- Email: [email protected]
Extracellular concentrations of amino acid transmitters in ventral hippocampus during and after the development of kindling
Brain Research, 540 (1991) 315-318
315
Elsevier BRES 24515
Extracellular concentrations of amino acid transmitters in ventral hippocampus during and after the development of kindling W . Q . Z h a n g 1, P . M . H u d s o n 1, T . J . S o b o t k a 2, J . S . H o n g I a n d H . A .
Tilson 1
1Laboratory of Molecular and Integrative Neuroscience, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 (U.S.A.) and 2Neurobehavioral Toxicology, Federal Drug Administration, Washington, DC 20204 (U.S.A.)
(Accepted 23 October 1990) Key words: Kindling; Ventral hippocampus; Brain dialysis; Amino acid
This study examined the possible involvement of amino acid release from ventral hippocampus in the establishment and maintenance of kindling in rats. Release of amino acids from ventral hippocampus was measured by microdialysis coupled with high-performance liquid chromatography. Samples were obtained by microdialysisperfusion of freely moving animals receivingdeep prepiriform cortex (DPC) electrical stimulation. Samples of perfusate were collected before, during and after kindling was established. DPC kindling stimulation significantly increased concentrations of glutamate (Glu) and glycine (Gly) in perfusate from ventral hippocampus during kindling. Increased basal release of Glu was evident up to 30 days after the last electrical stimulation. We conclude that release of Glu and Gly in the ventral hippocampus may play an important role during establishment, but not in maintenance of kindling. Kindling is a widely accepted animal model of epilepsy in which repeated subconvulsive electrical stimulation results in progressively more intense seizures and eventually generalized motor convulsions 9-11'18J9'26"27. Once the kindled state has been achieved, the increased sensitivity to subconvulsive stimulation is persistent 1°'27. The neural mechanisms responsible for the kindling phenomenon have been extensively investigated. Changes in levels of calcium binding protein 1, protein phosphorylation 24, and neurotransmitter receptor function 21'23"29'30 have been reported in kindled animals. Recent research has begun to explore the role that putative amino acid neurotransmitters may play in epilepsy and the kindling phenomenon. For example, the release of excitatory amino acids (EAA) has been postulated to be involved in the generation and expression of epileptic seizures 3'5'7"15"17"31-33.In addition, it was reported that electroconvulsive shocks also increased the release of E A A measured by in vivo microdialysis 14. Subsequent research has found a change in the number of quisqualate-sensitive glutamate receptor sites during the kindling process 29, while changes in EAA receptors have been associated with long-term potentiation 4 and kindling-like phenomena 6,8'~3'21"23. The objective of the present series of experiments was to investigate changes in the release of amino acids. including glutamate, glutamine, taurine, glycine, and aspartate, during and after development of electricalinduced kindling in the rat. These experiments utilized a
microdialysis procedure, which allows for the continuous monitoring of neurotransmitter concentrations in the perfusate obtained from conscious and freely moving animals TM. Electrical stimulation of the deep prepiriform cortex was used to produce kindling, while the ventral hippocampus was perfused using dialysis probes. The ventral hippocampus was chosen because of its known importance in the development of seizures 16. Male Fischer-344 rats (Charles River Breeding Co., Raleigh, NC) weighing 250-300 g at the time of surgery were anesthetized with chloral hydrate (350 mg/kg, i.p.) and implanted unilaterally with a single isolated bipolar stainless steel electrode (0.25 mm diameter). The incisor bar was set 5 mm above the intraural line and the coordinates for the right deep prepiriform cortex (DPC) were 3.8 mm anterior and 2.5 mm lateral to bregma, and 7.5 mm below the surface of the skull. The electrode plug was firmly anchored to the skull with miniature screws and dental acrylic cement. At least 7 days later, a dialysis loop 37 was implanted stereotaxically in the left ventral hippocampus using the following coordinates: 5.8 mm posterior and 5.0 mm lateral to the midline, and 7.5 mm below vertical from dura with the incisor bar set at 3.3 mm below the intraural line. The dialysis probe was connected by polyethylene tubing (PE10) to a swivel and Harvard infusion pump. The dialysis tubes were perfused continuously with artificial cerebral spinal fluid pumped through 0.22 mm Millipore syringe filters at a constant rate of 2/~l/min. Dialysis tubes
Correspondence: J.S. Hong, LMIN, NIEHS, P.O. Box 12233, Research Triangle Park, NC 27709, U.S.A.
