Developmentally induced microencephalopathy in guinea pigs -embryonic glial cell activation marks selective neuronal death

We have recently shown that in utero treatment of guinea pigs with the DNA methylating substance methylazoxymethanol acetate (MAM) on gestation day (GD) 24 results in neocortical microencephalopathy, increased protein kinase C activity and altered processing of the amyloid precursor protein in neocortex of the offsprings. In order to identify the primary neuronal lesions produced by MAM-treatment, we mapped the 5-bromo-2'-deoxyuridine (BrdU)-incorporation in dividing neurons on GD 24 and we followed the effects of MAM-treatment on GD 24 on embryonic immediate early gene expression and on glial cell activation. BrdU injected on GD 24 labeled many neurons of the ventricular zone and of the intermediate zone but only scattered neurons of the cortical plate. When time-mated guinea pigs were injected intraperitoneally with MAM on GD 24, we observed the activation of microglial cells in the ventricular/intermediate zone and the appearence of astrocytes between the intermediate zone and the cortical plate, 48 h after intoxification. The activation of glial cells was accompanied by the neuronal expression of c-Fos but not of c-Jun in the ventricular/intermediate zone. Based on our observations on BrdU-incorporation and on the morphological outcome of MAM treatment in the juvenile guinea pig, our data presented here indicate that selective neurodegeneration during development induces the activation of both phagocytotic microglial cells and of astrocytes which might trophically support damaged neurons surviving this lesion procedure.     

NMDA receptor activation induces glutamate release through nitric oxide synthesis in guinea pig dentate gyrus

We tested the hypothesis that the release of glutamate following activation of N-methyl-d-aspartate (NMDA) receptors is mediated by nitric oxide (NO) production, using slices of the guinea pig hippocampus. The NMDA-induced glutamate release from slices of dentate gyrus or CA1, which was both concentration-dependent and Ca²⁺-dependent, was also Mg²⁺-sensitive and abolished by MK-801, a selective non-competitive NMDA receptor antagonist. In dentate gyrus, the NMDA-induced glutamate release was inhibited non-significantly by tetrodotoxin, whereas the NO synthase (NOS) inhibitor N^G-nitro-l-arginine (l-NNA) blocked the NMDA-induced release of glutamate in a concentration-dependent manner, but not a high K⁺-evoked release of glutamate. In addition, the l-NNA blockade of NMDA-induced release of glutamate was recovered by pretreatment with l-arginine, the normal substrate for NOS. These results suggest that activation of NMDA receptors in dentate gyrus, as well as subsequent Ca²⁺ fluxes, is required for the neuronal glutamate release mediated by NO production. On the other hand, the NMDA-evoked glutamate release from CA1 region was tetrodotoxin-sensitive and was not inhibited by l-NNA, thereby suggesting that activation of NMDA receptors in CA1 results in increased glutamate release in an NO-independent manner. Taken together, the NMDA receptor-mediated neuronal release of glutamate from the guinea pig dentate gyrus likely involves the recruitment of NOS activity.     

Astrocytic glutamate uptake is slow and does not limit neuronal NMDA receptor activation in the neonatal neocortex

Glutamate uptake by astrocytes controls the time course of glutamate in the extracellular space and affects neurotransmission, synaptogenesis, and circuit development. Astrocytic glutamate uptake has been shown to undergo post‐natal maturation in the hippocampus, but has been largely unexplored in other brain regions. Notably, glutamate uptake has never been examined in the developing neocortex. In these studies, we investigated the development of astrocytic glutamate transport, intrinsic membrane properties, and control of neuronal NMDA receptor activation in the developing neocortex. Using astrocytic and neuronal electrophysiology, immunofluorescence, and Western blot analysis we show that: (1) glutamate uptake in the neonatal neocortex is slow relative to neonatal hippocampus; (2) astrocytes in the neonatal neocortex undergo a significant maturation of intrinsic membrane properties; (3) slow glutamate uptake is accompanied by lower expression of both GLT‐1 and GLAST; (4) glutamate uptake is less dependent on GLT‐1 in neonatal neocortex than in neonatal hippocampus; and (5) the slow glutamate uptake we report in the neonatal neocortex corresponds to minimal astrocytic control of neuronal NMDA receptor activation. Taken together, our results clearly show fundamental differences between astrocytic maturation in the developing neocortex and hippocampus, and corresponding changes in how astrocytes control glutamate signaling. GLIA 2015;63:1784–1796     

