Calcium-dependent mitochondrial superoxide modulates nuclear CREB phosphorylation in hippocampal neurons

We report evidence that mitochondrially produced superoxide (O(2)(-)) is involved in signaling in hippocampal neurons by examining the relationship between strong but physiological increases in cytosolic free Ca(2+), mitochondrial calcium accumulation, O(2)(-) production, and CREB phosphorylation. Strong depolarization-induced Ca(2+) entry through NMDA or L-type Ca(2+) channels evoked large Ca(2+) transients, a sustained increase in O(2)(-), and a large rise in nuclear CaM and pCREB. Under these conditions, inhibition of mitochondrial Ca(2+) uptake and consequent O(2)(-) production suppressed Ca(2+) entry-induced pCREB elevation, indicating that O(2)(-) produced by mitochondria supports CREB phosphorylation. Similarly, inhibiting mitochondrial respiration blocked O(2)(-) production and also depressed the elevation of pCREB. Blocking calcineurin reversed this depression. We conclude that strong Ca(2+) entry promotes mitochondrial calcium accumulation and the subsequent enhancement of mitochondrial O(2)(-) production, which in turn prolongs the lifetime of pCREB by suppressing calcineurin-dependent pCREB dephosphorylation.     

辣椒素对大鼠骶髓后连合核神经元AMPA受体所介导电流的抑制作用

目的研究辣椒素（CAP）在急性分离的大鼠骶髓后连合核（SDCN）神经元的药理学特性。方法采用制霉菌素穿孔膜片钳全细胞记录方法。结果当钳制电压为-40mV时，在所有被检测的SDCN神经元上给予CAP不引起任何电流；CAP（10～3000μmol／L）可以呈浓度依赖方式可逆性的抑制海人藻酸（KA）所激活的AMPA受体介导的电流，IC50为（60．7±19．2）μmol/L；该抑制作用呈电压非依赖性。结论CAP在大鼠SDCN神经元可对AMPA受体激活的电流产生抑制作用，此作用可能和内脏痛的调节有关。     

Increased activity of surviving locus ceruleus neurons in Alzheimer's disease

In Alzheimer's disease (AD) there is neuronal loss in the locus ceruleus (LC), and the noradrenergic system may be even more affected in depressed AD patients. However, this neuronal loss may go together with an increase in activity of the remaining noradrenergic neurons. We prospectively evaluated 16 AD patients (6 depressed, 5 transiently depressed, and 5 nondepressed) and 10 controls. We determined norepinephrine and its metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG) in various brain areas, and compared these data with previously established neuron numbers in the LC in the same patients. We could not confirm earlier studies reporting lower norepinephrine concentrations in depressed than in nondepressed dementia patients. The mean norepinephrine concentrations in AD patients were significantly lower than those in control patients, whereas the mean concentrations of MHPG were not different. Moreover, we found significant inverse relationships between the number of remaining pigmented LC neurons and the MHPG/norepinephrine ratio in the frontal cortex and LC. These data are the first to provide direct evidence for the hypothesis that remaining LC neurons are activated to compensate for decreased cerebral norepinephrine levels in AD, by demonstrating that the MHPG/norepinephrine ratio is significantly higher in AD, indicating increased metabolism. Ann Neurol 1999;45:82–91     

Interleukin-1 beta modulates AMPA receptor expression and phosphorylation in hippocampal neurons

Interleukin (IL)-1beta is a pro-inflammatory cytokine involved in modulating inflammation and stress responses in the brain. Central administration of IL-1beta impairs both memory functions and long-term potentiation (LTP) induction. However, the molecular events responsible for the downstream effects of IL-1beta are not fully understood. Given the potential regulatory role of IL-1beta in LTP, we assessed whether IL-1beta influences surface expression and phosphorylation of glutamate receptors. We found that IL-1beta, but not IL-10 or tumour necrosis factor (TNF)-alpha, down-regulated the surface expression and Ser831 phosphorylation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR1. Agents that block IL-1beta receptor activity abolished these effects. In contrast, no change in the surface expression of the N-methyl-d-aspartate (NMDA) receptor subunit NR1 was observed. The inhibition of NMDA receptor activity or depletion of extracellular calcium blocked IL-1beta effects on GluR1 phosphorylation and surface expression. NMDA-mediated calcium influx was also regulated by IL-1beta. These findings suggest that IL-1beta selectively regulates AMPA receptor phosphorylation and surface expression through extracellular calcium and an unknown mechanism involving NMDA receptor activity.     

