Excitatory amino acid neurotoxicity in the hippocampal slice preparation

The effect of sustained activation of excitatory amino acid receptors on neuronal survival was studied using slices of adult rat hippocampus and light and electron microscopy. Kainate, N-methyl-D-aspartate, quisqualate, and ibotenate all produce signs of severe neurotoxicity within 90 min. Neuronal damage occurs in the form of perikaryal and dendritic swelling, cytoplasmic and nucleoplasmic disintegration, and plasma and nuclear membrane ruffling and collapse. The toxicity is restricted to intrinsic neuronal somata, dendrites and spines, while afferent axons, boutons and glia are spared. Although damage is generally distributed throughout all areas of hippocampus, kainate has little effect on pyramidal neurons in the CA2 region. Quantitative analysis of neuronal survival indicates that agonists induce dose-dependent damage over concentration ranges known to be excitatory. Based on selective antagonism by DL-aminophosphonoheptanoate and the patterns of damage produced by each, N-methyl-D-aspartate, kainate, and quisqualate trigger neurotoxicity by acting on distinct receptor classes. It is concluded that, in hippocampal slices, excitatory amino acids induce neurotoxicity in a similar manner to their actions in vivo. The results support the hypothesis that hippocampal neurotoxicity is initiated by excessive excitation, and provide another example of the capacity of adult hippocampal neurons for rapid structural modification.     

Corticosterone and phenytoin reduce neuronal nitric oxide synthase messenger RNA expression in rat hippocampus.

The production and release of the corticosteroids, namely the glucocorticoids and the mineralocorticoids, are regulated by various stimuli, including stress. Previous studies from our laboratory have shown that chronic exposure to stress or to stress levels of glucocorticoids produces atrophy of the apical dendrites of CA3 pyramidal neurons in the hippocampus. This stress-induced dendritic remodeling is blocked by the anti-epileptic drug phenytoin, which suppresses glutamate release, and also by N-methyl-D-aspartate receptor antagonists. These results suggest an interaction between glucocorticoids and excitatory amino acids in the development of stress-induced atrophy of CA3 pyramidal neurons. Since nitric oxide is proposed to play an important role in mediating both the physiological and pathophysiological actions of excitatory amino acids, we examined the regulation of neuronal nitric oxide synthase messenger RNA expression by corticosterone and phenytoin in the rat hippocampus. The expression of neuronal nitric oxide synthase messenger RNA in hippocampal pyramidal neurons and granule neurons of the dentate gyrus was unaffected by 21-day administration of corticosterone (40 mg/kg), phenytoin (40 mg/kg) or the combination of corticosterone and phenytoin. However, in hippocampal interneurons, corticosterone/ phenytoin co-administration led to a significant reduction in neuronal nitric oxide synthase messenger RNA levels when compared with vehicle controls. These results suggest that, during exposure to stress levels of corticosterone, phenytoin inhibits glucocorticoid-induced atrophy of CA3 pyramidal neurons by reducing neuronal nitric oxide synthase expression in hippocampal interneurons. Moreover, these results may provide another example of synaptic plasticity in the hippocampus mediated by nitric oxide synthase.     

Long-term potentiation of guinea pig mossy fiber responses is not blocked by N-methyl D-aspartate antagonists

Receptors preferentially activated by the excitatory amino acid N-methyl-D-aspartate (NMDA) do not mediate synaptic transmission in the hippocampus but are involved in initiating long-term potentiation (LTP) in hippocampal region CA1. We have examined the role of NMDA receptors in LTP of the commissural/associational and mossy fiber pathways to region CA3 pyramidal neurons. In the commissural/associational pathway, NMDA receptor blockers did not reduce synaptic responses but reversibly blocked the induction of LTP. In contrast, NMDA receptor blockers had no effect on mossy fiber LTP. These results suggest that induction of commissural/associational LTP differs from mossy fiber LTP, although the mechanisms underlying expression of LTP along these pathways could be similar. Kynurenate and L-2-amino-4-phosphonobutyrate, which potently reduce mossy fiber responses, also did not block induction of mossy fiber LTP.     