316
i[
were anchored to the skull using dental acrylic cement. In the first e x p e r i m e n t , 24 h after implantation of the dialysis loop, 4 consecutive 1-h fractions were collected in microcentrifuge tubes containing 6 ~1 of 2.0 N perchloric acid. These samples were used to calculate the baseline release rate prior to stimulation. W h e n a relatively constant flow rate was o b t a i n e d , stimulation of the D P C began. Electrical stimulation (400/~A) was p r o d u c e d by a Grass-88 stimulator delivered through a modified constant current output. The stimulus consisted of a 1 s train of 50-Hz biphasic square wave current with 1 ms duration. In this e x p e r i m e n t , stimuli were delivered once per hour until kindling occurred. A t the time of stimulation, rats were rated for severity of seizure using a scale similar to that described by Racine 26 (chewing, stage l; head-nodding, stage 2; unilateral limbic clonus, state 3; rearing with bilateral forelimb clonus, stage 4; and bilateral forelimb clonus with rearing and falling, stage 5); each rating was associated with the perfusate sample collected at the time. Perfusions were t e r m i n a t e d after reaching stage 5. In a second e x p e r i m e n t , stimulating electrodes were implanted in the right D P C as described previously. Kindling was p r o d u c e d by stimulation twice daily for 8-10 days at intervals of at least 8 h, until 3 consecutive stimuli e v o k e d kindled seizures at stage 5. A f t e r the last kindled seizure, each animal was paired with a weightand age-matched control animal also implanted with an electrode, but not stimulated. One group of rats was i m p l a n t e d with dialysis tubes in the left ventral hippocampus. Twenty-four hours later, these animals were perfused for 8 consecutive hours. A n o t h e r group of rats was perfused at 30 days after the last stimulus, while a third group was perfused 90 days after the last stimulus. This e x p e r i m e n t was designed to d e t e r m i n e alterations in
m--e--~
I
O0
__
I
I
5 10 15 Number of shmulotions
I
20
Fig. I. Development of kindling following stimulation of the deep
prepiriform cortex while the left ventral hippocampus was perfused using a microdialysis tube. Rats were stimulated once per hour. Data are average seizure ratings for 10 rats. Stage 5 indicates bilateral forelimb clonus with rearing and falling.
basal release amino acids at various times after kindling had occurred. These animals were not stimulated at the time of the perfusion. Perfusate was collected for 8 h and the average amino acid content was d e t e r m i n e d . D a t a from this e x p e r i m e n t are displayed as a percentage of the unstimulated control group. D a t a analysis was p e r f o r m e d on the actual concentrations of amino acids collected in the perfusate. The samples of perfusate were analyzed for amino acid content (including aspartate, glutamate, glutamine, glycine, and taurine) using an ion exchange column procedure described elsewhere 2°. The data for amino acid content were based on integrated p e a k areas and calculated using simple linear interpolation. Changes in extracellular amino acid concentrations were subjected to analysis of variance. Post-hoc comparisons between groups were m a d e using Fisher's least significant difference test 35. The level of significance was considered to be P < 0.05.
Gin
Glu
2°°F-TT., 1~0~
l o o k - - r a m -
.
.
.
.
-
.
: ofiili 0
rOOp i
-~
Asp
--
1 2 3 4 5 Seizure stuge + Gly
1 2 3 4 5 Seizure stage Tau
4~
i 1 0 0 -. . . . . . . . . . . .
~o~i oi- 1 2 3 4 5 Seizure s t o g e
1
2
3 4
5
Seizure str]ge
I
2 3 4 5 Seizure stage
Fig. 2. Extracellular concentrations of amino acids from ventral hippocampus contralateral to the electrode during the process of kindling. Data are averages of l0 animals per stage calculated as a percent change from baseline release prior to stimulation. Baseline rates were 2210 + 283, 86 + 6, 2170 + 315,227 + 40, and 470 + 81 pmol/h for taurine, aspartate, glutamate and glycine, respectively. Asterisks indicate significant difference from baseline rate. *P < 0.05, **P < 0.01, ***P < 0.001.
317
15o~
'~
nl fll 2oo
oV
100
~
50
1001-
,o l-
Ii nl Ii Tau
Asp
Gin
Glu
mm
Control Kindled 1 - Day
Gly
150
0 2oo~
30- Days Tau
Asp
Gin
Glu
Gly - -
150~ I00 F
nl nl nl Ii o_oo. Tau
Asp
Gin
Glu
Gly
Fig. 3. Basal extracellular concentrations of amino acids from
ventral hippocampus 1, 30 and 90 days after the last stimulation producing kindling. Data are average percentages of the 8 h perfusion values from control rats that were implanted with electrodes, but not stimulated. *P < 0.05, **P < 0.01.
