Brain Research, 540 (1991) 297-301
297
Elsevier BRES 24509
Turtle cortical neurons survive glutamate exposures that are lethal to mammalian neurons Andrea M. Wilson and Arnold R. Kriegstein 1 Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA 94305 (U.S.A.) (Accepted 23 October 1990)
Key words: Glutamate neurotoxicity; Excitotoxicity; Anoxia; Hypoxia; Reptile; Glutamate receptor Glutamate is an excitatory neurotransmitter in turtle and mammalian cortex. In high concentrations it is toxic to mammalian neurons and is an important mediator in the pathway that leads to neuronal death from anoxia. Turtle neurons are remarkably resistant to anoxic injury and we sought to determine whether part of this resistance could be attributed to the sensitivity of turtle neurons to glutamate toxicity. Embryonic turtle cortical neurons were grown for 25 days in dissociated cell culture using a modification of a method developed for murine cortical cell culture. Turtle neurons in dissociated culture were found to express glutamate receptors which include both N-methyl-o-aspartate (NMDA) and non-NMDA receptor types. Remarkably, these neurons survive 5 minute exposures to glutamate in concentrations up to 3 mM, doses 30 times the LDs0 and 6 times the LD100for mouse cortical neurons ~2. Elucidating the mechanism for this resistance may suggest new strategies for brain protection.
It has long been known that mammalian cortex is exquisitely sensitive to anoxia. Recently, glutamate has been identified as a mediator in the pathway that leads to neuronal death. This process, called glutamate neurotoxicity, is postulated to occur by the excessive or inappropriate release of glutamate which generates a lethally high extracellular concentration of glutamate 11"15"27"31'32. Consistent with this hypothesized mechanism, glutamate has also been proposed to mediate cell death that is triggered by a variety of other causes including hypoglycemia, trauma, and several neurodegenerative disorders17.19,21,22,27.28.29,34. Diving turtles, unlike mammals, have evolved adaptations that enable them to survive long periods of anoxia 2. These adaptations may include the ability of turtle brain tissue to utilize anaerobic metabolism 3'4'25'26'3°, to modulate its metabolic rate 8"18"33, or to selectively preserve ion transport ~4. Since glutamate functions as a transmitter in turtle cortex, it may participate in the pathophysiology of anoxic brain damage in turtles. However, we found that turtle neurons are not killed by brief exposures to glutamate at least 30 times the LDso for mouse cortical neurons 12. This is consistent with the hypothesis that turtles resist anoxia, at least in part, because turtle neurons resist glutamate toxicity. Unravelling the mechanism of resistance to glutamate injury could provide insights that may lead tO new strategies for brain protection. In these experiments dissociated neurons were used in
order to control precisely the extracellular concentration of glutamate and the duration of glutamate exposure, variables which are difficult to manipulate in cortical slices. In addition, this experimental system was designed to conform to the system used by Choi 9 and others in order to permit direct comparison between results from turtle and mouse cortical neurons. Cultures of dissociated turtle cortical neurons (Pseudemys scripta) were prepared by a modification of the technique of Dichter 13. Cerebral cortex from embryos at stage 20 or 21 (staged according to the morphologic criteria of Y n t e m a 35) were dissected with sterile technique to yield cortical fragments isolated from the septum medially and the dorsal ventricular ridge laterally. Cortical tissue from approximately 40 embryos was pooled, minced, incubated in 0.08% acetylated trypsin for 50-75 min at 35 °C, and dissociated by trituration. The single-cell suspension was plated on Primaria multi-well plates (1.5 x 107/mm) in plating medium, consisting of Eagle's minimal essential medium (MEM) supplemented with heat-inactivated horse serum (10%), fetal bovine serum (10%), glutamine (2 mM, added the week of use), glucose (21 mM), and bicarbonate (26 mM) yielding a final osmolarity of 290-310 mOsm. Cultures were maintained at 30 °C in a humidified 5% CO2 atmosphere. After 48 h the plating medium was replaced with growth medium (plating medium lacking fetal bovine serum). Every 3 days; the medium was replaced with fresh growth medium. Growth medium
Correspondence: A.R. Kriegstein, Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA 94305, U.S.A. 0006-8993/91/$1/3.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
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Fig. 1. Cultured neurons exhibit spontaneous synaptic activity and the presence of glutamate receptors. A: spontaneous synaptic activity recorded from a turtle cortical neuron after 25 days in culture. The cell was voltage-clamped to the potentials indicated by the whole-cell voltage clamp technique. Spontaneous excitatory and inhibitory synaptic currents are visible and distinguishable by their different reversal potentials. B: the response to focally applied glutamate (1 raM), demonstrating both the fast, non-NMDA receptor mediated current and the slow NMDA-mediated current. Thus EAA receptors are present in the membranes of the cultured cells.
