Activity-dependent regulation of the subcellular localization of neuronal calcium sensor-1 in the avian cochlear nucleus

Neurons in the avian cochlear nucleus, nucleus magnocellularis (NM), are highly sensitive to manipulations of afferent input, and removal of afferent activity through cochlear ablation results in the death of approximately 20-40% of ipsilateral NM neurons. The intracellular cascades that determine whether an individual NM neuron will die or survive are not fully understood. One early event observed in NM following deafferentation is a rapid rise in intracellular calcium concentration. In most cellular systems, the activity of calcium-binding proteins is believed to accommodate calcium influx. The calcium-binding protein, neuronal calcium sensor-1 (NCS-1), is an intracellular neuronal calcium sensor belonging to the EF-hand superfamily. NCS-1 has been implicated in calcium-dependent regulation of signaling cascades. To evaluate NCS-1 action in NM neurons, the localization of NCS-1 protein was examined. Double-label immunofluorescence experiments revealed that NCS-1 expression is evident in both the presynaptic nerve terminal and postsynaptic NM neuron. The postsynaptic expression of NCS-1 typically appears to be closely associated with the cell membrane. This close proximity of NCS-1 to the postsynaptic membrane could allow NCS-1 to function as a modulator of postsynaptic signaling events. Following deafferentation, NM neurons were more likely to show diffuse cytoplasmic NCS-1 labeling. This increase in the number of cells showing diffuse cytoplasmic labeling was observed 12 and 24 h following cochlea ablation, but was not observed 4 days following surgery. This activity-dependent regulation of NCS-1 subcellular localization suggests it may be associated with, or influenced by, processes important for the survival of NM neurons.     

Lithium increases bcl-2 expression in chick cochlear nucleus and protects against deafferentation-induced cell death

Approximately 20-30% of neurons in the avian cochlear nucleus (nucleus magnocellularis) die following deafferentation (i.e. deafness produced by cochlea removal) and the remaining neurons show a decrease in soma size. Cell death is generally accepted to be a highly regulated process involving various pro-survival and pro-death molecules. One treatment that has been shown to modify the expression of these molecules is chronic administration of lithium. The present experiments examined whether lithium treatment can protect neurons from deafferentation-induced cell death. Post-hatch chicks were treated with LiCl or saline for 17 consecutive days, beginning on the day of hatching. On the 17th day, a unilateral cochlea ablation was performed. Five days following surgery, the nucleus magnocellularis neurons were counted stereologically on opposite sides of the same brains. Lithium reduced deafferentation-induced cell death by more than 50% (9.8% cell death as compared with 22.4% in saline-treated subjects). Lithium did not affect cell number on the intact side of the brain. Lithium also did not prevent the deafferentation-induced decrease in soma size, suggesting a dissociation between the mechanisms involved in the afferent control of soma size and those involved in the afferent control of cell viability. A possible mechanism for lithium's neuroprotective influence was examined in a second set of subjects. Previous studies suggest that the pro-survival molecule, bcl-2, may play a role in regulating cell death following deafferentation. Tissues from lithium- and saline-treated subjects were examined using immunocytochemistry. Chronic administration of lithium dramatically increased the expression of bcl-2 protein in nucleus magnocellularis neurons. These data suggest that lithium may impart its neuroprotective effect by altering the expression of molecules that regulate cell death.     

The influence of chronic lithium administration on deafferentation-induced cellular changes in the chick cochlear nucleus

The avian brainstem serves as a useful model system to address the question of how afferent activity influences viability of target neurons. Approximately 20-30% of neurons in the chick cochlear nucleus, nucleus magnocellularis (NM) die following deafferentation (i.e. deafness produced by cochlea removal). Previous studies have identified cellular events that occur within hours following cochlea removal, which are thought to lead to the ultimate death of NM neurons. We have recently shown that chronic lithium treatment increases neuronal survival following deafferentation. To assess where in the cell death cascade lithium is having its effect, we evaluated some of the early deafferentation-induced cellular changes in NM neurons. Lithium did not affect deafferentation-induced changes that occur across the entire population of NM neurons. There were still deafferentation-induced increases in intracellular calcium concentrations and early changes in the ribosomes, as indicated by Y10b immunolabeling. Lithium did, however, affect changes that are believed to be indicative of the subpopulation of NM neurons that will eventually die. Ribosomes recovered in all of the deafferented NM neurons (as assessed by Y10b labeling) by 10 h following cochlea removal in subjects pretreated with lithium, while a subpopulation of the NM neurons in saline-treated subjects showed dramatic reduction in Y10b labeling at that time. Lithium treatment also prevented the robust upregulation of b cell leukemia/lymphoma-2 (Bcl-2) mRNA that is observed in a subpopulation of deafferented NM neurons 6 h following cochlea removal.     

