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Reduction of A1 adenosine receptors in rat hippocampus after kainic acid-induced limbic seizures
Neuroscience Letters 284 (2000) 49±52
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Reduction of A1 adenosine receptors in rat hippocampus after kainic acid-induced limbic seizures Antigoni Ekonomou a, GuÈnther Sperk b, George Kostopoulos a, Fevronia Angelatou a,* a
Department of Physiology, School of Medicine, University of Patras, 265 00 Patras, Greece b Department of Pharmacology, University of Innsbruck, Austria
Received 4 February 2000; received in revised form 28 February 2000; accepted 28 February 2000
Abstract In a temporal lobe epilepsy (TLE) model induced by kainic acid (KA), we examined the effect of limbic seizures on A1 adenosine receptor distribution in hippocampus and cortex. By using quantitative autoradiography, we determined a progressive decrease in A1 receptor density in CA1 and CA3 regions of hippocampus, which coincided in time with the degenerating process of hippocampal pyramidal cells. This result indicates that a great amount of A1 receptors are located postsynaptically on pyramidal cell dendrites. No difference in A1 receptor density was observed in the inner compared to the outer molecular layer of dentate gyrus, or in the infrapyramidal band compared to the outer layer of stratum oriens of CA3. This could indicate that the newly sprouted mossy ®ber glutamatergic terminals do not contain A1 receptors, thus lacking a restrain in the release of glutamate. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: A1 adenosine receptors, Limbic seizures, Kainic acid, Rat
Adenosine is a major negative neuromodulator of central nervous system synaptic activity [9,10]. Acting on its A1 receptors, endogenous adenosine can exert anticonvulsant, as well as neuroprotective effects [10,20,24]. In this frame of action, it has been reported that adenosine can suppress epileptic activity [6] and its levels are elevated signi®cantly during seizures [7]. In addition, acute tonic clonic seizures can increase the density of A1 adenosine receptors, thus enhancing its inhibitory function [2,15,17]. Seizures can also upregulate the adenosine uptake sites, thus controlling the extracellular adenosine levels [16]. Furthermore, status epilepticus (SE) may be caused by loss of adenosine anticonvulsant mechanisms, since A1 adenosine receptor antagonists facilitate the development and increase the severity of convulsive activity of SE, while endogenous adenosine is shown to prevent and suppress it [24]. Administration of kainic acid (KA) to experimental animals induces strong and long lasting convulsions, resembling SE in man [24]. The KA model constitutes one of the most established models of temporal lobe epilepsy (TLE), since the behavioral, electrophysiological, biochemical and histopathological symptoms are reminiscent of those described in patients with TLE [3]. In this model, activation of * Corresponding author. Tel.: 130-61-992389; fax: 130-61997215. E-mail address: [email protected] (F. Angelatou).
A1 adenosine receptors with adenosine analogs has been shown to suppress KA seizures [25] and to protect neurons in hippocampus, basolateral amygdaloid nucleus and piriform cortex from damage [11]. Since endogenous adenosine has been shown to play a signi®cant anticonvulsant as well as neuroprotective role, it was of interest to investigate the possible involvement of A1 adenosine receptors in TLE. With this perspective, we have studied the effect of limbic seizures on the regional density of A1 adenosine receptors in rat hippocampus and somatosensory cortex using quantitative autoradiography at various time intervals after the KA injection. Male Sprague±Dawley rats (250±350 g) were injected with 10 mg/kg KA i.p. in buffered saline or with the corresponding amount of saline (controls) [22]. The animals showing the entire behavioral spectrum including sustained limbic seizures were decapitated at different time points after the KA injection (2, 3, 4, 6, 12, 24 h: short term experiments and 48 h, 8 and 30 days: long term experiments). The brains were cut in coronal sections of 20 mm. The A1 receptor binding assay using the radioligand [ 3H]cyclohexyladenosine (12 nM [ 3H]CHA, s.a. 34.4 Ci/mmol, NEN), was performed as previously described [8]. Dried sections were exposed to tritium-sensitive ®lm ([ 3H] Hyper®lm, Amersham) along with tritium standards ([ 3H] microscales, Amersham). Quanti®cation analysis of the autoradiographic images was performed by using an MCID (M1) image
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 00 95 4- X