姜黄素对AMPA/KA受体介导大鼠海马神经元钙内流的影响

目的 探讨姜黄素对α-氨基-3-羧基-5-甲基异恶唑-4-丙酸(AMPA)/海人酸(KA)受体介导大鼠海马神经元钙内流的影响.方法 选用胚胎17dSD鼠分离海马,离体培养海马神经元,借助活体钙荧光染色和激光共聚焦钙成像技术观察100μmol/LKA刺激海马神经元内钙的变化,不同浓度(5、10、15、30、50 μmol/L)姜黄素预孵育海马神经元30min对100μmol/L KA刺激下细胞内钙变化的影响,15 μmol/L姜黄素对不同浓度(10、30、50、100、200、300 μmol/L)KA刺激海马神经元内钙变化的影响.应用钴染色技术观察(30、100 μmol/L KA)刺激后海马神经元钴阳性染色细胞变化.姜黄素预孵育30min对KA刺激导致钴阳性染色细胞变化的影响.结果 不同浓度姜黄素预孵育30 min均可以明显缓解100 μmol/L或30 μmol/L KA导致的细胞内钙升高程度.差异均有统计学意义(P<0.05),其中15 μmol/L姜黄素作用最为明显.30μmol/L或100 μmol/LKA刺激均可以引起海马神经元钴染色阳性细胞增加,15 μmol/L姜黄素预处理30 min后明显减少钴染色阳性细胞,差异有统计学意义(P<0.05),而其他浓度(5 μmol/L或30 μmol/L)姜黄素未见明显影响.结论 一定浓度的姜黄素可以影响AMPA/KA受体介导大鼠海马神经元钙内流.这可能是姜黄素抗癫痫作用的一个机制.     

Transient Ca2+-permeable AMPA receptors in postnatal rat primary auditory neurons

Fast excitatory transmission in the nervous system is mostly mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors whose subunit composition governs physiological characteristics such as ligand affinity and ion conductance properties. Here, we report that AMPA receptors at inner hair cell (IHC) synapses lack the GluR2 subunit and are transiently Ca2+-permeable before hearing onset as evidenced using agonist-induced Co2+ accumulation, Western blots and GluR2 confocal microscopy in the rat cochlea. AMPA (100 microM) induced Co2+ accumulation in primary auditory neurons until postnatal day (PND) 10. This accumulation was concentration-dependent, strengthened by cyclothiazide (50 microM) and blocked by GYKI 52466 (80 microM) and Joro spider toxin (1 microM). It was unaffected by D-AP5 (50 microM), and it could not be elicited by 56 mM K+ or 1 mM NMDA + 10 microM glycine. Western blots showed that GluR1 immunoreactivity, present in homogenates of immature cochleas, had disappeared by PND12. GluR2 immunoreactivity was not detected until PND10 and GluR3 and GluR4 immunoreactivities were detected at all the ages examined. Confocal microscopy confirmed that the GluR2 immunofluorescence was not located postsynaptically to IHCs before PND10. In conclusion, AMPA receptors on maturing primary auditory neurons differ from those on adult neurons. They are probably composed of GluR1, GluR3 and GluR4 subunits and have a high Ca2+ permeability. The postsynaptic expression of GluR2 subunits may be continuously regulated by the presynaptic activity allowing for variations in the Ca2+ permeability and physiological properties of the receptor.     

Potassium chloride depolarization mediates CREB phosphorylation in striatal neurons in an NMDA receptor-dependent manner

Potassium chloride (KCl)-depolarization has been used to study the properties of L-type Ca²⁺ channel-mediated signal transduction in hippocampal neurons. Calcium influx through L-type Ca²⁺ channels stimulates a second messenger pathway that transactivates genes under the regulatory control of the Ca²⁺- and cyclic AMP-responsive element (CRE). Here, we show that in striatal neurons, but not in hippocampal neurons, CRE binding protein (CREB) phosphorylation and CRE-mediated gene expression after KCl-depolarization depends on functional NMDA receptors. This difference in NMDA receptor dependence is not due to different properties of L-type Ca²⁺ channels in either neuronal type, but rather to different neuron-intrinsic properties. Despite this variation, the second messenger pathway activated by KCl requires Ca²⁺/calmodulin (CaM) kinase for CREB phosphorylation in both neuronal types. We conclude that depolarization by KCl works differently in striatal and hippocampal neurons.     

