Francesco M . Rossi | Universidad de la República (Uruguay) (original) (raw)
Papers by Francesco M . Rossi
The Journal of Neuroscience, 2002
The Journal of Neuroscience, 2003
Nicotinic acetylcholine receptors (nAChRs) expressed by dopaminergic (DA) neurons have long been ... more Nicotinic acetylcholine receptors (nAChRs) expressed by dopaminergic (DA) neurons have long been considered as potential therapeutic targets for the treatment of several neuropsychiatric diseases, including nicotine and cocaine addiction or Parkinson's disease. However, DA neurons express mRNAs coding for most, if not all, neuronal nAChR subunits, and the subunit composition of functional nAChRs has been difficult to establish. Immunoprecipitation experiments performed on mouse striatal extracts allowed us to identify three main types of heteromeric nAChRs (␣42*, ␣62*, and ␣4␣62*) in DA terminal fields. The functional relevance of these subtypes was then examined by studying nicotine-induced DA release in striatal synaptosomes and recording ACh-elicited currents in DA neurons from ␣4, ␣6, ␣4␣6, and 2 knockout mice. Our results establish that ␣62* nAChRs are functional and sensitive to ␣-conotoxin MII inhibition. These receptors are mainly located on DA terminals and consistently do not contribute to DA release induced by systemic nicotine administration, as evidenced by in vivo microdialysis. In contrast, (non␣6)␣42* nAChRs represent the majority of functional heteromeric nAChRs on DA neuronal soma. Thus, whereas a combination of ␣62* and ␣42* nAChRs may mediate the endogenous cholinergic modulation of DA release at the terminal level, somato-dendritic (non␣6)␣42* nAChRs most likely contribute to nicotine reinforcement.
The FASEB Journal, 2002
The incidence of neurological disabilities ascribable to perinatal injury is rising in Western co... more The incidence of neurological disabilities ascribable to perinatal injury is rising in Western countries, raising ethical and financial problems. No curative treatments are available. The pathophysiology of brain lesions of hypoxic-ischemic or inflammatory origin involves various neurotransmitters or neuromodulators. Among these, glutamate plays a key role. By overactivating N-methyl-D-aspartate receptors, it triggers the excitotoxic cascade. Although addictive, nicotine prevents excitotoxic neuronal death in adult animals. Its potential neuroprotective effects have not been evaluated in neonates. We found that nicotine is neuroprotective in vivo, in a murine model of neonatal excitotoxic brain injury, and in vitro, in primary cultures of cortical neurons. We investigated the respective roles in nicotine-related neuroprotection of the two dominant nicotinic acetylcholine receptor (nAChR) isoforms, namely, alpha4beta2 (heteropentameric) and alpha7 (homopentameric). Inhibition of alpha4beta2, either pharmacological (i.e., an alpha4beta2 nAChR antagonist) or molecular (beta2-/- knockout mice), abolished the protective effect of nicotine in vivo and in vitro, suggesting the involvement of alpha4beta2 nAChR in neonatal nicotine-related neuroprotection. In contrast, activation of alpha7 nAChR, which is protective in adult animals, was deleterious in our neonatal model, whereas its blockade, either pharmacological or molecular (alpha7-/- knockout mice) provided neuroprotection. Neuroprotective strategies must consider these opposite properties of distinct nAChR isoforms in neonates.
Proceedings of the National Academy of Sciences, 2001
In the mammalian visual system the formation of eye-specific layers at the thalamic level depends... more In the mammalian visual system the formation of eye-specific layers at the thalamic level depends on retinal waves of spontaneous activity, which rely on nicotinic acetylcholine receptor activation. We found that in mutant mice lacking the β2 subunit of the neuronal nicotinic receptor, but not in mice lacking the α4 subunit, retinofugal projections do not segregate into eye-specific areas, both in the dorso-lateral geniculate nucleus and in the superior colliculus. Moreover, β2−/− mice show an expansion of the binocular subfield of the primary visual cortex and a decrease in visual acuity at the cortical level but not in the retina. We conclude that the β2 subunit of the nicotinic acetylcholine receptor is necessary for the anatomical and functional development of the visual system.
