Cellular mechanisms of burst firing-mediated long-term depression in rat neocortical pyramidal cells

During wakefulness and sleep, neurons in the neocortex emit action potentials tonically or in rhythmic bursts, respectively. However, the role of synchronized discharge patterns is largely unknown. We have recently shown that pairings of excitatory postsynaptic potentials (EPSPs) and action potential bursts or single spikes lead to long-term depression (burst-LTD) or long-term potentiation, respectively. In this study, we elucidate the cellular mechanisms of burst-LTD and characterize its functional properties. Whole-cell patch-clamp recordings were obtained from layer V pyramidal cells in somatosensory cortex of juvenile rats in vitro and composite EPSPs and EPSCs were evoked extracellularly in layers II/III. Repetitive burst-pairings led to a long-lasting depression of EPSPs and EPSCs that was blocked by inhibitors of metabotropic glutamate group 1 receptors, phospholipase C, protein kinase C (PKC) and calcium release from the endoplasmic reticulum, and that required an intact machinery for endocytosis. Thus, burst-LTD is induced via a Ca2+- and phosphatidylinositol-dependent activation of PKC and expressed through phosphorylation-triggered endocytosis of AMPA receptors. Functionally, burst-LTD is inversely related to EPSP size and bursts dominate single spikes in determining the sign of synaptic plasticity. Thus burst-firing constitutes a signal by which coincident synaptic inputs are proportionally downsized. Overall, our data thus suggest a mechanism by which synaptic weights can be reconfigured during non-rapid eye movement sleep.

Spine Ca2+ signaling in spike-timing-dependent plasticity

Calcium is a second messenger, which can trigger the modification of synaptic efficacy. We investigated the question of whether a differential rise in postsynaptic Ca2+ ([Ca2+]i) alone is sufficient to account for the induction of long-term potentiation (LTP) and long-term depression (LTD) of EPSPs in the basal dendrites of layer 2/3 pyramidal neurons of the somatosensory cortex. Volume-averaged [Ca2+]i transients were measured in spines of the basal dendritic arbor for spike-timing-dependent plasticity induction protocols. The rise in [Ca2+]i was uncorrelated to the direction of the change in synaptic efficacy, because several pairing protocols evoked similar spine [Ca2+]i transients but resulted in either LTP or LTD. The sequence dependence of near-coincident presynaptic and postsynaptic activity on the direction of changes in synaptic strength suggested that LTP and LTD were induced by two processes, which were controlled separately by postsynaptic [Ca2+]i levels. Activation of voltage-dependent Ca2+ channels before metabotropic glutamate receptors (mGluRs) resulted in the phospholipase C-dependent (PLC-dependent) synthesis of endocannabinoids, which acted as a retrograde messenger to induce LTD. LTP required a large [Ca2+]i transient evoked by NMDA receptor activation. Blocking mGluRs abolished the induction of LTD and uncovered the Ca2+-dependent induction of LTP. We conclude that the volume-averaged peak elevation of [Ca2+]i in spines of layer 2/3 pyramids determines the magnitude of long-term changes in synaptic efficacy. The direction of the change is controlled, however, via a mGluR-coupled signaling cascade. mGluRs act in conjunction with PLC as sequence-sensitive coincidence detectors when postsynaptic precede presynaptic action potentials to induce LTD. Thus presumably two different Ca2+ sensors in spines control the induction of spike-timing-dependent synaptic plasticity.

Effects of prolonged endotoxemia on liver, skeletal muscle and kidney mitochondrial function

