Brain Research, 562 (1991) 285-290 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS (1(/0689939117086C
285
BRES 17086
Increased density of excitatory amino acid transport sites in the hippocampal formation following an entorhinal lesion Kevin J. A n d e r s o n 1, Richard J. Bridges 3 and Carl W. C o t m a n 2 I Departments ()f Physiological Sciences and Neuroscience, University of Florida, Gainesville, FL 32610 (U.S.A.) and Departments of 2Psychobiology and 3Neurology, University of California, lrvine, lrvine, CA 92717 (U.S.A.)
(Accepted 4 June 199l) Key words: Transport; L-Glutamate; D-Aspartate: Autoradiography
High affinity transport of excitatory amino acids such as L-glutamate into astrocytes is necessary for the termination of its excitatory signal and the prevention of its excitotoxic effects. The removal of glutamate from the synaptic cleft is carried out by both sodium- and chloridedependent systems. Both sodium-dependent D-[3H]aspartate and chloride-dependent L-[[3H]glutamate binding were found to increase in the dentate gyrus molecular layer of rats following an entorhinal lesion. The increased binding reached a maximum at 5 and 7 days postlesion and returned to normal by 12 days postlesion. No changes in binding were observed at long time points postlesion. This increased ability to transport glutamate may be a compensatory response to protect the remaining neurons from the excitotoxic conditions that accompany neuronal degeneration. INTRODUCTION The neurotoxic properties of excitatory amino acids ( E A A ) , such as L-glutamate, necessitate the precise regulation of their extracellular concentrations in the CNS. Excitotoxic death attributable to excessive levels of glutamate or related excitatory agonists (e.g. L-aspartate) is currently thought to be a contributing factor in the neuronal loss associated with a wide spectrum of neurological insults (e.g. ischemia, hypoglycemia, epilepsy, Huntington's disease) 21'22'29. The amount of glutamate present in the synaptic cleft should be a direct function of its rate of release and its rate of uptake. I n d e e d , the termination of glutamate's excitatory signal is thought to be d e p e n d e n t upon its rapid removal from the synaptic cleft by high-affinity uptake into neurons and glia. in this respect, any conditions that alter the rate at which the excitatory transmitter is cleared from the cleft, whether the result of decreased transport or excessive substrate, could produce levels of glutamate sufficient to initiate an excitotoxic response and neuronal loss. Alternatively, an increase in transport capacity of a region may represent a protective mechanism, whereby increased concentrations of substrate may be m o r e efficiently removed from the extracellular space. A t present, evidence suggests that at least two distinct transport systems may participate in the regulation of
extracellular glutamate levels in the CNS. These two systems, each of which exhibits a characteristic pharmacology and anatomic distribution, are most easily distinguished on the basis of their ionic dependencies: one is s o d i u m - d e p e n d e n t , the other c h l o r i d e - d e p e n d e n t z. The s o d i u m - d e p e n d e n t glutamate system is the more thoroughly characterized of the two, having been well defined kinetically and pharmacologically in synaptosomes, tissue slices, and astrocytes 7'9'~3. In contrast, much less is known about the c h l o r i d e - d e p e n d e n t system, as it was initially identified during the course of L-[3H]glutamate binding studies and not in assays directly examining uptake. Subsequent biochemical and pharmacological characterization of this c h l o r i d e - d e p e n d e n t binding suggested the process represented the interaction of L-[3H]gluta mate with a transport system, as o p p o s e d to a novel excitatory amino acid receptor 3'8'14'19. More recent studies have d e m o n s t r a t e d c h l o r i d e - d e p e n d e n t uptake of L-glutamate into both synaptosomes and an astrocytoma cell line 28"3°. It has been further d e m o n s t r a t e d that membranes p r e p a r e d from cultured astrocytes also possess c h l o r i d e - d e p e n d e n t glutamate binding sites with a similar pharmacology to that of the astrocytoma system and that these binding sites can be visualized in rat brain slices by L-[3H]glutamate a u t o r a d i o g r a p h y 4,5. Thus, it appears the uptake of L-glutamate is attributable to the cumulative activity of both the s o d i u m - d e p e n d e n t and
Correspondence." K.J. Anderson, Physiological Sciences, Box J-144, JHMHC, University of Florida, Gainesville, FL 32610-0144, U.S.A.
286 c h l o r i d e - d e p e n d e n t transport systems present on neurons and/or glia. In this study we have used radioligand autoradiography to quantitate the density of glutamate transport sites in the h i p p o c a m p u s of rats that had b e e n given an entorhinal lesion. This animal m o d e l of neuronal injury is well known for its ability to trigger a series of complex sprouting responses in the d e n e r v a t e d d e n t a t e gyrus molecular layer (for review see ref. 6). D-[aH]Aspartate and L-[3H]glutamate binding were used to selectively evaluate the respective changes in the sodium- and chlorided e p e n d e n t u p t a k e systems. We r e p o r t that the density of both of these binding sites in the molecular layer of the d e n t a t e gyrus exhibit a transient increase that p e a k s 5 - 7 days postlesion and returns to control levels by 12 days. This increase in the density of the transport sites parallels an increased n u m b e r of reactive astrocytes in the d e n e r v a t e d area. A s the presence of s o d i u m - d e p e n d e n t and c h l o r i d e - d e p e n d e n t transport sites potentially reflects the ability to r e m o v e extracellular glutamate, an increase in binding in response to a lesion may represent a mechanism for buffering the possible accumulation of excitotoxins in a d a m a g e d brain region.
