Glutamate-mediated excitotoxicity in neonatal hippocampal neurons is mediated by mGluR-induced release of Ca++ from intracellular stores and is prevented by estradiol

Hypoxic/ischemic (HI) brain injury in newborn full-term and premature infants is a common and pervasive source of life time disabilities in cognitive and locomotor function. In the adult, HI induces glutamate release and excitotoxic cell death dependent on NMDA receptor activation. In animal models of the premature human infant, glutamate is also released following HI, but neurons are largely insensitive to NMDA or AMPA/kainic acid (KA) receptor-mediated damage. Using primary cultured hippocampal neurons we have determined that glutamate increases intracellular calcium much more than kainic acid. Moreover, glutamate induces cell death by activating Type I metabotropic glutamate receptors (mGluRs). Pretreatment of neurons with the gonadal steroid estradiol reduces the level of the Type I metabotropic glutamate receptors and completely prevents cell death, suggesting a novel therapeutic approach to excitotoxic brain damage in the neonate.     

Striatal degeneration induced by mitochondrial blockade is prevented by biologically delivered NGF

Consistent with the notion that a defect in cellular energy metabolism is a cause of human neurodegenerative disease, systemic treatment with the mitochondrial complex II inhibitor 3-nitropropionic acid (3-NPA) can model the striatal neurodegeneration seen in Huntington's disease. Previously, we have found that nerve growth factor (NGF), delivered biologically by the implantation of a genetically altered fibroblast cell-line, can protect locally against striatal degeneration induced by infusions of high doses of glutamate receptor agonists. We now report that implantation of NGF-secreting fibroblasts reduces the size of adjacent striatal 3-NPA lesions by an average of 64%. We conclude that biologically delivered NGF protects neurons against excitotoxicity and mitochondrial blockade—both energy-depleting processes—implying that appropriate neurotrophic support in the adult brain could protect against neurodegenerative diseases caused in part by energy depletion. © 1993 Wiley-Liss, Inc.     

Pain hypersensitivity induced by paradoxical sleep deprivation is not due to altered binding to brain mu-opioid receptors

Previous studies have established a relationship between sleep disruption and pain, and it has been suggested that hyperalgesia induced by paradoxical sleep deprivation (PSD) could be due to a reduction of opioidergic neurotransmission in the brain. In the present study rats deprived of sleep for 96 h as well as rats allowed to recover for 24h after PSD and normal controls received vehicle or morphine (2.5, 5 and 10 mg/kg, i.p.) and were tested on a hot plate 1h later. Quantitative receptor autoradiography was used to map alterations in binding to brain mu-opioid receptors in separate groups. Results demonstrated that PSD induced a significant reduction in thermal pain threshold, as measured by paw withdrawal latencies. This effect did not return to baseline control values after 24h of sleep recovery. The usual analgesic effect of morphine was observed in the control group but not in PSD or rebound groups except at the highest dose (10 mg/kg). Binding of [3H]DAMGO to mu sites did not differ significantly among the three groups in any of the 33 brain regions examined. These results do not exclude the participation of the opioid system in PSD-induced pain hypersensitivity since sleep-deprived rats were clearly resistant to morphine. However, the fact no changes were seen in [3H]DAMGO binding indicates that mechanisms other than altered mu-opioid binding must be sought to explain the phenomenon.     

Death of cultured human neuroblastoma cells induced by HIV-1 gp120 is prevented by NMDA receptor antagonists and inhibitors of nitric oxide and cyclooxygenase

