l-α-aminoadipate causes astrocyte pathology with negative impact on mouse hippocampal synaptic plasticity and memory.

Increasing evidence shows that astrocytes, by releasing and uptaking neuroactive molecules, regulate synaptic plasticity, considered the neurophysiological basis of memory. This study investigated the impact of l-α-aminoadipate (l-AA) on astrocytes which sense and respond to stimuli at the synaptic level and modulate hippocampal long-term potentiation (LTP) and memory. l-AA selectivity toward astrocytes was proposed in the early 70's and further tested in different systems. Although it has been used for impairing the astrocytic function, its effects appear to be variable in different brain regions. To test the effects of l-AA in the hippocampus of male C57Bl/6 mice we performed two different treatments (ex vivo and in vivo) and took advantage of other compounds that were reported to affect astrocytes. l-AA superfusion did not affect the basal synaptic transmission but decreased LTP magnitude. Likewise, trifluoroacetate and dihydrokainate decreased LTP magnitude and occluded the effect of l-AA on synaptic plasticity, confirming l-AA selectivity. l-AA superfusion altered astrocyte morphology, increasing the length and complexity of their processes. In vivo, l-AA intracerebroventricular injection not only reduced the astrocytic markers but also LTP magnitude and impaired hippocampal-dependent memory in mice. Interestingly, d-serine administration recovered hippocampal LTP reduction triggered by l-AA (2 h exposure in hippocampal slices), whereas in mice injected with l-AA, the superfusion of d-serine did not fully rescue LTP magnitude. Overall, these data show that both l-AA treatments affect astrocytes differently, astrocytic activation or loss, with similar negative outcomes on hippocampal LTP, implying that opposite astrocytic adaptive alterations are equally detrimental for synaptic plasticity.

The lysine derivative aminoadipic acid, a biomarker of protein oxidation and diabetes-risk, induces production of reactive oxygen species and impairs trypsin secretion in mouse pancreatic acinar cells.

We have examined the effects of α-aminoadipic acid, an oxidized derivative from the amino acid lysine, on the physiology of mouse pancreatic acinar cells. Changes in intracellular free-Ca2+ concentration, the generation of reactive oxygen species, the levels of carbonyls and thiobarbituric-reactive substances, cellular metabolic activity and trypsin secretion were studied. Stimulation of mouse pancreatic cells with cholecystokinin (1 nM) evoked a transient increase in [Ca2+]i. In the presence of α-amoniadipic acid increases in [Ca2+]i were observed. In the presence of the compound, cholecystokinin induced a Ca2+ response that was smaller compared with that observed when cholecystokinin was applied alone. Stimulation of cells with cholecystokinin in the absence of Ca2+ in the extracellular medium abolished further mobilization of Ca2+ by α-aminoadipic acid. In addition, potential pro-oxidant conditions, reflected as increases in ROS generation, oxidation of proteins and lipids, were noted in the presence of α-aminoadipic acid. Finally, the compound impaired trypsin secretion induced by the secretagogue cholecystokinin. We conclude that the oxidized derivative from the amino acid lysine induces pro-oxidative conditions and the impairment of enzyme secretion in pancreatic acinar cells. α-aminoadipic acid thus creates a situation that could potentially lead to disorders in the physiology of the pancreas.

Inhibitors of the NMDA-Nitric Oxide Signaling Pathway Protect Against Neuronal Atrophy and Synapse Loss Provoked by l-alpha Aminoadipic Acid-treated Astrocytes.