MATERIALS AND METHODS Sprague-Dawley rats (4-6 months of age) were anesthetized with pentobarbital (50 mg/kg) and subjected to a unilateral, electrolytic entorhinal lesion as previously described 24. After 2, 5, 7, 12, 18, 30, 60, 90 or 120 days postlesion (all time points n = 2, except 5 (n = 10) and 7 (n = 10) days postlesion) the animals were killed by decapitation and the brains rapidly removed and frozen on dry ice. The brains were sectioned (6/~m) on a cryostat and thaw-mounted on gelatin-subbed slides. Sections were processed for L-[3H]glutamate and D-[3H]aspartate autoradiography (see below). Alternate sections (20 k~m) were processed for glial fibrillary acidic protein (GFAP) immunoreactivity. In addition to the youngadult animals above, aged subjects (24-26 months old, n = 4) were subjected to a unilateral entorhinal lesion and killed at 7 days postlesion. When sectioned, the brain from an aged subject was mounted adjacent to the brain from a young-adult subject (also 7 days postlesion). Thus, each aged subject was cut and processed for autoradiography in parallel with a young-adult subject. The autoradiographic procedures were carried out using a methodology similar to that previously described for EAA receptor studies that had been modified to optimize binding to the transport systems 2"4A6A7. Briefly, 6-/~m sections of the tissue were incubated with either 100 nM D-[3H]aspartate (NEN, spec. act.: 15 ci/mmol) in a sodium-containing buffer (50 mM Tris-chloride, 300 mM NaCI, pH 7.4) or 100 nM L-[3H]glutamate (NEN, spec. act. 50.9 Ci/ mmol) in sodium-free, chloride-containing buffer (50 mM Tris-chloride, 1 mM calcium, pH 6.9) to assess the sodium- and chloridedependent sites, respectively. To selectively quantitate binding and avoid the complications that could be introduced by the sequestration of radioligand that had been transported through these systems, the incubations with radioligand were carried out at 0-2 °C for l0 min. Binding selectivity for o-[3H]aspartate sites was maintained by virtue of its sodium-dependency, as there is no appreciable binding in sodium-free/chloride-containing buffer 2"ll'tS. Furthermore, o-[3H]aspartate does not interact with any of the known EAA receptors2. In the case of the chloride-dependent glutamate
sites, specificity was achieved by including a mixture of N-methyl-. D-aspartate (NMDA), kainate (KA), and a-amino-3-hydroxy-5-mcthylisoxazole-4-propionate (AMPA) in the incubation buffer at concentrations (100 /tM each) sufficient to block binding to the well-characterized EAA receptors. Non-specific binding was determined by the addition of 100 ~M glutamate (chloride-dependent glutamate binding) or llX}/~M D,L-threo-fl-hydroxyaspartatc (D-aspartate binding). The sections were rinsed in four changes of icecold buffer for a total of 30 s and rapidly dricd under an air stream. Sections and methacrylate tritium standards (Amersham) were opposed to tritium-sensitive film (LKB) and exposed for 4 weeks. Autoradiograms were analyzed on a microcomptiter imaging device (MC1D) system (Imaging Research, Inc.. St. Catherines, Ont.). Statistical comparisons were made using a Mann-Whitney U-test. Parallel 20/~m sections were processed for GFAP immunoreactivity by the avidin-biotin-horseradish peroxidase method. Briefly, frozen sections were allowed to air dry and tixed with 4% buffered formalin. Sections were rinsed extensively and stained using a monoclonal antibody directed against human GFAP (BoeringerMannheim) and an ABC kit for mouse lgCs (Vector Labs). Sections were reacted with 3,3" diaminobcnzidine containing 0.01% H202, rinsed well in PBS, cleared in alcohols and xylene and coverslipped.
RESULTS Similar to the distribution of E A A receptors, the h i p p o c a m p u s of normal rats exhibits high levels of binding to both transport sites (Fig. l, also see ref. 2). Sod i u m - d e p e n d e n t D-[3H]aspartate binding was enriched in the molecular layer of the d e n t a t e gyrus (4763 + 481 fmol/mg protein), stratum r a d i a t u m , and stratum oriens, particularly in area CA1 (averaging 5312 + 250 fmol/mg protein). Very little D-aspartate b i n d i n g was found in the stratum lucidum, stratum granulosum or stratum pyramidale. The molecular layer of the d e n t a t e gyrus also contains one of the highest densities of chloride-dependent L-[3H]glutamate binding sites in the CNS (averaging 1067 -+ 19 fmol/mg protein). Lower levels of binding were found in the stratum r a d i a t u m and oriens of area C A 1 , while very little binding was seen over the stratum lucidum, stratum p y r a m i d a l e and stratum granulosum. Following entorhinal ablation, a transient increase in both c h l o r i d e - d e p e n d e n t L-glutamate and s o d i u m - d e p e n dent D-aspartate binding was o b s e r v e d in the d e n t a t e gyrus molecular layer. A t 5 days postlesion, a statistically significant 35-38% ( P ~< 0.01) increase in binding to both sites was observed in the outer two-thirds to three-fourths of the dentate gyrus molecular layer, the zone of deafferentation (Fig. 1). W h e n binding levels were c o m p a r e d to the n o n q e s i o n e d side, D-aspartate binding increased to 6473 + 303 fmol/mg protein and c h l o r i d e - d e p e n d e n t glutamate binding increased to 1473 _+ 97 fmol/mg protein. This increased binding persisted at 7 days, but r e t u r n e d to control values by 12 days postlesion. No changes in the levels of binding to either site relative to the controls were d e t e c t e d at longer postlesion time points (30. 60 and 120 days).