50
A. Ekonomou et al. / Neuroscience Letters 284 (2000) 49±52
analysis system (Imaging Res. Inc., Canada). Neuronal cell loss was evaluated by light microscopy after Nissl staining of the same sections on which the receptor binding assay had been performed. No signi®cant change in A1 receptor binding in any of the subregions of hippocampus or somatosensory cortex could be detected at the short term experiments (two to eight animals per time point, three adjacent sections per animal). At long term, a signi®cant decrease in A1 receptors in CA1 and CA3 regions of hippocampus as well as in hilus of dentate gyrus (DG), ranging from 19 to 32% was observed, starting at 48 h after the KA injection (Table 1, Fig. 1B). At the same time point, a smaller (15%) but signi®cant reduction in the inner and outer molecular layer (ML) of the DG could be detected (Table 1, Fig. 1B). Light microscopy revealed a pyramidal cell loss in CA1 and CA3 regions of hippocampus (Fig. 1B) at 48 h after KA injection. Eight days after limbic seizures, the decreases remained mainly at the same level, being slightly larger (33%) in CA1 region (Table 1, Fig. 1C). Thirty days after seizures, the decreases in A1 receptor density became substantially larger reaching in CA1 region 70%, in CA3 40% and in the inner and outer ML of DG 24% compared to control animals (Table 1, Fig. 1D1). At that time point (30 days after seizures), the Nissl-stained sections showed an evidently more severe neuronal cell loss compared to the corresponding ones of 48 h after the KA injection in both subregions,
which was more prominent in CA1 area (Fig.1D1). These observations are in accordance with previous histopathological data [22]. However, it must be noted, that the degree of cell degeneration varied among the epileptic animals of 30 days after seizures and in two animals out of eight tested it was not detectable in regions CA1 and CA3. In the example of Fig. 1D2, the intact appearance of Nissl stained pyramidal neurons corresponds well to the calculated small (15% in CA1) or no decrease (0% in CA3) of A1 receptor binding. Indeed, in each animal examined at any time point, the degree of A1 receptor binding decrease coincided well with the degree of neuronal degeneration in each hippocampal subregion. The mean values of A1 receptor binding were not larger in areas where sprouting of the mossy ®bers occurs [12,18] (i.e. the inner compared to the outer ML, or in the infrapyramidal band -inner third- compared to the outer layer of CA3 stratum oriens) at any time point in the long term experiments in the epileptic animals (Table 1). In cortex, no change in A1 receptor density could be detected in any of its layers at long term experiments (Table 1, Fig. 1). In the present study we have investigated, for the ®rst time, the effect of limbic seizures induced by administration of KA on the regional density of A1 adenosine receptors in dorsal hippocampus and somatosensory cortex in the rat brain.
Table 1 Speci®c [ 3H]CHA (12 nM) binding in hippocampus and cortex of KA-treated epileptic animals compared to controls (long term experiments) a Region
[ 3H]CHA binding (fmoles\mg tissue) Time after the injection Control
48 h
Control
8 days
Control
30 days
457 ^ 21 510 ^ 6
345 ^ 23 (24% # )* 411 ^ 23 (19% # )*
469 ^ 16 529 ^ 14
320 ^ 11 (32% # )** 352 ^ 8 (33% # )**
457 ^ 21 510 ^ 6
145 ^ 42 (68% # )** 152 ^ 50 (70% # )**
Hippocampus CA1 Stratum oriens Stratum radiatum CA3 Stratum oriens Infrapyramidal band Outer layer of stratum oriens Stratum radiatum Dentate Gyrus Molecular layer Inner molecular layer Outer molecular layer Hilus
343 ^ 8 346 ^ 8 380 ^ 6
239 ^ 5 (30% # )** 240 ^ 6 (31% # )** 291 ^ 16 (23% # )**
249 ^ 7 259 ^ 10 298 ^ 4
183 ^ 15 (26% # )* 183 ^ 14 (29% # )* 213 ^ 18 (28% # )*
343 ^ 8 346 ^ 8 361 ^ 9
206 ^ 35 (40% # )* 209 ^ 36 (40% # )* 235 ^ 37 (35% # )*
344 ^ 3 370 ^ 8 290 ^ 5
292 ^ 8 (15% # )* 317 ^ 12 (14% # )* 198 ^ 10 (32% # )**
350 ^ 5 380 ^ 8 233 ^ 4
304 ^ 13 (13% # )* 324 ^ 11 (15% # )* 193 ^ 3 (17% # )*
344 ^ 3 370 ^ 8 290 ^ 5
261 ^ 23 (24% # )* 285 ^ 25 (23% # )* 256 ^ 29 (12% # )
Cortex Somatosensory Layers I-III IV V±VI
230 ^ 7 282 ^ 8 227 ^ 9
214 ^ 8 289 ^ 6 244 ^ 6
199 ^ 2 284 ^ 14 222 ^ 6
207 ^ 1 261 ^ 7 204 ^ 3
230 ^ 7 282 ^ 8 227 ^ 9
204 ^ 16 264 ^ 19 225 ^ 16
a
Data are mean ^ SE obtained from three to eight animals per time point (measurements are derived from three adjacent sections per animal). Statistically signi®cant differences compared to the respective controls are indicated as *P , 0:05, **P , 0:005 (Unpaired Student's t-test). Numbers in parentheses indicate the percentage of decrease.