REM sleep deprivation reduces auditory evoked inhibition of dorsolateral pontine neurons

In many dorsolateral pontine neurons, auditory stimulation produces an initial excitation followed by a sustained inhibition. We now report that rapid eye movement (REM) sleep deprivation, for periods of from 22-48 h, reduced this auditory evoked inhibition of unit discharge. Inhibition returned to baseline levels after recovery REM sleep. Prior work indicates that the auditory evoked inhibition seen in noradrenergic cells in this region is partially mediated by norepinephrine. We hypothesize that the reduction in inhibition that we see is a consequence of either downregulation/desensitization of norepinephrine receptors or reduced norepinephrine release resulting from REM sleep deprivation.     

Sensory deprivation regulates the development of the hyperpolarization-activated current in auditory brainstem neurons

Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels are highly expressed in the superior olivary complex, the primary locus for binaural information processing. This hyperpolarization-activated current ( I _h) regulates the excitability of neurons and enhances the temporally precise analysis of the binaural acoustic cues. By using the whole-cell patch-clamp technique, we examined the properties of I _h current in neurons of the lateral superior olive (LSO) and the medial nucleus of the trapezoid body (MNTB) before and after hearing onset. Moreover, we tested the hypothesis that I _h currents are actively regulated by sensory input activity by performing bilateral and unilateral cochlear ablations before hearing onset, resulting in a chronic auditory deprivation. The results show that after hearing onset, I _h currents are rapidly upregulated in LSO neurons, but change only marginally in neurons of the MNTB. We also found a striking difference in maximal current density, voltage dependence and activation time constant between the LSO and the MNTB in mature-like animals. Following bilateral cochlear ablations before hearing onset, the I _h currents were scaled up in the LSO and scaled down in the MNTB. Consequently, in the LSO this resulted in a depolarized resting membrane potential and a lower input resistance of these neurons. This type of activity-dependent homeostatic change could thus result in an augmented response to the remaining inputs.     

Neurochemical evidence that AMPA receptor-mediated tonic inhibition of hypothalamic dopaminergic neurons occurs via activation of inhibitory interneurons

Blockade of -amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors with 6-nitro-7-sulfamoyl-benzo-[f]quinoxaline-2,3(1H,4H)-dione (NBOX) activates tuberoinfundibular (TIDA) and periventricular-hypophysial dopaminergic (PHDA) neurons suggesting that these neurons are tonically inhibited by endogenous excitatory amino acid neurotransmitters acting at AMPA receptors. The purpose of the present study was to identify prospective inhibitory neurotransmitters mediating this effect by examining the ability of γ-aminobutyric acid (GABA)_A and κ-opioid receptor agonists to reverse the stimulatory effects of NBQX on the activity of these neurons (estimated from concentrations of 3,4-dihydroxyphenylacetic acid [DOPAC] in the median eminence and intermediate lobe of the pituitary). The GABA_A receptor agonist isoguvacine prevented the NBQX-induced increase in DOPAC concentrations in the median eminence but not in the intermediate lobe. The κ-opioid receptor agonist U-50,488 had no effect in the median eminence, but attenuated the NBQX-induced increase of DOPAC concentrations in the intermediate lobe. These results suggest that excitatory amino acid neurotransmitters activate AMPA receptors and increase release of GABA, which by acting at GABA, which by acting at GABA_A receptors tonically inhibits TIDA neurons. On the other hand, AMPA receptor-mediated tonic inhibition of PHDA neurons occurs, at least in part, by a mechanism involving endogenous κ-opioids.     

Selective retrograde transport of nipecotic acid, a GABA analog, labels a subpopulation of gerbil olivocochlear neurons.

Perfusion of the gerbil cochlea with micromolar quantities of 3H-gamma-aminobutyric acid (GABA) results in rapid, selective labeling of 50-60% of the olivocochlear (OC) efferent terminals on afferent dendrites beneath the inner hair cells, and all of the efferent terminals beneath the outer hair cells. In order to identify the neurons from which these GABA-accumulating terminals originate, the cell bodies were localized by using retrograde transport of 3H-nipecotic acid, a metabolically inert GABA analog. With survival times of 6-30 hours after cochlear injection, myelinated OC efferent fibers and cell bodies were well labeled, with the greatest number being labeled at 12-18 hours. All of the labeled neurons belonged to the medial OC system, and no lateral OC neurons were labeled. It is concluded that the GABA-accumulating endings in the gerbil cochlea arise from medial OC neurons, and therefore that medial OC efferent neurons in this species project to both inner and outer hair cell regions.     