Influence of dizocilpine (MK-801) on neurotoxic effect of dexamethasone: behavioral and histological studies

Elevated levels of endogenous glucocorticoids (GCs) or prolonged treatment with high doses of dexamethasone (DEX) or other GCs preparations are frequently associated with psychosis as well as cognitive deficits, such as the impairment of memory and learning. GCs potentiate stress or ischemia-induced accumulation of excitatory amino acids in the extracellular space of hippocampus. The antagonism of glutamate receptors may potentially improve safety profile of therapy with GCs. The purpose of the present study was to investigate the effect of dizocilpine maleate (MK-801), non-competitive NMDA glutamate receptor antagonist, on neurotoxic effect of the prolonged treatment with the high dose of DEX. The results showed that DEX (120 mg/kg/day for 7 days) impaired the long-term memory and the motor coordination, reduced the body weight and induced the lethality of mice. The morphological and ultrastructural study have confirmed damage to hippocampal neurons especially in the CA3 region after the prolonged treatment with DEX alone. Damaged pyramidal neurons showed robust changes in the shape of the nucleus and cytoplasm condensation. MK-801 alone (at non-toxic dose of 0.3 mg/kg/day), changed neither the behavior of mice nor morphology of the hippocampal neurons. However, it did not prevent the neurotoxic effects of DEX. On the contrary, it intensified DEX-induced neurotoxicity.     

姜黄素对TNF-α损伤大鼠海马神经元的功能性保护作用及机制

目的:观察姜黄素对TNF-α损伤大鼠海马神经元的功能性保护作用并探讨其机制。方法:应用离体脑片记录技术,记录大鼠海马CA1区的兴奋性突触后电位(EPSP),给予Schaffer侧支高频刺激(HFS)诱发长时程增强(LTP),观察不同药物处理组EPSP起始斜率的变化情况。结果:与对照组相比,TNF-α和NMDA(N-甲基D-天冬氨酸)对大鼠海马脑片LTP产生明显的抑制作用(P0.05);而姜黄素可以部分拮抗TNF-α和NMDA对海马脑片LTP的抑制作用,与对照组相比无显著差异(P0.05);TNF-α、姜黄素和NMDA对大鼠海马神经元的基础突触传递没有显著影响。结论:姜黄素对TNF-α损伤的大鼠海马神经元有功能性保护作用,其机制可能是姜黄素部分拮抗TNF-α诱导的神经元细胞膜上的NMDA受体过度激活,维持神经元的长时程增强。     

Blockade of N-methyl-D-aspartate response in enzyme-treated rat hippocampal neurons

The responses to excitatory and inhibitory amino acids have been investigated in isolated pyramidal cells from young and adult rat hippocampus using internal perfusion and 'concentration-clamp' techniques. The neurons dissociated in a purely mechanical way were sensitive to all excitatory amino acids (glutamate, kainate, quisqualate and N-methyl-D-aspartate (NMDA] and inhibitory amino acids (glycine, taurine and gamma-aminobutyric acid). The NMDA response was dramatically potentiated by adding glycine at threshold concentration (10(-6) M). The enzyme treatment of hippocampal slices selectively removed the NMDA sensitivity but did not alter all other pharmacological properties of voltage- and agonist-gated ion channels.     

The kainate/quisqualate receptor antagonist, CNQX, blocks the fast component of spontaneous epileptiform activity in organotypic cultures of rat hippocampus

Intracellular recordings were made from CA1 pyramidal neurones of hippocampus maintained in organotypic culture. Both spontaneous interictal and ictal epileptiform activity was observed. CNQX, an antagonist at kainate/quisqualate but not at N-methyl-D-aspartate (NMDA)-sensitive excitatory amino acid receptors depressed but did not abolish spontaneous epileptiform activity. Addition of the specific NMDA receptor antagonist D-2-amino-5-phosphonovalerate (D-APV) abolished the remaining activity. Similar effects were observed on electrically evoked excitatory post synaptic potentials (EPSPs). This suggests a role for endogenous excitatory amino acids acting at both kainate/quisqualate and NMDA sensitive excitatory amino acid receptors in the generation and maintainance of epileptiform activity within these organotypic cultures.     