Afferent regulation of cytochrome-c and active caspase-9 in the avian cochlear nucleus

During development, a subpopulation (approximately 30%) of neurons in the avian cochlear nucleus, nucleus magnocellularis (NM), dies following removal of the cochlea. It is clear that neuronal activity coming from the auditory nerve provides trophic support critical for cell survival in the NM. Several aspects of the intracellular signaling cascades that regulate apoptosis have been defined for naturally occurring, or programmed cell death, in neurons. These intracellular cascades involve the extrusion of cytochrome-c from the mitochondria into the cytosol and the subsequent activation of proteolytic caspase cascades, which ultimately act on substrates that lead to the death of the cell. In contrast, the intracellular signaling cascades responsible for deafferentation-induced cell death are not fully understood. In the present series of experiments, the potential extrusion of cytochrome-c from the mitochondria into the cytosol, and the activation of caspases were examined in the NM following deafferentation. Cytochrome-c immunoreactivity increased within 6 h following deafferentation and persisted for at least 3–5 days following surgery. However, cytochrome-c was not detectable within immunoprecipitates obtained from cytosolic fractions of deafferented NM neurons. This suggests that the increased immunoreactivity of cytochrome-c is related to mitochondrial proliferation. As a positive control, cytochrome-c was detected in cytosolic fractions of deafferented NM neurons treated with kainic acid, a substance known to cause cytochrome-c release into the cytosol. In addition, immunoreactivity for downstream active caspase-9 did increase following cochlea ablation. This increase was observed within 3 h following cochlea removal, but was not observed 4 days following surgery, a time point after the dying population of NM neurons have already degenerated. Together, these findings suggest that deafferentation of NM neurons results in caspase activation, but this activation may be cytochrome-c independent.     

Synaptic excitation of the second and third order auditory neurons in the avian brain stem

Synaptic potentials were examined in the second- and third-order auditory neurons of nucleus magnocellularis and nucleus laminaris in the chick. Brain stems of mature chick embryos were explanted and maintained in vitro for 4 to 8 h. Field potentials, extracellular spike potentials and intracellular potentials evoked by 8th-nerve stimulation were examined. Eighth-nerve stimulation reliability elicited four identifiable field potentials which could be attributed to: (i) the afferent volley of the 8th-nerve axons, (ii) postsynaptic responses of n. magnocellularis neurons, and (iii) ipsilaterally and, (iv) contralaterally-evoked n. laminaris postsynaptic responses. Intracellular-recorded postsynaptic potentials were characterized by a rapid rise time and short duration. They were apparently monosynaptic with a synaptic delay of 0.4 ms. In each n. magnocellularis neuron the 'fast' excitatory postsynaptic potentials were composed of 1 to 3 all-or-none components. 'Slow' excitatory postsynaptic potentials were characterized by a longer latency, a longer duration and graded amplitude variation in proportion to the intensity of 8th-nerve stimulation. Both 'fast' and 'slow' excitatory postsynaptic potentials had similar reversal potentials. Since the 8th nerve makes monosynaptic connection with n. magnocellularis neurons, it is likely that at this synapse the 'fast' excitatory postsynaptic potentials were produced, while the 'slow' potential may be attributable to the convergence of many boutonal synapses of unknown origin. Intracellular injections of horseradish peroxidase into n. magnocellularis revealed that its efferents bifurcate below the nucleus and send one axon to the contralateral n. laminaris while the other axon forms a highly divergent projection to the ipsilateral laminar nucleus. The intracellular records obtained from n. laminaris are consistent with this anatomical finding in that graded excitatory postsynaptic potentials were elicited by 8th-nerve stimulation.     