The impact of treating astrocytes with the astrocytic toxin l-alpha amino adipic acid (L-AAA) on neuronal outgrowth, complexity and synapse formation was assessed, using a model of astrocyte-neuronal interaction. Treatment of rat primary cortical neurons with conditioned media (CM) derived from astrocytes treated with L-AAA reduced neuronal complexity and synapse formation. L-AAA provoked a reduction in the expression of glial fibrillary acid protein (GFAP) and a reduction in ATP-linked mitochondrial respiration in astrocytic cells. As the NMDA-R/PSD-95/NOS signaling pathway is implicated in regulating the structural plasticity of neurons, treatment of neuronal cultures with the neuronal nitric oxide synthase (nNOS) inhibitor 1-[2-(trifluoromethyl)phenyl] imidazole (TRIM) [100 nM] was assessed and observed to protect against L-AAA-treated astrocytic CM-induced reduction in neuronal complexity and synapse loss. Treatment with the NMDA-R antagonist ketamine protected against the CM-induced loss of synapse formation whereas the novel PSD-95/nNOS inhibitors 2-((1H-benzo[d] [1,2,3]triazol-5-ylamino) methyl)-4,6-dichlorophenol (IC87201) and 4-(3,5-dichloro-2-hydroxy-benzylamino)-2-hydroxybenzoic acid (ZL006) protected against synapse loss with partial protection against reduced neurite outgrowth. Furthermore, L-AAA delivery to the pre-limbic cortex (PLC) of mice was found to increase dendritic spine density and treatment with ZL006 reduced this effect. In summary, L-AAA-induced astrocyte impairment leads to a loss of neuronal complexity and synapse loss in vitro and increased dendritic spine density in vivo that may be reversed by inhibitors of the NMDA-R/PSD-95/NOS pathway. The results have implications for understanding astrocytic-neuronal interaction and the search for drug candidates that may provide therapeutic approaches for brain disorders associated with astrocytic histopathology.

Evidence supporting a role for astrocytes in the regulation of cognitive flexibility and neuronal oscillations through the Ca2+ binding protein S100ß.

The medial prefrontal cortex (mPFC) is important for cognitive flexibility, the ability to switch between two task-relevant dimensions. Changes in neuronal oscillations and alterations in the coupling across frequency ranges have been correlated with attention and cognitive flexibility. Here we show that astrocytes in the mPFC of adult male Sprague Dawley rats, participate in cognitive flexibility through the astrocyte-specific Ca2+ binding protein S100ß, which improves cognitive flexibility and increases phase amplitude coupling between theta and gamma oscillations. We further show that reduction of astrocyte number in the mPFC impairs cognitive flexibility and diminishes delta, alpha and gamma power. Conversely, chemogenetic activation of astrocytic intracellular Ca2+ signaling in the mPFC enhances cognitive flexibility, while inactivation of endogenous S100ß among chemogenetically activated astrocytes in the mPFC prevents this improvement. Collectively, our work suggests that astrocytes make important contributions to cognitive flexibility and that they do so by releasing a Ca2+ binding protein which in turn enhances coordinated neuronal oscillations.

Sex-Dependent Glial Signaling in Pathological Pain: Distinct Roles of Spinal Microglia and Astrocytes.

Increasing evidence suggests that spinal microglia regulate pathological pain in males. In this study, we investigated the effects of several microglial and astroglial modulators on inflammatory and neuropathic pain following intrathecal injection in male and female mice. These modulators were the microglial inhibitors minocycline and ZVEID (a caspase-6 inhibitor) and the astroglial inhibitors L-α-aminoadipate (L-AA, an astroglial toxin) and carbenoxolone (a connexin 43 inhibitor), as well as U0126 (an ERK kinase inhibitor) and D-JNKI-1 (a c-Jun N-terminal kinase inhibitor). We found that spinal administration of minocycline or ZVEID, or Caspase6 deletion, reduced formalin-induced inflammatory and nerve injury-induced neuropathic pain primarily in male mice. In contrast, intrathecal L-AA reduced neuropathic pain but not inflammatory pain in both sexes. Intrathecal U0126 and D-JNKI-1 reduced neuropathic pain in both sexes. Nerve injury caused spinal upregulation of the astroglial markers GFAP and Connexin 43 in both sexes. Collectively, our data confirmed male-dominant microglial signaling but also revealed sex-independent astroglial signaling in the spinal cord in inflammatory and neuropathic pain.