A. Ekonomou et al. / Neuroscience Letters 284 (2000) 49±52
Fig. 1. Digitized autoradiographic images from coronal brain sections showing the distribution of [ 3H]CHA binding sites, as detected by quantitative autoradiography (left column) and their corresponding Nissl-stained sections (right column) from control and KA-treated rats in different time points after KA injection. Non-speci®c binding represented less than 5% of total binding. (A) Control animals, (B) 48 h, (C) 8 days and (D1), (D2) 30 days after KA injection. Note the progressive neurodegeneration in CA1 and CA3 subregions of hippocampus along with the decrease in A1 receptor density in same regions, which was observed in the majority of animals (A, B, C, D1). In two/eight animals, (example in D2) neither degeneration nor change in receptor density was observed. DG, hippocampal dentate gyrus; SS, somatosensory cortex.
Limbic seizures induced by KA cause brain damage, the most severely affected areas being hippocampus, entorhinal and piriform cortices and amygdala. Within the hippocampus, CA3 and CA1 pyramidal neurons and interneurons of hilus of the DG (mossy cells and somatostatin-containing neurons) appear consistently damaged, whereas granule cells of the DG and the pyramidal cells of CA2 subregion are spared [3,21,23]. In this study, the decreases observed in A1 receptor density in the hippocampal subregions, seem to be related
51
to speci®c neuronal degeneration in these areas, rather than to be directly induced by the convulsive activity of SE. We favor this interpretation because of the regional and temporal correlation between A1 receptor decrease and the pyramidal cell degeneration, according to our autoradiographic and histological data. The signi®cant reductions in A1 receptor binding occur in CA1 and CA3 regions ®rst 48 h after seizures, at the time point when the pyramidal cell degeneration is ®rst detectable in these hippocampal areas [22] and become substantially larger thirty days after seizures, when severe cell degeneration is seen. This fact con®rms recent data [14], showing that a great amount of A1 receptors is located postsynaptically on the dendrites of pyramidal cells in CA1 and CA3 areas. The substantially lower density in A1 receptors seen thirty days after seizures in CA1 compared to CA3 region could additionally be related to an attenuation of the glutamatergic input to CA1 subregion, originating from the degenerating CA3 neurons (Schaffer collaterals). The terminals of Schaffer collaterals are endowed by A1 adenosine receptors controlling thus the release of glutamate [4]. Previous results in our laboratory have shown that, tonicclonic seizures induced by acute administration of pentylenetetrazole (PTZ) elicit an upregulation of A1 adenosine receptors (within 1 h after seizures) in hippocampal and cortical membranes [16]. Limbic seizures of SE type do not seem to elicit up- or down- regulation of A1 receptors at least at the time points of the short term experiments, i.e. 2±24 h, that we examined. The difference may be attributed to the fact that PTZ-induced seizures differ substantially from those in TLE models [3]. Hilar cells express A1 adenosine receptors [14] and degenerate after KA seizures [22]. This may explain the demonstrated here reduction of hilar adenosine receptor density. The slight but signi®cant reduction in A1 receptor binding seen 48 h after KA in the ML of DG could also be due to degeneration of hilar cells, mainly glutamatergic mossy cells, which form synaptic contacts with granule cell dendrites in the ML of DG and with GABAergic interneurons in hilus [21]. The relatively larger decrease in binding seen in ML 30 days after seizures could be additionally due to the degeneration of the glutamatergic input of the perforant path, originating from the entorhinal cortex [22] the endings of which also contain A1 adenosine receptors [5]. Glutamate release at mossy ®ber-CA3 terminals is regulated presynaptically by A1 adenosine receptors [1,13]. Under our experimental conditions, in the KA limbic seizures model, mossy ®bers sprout and make functional synaptic connections with dendrites of granule cells in the inner ML, as well as with dendrites of pyramidal cells in the infrapyramidal band of CA3 stratum oriens, contributing thus to perpetuation of seizure activity [12,18,21]. Our results, showing that A1 receptor density is not greater in the inner compared to the outer ML of DG or in the infrapyramidal band compared to the outer layer of CA3 stratum oriens, indicate that the newly sprouted mossy ®bers may not
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A. Ekonomou et al. / Neuroscience Letters 284 (2000) 49±52