Beta-amyloid25-35 inhibits glutamate uptake in cultured neurons and astrocytes: modulation of uptake as a survival mechanism

Glutamate transporters are vulnerable to oxidants resulting in reduced uptake function. We have studied the effects of beta-amyloid(25-35) (beta A(25-35)) on [(3)H]-glutamate uptake on cortical neuron or astrocyte cultures in comparison with a scrambled peptide (SCR) and dihydrokainic acid (DHK), a prototypic uptake inhibitor. beta A(25-35) was more potent than DHK in inhibiting glutamate uptake and the effects of both were more marked on astrocytes than on neurons. At 24 h, beta A(25-35) dose-dependently (0.5-15 microM) increased glutamate levels in media from neuron cultures. DHK only enhanced extracellular glutamate at the highest concentration tested (2500 microM). beta A(25-35) induced gradual neurotoxicity (0.1-50 microM) over time. Exposure to beta A(25-35) resulted in increased uptake in astrocytes (0.25-5 microM) and neurons (0.5-15 microM) surviving its toxic effects. However, exposure to DHK (2.5-2500 microM) did not induce neurotoxicity nor modulated uptake. These results indicate that, while inhibition of glutamate uptake may be involved in the neurotoxic effects of beta A(25-35), enhancement of uptake may be a survival mechanism following exposure to beta A(25-35).     

Impaired glutamate uptake and EAAT2 downregulation in an enterovirus chronically infected human glial cell line

Rapid and efficient uptake of glutamate via the high-affinity glutamate transporter EAAT2 is important for limiting glutamate-mediated excitotoxicity involved in neuronal death. Furthermore, there is evidence of altered glutamate uptake and catabolism in motor neuron diseases. Such a defect has been reported in amyotrophic lateral sclerosis, the major motor neuron disease, and was associated with impairment in EAAT2 processing. We recently reported the presence of enterovirus genome specifically in the anterior horn of amyotrophic lateral sclerosis cases, suggesting the involvement of a chronic/persistent enterovirus infection in amyotrophic lateral sclerosis. To investigate a putative link between enterovirus infection and the glutamate-mediated excitotoxicity observed in amyotrophic lateral sclerosis, we developed an in vitro model consisting of a human glial cell line infected with ECHOvirus 6, one of the enteroviruses with sequences closely related to those detected in patients with amyotrophic lateral sclerosis. In these glial cells, an ECHOvirus 6 chronic infection was established, resulting in altered extracellular glutamate uptake. This correlated with an aberrant splicing of the EAAT2 pre-messenger ribonucleic acid and a significant loss of EAAT2 protein expression, similar to that observed in patients with amyotrophic lateral sclerosis. These results provide convincing evidence that an enterovirus chronic/persistent infection may alter glial glutamate uptake and catabolism. As enteroviruses are extremely common human pathogens, they may act as a trigger in the development of certain motor neuron diseases, such as amyotrophic lateral sclerosis.     