Neuroscience, 1999
Neurotrophins play a crucial role in the development and activity-dependent plasticity of the vis... more Neurotrophins play a crucial role in the development and activity-dependent plasticity of the visual cortex [Berardi N. et al. (1994) Proc. natn. Acad. Sci. U.S.A. 91, 684-688; Bonhoeffer T. (1996) Curr. Opin. Neurobiol. 6, 119-126; Cellerino A. and Maffei L. (1996) Prog. Neurobiol. 49, 53-71; Domenici L. et al. (1994) NeuroReport 5, 2041-2044; Galuske R. A. W. et al (1996) Eur. J. Neurosci. 8, 1554-1559; Katz L. C. and Shatz C. J. (1996) Science 274, 1133-1138; Maffei L. et al. (1992) J. Neurosci. 12, 4651-4662; Pizzorusso T. and Maffei L. (1996) Curr. Opin. Neurol. 9, 122-125; Thoenen H. (1995) Science 270, 593-598]. As a possible mechanism of action, it has been postulated that the activity-dependent expression of neurotrophins by cortical cells could regulate synapse stabilization during the first period of postnatal life (critical period). Indeed, brain-derived neurotrophic factor messenger RNA expression in the visual cortex is regulated by neuronal activity as well as during development [Castrén E. et al. (1992) Proc. natn. Acad. Sci. U.S.A. 89, 9444-9448]. Moreover, we showed that monocular deprivation decreases brain-derived neurotrophic factor messenger RNA levels in the visual cortex receiving input from the deprived eye [Bozzi Y. et al. (1995) Neuroscience 69, 1133-1144]. What is missing, however, is the demonstration that brain-derived neurotrophic factor protein expression follows that of brain-derived neurotrophic factor messenger RNA. The aim of the present study is to fill this important gap in order to support the hypothesis that brain-derived neurotrophic factor is fundamental in the plasticity of the visual cortex. We found that brain-derived neurotrophic factor immunoreactivity peaks during the critical period and that it is preferentially localized in layers II-III and V-VI. We also demonstrated that monocular deprivation determines a decrease of brain-derived neurotrophic factor immunoreactivity exclusively in the visual cortex contralateral to the deprived eye. Our results support the proposed role for brain-derived neurotrophic factor in the development and activity-dependent plasticity of the visual cortex [Cabelli R. J. et al. (1995) Science 267, 1662-1666].
Neuron, 2003
activity. Between postnatal day 1 (P1) and P10 in this species, spontaneous activity is mediated ... more activity. Between postnatal day 1 (P1) and P10 in this species, spontaneous activity is mediated by nicotinic cholinergic transmission (Feller et al., 1996; Penn et al., 1998) and, in the form of either action potential firing (Meister et al., 1991) or transient influxes of calcium (e.g.,
European Journal of Neuroscience, 1999
It has been recently shown that intraventricular injections of nerve growth factor (NGF) prevent ... more It has been recently shown that intraventricular injections of nerve growth factor (NGF) prevent the effects of monocular deprivation in the rat. We have tested the localization and the molecular nature of the NGF receptor(s) responsible for this effect by activating cortical trkA receptors in monocularly deprived rats by cortical infusion of a specific agonist of NGF on trkA, the bivalent antirat trkA IgG (RTA-IgG). TrkA protein was detected by immunoblot in the rat visual cortex during the critical period. Rats were monocularly deprived for 1 week (P21-28) and RTA-IgG or control rabbit IgG were delivered by osmotic minipumps. The effects of monocular deprivation on the ocular dominance of visual cortical neurons were assessed by extracellular single cell recordings. We found that the shift towards the ipsilateral, non-deprived eye was largely prevented by RTA-IgG. Infusion of RTA-IgG combined with antibody that blocks p75 NTR (REX), slightly reduced RTA-IgG effectiveness in preventing monocular deprivation effects. These results suggest that NGF action in visual cortical plasticity is mediated by cortical TrkA receptors with p75 NTR exerting a facilitatory role.