PEDF counteracts DL-α-aminoadipate toxicity and rescues gliotoxic damages in RPE-free chicken retinal explants.

Gliotoxic responses complicate human eye diseases, the causes of which often remain obscure. Here, we activated Müller cells (MCs) by the gliotoxin DL-α-aminoadipate (AAA) and assayed possible protective effects by pigment epithelium-derived factor (PEDF) in RPE-free retinal explants of the E6 chick embryo. These models are suited to analyze gliotoxic reactions in vitro, since the avian retina contains only Müller cells (MCs) as glial components, and the RPE-free explants are devoid of a major PEDF source. ChAT- and AChE-immunohistochemistry (IHC) revealed that AAA treatment disrupted the differentiation of cholinergic amacrine cells in the inner plexiform layer. At the applied concentration of 1 mM AAA, apoptosis of MCs was slightly increased, as shown by TUNEL and caspase-3 activity assays. Concomitantly, cell-free gaps emerged in the middle of the retina, where MCs were swollen and amassed glutamine synthetase (shown by GS and Vimentin IHC). AAA treatment strongly activated MCs, as shown by GFAP IHC, and by an increase of stress-related catalase activity. Remarkably, nearly all effects of AAA on MCs were effectively counter-balanced by 50 ng/ml PEDF co-treatment, as also shown by RT-PCR. These findings suggest that supplementation with PEDF can protect the retina against gliotoxic attacks. Further studies should establish whether PEDF similarly protects a gliotoxic human retina.

Antidepressant-like effect of the mGluR5 antagonist MTEP in an astroglial degeneration model of depression.

The glutamatergic predominance in the excitatory-inhibitory balance is postulated to be involved in the pathogenesis of depression. Such imbalance may be induced by astrocyte ablation which reduces glutamate uptake and increases glutamate level in the synaptic cleft. In the present study, we tried to ascertain whether astroglial degeneration in the prefrontal cortex could serve as an animal model of depression and whether inhibition of glutamatergic transmission by the mGluR5 antagonist MTEP could have antidepressant potential. Astrocytic toxins l-or dl-alpha-aminoadipic acid (AAA), 100µg/2µl, were microinjected, bilaterally into the rat medial prefrontal cortex (PFC) on the first and second day of experiment. MTEP (10mg/kg) or imipramine (30mg/kg) were administered on the fifth day. Following administration of MTEP or imipramine the forced swim test (FST) was performed for assessment of depressive-like behavior. The brains were taken out for analysis on day eight. The astrocytic marker, glial fibrillary acidic protein (GFAP) was quantified in PFC by Western blot method and by stereological counting of immunohistochemically stained sections. Both l-AAA and dl-AAA induced a significant increase in immobility time in the FST. This effect was reversed by imipramine, which indicates depressive-like effects of these toxins. A significant decrease in GFAP (about 50%) was found after l-AAA. Both the behavioral and GFAP level changes were prevented by MTEP injection. The obtained results indicate that the degeneration of astrocytes in the PFC by l-AAA may be a useful animal model of depression and suggest antidepressant potential of MTEP.

Gliotoxin-induced swelling of astrocytes hinders diffusion in brain extracellular space via formation of dead-space microdomains.