contain A1 receptors, which would restrain the release of glutamate. Such a defect would facilitate epileptic activity, since even the newly synthesized adenosine by the enzyme 5 0 -nucleotidase appearing on the sprouted mossy ®bers [19], would not be functional presynaptically. If con®rmed, this ®nding may point to a novel factor contributing to epileptogenesis in TLE. The lack of any changes in A1 adenosine receptor density at any of the cortical layers tested is in line with histopathological data showing no neuronal damage in cortex in this TLE model [22]. In conclusion, the progressive reductions in A1 receptor density coincide in time with the degenerating process of pyramidal cells in these regions, indicating that a great amount of A1 receptors is located postsynaptically on the dendrites of pyramidal cells in CA1 and CA3 areas. Since areas where an evident sprouting of mossy ®bers occurs (inner ML of DG and infrapyramidal band of CA3 stratum oriens) do not show greater A1 receptor density compared to the outer ML and outer stratum oriens respectively, this may indicate that newly sprouted mossy ®ber terminals are not endowed by A1 receptors which would restrain the release of glutamate, thus facilitating epileptic activity. A. Ekonomou is a fellow of the National Fellowship Foundation. This study was supported by a grant (Karatheodoris) from the Research Committee of the University of Patras, Greece. [1] Alzheimer, C., Sutor, B. and Bruggencate, G., Disinhibition of hippocampal CA3 neurons induced by suppression of an adenosine A1 receptor-mediated inhibitory tonus: pre- and postsynaptic components, Neuroscience, 57 (1993) 565± 575. [2] Angelatou, F., Pagonopoulou, O. and Kostopoulos, G., Changes in seizure latency correlate with alterations in A1 adenosine receptor binding during daily repeated pentylentetrazol-induced convulsions in different mouse brain areas, Neurosci. Lett., 132 (1991) 203±206. [3] Ben-Ari, Y., Limbic seizure and brain damage induced by kainic acid: mechanisms and relevance to human temporal lobe epilepsy, Neuroscience, 14 (1985) 375±403. [4] Corradetti, R., LoConte, G., Moroni, F., Passani, M.B. and Pepeu, G., Adenosine decreases aspartate and glutamate release from rat hippocampal slices, Eur. J. Pharmacol., 104 (1984) 19±26. [5] Dragunow, M., Murphy, K., Leslie, R.A. and Robertson, H.A., Localization of adenosine A1-receptors to the terminals of the perforant path, Brain Res., 462 (1988) 252±257. [6] Dunwiddie, T.V. and Worth, T.J., Sedative and anticonvulsant effects of adenosine analogs in mouse and rat, J. Pharmacol. Exp. Ther., 220 (1982) 70±76. [7] During, M.J. and Spencer, D.P., Adenosine: a potential mediator of seizure arrest and postictal refractoriness, Ann. Neurol., 32 (1992) 618±624. [8] Ekonomou, A., Angelatou, F., Vergnes, M. and Kostopoulos, G., Lower density of A1 adenosine receptors in nucleus reticularis thalami in rats with genetic absence epilepsy, NeuroReport, 9 (1998) 2135±2140. [9] Fredholm, B.B., Adenosine receptors in the central nervous system, News. Physiol. Sci., 10 (1995) 122±128.
[10] Kostopoulos, G., Adenosine: a molecule for synaptic homeostasis? Evolution of current concepts on the physiological and pathological roles of adenosine, In M. Avoli, R. Dykes, P. Gloor, and T. Reader (Eds.), Neurotransmitters and Cortical Function. From Molecules to Mind, Plenum Press, USA, 1988, pp. 415±435. [11] MacGregor, D.G., Graham, D.I., Jones, P.A. and Stone, T.W., Protection by an adenosine analogue against kainate-induced extrahippocampal neuropathology, Gen. Pharmacol., 31 (1998) 223±238. [12] Marksteiner, J., Ortler, M., Bellmann, R. and Sperk, G., Neuropeptide Y biosynthesis is markedly induced in mossy ®bers during temporal lobe epilepsy of the rat, Neurosci. Lett., 112 (1990) 143±148. [13] Muzzolini, A., Bregola, G., Bianchi, C., Beani, L. and Simonato, M., Characterization of glutamate and [ 3H]-aspartate out¯ow from various in vitro preparations of the rat hippocampus, Neurochem. Int, 31 (1997) 113±124. [14] Ochiishi, T., Chen, L., Yukawa, A., Satoh, Y., Sekino, Y., Arai, T., Nakata, H. and Miyamoto, H., Cellular localization of adenosine A1 receptors in rat forebrain: immunohistochemical analysis using adenosine A1 receptor-speci®c monoclonal antibody, J. Comp. Neurol., 411 (1999) 301± 316. [15] Pagonopoulou, O., Angelatou, F. and Kostopoulos, G., Effect of Pentylentetrazol-induced seizures on A1 Adenosine receptor regional density in the mouse brain: a quantitative autoradiographic study, Neuroscience, 56 (1993) 711±716. [16] Pagonopoulou, O. and Angelatou, F., Time development and regional distribution of [ 3H]Nitrobenzylthioinosine adenosine uptake sites binding in