and plating medium contained only the glutamate present in the serum; based on the supplier's measurements of serum glutamate levels, the initial glutamate concentration in growth medium was less than 20/tM. Cells were cultured until they exhibited large, round bodies with phase-bright cell membranes characteristic of mature neurons; this typically required 25-35 days. Culture material was very difficult to obtain for two reasons. First, turtle embryos were only available 3 months of the year, during the egg-laying season. Second, it was necessary to pool the cortices of many embryos in order to achieve a reasonable density of neurons and this increased the rate of contamination of the cultures. Morphologically mature neurons were examined for spontaneous and evoked activity. Whole cell voltage clamp recordings 16 were obtained from mature cultured neurons (n = 4) using methods described previously for turtle cortical neurons in vitro 6, and both spontaneous and evoked currents were monitored. As shown in Fig. 1, with the cell held at -70 mV (near the resting potential) frequent spontaneous inward currents were seen corresponding to spontaneous postsynaptic currents (PSCs). W h e n the membrane was depolarized, for example to -30 mV as shown in Fig. 1A, both inward and outward currents could be resolved. The inward going events had the reversal potentials characteristic of cation mediated currents and correspond to spontaneous excitatory PSCs, while the outward going events became
inward at -50 mV (the chloride equilibrium potential based on the chloride concentration inside the patch electrode) and correspond to inhibitory PSCs mediated by chloride s . Thus spontaneous and evoked neuronal activity was present in morphologically mature dissociated neurons. Cultured mouse cortical neurons have a period of glutamate insensitivity that resolves with the development of mature neuronal features (by 14 days in culture) v, and turtle neurons in culture may have a similar period of glutamate insensitivity. In order to demonstrate the presence of glutamate receptors, cultured neurons were tested for their responsiveness to glutamate. Exogenously applied glutamate produced a large long-lasting inward current as shown in Fig. IB for a cell held at -70 mV. The glutamate-induced current was comparable in size to that seen with dissociated mouse neurons in culture a4. When applied to turtle neurons in cortical slices, glutamate activates both a fast current due to the activation of non-N-methyl-D-aspartate ( n o n - N M D A ) receptors and a slower, N M D A - m e d i a t e d current 5. In the present experiments, glutamate applied to neurons produced a current that had both features. In addition, the slower, presumed N M D A mediated current was accompanied by a significant increase in the background current fluctuations that correspond to the rapid opening and closing of glutamate activated channels. Fluctuation analysis I performed on the slow current produced a calculated mean channel open time of 3.6 milliseconds, in close agreement with the value previously obtained for the open time of the N M D A channel in turtle cortical neurons 7. Dissociated turtle cortical neurons in culture therefore express excitatory amino acid ( E A A ) receptors that respond to focally applied glutamate and demonstrate a current consistent with the presence of both N M D A and n o n - N M D A receptors. Neurons that had been in culture for at least 25 days were then tested for sensitivity to glutamate induced injury. To expose the neurons to a brief pulse of glutamate, cells were washed twice in a control solution of (in raM): NaC1 120, KCi 5.4, MgCl 2 0.8, CaCI 2 1.8, glucose 15, and H E P E S 20, at room temperature in room air. A n additional exchange was done with either control solution containing glutamate or control solution alone. After a 5 min exposure, the solution was removed and control solution was exchanged an additional 5 times to wash out all glutamate (effective dilution > 600-fold). Cultures were then placed in a 35 °C, 5% CO2 atmosphere incubator for an additional 24 h. Cultures were exposed to a 5 min pulse of glutamate to permit direct comparison with results obtained in mammalian cortical neurons. Under conditions very similar to those above, the
299 TABLE I Turtle cortical neuron survival after exposure to glutamate
There is only a small difference between percentage of neurons surviving exposure to control solution or to 3 mM glutamate. The number (and percent) of intact neurons from representative microscopicfields was assayed by cellular morphologyor trypan blue exclusion prior to, 5 rain after, and 24 h after exposure to control solution or 3 mM glutamate solution (n = 2 for each condition). The mean and standard deviation (SD) of the percent of neurons surviving in control and glutamate-exposed cultures is included. The difference between 0 and 3 mM glutamate exposure in percent surviving neurons is not statistically significant after 5 minutes (P > 0.1, two tail t test), or after 24 h (0.1 > P > 0.05, two-tail t test). Condition
Pre-exposure
5 min post
24 h post
Control
51 (100%) 59 (100%)
51 (100%) 57 (97%)
50 (98%) 57 (97%)
98.5 + 2.1
97.5 + 0.7
61 (95%) 32 (91%)
55 (86%) 26 (74%)
93.0 + 2.8
80.0 + 8.5
Mean % + S.D. 3 mM Glu Mean % ___S.D.
