Maternal milk as methylmercury source for suckling mice: neurotoxic effects involved with the cerebellar glutamatergic system.

Methylmercury (MeHg) is a highly neurotoxic compound and several studies have reported intoxication signs in children whose mothers were exposed to this environmental toxicant. Although it is well established that the in utero exposure to MeHg causes neurological deficits in animals and humans, there is no evidence of the exclusive contribution of lactational exposure to MeHg as a possible cause of neurotoxicity in the offspring. In this study, we investigated the exclusive contribution of MeHg exposure through maternal milk on biochemical parameters related to the glutamatergic homeostasis (glutamate uptake by slices) and to the oxidative stress (total and nonprotein sulfhydryl groups, nonprotein hydroperoxides, glutathione peroxidase and catalase activities) in the cerebellum of suckling mice (Swiss albino). The same parameters were also evaluated in the cerebellum of mothers. Our results showed, for the first time, that lactational exposure to MeHg caused a high percent of inhibition (50%) on glutamate uptake by cerebellar slices in pups. Contrarily, this effect was not observed in mothers, which were submitted to a direct oral exposure to MeHg (15 mg/l in drinking water). In addition, behavioral/functional changes were observed in the weaning mice exposed to MeHg. It was observed an increase in the levels of nonprotein hydroperoxides in cerebellum, and this increase was negatively correlated to the glutamate uptake by cerebellar slices. This study indicates that (1) the exposure of lactating mice to MeHg causes inhibition of the glutamate uptake by cerebellar slices in the offspring; (2) this inhibitory effect seems to be related to increased levels of hydroperoxide.     

Is chemical neurotransmission altered specifically during methylmercury-induced cerebellar dysfunction?

Methylmercury (MeHg) is an important environmental neurotoxicant that is present in seafood and affects the developing and mature nervous system. The neurotoxicity induced by MeHg is a concern, particularly for fish-eating populations and pregnant or nursing women. During MeHg-induced neurotoxicity, degeneration of the granule cell layer in the cerebellum occurs, which leads to deficits in motor function. I suggest that the action of MeHg on specific neurotransmitter receptors contributes to the selective vulnerability of granule cells. MeHg appears to stimulate M(3) muscarinic acetylcholine receptors and to inhibit GABA(A) receptor subtypes preferentially on cerebellar granule cells. This could lead to the loss of tonic inhibition of granule cells as a result of antagonism of GABA(A) receptors, and a M(3)-receptor-mediated increase in the intracellular concentration of Ca(2+) and block of a K(+)-dependent leak current. The net result would be increased spontaneous release of glutamate, which, coupled with a MeHg-induced impairment of glutamate uptake by astrocytes, could cause Ca(2+)-mediated cytotoxicity.     

Effects of the seafood toxin domoic acid on glutamate uptake by rat astrocytes.

Pronounced glutamic acid uptake was observed after only 15 min with glutamate concentrations of 60 nmol/mg protein when astrocytes were incubated with 1 mM glutamic acid. The uptake increased with time to a steady-state glutamate level of above 160 nmol/mg protein by 45 min. The uptake was energy dependent. Reduced temperature (0 degrees C) and ouabain (100 microM) inhibited uptake by 86.7% (P<0.001; n=18) and 84.4% (P<0.001; n=18), respectively, when compared with controls. After exposure of astrocytes to glutamate (1 mM) in the incubation medium, in the presence of domoic acid (10 and 100 microM) at 5 and 60 min, domoic acid (10 microM) elevated glutamate uptake by 64.0% (P<0.05; n=34) at 5 min but decreased glutamate uptake by 47.8% (P<0.01; n=19) at 60 min compared with controls. A higher dose of domoic acid (100 microM) decreased glutamate uptake by 49.6% (P<0.01; n=20) and 61.3% (P<0.001; n=20) at 5 and 60 min, respectively, compared with controls. This study suggests that domoic acid may induce neurotoxicity because of the failure of astrocytes to remove extracellular glutamate. This may contribute to excitotoxic injury.     

