Secreted phospholipase A2 potentiates glutamate-induced calcium increase and cell death in primary neuronal cultures

Secreted phospholipases A2 (sPLA2s) modulate neuronal survival and neurotransmitter release. Here we show that sPLA2 (group III) synergistically increases glutamate-induced cell death and intracellular calcium ([Ca2+]i) in cultured primary cortical and hippocampal neurons. Whereas 1 microM glutamate elicited transient [Ca2+]i increases in all neurons that recovered 66% to baseline, 25 ng/ml sPLA2 pretreatment resulted in sustained [Ca2+]i increases, with only 5% recovery. At 250 nM glutamate, 25% of neurons failed to respond, and the average recovery time was 101 +/- 12 sec; sPLA2 increased recovery time to 158 +/- 6 sec, and only 2% of cells failed to respond. Both the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 and the calcium-channel blocker cobalt inhibited this effect. Experiments with the glutamate uptake inhibitor L-trans-pyrollidine-2,4-dicarboxylic acid (2.5 microM) indicated that glutamate uptake sites are not a likely modulation point by sPLA2, whereas arachidonic acid (AA) potentiated calcium responses to glutamate. Thus the enhancement of glutamate-induced [Ca2+]i increases by sPLA2 may be due to modulation at NMDA receptors and/or calcium channels by AA. These results indicate that sPLA2 affects neuronal responses to both nontoxic (0.1-10 microM) and toxic (=25 microM) concentrations of glutamate, implicating this enzyme in neuronal functions in pathology.     

Effects of hippocampal lesioning on experimental periodontitis in Wistar rats

The hippocampus, which is a brain structure involved in learning and memory processes, plays a key role in the feedback regulation of the hypothalamic-pituitary-adrenal (HPA) axis and autonomic sympathetic nervous system, and the subsequent secretion of immuno-modulatory hormones in response to pathogenic microorganisms. Dysregulation of these brain-neuroendocrine-immune regulatory networks, which act in concert to maintain homeostasis, is found to be of critical importance to the host defence against pathogens, as well as susceptibility to diseases, including periodontal disease. The present study was designed to determine the effects of hippocampal lesioning on the progression of periodontitis. Experimental ligature-induced periodontitis was induced in 16 Wistar rats, which were bilaterally lesioned in their hippocampal region with an aspiration technique that is well documented to impair learning and memory, as well as in 15 sham-operated control rats. The disease progression was evaluated radiographically and histometrically. The results revealed that the hippocampal lesioned rats developed significantly more destruction of the periodontium than did the sham-operated controls. This finding supports recent studies that indicate that inappropriate brain-neuroendocrine regulation of inflammatory responses to infectious agents may play an important role in disease susceptibility and progression.     

Protection of striatal neurons by joint blockade of D1 and D2 receptor subtypes in an in vitro model of cerebral hypoxia

Massive increases in extracellular dopamine have been reported in the ischemic rodent striatum, implicating this neurotransmitter in toxic events. We have examined whether dopamine receptor antagonists are protective against hypoxic insult, using brain slices containing the rostral striatum obtained from adult male C57/BLIcrfa(t) mice. Slices were subjected in vitro to 20 min nitrogen hypoxia, with or without addition of: (i) 50 microM haloperidol (D2 receptor antagonist and sigma ligand), (ii) 10 microM SCH23390 (selective D1 receptor antagonist), (iii) 10 microM eticlopride (selective D2 receptor antagonist), (iv) 10 microM SCH23390 and 10 microM eticlopride in combination, and (v) 10 microM MK-801 (noncompetitive NMDA receptor antagonist). Subsequently, slices were reoxygenated, fixed 2 h postinsult, and processed for light microscopy. Damage was assessed by calculating pyknotic profiles as a percentage of total neuronal profiles present. No pyknotic profiles were detected in normoxic control tissue, but this phenotype predominated in most slices subject to hypoxia alone (60.1 +/- 30.6% pyknotic profiles). Marked protection was produced by haloperidol (7.1 +/- 7.6%, P = 0.002), MK-801 (8.6 +/- 6.9%, P = 0.007), and the combined application of SCH23390 and eticlopride (5.9 +/- 9.4%, P = 0.001). No protection was demonstrated for SCH23390 or eticlopride when applied separately. These data suggest that hypoxic damage in the rostral mouse striatum is mediated via NMDA, D1, and D2 receptors. Protection against hypoxic damage by dopamine receptor antagonists requires the combined blockade of both classes of dopamine receptor.     