Delayed treatment with nimesulide reduces measures of oxidative stress following global ischemic brain injury in gerbils

Metabolism of arachidonic acid by cyclooxygenase is one of the primary sources of reactive oxygen species in the ischemic brain. Neuronal overexpression of cyclooxygenase-2 has recently been shown to contribute to neurodegeneration following ischemic injury. In the present study, we examined the possibility that the neuroprotective effects of the cyclooxygenase-2 inhibitor nimesulide would depend upon reduction of oxidative stress following cerebral ischemia. Gerbils were subjected to 5 min of transient global cerebral ischemia followed by 48 h of reperfusion and markers of oxidative stress were measured in hippocampus of gerbils receiving vehicle or nimesulide treatment at three different clinically relevant doses (3, 6 or 12 mg/kg). Compared with vehicle, nimesulide significantly (P<0.05) reduced hippocampal glutathione depletion and lipid peroxidation, as assessed by the levels of malondialdehyde (MDA), 4-hydroxy-alkenals (4-HDA) and lipid hydroperoxides levels, even when the treatment was delayed until 6 h after ischemia. Biochemical evidences of nimesulide neuroprotection were supported by histofluorescence findings using the novel marker of neuronal degeneration Fluoro-Jade B. Few Fluoro-Jade B positive cells were seen in CA1 region of hippocampus in ischemic animals treated with nimesulide compared with vehicle. These results suggest that nimesulide may protect neurons by attenuating oxidative stress and reperfusion injury following the ischemic insult with a wide therapeutic window of protection.     

Regulation of glutamatergic and GABAergic neurotransmission in the chick nucleus laminaris: role of N-type calcium channels

Neurons in the chicken nucleus laminaris (NL), the third order auditory nucleus involved in azimuth sound localization, receive bilaterally segregated (ipsilateral vs contralateral) glutamatergic excitation from the cochlear nucleus magnocellularis and GABAergic inhibition from the ipsilateral superior olivary nucleus (SON). Here, I investigate the voltage-gated calcium channels (VGCCs) that trigger the excitatory and the inhibitory transmission in the NL. Whole-cell recordings were performed in acute brainstem slices. The excitatory transmission was predominantly mediated by N-type VGCCs, as the specific N-type blocker ω-Conotoxin-GVIA (ω-CTx-GVIA, 1–2.5 μM) inhibited excitatory postsynaptic currents (EPSCs) by ∼90%. Blockers for P/Q- and L-type VGCCs produced no inhibition, and blockade of R-type VGCCs produced a small inhibition. In individual cells, the effect of each VGCC blocker on the EPSC elicited by activation of the ipsilateral input was the same as that on the EPSC elicited by activation of the contralateral input, and the two EPSCs had similar kinetics, suggesting physiological symmetry between the two glutamatergic inputs to single NL neurons. The inhibitory transmission in NL neurons was almost exclusively mediated by N-type VGCCs, as ω-CTx-GVIA (1 μM) produced a ∼90% reduction of inhibitory postsynaptic currents, whereas blockers for other VGCCs produced no inhibition. In conclusion, N-type VGCCs play a dominant role in triggering both the excitatory and the inhibitory transmission in the NL, and the presynaptic VGCCs that mediate the two bilaterally segregated glutamatergic inputs to individual NL neurons are identical. These features may play a role in optimizing coincidence detection in NL neurons.     

Metabolism and functions of glutathione in brain

The tripeptide glutathione is the thiol compound present in the highest concentration in cells of all organs. Glutathione has many physiological functions including its involvement in the defense against reactive oxygen species. The cells of the human brain consume about 20% of the oxygen utilized by the body but constitute only 2% of the body weight. Consequently, reactive oxygen species which are continuously generated during oxidative metabolism will be generated in high rates within the brain. Therefore, the detoxification of reactive oxygen species is an essential task within the brain and the involvement of the antioxidant glutathione in such processes is very important. The main focus of this review article will be recent results on glutathione metabolism of different brain cell types in culture. The glutathione content of brain cells depends strongly on the availability of precursors for glutathione. Different types of brain cells prefer different extracellular glutathione precursors. Glutathione is involved in the disposal of peroxides by brain cells and in the protection against reactive oxygen species. In coculture astroglial cells protect other neural cell types against the toxicity of various compounds. One mechanism for this interaction is the supply by astroglial cells of glutathione precursors to neighboring cells. Recent results confirm the prominent role of astrocytes in glutathione metabolism and the defense against reactive oxygen species in brain. These results also suggest an involvement of a compromised astroglial glutathione system in the oxidative stress reported for neurological disorders.     