The cytotoxic effects of the human immunodeficiency virus type 1 (HIV-1) coat protein gp120 were studied in human CHP100 neuroblastoma cell cultures. Incubation of neuroblastoma cultures with gp120 (1 pM-10 nM) induces cell death which is not concentration-related. The significant cell death evoked by 10 pM gp120 was prevented by neutralization of the viral protein with a monoclonal anti-gp120 (IgG) antibody. In addition, gp120-induced cytotoxicity was inhibited by [DL-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid] (CGP37849; 100 μM), [(±)-3R1, 4as1, 6R1, 8aR1-6-(phosphonomethyl) decahydro-isoquinoline-3-carboxylic acid] (LY274614; 100 μM), MK801 (dizocilpine; 200 nM) and 7-chloro kynurenic acid (100 μM), selective antagonists of the NMDA receptor complex; by contrast, (6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 100 μM), a non-NMDA antagonist, was ineffective. Prevention of the lethality elicited by the HIV-1 coat protein was also obtained by incubating neuroblastoma cells with gp120 in Ca²⁺-free medium. The lethal effects induced by gp120 involve activation of L-arginine-nitric oxide (NO) pathway since these were prevented by haemoglobin (10 μM), a NO-trapping agent, and by D-arginine (1 mM), the less active enantiomer of the endogenous precursor of NO synthesis. Cytoprotection was also afforded by N^ω-nitro-L-arginine methyl ester (L-NAME; 200 μM), an inhibitor of NO synthase, and this was reversed by L-arginine (1 mM). Interestingly, indomethacin and flufenamic acid (10 μM), two inhibitors of cyclooxygenase, protected neuroblastoma cells from death induced by gp120. Furthermore, indomethacin prevented the neuroblastoma cell death evoked by exposure of cultures to sodium nitroprusside (SNP; 0.2–1.6 mM), a NO donor. Finally significant cytotoxic effects were observed after incubation of neuroblastoma cells with prostaglandin E₂ (0.1–10 μM). In conclusion, the present data suggest that death of human CHP100 neuroblastoma cells in culture produced by gp120 involves NO and PGE₂ production.     

Survival of motoneurons following axotomy is enhanced by lactation or by progesterone treatment

It was shown previously that neuron loss induced by nerve transection in the hypoglossal and facial motor nuclei of female rats could be reduced by long-term testosterone treatment following axotomy. In the present study, it is reported that such neuron loss could also be attenuated by lactation-related hormones and by progesterone treatment following axotomy.     

The trophic effect of beta-amyloid 25-35 peptide is not mediated by NK1 or bombesin receptors.

beta-Amyloid peptide and spantide have previously been described to have trophic effects on hippocampal neurones in vitro. We report here that bombesin and [Pro9]-substance P also show a neurotrophic effect on cultured hippocampal neurones. The neurotrophic effect of spantide or a beta-amyloid fragment containing amino acids 25 to 35 was not blocked by addition of the NK1 receptor agonist, substance P or the nonpeptide NK1 antagonist, RP 67580. For the bombesin-related peptides, the antagonist [Tyr4-DPhe12]-bombesin also provokes a trophic response, but the agonist alytesin and the antagonist [Leu13-psi-(CH2NH) Leu14]-bombesin have no effect on neurite growth. These results suggest that the observed trophic responses are unlikely to be mediated by a classical NK1 or bombesin receptor.     

Natriuresis, not seizures, induced by cholinergic stimulation of the locus coeruleus is affected by forebrain lesions and water deprivation

Two groups of rats with electrolytic lesions of the medial and upper septal area (MUL) or, alternatively, of the anteroventral portion of the third ventricle (AV3V) and a third group of sham-operated rats were water loaded and received three carbachol injections into the locus coeruleus according to the following schedule: 1) prelesion, 2) on the second postlesion day and 3) on the seventh postlesion day. Both MUL and AV3V lesions inhibited the carbachol-induced natriuresis on the second postlesion day. Recovery was almost complete after MUL but not after AV3V lesion on the seventh day. Water deprivation also reduced the carbachol-induced natriuresis but passive hydration of AV3V animals did not avoid the impairment induced by the lesion. Transient seizure phenomena such as clonic convulsions, salivation and analgesia subsequent to carbachol injection were not altered by the lesions.     

Alpha-synuclein protein is not scavenged in neuronal loss induced by kainic acid or focal ischemia

Alpha-synuclein, a presynaptic protein, is markedly included in Lewy bodies (LB) in Parkinson's and LB diseases. In this study, neuronal loss and the activation of glial cells such as microglia and astrocytes were induced by neurodegenerative insults such as the injection of kainic acid and occlusion of the middle cerebral artery. In contrast, immunoreactivity for alpha-synuclein did not change even at 7 days after these insults. These results suggest that alpha-synuclein protein may be so scarcely scavenged by glial cells that it readily condenses in neurodegenerative regions.     

