Extrasynaptic NMDA Receptors : Mediators of Excitotoxic Cell Death

The N-methyl-D-aspartate (NMDA) type of glutamate receptor is a calcium-permeable ion channel with important functions in the physiology and pathology of the mammalian brain. NMDA receptors are critical for long-lasting, activity-induced changes in synaptic transmission, a process thought to be involved in learning and memory. NMDA receptors also control neuronal survival and cell death. How can the biological consequences of NMDA receptor activation be so diametrically opposed? The outcome of NMDA receptor activation appears to be determined by its localization. Stimulation of synaptic NMDA receptors (by synaptically-released glutamate) activates gene expression mediated by the transcription factor, cAMP-response element-binding-protein (CREB) and induces pro-survival events. In contrast, calcium flux through extrasynaptic NMDA receptors overrides these functions, shutting off CREB activity, and causing mitochondrial dysfunction and cell death. These differences in the biological response are likely due to differences in the intracellular signaling complexes associated with synaptic vs. extrasynaptic NMDA receptors. As extrasynaptic NMDA receptors are thought to be activated following hypoxic/ischemic insults, specific blockade of extrasynaptic NMDA receptors or its signaling complex may efficiently reduce neuron loss following stroke

A quantitative method to assess extrasynaptic NMDA receptor function in the protective effect of synaptic activity against neurotoxicity

<p>Abstract</p> <p>Background</p> <p>Extrasynaptic NMDA receptors couple to a CREB shut-off pathway and cause cell death, whereas synaptic NMDA receptors and nuclear calcium signaling promote CREB-mediated transcription and neuronal survival. The distribution of NMDA receptors (synaptic versus extrasynaptic) may be an important parameter that determines the susceptibility of neurons to toxic insults. Changes in receptor surface expression towards more extrasynaptic NMDA receptors may lead to neurodegeneration, whereas a reduction of extrasynaptic NMDA receptors may render neurons more resistant to death. A quantitative assessment of extrasynaptic NMDA receptors in individual neurons is needed in order to investigate the role of NMDA receptor distribution in neuronal survival and death.</p> <p>Results</p> <p>Here we refined and verified a protocol previously used to isolate the effects of extrasynaptic NMDA receptors using the NMDA receptor open channel blocker, MK-801. Using this method we investigated the possibility that the known neuroprotective shield built up in hippocampal neurons after a period of action potential bursting and stimulation of synaptic NMDA receptors is due to signal-induced trafficking of extrasynaptic NMDA receptors or a reduction in extrasynaptic NMDA receptor function. We found that extrasynaptic NMDA receptor-mediated calcium responses and whole cell currents recorded under voltage clamp were surprisingly invariable and did not change even after prolonged (16 to 24 hours) periods of bursting and synaptic NMDA receptor activation. Averaging a large number of calcium imaging traces yielded a small (6%) reduction of extrasynaptic NMDA receptor-mediated responses in hippocampal neurons that were pretreated with prolonged bursting.</p> <p>Conclusion</p> <p>The slight reduction in extrasynaptic NMDA receptor function following action potential bursting and synaptic NMDA receptor stimulation could contribute to but is unlikely to fully account for activity-dependent neuroprotection. Other factors, in particular calcium signaling to the nucleus and the induction of survival promoting genes are more likely to mediate acquired neuroprotection.</p

Cellular and nuclear morphology…and calcium signaling: revealing the interplay between structure and function