Sex,stress and the hippocampus: allostasis,allostatic load and the aging process

The adaptive responses of the body that maintain homeostasis in response to stressors can be called "allostasis", meaning "achieving stability through change". Mediators produced by the immune system, autonomic nervous system (ANS) and hypothalamo-pituitary-adrenal(HPA) axis produce allostasis. The brain also shows allostasis, involving the activation of nerve cell activity and the release of neurotransmitters. When the individual is challenged repeatedly or when the allostatic systems remain turned on when no longer needed, the mediators of allostasis can produce a wear and tear on the body and brain that has been termed "allostatic load". Examples of allostatic load include the accumulation of abdominal fat, the loss of bone minerals and the atrophy of nerve cells in the hippocampus. Studies of the hippocampus as a target of stress and sex hormones have revealed a considerable degree of structural plasticity and remodeling in the adult brain that differs between the sexes. Three forms of hippocampal structural plasticity are affected by circulating hormones: (1) repeated stress causes remodeling of dendrites in the CA3 region; (2) different modalities of stress suppress neurogenesis of dentate gyrus granule neurons; (3) ovarian steroids regulate synapse formation during the estrous cycle of female rats. All three forms of structural remodeling of the hippocampus are mediated by hormones working in concert with excitatory amino acids (EAA) and NMDA receptors. EAA and NMDA receptors are also involved in neuronal death that is caused in pyramidal neurons by seizures, by ischemia and by severe and prolonged psychosocial stress. The aging brain seems to be more vulnerable to such effects, although there are considerable individual differences in vulnerability that can be developmentally determined. Moreover, the brain retains considerable resilience in the face of stress, and estrogens appear to play a role in this resilience. "Resilience is an example of successful allostasis in which wear and tear is minimized, and estrogens exemplify the type of agent that works against the allostatic load associated with aging." This review discusses the current status of work on underlying mechanisms for protection and damage.     

TrkB but not trkC receptors are necessary for postnatal maintenance of hippocampal spines

Dendritic spines are major sites of excitatory synaptic transmission and changes in their densities have been linked to alterations in learning and memory. The neurotrophins brain-derived neurotrophic factor and neurotrophin-3 and their receptors, trkB and trkC, are thought to be involved in learning, memory and long-term potentiation (LTP). LTP is known to induce trkB and trkC gene expression as well as spinogenesis in the hippocampus. In the aging hippocampus, declines in trkB and trkC mRNA levels may underlie, at least in part, impairments in spatial memory and reductions in spine densities. To determine the significance of trkB and trkC for the maintenance of dendritic spines, we have analyzed Golgi-impregnated hippocampi of adult and aged mice heterozygous for trkB, trkC, or both along with respective wildtype littermates. Deletion of one allele of trkB, but not trkC, significantly reduces spine densities of CA1 pyramidal neurons in both adult and aged mice, as compared to age-matched controls. This indicates that trkB, but not trkC, receptors are necessary for the maintenance of hippocampal spines during postnatal life.     

Ketamine selectively suppresses synchronized afterdischarges in immature hippocampus

The role of excitatory amino acid neurotransmission in epileptogenesis was investigated in the developing hippocampus. Bath application of ketamine blocked penicillin-induced, synchronized afterdischarges in immature rat CA3 hippocampal neurons. Ketamine also decreased the duration of the preceding intracellularly recorded depolarization shift but had no measurable effect on the resting membrane potential or input impedance of pyramidal cells. Concentrations of ketamine that blocked afterdischarge generation dramatically depressed intracellular depolarizations produced by iontophoretic application of N-methyl-D-aspartate (NMDA) but not quisqualate. The effects of the NMDA antagonist 2-amino-7-phosphonoheptanoic acid on epileptiform discharges were identical to those of ketamine. These results suggest that an endogenous excitatory amino acid acting on an NMDA receptor plays a key role in the pronounced capacity of immature hippocampus for seizures.     