Galanin is an autocrine myelin and oligodendrocyte trophic signal induced by leukemia inhibitory factor

In order to further investigate the molecular mechanisms that regulate oligodendrocyte (OC) survival, we utilized microarrays to characterize changes in OC gene expression after exposure to the cytokines neurotrophin3, insulin, or leukemia inhibitory factor (LIF) in vitro. We identified and validated the induction and secretion of the neuropeptide galanin in OCs, specifically in response to LIF. We next established that galanin is an OC survival factor and showed that autocrine or paracrine galanin secretion mediates LIF‐induced OC survival in vitro. We also revealed that galanin is up‐regulated in OCs in the cuprizone model of central demyelination, and that oligodendroglial galanin expression is significantly regulated by endogenous LIF in this context. We also showed that knock‐out of galanin reduces OC survival and exacerbates callosal demyelination in the cuprizone model. These findings suggest a potential role for the use of galanin agonists in the treatment of human demyelinating diseases. GLIA 2015;63:1005–1020     

REM sleep enhancement induced by sensory stimulation is prevented by kainic acid lesion of the pontine reticular formation

It has been shown that auditory or somatic stimulation during rapid eye movement (REM) sleep is capable of producing a significant increase in ponto-geniculo-occipital (PGO) spike density as well as in REM sleep duration. The purpose of this study was to determine the role of the medial pontine reticular formation (PRF) in mediating such increase of REM sleep duration. After a baseline recording whereby on the same recording day the control and the stimulus (auditory or somatic) alternated with each REM, a group of cats was lesioned with kainic acid in the PRF. The sleep-wake cycle was recorded again on days 15, 30 and 45 post-lesion, following the same procedure. The results showed no changes in REM sleep duration and PGO spike density in the lesioned animals. However, when sensory stimulation was applied it was ineffective in producing REM sleep enhancement, although it was able to increase PGO spike density. These findings suggest that the effects of sensory stimulation on REM sleep duration are accomplished through the PRF, probably by inducing an increase in the excitability levels of such neurons, and further suggests that PGO spike density and REM duration are independent of each other.     

Postnatal development of GFAP in mouse visual cortex is not affected by light deprivation

Mammalian visual cortex is immature at birth and develops gradually during defined postnatal temporal windows. In the present work, we studied the maturation of astrocytes in developing mouse visual cortex (VC). The cellular distribution and the level of glial fibrillary acidic protein (GFAP) were analyzed by immunohistochemistry and Western blotting. Experiments were performed at different postnatal ages: postnatal day 12 (P12), before eye opening; P24, corresponding roughly to the peak of the critical period for monocular deprivation, and P60, after the end of the critical period. At P12, GFAP immunoreactivity (IR) was distributed throughout all cortical layers. At P24, there was a prominent localization of GFAP IR in layers I, II, and VI, while cortical layers III, IV, and V contained no longer GFAP IR cells. No differences were found in GFAP IR between P24 and P60. Western blot analysis revealed a reduction of GFAP expression in the VC at P24 with respect to P12 and no significant difference between P60 and P24. These results show that GFAP expression is modulated during early postnatal development. To know whether visual experience influences the maturation pattern of GFAP expression, mice were dark-reared from P12 to P24. Dark rearing did not change the distribution and the expression of GFAP. Our results indicate that maturation of GFAP expression occurs early in postnatal development in mouse VC. In addition, we showed that GFAP development is not affected by visual deprivation.     