Transforming growth factor-beta2 modulates synaptic efficacy and plasticity and induces phosphorylation of CREB in hippocampal neurons

Transforming growth factor-betas (TGF-betas) are widely expressed and play roles as multifunctional growth factors and regulators of key events in development, disease, and repair. However, it is not known whether TGF-betas affect the plasticity of hippocampal neurons. As a first step to address this issue, we examined whether TGF-beta2 modulated the electrophysiological and biochemical properties of cultured hippocampal neurons. We found that prolonged 24 h treatment with TGF-beta2 induced facilitation of evoked postsynaptic currents (ePSCs). This facilitation was associated with a decrease in short-term synaptic depression of ePSCs and increases in both the amplitude and frequency of spontaneous miniature postsynaptic currents (mPSCs). The long-term changes of ePSCs and mPSCs may be associated with cAMP response element-binding protein (CREB), which has been previously implicated in long-term potentiation. Immunofluorescence techniques and Western blot analysis both revealed that TGF-beta2 enhanced the phosphorylation of CREB. Together, these results suggest that TGF-beta2 may play a role in the cascade of events underlying long-term synaptic facilitation in hippocampus, and that CREB may be an important mediator of these effects.     

Ivermectin inhibits AMPA receptor-mediated excitotoxicity in cultured motor neurons and extends the life span of a transgenic mouse model of amyotrophic lateral sclerosis

alpha-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-mediated excitotoxicity contributes to the selective motor neuron death in amyotrophic lateral sclerosis (ALS). In this study, we investigated the effect of P2 receptor-influencing substances on kainate-induced motor neuron death in an in vitro model for AMPA receptor-mediated excitotoxicity. Complete protection was found after preincubation of the motor neurons with ivermectin or Cibacron Blue 3G-A. Preincubation with both P2X4 modulators did not influence the number or Ca2+ permeability of the AMPA receptors and addition during kainate stimulation alone had no effect. Preincubation with a low concentration of ATP, the natural agonist of the P2X4 receptor, also protected the motor neurons against a subsequent excitotoxic stimulation, while high concentrations of ATP were toxic. Moreover, ivermectin increased the toxicity of low ATP concentrations, indicating that ivermectin can potentiate the effect of ATP on its receptor. Ivermectin and ATP also protected against hypoxia/hypoglycemia. To further investigate the relevance of these findings for ALS, we treated SOD1(G93A)-mice, a transgenic animal model for familial ALS, with ivermectin. This resulted in an extension of the life span of these mice with almost 10%. We conclude that ivermectin induces a mechanism in motor neurons, in vivo and in vitro, that protects against subsequent excitotoxic insults. Our in vitro data indicate that this protective mechanism is due to the potentiation by ivermectin of an effect of ATP mediated by the P2X4 receptor.     

Impaired modulation of AMPA receptors by calcium-dependent processes in streptozotocin-induced diabetic rats

The mechanisms by which diabetes impairs cognitive function are not well-established. In the present study, we determined the electrophysiological and biochemical nature of disturbances in the mechanism of long-term potentiation (LTP) in diabetic rats. As previously reported, the administration of streptozotocin (STZ) was found to reduce the magnitude of LTP in the CA1 region of the hippocampus, while the same treatment did not interact with the capacity of the hippocampus to generate long-term depression induced by low-frequency stimulation. In addition, STZ treatment did not modify the component of excitatory postsynaptic potentials mediated by activation of the N-methyl-

Full-size image

-aspartate (NMDA) subtype of glutamate receptors, suggesting that NMDA receptor function remained intact in STZ-treated slices. At the biochemical level, the capacity of calcium to increase [³H](RS)-α-amino-3-hydroxy-5-methylisoxazole propionic acid (³H-AMPA) binding to glutamate/AMPA receptors in rat brain tissue sections was markedly affected in most regions of the hippocampus of STZ-treated rats. Moreover, changes in ³H-AMPA binding properties elicited by both exogenous phospholipase A₂ and melittin, a potent activator of endogenous phospholipases, were also altered in synaptoneurosomes from diabetic rats. Taken together, the present data suggest that the loss of LTP maintenance in STZ-treated rats is more likely the result of disruption of calcium-dependent processes that are suspected to modulate postsynaptic AMPA receptors during synaptic potentiation. Understanding the biochemical factors participating in the impairment of AMPA receptor modulation might provide important clues revealing the very basis of memory deficits in diabetes.     