Rats receiving electrical stimulation once per hour in the right DPC while being perfused in the left ventral hippocampus gradually developed kindling (Fig. 1). The perfusion procedure did not appear to affect the rate of kindling as long as the perfusion was done contralateral to the side of the stimulation. Rats required an average of 15-17 stimulations to reach stage 5, a value similar to that reported elsewhere by our laboratory 36. Fig. 2 shows the extracellular concentrations of the amino acids at various seizure stages during the kindling process. Relative to prestimulation baseline, glutamate was increased significantly during all stages of kindling, while aspartate was increased significantly only during stage 1. Glycine was increased during stages 1, 2 and 4, while taurine was increased during stages 1 and 4. The only amino acid that was decreased relative to prestimulation baseline was glutamine, which was decreased during stages 3, 4 and 5. In control rats receiving no stimulation, the rate of release was relatively constant. Fig. 3 summarizes the persistence of some of the effects observed after reaching stage 5 of kindling. Significant increases in the basal release of glutamate were evident 1 and 30 days after the last stimulation. A significant increase in the basal release of glycine was seen 30 days after the last stimulation. There were no changes in the basal release of amino acids 90 days after the last stimulation. These results indicate that electrical stimulation of the DPC can result in significant alterations of extracellular levels in amino acids from the contralateral ventral hippocampus. Significant increases in glutamate in the perfusate was evident during all 5 stages of kindling and
persisted for up to 30 days after the last stimulation. Changes in aspartate and glutamine seen during the course of kindling did not persist at any time after stimulation had ceased. Significant increases in glycine were observed during kindling and increases in basal release of glycine were seen 30 days after kindling. Several reports have suggested a role for glutamate in the development of kindling 5'6'12'21. Our data are consistent with a recent report suggesting that K÷-evoked release of glutamate is increased in the hippocampal slices of rats kindled by entorhinal cortical stimulation 7. Another report found an increased release of glutamate in lateral ventricular perfusate following amygdaloid stimulation 25. These data suggest that kindling may be associated with a presynaptic mechanism dependent upon glutamate release in areas such as the hippocampus. Work reported by other laboratories has also indicated postsynaptic alterations may occur in glutamate receptors following kindling 6'8'21'23. Glutamate is thought to be stored in a non-cytoplasmic compartment of presynaptic nerve terminals and released in a Ca2+-dependent manner by membrane depolarization 22'28'33. Since release of glutamate is known to reflect an alteration in synaptic mechanisms, the increase in glutamate in the perfusate may be due to neuronal depolarization coupled with an inhibition of neuronal or glia reuptake. The enhancement of extracellular levels of glutamate may be associated with an increased neural excitability in the ventral hippocampus during and for up to 30 days after kindling. Although enhanced basal release of glutamate persists up to 30 days after kindling, the effect was not present 90 days later. Therefore, a permanent enhanced sensitivity to electrical stimulation cannot be attributed to a chronic change in basal glutamate release in the ventral hippocampus. However, the possibility still exists that elevated extracellular concentration of glutamate seen during the first 30 days after kindling may trigger some neurochemical changes which may be related to a permanent alteration in seizure threshold. The present results suggest that glutamate release in the ventral hippocampus may play an important role in the development, but not long-term persistence of kindling. The hippocampus has a high concentration of glutamate-containing fibers and there are considerable data suggesting a role of excitatory amino'acids such as glutamate in the generation of kindling in this region of the brain. Additional studies will be necessary to determine the role, if any, of the ventral hippocampus in the persistence of the kindling phenomenon. The authors wish to thank Mrs. Loretta Moore for the editorial assistance in the preparation of this manuscript.
318
1 Baimbridge, K.G. and Miller, J.J., Hippocampal calciumbinding protein during commissural kindling-induced epileptogenesis: progressive decline and effects of anticonvulsants, Brain Research, 324 (1984) 85-90. 2 Benveniste, H., Brain microdialysis, J. Neurochem., 52 (1989) 1667-1679. 3 Burnham, W.M., Racine, R.J. and Okazaki, M.O. In J.A. Wada (Ed.), Kindling Mechanism: H. Biochemical Studies, Kindling 3, Raven Press, New York, 1986, pp. 283-299. 4 Collingridge, G.L. and Bliss, T.V.P., NMDA receptor - their role in long-term potentiation, Trends Neurosci., 10 (1987) 288-297. 5 Croucher, M.J., Bradford, H.E, Sunter, D.C. and Watkins, J.C., Inhibition of the development of electrical kindling of the prepyriform cortex by daily focal injection of excitatory amino acid antagonists, Eur. J. Pharmacol., 152 (1988) 29-38. 6 Dingledine, R., Hynes, M.A. and King, G.L., Involvement of N-methyl-D-aspartate receptors in epileptiform bursting in the rat hippocampal slices, J. Physiol., 380 (1986) 175-189. 