287
Fig. I. Changcs in sodium-dependent D-[3H]aspartate binding (A) and chloride-dependent L-[3H]glutamate binding (B) in the hippocampal formation following an cntorhinal lesion. Both are shown 7 days postlesion and both are shown with the lesion side to the left. Autoradiograms arc color-coded so that red indicates high amount of binding and blue-black indicates low levels of binding. Calibration scales indicate specific binding in pmol/mg protein. Note the increase in binding in the molecular layer of the dentate gyrus relative to the non-lesioned (right) side for both sites.
288
Fig. 2. GFAP immunoreaetivity in the hippocampal formation following unilateral entorhinal lesion (7 days postfesiOn), Hippocampal dentate gyrus ipsilateral to the entorhinal lesion is shown in (A) while the dentate gyrus contralateral to the l ~ o n is shown in (B). Note thc coarse, reactive GFAP,p~itive astroeytes seen in the inner and outer molecular layers of the dentate gyrus ipsilateral to the entorhinal lesion. Lines show approximate borders of dentate gyrus laminae. Abbreviations: GC, granule celt layer: IMLi inner molecular layer: OML outer molecular layer, HiF, h ~ a m p a t fissure. Bar (lower right corner) = I00 ktm.
Further characterization of the response of the transport sites to an entorhinal lesion revealed that the observed increase in binding was sensitive to xylene extraction. Thus, when the tissue slices are pre-incubated in xylene for 10 rain at room temperature, no significant differences in either o-[3H]aspartate or L-[3H]gtutamate binding were observed between lesion and non-lesioned sides. We have previously employed this procedure to "delipidate" tissue sections and ensure the disruption of membrane compartments that could potentially sequester ligand 2. In these previous studies, xylene pretreatment was effective in ameliorating the process of ligand sequestration in brain slices incubated at 30 °C. In the present study we have relied on temperature-sensitivity (iacubation at 0 - 2 °(2) and short incubation times to prevent sequestration and allow binding to be selectively quantitated. However, the differential sensitivity of the lesion-induced transport sites to xylene extraction suggests that there are differences between the newly expressed sites and those that were present prelesion, possibly reflecting the way these sites are embedded in the membrane. The examination of alternate hippocampal sections indicated that the increase in the binding to transport sites occurred in parallel with the appearance of reactive astrocytes (Fig. 2). Swollen and coarse astrocyte processes, as revealed by G F A P immunoreactivity, were first observed at 5 days postlesion. The distribution of swollen astrocytes was largely confined to the denervated zone. although astrocytes in the inner molecular layer, which is not subject to the loss of entorhinal terminals, also exhibited some cells which appeared reactive. In contrast
to the transient change observed for the chloride-dependent e-glutamate and sodium-dependent D-aspartate binding, which returned to control levels by 12 days, the astrocytes in the molecular layer of the dentate gyrus remained "reactive" in appearance at even the longest time points postlesion (120 days). The entorhinal lesion-induced change in the transport site density was also observed in aged rats. At 7 days postlesion, the increase in binding to both sites in the dentate gyrus molecular layer of aged subjects was similar to that seen in young-adult subjects (i.e. sodium-dependent D-aspartate sites: 3939 ~ 232 to 5511 _-c_ 201 fmol/mg protein and chloride~dependent L-glutamate binding: 1095 - 25 to 1460 +-- 140 fmol/mg protein). DISCUSSION The rapid removal of glutamate from the synaptic cleft into presynaptic terminals and astrocytes is thought to be a key step in terminating its excitatory signal and. in the case of glial uptake, the recycling of transmitter via the glutamine cycle 25. With an increasing amount of evidence indicating that excitotoxicity is a c o m m o n underlying mechanism in a wide spectrum of neuropathologles, the transport of glutamate takes on even greater significance as a protective mechanism. Comparisons of E A A receptor and transport system distribution suggest that areas with a high receptor/transport site ratio may be more vulnerable to excitotoxic injury-'. For example. in the hippocampus, both area CA1 and the dentate granule molecular layer exhibit an enriched distribution of E A A receptors, whereas area CA1 has a relatively
289 low density of transport sites when compared to the dentate granule molecular layer. It is notable, in this regard, that area CA1 pyramidal cells are substantially more vulnerable to excitotoxic injury than the dentate granule cells 15'2°'29. Although a large number of factors may influence the susceptibility of a region to excitotoxic injury, the transport capacity of a specific region may be an important factor in its vulnerability to excitotoxicity. The present study suggests that the CNS may be able to regulate the levels of excitatory amino acid uptake in response to injury. The increase in binding to these transport sites paralleled initial morphological changes in astrocytes within the denervated zone. These changes included an increase in GFAP immunoreactivity and a thickening of astrocytic processes, as well as a possible increase in cell number ~°'23. The characteristic response of astroglia to injury is also thought to include an increase in the phagocytotic ability of these cells. Furthermore, this potentially protective mechanism appears to remain intact even in the aged brain, in contrast to agerelated deficits that may contribute to impairments in reactive synaptogenesis 1"~2. Thus, the observed increase in E A A transport site density may represent another injury-induced functional change in astroglia. Previously, Taxt and Storm-Mathisen demonstrated a decrease in sodium-dependent D-aspartate uptake in the hippocampus following an entorhinal lesion and concluded that this finding was consistent with the loss of uptake into degenerating presynaptic terminals 26. The apparent difference between this decrease and our demonstration of an increase in transport site binding most probably reflects differences in techniques used and the actual processes being measured. In the earlier study, D-aspartate uptake was followed at 25 °C for 10-30 min in relatively thick slices, (150/~m) after which the tissue was rinsed, fixed in glutaraldehyde, and exposed to film (only 50% of the radioligand was retained by fixation). These conditions would be expected to enhance the visualization of radioligand that had been sequestered by surviving cells in much larger and relatively stable pools (i.e. within cytoplasmic pools, synaptic vesicles). Our autoradiographic techniques, on the other hand, examined only that radioligand that was bound to membrane transport sites. Furthermore, it appears as if the two procedures may be examining different pools. Taxt and StormMathisen concluded that their technique selectively examined transport into nerve endings and that uptake into glia was probably not present to a significant degree.