INTRODUCTION: Sepsis may impair mitochondrial utilization of oxygen. Since hepatic dysfunction is a hallmark of sepsis, we hypothesized that the liver is more susceptible to mitochondrial dysfunction than the peripheral tissues, such as the skeletal muscle. We studied the effect of prolonged endotoxin infusion on liver, muscle and kidney mitochondrial respiration and on hepatosplanchnic oxygen transport and microcirculation in pigs. METHODS: 20 anesthetized pigs were randomized to receive endotoxin or saline infusion for 24 hours. Muscle, liver and kidney mitochondrial respiration was assessed. Cardiac output (thermodilution), carotid, superior mesenteric and kidney arterial, portal venous (ultrasound Doppler) and microcirculatory blood flow (laser Doppler) were measured, and systemic and regional oxygen transport and lactate exchange were calculated. RESULTS: Endotoxin infusion induced hyperdynamic shock and impaired the glutamate- and succinate-dependent mitochondrial respiratory control ratio (RCR) in the liver (glutamate: endotoxemia: median [range] 2.8 [2.3-3.8] vs. controls: 5.3 [3.8-7.0]; p<0.001; succinate: endotoxemia: 2.9 [1.9-4.3] vs. controls: 3.9 [2.6-6.3] p=0.003). While the ADP:O ratio was reduced with both substrates, maximal ATP production was impaired only in the succinate-dependent respiration. Hepatic oxygen consumption and extraction, and liver surface laser Doppler blood flow remained unchanged. Glutamate-dependent respiration in the muscle and kidney was unaffected. CONCLUSIONS: Endotoxemia reduces the efficiency of hepatic but neither skeletal muscle nor kidney mitochondrial respiration, independent of regional and microcirculatory blood flow changes.

Effect of endotoxin, dobutamine and dopamine on muscle mitochondrial respiration in vitro

INTRODUCTION: Mitochondrial respiration is impaired during endotoxemia. While catecholamines are frequently used in sepsis, their effects on mitochondrial function are controversial. We assessed effects of dobutamine and dopamine endotoxin on isolated muscle mitochondria. MATERIALS AND METHODS: Sternocleidomastoid muscle mitochondria were isolated from six anesthetized pigs. Each sample was divided into six different groups. Three groups were incubated with endotoxin, three with vehicle. After 1 h, dopamine and dobutamine at final concentrations of 100 microM were added to the vehicle and endotoxin groups. After 2 h, state 3 and 4 respiration rates were determined for all mitochondrial complexes. Oxygen consumption was determined with a Clark-type electrode. RESULTS: Endotoxin increased glutamate-dependent state 4 respiration from 9.3 +/- 3.6 to 31.9 +/- 9.1 (P = 0.001) without affecting state 3 respiration. This reduced the efficiency of mitochondrial respiration (RCR; state 3/state 4, 9.9 +/- 1.9 versus 3.6 +/- 0.6; P < 0.001). The other complexes were unaffected. Catecholamine partially restored the endotoxin-induced increase in complex I state 4 respiration rate (31.9 +/- 9.1 versus 17.1 +/- 6.4 and 20.1 +/- 12.2) after dopamine and dobutamine, respectively (P = 0.007), and enhanced the ADP:O ratio (P = 0.033). CONCLUSIONS: Dopamine and dobutamine enhanced the efficiency of mitochondrial respiration after short-term endotoxin exposure.

Effects of lamotrigine alone and in combination with MK-801, phenobarbital, or phenytoin on cell death in the neonatal rat brain

The neonatal rat brain is vulnerable to neuronal apoptosis induced by antiepileptic drugs (AEDs), especially when given in combination. This study evaluated lamotrigine alone or in combination with phenobarbital, phenytoin, or the glutamate antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) for a proapoptotic action in the developing rat brain. Cell death was assessed in brain regions (striatum, thalamus, and cortical areas) of rat pups (postnatal day 8) by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, 24 h after acute drug treatment. Lamotrigine alone did not increase neuronal apoptosis when given in doses up to 50 mg/kg; a significant increase in cell death occurred after 100 mg/kg. Combination of 20 mg/kg lamotrigine with 0.5 mg/kg MK-801 or 75 mg/kg phenobarbital resulted in a significant increase in TUNEL-positive cells, compared with MK-801 or phenobarbital treatment alone. A similar enhancement of phenytoin-induced cell death occurred after 30 mg/kg lamotrigine. In contrast, 20 mg/kg lamotrigine significantly attenuated phenytoin-induced cell death. Lamotrigine at 10 mg/kg was without effect on apoptosis induced by phenytoin. Although the functional and clinical implications of AED-induced developmental neuronal apoptosis remain to be elucidated, our finding that lamotrigine alone is devoid of this effect makes this drug attractive as monotherapy for the treatment of women during pregnancy, and for preterm or neonatal infants. However, because AEDs are often introduced as add-on medication, careful selection of drug combinations and doses may be required to avoid developmental neurotoxicity when lamotrigine is used in polytherapy.