One of the hallmarks of numerous life-threatening and debilitating brain diseases is cellular swelling that negatively impacts extracellular space (ECS) structure. The ECS structure is determined by two macroscopic parameters, namely tortuosity (λ) and volume fraction (α). Tortuosity represents hindrance imposed on the diffusing molecules by the tissue in comparison with an obstacle-free medium. Volume fraction is the proportion of tissue volume occupied by the ECS. From a clinical perspective, it is essential to recognize which factors determine the ECS parameters and how these factors change in brain diseases. Previous studies demonstrated that dead-space (DS) microdomains increased λ during ischemia and hypotonic stress, as these pocket-like structures transiently trapped diffusing molecules. We hypothesize that astrocytes play a key role in the formation of DS microdomains because their thin processes have concave shapes that may elongate as astrocytes swell in these pathologies. Here we selectively swelled astrocytes in the somatosensory neocortex of rat brain slices with a gliotoxin DL-α-Aminoadipic Acid (DL-AA), and we quantified the ECS parameters using Integrative Optical Imaging (IOI) and Real-Time Iontophoretic (RTI) diffusion methods. We found that α decreased and λ increased during DL-AA application. During recovery, α was restored whereas λ remained elevated. Increase in λ during astrocytic swelling and recovery is consistent with the formation of DS microdomains. Our data attribute to the astrocytes an important role in determining the ECS parameters, and indicate that extracellular diffusion can be improved not only by reducing the swelling but also by disrupting the DS microdomains.

Astrocyte pathology in the prefrontal cortex impairs the cognitive function of rats.

Interest in astroglial cells is rising due to recent findings supporting dynamic neuron-astrocyte interactions. There is increasing evidence of astrocytic dysfunction in several brain disorders such as depression, schizophrenia or bipolar disorder; importantly these pathologies are characterized by the involvement of the prefrontal cortex and by significant cognitive impairments. Here, to model astrocyte pathology, we injected animals with the astrocyte specific toxin L-α-aminoadipate (L-AA) in the medial prefrontal cortex (mPFC); a behavioral and structural characterization two and six days after the injection was performed. Behavioral data shows that the astrocyte pathology in the mPFC affects the attentional set-shifting, the working memory and the reversal learning functions. Histological analysis of brain sections of the L-AA-injected animals revealed a pronounced loss of astrocytes in the targeted region. Interestingly, analysis of neurons in the lesion sites showed a progressive neuronal loss that was accompanied with dendritic atrophy in the surviving neurons. These results suggest that the L-AA-induced astrocytic loss in the mPFC triggers subsequent neuronal damage leading to cognitive impairment in tasks depending on the integrity of this brain region. These findings are of relevance to better understand the pathophysiological mechanisms underlying disorders that involve astrocytic loss/dysfunction in the PFC.

Aldehyde dehydrogenase 7A1 (ALDH7A1) attenuates reactive aldehyde and oxidative stress induced cytotoxicity.

Mammalian aldehyde dehydrogenase 7A1 (ALDH7A1) is homologous to plant ALDH7B1 which protects against various forms of stress such as increased salinity, dehydration and treatment with oxidants or pesticides. Deleterious mutations in human ALDH7A1 are responsible for pyridoxine-dependent and folinic acid-responsive seizures. In previous studies, we have shown that human ALDH7A1 protects against hyperosmotic stress presumably through the generation of betaine, an important cellular osmolyte, formed from betaine aldehyde. Hyperosmotic stress is coupled to an increase in oxidative stress and lipid peroxidation (LPO). In this study, cell viability assays revealed that stable expression of mitochondrial ALDH7A1 in Chinese hamster ovary (CHO) cells provides significant protection against treatment with the LPO-derived aldehydes hexanal and 4-hydroxy-2-nonenal (4HNE) implicating a protective function for the enzyme during oxidative stress. A significant increase in cell survival was also observed in CHO cells expressing either mitochondrial or cytosolic ALDH7A1 treated with increasing concentrations of hydrogen peroxide (H(2)O(2)) or 4HNE, providing further evidence for anti-oxidant activity. In vitro enzyme activity assays indicate that human ALDH7A1 is sensitive to oxidation and that efficiency can be at least partially restored by incubating recombinant protein with the thiol reducing agent ß-mercaptoethanol (BME). We also show that after reactivation with BME, recombinant ALDH7A1 is capable of metabolizing the reactive aldehyde 4HNE. In conclusion, ALDH7A1 mechanistically appears to provide cells protection through multiple pathways including the removal of toxic LPO-derived aldehydes in addition to osmolyte generation.