Role of glutathione in determining the differential sensitivity between the cortical and cerebellar regions towards mercury-induced oxidative stress

Certain discrete areas of the CNS exhibit enhanced sensitivity towards MeHg. To determine whether GSH is responsible for this particular sensitivity, we investigated its role in MeHg-induced oxidative insult in primary neuronal and astroglial cell cultures of both cerebellar and cortical origins. For this purpose, ROS and GSH were measured with the fluorescent indicators, CMH₂DCFDA and MCB. Cell associated-MeHg was measured with ¹⁴C-radiolabeled MeHg. The intracellular GSH content was modified by pretreatment with NAC or DEM. For each of the dependent variables (ROS, GSH, and MTT), there was an overall significant effect of cellular origin, MeHg and pretreatment in all the cell cultures. A trend towards significant interaction between origin × MeHg × pretreatment was observed only for the dependent variable, ROS (astrocytes p = 0.056; neurons p = 0.000). For GSH, a significant interaction between origin × MeHg was observed only in astrocytes (p = 0.030). The cerebellar cell cultures were more vulnerable (astrocytes_mean = 223.77; neurons_mean = 138.06) to ROS than the cortical cell cultures (astrocytes_mean = 125.18; neurons_mean = 107.91) for each of the tested treatments. The cell associated-MeHg increased when treated with DEM, and the cerebellar cultures varied significantly from the cortical cultures. Non-significant interactions between origin × MeHg × pretreatment for GSH did not explain the significant interactions responsible for the increased amount of ROS produced in these cultures. In summary, although GSH modulation influences MeHg-induced toxicity, the difference in the content of GSH in cortical and cerebellar cultures fails to account for the increased ROS production in cerebellar cultures. Hence, different approaches for the future studies regarding the mechanisms behind selectivity of MeHg have been discussed.     

Participation of N-methyl-D-aspartate receptors on methylmercury-induced DNA damage in rat frontal cortex

Methylmercury (MeHg) inhibits glutamate uptake by astrocytes, which can contribute to neuronal loss through excitotoxicity. We explored the extent at which this phenomenon is involved in MeHg-induced DNA damage in the rat cortex. MeHg amounts that increase extracellular glutamate (1.5, 7.5 and 15 nmol, according to previous reports) were stereotaxically injected in the frontal cortex of adult rats before DNA-damage determination by means of a quantitative TUNEL assay. After either 24 or 48 h, the cortex of all exposed animals showed significant increments of damaged DNA, compared with rats that only received sterile saline. In parallel experiments, we found that the administration of a non competitive NMDA receptor antagonist (MK-801, 10 mg/kg, i.p.) 1 h before MeHg injection, significantly reduced DNA damage. These results demonstrate that activation of NMDA receptors contributes importantly to MeHg neurotoxicity.     

Organotins disrupt components of glutamate homeostasis in rat astrocyte cultures.

A rat cortical astrocyte preparation was used to investigate the effects of organotins on glutamate regulation by astrocytes. Exposure of astrocytes to low levels of organotins produced significant changes in two key components of glutamate homeostasis: glutamine synthetase (CS) activity and the high-affinity transport of L-glutamate. Trimethyltin (TMT), triethyltin (TET), and triphenyltin (TPT) exhibited differential abilities to reduce GS activity and glutamate uptake. Cultures incubated with 1 microM TET or TPT, but not TMT, exhibited a marked decrease in GS activity. Exposure to TET or TPT also produced a significant decrease in glutamate transport activity that was not observed with TMT. These declines in activity were not attributable to cell loss as measured by MTT reduction and lactate dehydrogenase (LDH) leakage. Since the loss of GS activity and transporter activity was not seen with acute organotin exposure, it is most likely attributable to a decreased presence of fully functioning protein. While the attenuation of GS and glutamate transporter activities by organotins does not match their pattern of neurotoxicity, the results indicate the potential for subtoxic concentrations of these compounds to increase extracellular glutamate and interact with other excitotoxic episodes.     

Prevention of glutamate neurotoxicity in cultured neurons by 3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran (CR-6), a scavenger of nitric oxide.