7 Geula, C., Jarvie, P.A., Logan, T.C. and Slevin, "12, Long-term enhancement of K-evoked release of e-glutamate in entorhinal kindled rats, Brain Research, 442 (1988) 368-372. 8 Gilbert, M.E., The NMDA-receptor antagonist, MK801, suppresses limbic kindling and kindled seizures, Brain Research, 463 (1988) 90-99. 9 Goddard, G.V., Development of epileptic seizures through brain stimulation at low intensity, Nature, 214 (1967) 1020-1021. 10 Goddard, G.V., McIntyre, D.C. and Leech, G.K., A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol., 25 (1969) 295-330. 11 Goddard, G.V. and Douglas, R.M., Does the engram of kindling model the engram of normal long-term memory? Can. J. Neurol. Sci., 2 (1975) 385-394. 12 Herron, C.E., Williamson, R. and Collingridge, G.L., A selective N-methyl-D-aspartate antagonist depresses epileptiform activity in rat hippoeampal slices, Neurosci. Lett., 61 (1985) 255-260. 13 Hwa, G.G.C. and Avoli, M., NMDA receptor antagonist CCP and MK-801 partially suppress the epileptiform discharges induced by the convulsant drug bicuculline in the rat neocortex, Neurosci. Lett., 98 (1989) 189-193. 14 Korf, J. and Veneman, K,, Amino acids in rat striatal dialysates: methodological aspects changes after electroconvulsive shock, J. Neurochem., 45 (1985) 1341-1344. 15 Leach, M,J., Marden, C.M., Miller, A.A., O'Donnel, R.H. and Weston, S.B., Changes in cortical amino acids during electrical kindling in rats, Neuropharmacology, 24 (1985) 937-940. 16 Lee, RH.K., Obie, J. and Hong, J.S., Intrahippocampal injections of a specific mu receptor ligand PL017 produced generalized convulsions in rats, Brain Research, 441 (1988) 381-385. 17 Lehmann, A., Sandberg, M. and Huxtable, R.J., In vivo release of neuroactive amines and amino acids from the hippocampus of seizure-resistant and seizure-susceptible rats, Neurochem. Int., 8 (1986) 513-520. 18 McNamara, J.O., Bonhaus, D.W., Crain, B.J., Gellman, R.L., Giaccbino, J.L. and Shin, C., The kindling model of epilepsy: a critical review, CRC Crit. Rev. Clin. Neurobiol., 1 (1985) 341-393. 19 McNamara, J.O., Bonhaus, D.W., Crain, B.J., Gelmann, R.L. and Shin, C. In J.A. Wada (Ed.), An Approach to Elucidating the Network of Brain Structures Underlying Kindling, Kindling 3, Raven Press, New York, 1986, pp. 125-138. 20 Mitchell, C.L., Grimes, L., Hudson, P.M. and Hong, J.S.,
21
22
23
24
25
26
27 28
29
30
3l
32
33
34
35 36
37
Stimulation of the perforant path alters hippocampat level ol opioid peptides, glutamine and GABA, Brain Research, 435 (1987) 343-347. Mody, I. and Heinemann, U., NMDA receptors of dentate gyrus granule cells participate in synaptic transmission following kindling, Nature, 326 (19871) 701-704. Nadler, J.V.. Vaca, K.W., White, F.W., Lynch, G.S. and Cotman, C.W., Aspartate and glutamate as possible transmitters of excitatory hippocampal afferents, Nature, 260 (1976) 538-540. Okazaki, M.M., McNamara, J.O. and Nadler, J.V., N-methylD-aspartate receptor autoradiography in rat brain after angular bundle kindling, Brain Research, 482 (1989) 359-364. Patel, J., Marangos, P.J., Contel, N., Gardner, G. and Post, R.M., Increased phosphorylation of membrane protein consequent to amygdaloid kindling, J. Neurochem., 43 (1984) 169173. Peterson, D.W., Collins, J.F. and Bradford, H.F., The kindled amygdala model of epilepsy: anticonvulsant action of amino acid antagonists, Brain Research, 275 (1983) 169-172. Racine, R.J., Modification of seizure activity by electrical stimulation. II. Motor seizure, Electroencephalogr. Clin. Neurophysiol., 32 (1972) 281-294. Racine, R.J., Kindling: the first decade, Neurosurgery, 3 (1978) 234-252. Sandoval, M.E., Horch, P. and Cotman, C.W., Evaluation of glutamate as a hippocampal neurotransmitter: glutamate uptake and release from synaptosomes, Brain Research, t42 (1978) 285-299. Savage, D.D., Werling, L.L., Nadler, J.V. and McNamara, J.O., Selective and reversible increase in the number of a quisqualate-sensitive glutamate binding site on hippocampal synaptic membranes after angular bundle kindling, Brain Research, 607 (1984) 332-335. Savage, D.D., Dasheiff, R.M. and McNamara, J.O., Kindled seizure-induced reduction of muscarinic cholinergic receptors in rat hippocampal formation: evidence for localization to dentate granule cells, J. Comp. Neurol., 221 (1983) 106-112. Slevin, J.T., Kasarkis, E.J., Vanaman, T.C. and Zurin, M., Excitatory amino acids and divalent cations in the kindling model of epilepsy. In R. Schwarcz and Y. Ben-Ari (Eds.), Excitatory Amino Acids and Epilepsy, Plenum, New York, 1986, pp. 587-598. Spencer, H.J., Tominez, G. and Halpern, B., Mass spectrographic analysis of stimulated release of endogenous amino acids from rat hippocampal slices, Brain Research, 212 (1981) 194197. Vezzani, A., Wu, H.Q., Moneta, E. and Samanin, R., Role of the N-methyl-D-aspartate-type receptors in the development and maintenance of hippocampal kindling in rats, Neurosci. Lett., 87 (1988) 63-68. Westerink, B.H.C., Damsma, G., Rollema, H., De Vries, J.B. and Horn, A.S., Scope and limitations of in vivo brain dialysis: a comparison of its application to various transmitter systems, Life Sci., 411 (1987) 1763-1776. Winer, BA., Statistical Principles in Experimental Design, McGraw-Hill, New York, 1962. Xie, C.W., Lee, P.H.K., Douglass, J., Crain, B. and Hong, J.S., Deep prepyriform cortex kindling differentially alters the levels of prodynorphin mRNA in rat hippocampus and striatum, Brain Research, 495 (1989) 156-160. Zhang, W., Tilson, H., Stachowiak, M.K. and Hong, J.S., Repeated haloperidol administration changes basal release of striatal dopamine and subsequent response to haloperidol challenge, Brain Research, 484 (1989) 389-392.