Submacular DL-alpha-aminoadipic acid eradicates primate photoreceptors but does not affect luteal pigment or the retinal vasculature.

PURPOSE: Macular telangiectasia type 2 (MT2) is a condition of uncertain etiology characterized by retinal vascular abnormalities, depletion of luteal pigment, and photoreceptor loss. To model this condition, the authors recently used a purportedly glial-selective toxin, DL-α-aminoadipic acid (DL-α-AAA), to test the effect of Müller cell disruption on the blood-retinal barrier in rats. In this study, they investigated macular changes after subretinal injection of DL-α-AAA in monkeys. METHODS: Various doses of DL-α-AAA were injected beneath the macula in eight monkey eyes. Eyes were examined by multifocal electroretinography (mfERG), optical coherence tomography (OCT), fundus autofluorescence, color photography, and fluorescein angiography. Five months after injection, eyes were examined by histology and immunohistochemistry for changes in photoreceptors and the retinal glia. In vitro studies evaluated the effect of DL-α-AAA on 661W cone photoreceptor viability. RESULTS: Subretinal injection of DL-α-AAA resulted in virtually complete ablation of photoreceptors in the injected area, as shown by OCT and histology, and severely impaired mfERG responses. Müller cells, albeit activated, survived the injury. Macular pigment remained unchanged in the central fovea. Subretinal injection of DL-α-AAA did not induce vascular leakage, though it increased the fundus autofluorescence. DL-α-AAA had a dose-dependent toxic effect on 661W photoreceptors. CONCLUSIONS: Submacular injection of DL-α-AAA induced severe damage to photoreceptors but failed to eliminate Müller cells in monkeys. Central macular pigment persisted despite loss of photoreceptors, and the retinal vasculature was unaffected. These observations may have significance in studying the roles of different cellular components in the pathogenesis of MT2.

Retinal vascular changes after glial disruption in rats.

Glial dysfunction is found in a number of retinal vascular diseases but its link with blood-retinal barrier (BRB) breakdown remains poorly understood. The present study tested the hypothesis that glial dysfunction is a major contributor to the BRB breakdown that is a hallmark of retinal vascular diseases. We investigated the specificity of the purportedly selective glial toxin, DL-alpha-aminoadipic acid (DL-alpha-AAA) on different types of ocular cells in vitro and then tested the effect of glial disruption on retinal vasculature after intraocular injection of DL-alpha-AAA or siRNA targeting glutamine synthetase (GS) in rats. DL-alpha-AAA was toxic to astrocytes and Müller cells but not to other types of BRB-related cells in vitro. Subretinal injection of DL-alpha-AAA disrupted retinal glial cells, induced vascular telangiectasis and increased vascular permeability from 4 days to over 2 months post-injection. Vascular changes induced by DL-alpha-AAA were observed predominantly in regions of glial disruption, as reflected by reduced expression of GS and increased expression of glial fibrillary acidic protein and vimentin. Confocal microscopy showed changes in all three layers of the retinal vasculature, which co-localised with areas of Müller cell disruption. Double labeling immunohistochemistry revealed that retinal glial disruption after DL-alpha-AAA injection was accompanied by increased expression of vascular endothelial growth factor and reduced expression of the tight junction protein claudin-5. Intravitreal injection of GS siRNA induced similar changes in Müller cells and BRB breakdown. Our data are consistent with the hypothesis that glial dysfunction is a primary contributor to the BRB breakdown in retinal vascular diseases.

Roles of glia limitans astrocytes and carbon monoxide in adenosine diphosphate-induced pial arteriolar dilation in newborn pigs.