Time-dependent inhibition of hippocampal LTP in vitro following contextual fear conditioning in the rat

The effects of contextual fear‐learning on hippocampal synaptic excitability were investigated by means of high frequency tetanic stimulation (HFS) in rat brain slices (hippocampal CA1 region), prepared at different intervals (immediately, 24 h or 7 days) after a one‐trial contextual fear conditioning paradigm session. In the latter, rats that had previously received aversive electrical footshocks in the experimental apparatus exhibited freezing (the conditioned response) when placed again in the same apparatus (retrieval test). It was shown that contextual fear‐learning affects the hippocampal synaptic response. In fact, the HFS produced a decrease in the amplitude of short‐term (STP) and long‐term potentiation (LTP) when compared to control ‘naïve’ subject values. This decrease in STP amplitude could be observed only in slices prepared immediately after the training session. A decrease in the amplitude of long‐term but not short‐term potentiation was also observed at 24 h. At 7 days, no decreases in amplitude were observed. These modifications may be thought of as specifically associated with the learning process as they were not recorded in brain preparations from ‘shock‐only’ rats (i.e. those that received the same number of aversive stimuli of equal intensity as the conditioned group but with the shocks compressed temporally so that the shocked subjects could not associate nociceptive stimulation and surroundings – no conditioned freezing during retention testing). In ‘exploration’ preparations (brain slices from rats having only freely explored the experimental apparatus without receiving any adverse stimulation) a decrease in LTP amplitude was recorded only immediately after the training session, and STP was never modified. The synaptic response modifications do not appear to be due to presynaptic events, as they are not associated with paired‐pulse facilitation curve (PPF) modifications. The present results show that contextual fear conditioning and exploration of a novel environment (i) reduce the ability to induce synaptic plasticity; (ii) differentially influence STP and LTP and that (iii) the persistence of synaptic modifications depends on an animal's prior experience.     

Increased vulnerability to kainate-induced seizures in utrophin-knockout mice

Utrophin, the autosomal homologue of dystrophin, the Duchenne muscular dystrophy gene product, is a cytoskeletal protein found in many tissues. In muscle fibers, the level and localization of utrophin depend on their state of differentiation and innervation. Transgenic overexpression of utrophin prevents degeneration of dystrophin-deficient muscle fibers. In brain, in addition to its enrichment in blood vessels, utrophin is associated primarily with the plasma membrane of large sensory and motor brainstem neurons, suggesting a contribution to their structural stability. Here, we examined the role of utrophin for long-term survival of dentate granule cells, which become markedly hypertrophic in a mouse model of temporal lobe epilepsy. This morphogenetic change is induced several weeks after a unilateral intrahippocampal injection of kainic acid (KA), while mice experience chronic focal seizures. Using in situ hybridization and immunohistochemistry, we show that dispersion and hypertrophy of granule cells in KA-treated wildtype mice are accompanied by a strong and long-lasting expression of utrophin in somata and proximal dendrites. Utrophin knockout mice had a normal hippocampal cytoarchitecture but were more sensitive to KA-induced excitotoxicity, as shown by increased mortality and faster progression of the lesion. At 6 weeks post-KA, the numerical density of granule cells and thickness of the granule cell layer were significantly reduced ipsilaterally in mutant mice, indicating a profound reduction in total cell number in the absence of utrophin. These findings suggest that utrophin contributes to protect CNS neurons against pathological insults, in particular, stimuli leading to massive neuronal hypertrophy.     

Novel expression of AMPA-receptor subunit GluR1 on mossy cells and CA3 pyramidal neurons in the human epileptogenic hippocampus

Previous immunocytochemical investigations performed in our laboratory on the human hippocampus surgically resected for the treatment of mesial temporal lobe epilepsy (MTLE) have demonstrated an increased expression of the AMPA-receptor subunit GluR1 on neurons in the hilus and area CA3. Light microscopically, many of these neurons exhibited peculiar filamentous extensions and grape-like excrescences that protruded from their somata and proximal dendrites, suggesting that these neurons may be mossy cells and CA3 pyramidal neurons, respectively. The present electron microscopic study was carried out to further characterize these cells. The filamentous extensions were identified as dendrites from which spines often protruded, and the grape-like excrescences represented clusters of closely associated dendrites and spines. A variety of synapses were formed by the GluR1-positive profiles. These arrangements ranged from simple contacts between a single unlabelled axon terminal and a single labelled postsynaptic element, to complex contacts involving multiple unlabelled axon terminals and labelled postsynaptic elements. Many of the axon terminals involved in these arrangements were mossy fibre boutons. Thus, a large proportion of the GluR1-positive neurons were identified as hilar mossy cells and CA3 pyramidal neurons, cells hitherto thought to be absent or greatly reduced in the MTLE hippocampus. Taken together, these data suggest the presence of a highly efficient excitatory circuit involving AMPA receptors, mossy cells and CA3 pyramidal neurons in the sclerotic hippocampus. Such a circuit could be critically involved in the genesis and maintenance of temporal lobe epilepsy.     