European Journal of Neuroscience, 2003
The mesostriatal dopaminergic system influences locomotor activity and the reinforcing properties... more The mesostriatal dopaminergic system influences locomotor activity and the reinforcing properties of many drugs of abuse including nicotine. Here we investigate the role of the a4 nicotinic acetylcholine receptor (nAChR) subunit in mediating the effects of nicotine in the mesolimbic dopamine system in mice lacking the a4 subunit. We show that there are two distinct populations of receptors in the substantia nigra and striatum by using autoradiographic labelling with 125 I a-conotoxin MII. These receptors are comprised of the a4, b2 and a6 nAChR subunits and non-a4, b2, and a6 nAChR subunits. Non-a4 subunit-containing nAChRs are located on dopaminergic neurons, are functional and respond to nicotine as demonstrated by patch clamp recordings. In vivo microdialysis performed in awake, freely moving mice reveal that mutant mice have basal striatal dopamine levels which are twice as high as those observed in wild-type mice. Despite the fact that both wild-type and a4 null mutant mice show a similar increase in dopamine release in response to intrastriatal KCl perfusion, a nicotine-elicited increase in dopamine levels is not observed in mutant mice. Locomotor activity experiments show that there is no difference between wild-type and mutant mice in basal activity in both habituated and nonhabituated environments. Interestingly, mutant mice sustain an increase in cocaine-elicited locomotor activity longer than wild-type mice. In addition, mutant mice recover from depressant locomotor activity in response to nicotine at a faster rate. Our results indicate that a4-containing nAChRs exert a tonic control on striatal basal dopamine release, which is mediated by a heterogeneous population of nAChRs.
We found that deprivation of pattern vision in one eye, that leaves luminance detection performan... more We found that deprivation of pattern vision in one eye, that leaves luminance detection performance unaffected, is sufficient to reduce brain-derived neurotrophic factor (but not trkB) messenger RNA in the visual cortex of young and adult rats. Monocular deprivation by means of eyelids' suture was performed during or after the critical period and the cortical amount of brain-derived neurotrophic factor messenger RNA was analysed by in situ hybridization and RNAase protection after 15-30 days of deprivation. A reduction of brain-derived neurotrophic factor messenger RNA was observed in the visual cortex contralateral to the deprived eye in rats monocularly deprived during the critical period. The same reduction was also found in rats monocularly deprived after the end of the critical period, when anatomical or physiological signs of monocular deprivation are absent. The pharmacological blockade of retinal activity equally affected the expression of brain-derived neurotrophic factor messenger RNA in young and adults. Quantitative RNAase protection assays revealed that the cortical level of brain-derived neurotrophic factor messenger RNA was reduced to the same extent when intraocular injections of tetrodotoxin were performed within or after the critical period. A developmental study of brain-derived neurotrophic factor messenger RNA expression in rat visual cortex showed a marked increase around the time of natural eye-opening followed by a plateau from postnatal day 20 until adult age. Messenger RNA for the kinasic domain of brain-derived neurotrophic factor receptor (trkB) was found in the dorsal lateral geniculate nucleus and the visual cortex during development and in adults. Our results suggest that the reduction of brain-derived neurotrophic factor messenger RNA induced by monocular deprivation is related to the absence of pattern vision rather than to the competitive interactions that underlie the effects of monocular deprivation during the critical period.
Archives of Toxicology, 2007
Pb(II) is a neurotoxic pollutant that produces permanent cognitive deficits in children. Pb(II) c... more Pb(II) is a neurotoxic pollutant that produces permanent cognitive deficits in children. Pb(II) can modulate cell signaling pathways and cell viability in a variety of cell types. However, these actions are not well demonstrated on glial cells, which represent an important target for metals into the central nervous system. The present work was undertaken to determine the ability of Pb(II) in modulating the activity of mitogen activated protein kinases (MAPKs) in cultures of C6 rat glioma cells, a useful functional model for the study of astrocytes. Additionally, cell viability was analyzed by measurement of MTT reduction. Cells were exposed to lead acetate 0.1, 1, 10 microM for 24 and 48 h. MAPKs activation - in particular ERK1/2, p38(MAPK) and JNK1/2 - were analyzed by western blotting. Results showed that 10 microM Pb(II) treatment for 24 h caused a discrete stimulation of p38(MAPK) phosphorylation. However, 1 and 10 microM Pb(II) treatment for 48 h provoked a significant stimulation in the phosphorylation state of p38(MAPK) and JNK1/2. The phosphorylation state of ERK1/2 was not modified by any Pb(II) treatment. Moreover, data indicate that at 48 h treatment even 1 microM Pb(II) can be cytotoxic, causing impairment on cell viability. Therefore, depending on a long incubation period, a significant concomitant activation of p38(MAPK) and JNK1/2 by Pb(II) took place in parallel with the impairment of C6 glioma cells viability.