BACKGROUND AND PURPOSE: Astrocytes, neurons, and microvessels together form a neurovascular unit allowing blood flow to match neuronal activity. Adenosine diphosphate (ADP) is an important signaling molecule in the brain, and dilation in response to ADP is astrocyte-dependent in rats and newborn pigs. Carbon monoxide (CO), produced endogenously by catabolism of heme to CO, iron, and biliverdin via heme oxygenase, is an important cell-signaling molecule in the neonatal cerebral circulation. We hypothesize ADP stimulates CO production by glia limitans astrocytes and that this CO causes pial arteriolar dilation. METHODS: Experiments were performed using anesthetized piglet with closed cranial windows, and freshly isolated piglet astrocytes and microvessels. Astrocyte injury was caused by topical application of L-2-alpha aminoadipic acid (2 mmol/L, 5 hours). Cerebrospinal fluid was collected from under the cranial windows for measurement of ADP-stimulated CO production. CO was measured by gas chromatography-mass spectroscopy analysis. RESULTS: Before, but not after, astrocyte injury in vivo, topical ADP stimulated both CO production and dilation of pial arterioles. Astrocyte injury did not block dilation to isoproterenol or bradykinin. Chromium mesoporphyrin, an inhibitor of heme oxygenase, also prevented the ADP-induced increase in cerebrospinal fluid CO and pial arteriolar dilation caused by ADP, but not dilation to sodium nitroprusside. ADP also increased CO production by freshly isolated piglet astrocytes and cerebral microvessels, although the increase was smaller in the microvessels. CONCLUSIONS: These data suggest that glia limitans astrocytes use CO as a gasotransmitter to cause pial arteriolar dilation in response to ADP.

Spatiotemporal characteristics of astroglial death in the rat hippocampo-entorhinal complex following pilocarpine-induced status epilepticus.

Recently we reported that astroglial loss and subsequent gliogenesis in the dentate gyrus play a role in epileptogenesis following pilocarpine-induced status epilepticus (SE). In the present study we investigated whether astroglial damages in the hippocampo-entorhinal complex following SE are relevant to pathological or electrophysiological properties of temporal lobe epilepsy. Astroglial loss/damage was observed in the entorhinal cortex and the CA1 region at 4 weeks and 8 weeks after SE, respectively. These astroglial responses in the hippocampo-entorhinal cortex were accompanied by hyperexcitability of the CA1 region (impairment of paired-pulse inhibition and increase in excitability ratio). Unlike the dentate gyrus and the entorhinal cortex, CA1 astroglial damage was protected by conventional anti-epileptic drugs. alpha-Aminoadipic acid (a specific astroglial toxin) infusion into the entorhinal cortex induced astroglial damage and changed the electrophysiological properties in the CA1 region. Astroglial regeneration in the dentate gyrus and the stratum oriens of the CA1 region was found to originate from gliogenesis, while that in the entorhinal cortex and stratum radiatum of the CA1 region originated from in situ proliferation. These findings suggest that regional specific astroglial death/regeneration patterns may play an important role in the pathogenesis of temporal lobe epilepsy.

Glial loss in the prefrontal cortex is sufficient to induce depressive-like behaviors.

BACKGROUND: Postmortem studies have repeatedly found decreased density and number of glia in cortical regions, including the prefrontal and cingulate areas, from depressed patients. However, it is unclear whether this glial loss plays a direct role in the expression of depressive symptoms. METHODS: To address this question, we characterized the effects of pharmacologic glial ablation in the prefrontal cortex (PFC) of adult rats on behavioral tests known to be affected by stress or antidepressant treatments: sucrose preference test (SPT), novelty suppressed feeding test (NSFT), forced swim test (FST), and two-way active avoidance test (AAT). We established the dose and time course for the actions of an astrocyte specific toxin, L-alpha-aminoadipic acid (L-AAA), and compared the behavioral effects of this gliotoxin with the effects of an excitotoxic (ibotenate) lesion and to the effects of chronic stress. RESULTS: The results demonstrate that L-AAA infusions induced anhedonia in SPT, anxiety in NSFT, and helplessness in FST and AAT. These effects of L-AAA were similar to chronic unpredictable stress (CUS)-induced depressive-like behaviors in these tests. However, ibotenate-induced neurotoxic lesion of the PFC had no effect in these behavioral tests. CONCLUSIONS: The results demonstrate that glial ablation in the PFC is sufficient to induce depressive-like behaviors similar to chronic stress and support the hypothesis that loss of glia contributes to the core symptoms of depression.

