Effect of acute exposure to ammonia on glutamate transport in glial cells isolated from the salamander retina

A rise of brain ammonia level, as occurs in liver failure, initially increases glutamate accumulation in neurons and glial cells. We investigated the effect of acute exposure to ammonia on glutamate transporter currents in whole cell clamped glial cells from the salamander retina. Ammonia potentiated the current evoked by a saturating concentration of L-glutamate, and decreased the apparent affinity of the transporter for glutamate. The potentiation had a Michaelis-Menten dependence on ammonia concentration, with a K(m) of 1.4 mM and a maximum potentiation of 31%. Ammonia also potentiated the transporter current produced by D-aspartate. Potentiation of the glutamate transport current was seen even with glutamine synthetase inhibited, so ammonia does not act by speeding glutamine synthesis, contrary to a suggestion in the literature. The potentiation was unchanged in the absence of Cl(-) ions, showing that it is not an effect on the anion current gated by the glutamate transporter. Ammonium ions were unable to substitute for Na+ in driving glutamate transport. Although they can partially substitute for K+ at the cation counter-transport site of the transporter, their occupancy of these sites would produce a potentiation of < 1%. Ammonium, and the weak bases methylamine and trimethylamine, increased the intracellular pH by similar amounts, and intracellular alkalinization is known to increase glutamate uptake. Methylamine and trimethylamine potentiated the uptake current by the amount expected from the known pH dependence of uptake, but ammonia gave a potentiation that was larger than could be explained by the pH change, and some potentiation of uptake by ammonia was still seen when the internal pH was 8.8, at which pH further alkalinization does not increase uptake. These data suggest that ammonia speeds glutamate uptake both by increasing cytoplasmic pH and by a separate effect on the glutamate transporter. Approximately two-thirds of the speeding is due to the pH change.     

Immunohistochemical demonstration of glutamate dehydrogenase in astrocytes

A rabbit antiserum was raised against glutamate dehydrogenase (GDH) and applied immunohistochemically to rat brain. GDH-immunoreactive grains were observed mainly in small cells throughout the brain. In most of these cells, coexistence of GDH and glial fibrillary acidic protein was shown by an immunofluorescence method. The results suggest that GDH is primarily an astrocytic enzyme. Possible roles of astrocytic GDH in glutamate and ammonia metabolism are discussed.     

Presence of glial cells in the rat pineal gland: a light and electron microscopic immunohistochemical study

Immunoperoxidase methods for the demonstration of three glial antigens, vimentin, glial fibrillary acidic protein, and S-100 protein, were applied to routine-fixed paraffin sections of rat pineal gland. A pre-embedding electron microscope immunoperoxidase method was also used to study the ultrastructural localization of S-100 protein in pineal cells. Light and electron microscopic results showed the presence of these antigenic glial markers in the second pineal cell type. The term glial cell is proposed for the second of parenchymatous cell in rat pineal gland.     

Modes of protein and peptide uptake in the pineal gland of the Mongolian gerbil: An ultrastructural study

The possible existence of either a blood-brain barrier or a CSF-brain barrier was examined in the pineal gland of the Mongolian gerbil using the ultrastructural tracers, horseradish peroxidase (HRP) and microperoxidase (MP). The mechanism of protein and peptide transport within the pineal gland and its possible relationship to pineal concretions was also considered. Gerbils were injected with either MP or HRP intravenously (IV), or they received intraventricular (IVT) injections of HRP. The IV injections resulted in both MP and HRP movement through the endothelial cells of the gland by vesicular transport and by diffusion through the endothelial intercellular junctions. Following the IVT injections, the tracer was demonstrated in the subarachnoid space as well as in the glial cells associated with the periphery of the gland. In addition, after the IVT injections, rounded enlargements of the intercellular space that resembled canaliculi were filled with reaction product. In both cases (IVT and IV), the reaction product was localized to the perivascular space, to the glial cells and pinealocytes, and to the intercellular spaces. More importantly, there was specific localization of the tracers in the vacuolated pinealocytes and in the pineal concretions. The results of this study demonstrate several significant findings: (1) neither a blood-brain barrier nor a CSF-brain barrier exists in the pineal gland of the gerbil, (2) localization of the tracers in pineal concretions indicates a relationship between these structures and protein and peptide storage within the gland, and (3) the presence of the tracers within canaliculi after the IVT injections suggests a possible mode of secretion of pineal substances into the CSF.     

