Dopamine receptor-mediated regulation of neuronal "clock" gene expression.

Using a transgenic mice model (i.e. "clock" knockouts), clock transcription factors have been suggested as critical regulators of dopaminergic behaviors induced by drugs of abuse. Moreover, it has been shown that systemic administration of psychostimulants, such as cocaine and methamphetamine regulates the striatal expression of clock genes. However, it is not known whether dopamine receptors mediate these regulatory effects of psychostimulants at the cellular level. Primary striatal neurons in culture express dopamine receptors as well as clock genes and have been successfully used in studying dopamine receptor functioning. Therefore, we investigated the role of dopamine receptors on neuronal clock gene expression in this model using specific receptor agonists. We found an inhibitory effect on the expression of mClock and mPer1 genes with the D2-class (i.e. D2/D3) receptor agonist quinpirole. We also found a generalized stimulatory effect on the expression of clock genes mPer1, mClock, mNPAS2 (neuronal PAS domain protein 2), and mBmal1 with the D1-class (i.e. D1) receptor agonist SKF38393. Further, we tested whether systemic administration of dopamine receptor agonists causes similar changes in striatal clock gene expression in vivo. We found quinpirole-induced alterations in mPER1 protein levels in the mouse striatum (i.e. rhythm shift). Collectively, our results indicate that the dopamine receptor system may mediate psychostimulant-induced changes in clock gene expression. Using striatal neurons in culture as a model, further research is needed to better understand how dopamine signaling modulates the expression dynamics of clock genes (i.e. intracellular signaling pathways) and thereby influences neuronal gene expression, neuronal transmission, and brain functioning.

Melatonin receptor agonist ramelteon activates the extracellular signal-regulated kinase 1/2 in mouse cerebellar granule cells.

The melatonin receptors MT1 and MT2 take part in the regulation of the activity (i.e. phosphorylation) of extracellular-signal-regulated kinase (ERK1/2), an enzyme involved in neuroplasticity. Primary cultures of mouse and rat cerebellar granule cells (CGC), which express both MT1 and MT2 receptors, have been widely used as an in vitro model to study neuronal ERK1/2. A novel MT1/MT2 agonist, ramelteon, has recently become clinically available. In this study, we characterized its action on neuronal ERK1/2. We used CGC cultures prepared from the cerebella of wild-type mice (MT1/MT2 CGC) and MT1- and MT2-knockout (KO) mice (MT1 KO CGC and MT2 KO CGC, respectively), and we employed a Western blot assay to evaluate ERK1/2 phosphorylation. Ramelteon increased ERK1/2 phosphorylation not only in MT1/MT2 CGC but also in CGC expressing only one of the two melatonin receptors. In the MT1 KO CGC, the stimulatory effect of ramelteon was blocked by an MT2 antagonist, 4P-PDOT, whereas in the MT2 KO CGC, this effect of ramelteon was blocked by luzindole. Pertussis toxin treatment did not prevent ramelteon from activating ERK1/2 but pretreatment with a tyrosine kinase (Trk) inhibitor, K252a, did, suggesting that an activation of Trk may mediate melatonin-receptor dependent ERK1/2 activation. In conclusion, we showed for the first time that a clinically used MT1/MT2 agonist, ramelteon, is capable of activating neuronal ERK1/2.

RNAi and brain function: was McConnell on the right track?

RNA interference (RNAi), one of the hottest topics of molecular biology research today, has unique features that are eerily reminiscent of the phenomenon of "RNA-mediated memory transfer," a controversial line of work that was investigated with great enthusiasm in the 1960s. If not a coincidence, then this suggests taking a new look at RNA-mediated modulation of neural function and raises the possibility that RNAi might be one of the physiologic mechanisms that regulate long-term gene expression in the brain.

Effect of fluoxetine and cocaine on the expression of clock genes in the mouse hippocampus and striatum.