Neural precursor-derived astrocytes of wobbler mice induce apoptotic death of motor neurons through reduced glutamate uptake

In the present study, we investigated whether cultured astrocytes derived from adult neural precursor cells (NPCs) obtained from the subventricular zone (SVZ) of wobbler mice display metabolic traits of the wobbler astrocytes in situ and in primary culture. We also utilized NPC-derived astrocytes as a tool to investigate the involvement of astrocytes in the molecular mechanism of MND focusing on the possible alteration of glutamate reuptake since excitotoxicity glutamate-mediated may be a contributory pathway.NPC-derived wobbler astrocytes are characterized by high immunoreactivity for GFAP, significant decrease of glutamate uptake and reduced immunoreactivity for glutamate transporters GLT1 and GLAST. Spinal cord motor neurons obtained from healthy mouse embryos, when co-cultured with wobbler NPC-derived astrocytes, show reduced viability and morphologic alterations. These suffering motor neurons are caspase-7 positive, and treatment with anti-apoptotic drug V5 increases cell survival. Physical contact with wobbler astrocytes is not essential because purified motor neurons display reduced survival also when treated with the medium conditioned by wobbler NPC-derived astrocytes. Toxic levels of glutamate were revealed by HPLC assay in the extracellular medium of wobbler NPC-derived astrocytes, whereas the level of intracellular glutamate is reduced if compared with controls. Moreover, glutamate receptor antagonists are able to enhance motor neuron survival. Therefore, our results demonstrate that astrocytes derived from wobbler neural precursor cells display impaired glutamate homeostasis that may play a crucial role in motor neuron degeneration. Finally, the cultured astrocytes derived from NPCs of adult mice may offer a useful alternative in vitro model to study the molecular mechanisms involved in neurodegeneration.     

Modulation of glial glutamate transport through cell interactions with the extracellular matrix

Glial glutamate transport plays a pivotal role in maintaining glutamate homeostasis in the central nervous system. Expression of glutamate transporters is highly regulated during brain development, and a number of pathological conditions are associated with deficits in expression and/or function of glutamate transports. While several soluble factors have been shown to regulate the expression of glutamate transporter, the contribution of cell-cell interaction and cell-environmental interaction in the regulation of glutamate transport is unknown. Extracellular matrix (ECM) molecules are essential components in cell-cell and cell-environmental interactions, and the ECM has been shown to play critical role in normal development and during brain pathogenesis. We, therefore, investigated the possibility that ECM molecules may regulate astrocytic glutamate transport. Therefore, we cultured rat cortical astrocytes with different ECMs and determined expression levels of the two astrocytic glutamate transporters GLT-1 and GLAST by Western Blot and determined transporter activity through measurements of 3H-D-aspartate uptake. Astrocytes grown on poly-ornithine or poly-D/L-lysine showed approximately two-fold higher GLT-1 expression than sister cells grown on plastic dishes without ECM. Naturally occurring ECM's, including laminin and collagen, showed a dose-dependent regulation of GLT-1 protein expression. These effects were specific for GLT-1 as GLAST expression was unaffected by different ECMs. Surprisingly, however, none of the examined ECMs altered the apparent glutamate uptake activity. In probing blots side-by-side for expression of Na(+)/K(+)-ATPase, we found that ECMs affected expression of Na(+)/K(+)-ATPase and GLT-1 in a reciprocal fashion. Poly-ornithine, for example, enhanced GLT-1 expression, but reduced expression of Na(+)/K(+)-ATPase. Na(+) transport may, thus, be a limiting factor for glutamate uptake.     

Exacerbation of neuronal cell death by activation of group I metabotropic glutamate receptors: role of NMDA receptors and arachidonic acid release

Both ionotropic and metabotropic glutamate receptors have been implicated in the pathogenesis of neuronal injury. Activation of group I metabotropic glutamate receptors (mGluR) exacerbates neuronal cell death, whereas inhibition is neuroprotective. However, the mechanisms involved remain unknown. Activation of group I mGluR modulates multiple signal transduction pathways including stimulation of phosphoinositide hydrolysis, potentiation of NMDA receptor activity, and release of arachidonic acid. Here we demonstrate that whereas activation of group I mGluR by (S)-3,5-dihydroxyphenylglycine (DHPG) potentiates NMDA-induced currents and intracellular calcium increases in rat cortical neuronal cultures, partial effects of group I mGluR activation or inhibition on neuronal injury induced by oxygen-glucose deprivation remain despite NMDA receptor blockade. DHPG stimulation also increases basal arachidonic acid release from rat neuronal-glial cultures and potentiates injury-induced arachidonic acid release in these cultures. Thus, activation of group I mGluR may exacerbate neuronal injury through multiple mechanisms, which include positive modulation of NMDA receptors and enhanced release of arachidonic acid.     

