Fredrik Asztely

Extrasynaptic Glutamate Spillover in the Hippocampus: Dependence on Temperature and the Role of Active Glutamate Uptake

At excitatory synapses on CA1 pyramidal cells of the hippocampus, a larger quantal content is sensed by N-methyl-D-aspartic acid receptors (NMDARs) than by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). A novel explanation for this discrepancy is that glutamate released from terminals presynaptic to one cell can diffuse to and activate NMDARs, but not AMPARs, on a neighboring cell. If this occurs in the living brain, it could invalidate the view that glutamatergic synapses function as private communication channels between neurons. Here, we show that the discrepancy in quantal content mediated by the two receptors is greatly decreased at physiological temperature, compared with conventional recording conditions. This effect of temperature is not due to changes in release probability or uncovering of latent AMPARs. It is, however, partially reversed by the glutamate uptake inhibitor dihydrokainate. The results suggest that glutamate transporters play a critical role in limiting the extrasynaptic diffusion of glutamate, thereby minimizing cross-talk between neighboring excitatory synapses.

Extrasynaptic glutamate spillover in the hippocampus: evidence and implications

In the mammalian brain most excitatory transmission is mediated by glutamate binding to AMPA and NMDA receptors. These receptors have markedly different biophysical properties, and at synapses in the CAI region of the hippocampus they play complementary roles in long-term potentiation (LTP): while postsynaptic NMDA receptor activation is necessary for the induction of this form of plasticity, AMPA receptors play a larger role in its expression. Recent studies in hippocampal slices have revealed a further striking difference in the behaviour of the two receptor types: NMDA receptors consistently sense a larger number of quanta of glutamate released from presynaptic terminals than do AMPA receptors. Two alternative explanations for this are either that AMPA receptors are functionally silent at a proportion of synapses (although they can be uncovered by LTP), or that glutamate can spill over from neighbouring synapses and selectively activate NMDA (but not AMPA) receptors. Both of these competing hypotheses have extensive implications for the mechanisms of expression of LTP. Extrasynaptic glutamate diffusion appears to depend critically on the recording temperature, but if excitatory synapses are sufficiently close for cross-talk to occur under physiological conditions, it could have profound implications for the specificity of synaptic communication in the brain.

LTP of AMPA and NMDA Receptor–Mediated Signals: Evidence for Presynaptic Expression and Extrasynaptic Glutamate Spill-Over

We have addressed the expression of long-term potentiation (LTP) in hippocampal CA1 by comparing AMPA and NMDA receptor-(AMPAR- and NMDAR-) mediated postsynaptic signals. We find that potentiation of NMDAR-mediated signals accompanies LTP of AMPAR-mediated signals, and is associated with a change in variability implying an increase in quantal content. Further, tetanic LTP of NMDAR-mediated signals can be elicited when LTP of AMPAR-mediated signals is prevented. We propose that LTP is mainly expressed presynaptically, and that, while AMPARs respond only to glutamate from immediately apposed terminals, NMDARs also sense glutamate released from terminals presynaptic to neighboring cells. We also find that tetanic LTP increases the rate of depression of NMDAR-mediated signals by the use-dependent blocker MK-801, implying an increase in the glutamate release probability. These findings argue for a presynaptic contribution to LTP and for extrasynaptic spill-over of glutamate onto NMDARs.

Mouse hippocampal organotypic tissue cultures exposed to in vitro "ischemia" show selective and delayed CA1 damage that is aggravated by glucose.

Oxygen and glucose deprivation (OGD) in cell cultures is generally studied in a medium, such as artificial cerebrospinal fluid (CSF), with an ion composition similar to that of the extracellular fluid of the normal brain (2 to 4 mmol/L K+, 2 to 3 mmol/L Ca2+; pH 7.4). Because the distribution of ions across cell membranes dramatically shifts during ischemia, the authors exposed mouse organotypic hippocampal tissue cultures to OGD in a medium, an ischemic cerebrospinal fluid, with an ion composition similar to the extracellular fluid of the brain during ischemia in vivo (70 mmol/L K+, 0.3 mmol/L Ca2+; pH 6.8). In ischemic CSF, OGD induced a selective and delayed cell death in the CA1 region, as assessed by propidium iodide uptake. Cell death was glutamate receptor dependent since blockade of the N-methyl-D-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors mitigated cell damage. Hyperglycemia aggravates ischemic brain damage in vivo, whereas in vitro glucose in artificial CSF prevents oxygen deprivation-induced damage. The authors demonstrate that glucose in ischemic CSF significantly exacerbates cell damage after oxygen deprivation. This new model of in vitro “ischemia” can be useful in future studies of the mechanisms and treatment of ischemic cell death, including studies using genetically modified mice.