Use of excitotoxins to lesion the hippocampus: update

Although many of the problems associated with the use of conventional lesion techniques (aspiration, electrolytic, radiofrequency) can be avoided by employing focal injections of excitotoxins, experience gained over the past 12 years has shown that considerable care must be exercised with this newer method, to limit the cell loss to the intended area or structure. Of the toxins that have been used most often to selectively destroy the cells that comprise the hippocampus, ibotenic acid (IBO) and N-methyl-D-aspartate (NMDA) have proved to be nonspecific in their effects on different cell types and these toxins do not cause seizures. In contrast, focal injections of kainate (KA) and quisqualate result in damage that centers primarily in the CA3 pyramidal cell field and hilar cells in the dentate gyrus. In addition, there are obvious seizures and secondary distant damage involving a number of structures and areas associated with mediating seizure activity. Intrahippocampal injections of the toxin colchicine result in a preferential destruction of dentate granule cells but usually also lead to additional cell loss in adjacent areas. Attempts to limit cell loss to specific hippocampal subfields, using different toxins, have met with mixed success. Both the dosage of the agent and the volume injected are important in determining the extent of cell loss, but the volume of the toxin injected has been shown to be especially important in limiting the damage to the intended area. With the development of newer procedures (e.g., immunotoxins, gene knockouts, antisense) that permit more selective cell loss, it should be possible in the future to achieve a level of lesion control that has been lacking in the past. As with the use of excitotoxins, these newer approaches will require special care to limit the damage to the intended area and interpret the results obtained properly.     

Field potentials in the human hippocampus during the encoding and recognition of visual stimuli

Intracranial field potentials were recorded from electrodes implanted in the hippocampus in 12 epileptic patients. Potentials were elicited by stimuli presented during a delayed matching-to-sample test. Each trial began with a sample stimulus composed of a 3 x 3 grid of rectangular color patches. The sample was followed by a sequence of similar but task-irrelevant stimuli and the sequential presentation of two test stimuli, one of which was identical to the sample. Patients indicated their recognition of the test stimulus that matched the sample with a button press. High-amplitude negative potentials were consistently elicited by sample and test stimuli. Peak amplitudes occurred 300-500 ms after stimulus onset and were larger for the sample in all cases. The patterns of potential gradients observed between adjacent hippocampal contacts and the locations of maximal amplitudes, as verified by magnetic resonance imaging in seven patients, suggest that these potentials were produced by neuronal activity in posterior hippocampus. These field potentials appear to index a memory storage function engaged in response to events that will later be remembered. The hippocampal contribution to storing declarative memories can thus begin, in some circumstances, within the first half-second after the presentation of a to-be-remembered stimulus.     

PET imaging of ischemic neuronal death in the hippocampus of living monkeys

The aim of the present study was to visualize postischemic hippocampal neuronal death in the living monkey brain, using a high-resolution positron emission tomography (PET) and novel radioligands. In preceding papers, we reported on postischemic hippocampal neuronal death in a model of Japanese monkeys (Macaca fuscata) undergoing a 20-min complete whole-brain ischemia. Using the same model here, we investigated the in vivo bindings of two radiotracers, [11C]Ro15-4513 (a type II benzodiazepine receptor ligand) and [11C](+)3-MPB (a muscarinic cholinergic receptor ligand), in the hippocampus on day 7 after ischemia, as compared to the normal hippocampus. A significant decrease in the in vivo binding of [11C]Ro154513 and [11C(+)3-MPB was observed in the postischemic monkey hippocampus on day 7 after ischemia compared to controls. Light and electron microscopic analyses of postischemic CA1 neurons showed typical features of coagulation necrosis, as associated with a marked reduction of postsynaptic densities and presynaptic vesicles. These results suggest that semiquantification of hippocampal neuronal death is possible in the living primate brain using PET, and that the same procedures can be applied for evaluating neuronal cell loss in patients with ischemic injuries and/or dementia.     

Brain slice culture for analysis of developmental brain disorders with special reference to congenital cytomegalovirus infection

ABSTRACT  Cytomegalovirus (CMV) is the most significant infectious cause of congenital abnormalities of the central nervous system (CNS) with variation from the fatal cytomegalic inclusion disease to functional brain disorder. The phenotype and degree of the brain disorder depends on infection time during the developing stage, virulence, route of infection and the viral susceptibility of the cells. The pathogenesis of the CMV infection to the CNS seems to be strongly related to neural migration, neural death, cellular compositions and the immune system of the brain. To understand the complex mechanism of this disorder, we used organotypic brain slice cultures. In the brain slice culture system, migration of CMV-in-fected neuronal cells was observed, which reflects infectious dynamics in vivo . Neural progenitor cells or glial immature cells in the subventricular zone and marginal area are most susceptible to murine cytomegalovirus (MCMV) infection in this system. The susceptibility declined as the number of immature glial cells decreased with age. The immature glial cells proliferated in brain slice cultures during prolonged incubation, and the susceptibility to MCMV infection also increased in association with the proliferation of these cells. The brain slice from an immunocompromised mouse (Beige-SCID mouse) unexpectedly showed lower susceptibility than that of an immunocompetent mouse during any prolonged incubation. These results suggest that the number of immature glial cells might determine the susceptibility of CMV infection to the brain, independent of the immune system. We reviewed recent findings of CMV infection to the brain from the perspective of brain slice cultures and the possibility that this system could be a useful method to investigate mechanisms of congenital anomaly of the brain.