Glutamate neurotoxicity in cerebellar neurons in culture is mediated by excessive production of nitric oxide (NO). We anticipated that 3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran (CR-6) could act as a scavenger of NO since it contains a position (C-5) highly activated towards nitration reaction. The aim of this work was to assess whether CR-6 acts as an NO scavenger and prevents glutamate neurotoxicity in cultures of cerebellar neurons. It was shown that CR-6 reduced, in a dose-dependent manner, glutamate-induced formation of cGMP (EC50 approximately 15 microM) and prevented glutamate neurotoxicity. The protection was approximately 50% at 3-10 microM and nearly complete at 100 microM. CR-6 did not prevent glutamate-induced activation of NO synthase, but interfered with the glutamate-NO-cGMP pathway at a later step. CR-6 reduced the formation of cGMP induced by S-nitroso-N-acetylpenicillamine (SNAP), an NO-generating agent, indicating that CR-6 acts as a scavenger of NO in cultured neurons. This was further supported by experiments showing that in neurons treated with CR-6 and glutamate, the 5-nitro derivative of CR-6 was formed, as determined by GC-MS analyses. Moreover, in vitro incubation of CR-6 with SNAP also produced the 5-nitroderivative, thus confirming that CR-6 directly reacts with NO. The results reported indicate that CR-6 acts as an NO scavenger in neurons and prevents glutamate neurotoxicity.     

Comparative sensitivity of rat cerebellar neurons to dysregulation of divalent cation homeostasis and cytotoxicity caused by methylmercury

The objective of the present study was to determine the relative effectiveness of methylmercury (MeHg) to alter divalent cation homeostasis and cause cell death in MeHg-resistant cerebellar Purkinje and MeHg-sensitive granule neurons. Application of 0.5-5 microM MeHg to Purkinje and granule cells grown in culture caused a concentration- and time-dependent biphasic increase in fura-2 fluorescence. At 0.5 and 1 microM MeHg, the elevations of fura-2 fluorescence induced by MeHg were biphasic in both cell types, but significantly delayed in Purkinje as compared to granule cells. Application of the heavy-metal chelator, TPEN, to Purkinje cells caused a precipitous decline in a proportion of the fura-2 fluorescence signal, indicating that MeHg causes release of Ca(2+) and non-Ca(2+) divalent cations. Purkinje cells were also more resistant than granule cells to the neurotoxic effects of MeHg. At 24.5 h after-application of 5 microM MeHg, 97.7% of Purkinje cells were viable. At 3 microM MeHg there was no detectable loss of Purkinje cell viability. In contrast, only 40.6% of cerebellar granule cells were alive 24.5 h after application of 3 microM MeHg. In conclusion, Purkinje neurons in primary cultures appear to be more resistant to MeHg-induced dysregulation of divalent cation homeostasis and subsequent cell death when compared to cerebellar granule cells. There is a significant component of non-Ca(2+) divalent cation released by MeHg in Purkinje neurons.     

Acute exposure to methylmercury opens the mitochondrial permeability transition pore in rat cerebellar granule cells.

Cerebellar granule cells are preferentially targeted during methylmercury (MeHg) poisoning. Following acute MeHg exposure, granule cells in culture undergo an increase in intracellular Ca2+ concentration ([Ca2+]i) that apparently contributes to cell death. This effect consists of several temporally and kinetically distinct phases. The initial elevation arises from release of Ca2+(i) stores; the second phase results from entry of Ca2+(e). In these experiments, we tested the hypothesis that release of mitochondrial Ca2+ through the mitochondrial permeability transition pore (MTP) contributes to the initial release of Ca2+(i). Neonatal rat cerebellar granule cells in culture and single cell microfluorimetry were used to examine MeHg-induced changes in [Ca2+]i and mitochondrial membrane potential (Psi(m)). Pretreatment with the MTP inhibitor cyclosporin A (CsA, 5 microM) delayed the initial phase of increased [Ca2+]i induced by 0.2 and 0.5 microM MeHg, but not 1.0 microM MeHg. CsA (5 microM) also delayed the irreversible loss of Psi(m) induced by 0.5 microM MeHg. Ca2+(e) was not required for either effect, because the effect of CsA on the first phase increase in [Ca2+]i and loss of Psi(m) was not altered in nominally Ca2+-free buffer. Increasing concentrations of MeHg (0.2-2.0 microM) caused increasing incidence of cell death at 24 h postexposure. Treatment with CsA provided protection against cytotoxicity at lower MeHg concentrations (0.2-0.5 microM), but not at higher MeHg concentrations (1.0-2.0 microM). Thus, the MTP appears to play a significant role in the cellular effects following acute exposure of cerebellar granule neurons to MeHg.     