64 (100%) 35 (100%)
glutamate LDs0 for dissociated mouse cortical neurons is 50-100 /~M 12, so l~reliminary experiments were performed using test solutions containing glutamate concentrations of 10/~M, 100/aM, 300/tM, 500/~M and 1000/~M. However, visual examination revealed that turtle neurons were intact 24 h after 5 min exposure to even the highest concentration, demonstrating a relative resistance to glutamate neurotoxicity. Therefore, in subsequent experiments concentrations of 0 and 3 mM glutamate were used. In order to quantitate the effect of 3 mM glutamate on neuronal survival, culture dishes were examined under phase-contrast microscopy and a representative series of microscopic fields were photographed and marked prior to exposure to glutamate or control solution. Marked fields were photographed again 5 min after exposure and 24 h later. Cultures were then incubated in 0.4% trypan blue, a vital exclusion dye, for 5 min at room temperature and re-photographed. Quantitative assessment of cell death was made by comparison of the photographs of the marked fields; specific neurons present on the baseline photographs but either absent or labeled with Trypan blue in the 5 min and 24 h photographs were scored as dead cells. The percentage of surviving neurons was calculated for 4 culture wells and is shown in Table I. Five min after exposure to 0 or 3 mM glutamate, approximately 98% and 93% of the neurons were alive, respectively. This result is not statistically significant (P > 0.1, two-tail t-test) and suggests that turtle neurons are not killed
instantaneously by brief exposure to glutamate. However, a small proportion of neurons eventually succumbed. Twenty-four h after exposure to control solution approximately 98% of the neurons were alive. When 3 mM glutamate was used 80% of the neurons were alive after 24 h. This result is also not significant (0.1 > P > 0.05, two-tail t-test), but suggests that glutamate has some effect on turtle neurons. Larger numbers of cells might die with modulation of variables in this experimental system, for example by increasing the duration of exposure. The modest degree of excess cell death observed here differs substantially from results with murine neurons where fewer than 10% of cortical neurons survive 24 h after exposure to 3 mM glutamate under identical conditions including temperature 23. Fig. 2 presents examples of representative microscopic fields from one culture exposed to control solution and one to 3 mM glutamate as they appeared prior to, immediately after, and 24 h after control or glutamate exposure. Examination of the microscopic fields did reveal an effect of glutamate exposure: Neurons in the test but not the control solution appeared swollen and granular immediately after exposure. (Fig. 2, compare A2 and B2) However, the majority of neurons in the glutamate exposed culture regained a normal appearance after 24 h (Fig. 2, compare B1 and B3). Mammalian neurons also exhibit cytoplasmic swelling and increased granularity acutely after suffering glutamate neurotoxicity 1°'12"2°. In addition they typically progress to cellular disintegration l°'a2'z°, a fate not suffered by the majority of turtle neurons. In summary, dissociated turtle neurons tested after the development of mature features (25 days) demonstrated characteristic glutamate-induced excitatory currents. However, the majority of cultured neurons were protected from the toxic effects of short exposure to high concentrations of glutamate. In light of the excitatory action of glutamate on turtle neurons it is remarkable that only 20% of cultured dissociated neurons suffered glutamate toxicity at doses 6 times higher than the mammalian LD10o12. As mentioned above, a number of metabolic mechanisms have been postulated to account for the resistance of turtles to anoxia. However, it seems plausible that several adaptations have evolved to mediate this remarkable resiliency, and resistance to brief exposures to glutamate as demonstrated by these experiments, might play an important role in protecting the turtle from anoxic damage. The in vitro turtle cortex can be used to explore the cellular mechanism by which turtle neurons evade glutamate-induced injury and anoxic damage. Elucidation of this mechanism may suggest therapeutic interventions for human neurological diseases that involve glutamate neurotoxicity.