Mechanical injury to neuronal/glial cultures in microplates: Role of NMDA receptors and pH in secondary neuronal cell death

In vitro models of traumatic injury are useful adjuncts to animal models for studying mechanisms of post-traumatic cell death. Here we describe a new in vitro model in which reproducible levels of injury are delivered by a punch device that produces 28 parallel cuts in individual wells of 96-well microplates. Cell loss is measured by LDH assay or quantitative fluorometric assay for ethidium homodimer staining. Glial cultures show cell death restricted to the initial injury site, whereas neuronal/glial cultures demonstrate substantial spread of cell loss over time. We used this model to examine the role of pH and NMDA receptors in delayed post-traumatic injury. NMDA receptor blockade by dizocilpine (MK-801) or treatment with antisense oligodeoxynucleotides directed against NMDAR1 was neuroprotective. Decreased cell death was observed under acidic conditions whereas increased extracellular pH was associated with increased, MK-801 sensitive cell loss. Advantages of our model include: reproducible trauma induction; rapid measurements of cell injury; and use of 96-well microplates which reduce time and cost. This model appears to be well-suited for the study of selected mechanisms of post-traumatic neuronal injury as well as for screening potential neuroprotective agents. J. Neurosci. Res. 51:748–758, 1998. © 1998 Wiley-Liss, Inc.     

Depolarization or glutamate receptor activation blocks apoptotic cell death of cultured cerebellar granule neurons

Cerebellar granule neurons can be readily maintained in culture if depolarized with high concentrations of K⁺ or subtoxic concentrations of various excitatory amino acids. We now report that these depolarizing stimuli promote cerebellar granule neuron survival by blocking their programmed death via apoptosis. Cerebellar granule neurons maintained in depolarizing conditions and then changed to non-depolarizing conditions, exhibit the morphological and biochemical features of apoptosis, including cytoplasmic blebbing, condensation and aggregation of nuclear chromatin internucleosomal DNA fragmentation. Inhibitors of RNA or protein synthesis greatly attenuate cell death induced by non-depolarizing culture conditions. In contrast, cerebellar granule neurons, when exposed to fresh serum-containing medium or to high concentrations of glutamate, exhibit a delayed-type of neurotoxicity which is non-apoptotic in nature. Given the actions of excitatory amino acid receptor agonists in preventing apoptosis of cultured cerebellar granule neurons, we hypothesize that the functional innervation of postmigratory granule neurons during cerebellar development may prevent further elimination of these neurons by blocking their programmed death.     

Is taurine a hypothalamic neurotransmitter?: a model of the differential uptake and compartmentalization of taurine by neuronal and glial cell particles from the rat hypothalamus