Long-term drug-induced alterations in CNS gene expression may be responsible for some therapeutic effects, such as antidepressant action, as well as for psychopathological conditions, such as drug addiction and abuse. Transcription factors called "clock" genes can be affected by psychotropic drugs and may modify the expression pattern of other genes. In this study in mice, we investigated the delayed effects of single and repeated (i.e. 14 days) administration of the antidepressant fluoxetine and the psychostimulant cocaine on the brain expression of clock genes Period1, Period2, Period3, Clock, Bmal1, Cryptochrome1, Cryptochrome2, and NPAS2 (neuronal PAS domain protein 2), and their putative target gene, serotonin N-acetyltransferase. Mice were treated at ZT05 (lights on at 5:00 am; ZT00). Brain samples (i.e. hippocampus, striatum, and prefrontal cortex) were processed for a semi-quantitative mRNA assay. Repeated but not single treatment with either drug increased serotonin N-acetyltransferase expression in all areas tested. On the other hand, the expression of clock genes was differentially affected depending on the drug (i.e. fluoxetine and cocaine), treatment schedule (i.e. single and repeated), and brain area (i.e. hippocampus and striatum) tested. More pronounced changes were induced by repeated rather than single administrations of fluoxetine or cocaine. We propose that the effects of psychoactive drugs on clock transcription factors may mediate long-term drug-induced changes, possibly by regulating the expression of a second set of genes (i.e. clock-controlled genes).

Chronic fluoxetine administration increases the serotonin N-acetyltransferase messenger RNA content in rat hippocampus.

BACKGROUND: It has been proposed that up-regulation of cyclic adenosine monophosphate response element binding protein is a common action of chronic antidepressant treatments that may regulate specific target genes in the hippocampus. We hypothesized that the serotonin N-acetyltransferase (AA-NAT; EC 2.3.1.87) gene is one such target. AA-NAT leads to formation of N-acetylserotonin from serotonin, and in the pineal gland, to melatonin synthesis. We investigated whether hippocampal AA-NAT expression can be modified by chronic administration of fluoxetine to rats. METHODS: Male Brown-Norway rats were administered 5 mg/kg fluoxetine or its vehicle either once (acute) or once daily for 21 days (chronic). They were sacrificed 18 hours after the last injection, and their hippocampi were processed for a quantitative reverse-transcription/polymerase-chain reaction assay of AA-NAT and cyclophilin (cyc) messenger (m)RNAs. The results are expressed as AA-NAT/cyc ratios. RESULTS: Chronic but not acute fluoxetine administration resulted in about a fivefold increase in hippocampal AA-NAT mRNA. CONCLUSIONS: Up-regulation of extrapineal, e.g., hippocampal, AA-NAT expression may play a role in mediating the therapeutic action of antidepressant drugs.

Inflammatory 5-LOX mRNA and protein are increased in brain of aging rats.

5-Lipoxygenase (5-LOX) is the key enzyme in the synthesis of leukotrienes, inflammatory mediators of arachidonic acid. 5-LOX is also expressed in neurons (in particular in the hippocampus and the cerebellum), and it seems to be capable of promoting neurodegeneration. Recently, we observed greater 5-LOX mRNA content in the hippocampus of older (24 months) than younger (2 months) rats. In this study, we measured in the hippocampus and the cerebellum of younger and older male F344 rats the contents of: 5-LOX mRNA, FLAP (5-LOX activating protein) mRNA, and 5-LOX protein. By using a quantitative reverse transcription/polymerase chain reaction (PCR) (RT-PCR) with internal standards we found that 5-LOX but not FLAP mRNA content is greater (both in hippocampus and cerebellum) of older than younger rats. By using quantitative Western immunoblotting, we found a greater content of 5-LOX protein in the hippocampus and the cerebellum of older rats; we also established that the membrane/cytosol 5-LOX content ratio is larger in the brains of older than younger rats (statistically significant in the cerebellum). The latter can be considered an indication of 5-LOX translocation/activation during aging. Together these results suggest that aging increases both neuronal 5-LOX expression and protein translocation, and indicate that the 5-LOX system might play a significant role in the pathobiology of aging-associated neurodegenerative diseases.

Amiloride blocks glutamate-operated cationic channels and protects neurons in culture from glutamate-induced death.