The Journal of Neuroscience, 2002
The Journal of Neuroscience, 2003
Nicotinic acetylcholine receptors (nAChRs) expressed by dopaminergic (DA) neurons have long been ... more Nicotinic acetylcholine receptors (nAChRs) expressed by dopaminergic (DA) neurons have long been considered as potential therapeutic targets for the treatment of several neuropsychiatric diseases, including nicotine and cocaine addiction or Parkinson's disease. However, DA neurons express mRNAs coding for most, if not all, neuronal nAChR subunits, and the subunit composition of functional nAChRs has been difficult to establish. Immunoprecipitation experiments performed on mouse striatal extracts allowed us to identify three main types of heteromeric nAChRs (␣42*, ␣62*, and ␣4␣62*) in DA terminal fields. The functional relevance of these subtypes was then examined by studying nicotine-induced DA release in striatal synaptosomes and recording ACh-elicited currents in DA neurons from ␣4, ␣6, ␣4␣6, and 2 knockout mice. Our results establish that ␣62* nAChRs are functional and sensitive to ␣-conotoxin MII inhibition. These receptors are mainly located on DA terminals and consistently do not contribute to DA release induced by systemic nicotine administration, as evidenced by in vivo microdialysis. In contrast, (non␣6)␣42* nAChRs represent the majority of functional heteromeric nAChRs on DA neuronal soma. Thus, whereas a combination of ␣62* and ␣42* nAChRs may mediate the endogenous cholinergic modulation of DA release at the terminal level, somato-dendritic (non␣6)␣42* nAChRs most likely contribute to nicotine reinforcement.
The FASEB Journal, 2002
The incidence of neurological disabilities ascribable to perinatal injury is rising in Western co... more The incidence of neurological disabilities ascribable to perinatal injury is rising in Western countries, raising ethical and financial problems. No curative treatments are available. The pathophysiology of brain lesions of hypoxic-ischemic or inflammatory origin involves various neurotransmitters or neuromodulators. Among these, glutamate plays a key role. By overactivating N-methyl-D-aspartate receptors, it triggers the excitotoxic cascade. Although addictive, nicotine prevents excitotoxic neuronal death in adult animals. Its potential neuroprotective effects have not been evaluated in neonates. We found that nicotine is neuroprotective in vivo, in a murine model of neonatal excitotoxic brain injury, and in vitro, in primary cultures of cortical neurons. We investigated the respective roles in nicotine-related neuroprotection of the two dominant nicotinic acetylcholine receptor (nAChR) isoforms, namely, alpha4beta2 (heteropentameric) and alpha7 (homopentameric). Inhibition of alpha4beta2, either pharmacological (i.e., an alpha4beta2 nAChR antagonist) or molecular (beta2-/- knockout mice), abolished the protective effect of nicotine in vivo and in vitro, suggesting the involvement of alpha4beta2 nAChR in neonatal nicotine-related neuroprotection. In contrast, activation of alpha7 nAChR, which is protective in adult animals, was deleterious in our neonatal model, whereas its blockade, either pharmacological or molecular (alpha7-/- knockout mice) provided neuroprotection. Neuroprotective strategies must consider these opposite properties of distinct nAChR isoforms in neonates.
Proceedings of the National Academy of Sciences, 2001
In the mammalian visual system the formation of eye-specific layers at the thalamic level depends... more In the mammalian visual system the formation of eye-specific layers at the thalamic level depends on retinal waves of spontaneous activity, which rely on nicotinic acetylcholine receptor activation. We found that in mutant mice lacking the β2 subunit of the neuronal nicotinic receptor, but not in mice lacking the α4 subunit, retinofugal projections do not segregate into eye-specific areas, both in the dorso-lateral geniculate nucleus and in the superior colliculus. Moreover, β2−/− mice show an expansion of the binocular subfield of the primary visual cortex and a decrease in visual acuity at the cortical level but not in the retina. We conclude that the β2 subunit of the nicotinic acetylcholine receptor is necessary for the anatomical and functional development of the visual system.