300
Fig. 2. Glutamate induces acute changes in neuronal appearance but does not lead to widespread degeneration. Representative microscopic fields from cultures exposed to control solution (A) or 3 mM glutamate solution (B). Fields were photographed prior to exposure (A1, BI). immediately after a 5 min exposure (A2, B2), and 24 h later (A3, B3). Note swollen and granular appearance of cells immediately after exposure to glutamate (B2) and restoration of normal appearance 24 h later (B3).
We would like to thank Dennis Choi and Hanna Monyer for assistance with the glutamate protocols, and Mark Blanton who was instrumental in applying the whole cell voltage clamp recording
technique and performed fluctuation analysis. This work was supported by NS 12151 and NS 21223.
1 Anderson, C.R. and Stevens, C.F., Voltage clamp analysis of acetylcholine produced end-plate current fluctuations at frog neuromuscular junction, J. Physiol., 235 (1973) 655-691. 2 Belkin, D.A., Anoxia: tolerance in reptiles, Science, 139 (1962) 492-493. 3 Bennett, A.F. and Dawson, W.R., In C. Gans, W.R. Dawson (Eds.), Biology of Reptilia, Vol. 5, Physiology, Academic Press, New York, 1976, pp. 127-223. 4 Berkson, H., Physiological adjustments to prolonged diving in the Pacific green turtle (Chelonia mydas agassizzi), Comp. Biochem. Physiol., 18 (1966) 101-119. 5 Blanton, M.G., Development of Amino Acid Neurotransmitter Function in Embryonic Cerebral Cortex, Doctoral dissertation, 1990. 6 Blanton, M.G., Lo Turco, J.J. and Kriegstein, A.R., Whole cell recording from neurons in slices of reptilian and mammalian cerebral cortex, J. Neurosci. Methods, 30 (1989) 203-210. 7 Blanton, M.G., Lo Turco, J.J. and Kriegstein, A.R., Endogenous neurotransmitter activates N-methyl-o-aspartate (NMDA) receptors on differentiating neurons in embryonic cortex, Proc. Natl. Acad. Sci. U.S.A., 87 (1990) 8027-8032. 8 Brooks, S.P.J. and Storey, K.B., Anoxic brain function: molecular mechanisms of metabolic depression, FEBS Lett., 232 (1988) 214-216. 9 Choi, D.W., Glutamate neurotoxicity in cortical cell culture is calcium dependent, Neurosci. Lett., 58 (1985) 293-297. 10 Choi, D.W., Ionic dependence of glutamate neurotoxicity, J. Neurosci., 7 (1987) 369-379. 11 Choi, D.W., Koh, J.-y. and Peters, S., Pharmacology of glutamate neurotoxicity in cortical cell culture: attenuation by NMDA antagonists, J. Neurosci., 8 (1988) 185-196. 12 Choi, D.W.. Maulucci-Gedde, M. and Kriegstein, A.R., Glu-
tamate neurotoxicity in cortical cell culture, J. Neurosci., 7 (1987) 357-368. 13 Dichter, M.A., Rat cortical neurons in cell culture: Culture methods, cell morphology, electrophysiology, and synapse formation, Brain Research, 149 (1978) 279-293. 14 Feng, Z.-C., Rosenthal, M. and Sick, T.J., Suppression of evoked potentials with continued ion transport during anoxia in turtle brain, Am. J. Physiol., 255 (1988) R478-R484. 15 Goldberg, M.P., Weiss, J.H., Pham P-C. and Choi, D.W., N-methyl-D-aspartate receptors mediate hypoxic neuronal injury in cortical culture, J. Pharmacol. Exp. Ther., 243 (19871 784-791. 16 Hamill, O.P., Marty, A., Neher, E., Sakman, B. and Sigworth, F.J., Improved patch-clamp techniques for high-resolution current recordings from cells and cell-free membrane patches, Pfliigers Arch. Eur. J. Physiol., 391 (1981) 85-100. 17 Ingvar, M., Morgan, P.E and Auer, R.N., The nature and timing of excitotoxic neuronal necrosis in the cerebral cortex, hippocampus and thalamus due to flurothyl-induced status epilepticus, Acta Neuropathol., 75 (1988) 362-3691 18 Jackson, D.C., Metabolic depression and oxygen depletion in the diving turtle, J. Appl. Physiol., 24 (1968) 503-509. 19 Korf, J., Gransbergen, J.-B.P., Prenen, G.H.M. and Go, K.G., Cation shifts and excitotoxins in Alzheimer and Huntington disease and experimental brain damage, Prog. Brain Res., 70 (1986) 213-226. 2{) Lehmann, J., Ferkany, J.W., Schaeffer, P. and Coyle, J.T., Dissociation between the excitatory and 'excitotoxic' effects of quinolinic acid analogs on the striatal cholinergic interneuron, J. Pharmacol. Exp. Ther., 232 (1985) 873-882. 21 Martin, J.B. and Gusella, J.F., Huntington's disease: Pathogencsis and management, N. Engl. J. Med., 315 (1986) 1267-1276.