REFERENCES 1 Anderson, K.J., Scheff, S.W. and DeKosky, S.T., Reactive synaptogenesis in hippocampal area CA1 of aged and young adult
In contrast, the present demonstration that the increase in transport site binding parallels the appearance of reactive astrocytes suggests that glial cells are probably an important component of this response. Taken together, the results suggest that although the total uptake capacity of the dentate molecular layer may be decreased by denervation and subsequent loss of presynaptic terminals, some cells, particularly astrocytes, exhibit an increased density of the transport sites that may reflect their ability to contribute to the stabilization of extracellular glutamate levels in the area undergoing degeneration, repair and synaptic reorganization. Our current hypothesis proposes that a transient lesion-induced increase may represent a mechanism by which further damage to critical circuitry following loss of afferent input may be prevented. Such a model, however, raises a number of interesting questions regarding the balance between extracellular glutamate levels, receptor densities, transport capacity and the ability to maintain normal physiological function. Immediately following the loss of afferent input, the target neurons may be more vulnerable to excitotoxic insults. For instance, at 7 and 14 days after an entorhinal lesion, AMPA and N M D A receptor levels are not different from control values in the denervated zone 27. An enhanced ability to buffer the extracellular concentrations of EAAs may thus serve to limit secondary excitotoxicity resulting from runaway excitatory activity. From another perspective, an increase in E A A uptake could potentially lead to a decrease in the amount of transmitter available for normal synaptic transmission. It is also possible that an increase in E A A buffering capacity may, in some way facilitate the rebuilding of damaged circuitry, as the activity of sprouting and reactive synaptogenesis following an entorhinal lesion are known to reach peak levels at the same time as the increase in transport sites 6. Although more work is necessary to evaluate both the role of glutamate uptake as a protective mechanism and the physiological consequence of increased transport capacity, these findings highlight the delicate balance that must exist between each component of the E A A transmitter system to maintain properly functioning circuitry. Acknowledgements. This work was supported by NIA Grants AG00538, AG00096 (C.W.C.), AG08843 (K.J.A.), the American Health Assistance Foundation (UC 11394) and the American Federation for Aging Research. R.J.B. is a LEAD junior investigator (AG07918). Thanks to Jennifer Kahle for critical reading of this manuscript.
rats, J. Comp. Neurol., 252 (1986) 374-384. 2 Anderson, K.J., Monaghan, D.T., Bridges, R.J. and Cotman, C.W., Autoradiographic comparison of sodium-dependent and chloride-dependent glutamate transport sites, Neuroscience, 38
290 (1990) 311-322. 3 Bridges, R.J., Hearn, T., Monaghan, D.T. and Cotman, C.W., A comparison of 2-amino-4-phosphonobutyric acid lAP4) receptors and 3H-AP4 binding sites in the rat brain, Brain Research, 375 (1986) 204-209. 4 Bridges, R.J., Kesslak, J.P., Nieto-Sampedro, M., Broderick, J., Yu, J. and Cotman, C.W., An L-[3H]-glutamate binding site on glia: an autoradiographic study on implanted astrocytes, Brain Research, 415 (1987) 163-168. 5 Bridges, R.J., Nieto-Sampedro, M., Kadri, M. and Cotman, C.W., A novel chloride-dependent L-[3H]-glutamate binding site in astrocyte membranes, J. Neurochem., 48 (1987) 1709-1715. 6 Cotman, C.W. and Anderson, K.J., Synaptic plasticity and functional stabilization in the hippocampal formation: possible role in Alzheimer's disease. In S.G. Waxman lEd.), Advances in Neurology, Vol. 47: Functional Recovery in Neurological Disease, Raven, New York, 1988, pp. 313-336. 7 Danbolt, N.C., Pines, G. and Kanner, B.I., Purification and reconstitution of the sodium- and potassium-coupled glutamate transport glycoprotein from rat brain, Biochemistry, 29 (1990) 6734-6740. 8 Fagg, C.E. and Lanthorn, T.H., Pharmacological characterization of AP4 receptors, Br. J. Pharrnacol., 86 (1985) 743-746. 9 Fonnum, F., Glutamate: a neurotransmitter in the mammalian brain, J. Neurochem., 42 (1984) 1-11. 10 Gall, C., Rose, G. and Lynch, G., Proliferative and migratory activity of glial cells in the partially deafferented hippocampus, J. Comp. Neurol., 183 (1979) 539-548. 11 Greenamyre, J.T., Higgins, D.S. and Young, A.B., Sodiumdependent D-aspartate "binding" is not a measure of presynaptic uptake sites in an autoradiographic assay, Brain Research, 511 (1990) 310-314. 12 Hoff, S.F., Scheff, S.W., Benardo, L.S. and Cotman, C.W., Lesion-induced synaptogenesis in the dentate gyrus of aged rats. II. Demonstration of an impaired degeneration clearing response, J. Comp. Neurol., 205 (1982) 253-259. 13 Kanner, B.I. and Bendahan, A., Binding order of substrates to the sodium and potassium ion coupled L-glutamic acid transporter from rat brain, Biochemistry, 21 (1982) 6327-6330. 14 Kessler, M., Petersen, G., Vu, H.M., Baudry, M. and Lynch, G., Phe-Glu stimulated, chloride-dependent glutamate binding represents glutamate sequestration mediated by an exchange system, J. Neurochem., 48 (1987) 1191-1195. 15 Kirino, T., Selective vulnerability in the gerbil hippocampus following transient ischemia, Acta Neuropathol., 62 (1984) 201208. 16 Monaghan, D.T., Holets, V.L., Toy, D.W. and Cotman, C.W., Anatomical distributions of four pharmacologicallly distinct 3H-