315
Elsevier BRES 24515
Extracellular concentrations of amino acid transmitters in ventral hippocampus during and after the development of kindling W . Q . Z h a n g 1, P . M . H u d s o n 1, T . J . S o b o t k a 2, J . S . H o n g I a n d H . A .
Tilson 1
1Laboratory of Molecular and Integrative Neuroscience, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 (U.S.A.) and 2Neurobehavioral Toxicology, Federal Drug Administration, Washington, DC 20204 (U.S.A.)
(Accepted 23 October 1990) Key words: Kindling; Ventral hippocampus; Brain dialysis; Amino acid
This study examined the possible involvement of amino acid release from ventral hippocampus in the establishment and maintenance of kindling in rats. Release of amino acids from ventral hippocampus was measured by microdialysis coupled with high-performance liquid chromatography. Samples were obtained by microdialysisperfusion of freely moving animals receivingdeep prepiriform cortex (DPC) electrical stimulation. Samples of perfusate were collected before, during and after kindling was established. DPC kindling stimulation significantly increased concentrations of glutamate (Glu) and glycine (Gly) in perfusate from ventral hippocampus during kindling. Increased basal release of Glu was evident up to 30 days after the last electrical stimulation. We conclude that release of Glu and Gly in the ventral hippocampus may play an important role during establishment, but not in maintenance of kindling. Kindling is a widely accepted animal model of epilepsy in which repeated subconvulsive electrical stimulation results in progressively more intense seizures and eventually generalized motor convulsions 9-11'18J9'26"27. Once the kindled state has been achieved, the increased sensitivity to subconvulsive stimulation is persistent 1°'27. The neural mechanisms responsible for the kindling phenomenon have been extensively investigated. Changes in levels of calcium binding protein 1, protein phosphorylation 24, and neurotransmitter receptor function 21'23"29'30 have been reported in kindled animals. Recent research has begun to explore the role that putative amino acid neurotransmitters may play in epilepsy and the kindling phenomenon. For example, the release of excitatory amino acids (EAA) has been postulated to be involved in the generation and expression of epileptic seizures 3'5'7"15"17"31-33.In addition, it was reported that electroconvulsive shocks also increased the release of E A A measured by in vivo microdialysis 14. Subsequent research has found a change in the number of quisqualate-sensitive glutamate receptor sites during the kindling process 29, while changes in EAA receptors have been associated with long-term potentiation 4 and kindling-like phenomena 6,8'~3'21"23. The objective of the present series of experiments was to investigate changes in the release of amino acids. including glutamate, glutamine, taurine, glycine, and aspartate, during and after development of electricalinduced kindling in the rat. These experiments utilized a
microdialysis procedure, which allows for the continuous monitoring of neurotransmitter concentrations in the perfusate obtained from conscious and freely moving animals TM. Electrical stimulation of the deep prepiriform cortex was used to produce kindling, while the ventral hippocampus was perfused using dialysis probes. The ventral hippocampus was chosen because of its known importance in the development of seizures 16. Male Fischer-344 rats (Charles River Breeding Co., Raleigh, NC) weighing 250-300 g at the time of surgery were anesthetized with chloral hydrate (350 mg/kg, i.p.) and implanted unilaterally with a single isolated bipolar stainless steel electrode (0.25 mm diameter). The incisor bar was set 5 mm above the intraural line and the coordinates for the right deep prepiriform cortex (DPC) were 3.8 mm anterior and 2.5 mm lateral to bregma, and 7.5 mm below the surface of the skull. The electrode plug was firmly anchored to the skull with miniature screws and dental acrylic cement. At least 7 days later, a dialysis loop 37 was implanted stereotaxically in the left ventral hippocampus using the following coordinates: 5.8 mm posterior and 5.0 mm lateral to the midline, and 7.5 mm below vertical from dura with the incisor bar set at 3.3 mm below the intraural line. The dialysis probe was connected by polyethylene tubing (PE10) to a swivel and Harvard infusion pump. The dialysis tubes were perfused continuously with artificial cerebral spinal fluid pumped through 0.22 mm Millipore syringe filters at a constant rate of 2/~l/min. Dialysis tubes
Correspondence: J.S. Hong, LMIN, NIEHS, P.O. Box 12233, Research Triangle Park, NC 27709, U.S.A.