Influence of glial cells in the dopamine releasing effect resulting from the stimulation of striatal δ-opioid receptors

Recent data indicate that striatal dopamine release induced by stimulation of δ-opioid receptors is a consequence of glutamate release. However, glial cells, which mainly support glutamate uptake and are involved in glutamate signaling and potentially express δ-opioid receptors, could participate to this effect. The present study investigates the contribution of glial cells in the releasing effects of [d-Pen2, d-Pen5]-enkephalin (DPDPE) by using the gliotoxin l-α-aminoadipate (l-αAA). Initially, we evaluated the early influence of l-αAA local infusion (10 μg/μL) on dialysate levels of glutamate and dopamine under basal or DPDPE treatment conditions. l-αAA produced a significant increase of glutamate and dopamine in dialysates (+76% and +50% respectively) and the concomitant infusion of DPDPE (10 μM) significantly enhanced this effect in an additive manner (+110% and +44% respectively). Secondly, we assessed the DPDPE effects on striatal glutamate and dopamine dialysate levels, 2 days after an intra-striatal injection of l-αAA which produced destruction of glial cells. This lesion, decreasing the basal glutamate dialysate level as well as its tissue content (by 55% and 36% respectively), prevented the increase in glutamate and dopamine extracellular levels induced by DPDPE. This result confirmed that the DPDPE-induced dopamine release requires an initial glutamate release. However, this effect could reflect a major disruption of glutamatergic transmission caused by the toxin, as suggested by the local infusion of glutamine (2.5 mM) which, in lesioned rats, prevented the decrease in the basal extracellular content of glutamate and restored the DPDPE-induced increase in glutamate and dopamine dialysate levels. Therefore, these results indicate that, although glial cells are essential to maintain functional glutamatergic neurotransmission, they are not directly involved in the process by which stimulation of striatal δ-opioid receptors induces extracellular glutamate release and, consecutively, dopamine release.     

A histochemical study of aldehyde fuchsin-positive material and “high-esterase cells” in the pineal gland of the Mongolian gerbil

The pineal gland of the Mongolian gerbil consists of a superficial gland, stalk and deep pineal. The deep pineal differentiates postnatally. Histochemical studies of the superficial pineal gland indicate that it may be involved in the secretion of protein. Presumptive secretory material visualized by aldehyde fuchsin (AF) and chrome hematoxylin was observed along the course of blood vessels and among the pinealocytes. The distribution and texture of the AF-positive material was distinctive. It did not correspond to the pattern and texture of material stained with PAS, Sudan Black or acid orcein. Staining with AF was markedly reduced after incubation with trypsin, indicating that the AF-positive material is at least partially protein. The amount of stainable material increased with age. The AF-positive material was observed in what appeared to be interstitial or glial cells and processes, and in the processes of perivascular cells. Cells and fibrous processes with high non-specific esterase activity (“highesterase cells”) were observed among the pinealocytes and along the course of blood vessels. The distribution of the “high-esterase cells” and the morphology and texture of their esterase-containing processes were remarkably similar to the morphology and distribution of the material that stained with AF. It may be that the “high-esterase cells” contain AF-positive material. The “high-esterase cells” hydrolyzed both alpha-naphthyl acetate and alpha-naphthyl butyrate. The pinealocytes hydrolyzed only alpha-naphthyl acetate. The “high-esterase cells” appear to form a distinct class of cells within the superficial pineal gland. They are tentatively identified as a type of glial cell.     