Zinc supplementation prevents alcoholic liver injury in mice through attenuation of oxidative stress

Alcoholic liver disease is associated with zinc decrease in the liver. Therefore, we examined whether dietary zinc supplementation could provide protection from alcoholic liver injury. Metallothionein-knockout and wild-type 129/Sv mice were pair-fed an ethanol-containing liquid diet for 12 weeks, and the effects of zinc supplementation on ethanol-induced liver injury were analyzed. Zinc supplementation attenuated ethanol-induced hepatic zinc depletion and liver injury as measured by histopathological and ultrastructural changes, serum alanine transferase activity, and hepatic tumor necrosis factor-alpha in both metallothionein-knockout and wild-type mice, indicating a metallothionein-independent zinc protection. Zinc supplementation inhibited accumulation of reactive oxygen species, as indicated by dihydroethidium fluorescence, and the consequent oxidative damage, as assessed by immunohistochemical detection of 4-hydroxynonenal and nitrotyrosine and quantitative analysis of malondialdehyde and protein carbonyl in the liver. Zinc supplementation suppressed ethanol-elevated cytochrome P450 2E1 activity but increased the activity of alcohol dehydrogenase in the liver, without affecting the rate of blood ethanol elimination. Zinc supplementation also prevented ethanol-induced decreases in glutathione concentration and glutathione peroxidase activity and increased glutathione reductase activity in the liver. In conclusion, zinc supplementation prevents alcoholic liver injury in an metallothionein-independent manner by inhibiting the generation of reactive oxygen species (P450 2E1) and enhancing the activity of antioxidant pathways.     

黄喉鹀延髓听觉核团的定位研究

用HRP顺行轴突传递法研究鸣禽黄喉鵐的耳蜗神经元向延髓听觉核团的上行投射和定位.将HRP分别注入左右侧耳蜗内,1.在同侧听神经有束状的标记纤维并分别投射至延髓的角状核和巨细胞核;2.在角状核内有密集成簇的标记终末;3.在巨细胞核内有中等量的标记终末.结果表明:耳蜗纤维分别投射至巨细胞核和角状核,由这两对亚核组成延髓的耳蜗核,它是听觉上行通路中在脑内的第一级中继站,延髓的层状核并不接受耳蜗纤维的直接投射.     

Effects of treatment with microencapsulated monosialoganglioside GM1 on cortical and striatal acetylcholine release in rats with cortical devascularizing lesions

The present study shows a novel administration form of the monoganglioside GM1, which following microencapsulation in human serum albumin was topically applied on cortical regions damaged by devascularization in rats. The effects of microencapsulated GM1 on extracellular levels of acetylcholine, choline and dopamine in the cortex and in the striatum were analyzed using in vivo microdialysis. Cholinergic neurons in the nucleus basalis magnocellularis were studied immunohistochemically using monoclonal antibodies raised against choline acetyltransferase (ChAT). It was found that cortical devascularizing lesions produced a decrease in extracellular levels of cortical acetylcholine and choline, and retrograde morphological changes in cholinergic neurons in the nucleus basalis magnocellularis. GM1 promoted (1) recovery of the retrograde morphological changes produced by the decortication in the nucleus basalis magnocellularis and (2) a parallel increase in cortical acetylcholine release. No changes were observed in the striatum, nor on cortical or striatal dopamine levels simultaneously measured in the same perfusates.     