Mechanisms of neuroprotective effects of therapeutic acetylcholinesterase inhibitors used in treatment of Alzheimer's disease

Donepezil, galanthamine, and tacrine are therapeutic acetylcholinesterase (AChE) inhibitors used for the treatment of Alzheimer's disease. The aim of this paper is to review recent findings on their neuroprotective properties and the mechanisms of neuroprotection against glutamate neurotoxicity in rat cortical neurons. First, the hallmark of neurotoxicity induced by two different glutamate treatment conditions was examined, revealing that acute glutamate treatment (1 mM, 10 min) induces necrotic neuronal death and that moderate glutamate treatment (100 microM, 24 hr) induces apoptotic neuronal death. Next, we showed that therapeutic AChE inhibitors protect cortical neurons from glutamate neurotoxicity in a time- and concentration-dependent manner. We examined the mechanism of this neuroprotective effect and found that the neuroprotective effects against both acute and moderate glutamate treatments are mediated through nicotinic acetylcholine receptors (nAChRs), or more specifically, the effects of donepezil and galanthamine are mediated through alpha4- and alpha7-nAChR. We also showed that donepezil and galanthamine protect cortical neurons against acute glutamate treatment-induced neurotoxicity at steps before, and that tacrine protects at steps after, nitric oxide radical formation. On the other hand, the neuroprotective effects of donepezil and galanthamine, but not of tacrine, against neurotoxicity induced by moderate glutamate treatment were mediated through the phosphatidylinositol 3-kinase-Akt pathway. These findings unveiled the hitherto unknown neuroprotective effects of therapeutic AChE inhibitors and provided valuable insights into its neuroprotective mechanisms. They may very likely form the basis for a novel treatment strategy against Alzheimer's disease.     

Evaluation of L-glutamate clearance capacity of cultured rat cortical astrocytes

Astrocytes have a function in the uptake of excitatory neurotransmitter L-glutamate via the glutamate transporter. To evaluate the L-glutamate clearance capacity of astrocytes, we developed a colorimetric method for the determination of L-glutamate concentration and measured changes in extracellular L-glutamate concentration in rat cortical astrocyte cultures. When L-glutamate (50-200 microM) was added to astrocyte cultures and incubated for 1-8 h, the extracellular L-glutamate concentration declined with time. When L-glutamate was mixed with astrocyte culture supernatants only, no significant change in L-glutamate concentration was observed, ruling out the possibility that L-glutamate is spontaneously or enzymatically degraded in the extracellular space. Alternatively, the time-dependent decline of extracellular L-glutamate concentration was blocked by the presence of glutamate uptake inhibitors, indicating that the glutamate uptake system of astrocytes plays a major role in the clearance of extracellular L-glutamate.     

Acute cytotoxic effects of mercuric compounds in cultured astrocytes prepared from cerebral hemisphere and cerebellum of newborn rats

We investigated acute cytotoxic effects and Hg accumulation after exposure to methylmercury (MeHg) or Hg(2+) in the presence or absence of serum in cultured astrocytes prepared from the cerebral hemisphere or cerebellum of newborn rats. Dose-related changes in viable cell numbers after exposure to mercuric compounds were not different between astrocytes from both regions under the specified conditions. Accumulation of each compound for 3 h was similar in both astrocytes but that for 24 h became different, especially that of Hg(2+). In both astrocytes, susceptibility to the respective compounds was higher in the order of those exposed immediately after, without, and 24 h after changing the serum-containing medium to a serum-free defined medium (SFDM). Accumulation for 3 h was higher in the respective astrocytes exposed to MeHg or Hg(2+) immediately after being maintained in SFDM than in those exposed 24 h after. These results suggest that accumulation of mercuric compounds up to 3 h strongly correlates with susceptibility, at least when maintained in SFDM. Astrocytic morphology changed to a satellite shape after the medium change to SFDM particularly in cerebellar astrocytes but only a few in cerebral hemisphere astrocytes, and it was reverted to a polygonal shape by MeHg but not Hg(2+) at 3 microM. The present results suggest that although some properties such as morphological changes and Hg accumulation are different between cerebral hemisphere and cerebellar astrocytes, these differences are not simply reflected by susceptibility to the acute cytotoxicity of mercuric compounds.     