Enhanced intrinsic excitability and EPSP-spike coupling accompany enriched environment-induced facilitation of LTP in hippocampal CA1 pyramidal neurons

Environmental enrichment (EE) is a well-established paradigm for studying naturally occurring changes in synaptic efficacy in the hippocampus that underlie experience-induced modulation of learning and memory in rodents. Earlier research on the effects of EE on hippocampal plasticity focused on long-term potentiation (LTP). Whereas many of these studies investigated changes in synaptic weight, little is known about potential contributions of neuronal excitability to EE-induced plasticity. Here, using whole-cell recordings in hippocampal slices, we address this gap by analyzing the impact of EE on both synaptic plasticity and intrinsic excitability of hippocampal CA1 pyramidal neurons. Consistent with earlier reports, EE increased contextual fear memory and dendritic spine density on CA1 cells. Furthermore, EE facilitated LTP at Schaffer collateral inputs to CA1 pyramidal neurons. Analysis of the underlying causes for enhanced LTP shows EE to increase the frequency but not amplitude of miniature excitatory postsynaptic currents. However, presynaptic release probability, assayed using paired-pulse ratios and use-dependent block of N-methyl-d-aspartate receptor currents, was not affected. Furthermore, CA1 neurons fired more action potentials (APs) in response to somatic depolarization, as well as during the induction of LTP. EE also reduced spiking threshold and after-hyperpolarization amplitude. Strikingly, this EE-induced increase in excitability caused the same-sized excitatory postsynaptic potential to fire more APs. Together, these findings suggest that EE may enhance the capacity for plasticity in CA1 neurons, not only by strengthening synapses but also by enhancing their efficacy to fire spikes-and the two combine to act as an effective substrate for amplifying LTP.     

The actions of 2-hydroxy-saclofen at presynaptic GABAB receptors in the rat hippocampus

The actions of 2-hydroxy-saclofen (2-OH-S), a recently developed analog of baclofen, were studied at presynaptic GABAB receptors in the rat hippocampal slice. Baclofen (0.5-20 microM) reduces the amplitude of excitatory postsynaptic potentials (EPSPs) recorded from hippocampal CA1 pyramidal neurons. In the presence of 200-500 microM 2-OH-S, the synaptic depressant action of baclofen is significantly reduced. These data show that 2-OH-S is an effective antagonist at presynaptic GABAB receptors on excitatory terminals in the hippocampus.     

Hebbian LTP in feed-forward inhibitory interneurons and the temporal fidelity of input discrimination

Cortical information processing requires a delicate balance of excitatory and inhibitory signaling. How is this balance preserved during hippocampal memory encoding, which involves NMDA receptor-dependent long term potentiation (LTP)? This form of LTP occurs at synapses between pyramidal neurons but has not been detected in feed-forward inhibitory interneurons. We show that paired pre- and postsynaptic activity evokes pathway-specific LTP in half of rat stratum radiatum interneurons if cytoplasmic integrity is preserved. LTP occurs in aspiny feed-forward interneurons and propagates to pyramidal neurons as an enhancement of disynaptic inhibition. We also show that when LTP is restricted to synapses on pyramidal neurons, the temporal fidelity of synaptic integration and action potential generation in pyramidal cells is compromised. However, when LTP also occurs at synapses on feed-forward interneurons, temporal fidelity is preserved. We propose that Hebbian LTP at synapses driving disynaptic inhibition is necessary to maintain information processing without degradation during memory encoding.     

GluR5 kainate receptor activation in interneurons increases tonic inhibition of pyramidal cells

We studied the modulation of GABAergic inhibition by glutamate and kainate acting on GluR5-containing kainate receptors in the CA1 hippocampal region. Glutamate, kainate or ATPA, a selective agonist of GluR5-containing receptors, generates an inward current in inhibitory interneurons and cause repetitive action potential firing. This results in a massive increase of tonic GABAergic inhibition in the somata and apical dendrites of pyramidal neurons. These effects are prevented by the GluR5 antagonist LY 293558. Electrical stimulation of excitatory afferents generates kainate receptor-mediated excitatory postsynaptic currents (EPSCs) and action potentials in identified interneurons that project to the dendrites and somata of pyramidal neurons. Therefore glutamate acting on kainate receptors containing the GluR5 subunit may provide a protective mechanism against hyperexcitability.     