The diuretic amiloride has been suggested as a specific inhibitor of T-type neuronal Ca2+ channels. The effects of amiloride on glutamate receptor-gated cationic channels and glutamate-induced. Ca2(+)-dependent neuronal death were investigated in primary neuronal cultures from neonatal rats. In primary cultures of cerebellar granule neurons of the rat, receiving 50 microM glutamate for 15 min, at 22 degrees C, in the absence of Mg2+, about 80% of neurons were killed in about 24 hr. Exposure of neurons to such a pulse of glutamate, in the presence of various concentrations of amiloride, resulted in a dose-dependent protection from neurotoxicity (EC50 300 microM, complete protection 1 mM). In voltage-clamped cortical and cerebellar neurons of neonatal rats in primary culture, 100 microM amiloride diminished (by about 25%) glutamate- and/or NMDA-evoked cationic currents, recorded in the whole-cell mode. About 80% of the NMDA-(20 microM) stimulated current was inhibited by 700 microM amiloride. The inhibitory effect of amiloride was not voltage-dependent. In outside-out membrane patches, excised from granule cells and held at -50 mV, 100 microM amiloride changed the NMDA-elicited single channel activity into a fast flickering between the open and closed states. The noise analysis of the data revealed that, although resembling the Mg2(+)-induced flickering, the amiloride-induced channel block was more similar to the effects described for the action of local anaesthetics on the nicotinic cholinergic channel. The pharmacological relevance of this action of amiloride requires further characterization; the data point out the necessity of a cautious use of amiloride in studying neuronal function.

Inhibition of glutamate-induced cell death by sodium nitroprusside is not mediated by nitric oxide.

Pretreatment of primary cultures of cerebellar granule cells with sodium nitroprusside (SNP) protected these neurons from delayed death induced by glutamate and N-methyl-D-aspartate (NMDA). This neuroprotective effect was not mimicked by S-nitroso-N-acetylpenicillamine (SNAP) which like SNP stimulates guanylate cyclase via a nitric oxide (NO) related mechanism. In contrast, neuroprotection was achieved with potassium ferrocyanide, a compound structurally related to SNP, but devoid of NO. On the other hand, kainate-induced neurotoxicity was not protected but potentiated by SNP. This effect of SNP was not mimicked by SNAP, potassium ferrocyanide and potassium ferricyanide. We conclude that neuroprotective properties of SNP on glutamate- and NMDA-induced neurotoxicity are not due to the release of NO and activation of guanylate cyclase, but are determined by the ferrocyanide portion of the SNP molecule.

Protective effect of melatonin against hippocampal DNA damage induced by intraperitoneal administration of kainate to rats.

The pineal hormone melatonin protects neurons in vitro from excitotoxicity mediated by kainate-sensitive glutamate receptors and from oxidative stress-induced DNA damage and apoptosis. Intraperitoneal injection on kainate into experimental animals triggers DNA damage in several brain areas, including the hippocampus. It is not clear whether melatonin is neuroprotective in vivo. In this study, we tested the in vivo efficacy of melatonin in preventing kainate-induced DNA damage in the hippocampus of adult male Wistar rats. Melatonin and kainate were injected i.p. Rats were killed six to 72 h later and their hippocampi were examined for evidence of DNA damage (in situ dUTP-end-labeling, i.e. TUNEL staining) and for cell viability (Nissl staining). Quantitative assay was performed using computerized image analysis. At 48 and 72 h after kainate we found TUNEL-positive cells in the CA1 region of the hippocampus; in the adjacent sections that were Nissl-stained, we found evidence of cell loss. Both the number of TUNEL-positive cells and the loss of Nissl staining were reduced by i.p. administration of melatonin (4 x 2.5 mg/kg; i.e. 20 min before kainate, immediately after, and 1 and 2 h after the kainate). Our results suggest that melatonin might reduce the extent of cell damage associated with pathologies such as epilepsy that involve the activation of kainate-sensitive glutamate receptors.

Time course, localization and pharmacological modulation of immediate early inducible genes, brain-derived neurotrophic factor and trkB messenger RNAs in the rat brain following photochemical stroke.