Although taurine has been postulated to be a neurotransmitter or neuromodulator in the mammalian CNS, little is known concerning its role in brain function. Evidence suggesting that taurine may influence endocrine and homeostatic mechanisms via the hypothalamus resulted in our investigations into its function in this brain region.The main objectives of the research were to characterize the specific binding, uptake, and release of taurine in the hypothalamus. A specific aim was to examine the proposed neurotransmitter role for taurine in the hypothalamus. This was accomplished by comparing the characteristics and properties of the binding, uptake, and release of taurine with those for the classical neurotransmitters which satisfy the criteria for a neurotransmitter. On such a comparative basis, the characteristics of taurine uptake satisfy the neurotransmitter criterion of inactivation of taurine in the hypothalamus. However, the observed characteristics of taurine binding and release in the hypothalamus do not satisfy the respective neurotransmitter criteria of specific receptors and Ca²⁺-dependent evoked release. Therefore, solely on the basis of the experimental observations reported herein, we must conclude that taurine apparently does not function as a neurotransmitter in the hypothalamus.Two uptake systems were found in the P₂ fraction, a high affinity uptake system and a low affinity uptake system. Uptake systems for taurine have previously been reported in glial and nerve cell homogenates, and therefore, because of the known contamination of crude synaptosomal preparations with glial particles, we sought to determine the cellular origin of the two taurine uptake systems in our crude preparation. Using a variety of diverse biochemical techniques such as hypo-osmotic shock, release experiments and Arrhenius plots, we determined that physical changes of the media or depolarizing stimuli which would influence neuronal and glial cell particles differently, also had differing effects on high and low affinity taurine uptake or its release from the respective uptake compartments. We conclude that the high affinity taurine uptake system/compartment is located on/in neuronal membranes/particles and that the low affinity taurine uptake system/compartment is located on/in glial membranes/particles. Such a model for the differential cellular transport and compartmentalization of taurine into neuronal and glial cells has important implications concerning its possible role in the CNS.     

N-Methyl--aspartate receptor activation results in regulation of extracellular signal-regulated kinases by protein kinases and phosphatases in glutamate-induced neuronal apototic-like death

Extracellular signal-regulated kinases (ERK1/ERK2) have been shown transiently activated and involved in excitotoxicity. We searched for upstream molecules responsible for the regulation of glutamate-induced ERK1/ERK2 activation and ERK1/ERK2-mediated apototic-like death in cultured rat cortical neurons. ERK1/ERK2 activation (monitored by anti-active ERK1/ERK2 antibody) was almost completely prevented by blockage of NMDA receptor (NMDA-R) or elimination of extracellular Ca²⁺, but not any other glutamate receptor or L-type voltage-gated Ca²⁺ channel. It was prevented largely by inhibition of protein kinase C (PKC), protein-tyrosine kinases (PTK), respectively, but mildly by that of CaM kinase II. Combined inhibition of CaM kinase II (but not PTK) and PKC had an additive effect. Reversion of ERK1/ERK2 activation was largely prevented by inhibition of protein phosphatase (PP) 1 or protein tyrosine phosphatase (PTP). Combined inhibition of PP 1 and PTP had no additive effect. Glutamate-induced apoptotic-like death (determined by DAPI staining) was largely prevented by inhibition of NMDA-R, PKC, CaM kinase II, PTK and MEK1/MEK2 (ERK1/ERK2 kinase), respectively. Combined inhibition of CaM kinase II (but not PKC or PTK) and MEK1/MEK2 had an additive effect. Glutamate-induced apoptotic-like death was promoted by inhibition of PP1 and PTP, respectively. The above results suggested that in glutamate-induced cortical neurotoxicity ERK1/ERK2 activation be mainly mediated by NMDA-R. Subsequently, a pathway dependent on both PKC and PTK was mainly involved, which was also mainly responsible for ERK1/ERK2-mediated apoptotic-like death, and a CaM kinase II-dependent pathway was relatively mildly involved. Reversion of ERK1/ERK2 activation was mainly mediated by a pathway dependent on both PP1 and PTP, which might be involved in the restrain of glutamate-induced neurotoxicity.     

Lack of PSD-95 drives hippocampal neuronal cell death through activation of an alpha CaMKII transduction pathway