Ebselen protects against methylmercury-induced inhibition of glutamate uptake by cortical slices from adult mice

Methylmercury (MeHg) is a highly neurotoxic compound and the inhibition of glutamate uptake by astrocytes has been pointed as an important mechanism involved in MeHg-induced glutamate excitotoxicity. We examined the effect of oral exposure to MeHg (10 and 40 mg/l in drinking water) on glutamate uptake by brain cortical slices of adult mice. Moreover, the possible protective role of ebselen (20 mg/kg, subcutaneously) against MeHg effect was also examined. In addition, it was measured the glutathione peroxidase and catalase activities in mice brain. Our results demonstrated, for the first time, that in vivo exposure to MeHg causes a dose-dependent decrease in glutamate uptake and that ebselen, which did not affect the uptake per se, reverted this effect. MeHg decreased glutathione peroxidase activity and increased catalase activity, effects which were also prevented by ebselen. These results may indirectly indicate that: (i) the in vivo inhibitory effect of MeHg on glutamate uptake could be probably related to overproduction of H(2)O(2); (ii) the protective effect of ebselen on MeHg-induced inhibition of glutamate uptake could be related to its ability to detoxify H(2)O(2).     

N-methyl-D-aspartate exposure blocks glutamate toxicity in cultured cerebellar granule cells.

Exposure of cultured cerebellar granule cells to glutamate results in a concentration-dependent (EC50 = 22.7 +/- 0.4 microM) and delayed (24-72 hr) neurotoxicity, which is blocked by the specific N-methyl-D-aspartate (NMDA) receptor antagonists 2-amino-5-phosphovalerate and MK-801 but is unaffected by the non-NMDA receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione and 6,7-dinitroquinoxaline-2,3-dione. Although glutamate toxicity in these cells is mediated by the NMDA subtype of glutamate receptor, pretreatment of cerebellar granule cells with subtoxic concentrations of NMDA markedly antagonizes the neurotoxic actions of glutamate, with an IC50 of 55 +/- 4 microM. The neuroprotective effect of NMDA requires a preincubation time of approximately 120 min to be fully manifested and does not require the presence of NMDA during glutamate exposure. These data demonstrate that NMDA receptors mediate both neurotoxicity and neuroprotection in cerebellar granule cells. Among four glutamate receptor agonists tested (NMDA, quisqualate, ibotenate, and kainate), only NMDA was able to provide a robust neuroprotection against glutamate toxicity. Quisqualate was neither neurotoxic nor neuroprotective, whereas ibotenate, which was nontoxic by itself, induced a small degree of neuroprotection. In contrast, kainate, which was neurotoxic to cerebellar granule cells, also provided considerable neuroprotection against glutamate toxicity. Because preincubation of cerebellar granule cells with NMDA fails to alter NMDA receptor-mediated phosphoinositide hydrolysis or the specific binding of [3H]MK-801 to NMDA receptors, it appears that the neuroprotective effects of NMDA are not due to NMDA receptor desensitization.     

