because its subunit proteins can exist in immature and preassembled forms, but may execute cellular roles irrelevant to gamma-site cleavage. In this study, we characterized [3H]-L-685,458 as a radiotracer for the detection of active gamma-secretase in adult rat brain. In vitro autoradiography indicated that [3H]-L-685,458 binding was saturatable, displaceable by peptidomimetic and small molecule gamma-secretase inhibitors, and exhibited rapid association and dissociation kinetics. In cultured hippocampal slices, [3H]-L-685,458 binding density correlated with Abeta reduction following in-dish dosing of this radioligand or a non-radioactive gamma-secretase inhibitor. [3H]-L-685,458 binding sites in the adult brain were differentially distributed across regions and laminas, with heavy binding localized to the olfactory glomeruli, hippocampal CA3 and cerebellar molecular layer, and moderate binding in the cerebral cortex, amygdala and selected subcortical regions. All of these regions showed labeling for presenilin-1 N-terminal fragments (PS1-NTFs). A distinct correlation of dense binding sites with abundant presence of PS1-NTFs was verified in hippocampal mossy fiber terminals and olfactory bulb glomeruli, suggestive of a rich expression of gamma-secretase in the synapses at these locations that are characteristic of dynamic plasticity. Together, [3H]-L-685,458 is an excellent radiotracer for mapping active gamma-secretase complex, and may serve as a useful tool for studying the enzyme in vivo and in vitro.

Plastic changes and disease-modifying effects of scopolamine in the pilocarpine model of epilepsy in rats.

PURPOSE: We describe the use of a clinically relevant pharmacological intervention that alters the clinical history of status epilepticus (SE)-induced spontaneous recurrent seizures (SRS) in the pilocarpine model and the possible plastic changes underlying such an effect. METHODS: Two hours after pilocarpine-induced SE (320-350 mg/kg, i.p.), rats received scopolamine 1-2 mg/kg i.p. or saline, every 6 h for 3 days. After that, osmotic minipumps were implanted for continuous delivery of scopolamine or saline for an additional 14 days. Animals were video-monitored for 12 h/week during the following 3-month period for the occurrence of SRS and, thereafter, were perfused, processed, and coronal brain sections were stained for acetylcholinesterase (AChE) and for the presence of supragranular mossy fibers (Timm). RESULTS: Treatment with scopolamine led to significantly fewer SRS. Staining for AChE in the dentate gyrus was significantly more intense in naïve animals. The scopolamine group had the least intense AChE staining of all groups. However, regression analysis of the AChE staining for this group did not correlate with the presence or absence of SRS, or the latency or frequency of SRS. Supragranular mossy fiber sprouting developed in all animals experiencing pilocarpine-induced SE, irrespective of whether or not they were treated with scopolamine. CONCLUSIONS: Pilocarpine-induced SE in the presence of scopolamine might produce animals that, despite mossy fiber sprouting, were not seen to exhibit spontaneous seizures. In addition, our data suggest that the encountered changes in the AChE staining in the dentate gyrus that followed treatment with scopolamine do not help to explain its disease-modifying effects.

Structural and behavioural consequences of double deficiency for creatine kinases BCK and UbCKmit.

The cytosolic brain-type creatine kinase (BCK) isoform and the mitochondrial ubiquitous creatine kinase (UbCKmit) isoform are both important for the maintenance and distribution of cellular energy in neurons and astrocytes. Previously, we reported that mice deficient for BCK or UbCKmit each showed a surprisingly mild phenotype, probably due to reciprocal functional compensation by the remaining creatine kinase. This study shows that adult male mice lacking both creatine kinase isoforms (CK--/-- double knockout mice) have a reduced body weight, and demonstrate a severely impaired spatial learning in both a dry and a wet maze, lower nestbuilding activity and diminished acoustic startle reflex responses when compared to age-matched male wildtype mice with the same genetic background. In contrast, their visual and motor functions, exploration behaviour, prepulse inhibition and anxiety-related responses were not changed, suggesting no global deficit in sensorimotor function, hearing or motivation. Morphological analysis of CK--/-- double knockout brains revealed a reduction of approximately 7% in wet brain weight and hippocampal size, a approximately 15% smaller regio-inferior and relatively larger supra-pyramidal, and intra-infra-pyramidal mossy fiber areas. These results suggest that lack of both brain specific creatine kinase isoforms renders the synaptic circuitry in adult brain less efficient in coping with sensory or cognitive activity related challenges.

Changes in trkB-ERK1/2-CREB/Elk-1 pathways in hippocampal mossy fiber organization after traumatic brain injury.

Traumatic brain injury (TBI) leads to mossy fiber reorganization, which is considered to be a causative factor in the development of temporal lobe epilepsy. However, the underlying mechanism is not fully understood. Emerging evidence suggests that TrkB-ERK1/2-CREB/Elk-1 pathways are highly related to synaptic plasticity. This study used the rat fluid-percussion injury model to investigate activation of TrkB-ERK1/2-CREB/Elk-1 signaling pathways after TBI. Rats were subjected to 2.0-atm parasagittal TBI followed by 30 minutes, 4 hours, 24 hours, and 72 hours of recovery. After TBI, striking activation of TrkB-ERK1/2-CREB/Elk-1 signaling pathways in mossy fiber organization were observed with confocal microscopy and Western blot analysis. ERK1/2 was highly phosphorylated predominantly in hippocampal mossy fibers, whereas TrkB was phosphorylated both in the mossy fibers and the dentate gyrus region at 30 minutes and 4 hours of recovery after TBI. CREB was also activated at 30 minutes, peaked at 24 hours of recovery, and returned to the control level at 72 hours of recovery in dentate gyrus granule cells. Elk-1 phosphorylation was seen in CA3 neurons at 4 hours after TBI. The results suggest that the signaling pathways of TrkB-ERK1/2-CREB/Elk-1 are highly activated in mossy fiber organization, which may contribute to mossy fiber reorganization seen after TBI.