the mouse brain after acute pentylenetetrazol-induced seizures, J. Neurosci. Res., 53 (1998) 433±442. [17] Psarropoulou, C., Matsokis, N., Angelatou, F. and Kostopoulos, G., Pentylentetrazol-induced seizures decrease aaminoboutyric acid-mediated recurrent inhibition and enhance adenosine-mediated depression, Epilepsia, 35 (1994) 12±19. [18] Represa, A. and Ben-Ari, Y., Kindling is associated with the formation of novel mossy ®bre synapses in the CA3 region, Exp. Brain Res., 92 (1992) 69±78. [19] Schoen, S.W., Ebert, U. and Loscher, W., 5 0 -Noucleotidase activity of mossy ®bers in the dentate gyrus of normal and epileptic rats, Neuroscience, 93 (1999) 519±526. [20] Schubert, P., Ogata, T., Marchini, C., Ferroni, S. and Rudolphi, K., Protective mechanisms of adenosine in neurons and glial cells, Ann. N.Y. Acad. Sci. USA, 825 (1997) 1±10. [21] Sperk, G., Kainic acid seizures in the rat, Prog. Neurobiol., 42 (1994) 1±32. [22] Sperk, G., Lassman, H., Baran, H., Kish, S.J., Seitelberger, F. and Hornykiewicz, O., Kainic acid induced seizures: neurochemical and histopathological changes, Neuroscience, 10 (1983) 1301±1315. [23] Sperk, G., Schwarzer, C., Ho¯ehner, J., Tsunashima, K., Kirchmair, E. and Vezzani, A., Neuropeptides as markers for functional changes in the hippocampus of epileptic rats, In N. Kato (Ed.), The Hippocampus: Functions and Clinical Relevance, Elsevier, Amsterdam, 1996, pp. 415±435. [24] Young, D. and Dragunow, M., Status epilepticus may be caused by loss of adenosine anticonvulsant mechanisms, Neuroscience, 58 (1994) 245±261. [25] Zhang, G., Franklin, P.H. and Murray, T.F., Anticonvulsant effects of N-ethylcarvoxamidoadenosine against kainic acid induced behavioral seizures in the rat prepiriform cortex, Neurosci. Lett., 114 (1990) 345±350.
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Reduction of A1 adenosine receptors in rat hippocampus after kainic acid-induced limbic seizures Antigoni Ekonomou a, GuÈnther Sperk b, George Kostopoulos a, Fevronia Angelatou a,* a
Department of Physiology, School of Medicine, University of Patras, 265 00 Patras, Greece b Department of Pharmacology, University of Innsbruck, Austria
Received 4 February 2000; received in revised form 28 February 2000; accepted 28 February 2000
Abstract In a temporal lobe epilepsy (TLE) model induced by kainic acid (KA), we examined the effect of limbic seizures on A1 adenosine receptor distribution in hippocampus and cortex. By using quantitative autoradiography, we determined a progressive decrease in A1 receptor density in CA1 and CA3 regions of hippocampus, which coincided in time with the degenerating process of hippocampal pyramidal cells. This result indicates that a great amount of A1 receptors are located postsynaptically on pyramidal cell dendrites. No difference in A1 receptor density was observed in the inner compared to the outer molecular layer of dentate gyrus, or in the infrapyramidal band compared to the outer layer of stratum oriens of CA3. This could indicate that the newly sprouted mossy ®ber glutamatergic terminals do not contain A1 receptors, thus lacking a restrain in the release of glutamate. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: A1 adenosine receptors, Limbic seizures, Kainic acid, Rat
Adenosine is a major negative neuromodulator of central nervous system synaptic activity [9,10]. Acting on its A1 receptors, endogenous adenosine can exert anticonvulsant, as well as neuroprotective effects [10,20,24]. In this frame of action, it has been reported that adenosine can suppress epileptic activity [6] and its levels are elevated signi®cantly during seizures [7]. In addition, acute tonic clonic seizures can increase the density of A1 adenosine receptors, thus enhancing its inhibitory function [2,15,17]. Seizures can also upregulate the adenosine uptake sites, thus controlling the extracellular adenosine levels [16]. Furthermore, status epilepticus (SE) may be caused by loss of adenosine anticonvulsant mechanisms, since A1 adenosine receptor antagonists facilitate the development and increase the severity of convulsive activity of SE, while endogenous adenosine is shown to prevent and suppress it [24]. Administration of kainic acid (KA) to experimental animals induces strong and long lasting convulsions, resembling SE in man [24]. The KA model constitutes one of the most established models of temporal lobe epilepsy (TLE), since the behavioral, electrophysiological, biochemical and histopathological symptoms are reminiscent of those described in patients with TLE [3]. In this model, activation of * Corresponding author. Tel.: 130-61-992389; fax: 130-61997215. E-mail address: [email protected] (F. Angelatou).