Poster presentation: Calcium plays a pivotal role in relaying electrical signals of the cell to subcellular compartments, such as the nucleus. Since this one ion type is used by the cell for many processes a neuron needs to establish finely tuned calcium pathways in order to be able to differentiate multiple tasks, [1-3]. While it is known that neurons can actively change their shape upon neuronal activity, [4-7], we here present novel findings of activity-regulated nuclear morphology, [8,9]. With the help of an experimental and computational modeling approach, we show that hippocampal neurons can change the previously spherical shape of their nuclei to complex and infolded morphologies. This morphology regulation is demonstrated to be regulated by NMDA-receptor gated calcium, while synaptic and extra-synaptic NMDA-receptors elicit opposing effects on nuclear morphology, [8]. The structural alterations of the cell nucleus have significant effects on nuclear calcium dynamics. Compartmentalization of the nucleus, due to membrane infoldings, changes calcium frequencies, amplitudes and spatial distributions, [8,10]. Since these parameters have been shown to control downstream events towards gene transcription, [11,12], the results elucidate the cellular control of nuclear function with the help of morphology modulation. With respect to processes downstream of calcium, we show that histone H3 phosphorylation is closely linked to nuclear morphology. Investigating the nuclear morphologies of hippocampal neurons, two major classes were identified [9,10]. One class contains non-infolded nuclei that have the function of calcium signal integrators, while the other class contains highly infolded nuclei, which function as frequency detectors of nuclear calcium, [10]. Extending this interdisciplinary approach of investigating structure/function relationships in neurons, the effects of cellular morphology – as well as the morphology of the endoplasmic reticulum and other organelles – on neuronal calcium signals is currently being investigated. This endeavor makes use of highly detailed, three-dimensional models of neuronal calcium dynamics, including the three-dimensional morphology of the cell and its organelles

A transcription-dependent increase in miniature EPSC frequency accompanies late-phase plasticity in cultured hippocampal neurons

<p>Abstract</p> <p>Background</p> <p>The magnitude and longevity of synaptic activity-induced changes in synaptic efficacy is quantified by measuring evoked responses whose potentiation requires gene transcription to persist for more than 2-3 hours. While miniature EPSCs (mEPSCs) are also increased in amplitude and/or frequency during long-term potentiation (LTP), it is not known how long such changes persist or whether gene transcription is required.</p> <p>Results</p> <p>We use whole-cell patch clamp recordings from dissociated hippocampal cultures to characterise for the first time the persistence and transcription dependency of mEPSC upregulation during synaptic potentiation. The persistence of recurrent action potential bursting in these cultures is transcription-, translation- and NMDA receptor-dependent thus providing an accessible model for long-lasting plasticity. Blockade of GABA_A-receptors with bicuculline for 15 minutes induced action potential bursting in all neurons and was maintained in 50-60% of neurons for more than 6 hours. Throughout this period, the frequency but neither the amplitude of mEPSCs nor whole-cell AMPA currents was markedly increased. The transcription blocker actinomycin D abrogated, within 2 hours of burst induction, both action potential bursting and the increase in mEPSCs. Reversible blockade of action potentials during, but not after this 2 hour transcription period suppressed the increase in mEPSC frequency and the recovery of burst activity at a time point 6 hours after induction.</p> <p>Conclusion</p> <p>These results indicate that increased mEPSC frequency persists well beyond the 2 hour transcription-independent phase of plasticity in this model. This long-lasting mEPSC upregulation is transcription-dependent and requires ongoing action potential activity during the initial 2 hour period but not thereafter. Thus mEPSC upregulation may underlie the long term, transcription-dependent persistence of action potential bursting. This provides mechanistic insight to link gene candidates already identified by gene chip analysis to long lasting plasticity in this in vitro model.</p

Adeno-associated virus (AAV)-mediated suppression of Ca2+/calmodulin kinase IV activity in the nucleus accumbens modulates emotional behaviour in mice

<p>Abstract</p> <p>Background</p> <p>Calcium/calmodulin-dependent protein kinase IV (CaMKIV) controls activity-dependent gene transcription by regulating the activity of the cyclic AMP response element binding protein (CREB). This signaling pathway is involved in gating emotional responses in the CNS but previous studies did not address the potential roles of CaMKIV in discrete brain regions. In the present study, we aimed at specifically dissecting the role of CaMKIV in the nucleus accumbens of adult mice.</p> <p>Results</p> <p>We used recombinant adeno-associated virus (rAAV)-mediated gene transfer of a dominant-negative CaMKIV variant (rAAV-dnCaMKIV) to inhibit endogenous CaMKIV in the nucleus accumbens. rAAV-dnCaMKIV treated animals were subjected to a battery of tests including, prepulse inhibition of the acoustic startle response, open field, social interaction and anxiety-related behaviour. We found that basal locomotor activity in the open field, and prepulse inhibition or startle performance were unaltered in mice infected with rAAV-dnCaMKIV in the nucleus accumbens. However, anxiogenic effects were revealed in social interaction testing and the light/dark emergence test.</p> <p>Conclusion</p> <p>Our findings suggest a modulatory role of CaMKIV in the nucleus accumbens in anxiety-like behaviour but not sensorimotor gating.</p