316
i[
were anchored to the skull using dental acrylic cement. In the first e x p e r i m e n t , 24 h after implantation of the dialysis loop, 4 consecutive 1-h fractions were collected in microcentrifuge tubes containing 6 ~1 of 2.0 N perchloric acid. These samples were used to calculate the baseline release rate prior to stimulation. W h e n a relatively constant flow rate was o b t a i n e d , stimulation of the D P C began. Electrical stimulation (400/~A) was p r o d u c e d by a Grass-88 stimulator delivered through a modified constant current output. The stimulus consisted of a 1 s train of 50-Hz biphasic square wave current with 1 ms duration. In this e x p e r i m e n t , stimuli were delivered once per hour until kindling occurred. A t the time of stimulation, rats were rated for severity of seizure using a scale similar to that described by Racine 26 (chewing, stage l; head-nodding, stage 2; unilateral limbic clonus, state 3; rearing with bilateral forelimb clonus, stage 4; and bilateral forelimb clonus with rearing and falling, stage 5); each rating was associated with the perfusate sample collected at the time. Perfusions were t e r m i n a t e d after reaching stage 5. In a second e x p e r i m e n t , stimulating electrodes were implanted in the right D P C as described previously. Kindling was p r o d u c e d by stimulation twice daily for 8-10 days at intervals of at least 8 h, until 3 consecutive stimuli e v o k e d kindled seizures at stage 5. A f t e r the last kindled seizure, each animal was paired with a weightand age-matched control animal also implanted with an electrode, but not stimulated. One group of rats was i m p l a n t e d with dialysis tubes in the left ventral hippocampus. Twenty-four hours later, these animals were perfused for 8 consecutive hours. A n o t h e r group of rats was perfused at 30 days after the last stimulus, while a third group was perfused 90 days after the last stimulus. This e x p e r i m e n t was designed to d e t e r m i n e alterations in
m--e--~
I
O0
__
I
I
5 10 15 Number of shmulotions
I
20
Fig. I. Development of kindling following stimulation of the deep
prepiriform cortex while the left ventral hippocampus was perfused using a microdialysis tube. Rats were stimulated once per hour. Data are average seizure ratings for 10 rats. Stage 5 indicates bilateral forelimb clonus with rearing and falling.
basal release amino acids at various times after kindling had occurred. These animals were not stimulated at the time of the perfusion. Perfusate was collected for 8 h and the average amino acid content was d e t e r m i n e d . D a t a from this e x p e r i m e n t are displayed as a percentage of the unstimulated control group. D a t a analysis was p e r f o r m e d on the actual concentrations of amino acids collected in the perfusate. The samples of perfusate were analyzed for amino acid content (including aspartate, glutamate, glutamine, glycine, and taurine) using an ion exchange column procedure described elsewhere 2°. The data for amino acid content were based on integrated p e a k areas and calculated using simple linear interpolation. Changes in extracellular amino acid concentrations were subjected to analysis of variance. Post-hoc comparisons between groups were m a d e using Fisher's least significant difference test 35. The level of significance was considered to be P < 0.05.
Gin
Glu
2°°F-TT., 1~0~
l o o k - - r a m -
.
.
.
.
-
.
: ofiili 0
rOOp i
-~
Asp
--
1 2 3 4 5 Seizure stuge + Gly
1 2 3 4 5 Seizure stage Tau
4~
i 1 0 0 -. . . . . . . . . . . .
~o~i oi- 1 2 3 4 5 Seizure s t o g e
1
2
3 4
5
Seizure str]ge
I
2 3 4 5 Seizure stage
Fig. 2. Extracellular concentrations of amino acids from ventral hippocampus contralateral to the electrode during the process of kindling. Data are averages of l0 animals per stage calculated as a percent change from baseline release prior to stimulation. Baseline rates were 2210 + 283, 86 + 6, 2170 + 315,227 + 40, and 470 + 81 pmol/h for taurine, aspartate, glutamate and glycine, respectively. Asterisks indicate significant difference from baseline rate. *P < 0.05, **P < 0.01, ***P < 0.001.
317
15o~
'~
nl fll 2oo
oV
100
~
50
1001-
,o l-
Ii nl Ii Tau
Asp
Gin
Glu
mm
Control Kindled 1 - Day
Gly
150
0 2oo~
30- Days Tau
Asp
Gin
Glu
Gly - -
150~ I00 F
nl nl nl Ii o_oo. Tau
Asp
Gin
Glu
Gly
Fig. 3. Basal extracellular concentrations of amino acids from
ventral hippocampus 1, 30 and 90 days after the last stimulation producing kindling. Data are average percentages of the 8 h perfusion values from control rats that were implanted with electrodes, but not stimulated. *P < 0.05, **P < 0.01.