Temporal profile of CREB phosphorylation after focal ischemia in rat brain.

The phosphorylation of cAMP response element binding protein (CREB) in the rat brain was examined immunohistochemically at 3.5 h, 12 h, 24 h and 48 h of recirculation after focal ischemia induced by occlusion of the middle cerebral artery for 1.5 h. Brain sections were stained with affinity purified anti-phosphorylated CREB antibody. The ischemic core revealed a significant, but transient increase in number of phosphorylated CREB-positive cells at 3.5 h of recirculation, followed by a rapid decrease during the subsequent period. In the peri-ischemia area, the number of phosphorylated CREB-positive cells showed a more marked increase as compared to that in the ischemic core at 3.5 h of recirculation, and the increase continued until 48 h of recirculation with a tendency for gradual decline. Persistent enhancement of CREB phosphorylation may thus be closely related to the neuronal viability and neuroprotective mechanisms, whereas rapid disappearance of CREB phosphorylation may clearly precede neuronal death.     

Activation of a subpopulation of orexin/hypocretin-containing hypothalamic neurons by GABAA receptor-mediated inhibition of the nucleus accumbens shell, but not by exposure to a novel environment

Gamma-amino butyric acid (GABA)A receptor stimulation in the nucleus accumbens shell produces intense hyperphagia in rats and increases Fos expression in the lateral hypothalamus. To explore the involvement of hypothalamic orexin/hypocretin- or melanin concentrating hormone-immunoreactive neurons in this effect, the GABAA agonist, muscimol (0, 50 ng), was infused directly into the nucleus accumbens shell of rats; 90 min later, their brains were collected and subsequently processed for immunohistochemistry. A group exposed to a novel environment was included to evaluate the specificity of Fos expression changes with regard to general arousal. Alternating sections through the hypothalamus were double-stained for orexin/hypocretin-Fos or melanin concentrating hormone-Fos combinations. Intra-accumbens shell muscimol treatment significantly increased the percentage of orexin/hypocretin-containing neurons expressing Fos in the lateral, but not medial, portion of the perifornical/lateral hypothalamic area. Regardless of treatment condition, greater percentages of orexin/hypocretin-containing neurons in the medial portion of the hypothalamus expressed Fos relative to cells located more laterally. None of the manipulations increased Fos expression in melanin concentrating hormone-immunoreactive neurons. Muscimol treatment also markedly increased Fos expression in the arcuate nucleus, which connects reciprocally to the lateral/perifornical hypothalamic area. Thus, orexin/hypocretin-containing neurons in lateral sectors of the hypothalamus, along with cells in the arcuate nucleus, display phasic increases in Fos expression after an orexigenic pharmacological manipulation of the nucleus accumbens shell, but to a lesser degree after the heightened arousal associated with exposure to a novel environment.     

Pentylenetetrazole-induced bursting activity and cellular protein phosphorylation in snail neurons

To elucidate the intracellular mechanism of seizure discharge, phosphorylation of cellular protein during pentylenetetrazole (PTZ)-induced bursting activity in snail neurons was investigated. PTZ markedly enhanced the phosphorylation of proteins of 34,000 and 50,000 molecular weight. Similar effects were observed by application of a calcium ionophore, A23187. Calmodulin antagonist, N-(6-aminohexyl)-5-chloronaphthalenesulfonamide hydrochloride (W-7), inhibited the PTZ-induced increased phosphorylation of these two proteins. Dibutyryl cyclic AMP and iosobutylmethylxanthine (IBMX) showed no significant effect on the phosphorylation pattern. Bath application of the calcium ionophore produced bursting activity followed by long-lasting hyperpolarization. Bath application of W-7 completely inhibited the PTZ-induced bursting activity. These results suggest that the phosphorylation of proteins of 34,000 and 50,000 in molecular weight is related to the generation of bursting activity by PTZ.