Brain metabolism in Alzheimer disease and vascular dementia assessed by in vivo proton magnetic resonance spectroscopy

Proton magnetic resonance spectroscopy (MRS) allows the assessment of various cerebral metabolites non-invasively in vivo. Among 1H MRS-detectable metabolites, N-acetyl-aspartate and N-acetyl-aspartyl-glutamate (tNAA), trimethylamines (TMA), creatine and creatine phosphate (tCr), inositol (Ins) and glutamate (Gla) are of particular interest, since these moieties can be assigned to specific neuronal and glial metabolic pathways, membrane constituents, and energy metabolism. In this study on 94 subjects from a memory clinic population, 1H MRS results (single voxel STEAM: TE 20 ms, TR 1500 ms) on the above metabolites were assessed for five different brain regions in probable vascular dementia (VD), probable Alzheimer's disease (AD), and age-matched healthy controls. In both VD and AD, ratios of tNAA/tCr were decreased, which may be attributed to neuronal atrophy and loss, and Ins/tCr-ratios were increased indicating either enhanced gliosis or alteration of the cerebral inositol metabolism. However, the topographical distribution of the metabolic alterations in both diseases differed, revealing a temporoparietal pattern for AD and a global, subcortically pronounced pattern for VD. Furthermore, patients suffering from vascular dementia (VD) had remarkably enhanced TMA/tCr ratios, potentially due to ongoing degradation of myelin. Thus, the metabolic alterations obtained by 1H MRS in vivo allow insights into the pathophysiology of the different dementias and may be useful for diagnostic classification.

Clindamycin is neuroprotective in experimental Streptococcus pneumoniae meningitis compared with ceftriaxone

In animal models of Streptococcus pneumoniae meningitis, rifampin is neuroprotective in comparison to ceftriaxone. So far it is not clear whether this can be generalized for other protein synthesis-inhibiting antimicrobial agents. We examined the effects of the bactericidal protein synthesis-inhibiting clindamycin (n = 12) on the release of proinflammatory bacterial components, the formation of neurotoxic compounds and neuronal injury compared with the standard therapy with ceftriaxone (n = 12) in a rabbit model of pneumococcal meningitis. Analysis of the CSF and histological evaluation were combined with microdialysis from the hippocampal formation and the neocortex. Compared with ceftriaxone, clindamycin reduced the release of lipoteichoic acids from the bacteria (p = 0.004) into the CSF and the CSF leucocyte count (p = 0.011). This led to lower extracellular concentrations of hydroxyl radicals (p = 0.034) and glutamate (p = 0.016) in the hippocampal formation and a subsequent reduction of extracellular glycerol levels (p = 0.018) and neuronal apoptosis in the dentate gyrus (p = 0.008). The present data document beneficial effects of clindamycin compared with ceftriaxone on various parameters linked with the pathophysiology of pneumococcal meningitis and development of neuronal injury. This study suggests neuroprotection to be a group effect of bactericidal protein synthesis-inhibiting antimicrobial agents compared with the standard therapy with beta-lactam antibiotics in meningitis.

The end1 gene of Schizosaccharomyces pombe coding for a DNase is identical with the pnu1 gene coding for an RNase

The DNA nuclease activity encoded by the end1 gene, and its inactivation by mutation, was described in connection with the characterization of DNA topoisomerases in the fission yeast Schizosaccharomyces pombe (Uemura and Yanagida, 1984). Subsequently, end1 mutant strains were used for the preparation of cell extracts for the study of enzymes and intermediates involved in DNA metabolism. The molecular identification of the end1 gene and its identity with the pnu1 gene is presented. The end1-458 mutation alters glycine to glutamate in the conserved motif TGPYLP. The pnu1 gene codes for an RNase that is induced by nitrogen starvation (Nakashima et al., 2002b). Thus, the End1/Pnu1 protein, like related mitochondrial proteins in other organisms, is an example of a sugar-non-specific nuclease. The analysis of strains carrying a pnu1 deletion revealed no defects in meiotic recombination and spore viability.

