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Development of changes in endogenous GABA release during kindling epileptogenesis in rat hippocampus
Brain Research, 545 (1991) 33-40 © 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0006-8993/91/$03.50 ADONIS 000689939116450Q
33
BRES 16450
Development of changes in endogenous GABA release during kindling epileptogenesis in rat hippocampus W. Kamphuis, E. Huisman, M.J. Veerman and F.H. Lopes da Silva Department of Experimental Zoology, University of Amsterdam, Amsterdam (The Netherlands) (Accepted 23 October 1990)
Key words: Kindling: 7-Aminobutyric acid; Hippocampus; Neurotransmitter release; ~,-Aminobutyrc acid uptake; Epilepsy; SK&F 89776-A; Glutamate
The calcium-dependent y-aminobutyric acid (GABA) and glutamate release from rat hippocampal CA1 slices, evoked by a 1-min depolarization with 50 mM K +, was investigated in different stages of kindling epileptogenesis. Kindling was induced by tetanic stimulation of the Schaffer collateral]commissural pathway. In agreement with our previous results, we found a significantly increased calcium-dependent GABA release compared to that of implanted controls, in a group of fully kindled animals 1 day after the last seizure and also 25-36 days after the last seizure. In addition, we found that the increase in GABA release was associated with late phases of kindling epileptogenesis since no significant alterations were found in partly kindled animals that had received only 6 kindling stimulations while a significant increase was apparent in animals that had received 14 tetanic stimuli. When the release protocol was carried out in the presence of SK&F 89776-A, a blocker of the GABA uptake carder, an additional amount of GABA was found after depolarization. This additional amount of GABA, reflecting the amount of GABA taken up under conditions without blocker, was in kindled animals not different from controls which demonstrates that a reduced GABA uptake does not account for the observed enhanced release in kindled animals. The calcium-dependent release of glutamate evoked by 1 min of high potassium depolarization was not significantly changed in the kindled groups. Only after prolonged depolarization during 4 subsequent minutes a significant increase in animals of the fully kindled group and at long-term after kindling was observed. The threshold K + concentration for eliciting a calcium-dependent release of GABA and glutamate, was not changed in the kindled animals. The results reported here indicate that the late stages of kindling development and the long-lasting enhanced seizure sensitivity are associated with an enhanced GABA exocytosis, possibly to counteract a long-lasting postsynaptic reduction of GABA receptor sensitivity.
INTRODUCTION R e p e a t e d focal electrical tetanic stimulation of the brain, once o r twice daily, results in a lengthening of the elicited epileptiform activity and a progression of the b e h a v i o r a l seizure response. This process of epileptogenesis, resulting in a long-lasting e n h a n c e d seizure sensitivity, is called kindling 6'14'22. O n e of the possible neurobiological mechanisms underlying kindling epileptogenesis is an i m p a i r m e n t of y - a m i n o b u t y r i c acid ( G A B A ) m e d i a t e d recurrent inhibition 14. In accordance with this hypothesis, it was shown that kindling leads to a gradual reduction of paired-pulse depression of local e v o k e d field potentials in the C A 1 a r e a of the rat h i p p o c a m p u s 12'17 and to a d e c r e a s e d sensitivity of h i p p o c a m p a l p y r a m i d a l cells to G A B A 16. W i t h i m m u n o c y t o c h e m i c a l techniques, it was investigated w h e t h e r the o b s e r v e d decrease of inhibitory control was caused by a r e d u c e d cellular G A B A content of local interneurons. H o w e v e r , our studies revealed an
e n h a n c e d immunoreactivity of G A B A e r g i c s o m a t a in CA1 during the initial stages of kindling xl and a reduction only at long t e r m after the last seizure in fully kindled animals 13. M o r e o v e r , at the synaptic level evidence was found for an increased c a l c i u m - d e p e n d e n t release of G A B A from CA1 subslices in fully k i n d l e d animals 15. The latter result led us to suggest that an increased exocytosis of G A B A in the initial phase of kindling acquisition m a y l e a d to a r e d u c e d G A B A r e c e p t o r sensitivity and consequently to a r e d u c e d paired-pulse inhibition. In the present r e p o r t we describe a new series of release e x p e r i m e n t s c a r d e d out to clarify the following 3 issues regarding changes in G A B A release. (1) To c o m p r e h e n d the relation b e t w e e n the electrophysiological alterations and the previously o b s e r v e d changes in G A B A release, we studied with an i m p r o v e d technique the release of G A B A at 4 different phases of kindling epileptogenesis. Simultaneously, the release of glutamate, the main excitatory n e u r o t r a n s m i t t e r in C A 1 , was
Correspondence: W. Kamphuis, Department of Experimental Zoology, University of Amsterdam, Kruislaan 320, 1098 SM Amsterdam, The Netherlands.