Cu,Zn Superoxide Dismutase of Mycobacterium tuberculosis Contributes to Survival in Activated Macrophages That Are Generating an Oxidative Burst

Macrophages produce reactive oxygen species and reactive nitrogen species that have potent antimicrobial activity. Resistance to killing by macrophages is critical to the virulence of Mycobacterium tuberculosis. M. tuberculosis has two genes encoding superoxide dismutase proteins, sodA and sodC. SodC is a Cu,Zn superoxide dismutase responsible for only a minor portion of the superoxide dismutase activity of M. tuberculosis. However, SodC has a lipoprotein binding motif, which suggests that it may be anchored in the membrane to protect M. tuberculosis from reactive oxygen intermediates at the bacterial surface. To examine the role of the Cu,Zn superoxide dismutase in protecting M. tuberculosis from the toxic effects of exogenously generated reactive oxygen species, we constructed a null mutation in the sodC gene. In this report, we show that the M. tuberculosis sodC mutant is readily killed by superoxide generated externally, while the isogenic parental M. tuberculosis is unaffected under these conditions. Furthermore, the sodC mutant has enhanced susceptibility to killing by gamma interferon (IFN-gamma)-activated murine peritoneal macrophages producing oxidative burst products but is unaffected by macrophages not activated by IFN-gamma or by macrophages from respiratory burst-deficient mice. These observations establish that the Cu,Zn superoxide dismutase contributes to the resistance of M. tuberculosis against oxidative burst products generated by activated macrophages.     

Lipid peroxidation and the protective effect of physical exercise on breast cancer

Physical exercise has been found to decrease the risk of breast cancer by undefined means. In our prior communication, we proposed lipid peroxidation to be a relevant mechanism for breast cancer protection associated with many established protective factors such as parity, oophorectomy, menopause, physical exercise, etc. [Gago-Dominguez M, Castelao J, Pike MC, Sevanian A, Haile RW. Role of lipid peroxidation in the epidemiology and prevention of breast cancer. Cancer Epidemiol Biomark Prevent 2005;14:2829-39]. In the present communication, we examine in detail the physical exercise-breast cancer relationship in light of the lipid peroxidation mechanism. We provide additional supporting evidence for the hypothesis that oxidative stress-induced apoptosis may be a mechanism responsible, at least in part, for the protective effect of exercise in breast cancer. Specifically, we describe (1) the sources of free radicals occurring during exercise, (2) existing experimental data on physical exercise, lipid peroxidation and cancer, (3) existing supporting data implicating exercise-induced reactive oxygen species (ROS) as the mechanism responsible for increased apoptosis in different cell systems, and (4) changes in the antioxidant enzymes glutathione S transferases (GSTs), superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT) that occur after physical exercise, which are believed to be a physiologic response to oxidative stress induced by physical exercise.     

NGF prevents further atrophy of cholinergic cells of the nucleus basalis due to cortical infarction in adult post-hypothyroid rats but does not restore cell size compared to euthroid rats

We have tested the hypotheses that nerve growth factor treatment in adult post-hypothyroid rats can: (1) restore cross-sectional area of cholinergic cells of the nucleus basalis and (2) prevent further atrophy of these neurons following cortical infaction. In addition, we assessed the expression of p75^NGFR and p140^trkA mRNAs in the nucleus basalis cells of post-hypothyroid rats. Rats were rendered hypothyroid by the addition of propylthiouracil to their diet beginning on embryonic day 19 until the age of 1 month. At this time both the pups and their dams continued to receive 0.05% propylthiouracil in their diet and the pups were thyroidectomized. At 60 days, propylthiouracil treatment was interrupted and thyroxine levels were restored to normal by daily subcutaneous administration of physiological levels of thyroxine. Morphometric analysis identified atrophied nucleus basalis magnocellularis cholinergic cells at two ages, days 75 and 105, identified by in situ hybridization for p75^NGFR and p140^trkA mRNAs in methylene blue stained cells (day 75) and choline acetyltransferase immunostaining (day 105). The mean number of silver grains (pixels) per μm² (mean±S.E.M.) of cell body cross-sectional area for p75^NGFR mRNA in the nucleus basalis magnocellularis of euthyroid rats was 3.43±0.89, which was not statistically different from post-hypothyroid animals (4.02±1.07). A similar finding was noted for p140^trkA mRNA: mean number of grains in the euthyroid group was 5.54±0.96 and was not statistically different from the post-hypothyroid group (6.32±1.45). Nerve growth factor treatment in adulthood (between days 75 and 82) did not restore cross-sectional area from early thyroid deprivation. However, it prevented further atrophy of nucleus basalis magnocellularis neurons following cortical devascularization inflicted in adulthood (day 75).     