L-glutamate binding sites, Nature, 306 (1983) 17~-179. 17 Monaghan, D.T., Yao, D. and (k)tman, C.W., t_-[~H]glutamate binds to kainate-, NMDA-, and AMPA-sensitive binding sites: an autoradiographic analysis, Brain Research, 340 (1985) 378-383. 18 Parsons, B. and Rainbow, "I'.C~, Quantitative autoradiography of sodium-dependent D-[3H]aspartate binding sites in rat brain, Neurosci. Lett., 36 (1983) 9-12. 19 Pin, J.P., Bockaert, J. and Recasens, M., The Ca~"/Ct dependent L-[3H] glutamate binding: a new receptor or a particular transport process? FEBS Lett., 175 (1984) 31-36. 20 Pulsinelli, W.A., Brierley, J.B. and Plum, F., Temporal profile of neuronal damage in a model of transient forebrain ischemia, Ann. Neurol., 11 (1982)491-498. 21 Rothman, S.M. and Olney, J.W., Glutamate and the pathology of hypoxic/ischemic brain damage, Ann. NeuroL, 19 (1986) 105-111. 22 Rothman, S.M. and Olney, J.W.. Excitotoxicity and the NMDA receptor, Trends Neurosei., 10 (1987) 299-302. 23 Rose, G., Lynch, G. and Cotman, C.W., Hypertrophy and redistribution of astrocytes in the deafferented dentate gyrus. Brain Res. Bull., 1 (1976)87-92 24 Scheff, S.W., Bernardo, L.S. and Cotman, C.W., Decline in reactive fiber growth in the dentate gyrus of aged rats compared to young adult rats following entorhinat cortex removal, Brain Research, 199 (1980) 21-38. 25 Shank, R.P. and Aprison, M.H., Present status and significance of the glutamine cycle in neural tissues, Life Sci.. 28 (t981) 837-842. 26 Taxt, T. and Storm-Mathisen, J., Uptake of D-aspartatc and L-glutamate in excitatory axon terminals in hippocampus: autoradiographic and biochemical comparison with 7-aminobutyratc and other amino acids in normal rats and in rats with lesions. Neuroscience, 11 (1984) 79-1(10. 27 Ulas, J., Monaghan, D.T. and Cotman, C.W., Plastic response of hippocampal excitatory amino acid receptors to deafferentation and reinnervation., Neuroscience, 34 (1990) 9-171 28 Waniewski, R.A. and Martin, D.L., Characterization of L-glutamic acid transport by glioma cells in culture: evidence for sodium-independent, chloride-dependent high affinity influx. J. Neurosci., 4 (1984) 2237-2246. 29 Wieloch, "12, Endogenous excitotoxins as possible mediators ot ischemic and hypoglycemic brain damage, Prog. Brain Res., 63 (1985) 69-85. 30 Zaczek, R., Balm, M., Arlis, S., l)rucker, H. and Coyle, J.rl'Quisqualate-sensitive, chloride-dependent transport of glutamate into rat brain synaptosomcs. J. Neurosci. Res., 18 (1987) 425-431.
285
BRES 17086
Increased density of excitatory amino acid transport sites in the hippocampal formation following an entorhinal lesion Kevin J. A n d e r s o n 1, Richard J. Bridges 3 and Carl W. C o t m a n 2 I Departments ()f Physiological Sciences and Neuroscience, University of Florida, Gainesville, FL 32610 (U.S.A.) and Departments of 2Psychobiology and 3Neurology, University of California, lrvine, lrvine, CA 92717 (U.S.A.)
(Accepted 4 June 199l) Key words: Transport; L-Glutamate; D-Aspartate: Autoradiography
High affinity transport of excitatory amino acids such as L-glutamate into astrocytes is necessary for the termination of its excitatory signal and the prevention of its excitotoxic effects. The removal of glutamate from the synaptic cleft is carried out by both sodium- and chloridedependent systems. Both sodium-dependent D-[3H]aspartate and chloride-dependent L-[[3H]glutamate binding were found to increase in the dentate gyrus molecular layer of rats following an entorhinal lesion. The increased binding reached a maximum at 5 and 7 days postlesion and returned to normal by 12 days postlesion. No changes in binding were observed at long time points postlesion. This increased ability to transport glutamate may be a compensatory response to protect the remaining neurons from the excitotoxic conditions that accompany neuronal degeneration. INTRODUCTION The neurotoxic properties of excitatory amino acids ( E A A ) , such as L-glutamate, necessitate the precise regulation of their extracellular concentrations in the CNS. Excitotoxic death attributable to excessive levels of glutamate or related excitatory agonists (e.g. L-aspartate) is currently thought to be a contributing factor in the neuronal loss associated with a wide spectrum of neurological insults (e.g. ischemia, hypoglycemia, epilepsy, Huntington's disease) 21'22'29. The amount of glutamate present in the synaptic cleft should be a direct function of its rate of release and its rate of uptake. I n d e e d , the termination of glutamate's excitatory signal is thought to be d e p e n d e n t upon its rapid removal from the synaptic cleft by high-affinity uptake into neurons and glia. in this respect, any conditions that alter the rate at which the excitatory transmitter is cleared from the cleft, whether the result of decreased transport or excessive substrate, could produce levels of glutamate sufficient to initiate an excitotoxic response and neuronal loss. Alternatively, an increase in transport capacity of a region may represent a protective mechanism, whereby increased concentrations of substrate may be m o r e efficiently removed from the extracellular space. A t present, evidence suggests that at least two distinct transport systems may participate in the regulation of
extracellular glutamate levels in the CNS. These two systems, each of which exhibits a characteristic pharmacology and anatomic distribution, are most easily distinguished on the basis of their ionic dependencies: one is s o d i u m - d e p e n d e n t , the other c h l o r i d e - d e p e n d e n t z. The s o d i u m - d e p e n d e n t glutamate system is the more thoroughly characterized of the two, having been well defined kinetically and pharmacologically in synaptosomes, tissue slices, and astrocytes 7'9'~3. In contrast, much less is known about the c h l o r i d e - d e p e n d e n t system, as it was initially identified during the course of L-[3H]glutamate binding studies and not in assays directly examining uptake. Subsequent biochemical and pharmacological characterization of this c h l o r i d e - d e p e n d e n t binding suggested the process represented the interaction of L-[3H]gluta mate with a transport system, as o p p o s e d to a novel excitatory amino acid receptor 3'8'14'19. More recent studies have d e m o n s t r a t e d c h l o r i d e - d e p e n d e n t uptake of L-glutamate into both synaptosomes and an astrocytoma cell line 28"3°. It has been further d e m o n s t r a t e d that membranes p r e p a r e d from cultured astrocytes also possess c h l o r i d e - d e p e n d e n t glutamate binding sites with a similar pharmacology to that of the astrocytoma system and that these binding sites can be visualized in rat brain slices by L-[3H]glutamate a u t o r a d i o g r a p h y 4,5. Thus, it appears the uptake of L-glutamate is attributable to the cumulative activity of both the s o d i u m - d e p e n d e n t and