Neuroscience, 1999
Neurotrophins play a crucial role in the development and activity-dependent plasticity of the vis... more Neurotrophins play a crucial role in the development and activity-dependent plasticity of the visual cortex [Berardi N. et al. (1994) Proc. natn. Acad. Sci. U.S.A. 91, 684-688; Bonhoeffer T. (1996) Curr. Opin. Neurobiol. 6, 119-126; Cellerino A. and Maffei L. (1996) Prog. Neurobiol. 49, 53-71; Domenici L. et al. (1994) NeuroReport 5, 2041-2044; Galuske R. A. W. et al (1996) Eur. J. Neurosci. 8, 1554-1559; Katz L. C. and Shatz C. J. (1996) Science 274, 1133-1138; Maffei L. et al. (1992) J. Neurosci. 12, 4651-4662; Pizzorusso T. and Maffei L. (1996) Curr. Opin. Neurol. 9, 122-125; Thoenen H. (1995) Science 270, 593-598]. As a possible mechanism of action, it has been postulated that the activity-dependent expression of neurotrophins by cortical cells could regulate synapse stabilization during the first period of postnatal life (critical period). Indeed, brain-derived neurotrophic factor messenger RNA expression in the visual cortex is regulated by neuronal activity as well as during development [Castrén E. et al. (1992) Proc. natn. Acad. Sci. U.S.A. 89, 9444-9448]. Moreover, we showed that monocular deprivation decreases brain-derived neurotrophic factor messenger RNA levels in the visual cortex receiving input from the deprived eye [Bozzi Y. et al. (1995) Neuroscience 69, 1133-1144]. What is missing, however, is the demonstration that brain-derived neurotrophic factor protein expression follows that of brain-derived neurotrophic factor messenger RNA. The aim of the present study is to fill this important gap in order to support the hypothesis that brain-derived neurotrophic factor is fundamental in the plasticity of the visual cortex. We found that brain-derived neurotrophic factor immunoreactivity peaks during the critical period and that it is preferentially localized in layers II-III and V-VI. We also demonstrated that monocular deprivation determines a decrease of brain-derived neurotrophic factor immunoreactivity exclusively in the visual cortex contralateral to the deprived eye. Our results support the proposed role for brain-derived neurotrophic factor in the development and activity-dependent plasticity of the visual cortex [Cabelli R. J. et al. (1995) Science 267, 1662-1666].
Neuron, 2003
activity. Between postnatal day 1 (P1) and P10 in this species, spontaneous activity is mediated ... more activity. Between postnatal day 1 (P1) and P10 in this species, spontaneous activity is mediated by nicotinic cholinergic transmission (Feller et al., 1996; Penn et al., 1998) and, in the form of either action potential firing (Meister et al., 1991) or transient influxes of calcium (e.g.,
European Journal of Neuroscience, 1999
It has been recently shown that intraventricular injections of nerve growth factor (NGF) prevent ... more It has been recently shown that intraventricular injections of nerve growth factor (NGF) prevent the effects of monocular deprivation in the rat. We have tested the localization and the molecular nature of the NGF receptor(s) responsible for this effect by activating cortical trkA receptors in monocularly deprived rats by cortical infusion of a specific agonist of NGF on trkA, the bivalent antirat trkA IgG (RTA-IgG). TrkA protein was detected by immunoblot in the rat visual cortex during the critical period. Rats were monocularly deprived for 1 week (P21-28) and RTA-IgG or control rabbit IgG were delivered by osmotic minipumps. The effects of monocular deprivation on the ocular dominance of visual cortical neurons were assessed by extracellular single cell recordings. We found that the shift towards the ipsilateral, non-deprived eye was largely prevented by RTA-IgG. Infusion of RTA-IgG combined with antibody that blocks p75 NTR (REX), slightly reduced RTA-IgG effectiveness in preventing monocular deprivation effects. These results suggest that NGF action in visual cortical plasticity is mediated by cortical TrkA receptors with p75 NTR exerting a facilitatory role.