Glial-toxin-mediated disruption of spinal cord locomotor network function and its modulation by 5-HT.

While it is established that glial cells actively influence neuronal and synaptic properties, the functional effects of glial-neuronal interactions are still not well understood. To address the role of glia at the network level we have examined the effects of the specific gliotoxin L-aminoadipic acid on the locomotor network output and cellular and synaptic properties in the lamprey spinal cord. The gliotoxic effect of aminoadipic acid was associated with a specific depolarization of glial cells. Aminoadipic acid depolarized the membrane potential of spinal cord neurons, suggesting a functional link between glia and neurons. The depolarization was significantly reduced by glutamate receptor antagonists in adults, but by gap junction blockers in larvae, suggesting a developmental difference in glial-neuronal interactions. Aminoadipic acid also reduced the amplitude of monosynaptic excitatory postsynaptic potentials (EPSPs), an effect that was not associated with changes in the presynaptic release probability or postsynaptic response to glutamate. These cellular and synaptic effects of aminoadipic acid were associated with disruption of the locomotor network output. This could not be accounted for by changes in glutamate uptake or potassium buffering by glia, suggesting a direct role for glia in the network. Interestingly, we found that the aminoadipic acid-evoked disruption of network activity and reduction of monosynaptic EPSP amplitudes did not occur in the presence of the endogenous spinal modulator 5-HT. These results thus provide evidence for an active functional role for glial cells in spinal cord locomotor networks, and suggest a potential glial modulatory effect of 5-HT.

Might astrocytes play a role in maintaining the seizure-prone state?

The amygdala-kindling model is used to study complex partial epilepsy with secondary generalization. The present study was designed to (A) quantify astrocytic changes in the piriform cortex of amygdala-kindled subjects over time and (B) investigate the role that astrocytes might play in maintaining the seizure-prone state. In Study A, once the experimental subjects reached five stage 5 seizures, stimulation was stopped, and both kindled and control rats were allowed to survive for the interval appropriate to their group (7, 18, 30, or 90 days). Following each interval, the kindled and control animals were given 10 intraperitoneal injections of bromodeoxyuridine (BrdU) and sacrificed 24 h following the last injection. Significantly higher numbers of dividing astrocytes (identified by co-labeling for BrdU and to one of the astrocytic intermediate filament proteins glial fibrillary acidic protein or vimentin) were found in the kindled brains. All kindled groups had significantly higher numbers of double-labeled cells on the side contralateral to the stimulation site, except for those in the 90 day survival group. In Study B, rats were implanted with chemotrodes, were kindled as in Study A, and were subsequently infused with either saline or with L alpha-AA (to lesion astrocytes) during a further 25 stimulations (1/day). L alpha-AA infused rats had significantly diminished levels of behavioral seizures, higher after discharge thresholds, lower after discharge durations, and decreased numbers of double-labeled astrocytes in piriform cortex than did saline infused rats. Together, the data indicate that astrocytes may play a role in maintaining the seizure-prone state.

The glio-toxic mechanism of alpha-aminoadipic acid on cultured astrocytes.

The mechanism of action of the glutamate analogue alpha-aminoadipic (AAA) acid was investigated in terms of its toxicity to cultured astrocytes. AAA was more toxic to type 1 astrocytes than type 2 astrocytes. Also the higher toxicity of the L-isomer as compared to the D-isomer was seen on type 1 astrocytes but not type 2. The toxicity of AAA can be reduced by co-culture of type 1 astrocytes with microglia. This inhibition may be due to glutamate release by microglia. No such effect is seen for type 2 astrocytes. The major uptake route for AAA by type 1 astrocytes is through the sodium dependent glutamate port. Both isomers of AAA are toxic to dividing astrocytes. The D-isomer appears to be toxic only for mitotic cells. The mechanism of this toxicity is protein synthesis dependent. It is suggested that AAA is toxic to mitotic astrocytes by interference with protein synthesis needed for cell division. D-AAA as opposed to L-AAA may prove a valuable tool for investigation of astrocyte proliferation in development and disease.