Rats receiving electrical stimulation once per hour in the right DPC while being perfused in the left ventral hippocampus gradually developed kindling (Fig. 1). The perfusion procedure did not appear to affect the rate of kindling as long as the perfusion was done contralateral to the side of the stimulation. Rats required an average of 15-17 stimulations to reach stage 5, a value similar to that reported elsewhere by our laboratory 36. Fig. 2 shows the extracellular concentrations of the amino acids at various seizure stages during the kindling process. Relative to prestimulation baseline, glutamate was increased significantly during all stages of kindling, while aspartate was increased significantly only during stage 1. Glycine was increased during stages 1, 2 and 4, while taurine was increased during stages 1 and 4. The only amino acid that was decreased relative to prestimulation baseline was glutamine, which was decreased during stages 3, 4 and 5. In control rats receiving no stimulation, the rate of release was relatively constant. Fig. 3 summarizes the persistence of some of the effects observed after reaching stage 5 of kindling. Significant increases in the basal release of glutamate were evident 1 and 30 days after the last stimulation. A significant increase in the basal release of glycine was seen 30 days after the last stimulation. There were no changes in the basal release of amino acids 90 days after the last stimulation. These results indicate that electrical stimulation of the DPC can result in significant alterations of extracellular levels in amino acids from the contralateral ventral hippocampus. Significant increases in glutamate in the perfusate was evident during all 5 stages of kindling and
persisted for up to 30 days after the last stimulation. Changes in aspartate and glutamine seen during the course of kindling did not persist at any time after stimulation had ceased. Significant increases in glycine were observed during kindling and increases in basal release of glycine were seen 30 days after kindling. Several reports have suggested a role for glutamate in the development of kindling 5'6'12'21. Our data are consistent with a recent report suggesting that K÷-evoked release of glutamate is increased in the hippocampal slices of rats kindled by entorhinal cortical stimulation 7. Another report found an increased release of glutamate in lateral ventricular perfusate following amygdaloid stimulation 25. These data suggest that kindling may be associated with a presynaptic mechanism dependent upon glutamate release in areas such as the hippocampus. Work reported by other laboratories has also indicated postsynaptic alterations may occur in glutamate receptors following kindling 6'8'21'23. Glutamate is thought to be stored in a non-cytoplasmic compartment of presynaptic nerve terminals and released in a Ca2+-dependent manner by membrane depolarization 22'28'33. Since release of glutamate is known to reflect an alteration in synaptic mechanisms, the increase in glutamate in the perfusate may be due to neuronal depolarization coupled with an inhibition of neuronal or glia reuptake. The enhancement of extracellular levels of glutamate may be associated with an increased neural excitability in the ventral hippocampus during and for up to 30 days after kindling. Although enhanced basal release of glutamate persists up to 30 days after kindling, the effect was not present 90 days later. Therefore, a permanent enhanced sensitivity to electrical stimulation cannot be attributed to a chronic change in basal glutamate release in the ventral hippocampus. However, the possibility still exists that elevated extracellular concentration of glutamate seen during the first 30 days after kindling may trigger some neurochemical changes which may be related to a permanent alteration in seizure threshold. The present results suggest that glutamate release in the ventral hippocampus may play an important role in the development, but not long-term persistence of kindling. The hippocampus has a high concentration of glutamate-containing fibers and there are considerable data suggesting a role of excitatory amino'acids such as glutamate in the generation of kindling in this region of the brain. Additional studies will be necessary to determine the role, if any, of the ventral hippocampus in the persistence of the kindling phenomenon. The authors wish to thank Mrs. Loretta Moore for the editorial assistance in the preparation of this manuscript.
318
1 Baimbridge, K.G. and Miller, J.J., Hippocampal calciumbinding protein during commissural kindling-induced epileptogenesis: progressive decline and effects of anticonvulsants, Brain Research, 324 (1984) 85-90. 2 Benveniste, H., Brain microdialysis, J. Neurochem., 52 (1989) 1667-1679. 3 Burnham, W.M., Racine, R.J. and Okazaki, M.O. In J.A. Wada (Ed.), Kindling Mechanism: H. Biochemical Studies, Kindling 3, Raven Press, New York, 1986, pp. 283-299. 4 Collingridge, G.L. and Bliss, T.V.P., NMDA receptor - their role in long-term potentiation, Trends Neurosci., 10 (1987) 288-297. 5 Croucher, M.J., Bradford, H.E, Sunter, D.C. and Watkins, J.C., Inhibition of the development of electrical kindling of the prepyriform cortex by daily focal injection of excitatory amino acid antagonists, Eur. J. Pharmacol., 152 (1988) 29-38. 6 Dingledine, R., Hynes, M.A. and King, G.L., Involvement of N-methyl-D-aspartate receptors in epileptiform bursting in the rat hippocampal slices, J. Physiol., 380 (1986) 175-189. 