34
w e c a r r i e d o u t e x p e r i m e n t s in t h e p r e s e n c e o f S K & F
kindled state, evidenced by the occurrence of class 5 generalized tonic-clonic convulsions27, the release was investigated in two subgroups at different intervals after the last elicited convulsion. Fully kindled (FK)-group (n = 6). Kindled animals studied 24 h after the last class 5 seizure, together with 5 rats of the CON-group. Kindled animals of this group experienced a mean of 27 afterdischarges. A mean of 5 generalized tonic-clonic seizures (stage 5) had been elicited. Long-term (LT)-group (n = 7). Kindled animals studied 25-36 days after the last seizure, together with 7 animals of the CON group. A mean of 31 afterdischarges were evoked in these kindled animals. A mean of 5 generalized seizures (stage 5) were elicited.
8 9 7 7 6 - A , a specific b l o c k e r o f t h e G A B A u p t a k e carr i e r 26'31'32. T h i s s e l e c t i v e a n d p o t e n t d r u g h a s t h e a d v a n -
Amino acid release procedures
m e a s u r e d . (2) T h e r e l e a s e m e t h o d u s e d a c t u a l l y d e t e r mines
the
efflux
of
amino
acids
from
slices.
This
corresponds to the net difference between the quantity of a m i n o acid r e l e a s e d f r o m t h e i n t r a c e l l u l a r c o m p a r t m e n t and the quantity removed from the extracellular space by u p t a k e p r o c e s s e s . T h e r e f o r e , a c h a n g e in t h e effiux m a y b e d u e t o a c h a n g e in r e l e a s e a n d / o r u p t a k e p r o c e s s e s . T o be able to differentiate between these two possibilities,
t a g e o v e r n i p e c o t i c acid, p r e v i o u s l y u s e d b y us t o i n h i b i t the
GABA
uptake
m e c h a n i s m 1°'15,
in
that
it is
a
n o n - c o m p e t i t i v e b l o c k e r . S i n c e S K & F is n o t t a k e n u p b y t h e t i s s u e it d o e s n o t d i s t u r b t h e i n t r a c e l l u l a r G A B A homeostasis
and
is w i t h o u t
effect on the
basal and
K + - e v o k e d r e l e a s e o f [ 3 H ] G A B A f r o m s y n a p t o s o m e s 26. (3) T h e e n l a r g e d G A B A
r e l e a s e f o u n d p r e v i o u s l y in fully
kindled animals may be the result of a lowered threshold K ÷ - c o n c e n t r a t i o n f o r eliciting a c a l c i u m - d e p e n d e n t rel e a s e . I n o r d e r to a d d r e s s this p o s s i b i l i t y , t h e t h r e s h o l d was determined GABA
by measuring the calcium-dependent
release over a range of K+-concentrations.