Role of cyclic GMP in glutamate neurotoxicity in primary cultures of cerebellar neurons

The role of cGMP in the mediation of glutamate neurotoxicity remains controversial. Some reports indicate that cGMP mediates glutamate neurotoxicity while others indicate that cGMP is neuroprotective. We have studied the role of cGMP in the mediation of glutamate and nitric oxide neurotoxicity in primary cultures of cerebellar neurons. Inhibition of soluble guanylate cyclase prevents glutamate and nitric oxide neurotoxicity. There is a good correlation between inhibition of cGMP formation and neuroprotection. Moreover 8-Br-cGMP, a cell permeable analog of cGMP, induced neuronal death. These results indicate that increased intracellular cGMP is involved in the mechanism of neurotoxicity. Inhibitors of phosphodiesterase did not increase intracellular cGMP but increased the content of cGMP in the extracellular medium and prevented glutamate neurotoxicity. Moreover, addition of cGMP to the extracellular medium also prevented glutamate neurotoxicity in cerebellar neurons in culture. These results are compatible with a neurotoxic effect of increased intracellular cGMP and a neuroprotective effect of increased extracellular cGMP.     

Protective effect of fangchinoline on cyanide-induced neurotoxicity in cultured rat cerebellar granule cells

The present study was performed to examine the effect of fangchinoline, a bis- benzylisoquinoline alkaloid, which exhibits the characteristics of a Ca2+ channel blocker, on cyanide-induced neurotoxicity using cultured rat cerebellar granule neurons. NaCN produced a concentration-dependent reduction of cell viability, which was blocked by MK-801, an N-methyl-D-aspartate (NMDA) receptor antagonist, verapamil, L-type Ca2+ channel blocker, and L-NAME, a nitric oxide synthase inhibitor. Pretreatment with fangchinoline over a concentration range of 0.1 to 10 microM significantly decreased the NaCN-induced neuronal cell death, glutamate release into medium, and elevation of [Ca2+]i and oxidants generation. These results suggest that fangchinoline may mitigate the harmful effects of cyanide-induced neuronal cell death by interfering with [Ca2+]i influx, due to its function as a Ca2+ channel blocker, and then by inhibiting glutamate release and oxidants generation.     

Treatment of hippocampal neurons with cyclosporin A results in calcium overload and apoptosis which are independent on NMDA receptor activation

Calcineurin is a ubiquitous calcium/calmodulin dependent protein phosphatase that has been shown to regulate the activity of ion channels, glutamate release, and synaptic plasticity. In the present study we show that CsA, a specific inhibitor of calcineurin, affects the survival of cultures developed from hippocampal dentate gyrus. Mixed neuronal-glial cultures exposed to 8 - 40 microM CsA undergo cell death characterized by apoptotic changes in cellular and nuclear morphology. TUNEL-positive staining was observed only in neurons that developed pyknotic morphology after treatment with 8 microM CsA for 24 - 72 h. Immunocytochemical staining with an anti-GFAP monoclonal antibody revealed that astrocytes from mixed neuronal/glial cultures were unaffected by exposure to CsA at doses toxic for neurons and all TUNEL-positive cells were neurons. MK-801, a noncompetitive inhibitor of glutamate receptor, does not inhibit the appearance of TUNEL-positive neurons and apoptotic changes in nuclear morphology. Preincubation of cells with 8 microM CsA increased basal intracellular calcium level in time dependent manner and decreased relative calcium response to glutamate. Application of 1 microM MK-801 had no effect on CsA-induced changes in Ca(2+) level. Our findings suggest that the neuronal death after CsA treatment is not a result of glutamate excitotoxicity and the increase in intracellular calcium concentration in neurons is not dependent on calcium influx via NMDA channel.     

How do rat cortical cells cultured with aluminum die: necrosis or apoptosis?

Aluminum (Al) exposure has been implicated as the cause of neural cells loss in several neurodegenerative diseases. Therefore, defining the mechanism of neural cell death in Al toxicity and degenerative diseases might lead to the development of therapeutic agents which promote neural cell survival. Furthermore, knowledge of cell death pathways might facilitate the discovery of treatments for neurodegeneration. However, the death mode of neural cells triggered by Al has not been firmly established. The present study focuses on understanding the pathway of cells death in cultured cortical cells treated with Al. Primary neurons cultured alone, astrocytes cultured alone, and neuron/astrocyte co-cultures obtained from newborn rats were incubated with Al at the concentrations of 0, 0.5, 1.0, or 2.0 mM for 72 h. Morphological changes were observed with an inverted phase microscope, a fluorescent microscope, and an electron microscope. Simultaneously, the rate of apoptosis was quantified with flow cytometry. Morphological characteristics of apoptosis such as cell shrinkage, aggregation and fragmentation of chromatin, membrane buds, and formation of membrane-bound apoptotic bodies were observed in Al-treated neurons, while none of these characteristics were found in Al-treated astrocytes. Quantitative results of apoptotic rates detected with flow cytometry indicated a typical apoptosis progression in neurons at various dosages. A concentration-dependent relationship between Al concentration and apoptotic rates confirmed that apoptosis is the prominent cause of cell death in primary cultured neurons, even at a concentration lower than 2 mM. Both necrosis and apoptosis are evident in neuron/astrocyte co-cultures, but the intensity of apoptosis is much less compared with that of neurons, suggesting that astrocytes may be especially important for neuronal survival in the presence of Al.     