M1 and M4 receptors modulate hippocampal pyramidal neurons

Acetylcholine (ACh), acting at muscarinic ACh receptors (mAChRs), modulates the excitability and synaptic connectivity of hippocampal pyramidal neurons. CA1 pyramidal neurons respond to transient ("phasic") mAChR activation with biphasic responses in which inhibition is followed by excitation, whereas prolonged ("tonic") mAChR activation increases CA1 neuron excitability. Both phasic and tonic mAChR activation excites pyramidal neurons in the CA3 region, yet ACh suppresses glutamate release at the CA3-to-CA1 synapse (the Schaffer-collateral pathway). Using mice genetically lacking specific mAChRs (mAChR knockout mice), we identified the mAChR subtypes responsible for cholinergic modulation of hippocampal pyramidal neuron excitability and synaptic transmission. Knockout of M1 receptors significantly reduced, or eliminated, most phasic and tonic cholinergic responses in CA1 and CA3 pyramidal neurons. On the other hand, in the absence of other G(q)-linked mAChRs (M3 and M5), M1 receptors proved sufficient for all postsynaptic cholinergic effects on CA1 and CA3 pyramidal neuron excitability. M3 receptors were able to participate in tonic depolarization of CA1 neurons, but otherwise contributed little to cholinergic responses. At the Schaffer-collateral synapse, bath application of the cholinergic agonist carbachol suppressed stratum radiatum-evoked excitatory postsynaptic potentials (EPSPs) in wild-type CA1 neurons and in CA1 neurons from mice lacking M1 or M2 receptors. However, Schaffer-collateral EPSPs were not significantly suppressed by carbachol in neurons lacking M4 receptors. We therefore conclude that M1 and M4 receptors are the major mAChR subtypes responsible for direct cholinergic modulation of the excitatory hippocampal circuit.     

NMDA receptor-dependent long-term potentiation in mouse hippocampal interneurons shows a unique dependence on Ca2+/calmodulin-dependent kinases

Long-term potentiation (LTP) of excitatory synaptic transmission plays a major role in memory encoding in the cerebral cortex. It can be elicited at many synapses on principal cells, where it depends on Ca²⁺ influx through postsynaptic N -methyl- d -aspartic acid (NMDA) receptors. Ca²⁺ influx triggers phosphorylation of several kinases, in particular Ca²⁺/calmodulin-dependent kinase type II (CaMKII). Auto-phosphorylation of CaMKII is a key step in the LTP induction cascade, as revealed by the absence of LTP in hippocampal pyramidal neurons of αCaMKII T286A-mutant mice, where auto-phosphorylation of the α isoform at residue T286 is prevented. A subset of hippocampal interneurons mediating feed-forward inhibition also exhibit NMDA receptor-dependent LTP, which shows all the cardinal features of Hebbian LTP in pyramidal neurons. This is unexpected, because αCaMKII has not been detected in interneurons. Here we show that pathway-specific NMDA receptor-dependent LTP is intact in hippocampal inhibitory interneurons of αCaMKII T286A-mutant mice, although in pyramidal cells it is blocked. However, LTP in interneurons is blocked by broad-spectrum pharmacological inhibition of Ca²⁺/calmodulin-dependent kinases. The results suggest that non-α Ca²⁺/calmodulin-dependent kinases substitute for the α isoform in NMDA receptor-dependent LTP in interneurons.     

Two different forms of long-term potentiation at CA1–subiculum synapses

Distinct functional roles in learning and memory are attributed to certain areas of the hippocampus and the parahippocampal region. The subiculum as a part of the hippocampal formation is the principal target of CA1 pyramidal cell axons and serves as an interface in the information processing between the hippocampus and the neocortex. Subicular pyramidal cells have been classified as bursting and regular firing cells. Here we report fundamental differences in long-term potentiation (LTP) between both cell types. Prolonged high-frequency stimulation induced NMDA receptor-dependent LTP in both cell types. While LTP relied on postsynaptic calcium in regular firing neurons, no increase in postsynaptic calcium was required in bursting cells. Furthermore, paired-pulse facilitation revealed that the site of LTP expression was postsynaptic in regular firing neurons, while presynaptic in burst firing neurons. Our findings on synaptic plasticity in the subiculum indicate that regular firing and bursting cells represent two functional units with distinct physiological roles in processing hippocampal output.     