301 22 Monyer, H. and Choi, D.W., Morphinans attenuate cortical neuronal injury induced by glucose deprivation in vitro, Brain Research, 446 (1988) 144-148. 23 Monyer, H. and Choi, D.W., Personal communication. 24 Patneau, D.K. and Mayer, M.L., Structure-activity relationships for amino acid transmitter candidates acting at N-methylD-aspartate and quisqualate receptors, J. Neurosci., 10 (1990) 2385-2399. 25 Robin, E.D., Lewiston, N., Newman, A., Simon, L.M. and Theodore, J., Bioenergetic pattern of turtle brain and resistance to profound loss of mitochondrial ATP generation, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 3922-3926. 26 Robin, E.D., Vester, J.W., Murdaugh, H.V., and Millen, J.E., Prolonged anaerobiosis in a vertebrate: anaerobic metabolism in the freshwater turtle, J. Cell. Comp. Physiol., 63 (1964) 287-297. 27 Rothman, S.M. and Olney, J.W., Glutamate and the pathophysiology of hypoxic-ischemic brain damage, Ann. Neurol., 19 (1986) 105-111. 28 Rothman, S.M. and Olney, J.W., Excitotoxicity and the NMDA receptor, Trends Neurosci., 10 (1987) 299-302. 29 Sanchez-Prieto, J. and Gonzalez, P., Occurrence of a large
30 31 32 33
34 35
Ca2+-independent release of glutamate during anoxia in isolated nerve terminals (synaptosomes), J. Neurochem., 50 (1988) 1322-1324. Sick, T.J., Rosenthal, M., LaManna, J.C. and Lutz, P.L., Brain potassium ion homeostasis, anoxia, and metabolic inhibition in turtles and rats, Am. J. Physiol., 243 (1982) R281-R288. Siesjo, B.K. and Wieloch, T., Cerebral metabolism in ischaemia: Neurochemical basis for therapy, Br. J. Anaesth., 57 (1985) 47-62. Silverstein, F.S., Buchanan, K. and Johnston, M.V., Perinatal hypoxia-ischemia disrupts striatal high-affinity [3H] glutamate uptake into synaptosomes, J. Neurochem., 47 (1986) 1614-1619. Suarez, R.K., Doll, C.J., Buie, A.E., West, T.G., Funk, G.D. and Hochachka, P.W., Turtles and rats: a biochemical comparison of anoxia-tolerant and anoxia-sensitive brains, Am. J. Physiol., 257 (1989) R1083-R1088. Tecoma, E.S., Monyer, H., Goldberg, M.P. and Choi, D.W., Traumatic neuronal injury in vitro is attenuated by NMDA antagonists, Neuron, 2 (1989) 1541-1545. Yntema, C.L., A series of stages in the embryonic development of Chelydra serpentina, J. Morphol., 125 (1968) 219-252.
297
Elsevier BRES 24509
Turtle cortical neurons survive glutamate exposures that are lethal to mammalian neurons Andrea M. Wilson and Arnold R. Kriegstein 1 Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA 94305 (U.S.A.) (Accepted 23 October 1990)
Key words: Glutamate neurotoxicity; Excitotoxicity; Anoxia; Hypoxia; Reptile; Glutamate receptor Glutamate is an excitatory neurotransmitter in turtle and mammalian cortex. In high concentrations it is toxic to mammalian neurons and is an important mediator in the pathway that leads to neuronal death from anoxia. Turtle neurons are remarkably resistant to anoxic injury and we sought to determine whether part of this resistance could be attributed to the sensitivity of turtle neurons to glutamate toxicity. Embryonic turtle cortical neurons were grown for 25 days in dissociated cell culture using a modification of a method developed for murine cortical cell culture. Turtle neurons in dissociated culture were found to express glutamate receptors which include both N-methyl-o-aspartate (NMDA) and non-NMDA receptor types. Remarkably, these neurons survive 5 minute exposures to glutamate in concentrations up to 3 mM, doses 30 times the LDs0 and 6 times the LD100for mouse cortical neurons ~2. Elucidating the mechanism for this resistance may suggest new strategies for brain protection.