A1 adenosine receptors with adenosine analogs has been shown to suppress KA seizures [25] and to protect neurons in hippocampus, basolateral amygdaloid nucleus and piriform cortex from damage [11]. Since endogenous adenosine has been shown to play a signi®cant anticonvulsant as well as neuroprotective role, it was of interest to investigate the possible involvement of A1 adenosine receptors in TLE. With this perspective, we have studied the effect of limbic seizures on the regional density of A1 adenosine receptors in rat hippocampus and somatosensory cortex using quantitative autoradiography at various time intervals after the KA injection. Male Sprague±Dawley rats (250±350 g) were injected with 10 mg/kg KA i.p. in buffered saline or with the corresponding amount of saline (controls) [22]. The animals showing the entire behavioral spectrum including sustained limbic seizures were decapitated at different time points after the KA injection (2, 3, 4, 6, 12, 24 h: short term experiments and 48 h, 8 and 30 days: long term experiments). The brains were cut in coronal sections of 20 mm. The A1 receptor binding assay using the radioligand [ 3H]cyclohexyladenosine (12 nM [ 3H]CHA, s.a. 34.4 Ci/mmol, NEN), was performed as previously described [8]. Dried sections were exposed to tritium-sensitive ®lm ([ 3H] Hyper®lm, Amersham) along with tritium standards ([ 3H] microscales, Amersham). Quanti®cation analysis of the autoradiographic images was performed by using an MCID (M1) image
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 00 95 4- X
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A. Ekonomou et al. / Neuroscience Letters 284 (2000) 49±52
analysis system (Imaging Res. Inc., Canada). Neuronal cell loss was evaluated by light microscopy after Nissl staining of the same sections on which the receptor binding assay had been performed. No signi®cant change in A1 receptor binding in any of the subregions of hippocampus or somatosensory cortex could be detected at the short term experiments (two to eight animals per time point, three adjacent sections per animal). At long term, a signi®cant decrease in A1 receptors in CA1 and CA3 regions of hippocampus as well as in hilus of dentate gyrus (DG), ranging from 19 to 32% was observed, starting at 48 h after the KA injection (Table 1, Fig. 1B). At the same time point, a smaller (15%) but signi®cant reduction in the inner and outer molecular layer (ML) of the DG could be detected (Table 1, Fig. 1B). Light microscopy revealed a pyramidal cell loss in CA1 and CA3 regions of hippocampus (Fig. 1B) at 48 h after KA injection. Eight days after limbic seizures, the decreases remained mainly at the same level, being slightly larger (33%) in CA1 region (Table 1, Fig. 1C). Thirty days after seizures, the decreases in A1 receptor density became substantially larger reaching in CA1 region 70%, in CA3 40% and in the inner and outer ML of DG 24% compared to control animals (Table 1, Fig. 1D1). At that time point (30 days after seizures), the Nissl-stained sections showed an evidently more severe neuronal cell loss compared to the corresponding ones of 48 h after the KA injection in both subregions,
which was more prominent in CA1 area (Fig.1D1). These observations are in accordance with previous histopathological data [22]. However, it must be noted, that the degree of cell degeneration varied among the epileptic animals of 30 days after seizures and in two animals out of eight tested it was not detectable in regions CA1 and CA3. In the example of Fig. 1D2, the intact appearance of Nissl stained pyramidal neurons corresponds well to the calculated small (15% in CA1) or no decrease (0% in CA3) of A1 receptor binding. Indeed, in each animal examined at any time point, the degree of A1 receptor binding decrease coincided well with the degree of neuronal degeneration in each hippocampal subregion. The mean values of A1 receptor binding were not larger in areas where sprouting of the mossy ®bers occurs [12,18] (i.e. the inner compared to the outer ML, or in the infrapyramidal band -inner third- compared to the outer layer of CA3 stratum oriens) at any time point in the long term experiments in the epileptic animals (Table 1). In cortex, no change in A1 receptor density could be detected in any of its layers at long term experiments (Table 1, Fig. 1). In the present study we have investigated, for the ®rst time, the effect of limbic seizures induced by administration of KA on the regional density of A1 adenosine receptors in dorsal hippocampus and somatosensory cortex in the rat brain.