Exposure to the insecticide endosulfan induces liver morphology alterations and oxidative stress in fruit‐eating bats (Artibeus lituratus)

Exposure to pesticides may increase the generation of reactive oxygen species (ROS), leading to oxidation of cell membrane lipids and proteins. Although fruit bats are potentially exposed to pesticides during their entire lifespan, the impacts of this exposure are still poorly investigated. We examined the effects of low, commercially recommended concentrations (0, 1.05 and 2.1 g/l) of an organochlorine insecticide endosulfan (EDS) formulation on oxidative responses in the liver and kidneys of Neotropical fruit bats (Artibeus lituratus), as well as possible liver morphological alterations following a 35‐day oral exposure. Superoxide dismutase activity was significantly decreased upon exposure to 1.05 g/l of EDS in the liver and kidneys, catalase was decreased in the liver of 2.1 g/l EDS‐exposed bats, while glutathione S‐transferase was increased in the liver of 2.1 g/l EDS‐exposed bats. Protein carbonyls increased following the exposure to the highest EDS dose tested. Endosulfan‐induced morphological alterations in the liver included cell degeneration and cell death, with apparent cytoplasm lipid accumulation (steatosis) and pyknotic nuclei, karyolysis and deposit of collagen fibres. Our findings suggest that exposure to low concentrations of EDS induced a certain extent of oxidative damage in fruit bats, which may have led to liver morphological alterations.     

N-acetylcysteine amide (NACA) prevents retinal degeneration by up-regulating reduced glutathione production and reversing lipid peroxidation

Oxidative stress plays a critical role in accelerating retinal pigment epithelial dysfunction and death in degenerative retinal diseases, including age-related macular degeneration. Given the key role of oxidative stress-induced retinal pigment epithelial cell death and secondary photoreceptor loss in the pathogenesis of age-related macular degeneration, we hypothesized that a novel thiol antioxidant, N-acetylcysteine amide (NACA), might ameliorate cellular damage and subsequent loss of vision. Treatment of human retinal pigment epithelial cells with NACA protected against oxidative stress-induced cellular injury and death. NACA acted mechanistically by scavenging existing reactive oxygen species while halting production of reactive oxygen species by reversing lipid peroxidation. Furthermore, NACA functioned by increasing the levels of reduced glutathione and the phase II detoxification enzyme glutathione peroxidase. Treatment of mice exposed to phototoxic doses of light with NACA maintained retinal pigment epithelial cell integrity and prevented outer nuclear layer cell death as examined by histopathologic methods and rescued photoreceptor function as measured by electroretinography. These observations indicate that NACA protects against oxidative stress-induced retinal pigment epithelial and photoreceptor cell death in vitro and in vivo. The data suggest that NACA may be a novel treatment in rescuing retinal function and preventing vision loss secondary to retinal degenerative diseases, including age-related macular degeneration.     

Cellular mechanisms preventing sustained activation of cortex during subcortical high-frequency stimulation

Axonal excitation has been proposed as a key mechanism in therapeutic brain stimulation. In this study we examined how high-frequency stimulation (HFS) of subcortical white matter tracts projecting to motor cortex affects downstream postsynaptic responses in cortical neurons. Whole cell recordings were performed in the primary motor cortex (M1) and ventral thalamus of rat brain slices. In M1, neurons showed only an initial depolarization in response to HFS, after which the membrane potential returned to prestimulation levels. The prolonged suppression of excitation during stimulation was neither associated with GABAergic inhibition nor complete action potential failure in stimulated axons. Instead we found that HFS caused a depression of excitatory synaptic currents in postsynaptic neurons that was specific to the stimulated subcortical input. These data are consistent with the hypothesis that axonal HFS produces a functional deafferentation of postsynaptic targets likely from depletion of neurotransmitter.     