The role of mammalian ionotropic receptors in synaptic plasticity: LTP, LTD and epilepsy.

Abstract. Synaptic plasticity is the foremost candidate mechanism to explain the rapid acquisition of memories. In the mammalian brain, the NMDA subclass of glutamate receptors plays a central role in the induction of several forms of use-dependent plasticity. The finding that modifications in synaptic strength are largely expressed by receptors of the AMPA subclass has focused attention on molecular mechanisms that affect their function and targeting. Receptor plasticity has also been reported in pathological situations, notably in animal and human forms of epilepsy. Which of these changes are causally implicated in the generation of seizures, and which may be compensatory or neuroprotective adaptations, has not been fully resolved.

ENDOGENOUS NEUROTROPHIN-3 REGULATES SHORT-TERM PLASTICITY AT LATERAL PERFORANT PATH-GRANULE CELL SYNAPSES

In the adult brain, neurotrophin-3 (NT-3) is mainly localized in dentate granule cells, and its expression is decreased by various stimuli, e.g., seizure activity. We have examined the role of endogenous NT-3 for excitatory synaptic transmission at lateral perforant path–dentate granule cell synapses using hippocampal slices from NT-3 knock-out (+/−) and wild-type (+/+) mice. Paired-pulse facilitation (PPF) and also short-term synaptic plasticity induced by a brief, high-frequency train of afferent stimulation were reduced, but the expression of long-term potentiation was not affected in the NT-3+/− mice. Incubation of the slices with recombinant NT-3 reversed the deficit in PPF through a mechanism requiring de novoprotein synthesis, implying that the impaired short-term plasticity does not result from a developmental alteration. No changes of overall presynaptic release probability, measured by the progressive block of NMDA receptor-mediated synaptic currents by MK-801, or desensitization of AMPA receptors were detected. Because NT-3 expression is reduced after focal seizures, impaired short-term facilitation may represent a protective response that limits the propagation of epileptiform activity from the entorhinal cortex to the hippocampus.

Glucose but Not Lactate in Combination With Acidosis Aggravates Ischemic Neuronal Death In Vitro

Background and Purpose— Hyperglycemia aggravates brain damage in clinical stroke and in experimental in vivo models of cerebral ischemia. Elevated preischemic glucose levels, lactate production, and intracerebral acidosis correlate with increased brain damage. We have developed a murine hippocampal slice culture model of in vitro ischemia (IVI), suitable for studies of the mechanisms of neuronal death. In this model we investigated the individual contribution of glucose, pH, lactate, and combinations thereof as well as ionotropic glutamate receptor activation to the development of hyperglycemic ischemic cell death. Methods— Murine organotypic hippocampal slice cultures were exposed to IVI in a medium with an ionic composition similar to that of the extracellular fluid in the brain during ischemia in vivo. Cell death was assessed by propidium iodide uptake. Ionotropic glutamate receptor blockade was accomplished by d-2-amino-5-phosphonopentanoic acid (D-APV) or 2,3-dihydro-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX). Results— The combination of high glucose levels and acidosis (pH 6.8), but not acidosis per se or the combination of lactate and acidosis during IVI, exacerbated damage. Cell death after hyperglycemic IVI was not diminished by blockade of ionotropic glutamate receptors. Conclusions— Aggravation of brain damage by hyperglycemia in vivo can be reproduced in hippocampal slice cultures in vitro. Our results demonstrate that glucose per se, but not lactate, in combination with acidosis mediates the detrimental hyperglycemic effect through a mechanism independent of ionotropic glutamate receptors.

Afferent-specific modulation of short-term synaptic plasticity by neurotrophins in dentate gyrus

Neurotrophins modulate synaptic transmission and plasticity in the adult brain. We here show a novel feature of this synaptic modulation, i.e. that two populations of excitatory synaptic connections to granule cells in the dentate gyrus, lateral perforant path (LPP) and medial perforant path (MPP), are differentially influenced by the neurotrophins BDNF and NT‐3. Using field recordings and whole‐cell patch‐clamp recordings in hippocampal slices, we found that paired‐pulse (PP) depression at MPP–granule cell synapses was impaired in BDNF knock‐out (+/–) mice, but PP facilitation at LPP synapses to the same cells was not impaired. In accordance, scavenging of endogenous BDNF with TrkB–IgG fusion protein also impaired PP depression at MPP–granule cell synapses, but not PP facilitation at LPP–granule cell synapses. Conversely, in NT‐3+/– mice, PP facilitation was impaired at LPP–granule cell synapses whilst PP depression at MPP–granule cell synapses was unaffected. These deficits could be reversed by application of exogenous neurotrophins in an afferent‐specific manner. Our data suggest that BDNF and NT‐3 differentially regulate the synaptic impact of different afferent inputs onto single target neurons in the CNS.