It has long been known that mammalian cortex is exquisitely sensitive to anoxia. Recently, glutamate has been identified as a mediator in the pathway that leads to neuronal death. This process, called glutamate neurotoxicity, is postulated to occur by the excessive or inappropriate release of glutamate which generates a lethally high extracellular concentration of glutamate 11"15"27"31'32. Consistent with this hypothesized mechanism, glutamate has also been proposed to mediate cell death that is triggered by a variety of other causes including hypoglycemia, trauma, and several neurodegenerative disorders17.19,21,22,27.28.29,34. Diving turtles, unlike mammals, have evolved adaptations that enable them to survive long periods of anoxia 2. These adaptations may include the ability of turtle brain tissue to utilize anaerobic metabolism 3'4'25'26'3°, to modulate its metabolic rate 8"18"33, or to selectively preserve ion transport ~4. Since glutamate functions as a transmitter in turtle cortex, it may participate in the pathophysiology of anoxic brain damage in turtles. However, we found that turtle neurons are not killed by brief exposures to glutamate at least 30 times the LDso for mouse cortical neurons 12. This is consistent with the hypothesis that turtles resist anoxia, at least in part, because turtle neurons resist glutamate toxicity. Unravelling the mechanism of resistance to glutamate injury could provide insights that may lead tO new strategies for brain protection. In these experiments dissociated neurons were used in
order to control precisely the extracellular concentration of glutamate and the duration of glutamate exposure, variables which are difficult to manipulate in cortical slices. In addition, this experimental system was designed to conform to the system used by Choi 9 and others in order to permit direct comparison between results from turtle and mouse cortical neurons. Cultures of dissociated turtle cortical neurons (Pseudemys scripta) were prepared by a modification of the technique of Dichter 13. Cerebral cortex from embryos at stage 20 or 21 (staged according to the morphologic criteria of Y n t e m a 35) were dissected with sterile technique to yield cortical fragments isolated from the septum medially and the dorsal ventricular ridge laterally. Cortical tissue from approximately 40 embryos was pooled, minced, incubated in 0.08% acetylated trypsin for 50-75 min at 35 °C, and dissociated by trituration. The single-cell suspension was plated on Primaria multi-well plates (1.5 x 107/mm) in plating medium, consisting of Eagle's minimal essential medium (MEM) supplemented with heat-inactivated horse serum (10%), fetal bovine serum (10%), glutamine (2 mM, added the week of use), glucose (21 mM), and bicarbonate (26 mM) yielding a final osmolarity of 290-310 mOsm. Cultures were maintained at 30 °C in a humidified 5% CO2 atmosphere. After 48 h the plating medium was replaced with growth medium (plating medium lacking fetal bovine serum). Every 3 days; the medium was replaced with fresh growth medium. Growth medium
Correspondence: A.R. Kriegstein, Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA 94305, U.S.A. 0006-8993/91/$1/3.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
298 A 30
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Fig. 1. Cultured neurons exhibit spontaneous synaptic activity and the presence of glutamate receptors. A: spontaneous synaptic activity recorded from a turtle cortical neuron after 25 days in culture. The cell was voltage-clamped to the potentials indicated by the whole-cell voltage clamp technique. Spontaneous excitatory and inhibitory synaptic currents are visible and distinguishable by their different reversal potentials. B: the response to focally applied glutamate (1 raM), demonstrating both the fast, non-NMDA receptor mediated current and the slow NMDA-mediated current. Thus EAA receptors are present in the membranes of the cultured cells.