The PSD-95 protein family organizes the glutamatergic postsynaptic density and it is involved in the regulation of the excitatory signal at central nervous system synapses. We show here that PSD-95 deficiency by means of antisense oligonucleotides induces significant neuronal cell death within 24 h both in primary hippocampal cultures and in organotypic hippocampal slices. On the other hand, cultured cortical neurons are spared by PSD-95 antisense toxicity until they reach a NR2A detectable protein level (24 days in vitro). The neurotoxic event is characterized by increased alpha CaMKII association to NR2 regulatory subunits of NMDA receptor complex. As a direct consequence of alpha CaMKII association, we found increased GluR1 delivery to cell surface in cultured hippocampal neurons paralleled by AMPA-dependent increase in [Na+]I levels. In addition, both CaMKII specific inhibitor KN-93 and AMPA receptor antagonists CNQX and NBQX rescued neuronal survival to control values. On the other hand, both the NMDA channel blocker MK-801 and Dantrolene, an inhibitor of calcium release from ryanodine-sensitive endoplasmic reticulum stores, failed to have any effect on neuronal survival in PSD-95 deficient neurons. Thus, our data provide clues that PSD-95 reduced expression in neurons is responsible for neuronal vulnerability mediated by direct activation of alpha CaMKII transduction pathway in the postsynaptic compartment.     

Spermine induces cell death in cultured human embryonic cerebral cortical neurons through N-methyl-D-aspartate receptor activation

The polyamines putrescine, spermidine, and spermine play important roles in cell proliferation, differentiation, and modulation of ion channel receptors. However, the function of increased concentrations of these compounds in brain injury and disease is unclear, in that they have been proposed as being both neuroprotective and neurotoxic. The effects of spermine and putrescine were studied in human primary cerebral cortical cultures containing both neurons and glia. No toxic effects were induced at 8 days in vitro (DIV) by either of the two polyamines at concentrations ranging from 0.3 microM to 2 mM. However, when the oxidative metabolism of spermine that generates toxic byproducts was induced by the presence of fetal calf serum, spermine caused cellular death with an LC(50) of approximately 50 microM. At 14 DIV, the coapplication of spermine 2 mM and glutamate 5 mM induced neuron cell death, but the effect of applying both components separately was null. Both spermine and glutamate were toxic to older neurons (26-42 DIV cultures), and here the coapplication of glutamate was found always to intensify the effect of spermine. Spermine showed greater toxicity than glutamate in neurons. Another effect observed is that glutamate, but not spermine, induced astrocyte swelling. Spermine toxicity was inhibited by both MK801 and ifenprodil, indicating a mechanism involving N-methyl-D-aspartate (NMDA) receptor activation. Moreover, a strong spermine modulation of the NMDA receptor was demonstrated by the inhibition of glutamate toxicity by ifenprodil. Putrescine induced minor effects also as a neurotoxic agent. In conclusion, neuronal death by spermine can be induced by its toxic byproducts as well as through NMDA receptor action. The present results confirm the potentially harmful role of the polyamines in excitotoxicity-related human disorders.     

Glial and neuronal localization of ionotropic glutamate receptor subunit-immunoreactivities in the median eminence of female rats: GluR2/3 and GluR6/7 colocalize with vimentin, not with glial fibrillary acidic protein (GFAP)

Female rat median eminence was immunostained with anti-NR1, GluR1, GluR2/3, GluR6/7, or KA2. GluR2/3- and GluR6/7-immunoreactivities were detected in cells lining the basal portion of the third ventricle. To identify these cells as tanycytes, the median eminence was dual-immunostained with glutamate receptors and glial cytoskeletal marker proteins, such as vimentin or glial fibrillary acidic protein (GFAP). Both GluR2/3 and GluR6/7 were shown to colocalize with vimentin, not with GFAP. These results suggest the potential role for tanycytes in conducting glutamate signaling.     

Insulin-like growth factor 1 prevents neuronal cell death induced by corticosterone through activation of the PI3k/Akt pathway