成年牦牛松果体的光镜和电镜观察

目的 探讨成年牦牛松果体形态结构特征,为哺乳动物松果体形态结构研究积累资料.方法 光镜HE染色、浸银染色和透射电子显微镜技术.结果 光镜下,牦牛松果体由松果体细胞、少量的神经胶质细胞、毛细血管和神经等组成.电镜下,松果体细胞电子致密度低,细胞质内含丰富的线粒体、粗面内质网、滑面内质网、微管、微丝和核糖体;高尔基复合体数量极少,典型异质细胞器突触带呈球形,多位于质膜附近.神经胶质细胞的细胞质内含丰富的线粒体,其胞体突起呈球形膨大伸入到松果体细胞之间.松果体细胞以及神经胶质细胞间均存在突触和连接复合体.牦牛松果体内毛细血管为连续型,其远腹侧血管周围可见色素细胞.结论 成年牦牛松果体细胞内存在神经上皮样和腺上皮样2种细胞连接方式.毛细血管为连续型.松果体细胞内细胞器发达,但很少观察到高尔基复合体.     

Junctional systems in the pineal gland of the Wistar rat (Ratus ratus). A freeze-fracture and thin section study

Intercellular junctions between neighbouring pinealocytes, glial cells, glial cells and pinealocytes as well as between nerve endings and parenchymal cells of the pineal gland of Wistar rats were investigated on freeze-fracture replicas and thin sections by transmission electron microscopy. Gap junctions, tight junctions and the annular gap junctions have been revealed. In addition, chemical synapses between nerve endings and pinealocytes have been observed.     

Two components of the pineal organ in the mink (Mustela vison): their structural similarity to submammalian pineal complexes and calcification.

The pineal complex in the mink (Mustela vison) consists of a larger ventral and a smaller dorsal pineal. Both organs contain pinealocytes, neurons, glial cells, nerve fibers and synapses in an organization characteristic of nervous tissue. The cellular elements are arranged circularly around strait lumina. These lumina correspond to the photoreceptor spaces of submammalian pineals. A 9 + 0-type cilium marks the receptory pole of the pinealocytes which may form an inner-segment-like dendrite terminal in the pineal lumina. The cilia correspond to outer segments which form photoreceptor membrane multiplications in the pineal of submammalians and in certain insectivorous and mustelid mammals (bat, hedgehog, ferret). Axonal processes of the pinealocytes contain synaptic ribbons and terminate on intrapineal neurons of both organs. This pattern represents a neural efferentation of the pineal nervous tissue. The axonal processes of pinealocytes also form neurohormonal endings which pierce the perivascular limiting glial membrane in the ventral as well as in the dorsal pineal. The upper pineal ("epipineal") of the mink may correspond to the parapineal, frontal, or parietal organs of submammalian pineal complexes. Both pineals are encapsulated by the meningeal tissue of the brain stem. Afferent vasomotor axons of the meninges innervate smooth muscle cells of pineal arterioles. There are corpora arenacea in the pineal arachnoid and in the pineal nervous tissue, primarily in the ventral pineal. The localization of calcium ions detected around the membrane of pineal cells by pyroantimonate cytochemistry suggests membrane activity as the source of the calcium ions. The accumulation of calcium by the pinealocytes may be due to their neurosensory character. The mink is the first animal described to have both intrapineal and meningeal concrements like the human pineal.     

Ultrastructural characterization of glial cells in the rat pineal gland with special reference to the pineal stalk