Blockade of NMDA receptor subtype NR2B prevents seizures but not apoptosis of dentate gyrus neurons in bacterial meningitis in infant rats

BACKGROUND: Excitotoxic neuronal injury by action of the glutamate receptors of the N-methyl-d-aspartate (NMDA) subtype have been implicated in the pathogenesis of brain damage as a consequence of bacterial meningitis. The most potent and selective blocker of NMDA receptors containing the NR2B subunit is (R,S)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperid inepropanol (RO 25-6981). Here we evaluated the effect of RO 25-6981 on hippocampal neuronal apoptosis in an infant rat model of meningitis due to Streptococcus pneumoniae. Animals were randomized for treatment with RO 25-6981 at a dosage of either 0.375 mg (15 mg/kg; n = 28) or 3.75 mg (150 mg/kg; n = 15) every 3 h or an equal volume of sterile saline (250 microl; n = 40) starting at 12 h after infection. Eighteen hours after infection, animals were assessed clinically and seizures were observed for a period of 2 h. At 24 h after infection animals were sacrificed and brains were examined for apoptotic injury to the dentate granule cell layer of the hippocampus. RESULTS: Treatment with RO 25-6981 had no effect on clinical scores, but the incidence of seizures was reduced (P < 0.05 for all RO 25-6981 treated animals combined). The extent of apoptosis was not affected by low or high doses of RO 25-6981. Number of apoptotic cells (median [range]) was 12.76 [3.16-25.3] in animals treated with low dose RO 25-6981 (control animals 13.8 [2.60-31.8]; (P = NS) and 9.8 [1.7-27.3] (controls: 10.5 [2.4-21.75]) in animals treated with high dose RO 25-6981 (P = NS). CONCLUSIONS: Treatment with a highly selective blocker of NMDA receptors containing the NR2B subunit failed to protect hippocampal neurons from injury in this model of pneumococcal meningitis, while it had some beneficial effect on the incidence of seizures.

Multiple pathways of neuroprotection against oxidative stress and excitotoxic injury in immature primary hippocampal neurons

In the immature brain hydrogen peroxide accumulates after excitotoxic hypoxia-ischemia and is neurotoxic. Immature hippocampal neurons were exposed to N-methyl-D-aspartate (NMDA), a glutamate agonist, and hydrogen peroxide (H(2)O(2)) and the effects of free radical scavenging and transition metal chelation on neurotoxicity were studied. alpha-Phenyl-N-tert.-butylnitrone (PBN), a known superoxide scavenger, attenuated both H(2)O(2) and NMDA mediated toxicity. Treatment with desferrioxamine (DFX), an iron chelator, at the time of exposure to H(2)O(2) was ineffective, but pretreatment was protective. DFX also protected against NMDA toxicity. TPEN, a metal chelator with higher affinities for a broad spectrum of transition metal ions, also protected against H(2)O(2) toxicity but was ineffective against NMDA induced toxicity. These data suggest that during exposure to free radical and glutamate agonists, the presence of iron and other free metal ions contribute to neuronal cell death. In the immature nervous system this neuronal injury can be attenuated by free radical scavengers and metal chelators.

[In search of strategies for preventing brain damage as a sequela of bacterial meningitis]

Multiplication of bacteria within the central nervous system compartment triggers a host response with an overshooting inflammatory reaction which leads to brain parenchyma damage. Some of the inflammatory and neurotoxic mediators involved in the processes leading to neuronal injury during bacterial meningitis have been identified in recent years. As a result, the therapeutic approach to the disease has widened from eradication of the bacterial pathogen with antibiotics to attenuation of the detrimental effects of host defences. Corticosteroids represent an example of the adjuvant therapeutic strategies aimed at downmodulating excessive inflammation in the infected central nervous system. Pathophysiological concepts derived from an experimental rat model of bacterial meningitis revealed possible therapeutic strategies for prevention of brain damage. The insights gained led to the evaluation of new therapeutic modalities such as anticytokine agents, matrix metalloproteinase inhibitors, antioxidants, and antagonists of endothelin and glutamate. Bacterial meningitis is still associated with persistent neurological sequelae in approximately one third of surviving patients. Future research in the model will evaluate whether the neuroprotective agents identified so far have the potential to attenuate learning disabilities as a long-term consequence of bacterial meningitis.