MATERIALS AND METHODS
Implantation and kindling Male Wistar rats (200-250 g) were used in this study. Chronic indwelling electrodes were placed in CA1 area of the left dorsal hippocampus to enable kindling stimulations to this area. The details of this procedure were described previously 12. In short: under pentobarbital anesthesia, two bundles of stainless steel wire electrodes were implanted. The stimulation bundle was placed in the Schaffer-collaterai/commissural fiber pathway and the recording bundle was placed in stratum radiatum of CA1. Local evoked field potentials, elicited by test stimuli, were used to optimize the position of the electrodes. Electrodes were fixed in position with dental cement and placed in a connector plug. After at least 2 weeks of recovery, field potentials were recorded in the freely moving rat. The stimulation electrode that elicited the optimal evoked field potential in response to single pulse stimulation of the Schaffer collateral/commissural fiber pathway was selected and used to deliver kindling stimulations. A group of 49 implanted animals was divided into a nonstimulated control group (n = 18) and a group (n = 31) that received, twice daily, kindling stimulations at an intensity suprathreshold for the induction of an afterdischarge (200-300/zA, 50 Hz, 1 s). The animals of the control group (CON) were handled throughout the experimental period in a way comparable to the kindled rats but did not receive tetanic stimulations. The release of CA1 subslices was studied in the following groups of stimulated animals. 6AD-group (n = 9). Animals that had received 6 kindling stimulations. The release was studied 24 h after the last stimulation. All kindling stimulated animals showed at this early phase of the kindling process stage 1-2 behavioral seizures 27. Together with this group, 4 animals of the CON group were studied. 14AD-group (n = 9). Animals that had received 14 kindling stimulations were studied 24 h after the last stimulation. All stimulated animals in this group showed stage 2-3 seizures. Two control animals were studied together with the stimulated animals. When the remaining stimulated animals had acquired the fully
Rats were anesthetized with ether, decapitated and the brains were rapidly removed from the skull. Both hippocampi were dissected on an ice-cooled plate. From each hippocampus, 12 slices were cut along the septotemporal axis. Slices obtained from the ipsilateral and contralateral hemisphere in respect to the kindled side were collected in 95% 0 2 - 5 % CO 2 gassed Krebs-Ringer buffer at room temperature. Composition of the buffer in raM: NaCI 124, KCI 3.5, CaCI 2 2.0, MgSO 4 2.0, NaH2PO 4 1.25, NaHCO 3 26.0, D-glucose 10.0 with pH 7.4. The slices were divided at random in 4 sets of 6 slices. The CA1 area was dissected from the hippocampal slice to study specifically the release from this area 2'9'15. The CA1 subslices were placed in transfer chambers that were made from small tubes provided with a fine meshed nylon gauze bottom. At t = 0 min, the transfer chambers with the subslices laying on the meshed bottom were placed in a large reservoir filled with Krebs-Ringer buffer. All media in the release protocol were kept at a constant temperature of 34.4 °C. After a recovery period of 15 min (t = 0-15 min), the chambers were transferred to the wells of a 24-well tissue culture plate filled with 1000 #1 medium. From that moment onward, the chambers were transferred every 5 rain to the next row of wells filled with fresh medium. This method, based on a multiwell system described by Minc-Golomb 2a, allows a change over of the tissue from one medium to the next in a stepwise manner. To obtain an optimal exchange between the medium and the slices during the incubation, the submerged slices were gently shaken in the transfer chamber. Three different protocols were employed in this study; The standard protocol, the SK&F protocol and the potassium protocol. A schematic representation of the protocols is given in Fig. 1. Standard protocol. The basal release in normal Krebs-Ringer medium was determined from t = 40-45 min. The exocytotic release of G A B A and glutamate was stimulated by raising the extracellular K ÷ concentration from 3.5 to 50 mM. A first sample of the stimulated release was taken from the first minute (t = 45-46 min). Then the slices were transferred to the next well, also containing 50 mM K ÷, for a prolonged stimulation of 4 min, yielding a second sample (t = 46-50; see Fig. la). This design provides information both on the release during a short initial period of depolarization and on effects of a prolonged depolarization, comparable with the protocol used in our previous study (5 min incubation with 50 mM K+). SK&F protocol. Identical to the standard protocol but 10 #M SK&F 89976-A was added to all media from t = 35 min onward. Potassium protocol. Identical to the standard protocol but at t = 45 rain the K ÷ concentration was increased to 15 mM, followed after 1 min by a transfer to medium with 20 mM K ÷. Thereafter, the K ÷ concentration was increased with steps of 5 mM K ÷ to 50 mM K + (see Fig. lb). For each of above-mentioned release protocols two transfer chambers were used. In the first, the protocol was carried out with 2 mM Ca 2 present in all media to determine the total release. In the second, Ca 2 was replaced by 2 mM EGTA in all media from t = 35 min onward, to study the calcium-independent release. Since from one rat 4 transfer chambers could be filled, only two of the 3 protocols could be carried out for one particular animal. After completion of the protocol, the medium of the relevant
35
Standard / SK 8, F protocol 1' 50
Potassium protocol
2'-5'
--|
a
40
E
v
E v
30.