A focal, unilateral thrombotic stroke was produced in the rat sensorimotor cortex. The time course of expression and localization of the immediate early inducible genes: c-fos, c-jun, zif268; nerve growth factor, brain-derived neurotrophic factor and the related tyrosine kinase high-affinity receptor (trkB) messenger RNAs were studied by in situ hybridization. The levels of messenger RNAs for c-fos, zif268, brain-derived neurotrophic factor (but not nerve growth factor) and trkB were consistently increased in cortex ipsilaterally to the lesion, while c-jun messenger RNA content was only slightly increased. The brain-derived neurotrophic factor messenger RNA was increased from 2 to 18 h following the stroke, mainly in cells having a normal morphological appearance. The trkB messenger RNA displayed temporal and spatial increases similar to brain-derived neurotrophic factor messenger RNA. The time course and pattern of expression of immediate early inducible gene and trophic factor messenger RNAs did not clearly support a causal relationship between these two families of factors. The observed messenger RNA increases were greatly attenuated by the non-competitive N-methyl-D-aspartate-sensitive glutamate receptor antagonist (+)-5-methyl-10,11-dihydroxy-5H-dibenzo(a,d)cyclohepten-5,10-imine , but substantially unaffected by the non-N-methyl-D-aspartate receptor antagonist 2,3-dihydroxy-6-nitrosulphanoylbenzoquinoxaline. The results suggest a major contribution of N-methyl-D-aspartate-sensitive glutamate receptor activation to the transcriptionally directed events subsequent to stroke. Future studies should clarify the contribution of these processes to either the progression of neuronal degeneration or the establishment of protective compensatory responses.

5-Lipoxygenase and cyclooxygenase mRNA expression in rat hippocampus:early response to glutamate receptor activation by kainate.

Recent research has identified in central nervous system neurons the expression of two enzymes from the inflammatory pathway of the metabolism of arachidonic acid, the 5-lipoxygenase (5LOX) and the cyclooxygenase-2 (COX2). Expression of both enzymes appears to be upregulated during aging; upregulated 5LOX/COX2 expression in neurons may be responsible for the increased neuronal vulnerability to degeneration. Involvement of the excitatory neurotransmitter glutamate in aging-associated neurodegeneration has also been suggested. Stimulation of glutamate receptors by kainic acid (kainate) has been shown independently to affect the brain expression of 5LOX or COX2. Using a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay to measure the contents of mRNAs we found 3h after kainate injection (intraperitoneally; 10 mg/kg) increased mRNA levels of 5LOX and COX2, but not that of COX1 in the hippocampus of rats. Pretreatment with the COX2 inhibitor NS-398 (9 mg/kg, 1h prior to kainate) inhibited the kainate-stimulated increase of 5LOX and COX2 mRNA levels. Our results indicate that hippocampal expression of both 5LOX and COX2 increases rather promptly when glutamate receptors are stimulated by kainate. The mechanism of how NS-398 inhibits this action of kainate should be further investigated.

Apoptosis induced in neuronal cultures by either the phosphatase inhibitor okadaic acid or the kinase inhibitor staurosporine is attenuated by isoquinolinesulfonamides H-7, H-8, and H-9.

Protein phosphorylation is kept in balance by an orchestrated action of kinases and phosphatases; when this balance is lost, neuronal apoptosis may occur. Okadaic acid (OKA), a marine toxin that inhibits specifically protein phosphatases 1 and 2A (EC 3.1.3.16), and staurosporine, an inhibitor of protein kinase C (PKC; EC 2.7.1.37), induced apoptosis in primary cultures of rat cerebellar granule neurons. We assayed apoptosis by the DNA gel electrophoresis, by the in situ TUNEL assay, and by morphological appearance following propidium iodide staining. Cell viability was assessed by the Trypan blue assay. Both OKA- and staurosporine-induced neuronal apoptosis were prevented by a macromolecular synthesis inhibitor actinomycin D and by a group of isoquinolinesulfonamide kinase inhibitors (H-7, 1-[5-isoquinolinesulfonyl]-2-methylpiperazine; H-8, N-¿2-[methylamino]ethyl¿-5-isoquinolinesulfonamide; H-9, N-(2-aminoethyl)-5-isoquinolinesulfonamide, but not by inhibitors of PKC, cyclic-GMP- and cyclic-AMP-dependent kinases, calcium/calmodulin-dependent kinases, tyrosine kinases, or by antioxidants. We postulate that a common mechanism, possibly an increased protein phosphorylation, is responsible for apoptosis triggered by an inhibition of phosphatases 1 and 2A and PKC. Elucidating the isoquinolinesulfonamide-sensitive mechanism may help us find new therapies for neurodegenerative diseases that involve apoptosis.

Effects of the potential antidepressant dihydroergosine in rats forced to swim: influence on plasma corticosterone.