Correspondence." K.J. Anderson, Physiological Sciences, Box J-144, JHMHC, University of Florida, Gainesville, FL 32610-0144, U.S.A.
286 c h l o r i d e - d e p e n d e n t transport systems present on neurons and/or glia. In this study we have used radioligand autoradiography to quantitate the density of glutamate transport sites in the h i p p o c a m p u s of rats that had b e e n given an entorhinal lesion. This animal m o d e l of neuronal injury is well known for its ability to trigger a series of complex sprouting responses in the d e n e r v a t e d d e n t a t e gyrus molecular layer (for review see ref. 6). D-[aH]Aspartate and L-[3H]glutamate binding were used to selectively evaluate the respective changes in the sodium- and chlorided e p e n d e n t u p t a k e systems. We r e p o r t that the density of both of these binding sites in the molecular layer of the d e n t a t e gyrus exhibit a transient increase that p e a k s 5 - 7 days postlesion and returns to control levels by 12 days. This increase in the density of the transport sites parallels an increased n u m b e r of reactive astrocytes in the d e n e r v a t e d area. A s the presence of s o d i u m - d e p e n d e n t and c h l o r i d e - d e p e n d e n t transport sites potentially reflects the ability to r e m o v e extracellular glutamate, an increase in binding in response to a lesion may represent a mechanism for buffering the possible accumulation of excitotoxins in a d a m a g e d brain region.
MATERIALS AND METHODS Sprague-Dawley rats (4-6 months of age) were anesthetized with pentobarbital (50 mg/kg) and subjected to a unilateral, electrolytic entorhinal lesion as previously described 24. After 2, 5, 7, 12, 18, 30, 60, 90 or 120 days postlesion (all time points n = 2, except 5 (n = 10) and 7 (n = 10) days postlesion) the animals were killed by decapitation and the brains rapidly removed and frozen on dry ice. The brains were sectioned (6/~m) on a cryostat and thaw-mounted on gelatin-subbed slides. Sections were processed for L-[3H]glutamate and D-[3H]aspartate autoradiography (see below). Alternate sections (20 k~m) were processed for glial fibrillary acidic protein (GFAP) immunoreactivity. In addition to the youngadult animals above, aged subjects (24-26 months old, n = 4) were subjected to a unilateral entorhinal lesion and killed at 7 days postlesion. When sectioned, the brain from an aged subject was mounted adjacent to the brain from a young-adult subject (also 7 days postlesion). Thus, each aged subject was cut and processed for autoradiography in parallel with a young-adult subject. The autoradiographic procedures were carried out using a methodology similar to that previously described for EAA receptor studies that had been modified to optimize binding to the transport systems 2"4A6A7. Briefly, 6-/~m sections of the tissue were incubated with either 100 nM D-[3H]aspartate (NEN, spec. act.: 15 ci/mmol) in a sodium-containing buffer (50 mM Tris-chloride, 300 mM NaCI, pH 7.4) or 100 nM L-[3H]glutamate (NEN, spec. act. 50.9 Ci/ mmol) in sodium-free, chloride-containing buffer (50 mM Tris-chloride, 1 mM calcium, pH 6.9) to assess the sodium- and chloridedependent sites, respectively. To selectively quantitate binding and avoid the complications that could be introduced by the sequestration of radioligand that had been transported through these systems, the incubations with radioligand were carried out at 0-2 °C for l0 min. Binding selectivity for o-[3H]aspartate sites was maintained by virtue of its sodium-dependency, as there is no appreciable binding in sodium-free/chloride-containing buffer 2"ll'tS. Furthermore, o-[3H]aspartate does not interact with any of the known EAA receptors2. In the case of the chloride-dependent glutamate
sites, specificity was achieved by including a mixture of N-methyl-. D-aspartate (NMDA), kainate (KA), and a-amino-3-hydroxy-5-mcthylisoxazole-4-propionate (AMPA) in the incubation buffer at concentrations (100 /tM each) sufficient to block binding to the well-characterized EAA receptors. Non-specific binding was determined by the addition of 100 ~M glutamate (chloride-dependent glutamate binding) or llX}/~M D,L-threo-fl-hydroxyaspartatc (D-aspartate binding). The sections were rinsed in four changes of icecold buffer for a total of 30 s and rapidly dricd under an air stream. Sections and methacrylate tritium standards (Amersham) were opposed to tritium-sensitive film (LKB) and exposed for 4 weeks. Autoradiograms were analyzed on a microcomptiter imaging device (MC1D) system (Imaging Research, Inc.. St. Catherines, Ont.). Statistical comparisons were made using a Mann-Whitney U-test. Parallel 20/~m sections were processed for GFAP immunoreactivity by the avidin-biotin-horseradish peroxidase method. Briefly, frozen sections were allowed to air dry and tixed with 4% buffered formalin. Sections were rinsed extensively and stained using a monoclonal antibody directed against human GFAP (BoeringerMannheim) and an ABC kit for mouse lgCs (Vector Labs). Sections were reacted with 3,3" diaminobcnzidine containing 0.01% H202, rinsed well in PBS, cleared in alcohols and xylene and coverslipped.