E
20 0 12.
50b 45403530252015-
o--a
._1 a--m
I m--m
I
n
basal 3.5. o
--,
,Q'
3.5---
,~,
,
40
45 46 Time (rain)
5'0
0
~io
45
47
49
51
53
Time (min)
Fig. 1. a: schematic representation of the standard and the SK&F release protocol. Basal release was determined from t = 40-45 min in medium containing 3.5 mM K +. Thereafter, the tissue was transferred to medium containing 50 mM K +. To study the evoked amino acid in the first minute of stimulation, a sample was taken from 45-46 min (1'). After transfer to fresh medium also containing 50 mM K ÷, a sample was taken from 46--50 min (2'-5') to study the release after prolonged depolarization. The SK&F protocol was identical to the standard protocol but 10/~M SK&F was present in all media from t = 35 onward, b: schematic representation of the potassium protocol. Slices of CA1 were kept in normal medium until t = 45 min, then the tissue was transferred to 15 mM K ÷ . Thereafter, the K ÷ concentration was increased every minute with steps of 5 mM K ÷ to 50 mM K ÷.
steps was collected (700 #1) from the wells, an internal reference amino acid (homoserine) was added and the samples were stored frozen in the presence 0.1% TCA. The wet weight of the tissue in each chamber was determined and the tissue was frozen for later determination of the total amino acid content. For this purpose material was homogenized at 4 °C in aqua dest + 0.1% Triton X-100 (15 mg tissue/100 gl). A sample from this homogenate was diluted 1:10 in methanol 100% and the amino acid concentration was determined. H P L C amino acid analysis Amino acid analysis was carried out with an isocratic high performance liquid chromatography (HPLC) system using a reversed-phase column and a fluorometrical detection. Details of this method were given previously 14"28. Peak height amplitudes from baseline were measured. Calibration was carried out by means of GABA and glutamate standards with a concentration of 1.0/~M and the internal standard present in each release sample. Release under basal and stimulated conditions was calculated as pmol GABA/glutamate expressed per minute per mg tissue. RESULTS
C O N group. Release pattern o f G A B A
aspartate, glutamate, glutamine, serine, glycine, alanine, taurine and G A B A
from hippocampal subslices. A n
increase of the KCI concentration to 50 m M , e q u i m o l a r reduction
of NaCI, induced
with
an enhanced
release of aspartate, glutamate and G A B A while the of glutamine
groups of control animals studied at different times along with the 4 kindled groups. Th e controls were, therefore, pooled into a single control g r o u p for comparison with the e x p e r i m e n t a l groups. Th e basal G A B A release from subslices of CA1 region of the C O N group in normal m e d i u m was 0.3 + 0.1 p m o l / m i n / m g tissue ( m ean + S . E . M . , Table I). This basal release was c a l c i u m - i n d e p e n d e n t because it was not significantly changed when Ca 2÷ in the m e d i u m was
decreased.
In
the
absence
of
Mean G A B A release (pmol/mg tissue +_S. E. M.) 0]:6 CA1 subslices of the control group (CON) under basal and under stimulated conditions of the Standard protocol and of the S K & F protocol The evoked release is given for the first (1"/50K) and the subsequent 4 minutes (2"-5"/50K) of depolarization and is separated into the calcium-independent and the calcium-dependent release. All release values are expressed as pmol/min/mg tissue.
extracellular Ca 2÷, the e n h a n c e m e n t of glutamate and
Standard (n = 16)
G A B A was strongly reduced. It is important to note that
1 750K
we use in this report the term release to describe the effiux of amino acids; that is the quantity of amino acids released by the CAI subslices into the incubation media. In the present report the main emphasis will be on the release of G A B A
in controls
No significant differences w e r e f o u n d b e t w e e n the
TABLE I
T h e amino acid analysis revealed a basal release of
release
observed in the kindled groups is c o m p a r e d to that of the
since only a few kindling related
changes in the glutamate release w e r e found. First, the release characteristics o f G A B A in the control group for the 3 protocols are presented. Second, the release
SK&F (n = 12) 2"--5 750K 1 750K
2"--5 750K
Basal release 0.3+0.1 0.9+0.1"** Stimulated release CA 2+ independent 5.6+0.3 2.5+0.2 3.3+0.3* 3.3+0.2** CA 2÷ dependent fraction 13.4+0.5 14.9+0.6 41.7+1.5"** 30.0+0.8*** *P ~< 0.01, **P ~< 0.005, ***P < 0.001; Student's t-test with paired comparisons (n = 21) between standard and SK&F protocol of the same animal.