Corticosterone (CORT) is well known to induce neuronal damage in various brain regions including the hippocampus, but the precise mechanism(s) of action underlying these effects has yet to be fully established. Insulin-like growth factor-1 (IGF-1) is a trophic factor promoting cell survival by the activation of the phosphatidylinositide 3-kinase (PI3K)/Akt kinase pathway. We report that IGF-1 prevents neuronal cell death induced by CORT, likely via the stimulation of the PI3K/Akt pathway in primary hippocampal cultured neurons. CORT induced neuronal cell death at a minimal concentration of 50 nM. IGF-1 (10 nM) prevented cell death induced by CORT under serum-free conditions. The neuroprotective effect of IGF-1 was accompanied by reversal of the Akt pathway inhibition induced by CORT. The PI3 kinase inhibitor, LY29004, inhibited the neuroprotective effect of IGF-1 whereas the MEK (MAPK kinase) inhibitor PD98059, an upstream blocker of mitogen-activated protein (MAP) kinase, had no effect. These results suggest that IGF-1 can prevent neuronal cell death induced by CORT in hippocampal neurons by modulating the activity of the PI3K/Akt pathway.     

Intracellular calcium elevation during plateau potentials mediated by extrasynaptic NMDA receptor activation in rat hippocampal CA1 pyramidal neurons is primarily due to calcium entry through voltage‐gated calcium channels

We reported previously that plateau potentials mediated by extrasynaptic N‐methyl‐d ‐aspartate receptors (NMDARs) can be induced either by synaptic stimulation in the presence of glutamate transporter antagonist or by iontophoresis of NMDA in rat hippocampal CA1 pyramidal neurons. To examine whether the plateau potentials are accompanied by an elevation of intracellular Ca²⁺ and to determine the source of Ca²⁺ elevation, we performed Ca²⁺ imaging during the plateau potential. Neurons were loaded with Ca²⁺ indicator fluo‐4, and the plateau potentials were generated either synaptically in the presence of glutamate transporter antagonist or by iontophoretically applying NMDA. We have found that a transient elevation in intracellular Ca²⁺ accompanies the plateau potential. The synaptically induced plateau potential and the Ca²⁺ elevation were blocked by 5,7‐dichlorokynurenic acid (5,7‐dCK), an antagonist for the glycine‐binding sites of NMDAR. A mixture of Cd²⁺ and tetrodotoxin did not block NMDA‐induced plateau potentials, but completely abolished the accompanying Ca²⁺ elevation in both the presence and absence of Mg²⁺ ions in the bathing solution. The NMDA‐induced plateau potential was blocked by further adding 5,7‐dCK. Our results show that the NMDAR‐mediated plateau potential is accompanied by elevation of intracellular Ca²⁺ that is primarily caused by the influx of Ca²⁺ through voltage‐gated Ca²⁺ channels.     

Methimazole-induced cell death in rat olfactory receptor neurons occurs via apoptosis triggered through mitochondrial cytochrome c-mediated caspase-3 activation pathway

The administration of methimazole is known to induce cell death in rat olfactory receptor neurons (ORNs). We investigated whether this injury occurs via apoptosis or through necrosis and whether it involves the extrinsic or intrinsic pathway. Rats were intraperitoneally injected with vehicle (control) or 300 mg/kg methimazole. The experimental animals were also administered vehicle or a caspase-3 or caspase-9 inhibitor 30 min earlier. The administration of methimazole induced cell death predominantly in the mature ORNs and partially reduced olfactory sensitivity in the rats; the injured cells were TUNEL-positive and showed a nuclear staining pattern. This insult induced cytochrome c release from the mitochondria and a significant increase in the immunoreactivity of activated caspase-3 and caspase-9 as well as that of cleaved poly-ADP-ribose-polymerase; in addition, it caused a significant increase in the fluorogenic activity of caspase-3 and caspase-9. However, it did not affect the immunoreactivity of activated caspase-8 or the fluorogenic activity of caspase-8. Pretreatment with a caspase-3 or caspase-9 inhibitor nearly completely prevented the morphologic, biochemical, and functional changes induced by methimazole. These findings suggest strongly that methimazole-induced cell death in rat ORNs is predominantly apoptosis; moreover, the majority of this apoptotic cell death is triggered through mitochondrial cytochrome c-mediated caspase-3 activation pathway, and both caspase-3 and caspase-9 inhibitors can prevent methimazole-induced cell death in the ORNs.