Table 1 Speci®c [ 3H]CHA (12 nM) binding in hippocampus and cortex of KA-treated epileptic animals compared to controls (long term experiments) a Region
[ 3H]CHA binding (fmoles\mg tissue) Time after the injection Control
48 h
Control
8 days
Control
30 days
457 ^ 21 510 ^ 6
345 ^ 23 (24% # )* 411 ^ 23 (19% # )*
469 ^ 16 529 ^ 14
320 ^ 11 (32% # )** 352 ^ 8 (33% # )**
457 ^ 21 510 ^ 6
145 ^ 42 (68% # )** 152 ^ 50 (70% # )**
Hippocampus CA1 Stratum oriens Stratum radiatum CA3 Stratum oriens Infrapyramidal band Outer layer of stratum oriens Stratum radiatum Dentate Gyrus Molecular layer Inner molecular layer Outer molecular layer Hilus
343 ^ 8 346 ^ 8 380 ^ 6
239 ^ 5 (30% # )** 240 ^ 6 (31% # )** 291 ^ 16 (23% # )**
249 ^ 7 259 ^ 10 298 ^ 4
183 ^ 15 (26% # )* 183 ^ 14 (29% # )* 213 ^ 18 (28% # )*
343 ^ 8 346 ^ 8 361 ^ 9
206 ^ 35 (40% # )* 209 ^ 36 (40% # )* 235 ^ 37 (35% # )*
344 ^ 3 370 ^ 8 290 ^ 5
292 ^ 8 (15% # )* 317 ^ 12 (14% # )* 198 ^ 10 (32% # )**
350 ^ 5 380 ^ 8 233 ^ 4
304 ^ 13 (13% # )* 324 ^ 11 (15% # )* 193 ^ 3 (17% # )*
344 ^ 3 370 ^ 8 290 ^ 5
261 ^ 23 (24% # )* 285 ^ 25 (23% # )* 256 ^ 29 (12% # )
Cortex Somatosensory Layers I-III IV V±VI
230 ^ 7 282 ^ 8 227 ^ 9
214 ^ 8 289 ^ 6 244 ^ 6
199 ^ 2 284 ^ 14 222 ^ 6
207 ^ 1 261 ^ 7 204 ^ 3
230 ^ 7 282 ^ 8 227 ^ 9
204 ^ 16 264 ^ 19 225 ^ 16
a
Data are mean ^ SE obtained from three to eight animals per time point (measurements are derived from three adjacent sections per animal). Statistically signi®cant differences compared to the respective controls are indicated as *P , 0:05, **P , 0:005 (Unpaired Student's t-test). Numbers in parentheses indicate the percentage of decrease.
A. Ekonomou et al. / Neuroscience Letters 284 (2000) 49±52
Fig. 1. Digitized autoradiographic images from coronal brain sections showing the distribution of [ 3H]CHA binding sites, as detected by quantitative autoradiography (left column) and their corresponding Nissl-stained sections (right column) from control and KA-treated rats in different time points after KA injection. Non-speci®c binding represented less than 5% of total binding. (A) Control animals, (B) 48 h, (C) 8 days and (D1), (D2) 30 days after KA injection. Note the progressive neurodegeneration in CA1 and CA3 subregions of hippocampus along with the decrease in A1 receptor density in same regions, which was observed in the majority of animals (A, B, C, D1). In two/eight animals, (example in D2) neither degeneration nor change in receptor density was observed. DG, hippocampal dentate gyrus; SS, somatosensory cortex.
Limbic seizures induced by KA cause brain damage, the most severely affected areas being hippocampus, entorhinal and piriform cortices and amygdala. Within the hippocampus, CA3 and CA1 pyramidal neurons and interneurons of hilus of the DG (mossy cells and somatostatin-containing neurons) appear consistently damaged, whereas granule cells of the DG and the pyramidal cells of CA2 subregion are spared [3,21,23]. In this study, the decreases observed in A1 receptor density in the hippocampal subregions, seem to be related
51
to speci®c neuronal degeneration in these areas, rather than to be directly induced by the convulsive activity of SE. We favor this interpretation because of the regional and temporal correlation between A1 receptor decrease and the pyramidal cell degeneration, according to our autoradiographic and histological data. The signi®cant reductions in A1 receptor binding occur in CA1 and CA3 regions ®rst 48 h after seizures, at the time point when the pyramidal cell degeneration is ®rst detectable in these hippocampal areas [22] and become substantially larger thirty days after seizures, when severe cell degeneration is seen. This fact con®rms recent data [14], showing that a great amount of A1 receptors is located postsynaptically on the dendrites of pyramidal cells in CA1 and CA3 areas. The substantially lower density in A1 receptors seen thirty days after seizures in CA1 compared to CA3 region could additionally be related to an attenuation of the glutamatergic input to CA1 subregion, originating from the degenerating CA3 neurons (Schaffer collaterals). The terminals of Schaffer collaterals are endowed by A1 adenosine receptors controlling thus the release of glutamate [4]. Previous results in our laboratory have shown that, tonicclonic seizures induced by acute administration of pentylenetetrazole (PTZ) elicit an upregulation of A1 adenosine receptors (within 1 h after seizures) in hippocampal and cortical membranes [16]. Limbic seizures of SE type do not seem to elicit up- or down- regulation of A1 receptors at least at the time points of the short term experiments, i.e. 2±24 h, that we examined. The difference may be attributed