36
25"
& •
14AD+FK (n=5) LT (n=4) CON(n=8)
strated a small increase of the c a l c i u m - d e p e n d e n t stimulated release during p r o l o n g e d stimulation (Table I). In contrast, the calcium-independent stimulated release was reduced by a factor 0.45 ( P < 0.001, p a i r e d Student's t-test, Table I) during p r o l o n g e d depolarization. Inhibition of G A B A u p t a k e by 10 /~M S K & F was studied in 12 control animals. T h e results are p r e s e n t e d in Table I. The addition of S K & F d e a r l y e n h a n c e d the detected a m o u n t of G A B A u n d e r basal conditions. During stimulation, the presence of S K & F increased the detected a m o u n t of G A B A from the c a l c i u m - d e p e n d e n t release during the first minute of high K +, a p p r o x i m a t e l y 3-fold. During the following 4 min, the d e t e c t e d a m o u n t of c a l c i u m - d e p e n d e n t stimulated G A B A release was about 2-fold of that found without u p t a k e blocker. The calcium-independent fraction b e c a m e significantly smaller in the presence of S K & F during the first minute of high K +, but was slightly increased in the following 4 min. S K & F had no detectable effect on the release of glutamate (data not shown). Stepwise elevation of the K ÷ level by 5 m M every minute resulted after a threshold concentration in a gradual increase of the c a l c i u m - d e p e n d e n t stimulated G A B A release and reached a p l a t e a u at 45 m M K ÷ of 11-12 pmol/min/mg tissue issue (Fig. 2). In each control animal studied (n = 8), the threshold concentration of K + that e v o k e d a significant c a l c i u m - d e p e n d e n t fraction, was between 25 and 30 mM.
20" 15"
<[ 10 O
S 0 ~'5 ~o ~'s 3'o 3's 4'o ,,'s s o
(mM)
potassium concentration
Fig. 2. Potassium protocol; mean calcium-dependent fraction of GABA release (_+ S.E.M.) as a function of stepwise increased extracellular potassium concentration. Release is expressed as pmol GABA per minute per mg tissue. The animals of the 14AD (n = 2) and the FK groups (n = 3) were pooled. No animals of the 6AD group were studied with this protocol. The increases in the 14AD + FK group were significant for potassium concentrations greater or equal than 30 mM and for the LT group from 35 mM on (P <~ 0.05, Student's t-test).
r e p l a c e d by E G T A . In contrast, the release of G A B A , e v o k e d by 50 m M K +, was of p r e d o m i n a n t calciumd e p e n d e n t nature. To d e t e r m i n e the calcium-dependent stimulated release, the calcium-independent stimulated release d e t e r m i n e d u n d e r E G T A conditions, was subtracted from the total release d e t e r m i n e d in the presence of Ca 2÷. The protocols were designed in such a way that the release during the first minute of depolarization could be c o m p a r e d with the release e v o k e d during p r o l o n g e d stimulation in the following 4 min (Fig. 1). This demon-
Retained G A B A content in control and kindled groups The m e a n r e t a i n e d content of the CA1 slices of the C O N - g r o u p after the standard p r o t o c o l was 1084 + 38 p m o l G A B A p e r mg tissue (-- 100%). In comparison to
lO-a
35tb
08-
30
~,
25 2o
0.4-
T
-t-
~-
'~ E "" ~ 15 ~
T
~
j
-~ ~o
02
0.0
: "
T
CON 6AD 14AD FK
I
LT
.
.
.