European Journal of Neuroscience, 2003
The mesostriatal dopaminergic system influences locomotor activity and the reinforcing properties... more The mesostriatal dopaminergic system influences locomotor activity and the reinforcing properties of many drugs of abuse including nicotine. Here we investigate the role of the a4 nicotinic acetylcholine receptor (nAChR) subunit in mediating the effects of nicotine in the mesolimbic dopamine system in mice lacking the a4 subunit. We show that there are two distinct populations of receptors in the substantia nigra and striatum by using autoradiographic labelling with 125 I a-conotoxin MII. These receptors are comprised of the a4, b2 and a6 nAChR subunits and non-a4, b2, and a6 nAChR subunits. Non-a4 subunit-containing nAChRs are located on dopaminergic neurons, are functional and respond to nicotine as demonstrated by patch clamp recordings. In vivo microdialysis performed in awake, freely moving mice reveal that mutant mice have basal striatal dopamine levels which are twice as high as those observed in wild-type mice. Despite the fact that both wild-type and a4 null mutant mice show a similar increase in dopamine release in response to intrastriatal KCl perfusion, a nicotine-elicited increase in dopamine levels is not observed in mutant mice. Locomotor activity experiments show that there is no difference between wild-type and mutant mice in basal activity in both habituated and nonhabituated environments. Interestingly, mutant mice sustain an increase in cocaine-elicited locomotor activity longer than wild-type mice. In addition, mutant mice recover from depressant locomotor activity in response to nicotine at a faster rate. Our results indicate that a4-containing nAChRs exert a tonic control on striatal basal dopamine release, which is mediated by a heterogeneous population of nAChRs.
We found that deprivation of pattern vision in one eye, that leaves luminance detection performan... more We found that deprivation of pattern vision in one eye, that leaves luminance detection performance unaffected, is sufficient to reduce brain-derived neurotrophic factor (but not trkB) messenger RNA in the visual cortex of young and adult rats. Monocular deprivation by means of eyelids' suture was performed during or after the critical period and the cortical amount of brain-derived neurotrophic factor messenger RNA was analysed by in situ hybridization and RNAase protection after 15-30 days of deprivation. A reduction of brain-derived neurotrophic factor messenger RNA was observed in the visual cortex contralateral to the deprived eye in rats monocularly deprived during the critical period. The same reduction was also found in rats monocularly deprived after the end of the critical period, when anatomical or physiological signs of monocular deprivation are absent. The pharmacological blockade of retinal activity equally affected the expression of brain-derived neurotrophic factor messenger RNA in young and adults. Quantitative RNAase protection assays revealed that the cortical level of brain-derived neurotrophic factor messenger RNA was reduced to the same extent when intraocular injections of tetrodotoxin were performed within or after the critical period. A developmental study of brain-derived neurotrophic factor messenger RNA expression in rat visual cortex showed a marked increase around the time of natural eye-opening followed by a plateau from postnatal day 20 until adult age. Messenger RNA for the kinasic domain of brain-derived neurotrophic factor receptor (trkB) was found in the dorsal lateral geniculate nucleus and the visual cortex during development and in adults. Our results suggest that the reduction of brain-derived neurotrophic factor messenger RNA induced by monocular deprivation is related to the absence of pattern vision rather than to the competitive interactions that underlie the effects of monocular deprivation during the critical period.
Archives of Toxicology, 2007
Pb(II) is a neurotoxic pollutant that produces permanent cognitive deficits in children. Pb(II) c... more Pb(II) is a neurotoxic pollutant that produces permanent cognitive deficits in children. Pb(II) can modulate cell signaling pathways and cell viability in a variety of cell types. However, these actions are not well demonstrated on glial cells, which represent an important target for metals into the central nervous system. The present work was undertaken to determine the ability of Pb(II) in modulating the activity of mitogen activated protein kinases (MAPKs) in cultures of C6 rat glioma cells, a useful functional model for the study of astrocytes. Additionally, cell viability was analyzed by measurement of MTT reduction. Cells were exposed to lead acetate 0.1, 1, 10 microM for 24 and 48 h. MAPKs activation - in particular ERK1/2, p38(MAPK) and JNK1/2 - were analyzed by western blotting. Results showed that 10 microM Pb(II) treatment for 24 h caused a discrete stimulation of p38(MAPK) phosphorylation. However, 1 and 10 microM Pb(II) treatment for 48 h provoked a significant stimulation in the phosphorylation state of p38(MAPK) and JNK1/2. The phosphorylation state of ERK1/2 was not modified by any Pb(II) treatment. Moreover, data indicate that at 48 h treatment even 1 microM Pb(II) can be cytotoxic, causing impairment on cell viability. Therefore, depending on a long incubation period, a significant concomitant activation of p38(MAPK) and JNK1/2 by Pb(II) took place in parallel with the impairment of C6 glioma cells viability.