RESULTS Similar to the distribution of E A A receptors, the h i p p o c a m p u s of normal rats exhibits high levels of binding to both transport sites (Fig. l, also see ref. 2). Sod i u m - d e p e n d e n t D-[3H]aspartate binding was enriched in the molecular layer of the d e n t a t e gyrus (4763 + 481 fmol/mg protein), stratum r a d i a t u m , and stratum oriens, particularly in area CA1 (averaging 5312 + 250 fmol/mg protein). Very little D-aspartate b i n d i n g was found in the stratum lucidum, stratum granulosum or stratum pyramidale. The molecular layer of the d e n t a t e gyrus also contains one of the highest densities of chloride-dependent L-[3H]glutamate binding sites in the CNS (averaging 1067 -+ 19 fmol/mg protein). Lower levels of binding were found in the stratum r a d i a t u m and oriens of area C A 1 , while very little binding was seen over the stratum lucidum, stratum p y r a m i d a l e and stratum granulosum. Following entorhinal ablation, a transient increase in both c h l o r i d e - d e p e n d e n t L-glutamate and s o d i u m - d e p e n dent D-aspartate binding was o b s e r v e d in the d e n t a t e gyrus molecular layer. A t 5 days postlesion, a statistically significant 35-38% ( P ~< 0.01) increase in binding to both sites was observed in the outer two-thirds to three-fourths of the dentate gyrus molecular layer, the zone of deafferentation (Fig. 1). W h e n binding levels were c o m p a r e d to the n o n q e s i o n e d side, D-aspartate binding increased to 6473 + 303 fmol/mg protein and c h l o r i d e - d e p e n d e n t glutamate binding increased to 1473 _+ 97 fmol/mg protein. This increased binding persisted at 7 days, but r e t u r n e d to control values by 12 days postlesion. No changes in the levels of binding to either site relative to the controls were d e t e c t e d at longer postlesion time points (30. 60 and 120 days).
287
Fig. I. Changcs in sodium-dependent D-[3H]aspartate binding (A) and chloride-dependent L-[3H]glutamate binding (B) in the hippocampal formation following an cntorhinal lesion. Both are shown 7 days postlesion and both are shown with the lesion side to the left. Autoradiograms arc color-coded so that red indicates high amount of binding and blue-black indicates low levels of binding. Calibration scales indicate specific binding in pmol/mg protein. Note the increase in binding in the molecular layer of the dentate gyrus relative to the non-lesioned (right) side for both sites.
288
Fig. 2. GFAP immunoreaetivity in the hippocampal formation following unilateral entorhinal lesion (7 days postfesiOn), Hippocampal dentate gyrus ipsilateral to the entorhinal lesion is shown in (A) while the dentate gyrus contralateral to the l ~ o n is shown in (B). Note thc coarse, reactive GFAP,p~itive astroeytes seen in the inner and outer molecular layers of the dentate gyrus ipsilateral to the entorhinal lesion. Lines show approximate borders of dentate gyrus laminae. Abbreviations: GC, granule celt layer: IMLi inner molecular layer: OML outer molecular layer, HiF, h ~ a m p a t fissure. Bar (lower right corner) = I00 ktm.