. CON 6AD 14AD FK
LT
Fig. 3. Standard protocol, a: mean basal GABA release (_+ S.E.M.) of 6 CA1 subslices of the CON group and of the kindled groups. Release is expressed as pmol GABA per min per mg tissue. The mean release of the pooled animals of the 6AD, 14AD and FK groups (0.38 + 0.04) was larger than that of controls (P ~<0.031). b: mean calcium-dependent fraction of the stimulated GABA release under 50 mM K + conditions. The release is given for the first (1"/50K) and the subsequent 4 min (2"-5"/50K) of depolarization. Values are expressed as pmol GABA + S.E.M. per min per mg tissue. **P ~< 0.004, ***P < 0.001; Student's t-test, comparison between kindled and control animals. Number of observations: CON, n = 16; 6AD, n = 9; 14AD, n = 9; FK, n = 4; LT, n = 5.
37 35
!
E= =o
30 o~ E~ ¢1 "~U 25
o<
~o t-o
basal 1'/50K + SK&F 2'-5750K + SK&F
10 5
0
CC~
6AD
14AD
FK
LT
Fig. 4. S K & F protocol. Additionally detected amount of G A B A in comparison to the standard protocol for the CON and the kindled groups under basal and stimulated conditions. The excess G A B A obtained in the presence of S K & F 89776-A was calculated by subtracting the mean release in the standard protocol from the release, under similar conditions, in the presence of 10/~M S K & E The amounts for stimulated conditions arc given for the first (]'/50K) and the subsequent 4 rain (2"-5"/50K) of depolarization. Values are expressed as mean _+ S.E.M. per rain per mg tissue. Number of observations: CON, n = ]2; 6AD, n = 9; 14AD, n =
5;FK, n = 4;LT, n = 5. this control value, a significant increase was found in the 6AD (116%, P ~< 0.009, Student's t-test), the 14AD (123%, P ~< 0.001) and the FK group (121%, P ~< 0.008). No significant differences were found between these groups of kindled animals. The retained content of the LT group did not differ from that of the controls.
Development of GABA release in kindled groups The mean basal release from the control and the kindled groups is illustrated in Fig. 3a. As in the CON group, in all kindled groups the mean basal G A B A release was not different from the basal release when Ca 2+ in the medium was replaced by EGTA. The mean basal release of all the kindled groups studied 24 h after the last seizure was larger than the basal release of controls (Fig. 3a). However, the detected amount of G A B A in the basal release samples was close to the detection limit of the H P L C and this caused a large relative variation in the measurements. When the kindled group 6AD, 14AD and FK were pooled, we found a significant increase of the basal release (P ~< 0.031, Student's t-test). The values of the stimulated calcium-dependent G A B A release are presented in Fig. 3b. Comparison with the CON group ( = 100%) showed a highly significant increase in the 14AD (128%), the FK (199%) and the LT (146%) groups in the first minute of depolarization. These differences were also found in the following 4 rain of depolarization. In contrast to the release of the other kindled groups, the evoked G A B A release from the 6AD group gave no indication of an increase. It is important to emphasize that the animals of the 6AD
group showed clear signs of kindling acquisition; the animals responded with stage 1-2 behavioral seizures to the last tetanic stimulation and the duration of the afterdischarge became gradually longer from 21 + 4 s (mean + S.E.M., n --- 9) at session 1 to 33 + 2 s at session 6 (P -<-< 0.001, paired comparison). Moreover, field potential recordings indicated a reduction of paired-pulse depression at this phase of kindling development (data not shown) 12. The calcium-independent stimulated release in the kindled groups was not significantly different from the CON group.