In the present study the “interstitial” cells of the superficial pineal gland and the nonparenchymal cells of the pineal stalk in Sprague-Dawley rats were examined ultrastructurally with the aim of defining the cells more closely. The “interstitial” cells of the superficial pineal gland do not represent a homogeneous cell population. The most abundant cell type is the mononuclear phagocyte, most easily recognized by its dark appearance and its content of primary and conspicuous secondary lysosomes. Astrocytes can be distinguished by the typical appearance of their nuclei (i.e., a thin continuous rim of heterochromatin adjacent to the nuclear membrane), identical to that of astrocytes in the CNS. Depending on the absence or presence of glial filaments and their amount, a spectrum of astrocytic cells is present. Mature astrocytes with filaments throughout their cytoplasm are rare. Immature glial cells with few or no filaments predominate. In the vicinity of blood vessels pericytes are present. In view of the fact that the “interstitial” cells could generally be identified it is suggested to abandon the term interstitial for the cells in question. In the pineal stalk mature astrocytes predominate; they have some features in common with pinealocytes, i.e., the presence of intergrade endoplasmic reticulum and grumose bodies (lysosomes). Other unusual features are a relative abundance of coated pits and vesicles. Oligodendrocytes are restricted to the proximal part of the stalk, near the deep pineal, where myelinated axons are abundant. More distally a few Schwann cells were seen.     

Peripheral autonomic nerves of human pineal organ terminate on vessels, their supposed role in the periodic secretion of pineal melatonin

Nonvisual pineal and retinal photoreceptors are synchronizing circadian and circannual periodicity to the environmental light periods in the function of various organs. Melatonin of the pineal organ is secreted at night and represents an important factor of this periodic regulation. Night illumination suppressing melatonin secretion may result in pathological events like breast and colorectal cancer. Experimental works demonstrated the role of autonomic nerves in the pineal melatonin secretion. It was supposed that mammalian pineals have lost their photoreceptor capacity that is present in submammalians, and sympathetic fibers would mediate light information from the retina to regulate melatonin secretion. Retinal afferentation may reach the organ by central nerve fibers via the pineal habenulae as well. In our earlier works we have found that the pineal organ developing from lobular evaginations of the epithalamus differs from peripheral endocrine glands and is composed of a retina-like central nervous tissue that is comprised of cone-like pinealocytes, secondary pineal neurons and glial cells. Their autonomic nerves in submammalians as well as in mammalian animals do not terminate on pineal cells, rather, they run in the meningeal septa among pineal lobules and form vasomotor nerve endings. Concerning the adult human pineal there are no detailed fine structural data about the termination of autonomic fibers, therefore, in the present work we investigated the ultrastructure of the human pineal peripheral autonomic nerve fibers. It was found, that similarly to other parts of the brain, autonomic nerves do not enter the human pineal nervous tissue itself but separated by glial limiting membranes take their course in the meningeal septa of the organ and terminate on vessels by vasomotor endings. We suppose that these autonomic vasomotor nerves serve the regulation of the pineal blood supply according to the circadian and circannual changes of the metabolic activity of the organ and support by this effect the secretion of pineal neurohormones including melatonin.     

Differential immunocytochemical localization of calretinin in the pineal gland of three mammalian species

Summary Calcium plays an important role for signal transduction in the mammalian pineal organ. The regulation of the intracellular concentration of free calcium probably involves calcium-binding proteins of the calmodulin superfamily. In the present study, we have investigated the expression of calretinin, one member of this superfamily, in the pineal organ of hamsters, gerbils and guinea-pigs by means of immunochemical and immunocytochemical analyses with a calretinin-specific antiserum. In immunoblots this antibody recognized a single protein band of 29 kDa in the brain and pineal organ of all three mammalian species. Immunocytochemical investigations of serial semithin sections of plastic-embedded pineals revealed the constant occurrence of variable numbers of calretinin-positive cells throughout all glands. In order to identify the immunopositive cells precisely, adjacent sections were exposed to antibodies against various marker proteins of pineal cell types, i.e., synaptophysin, neuron-specific enolase, protein gene product 9.5, S-antigen, vimentin and S-100. By this approach, calretinin could be localized to vimentin-positive cells in the gerbil which are generally considered as interstitial glial cells. Likewise, calretinin-positive cells in the guinea-pig probably correspond to interstitial cells, taking into account their morphology and the lack of calretinin immunoreactivity in pinealocytes. The unusual expression of calretinin in astrocyte-like cells further supports the notion that pineal glial cells are endowed with peculiar properties. In contrast to gerbil and guinea-pig, a subpopulation of pinealocytes displayed calretinin immunoreactivity in the hamster. This finding adds to the hypothesis that in pinealocytes of some species calretinin plays a role in calcium-mediated signal transduction which eventually is linked to melatonin synthesis. Our results demonstrate that calretinin is a regular constituent of pineal glands in three mammalian species, but that its cellular localisation shows interspecific variation. This variation suggests that the protein is involved in diverse calcium-mediated functions in the mammalian pineal gland.     