and plating medium contained only the glutamate present in the serum; based on the supplier's measurements of serum glutamate levels, the initial glutamate concentration in growth medium was less than 20/tM. Cells were cultured until they exhibited large, round bodies with phase-bright cell membranes characteristic of mature neurons; this typically required 25-35 days. Culture material was very difficult to obtain for two reasons. First, turtle embryos were only available 3 months of the year, during the egg-laying season. Second, it was necessary to pool the cortices of many embryos in order to achieve a reasonable density of neurons and this increased the rate of contamination of the cultures. Morphologically mature neurons were examined for spontaneous and evoked activity. Whole cell voltage clamp recordings 16 were obtained from mature cultured neurons (n = 4) using methods described previously for turtle cortical neurons in vitro 6, and both spontaneous and evoked currents were monitored. As shown in Fig. 1, with the cell held at -70 mV (near the resting potential) frequent spontaneous inward currents were seen corresponding to spontaneous postsynaptic currents (PSCs). W h e n the membrane was depolarized, for example to -30 mV as shown in Fig. 1A, both inward and outward currents could be resolved. The inward going events had the reversal potentials characteristic of cation mediated currents and correspond to spontaneous excitatory PSCs, while the outward going events became
inward at -50 mV (the chloride equilibrium potential based on the chloride concentration inside the patch electrode) and correspond to inhibitory PSCs mediated by chloride s . Thus spontaneous and evoked neuronal activity was present in morphologically mature dissociated neurons. Cultured mouse cortical neurons have a period of glutamate insensitivity that resolves with the development of mature neuronal features (by 14 days in culture) v, and turtle neurons in culture may have a similar period of glutamate insensitivity. In order to demonstrate the presence of glutamate receptors, cultured neurons were tested for their responsiveness to glutamate. Exogenously applied glutamate produced a large long-lasting inward current as shown in Fig. IB for a cell held at -70 mV. The glutamate-induced current was comparable in size to that seen with dissociated mouse neurons in culture a4. When applied to turtle neurons in cortical slices, glutamate activates both a fast current due to the activation of non-N-methyl-D-aspartate ( n o n - N M D A ) receptors and a slower, N M D A - m e d i a t e d current 5. In the present experiments, glutamate applied to neurons produced a current that had both features. In addition, the slower, presumed N M D A mediated current was accompanied by a significant increase in the background current fluctuations that correspond to the rapid opening and closing of glutamate activated channels. Fluctuation analysis I performed on the slow current produced a calculated mean channel open time of 3.6 milliseconds, in close agreement with the value previously obtained for the open time of the N M D A channel in turtle cortical neurons 7. Dissociated turtle cortical neurons in culture therefore express excitatory amino acid ( E A A ) receptors that respond to focally applied glutamate and demonstrate a current consistent with the presence of both N M D A and n o n - N M D A receptors. Neurons that had been in culture for at least 25 days were then tested for sensitivity to glutamate induced injury. To expose the neurons to a brief pulse of glutamate, cells were washed twice in a control solution of (in raM): NaC1 120, KCi 5.4, MgCl 2 0.8, CaCI 2 1.8, glucose 15, and H E P E S 20, at room temperature in room air. A n additional exchange was done with either control solution containing glutamate or control solution alone. After a 5 min exposure, the solution was removed and control solution was exchanged an additional 5 times to wash out all glutamate (effective dilution > 600-fold). Cultures were then placed in a 35 °C, 5% CO2 atmosphere incubator for an additional 24 h. Cultures were exposed to a 5 min pulse of glutamate to permit direct comparison with results obtained in mammalian cortical neurons. Under conditions very similar to those above, the
299 TABLE I Turtle cortical neuron survival after exposure to glutamate
There is only a small difference between percentage of neurons surviving exposure to control solution or to 3 mM glutamate. The number (and percent) of intact neurons from representative microscopicfields was assayed by cellular morphologyor trypan blue exclusion prior to, 5 rain after, and 24 h after exposure to control solution or 3 mM glutamate solution (n = 2 for each condition). The mean and standard deviation (SD) of the percent of neurons surviving in control and glutamate-exposed cultures is included. The difference between 0 and 3 mM glutamate exposure in percent surviving neurons is not statistically significant after 5 minutes (P > 0.1, two tail t test), or after 24 h (0.1 > P > 0.05, two-tail t test). Condition
Pre-exposure
5 min post
24 h post
Control
51 (100%) 59 (100%)
51 (100%) 57 (97%)
50 (98%) 57 (97%)
98.5 + 2.1
97.5 + 0.7
61 (95%) 32 (91%)
55 (86%) 26 (74%)
93.0 + 2.8
80.0 + 8.5
Mean % + S.D. 3 mM Glu Mean % ___S.D.