Uptake of GABA in kindled animals A part of the G A B A released from the terminals is taken up by the active carder mechanism before it can reach the incubation medium in which the slices are kept. We estimated the amount taken up, by blocking the carder with SK&F and calculating the difference between the mean release found in the standard protocol and the release found in the presence of 10 /~M SK&E This additionally detected amount of G A B A under SK&F conditions, reflects the activity of the G A B A uptake carrier. The additionally detected amount was for the kindled groups under basal conditions not different from controls (Fig. 4). No significant, kindling related, change in the additionally detected amount of G A B A under high K ÷ conditions was encountered (Fig. 4). The small SK&F-induced decrease of the calciumindependent fraction during the first minute of high K ÷ in controls (Table I), was also found in the kindled groups. This decrease did not differ between kindled and control animals. Potassium threshold for calcium-dependent GABA release in kindled animals The results of the potassium protocol for the kindled groups are presented in Fig. 2. The threshold concentration for eliciting a calcium-dependent G A B A release was between 25 and 30 mM for all investigated kindled animals and is therefore not different from the threshold observed in control animals. The G A B A release of the kindled animals was larger compared to controls at 30 mM K ÷ and at all following K ÷ steps. The relative increase in kindled animals was about the same for the different K + concentrations. This increase in comparison to the CON group was significant from 30 mM onward for the pooled animals of the 14AD and the FK groups (n = 5) and from 35 mM onward for the LT group. Glutamate release During prolonged depolarization a significant increase of the mean calcium-dependent glutamate release, was
38 found in the FK and LT groups as compared to the CON group (CON, 21.9 + 1.3; vs FK, 30.8 + 1.6; P ~< 0.002, Student's t-test; and CON vs LT, 34.6 + 2.7; P ~< 0.044, one-tailed). In contrast, during the first minute, the increase measured in the mean calcium-dependent fraction of the FK (23.0 + 2.7) and the LT (25.6 _+ 2.5) groups was not statistically different from controls (20.3 + 1.8). With the potassium protocol the threshold for eliciting a calcium-dependent glutamate release was established between 30 and 40 mM KCI. Similar thresholds were found for kindled animals. DISCUSSION In the present study the effects of Schaffer collateral/ commissural pathway kindling on the release of the amino acid neurotransmitters G A B A and glutamate in CAI area were investigated. Our main finding was that the calcium-dependent, high K÷-evoked G A B A release was significantly enhanced in animals that had received 14 kindling stimulations, in fully kindled animals 24 h and 25-36 days after the last generalized seizure. The observed increase was found in the first minute of evoked release. Prolonged stimulation with high K ÷ showed also an enhanced release confirming our previous observations 15. In animals that had received 6 stimulations no increase was observed despite the fact that these animals showed clear manifestations of kindling epileptogenesis. From the observations reported here, follows the conclusion that kindling stimulations in CAI lead to a gradual increase in calcium-dependent stimulated G A B A release in the same region with an onset of this change between session 6 and session 12. The K÷-evoked, calcium-dependent net efflux of G A B A and glutamate from CA1 subslices in vitro, termed in this report release, is generally considered to provide information on the calcium-dependent exocytosis from synaptic vesicles in the terminals triggered by the high K÷-induced depolarization 2'23'31. It is important to note that in the intact system exocytotic release is likely to occur in milliseconds while our measurements are made on a time scale of 1 min. Notwithstanding this fact, the results reported here indicate that the late phases of kindling epileptogenesis are associated with a long-lasting enhanced exocytotic release of GABA. The retained total tissue content of G A B A was enlarged in the 6AD, the 14AD and the FK groups probably underlying the enhanced basal release from the cytosolic compartment via a calcium-independent mechanism. This alteration was not found in the LT group, suggesting that this change is seizure associated and different from the changes underlying the enhanced G A B A exocytosis 7.