to the fact that PTZ-induced seizures differ substantially from those in TLE models [3]. Hilar cells express A1 adenosine receptors [14] and degenerate after KA seizures [22]. This may explain the demonstrated here reduction of hilar adenosine receptor density. The slight but signi®cant reduction in A1 receptor binding seen 48 h after KA in the ML of DG could also be due to degeneration of hilar cells, mainly glutamatergic mossy cells, which form synaptic contacts with granule cell dendrites in the ML of DG and with GABAergic interneurons in hilus [21]. The relatively larger decrease in binding seen in ML 30 days after seizures could be additionally due to the degeneration of the glutamatergic input of the perforant path, originating from the entorhinal cortex [22] the endings of which also contain A1 adenosine receptors [5]. Glutamate release at mossy ®ber-CA3 terminals is regulated presynaptically by A1 adenosine receptors [1,13]. Under our experimental conditions, in the KA limbic seizures model, mossy ®bers sprout and make functional synaptic connections with dendrites of granule cells in the inner ML, as well as with dendrites of pyramidal cells in the infrapyramidal band of CA3 stratum oriens, contributing thus to perpetuation of seizure activity [12,18,21]. Our results, showing that A1 receptor density is not greater in the inner compared to the outer ML of DG or in the infrapyramidal band compared to the outer layer of CA3 stratum oriens, indicate that the newly sprouted mossy ®bers may not
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contain A1 receptors, which would restrain the release of glutamate. Such a defect would facilitate epileptic activity, since even the newly synthesized adenosine by the enzyme 5 0 -nucleotidase appearing on the sprouted mossy ®bers [19], would not be functional presynaptically. If con®rmed, this ®nding may point to a novel factor contributing to epileptogenesis in TLE. The lack of any changes in A1 adenosine receptor density at any of the cortical layers tested is in line with histopathological data showing no neuronal damage in cortex in this TLE model [22]. In conclusion, the progressive reductions in A1 receptor density coincide in time with the degenerating process of pyramidal cells in these regions, indicating that a great amount of A1 receptors is located postsynaptically on the dendrites of pyramidal cells in CA1 and CA3 areas. Since areas where an evident sprouting of mossy ®bers occurs (inner ML of DG and infrapyramidal band of CA3 stratum oriens) do not show greater A1 receptor density compared to the outer ML and outer stratum oriens respectively, this may indicate that newly sprouted mossy ®ber terminals are not endowed by A1 receptors which would restrain the release of glutamate, thus facilitating epileptic activity. A. Ekonomou is a fellow of the National Fellowship Foundation. This study was supported by a grant (Karatheodoris) from the Research Committee of the University of Patras, Greece. [1] Alzheimer, C., Sutor, B. and Bruggencate, G., Disinhibition of hippocampal CA3 neurons induced by suppression of an adenosine A1 receptor-mediated inhibitory tonus: pre- and postsynaptic components, Neuroscience, 57 (1993) 565± 575. [2] Angelatou, F., Pagonopoulou, O. and Kostopoulos, G., Changes in seizure latency correlate with alterations in A1 adenosine receptor binding during daily repeated pentylentetrazol-induced convulsions in different mouse brain areas, Neurosci. Lett., 132 (1991) 203±206. [3] Ben-Ari, Y., Limbic seizure and brain damage induced by kainic acid: mechanisms and relevance to human temporal lobe epilepsy, Neuroscience, 14 (1985) 375±403. [4] Corradetti, R., LoConte, G., Moroni, F., Passani, M.B. and Pepeu, G., Adenosine decreases aspartate and glutamate release from rat hippocampal slices, Eur. J. Pharmacol., 104 (1984) 19±26. [5] Dragunow, M., Murphy, K., Leslie, R.A. and Robertson, H.A., Localization of adenosine A1-receptors to the terminals of the perforant path, Brain Res., 462 (1988) 252±257. [6] Dunwiddie, T.V. and Worth, T.J., Sedative and anticonvulsant effects of adenosine analogs in mouse and rat, J. Pharmacol. Exp. Ther., 220 (1982) 70±76. [7] During, M.J. and Spencer, D.P., Adenosine: a potential mediator of seizure arrest and postictal refractoriness, Ann. Neurol., 32 (1992) 618±624. [8] Ekonomou, A., Angelatou, F., Vergnes, M. and Kostopoulos, G., Lower density of A1 adenosine receptors in nucleus reticularis thalami in rats with genetic absence epilepsy, NeuroReport, 9 (1998) 2135±2140. [9] Fredholm, B.B., Adenosine receptors in the central nervous system, News. Physiol. Sci., 10 (1995) 122±128.
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