Neurotoxicity of glia activated by gram-positive bacterial products depends on nitric oxide production

The present study examined the mechanism by which bacterial cell walls from two gram-positive meningeal pathogens, Streptococcus pneumoniae and the group B streptococcus, induced neuronal injury in primary cultures of rat brain cells. Cell walls from both organisms produced cellular injury to similar degrees in pure astrocyte cultures but not in pure neuronal cultures. Cell walls also induced nitric oxide production in cultures of astrocytes or microglia. When neurons were cultured together with astrocytes or microglia, the cell walls of both organisms became toxic to neurons. L-NAME, a nitric oxide synthase inhibitor, protected neurons from cell wall-induced toxicity in mixed cultures with glia, as did dexamethasone. In contrast, an excitatory amino acid antagonist (MK801) had no effect. Low concentrations of cell walls from either gram-positive pathogen added together with the excitatory amino acid glutamate resulted in synergistic neurotoxicity that was inhibited by L-NAME. The induction of nitric oxide production and neurotoxicity by cell walls was independent of the presence of serum, whereas endotoxin exhibited these effects only in the presence of serum. We conclude that gram-positive cell walls can cause toxicity in neurons by inducing the production of nitric oxide in astrocytes and microglia.

Neuroprotective effect of excitatory amino acid antagonist kynurenic acid in experimental bacterial meningitis

Sustained high-level exposure to glutamate, an excitatory amino acid neurotransmitter, leads to neuronal death. Kynurenic acid attenuates the toxic effects of glutamate by inhibition of neuronal excitatory amino acid receptors, including the N-methyl-D-aspartate subtype. To evaluate the role of glutamate in causing neuronal injury in a rat model of meningitis due to group B streptococci, animals were treated with kynurenic acid (300 mg/kg subcutaneously once daily) or saline beginning at the time of infection. Histopathologic examination after 24-72 h showed two distinct forms of neuronal injury, areas of neuronal necrosis in the cortex and injury of dentate granule cells in the hippocampus. Animals treated with kynurenic acid showed significantly less neuronal injury (P < .03) in the cortex and the hippocampus than did untreated controls. These results suggest an important contribution of glutamate to neurotoxicity in this animal model of neonatal meningitis.

Amino acids in cerebrospinal and brain interstitial fluid in experimental pneumococcal meningitis

Excitatory amino acids are increasingly implicated in the pathogenesis of neuronal injury induced by a variety of CNS insults, such as ischemia, trauma, hypoglycemia, and epilepsy. Little is known about the role of amino acids in causing CNS injury in bacterial meningitis. Several amino acids were measured in cerebrospinal fluid and in microdialysis samples from the interstitial fluid of the frontal cortex in a rabbit model of pneumococcal meningitis. Cerebrospinal fluid concentrations of glutamate, aspartate, glycine, taurine, and alanine increased significantly in infected animals. Among the amino acids with known excitatory or inhibitory function, interstitial fluid concentrations of glutamate were significantly elevated (by 470%). Alanine, a marker for anaerobic glycolysis, also increased in the cortex of infected rabbits. The elevated glutamate concentrations in the brain extracellular space suggest that excitotoxic neuronal injury may play a role in bacterial meningitis.

Concurrent monitoring of cerebral electrophysiology and metabolism after traumatic brain injury: an experimental and clinical study

Multiparameter cerebral monitoring has been widely applied in traumatic brain injury to study posttraumatic pathophysiology and to manage head-injured patients (e.g., combining O(2) and pH sensors with cerebral microdialysis). Because a comprehensive approach towards understanding injury processes will also require functional measures, we have added electrophysiology to these monitoring modalities by attaching a recording electrode to the microdialysis probe. These dual-function (microdialysis/electrophysiology) probes were placed in rats following experimental fluid percussion brain injuries, and in a series of severely head-injured human patients. Electrical activity (cell firing, EEG) was monitored concurrently with microdialysis sampling of extracellular glutamate, glucose and lactate. Electrophysiological parameters (firing rate, serial correlation, field potential occurrences) were analyzed offline and compared to dialysate concentrations. In rats, these probes demonstrated an injury-induced suppression of neuronal firing (from a control level of 2.87 to 0.41 spikes/sec postinjury), which was associated with increases in extracellular glutamate and lactate, and decreases in glucose levels. When placed in human patients, the probes detected sparse and slowly firing cells (mean = 0.21 spike/sec), with most units (70%) exhibiting a lack of serial correlation in the spike train. In some patients, spontaneous field potentials were observed, suggesting synchronously firing neuronal populations. In both the experimental and clinical application, the addition of the recording electrode did not appreciably affect the performance of the microdialysis probe. The results suggest that this technique provides a functional monitoring capability which cannot be obtained when electrophysiology is measured with surface or epidural EEG alone.