Behavioural, biochemical and histochemical effects of different neurotoxic amino acids injected into nucleus basalis magnocellularis of rats

Lesions of the nucleus basalis magnocellularis in rats have been used to investigate functions of the extrinsic cortical cholinergic system which originates from these neurons. These lesions also produce extensive non-specific subcortical damage and associated regulatory and neurological impairments, causing doubt about the specificity of consequent functional impairments. Here, nucleus basalis magnocellularis lesions made with four different neurotoxic amino acids (kainic acid, ibotenic acid, N-methyl-D-aspartate, and quisqualic acid) have been compared. Quisqualic acid produced less subcortical damage and lesser neurological and regulatory impairments than the other toxins at doses that produced comparable cholinergic deafferentation of the neocortex, as assessed both histologically and biochemically. This suggests that these impairments are non-specific rather than specific consequences of cholinergic cell loss. The effects on learning a spatial navigation task were more ambiguous, suggesting the involvement of both cholinergic and non-cholinergic systems. Impairment of a passive shock avoidance task was as great following quisqualic acid as the other neurotoxins, which may suggest a more direct relationship specifically with the decline in cortical cholinergic activity. It is concluded that in the absence of availability of a specific cholinergic neurotoxin, quisqualic acid produces less non-specific neuroanatomical and neurological side effects than the more widely used toxins N-methyl-D-aspartate, kainic acid or ibotenic acid.     

Heme oxygenase protects hippocampal neurons from ethanol-induced neurotoxicity

Ethanol has deleterious effects on neuronal cells both in vivo and in vitro, but the mechanisms are unknown. Here, treatment with increasing doses of ethanol (from 20 up to 600mM) decreased the viability of a mouse hippocampal neuroblastoma cell line, HT22. The glutathione concentration decreased and intracellular reactive oxygen species (ROS) increased in a dose-and time-dependent manner, suggesting that the neurotoxicity was due to oxidative stress. Expression of heme oxygenase (HO)-1, a redox regulator and heat shock protein, increased with time after ethanol treatment, but HO-2 was expressed constitutively. The addition of 5microM zinc protoporphyrin IX (ZnPP IX), a competitive HO inhibitor, with the ethanol further reduced cell viability and increased intracellular ROS, but these effects were reversed by co-treatment with 50nM bilirubin, a well-known antioxidant and a product of HO catalysis. These results suggest that HO has a protective role in hippocampal neurons as an intrinsic factor against ethanol-induced oxidative stress and the protection depends on the degree of oxidative stress.     

Deafferentation-induced caspase-3 activation and DNA fragmentation in chick cochlear nucleus neurons

Cochlea removal severs peripheral processes of cochlear ganglion cells and permanently abolishes afferent input to nucleus magnocellularis (NM) neurons. Deafferented chick NM neurons undergo a series of morphologic and metabolic changes, which ultimately trigger the death of 20%–40% of neurons. Previous studies suggested that this cell specific death involves activation of the intrinsic apoptotic pathway, including increased presence of cytochrome c and active caspase-9 in the cytoplasm of deafferented NM neurons. Interestingly, however, both markers were detected pan-neuronally, in both degenerating and surviving NM neurons [Wilkinson BL, Elam JS, Fadool DA, Hyson RL (2003) Afferent regulation of cytochrome-c and active caspase-9 in the avian cochlear nucleus. Neuroscience 120:1071–1079]. Here, we provide evidence for the increased appearance of late apoptotic indicators and describe novel characteristics of cell death in deafferented auditory neurons. Young broiler chickens were subjected to unilateral cochlea removal, and brainstem sections through NM were reacted for active caspase-3 and terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL). Caspase-3 activation is observed in the cytoplasm of both dying and surviving deafferented NM neurons 24 h to 7 days following cochlea removal, suggesting that caspase-3, usually considered an “executioner” of apoptotic death, may also function as a “modulator” of death. In addition, we find that TUNEL labeling of degraded DNA is observed in deafferented NM. In contrast to upstream apoptotic markers, however, TUNEL labeling is restricted to a subpopulation of deafferented neurons. Twelve hours following cochlea removal, TUNEL labeling is observed as punctate accumulations within nuclei. Twenty-four hours following cochlea removal, TUNEL accumulates diffusely throughout neuronal cytoplasm in those neurons likely to die. This cytoplasmic TUNEL labeling may implicate mitochondrial nucleic acid degradation in the selective death of some deafferented NM neurons. Our study examines the subcellular distributions of two prominent apoptotic mediators, active caspase-3 and TUNEL, relative to known histochemical markers, in deafferented NM; provides new insight into the apoptotic mechanism of cell death; and proposes a role for mitochondrial DNA in deafferentation-induced cell death.