Prolonged exposure to ammonia increases extracellular glutamate in cultured rat astrocytes

Abnormal alteration of brain function is a characteristic complication of hepatic encephalopathy in both acute and chronic liver failure. Previous studies suggest that the pathogenesis of hepatic encephalopathy involves chronic glial edema with subsequent alteration of glioneuronal communication, N-methyl-d-aspartate (NMDA) receptor activation, and oxidative/nitrosative stress. In the present study, we investigated extracellular glutamate levels in cultured astrocytes under prolonged exposure to ammonia. Using an enzyme-linked high-performance liquid chromatography assay to detect glutamate, prolonged (48 h) exposure of cultured astrocytes to ammonia resulted in a concentration- and time-dependent increase in extracellular glutamate. Similar increases were observed when ammonia-containing medium (pH 7.8) was adjusted to the pH of control medium (pH 7.4), indicating that the effect is not due to pH. Treatment of astrocytes with an antioxidant (l-ascorbic acid), an NADPH oxidase inhibitor (apocynin), a Ca²⁺ chelator (BAPTA-AM), an NMDA receptor antagonist (NK801), or a mitochondrial permeability transition inhibitor (cyclosporine A) suppressed the increase of extracellular glutamate in response to prolonged ammonia exposure. Prolonged exposure to ammonia increased extracellular glutamate through the NMDA receptor, increased intracellular Ca²⁺ levels, and upregulation of excitatory amino acids. The addition of ATP further increased extracellular glutamate levels in astrocytes subjected to prolonged ammonia treatment (5 mM, 48 h) in a dose-dependent manner. These results indicate that the deregulation of glutamate release from astrocytes may contribute to the dysfunction of glutamatergic neurons in patients with acute liver failure (ALF).     

Topographical relationships of synaptic ribbons in the pineal system of the vole (Microtus agrestis)

Summary In the pineal system of the vole (Microtus agrestis) both the superficial and the deep pineal exhibit a high percentage of synaptic ribbons lying in intimate contact with the cell membrane of the pinealocytes. At the sites of contact, densities resembling the presynaptic dense projections of synapses are arranged between the ribbons and the cell membrane. Opposite the sites of contact various elements were found. The quantitative estimation revealed that in the superficial pineal about 40% and in the deep pineal about 60% of the membrane-contacting ribbons are located opposite glial cells: in both organ parts about 18% of the membrane-contacting ribbons were found opposite adjacent pinealocytes. The location of ribbons at the perivascular space was almost exclusively found in the superficial pineal, while the cerebrospinal fluid-contacting area in the deep pineal exhibited ribbons in intimate contact with the lumen of the third ventricle. The heterogeneity of the topographical relationships would seem to indicate a diffuse functional effect of the synaptic ribbons in the mammalian pineal gland.     