The mitochondrial calcium uniporter is crucial for the generation of fast cortical network rhythms

The role of the mitochondrial calcium uniporter (MCU) gene (Mcu) in cellular energy homeostasis and generation of electrical brain rhythms is widely unknown. We investigated this issue in mice and rats using Mcu-knockout and -knockdown strategies in vivo and in situ and determined the effects of these genetic manipulations on hippocampal gamma oscillations (30–70 Hz) and sharp wave-ripples. These physiological network states require precise neurotransmission between pyramidal cells and inhibitory interneurons, support spike-timing and synaptic plasticity and are associated with perception, attention and memory. Absence of the MCU resulted in (i) gamma oscillations with decreased power (by >40%) and lower synchrony, including less precise neural action potential generation (‘spiking'), (ii) sharp waves with decreased incidence (by about 22%) and decreased fast ripple frequency (by about 3%) and (iii) lack of activity-dependent pyruvate dehydrogenase dephosphorylation. However, compensatory adaptation in gene expression related to mitochondrial function and glucose metabolism was not detected. These data suggest that the neuronal MCU is crucial for the generation of network rhythms, most likely by influences on oxidative phosphorylation and perhaps by controlling cytoplasmic Ca(2+) homeostasis. This work contributes to an increased understanding of mitochondrial Ca(2+) uptake in cortical information processing underlying cognition and behaviour

Muscarinic M1 receptors modulate endotoxemia-induced loss of synaptic plasticity

Septic encephalopathy is associated with rapid deterioration of cortical functions. Using magnetic resonance imaging (MRI) we detected functional abnormalities in the hippocampal formation of patients with septic delirium. Hippocampal dysfunction was further investigated in an animal model for sepsis using lipopolysaccharide (LPS) injections to induce endotoxemia in rats, followed by electrophysiological recordings in brain slices. Endotoxemia induced a deficit in long term potentiation which was completely reversed by apamin, a blocker of small conductance calcium-activated potassium (SK) channels, and partly restored by treatment with physostigmine (eserine), an acetylcholinesterase inhibitor, or TBPB, a selective M1 muscarinic acetylcholine receptor agonist. These results suggest a novel role for SK channels in the etiology of endotoxemia and explain why boosting cholinergic function restores deficits in synaptic plasticity. Drugs which enhance cholinergic or M1 activity in the brain may prove beneficial in treatment of septic delirium in the intensive care unit

Preconditioning doses of NMDA promote neuroprotection by enhancing neuronal excitability

Neuroprotection can be induced by low doses of NMDA, which activate both synaptic and extrasynaptic NMDA receptors. This is in apparent contradiction with our recent findings that extrasynaptic NMDA receptor signaling exerts a dominant inhibitory effect on prosurvival signaling from synaptic NMDA receptors. Here we report that exposure to low preconditioning doses of NMDA results in preferential activation of synaptic NMDA receptors because of a dramatic increase in action potential firing. Both acute and long-lasting phases of neuroprotection in the face of apoptotic or excitotoxic insults are dependent on this firing enhancement. Key mediators of synaptic NMDA receptor-dependent neuroprotection, phosphatidylinositol 3 kinase-Akt (PI3 kinase-Akt) signaling to Forkhead box subgroup O (FOXO) export and glycogen synthase kinase 3β (GSK3β) inhibition and cAMP response element-binding protein-dependent (CREB-dependent) activation of brain-derived neurotrophic factor (BDNF), can be induced only by low doses of NMDA via this action potential-dependent route. In contrast, NMDA doses on the other side of the toxicity threshold do not favor synaptic NMDA receptor activation because they strongly suppress firing rates below baseline. The classic bell-shaped curve depicting neuronal fate in response to NMDA dose can be viewed as the net effect of two antagonizing (synaptic vs extrasynaptic) curves: via increased firing the synaptic signaling dominates at low doses, whereas firing becomes suppressed and extrasynaptic signaling dominates as the toxicity threshold is crossed