L-DOPA neurotoxicity is prevented by neuroprotective effects of erythropoietin

The neurotoxicity of L-3,4-dihydroxyphenylalanine (L-DOPA), one of the most important drugs for the treatment of Parkinson's disease, still remains controversial, although much more data on L-DOPA neurotoxicity have been presented. Considering the well known neuroprotective effects of erythropoietin (EPO), the inhibitory effects of EPO on L-DOPA neurotoxicity need to be evaluated. Neuronally differentiated PC12 (nPC12) cells were treated with different concentrations of L-DOPA and/or EPO for 24h. Cell viability was evaluated using trypan blue, 4',6-diamidino-2-phenylindole (DAPI) and TUNEL staining, and cell counting. Free radicals and intracellular signaling protein levels were measured with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and Western blotting, respectively. L-DOPA reduced nPC12 cell viability at higher concentrations, but combined treatment with EPO and L-DOPA significantly restored cell viability. Free radicals and hydroxyl radical levels increased by L-DOPA were decreased after combined treatment of L-DOPA and EPO. Levels of survival-related intracellular signaling proteins decreased in nPC12 cells treated with 200 μM L-DOPA but increased significantly in cells treated with 200μM L-DOPA and 5 μM EPO. However, cleaved caspase-3, a death-related protein, increased in nPC12 cells treated with 200 μM L-DOPA but decreased significantly in cells treated with 200 μM L-DOPA and 5 μM EPO. Pretreatment with LY294002, a phosphatidylinositol 3-kinase inhibitor, prior to combined treatment with EPO and L-DOPA almost completely blocked the protective effects of EPO. These results indicate that EPO can prevent L-DOPA neurotoxicity by activating the PI3K pathway as well as reducing oxidative stress.     

Hyperlocomotion induced by dopamine or cholecystokinin + dopamine in the nucleus accumbens is not modified by chronic lithium treatment

1. Peptides such as cholecystokinin have been reported to modulate the effects of dopaminergic agonists on locomotion in rats. 2. The present experiments tested the possibility that lithium interacts with dopaminergic function through the same mechanism by which cholecystokinin potentiates dopaminergic function. 3. The results suggest that dietary lithium has no effect on the ability of either dopamine alone, or the combination of dopamine plus cholecystokinin, microinjected directly into the nucleus accumbens, to stimulate hyperlocomotion.     

Feeding induced by food deprivation is differentially reduced by G-protein alpha-subunit antisense probes in rats

The alpha 7 nicotinic acetylcholine receptor (nAChR) subunit can be assembled to form a homomeric-pentamer with high permeability to calcium. Although the expression of the alpha 7-nAChR has been demonstrated throughout the CNS, the neurochemical phenotype of neurons expressing alpha 7 remains to a large extent unknown. Using an antibody against the alpha 7 nAChR subunit, immunohistochemical staining was observed in rat dorsal raphe nucleus (DRN) and locus coeruleus (LC), serotonergic and noradrenergic brainstem nuclei, respectively. In both the DRN and LC, there appeared to be two histologically distinct alpha 7-expressing cell types as distinguished by size, i.e. large versus small diameter. In rats treated with either a serotonergic (5,7-dihydroxytryptamine) or noradrenergic (anti-dopamine-beta-hydroxylase saporin) neurotoxin, tryptophan hydroxylase and tyrosine hydroxylase immunostaining was abolished, respectively. Similarly, the alpha 7-positive large-diameter cells were no longer detectable, suggesting that these cells were serotonergic DRN and noradrenergic LC neurons. Indeed, double-labeling experiments revealed in the large cell types coexpression of alpha 7 with tryptophan hydroxylase in the DRN and with tyrosine hydroxylase in the LC of saline-treated rats. In contrast to the large-diameter cells, the alpha 7-positive small-diameter cells were neither serotonergic nor adrenergic, and were still detected in both the DRN and LC of lesioned rats. Moreover, cell counts revealed an increase number of these cells in lesioned rats with expression of alpha 7 in somal processes not seen in non-lesioned controls. Double labeling revealed coexpression of alpha 7 and GABA within the majority, but not all, of the toxin-resistant cells. The results of these studies suggest that both serotonergic and noradrenergic neurons express alpha 7 nAChRs. In addition, there appears to be a small-diameter cell-type in both the DRN and LC, possibly a GABAergic interneuron, expressing alpha 7 that may be regulated by neurotoxic injury.     