Cytologic features of the normal pineal gland on squash preparations

As primary pineal lesions are extremely rare, many surgical pathologists are unfamiliar with normal pineal cytologic features. We describe cytologic features of the normal pineal gland in patients of varying ages and identify common diagnostic pitfalls. We performed a retrospective review of pineal gland biopsies performed at our institution, where approximately 30,000 surgical specimens are accessioned yearly, for the last 23 years. Only two pineal gland biopsies were found. Although both cases were initially diagnosed as low‐grade gliomas on frozen section, the final diagnosis was benign pineal tissue based on light microscopy and immunohistochemistry results. Additionally, we performed squash preparations of five normal pineal gland autopsy specimens with Papanicolaou and Diff–Quik® (Dade Behring, Newark, DE) stains. Infant preparations were highly cellular smears composed of numerous, uniform, single cells with indistinct cytoplasm, small round‐to‐oval nuclei, fine chromatin, and absent nucleoli and calcifications. The vague microfollicular pattern mimicked a pineocytoma and the fine fibrillary background mimicked a glial neoplasm. Young adult smears were similar; however, microcalcifications were present with fewer background single cells. Older patients had much less cellular smears composed of small clusters of cells with fusiform‐to‐spindle nuclei, a fine chromatin pattern, and indistinct cytoplasmic borders. There were fewer background single cells and more microcalcifications. The cytologic features of the native pineal gland vary with age. Normal pineal tissue can be confused with a pineocytoma or low‐grade glioma. Familiarity with normal pineal gland cytological features will help to avoid a potential misdiagnosis. Diagn. Cytopathol. 2014;42:939–943. © 2014 Wiley Periodicals, Inc.     

Distribution of glutamate transporter 1 mRNA in the central nervous system of the pigeon (Columba livia)

Glutamate transporter 1 (GLT1) in glial cells removes glutamate that diffuses from the synaptic cleft into the extracellular space. Previously, we have shown the distribution of glutamatergic neurons in the central nervous system (CNS) of the pigeon. In the present study, we identified cDNA sequence of the pigeon GLT1, and mapped the distribution of the mRNA-expressing cells in CNS to examine whether GLT1 is associated with glutamatergic terminal areas. The cDNA sequence of the pigeon GLT1 consisted of 1889 bp nucleotides and the amino acids showed 97% and 87% identity to the chicken and human GLT1, respectively. In situ hybridization autoradiograms revealed GLT1 mRNA expression in glial cells and produced regional differences of GLT1 mRNA distribution in CNS. GLT1 mRNA was expressed preferentially in the pallium than the subpallium. Moderate expression was seen in the hyperpallium, Field L, mesopallium, and hippocampal formation. In the thalamus, moderate expression was found in the ovoidal nucleus, rotundal nucleus, triangular nucleus, and lateral spiriform nucleus, while the dorsal thalamic nuclei were weak. In the brainstem, the isthmic nuclei, optic tectum, vestibular nuclei, and cochlear nuclei expressed moderately, but the cerebellar cortex showed strong expression. Bergmann glial cells expressed GLT1 mRNA very strongly. The results indicate that cDNA sequence of the pigeon GLT1 is comparable with that of the mammalian GLT1, and a large number of GLT1 mRNA-expressing areas correspond with areas where AMPA-type glutamate receptors are located. Avian GLT1 in glial cells probably maintain microenvironment of glutamate concentration around synapses as in mammalian GLT1.     

Near-ultraviolet light perceived by the retina generates the signal suppressing melatonin synthesis in the chick pineal gland-an involvement of NMDA glutamate receptors

Exposure of dark-adapted chicks to near ultraviolet (UV-A) light significantly decreased melatonin (MEL) content and the activity of serotonin N-acetyltransferase (AA-NAT; the penultimate and key regulatory enzyme in MEL production) in the pineal glands. Significant reduction in MEL level and AA-NAT activity was also found in pineals of animals whose heads were covered with black opaque tape, an observation suggesting that in the chicken UV-A light perceived by the eyes alone is capable of affecting MEL synthesis in the pineal gland. Covering the chick's eyes, in addition to the head, totally blocked the studied UV-A action. Although SCH 23390 (a selective D1-dopamine receptor antagonist), injected directly into both eyes at a dose of 10 nmol/eye, prevented the decline in pineal AA-NAT activity produced by retinal illumination with white light, the drug did not modify the UV-A light-evoked decrease in the enzyme activity. MK-801 (a selective antagonist of NMDA glutamate receptors; 1 nmol/eye) abolished the suppressive action of UV-A light on pineal AA-NAT activity, but it was inactive in the case of white light. Intraocularly injected sulpiride and CNQX (selective antagonists of D2-dopamine and AMPA/kainite glutamate receptors, respectively) had no effect on the actions of both UV-A and white light (acting on the eyes only) on pineal AA-NAT activity. It is concluded that in the chick retinally perceived UV-A light generates a signal which suppresses MEL production in the pineal gland. At the level of the retina, such signal does not involve dopamine, but is dependent on the stimulation of NMDA glutamate receptors.     