7 Geula, C., Jarvie, P.A., Logan, T.C. and Slevin, "12, Long-term enhancement of K-evoked release of e-glutamate in entorhinal kindled rats, Brain Research, 442 (1988) 368-372. 8 Gilbert, M.E., The NMDA-receptor antagonist, MK801, suppresses limbic kindling and kindled seizures, Brain Research, 463 (1988) 90-99. 9 Goddard, G.V., Development of epileptic seizures through brain stimulation at low intensity, Nature, 214 (1967) 1020-1021. 10 Goddard, G.V., McIntyre, D.C. and Leech, G.K., A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol., 25 (1969) 295-330. 11 Goddard, G.V. and Douglas, R.M., Does the engram of kindling model the engram of normal long-term memory? Can. J. Neurol. Sci., 2 (1975) 385-394. 12 Herron, C.E., Williamson, R. and Collingridge, G.L., A selective N-methyl-D-aspartate antagonist depresses epileptiform activity in rat hippoeampal slices, Neurosci. Lett., 61 (1985) 255-260. 13 Hwa, G.G.C. and Avoli, M., NMDA receptor antagonist CCP and MK-801 partially suppress the epileptiform discharges induced by the convulsant drug bicuculline in the rat neocortex, Neurosci. Lett., 98 (1989) 189-193. 14 Korf, J. and Veneman, K,, Amino acids in rat striatal dialysates: methodological aspects changes after electroconvulsive shock, J. Neurochem., 45 (1985) 1341-1344. 15 Leach, M,J., Marden, C.M., Miller, A.A., O'Donnel, R.H. and Weston, S.B., Changes in cortical amino acids during electrical kindling in rats, Neuropharmacology, 24 (1985) 937-940. 16 Lee, RH.K., Obie, J. and Hong, J.S., Intrahippocampal injections of a specific mu receptor ligand PL017 produced generalized convulsions in rats, Brain Research, 441 (1988) 381-385. 17 Lehmann, A., Sandberg, M. and Huxtable, R.J., In vivo release of neuroactive amines and amino acids from the hippocampus of seizure-resistant and seizure-susceptible rats, Neurochem. Int., 8 (1986) 513-520. 18 McNamara, J.O., Bonhaus, D.W., Crain, B.J., Gellman, R.L., Giaccbino, J.L. and Shin, C., The kindling model of epilepsy: a critical review, CRC Crit. Rev. Clin. Neurobiol., 1 (1985) 341-393. 19 McNamara, J.O., Bonhaus, D.W., Crain, B.J., Gelmann, R.L. and Shin, C. In J.A. Wada (Ed.), An Approach to Elucidating the Network of Brain Structures Underlying Kindling, Kindling 3, Raven Press, New York, 1986, pp. 125-138. 20 Mitchell, C.L., Grimes, L., Hudson, P.M. and Hong, J.S.,
21
22
23
24
25
26
27 28
29
30
3l
32
33
34
35 36
37
Stimulation of the perforant path alters hippocampat level ol opioid peptides, glutamine and GABA, Brain Research, 435 (1987) 343-347. Mody, I. and Heinemann, U., NMDA receptors of dentate gyrus granule cells participate in synaptic transmission following kindling, Nature, 326 (19871) 701-704. Nadler, J.V.. Vaca, K.W., White, F.W., Lynch, G.S. and Cotman, C.W., Aspartate and glutamate as possible transmitters of excitatory hippocampal afferents, Nature, 260 (1976) 538-540. Okazaki, M.M., McNamara, J.O. and Nadler, J.V., N-methylD-aspartate receptor autoradiography in rat brain after angular bundle kindling, Brain Research, 482 (1989) 359-364. Patel, J., Marangos, P.J., Contel, N., Gardner, G. and Post, R.M., Increased phosphorylation of membrane protein consequent to amygdaloid kindling, J. Neurochem., 43 (1984) 169173. Peterson, D.W., Collins, J.F. and Bradford, H.F., The kindled amygdala model of epilepsy: anticonvulsant action of amino acid antagonists, Brain Research, 275 (1983) 169-172. Racine, R.J., Modification of seizure activity by electrical stimulation. II. Motor seizure, Electroencephalogr. Clin. Neurophysiol., 32 (1972) 281-294. Racine, R.J., Kindling: the first decade, Neurosurgery, 3 (1978) 234-252. Sandoval, M.E., Horch, P. and Cotman, C.W., Evaluation of glutamate as a hippocampal neurotransmitter: glutamate uptake and release from synaptosomes, Brain Research, t42 (1978) 285-299. Savage, D.D., Werling, L.L., Nadler, J.V. and McNamara, J.O., Selective and reversible increase in the number of a quisqualate-sensitive glutamate binding site on hippocampal synaptic membranes after angular bundle kindling, Brain Research, 607 (1984) 332-335. Savage, D.D., Dasheiff, R.M. and McNamara, J.O., Kindled seizure-induced reduction of muscarinic cholinergic receptors in rat hippocampal formation: evidence for localization to dentate granule cells, J. Comp. Neurol., 221 (1983) 106-112. Slevin, J.T., Kasarkis, E.J., Vanaman, T.C. and Zurin, M., Excitatory amino acids and divalent cations in the kindling model of epilepsy. In R. Schwarcz and Y. Ben-Ari (Eds.), Excitatory Amino Acids and Epilepsy, Plenum, New York, 1986, pp. 587-598. Spencer, H.J., Tominez, G. and Halpern, B., Mass spectrographic analysis of stimulated release of endogenous amino acids from rat hippocampal slices, Brain Research, 212 (1981) 194197. Vezzani, A., Wu, H.Q., Moneta, E. and Samanin, R., Role of the N-methyl-D-aspartate-type receptors in the development and maintenance of hippocampal kindling in rats, Neurosci. Lett., 87 (1988) 63-68. Westerink, B.H.C., Damsma, G., Rollema, H., De Vries, J.B. and Horn, A.S., Scope and limitations of in vivo brain dialysis: a comparison of its application to various transmitter systems, Life Sci., 411 (1987) 1763-1776. Winer, BA., Statistical Principles in Experimental Design, McGraw-Hill, New York, 1962. Xie, C.W., Lee, P.H.K., Douglass, J., Crain, B. and Hong, J.S., Deep prepyriform cortex kindling differentially alters the levels of prodynorphin mRNA in rat hippocampus and striatum, Brain Research, 495 (1989) 156-160. Zhang, W., Tilson, H., Stachowiak, M.K. and Hong, J.S., Repeated haloperidol administration changes basal release of striatal dopamine and subsequent response to haloperidol challenge, Brain Research, 484 (1989) 389-392.