Monitoring of glutamate‐induced excitotoxicity by mitochondrial oxygen consumption

Dysfunction of mitochondrial activity is often associated with the onset and progress of neurodegenerative diseases. Membrane depolarization induced by Na⁺ influx increases intracellular Ca²⁺ levels in neurons, which upregulates mitochondrial activity. However, overlimit of Na⁺ influx and its prolonged retention ultimately cause excitotoxicity leading to neuronal cell death. To return the membrane potential to the normal level, Na⁺/K⁺‐ATPase exchanges intracellular Na⁺ with extracellular K⁺ by consuming a large amount of ATP. This is a reason why mitochondria are important for maintaining neurons. In addition, astrocytes are thought to be important for supporting neighboring neurons by acting as energy providers and eliminators of excessive neurotransmitters. In this study, we examined the meaning of changes in the mitochondrial oxygen consumption rate (OCR) in primary mouse neuronal populations. By varying the medium constituents and using channel modulators, we found that pyruvate rather than lactate supported OCR levels and conferred on neurons resistance to glutamate‐mediated excitotoxicity. Under a pyruvate‐restricted condition, our OCR monitoring could detect excitotoxicity induced by glutamate at only 10 μM. The OCR monitoring also revealed the contribution of the N‐methyl‐D‐aspartate receptor and Na⁺/K⁺‐ATPase to the toxicity, which allowed evaluating spontaneous excitation. In addition, the OCR monitoring showed that astrocytes preferentially used glutamate, not glutamine, for a substrate of the tricarboxylic acid cycle. This mechanism may be coupled with astrocyte‐dependent protection of neurons from glutamate‐mediated excitotoxicity. These results suggest that OCR monitoring would provide a new powerful tool to analyze the mechanisms underlying neurotoxicity and protection against it.     

The reductions in sweetened milk intake induced by interleukin-1 and endotoxin are not prevented by chronic antidepressant treatment.

Administration of interleukin-1 (IL-1) and endotoxin (lipopolysaccharide, LPS) to rodents can decrease food intake, a behavioral response resembling the diminution of appetite observed in human depression. IL-1 and LPS are known to affect cerebral neurotransmission involving norepinephrine and serotonin, both of which have been implicated in feeding behavior and in the pharmacotherapy of depression in man. The ability of chronic antidepressant treatment to attenuate LPS-induced depressed feeding in rats has been cited as evidence that cytokines may be involved in human depression. Thus, we studied the effects of chronic treatment with the tricyclic antidepressant, imipramine, and the novel antidepressant, venlafaxine, on the sweetened milk intake challenged with intraperitoneally injected IL-1 beta and LPS. Chronic (from 2 to 8 weeks) treatment of the mice with imipramine (10 mg/kg once or twice daily) or venlafaxine (10 and 20 mg/kg/day) did not significantly alter the decreases in milk intake in response to mIL-1 beta or LPS. In some experiments, chronic imipramine slightly decreased body weight and slightly increased milk intake, but not food pellet intake. Venlafaxine had none of these effects. Analysis of variance did not indicate any significant interactions between the antidepressant and IL-1 or LPS treatments. These results indicate that chronic treatment with antidepressants does not significantly alter the responses to IL-1 or LPS in the mouse sweetened milk model of sickness behavior.     

NOX2 mediates apoptotic death induced by staurosporine but not by potassium deprivation in cerebellar granule neurons

Neuronal apoptotic death involves the participation of reactive oxygen species (ROS), but their sources have not been completely elucidated. Previous studies have demonstrated that the ROS‐producing enzyme NADPH oxidase is present in neuronal cells and that this enzyme could participate in the apoptotic neuronal death. Cerebellar granule neurons (CGN) undergo apoptosis when cells are transferred from a medium with 25 mM KCl (K25) to a 5 mM KCl (K5) medium or when they are treated with staurosporine (ST). Under these conditions, apoptotic death of CGN is dependent on ROS production. In this study, we evaluated the role of NOX2, an NADPH oxidase homolog, in the apoptotic death of CGN induced by two different conditions. In CGN from NOX2‐deficient (ko) mice, a significantly lower rate of apoptotic death occurs compared with wild‐type (wt) CGN. Also, caspase‐3 activation, NADPH oxidase activity, and superoxide anion production induced by ST were markedly lower in ko neurons than in wt CGN. In contrast to the case with ST, when CGN were treated with K5, no differences were observed between ko and wt cells in any of the parameters measured. However, all NADPH oxidase inhibitors tested noticeably reduced cell death and apoptotic parameters induced by K5 in both wt and ko CGN. These results suggest that NOX2 could be necessary for apoptotic death induced by ST, but not by K5, which could require other member of the NOX family in the apoptotic process. © 2009 Wiley‐Liss, Inc.     