Further characterization of the response of the transport sites to an entorhinal lesion revealed that the observed increase in binding was sensitive to xylene extraction. Thus, when the tissue slices are pre-incubated in xylene for 10 rain at room temperature, no significant differences in either o-[3H]aspartate or L-[3H]gtutamate binding were observed between lesion and non-lesioned sides. We have previously employed this procedure to "delipidate" tissue sections and ensure the disruption of membrane compartments that could potentially sequester ligand 2. In these previous studies, xylene pretreatment was effective in ameliorating the process of ligand sequestration in brain slices incubated at 30 °C. In the present study we have relied on temperature-sensitivity (iacubation at 0 - 2 °(2) and short incubation times to prevent sequestration and allow binding to be selectively quantitated. However, the differential sensitivity of the lesion-induced transport sites to xylene extraction suggests that there are differences between the newly expressed sites and those that were present prelesion, possibly reflecting the way these sites are embedded in the membrane. The examination of alternate hippocampal sections indicated that the increase in the binding to transport sites occurred in parallel with the appearance of reactive astrocytes (Fig. 2). Swollen and coarse astrocyte processes, as revealed by G F A P immunoreactivity, were first observed at 5 days postlesion. The distribution of swollen astrocytes was largely confined to the denervated zone. although astrocytes in the inner molecular layer, which is not subject to the loss of entorhinal terminals, also exhibited some cells which appeared reactive. In contrast
to the transient change observed for the chloride-dependent e-glutamate and sodium-dependent D-aspartate binding, which returned to control levels by 12 days, the astrocytes in the molecular layer of the dentate gyrus remained "reactive" in appearance at even the longest time points postlesion (120 days). The entorhinal lesion-induced change in the transport site density was also observed in aged rats. At 7 days postlesion, the increase in binding to both sites in the dentate gyrus molecular layer of aged subjects was similar to that seen in young-adult subjects (i.e. sodium-dependent D-aspartate sites: 3939 ~ 232 to 5511 _-c_ 201 fmol/mg protein and chloride~dependent L-glutamate binding: 1095 - 25 to 1460 +-- 140 fmol/mg protein). DISCUSSION The rapid removal of glutamate from the synaptic cleft into presynaptic terminals and astrocytes is thought to be a key step in terminating its excitatory signal and. in the case of glial uptake, the recycling of transmitter via the glutamine cycle 25. With an increasing amount of evidence indicating that excitotoxicity is a c o m m o n underlying mechanism in a wide spectrum of neuropathologles, the transport of glutamate takes on even greater significance as a protective mechanism. Comparisons of E A A receptor and transport system distribution suggest that areas with a high receptor/transport site ratio may be more vulnerable to excitotoxic injury-'. For example. in the hippocampus, both area CA1 and the dentate granule molecular layer exhibit an enriched distribution of E A A receptors, whereas area CA1 has a relatively
289 low density of transport sites when compared to the dentate granule molecular layer. It is notable, in this regard, that area CA1 pyramidal cells are substantially more vulnerable to excitotoxic injury than the dentate granule cells 15'2°'29. Although a large number of factors may influence the susceptibility of a region to excitotoxic injury, the transport capacity of a specific region may be an important factor in its vulnerability to excitotoxicity. The present study suggests that the CNS may be able to regulate the levels of excitatory amino acid uptake in response to injury. The increase in binding to these transport sites paralleled initial morphological changes in astrocytes within the denervated zone. These changes included an increase in GFAP immunoreactivity and a thickening of astrocytic processes, as well as a possible increase in cell number ~°'23. The characteristic response of astroglia to injury is also thought to include an increase in the phagocytotic ability of these cells. Furthermore, this potentially protective mechanism appears to remain intact even in the aged brain, in contrast to agerelated deficits that may contribute to impairments in reactive synaptogenesis 1"~2. Thus, the observed increase in E A A transport site density may represent another injury-induced functional change in astroglia. Previously, Taxt and Storm-Mathisen demonstrated a decrease in sodium-dependent D-aspartate uptake in the hippocampus following an entorhinal lesion and concluded that this finding was consistent with the loss of uptake into degenerating presynaptic terminals 26. The apparent difference between this decrease and our demonstration of an increase in transport site binding most probably reflects differences in techniques used and the actual processes being measured. In the earlier study, D-aspartate uptake was followed at 25 °C for 10-30 min in relatively thick slices, (150/~m) after which the tissue was rinsed, fixed in glutaraldehyde, and exposed to film (only 50% of the radioligand was retained by fixation). These conditions would be expected to enhance the visualization of radioligand that had been sequestered by surviving cells in much larger and relatively stable pools (i.e. within cytoplasmic pools, synaptic vesicles). Our autoradiographic techniques, on the other hand, examined only that radioligand that was bound to membrane transport sites. Furthermore, it appears as if the two procedures may be examining different pools. Taxt and StormMathisen concluded that their technique selectively examined transport into nerve endings and that uptake into glia was probably not present to a significant degree.
REFERENCES 1 Anderson, K.J., Scheff, S.W. and DeKosky, S.T., Reactive synaptogenesis in hippocampal area CA1 of aged and young adult
In contrast, the present demonstration that the increase in transport site binding parallels the appearance of reactive astrocytes suggests that glial cells are probably an important component of this response. Taken together, the results suggest that although the total uptake capacity of the dentate molecular layer may be decreased by denervation and subsequent loss of presynaptic terminals, some cells, particularly astrocytes, exhibit an increased density of the transport sites that may reflect their ability to contribute to the stabilization of extracellular glutamate levels in the area undergoing degeneration, repair and synaptic reorganization. Our current hypothesis proposes that a transient lesion-induced increase may represent a mechanism by which further damage to critical circuitry following loss of afferent input may be prevented. Such a model, however, raises a number of interesting questions regarding the balance between extracellular glutamate levels, receptor densities, transport capacity and the ability to maintain normal physiological function. Immediately following the loss of afferent input, the target neurons may be more vulnerable to excitotoxic insults. For instance, at 7 and 14 days after an entorhinal lesion, AMPA and N M D A receptor levels are not different from control values in the denervated zone 27. An enhanced ability to buffer the extracellular concentrations of EAAs may thus serve to limit secondary excitotoxicity resulting from runaway excitatory activity. From another perspective, an increase in E A A uptake could potentially lead to a decrease in the amount of transmitter available for normal synaptic transmission. It is also possible that an increase in E A A buffering capacity may, in some way facilitate the rebuilding of damaged circuitry, as the activity of sprouting and reactive synaptogenesis following an entorhinal lesion are known to reach peak levels at the same time as the increase in transport sites 6. Although more work is necessary to evaluate both the role of glutamate uptake as a protective mechanism and the physiological consequence of increased transport capacity, these findings highlight the delicate balance that must exist between each component of the E A A transmitter system to maintain properly functioning circuitry. Acknowledgements. This work was supported by NIA Grants AG00538, AG00096 (C.W.C.), AG08843 (K.J.A.), the American Health Assistance Foundation (UC 11394) and the American Federation for Aging Research. R.J.B. is a LEAD junior investigator (AG07918). Thanks to Jennifer Kahle for critical reading of this manuscript.
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