5,7-Dihydroxitryptamine toxicity to serotonergic neurons in serum free raphe cultures

5,7-Dihydroxytryptamine (5,7-DHT), is an experimentally widely used selective serotonergic neurotoxin, though the mechanisms of toxicity remain to be fully elucidated. In the present study, we evaluated 5,7-dihydroxitryptamine (5,7-DHT) induced serotonergic neurotoxicity in foetal raphe serum free cultures from the rat. For this purpose, a model of foetal raphe serum free neuronal cultures from the rat was established, containing about 16% serotonergic neurons and studied up to 3 months. Two weeks old raphe cultures were exposed to the serotonergic neurotoxin 5,7-DHT (concentration range 10-100 microM) for 72 h, after which the medium was replaced and neurotoxicity was evaluated by immunocitochemistry 1 week later. Lactate dehydrogenase release into the medium, 72 h after exposure to 5,7-DHT, showed a concentration-dependent neurotoxicity. To access morphologically the serotonergic toxicity tryptophan hydroxylase (TPH) was used as a specific marker of these neurons. Immunocitochemistry using TPH antisera showed a concentration-dependent serotonergic neurotoxicity induced by 5,7-DHT. Serotonergic neurons showed the typical pattern of "pruning" accompanied by axon terminals and dendrites loss, which were either partial or total. The axotomy induced by the neurotoxin was morphologically characteristic of retrograde axonal degeneration. Fluoxetine (0.1 microM) pre-treatment reduced 5,7-DHT-induced serotonergic neurotoxicity. These results indicate that the mechanism by which 5,7-DHT-induces serotonergic neurotoxicity is, at least partially, dependent on the toxin uptake by the serotonin transporter. Finally, we have established a robust model of primary raphe neuronal culture to evaluate serotonergic neurons development and the mechanisms of toxicity involving this neuronal population.     

Development of a neurotoxicity test-system, using human post-mitotic, astrocytic and neuronal cell lines in co-culture.

Astrocytes are essential for neuronal function and survival, so both cell types were included in a human neurotoxicity test-system to assess the protective effects of astrocytes on neurons, compared with a culture of neurons alone. The human NT2.D1 cell line was differentiated to form either a co-culture of post-mitotic NT2.N neuronal (TUJ1, NF68 and NSE positive) and NT2.A astrocytic (GFAP positive) cells (approximately 2:1 NT2.A:NT2.N), or an NT2.N mono-culture. Cultures were exposed to human toxins, for 4h at sub-cytotoxic concentrations, in order to compare levels of compromised cell function and thus evidence of an astrocytic protective effect. Functional endpoints examined included assays for cellular energy (ATP) and glutathione (GSH) levels, generation of hydrogen peroxide (H(2)O(2)) and caspase-3 activation. Generally, the NT2.N/A co-culture was more resistant to toxicity, maintaining superior ATP and GSH levels and sustaining smaller significant increases in H(2)O(2) levels compared with neurons alone. However, the pure neuronal culture showed a significantly lower level of caspase activation. These data suggest that besides their support for neurons through maintenance of ATP and GSH and control of H(2)O(2) levels, following exposure to some substances, astrocytes may promote an apoptotic mode of cell death. Thus, it appears the use of astrocytes in an in vitro predictive neurotoxicity test-system may be more relevant to human CNS structure and function than neuronal cells alone.