Corticotropin-releasing factor type-1 receptor mRNA is not induced in mouse hypothalamus by either stress or osmotic stimulation

In rats, acute stress substantially increases corticotropin-releasing factor (CRF) type 1 receptor (CRFR-1) mRNA expression in the paraventricular nucleus (PVN) and osmotic stimulation induces both CRF and CRFR-1 mRNA in magnocellular PVN and supraoptic nucleus (SON). However, these phenomena have not been analysed in other species. We compared CRF and CRFR-1 expression in rat and mouse hypothalamus. Male C57BL/6 mice and Wistar rats were exposed to acute restraint stress for 3 h, or to hypertonic saline ingestion for 7 days. Restraint stress increased CRF and c-fos mRNA expression in both rat and mouse PVN. CRFR-1 mRNA was barely detectable in controls, whereas restraint stress substantially increased CRFR-1 mRNA in rat PVN, but not in mouse. Hypertonic saline ingestion induced CRF mRNA in magnocellular PVN and SON of the rat, but did not alter CRF mRNA levels in mouse hypothalamus. CRFR-1 mRNA was also induced in magnocellular PVN and SON of the rat in response to osmotic stimulation, but not in mouse. Immunohistochemistry demonstrated that CRFR-1-like immunoreactivity (ir) was distributed within parvocellular and magnocellular PVN of mouse and rat. CRFR-1-ir in rat PVN was increased by acute stress and osmotic stimulation. By contrast, these treatments did not alter CRFR-1-ir in mouse PVN. Combined immunohistochemistry and in situ hybridization revealed that CRFR-1-ir was most frequently colocalized to CRF in mouse PVN, whereas only a small percentage of oxytocin and vasopressin-producing cells coexpressed CRFR-1-ir. These results indicate that (i) by contrast to rats, neither acute stress nor osmotic stimulation induces CRFR-1 mRNA expression in the mouse PVN; (ii) osmotic stimulation does not alter CRF mRNA expression in parvocellular and magnocellular neurones of mouse PVN; and (iii) acute stress increases c-fos and CRF mRNA to a similar degree in mouse and rat PVN. Thus, differences may exist between mouse and rat in the regulation of CRF and CRFR-1 gene expression in hypothalamus following stress and osmotic stimulation.     

Roles of Ras-Erk in apoptosis of PC12 cells induced by trophic factor withdrawal or oxidative stress

To understand the role of Ras-MAPK (mitogen-activated protein kinase) in trophic factor withdrawal- and oxidative stress-induced apoptotic cell death processes, undifferentiated rat pheochromocytoma PC12 cells and a PC12 variant cell line stably expressing the Ras dominant-negative mutant (M-M17-26) were subjected to serum withdrawal in the absence or presence of H₂O₂ treatment. The extent of cell death was analyzed by lactate dehydrogenase release, internucleosomal DNA fragmentation, and caspase-3 assays. Both serum with-drawal and H₂O₂ treatment induced apoptotic cell death in PC12 cells, and the extent of cell death was greatly enhanced in M-M17-26 cells. DNA fragmentation induced by serum withdrawal or H₂O₂ treatment was blocked completely by a general caspase inhibitor, Z-VAD-FMK. A selective MAPK kinase inhibitor, U0126, blocked the H₂O₂-induced phosphorylation of Erk1/2 (extracellular signal-regulated kinase) in PC12 cells and increased the levels of active caspase-3 in M-M17-26 under serum withdrawal or H₂O₂ treatment. In addition, the short-term H₂O₂ treatment (5–30 min) was sufficient to cause DNA fragmentation in M-M17-26 cells even though H₂O₂ was removed and cells were incubated in regular growth medium with complete serum for 24 h. However, similar, short-term H₂O₂ treatment of PC12 cells did not induce DNA fragmentation 24 h later. These results suggest that the Ras-Erk pathway is critical in mediating protection against apoptotic cell death induced by either trophic factor withdrawal or increased oxidative stress.