Ultrastructural study of the human pineal gland in aged patients including a centenarian

An ultrastructural study of human pineal glands obtained at autopsy from 7 patients older than 70 years was conducted in order to clarify the functional anatomy of the pineal in the aged. By light microscopy, the pineal glands from aged patients were parenchymatous and almost indistinguishable from those of younger controls. Electron microscopy of the pineal parenchymal cells revealed deep nuclear indentations, synaptic ribbons and ribbon fields, Golgi apparatus, lipofuscin granules and microtubular sheaves in all subjects, cilia with a 9 + 0 pattern in a few, and lamellated structures suggestive of the outer segment of photoreceptor cells very rarely. Microtubules were numerous in the cytoplasmic processes and bulbous endings. Fibrous astrocytes located between the pinealocytes showed long and thin cytoplasmic processes containing numerous glial filaments. Two types of nerve bouton were present in the pineal parenchyma, one of which contained clear vesicles forming synapse-like contacts with pinealocytes. There were no significant age-related changes in these features in a qualitative comparison with pineal glands from 5 adult patients younger than 70 years. These findings indicate that even in advanced age, the human pineal gland maintains some functions, such as intercellular communication and photoreception, in common with the pineal in lower vertebrates.     

Depolarization of cultured astrocytes by glutamate and aspartate

The excitatory transmitter substances glutamate and aspartate are known to have a depolarizing action on cultured CNS neurones, the depolarization being associated with an increase in membrane conductance. When the effects of these amino acids (at a concentration of 10^?4 M) were studied on the membrane potential and resistance of cultured glial cells, they also caused a depolarization of many astrocytes but without producing significant changes in membrane resistance. The majority of glial cells depolarized by glutamate and aspartate were lying in the vicinity of neurones in the dense zone of the cultures, whereas isolated astrocytes in the outgrowth zone were usually not affected by the amino acids. 4-Aminopyridine (5 mM), a substance known to block K⁺-conductance in various excitable membranes, reversibly reduced or abolished the depolarization caused by glutamate and aspartate on glial cells, but had no or only a small effect on the depolarization of neurones caused by these amino acids.These results suggest that the depolarization of glial cells by glutamate and aspartate is caused by an increase in the concentration of extracellular K⁺ which is released from neighbouring neurones during their activation by the amino acids.     

Alpha-aminoadipic acid blocks the Na+-dependent glutamate transport into acutely isolated Müller glial cells from guinea pig retina

The effect of the glial toxin α-aminoadipic acid (AAA) upon theNa⁺/glutamate cotransporter of acutely isolated guinea pig retinal glial cells was studied using the whole-cell voltage-clamp technique. Glutamate evoked an in ward current in these cells at negative holding potentials dependent on the presence of extracellular Na⁺ and intracellular K⁺. A reversal potential could not be found for the current. L-trans-Pyrrolidine-2.4-dicarboxylic acid (PDC), a blocker of Na⁺-dependent glutamate uptake, diminished the glutamate current also in our cells. Application of L-AAA also generated an inward current at negative holding potentials, without a reversal potential, being suppressed if extracellularNa⁺ or intracellular K⁺ was removed. The glutamate uptake blocker, PDC (200 μM), blocked the L-AAA (1 mM) current. Thus, L-AAA proved to be transported by the Na⁺/glutamate transporter of Müller cells. Hence, glutamate currents were diminished by L-AAA competitively with a K_m of 499 μM at a glutamate concentration of 10 μM. The Na⁺/glutamate uptake was less sensitive to DL- and D-AAA block. It is suggested that the blocking effect of AAA on Na⁺-dependent glutamate uptake into glial cells might be involved in the well known glia toxicity of this compound.