Müller cell involvement in methanol-induced retinal toxicity.

Methanol is an ocular toxicant which causes visual dysfunction often leading to blindness after acute exposure. The physiological and biochemical changes responsible for this toxicity are poorly understood. Previously, we reported that the folate-reduced (FR) rat is an animal model which mimics the characteristic human methanol toxicities. The present study examines the hypothesis that depletion of ATP after methanol administration is the initiating event in methanol-induced retinal toxicity. ATP is reduced in retinae of methanol-treated FR rats to the same extent as is seen in retinae of FR and folate-sufficient (FS) rats treated with the Müller cell (retinal glial cell) toxin alpha-aminoadipic acid. Changes in the electroretinogram and the response of Müller cells to a potassium stimulus are also similarly eliminated in methanol-treated FR rats and alpha-aminoadipic acid-treated FR and FS rats. These results suggest that the Müller cell may be the initial target in methanol-induced visual system toxicity.

Cystine/glutamate antiporter expression in retinal Müller glial cells: implications for DL-alpha-aminoadipate toxicity.

A cytotoxicity of glutamate or related amino acids (10 mM) mediated by a cystine/glutamate antiporter (system Xc) has recently been demonstrated in N18 neuroblastoma-rat retina hybrid (N18RE105) cells and C6 glioma cells. The antiporter usually transports glutamate outside and cystine inside, thereby maintaining cellular concentrations of glutathione. High concentrations of glutamate inhibit cystine uptake and lead to depletion of cellular levels of glutathione. Among related amino acids, DL-alpha-aminoadipic acid (DL-alpha-AAA), which is well known as a selective gliotoxin in the retina, is also toxic to these cells. However, this does not explain why DL-alpha-AAA acts gliospecifically on the retina. To answer this question we first examined the effects of DL-alpha-AAA on the [35S]cystine uptake with parental N18 neuroblastoma cells and rat retina of the hybrid cells. DL-alpha-AAA showed a competitive inhibition of [35S]cystine uptake in the rat retina but not in the N18 cells. Such a competitive inhibition of cystine uptake by DL-alpha-AAA could also be seen in the carp retina. The cystine uptake with carp retina was mainly Na(+)-independent and Cl(-)-dependent as already described as a characteristic ion dependency of the Xc antiporter. We next examined the effects of exogenous cystine on the glutamate release from the retina. Cystine (1 mM) actually induced a glutamate release approximately twice that of the control. Furthermore, the glutamate release induced by cystine was also Na(+)-independent and Cl(-)-dependent, and was blocked by DL-alpha-AAA. An autoradiogram of [35S]cystine uptake in the carp retina showed typical radial glial Müller cells. A large incorporation of [35S]cystine into retinal glutathione fraction was detected by a high pressure liquid chromatography method during a 1-4-h incubation. A significant or large decrease of retinal levels of glutathione was observed one day ater an intravitreal injection of 8 mumol DL-alpha-AAA or L-alpha-AAA, respectively. Buthionine sulfoximine (2.5 mumol), a specific inhibitor of glutathione synthesis, induced a large decrease of retinal levels of glutathione and a loss of electroretinographic b-wave 20-30 h after treatment. Taken together, our present data with rat and carp retinas strongly indicate that the expression of cystine/glutamate antiporter is enriched in the retina, particularly in the glial Müller cells which have a rapid turnover pool for glutathione. The gliotoxin DL-alpha-AAA inhibits cystine uptake through this antiporter on the glial cells and elicits reduction of cellular levels of glutathione.(ABSTRACT TRUNCATED AT 400 WORDS)