Brainstem and spinal projections of augmenting expiratory neurons in the rat

To examine the role of Ca(2+) entry through AMPA receptors in the pathogenesis of the ischemia-induced cell death of hippocampal neurons, we delivered cDNA of Q/R site-unedited form (GluR2Q) of AMPA receptor subunit GluR2 in the hippocampus by using an HVJ-liposome-mediated gene transfer technique. Two days prior to transient forebrain ischemia, we injected an HVJ-liposome containing cDNA of the GluR2Q-myc fusion gene into a rat unilateral hippocampus. In the absence of ischemic insult, overexpression of Ca(2+)-permeable GluR2Q did not cause any neurodegeneration in the cDNA-injected hippocampus. In ischemic rats, overexpression of Ca(2+)-permeable GluR2Q markedly promoted ischemic cell death of CA1 pyramidal neurons, while complete rescue of CA1 pyramidal neurons from ischemic damage occurred in the hippocampal hemisphere opposite the GluR2Q expression. Overexpression of the Q/R-site edited form (GluR2R) of subunit GluR2 did not affect the ischemia-induced damage of CA1 pyramidal neurons. From these results, we suggest that the Ca(2+)-permeability of AMPA receptors does not have a direct contribution to glutamate receptor-mediated neurotoxicity but has a promotive action in the evolution of ischemia-induced neurodegeneration of vulnerable neurons.     

Multiple forms of LTP in hippocampal CA3 neurons use a common postsynaptic mechanism.

We investigated long-term potentiation (LTP) at mossy fiber synapses on CA3 pyramidal neurons in the hippocampus. Using Ca2+ imaging techniques, we show here that when postsynaptic Ca2+ was sufficiently buffered so that [Ca2+]i did not rise during synaptic stimulation, the induction of mossy fiber LTP was prevented. In addition, induction of mossy fiber LTP was suppressed by postsynaptic injection of a peptide inhibitor of cAMP-dependent protein kinase. Finally, when ionotropic glutamate receptors were blocked, LTP depended on the postsynaptic release of Ca2+ from internal stores triggered by activation of metabotropic glutamate receptors. These results support the conclusion that mossy fiber LTP and LTP at other hippocampal synapses share a common induction mechanism involving an initial rise in postsynaptic [Ca2+].     

The linoleic acid derivative FR236924 facilitates hippocampal synaptic transmission by enhancing activity of presynaptic alpha7 acetylcholine receptors on the glutamatergic terminals

The present study aimed at understanding the effect of FR236924, a newly synthesized linoleic acid derivative with cyclopropane rings instead of cis-double bonds, on hippocampal synaptic transmission in both the in vitro and in vivo systems. FR236924 increased the rate of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor-mediated miniature excitatory postsynaptic currents, without affecting the amplitude, triggered by nicotine in CA1 pyramidal neurons of rat hippocampal slices, that is inhibited by GF109203X, a selective protein kinase C (PKC) inhibitor or alpha-bungarotoxin, an inhibitor of alpha7 acetylcholine (ACh) receptors. FR236924 stimulated glutamate release from rat hippocampal slices and in the hippocampus of freely behaving rats, and the effect was also inhibited by GF109203X or alpha-bungarotoxin. FR236924 induced a transient huge potentiation followed by a long-lasting potentiation in the slope of field excitatory postsynaptic potentials recorded from the CA1 region of rat hippocampal slices, and the latter effect was blocked by GF109203X or alpha-bungarotoxin. Likewise, the compound persistently facilitated hippocampal synaptic transmission in the CA1 region of the intact rat hippocampus. It is concluded from these results that FR236924 stimulates glutamate release by functionally targeting presynaptic alpha7 ACh receptors on the glutamatergic terminals under the influence of PKC, responsible for the facilitatory action on hippocampal synaptic transmission. This may provide evidence for a link between cis-unsaturated free fatty acids and presynaptic alpha7 ACh receptors in hippocampal synaptic plasticity.