64 (100%) 35 (100%)
glutamate LDs0 for dissociated mouse cortical neurons is 50-100 /~M 12, so l~reliminary experiments were performed using test solutions containing glutamate concentrations of 10/~M, 100/aM, 300/tM, 500/~M and 1000/~M. However, visual examination revealed that turtle neurons were intact 24 h after 5 min exposure to even the highest concentration, demonstrating a relative resistance to glutamate neurotoxicity. Therefore, in subsequent experiments concentrations of 0 and 3 mM glutamate were used. In order to quantitate the effect of 3 mM glutamate on neuronal survival, culture dishes were examined under phase-contrast microscopy and a representative series of microscopic fields were photographed and marked prior to exposure to glutamate or control solution. Marked fields were photographed again 5 min after exposure and 24 h later. Cultures were then incubated in 0.4% trypan blue, a vital exclusion dye, for 5 min at room temperature and re-photographed. Quantitative assessment of cell death was made by comparison of the photographs of the marked fields; specific neurons present on the baseline photographs but either absent or labeled with Trypan blue in the 5 min and 24 h photographs were scored as dead cells. The percentage of surviving neurons was calculated for 4 culture wells and is shown in Table I. Five min after exposure to 0 or 3 mM glutamate, approximately 98% and 93% of the neurons were alive, respectively. This result is not statistically significant (P > 0.1, two-tail t-test) and suggests that turtle neurons are not killed
instantaneously by brief exposure to glutamate. However, a small proportion of neurons eventually succumbed. Twenty-four h after exposure to control solution approximately 98% of the neurons were alive. When 3 mM glutamate was used 80% of the neurons were alive after 24 h. This result is also not significant (0.1 > P > 0.05, two-tail t-test), but suggests that glutamate has some effect on turtle neurons. Larger numbers of cells might die with modulation of variables in this experimental system, for example by increasing the duration of exposure. The modest degree of excess cell death observed here differs substantially from results with murine neurons where fewer than 10% of cortical neurons survive 24 h after exposure to 3 mM glutamate under identical conditions including temperature 23. Fig. 2 presents examples of representative microscopic fields from one culture exposed to control solution and one to 3 mM glutamate as they appeared prior to, immediately after, and 24 h after control or glutamate exposure. Examination of the microscopic fields did reveal an effect of glutamate exposure: Neurons in the test but not the control solution appeared swollen and granular immediately after exposure. (Fig. 2, compare A2 and B2) However, the majority of neurons in the glutamate exposed culture regained a normal appearance after 24 h (Fig. 2, compare B1 and B3). Mammalian neurons also exhibit cytoplasmic swelling and increased granularity acutely after suffering glutamate neurotoxicity 1°'12"2°. In addition they typically progress to cellular disintegration l°'a2'z°, a fate not suffered by the majority of turtle neurons. In summary, dissociated turtle neurons tested after the development of mature features (25 days) demonstrated characteristic glutamate-induced excitatory currents. However, the majority of cultured neurons were protected from the toxic effects of short exposure to high concentrations of glutamate. In light of the excitatory action of glutamate on turtle neurons it is remarkable that only 20% of cultured dissociated neurons suffered glutamate toxicity at doses 6 times higher than the mammalian LD10o12. As mentioned above, a number of metabolic mechanisms have been postulated to account for the resistance of turtles to anoxia. However, it seems plausible that several adaptations have evolved to mediate this remarkable resiliency, and resistance to brief exposures to glutamate as demonstrated by these experiments, might play an important role in protecting the turtle from anoxic damage. The in vitro turtle cortex can be used to explore the cellular mechanism by which turtle neurons evade glutamate-induced injury and anoxic damage. Elucidation of this mechanism may suggest therapeutic interventions for human neurological diseases that involve glutamate neurotoxicity.
300
Fig. 2. Glutamate induces acute changes in neuronal appearance but does not lead to widespread degeneration. Representative microscopic fields from cultures exposed to control solution (A) or 3 mM glutamate solution (B). Fields were photographed prior to exposure (A1, BI). immediately after a 5 min exposure (A2, B2), and 24 h later (A3, B3). Note swollen and granular appearance of cells immediately after exposure to glutamate (B2) and restoration of normal appearance 24 h later (B3).
We would like to thank Dennis Choi and Hanna Monyer for assistance with the glutamate protocols, and Mark Blanton who was instrumental in applying the whole cell voltage clamp recording
technique and performed fluctuation analysis. This work was supported by NS 12151 and NS 21223.
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