The potential antidepressant properties of the ergot alkaloid dihydroergosine (DHESN) were studied in rats forced to swim in a restricted space. DHESN (50 mg/kg, intraperitoneally) reduced the duration of immobility in rats forced to swim 1 hr after administration. This effect was still present after 48 hr, but 7 hr. In non-swimming rats, the same dose of DHESN increased the plasma corticosterone concentration when administered 1 hr prior to sacrifice, but was without effect 48 hr after administration. In rats forced to swim, DHESN elicited two opposite effects. One hr following a single administration, it increased plasma secretion of corticosterone, whereas administered 48 hr prior to forced swimming, it decreased plasma corticosterone. These results, along with our previous data, give evidence that DHESN might possess antidepressant properties.

Effect of dihydroergotoxine on the susceptibility of rats to convulsions produced by different convulsant agents.

The study was undertaken to test further whether diminished GABAergic transmission might be responsible for the increased susceptibility of rats to picrotoxin-induced convulsions. In rats kept individually in cages in a noise-free room, the time between the intraperitoneal injection of the convulsant agent and the onset of convulsions was measured. Acute and subacute treatment with low doses of dihydroergotoxine (0.01-1.0 mg/kg) increased the occurrence and decreased the latency of picrotoxin-induced convulsions. Acute administration of dihydroergotoxine, 1.0 mg/kg, caused convulsions in animals injected with the subconvulsive dose (3 mg/kg) of bicuculline and of 10.0 mg/kg dihydroergotoxine in animals injected with the subconvulsive dose (1.5 mg/kg) of strychnine. Some of the animals injected with the 100% convulsive dose of strychnine were protected by dihydroergotoxine pretreatment (1.0 mg/kg) as evidenced by the lower occurrence of convulsions and fewer animals dying, as well as by a delay in the appearance of convulsions at 10.0 mg/kg. These results together with the previous findings on the GABA system suggest that dihydroergotoxine potentiates the appearance of picrotoxin and bicuculline-induced convulsions by a diminution of GABAergic transmission.

Effect of diazepam on plasma corticosterone levels.

In light of the numerous but rather conflicting reports on the action of benzodiazepines upon the hypothalamo-hypophyseal-adrenal (HHA) axis activity, the effect of different doses of diazepam (0.1, 1.0 and 10.0 mg/kg) administered 15, 30, 60, 120 and 240 min before decapitation on plasma corticosterone level was studied in rats. While 0.1 mg/kg diazepam had no effect, 1.0 mg/kg diazepam decreased plasma corticosterone levels 30 and 60 min following drug administration. On the other hand, treatment with 10.0 mg/kg diazepam produced an increase in plasma corticosterone levels from 15-120 min following drug administration.

Sex differences in the sensitivity of CBA mice to convulsions induced by GABA antagonists are age-dependent.

The administration of the GABA-blocking agents picrotoxin and bicuculline to adult (2.5-3 months old) CBA/HZgr mice resulted in the appearance of convulsions, the occurrence and/or lethality of which was greater in males than in females. The latency of picrotoxin-induced convulsions was also shorter in male mice. Strychnine, a drug which induces convulsions by blocking glycine receptors was equally effective in producing convulsions in both male and female adult mice. Unlike adult mice, young (20 days old) or old (2 years old) mice fail to display sex differences following the picrotoxin administration. Accordingly, the observed sex differences in the sensitivity of CBA mice to administration of convulsive agents are specific for the GABA system and present only in sexually mature, but not in immature or old animals.

Effect of dihydroergosine (DHESN) on the serotoninergic system and behaviour: is DHESN a new antidepressive agent?

Acute (50.0 mg/kg) and repeated (0.1-10.0 mg/kg) administration of dihydroergosine (DHESN) to rats over 5 days lowered the concentration of 5-HIAA in the brain. DHESN given acutely increased the brain 5-HT in p-CPA-treated animals and diminished the probenecid-induced increase in brain 5-HIAA. In pargyline-treated rats DHESN enhanced the 5-HT/5-HIAA ratio. DHESN administered to rats repeatedly over 5 days decreased the level of 5-HT in blood platelets, and in vitro at concentrations of 10(-4) M and 10(-3) M inhibited the uptake of [14C]-5-HT in platelets. DHESN (10.0-100.0 mg/kg) potentiated the 5-HT syndrome produced in rats by pargyline and 5-HTP. This potentiation was blocked with cyproheptadine but not with haloperidol. DHESN (1.0 and 10.0 mg/kg) lowered the locomotor activity of rats and 10.0 mg/kg DHESN also reduced the duration of immobility in rats forced to swim in a restricted space. The results indicate that DHESN, like antidepressants, decreases the turnover of serotonin in the brain and potentiates the 5-HT-mediated behaviour. This might suggest that the drug should be further investigated for its potential antidepressive properties.

Early upregulation of hippocampal 5-lipoxygenase following systemic administration of kainate to rats.

5-Lipoxygenase (5-LO; arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) is the enzyme responsible for the first step in the formation of inflammatory leukotrienes from arachidonic acid. 5-LO is expressed in hippocampal neurons. Increased formation of leukotrienes was found in the hippocampus of rats in which seizures were induced by a glutamate receptor agonist, kainate. Expression of the 5-LO gene can be stimulated by vitamin D3 and suppressed by the pineal hormone melatonin. Here we hypothesize that kainate also stimulates 5-LO expression in the hippocampus. Kainate was injected intraperitoneally (10 mg/kg). Rats were sacrificed 3 hr later and their hippocampi were dissected and total RNA was extracted and processed for quantitative reverse transcription/polymerase chain reaction (RT-PCR) assay of 5-LO and cyclophilin (cyc) mRNAs. Mutated primers were used as internal standards to assay attomol quantities of these two specific mRNAs per microgram of total RNA. Fixed hippocampal slices were processed for 5-LO immunostaining and Nissl staining (assay of cell damage). Kainate induced about a 2.5-fold increase in 5-LO mRNA and triggered a redistribution of 5-LO like immunoreactivity from the pyramidal cell bodies into the dendrites of these neurons, particularly in the CA3 area. The results suggest that glutamate receptor-mediated signaling may modify the expression of neuronal 5-LO and that this enzyme might be involved in glutamate receptor-mediated neuronal plasticity and/or degeneration.

Opposite effects of pinealectomy and melatonin administration on brain damage following cerebral focal ischemia in rat.

The endocrine system has been recognized as an important factor that may contribute to the outcome of stroke. We tested in rats the hypothesis that the pineal gland and/or its hormone melatonin may affect the outcome of a transient cerebral arteries occlusion (CerAO). Reversible 90 min focal ischemia was produced using a three-vessel occlusion method. Surgically or sham pinealectomized rats were exposed to CerAO 15 days after surgery. Melatonin (4 x 2.5 mg/kg: 30 min prior to onset of CerAO, immediately after recirculation was established, and 1 and 2 hr later) or its vehicle were administered intraperitoneally. The outcome of CerAO was assessed by quantitative assay of DNA damage or by Nissl staining and measurement of the infarct volume. Pinealectomy increased both the extent of DNA damage and the infarct volume; administration of melatonin to pinealectomized rats reduced both these markers of brain injury. We propose that the pineal endocrine system may influence the outcome of stroke. The mechanism of action and the pathophysiological role of this system, e.g., in aging, should be further characterized.

In vivo protection against kainate-induced apoptosis by the pineal hormone melatonin: effect of exogenous melatonin and circadian rhythm.

We recently reported that the pineal hormone melatonin protected neuronal cultures from excitotoxicity mediated via kainate-sensitive glutamate receptors and from oxidative stress-induced apoptosis. It has been shown that in rats, a systemic administration of kainate induces apoptotic cell death in various brain regions. In this study, we assayed the extent of brain injury after intraperitoneal (i.p.) administration of 10 mg/kg kainate to rats, using the quantitative TUNEL technique and Nissl staining. We examined the role of melatonin on kainate-induced brain injury by (a) injecting melatonin (4 × 2.5 mg/kg i.p.) prior to and after kainate injection and (b) injecting kainate at the time of low circulating melatonin levels (day/light), and high melatonin levels (night/dark). The extent of kainate-triggered DNA damage and the loss of Nissl staining were lower in animals treated with melatonin, or when kainate was injected at night, i.e. in the presence of high endogenous levels of melatonin. Our results suggest that both the pharmacological use of melatonin and the circadian secretion of endogenous melatonin during the night may reduce the extent of excitotoxic brain injury. Further studies are needed to fully characterize the relevance of our findings for the treatment of progressive neurodegenerative processes which involve excitotoxicity and apoptotic neuronal death.