The presence of G A B A and glutamate/aspartate reuptake mechanisms in the tissue, in addition to technical limitations which only allow the detection of the 'overflow' of amino acid neurotransmitters from the slices, leads to an underestimation of the actual amount of neurotransmitter exocytotically released into the extracellular space. It is possible that the observed enhanced release of G A B A from the slices may be accounted for by a decreased uptake. This possibility is not excluded by the fact that the rise in K ÷ and the simultaneous lowering of Na ÷, to depolarize the tissue, leads to a partial suppression of Na÷-dependent G A B A uptake 1'3. This possibility was checked by blocking the G A B A uptake carrier with SK&F 89776-A 26'32. Our observations on the effects of SK&F on G A B A release are in agreement with the finding that SK&F enhanced the amount of G A B A detected after electrical or high potassium stimulated release of endogenous G A B A from cortical brain slices 31. In addition, we also found that SK&F decreased the calcium-independent, K÷-evoked, G A B A release during the first minute of depolarization which may be related to an inhibition of the reversal of the uptake carrier during depolarization 25' 28. The amount of G A B A additionally detected due to SK&F presence, representing the amount of G A B A taken up in the standard protocol, was unchanged in the kindled animals. Thus, the changes in G A B A release found in kindled animals are not different in the presence of the G A B A uptake blocker, indicating that the kindling related enhanced release originates from a calciumdependent pool likely from exocytotic origin. This interpretation is further supported by our recent release experiments using purified synaptosomal suspensions where re-uptake is negligible. The K÷-evoked calciumdependent G A B A release in the first minute of stimulation was enhanced in synaptosomes prepared from kindled animals 3°. The threshold K+-concentration for the calcium-dependent G A B A release was found to be between 25 and 30 mM KC12. In kindled animals this threshold was unaltered while the release was enhanced over the whole range of investigated K÷-concentrations, suggesting no clear change in the threshold level of depolarization that is needed to evoke exocytotic release in kindled animals. Concerning the release of glutamate, we found an enhanced glutamate release in the FK and LT groups. However, this change was clearly dependent upon a prolonged depolarization since the release during the first minute of stimulation was not significantly increased. An increased glutamate release from hippocampal slices, 1 month after the last seizure in entorhinal kindled rats, was demonstrated by Geula et al. 5 and Jarvie et al. 8 who used in their studies a long depolarization of 5 min with
39 55 mM KC1. The presence of a glutamate uptake mechanism 3, that may mask possible changes occurring in glutamate exocytosis during kindling epileptogenesis, prevents definite conclusions on basis of the present data. Data available in the literature are in agreement with the notion of an enhanced G A B A release in kindled animals. An increased K÷-stimulated (40 rain with 60 mM KCI) release of G A B A from hippocampal slices of entorhinal kindled rats, 24 h after the last convulsion, was demonstrated by Liebowitz et al. 19. Jarvie et al. 9 studied in entorhinal cortex kindled rats, 1 month after the last seizure, the K+-evoked (5 min with 55 mM KCI), calcium-dependent release of amino acids from 3 hippocampal regions. The mean G A B A release from one ipsiand contralateral side was enhanced by approximately 70% in CA1 and in CA3, but due to a large variability in their measurements these increases were not statistically significant 9. In contrast to our results, a decreased total G A B A level and a significant long-lasting decrease of the K÷-stimulated (30 min with 60 mM KCI), calcium-dependent G A B A release was found in hippocampal slices obtained from animals that were stimulated with a large number of rather long tetanic stimulations applied with a short interstimulus interval 18'2°. The electrical stimulation protocol in these studies is clearly different from the classical kindling paradigm used by us 6 and the differences with the results of experiments described in the present report can probably be attributed to the differences in electrical stimulation paradigm. At present it is not clear which mechanism is underlying the enhanced G A B A exocytosis. On the one hand, the changes may be located in the cascade of processes between the stimulus and the release of neurotransmitter; for instance they may be due to an enhanced calcium influx or an enhanced CaE÷/calmodulin-dependent protein kinase II activity24. On the other hand, structural changes resulting in a larger number of GABAergic terminals due to sprouting process may also contribute to the observed changes. The latter would also explain why the reduced number of GABA-immunoreactive somata present in CA1 at long-term after kindling is associated
with an enhanced release of G A B A 13. The fact that no alterations occur in the 6AD group merits special attention. At this phase of kindling an attenuation of paired-pulse depression in CA1 is already clearly present 12. On the basis of the results of our previous release study in fully kindled animals, we suggested that an enhanced G A B A exocytosis could lead to a G A B A receptor desensitization resulting in a reduced paired-pulse inhibition and in a decreased sensitivity of hippocampal pyramidal cells to G A B A 15'16. On the basis of the present results, we now have to conclude that the enhanced G A B A exocytosis cannot be responsible for the observed electrophysiological changes. It appears that the presynaptic changes are manifested only after the changes in the field potentials become evident which indicates that the enhanced G A B A exocytosis may be a compensatory response of the cells to counteract the decreased stability caused by a reduced postsynaptic GABAergic function. On the basis of field potential alterations in the kindled focus we put forward the suggestion that the long-lasting kindling induced hyperexcitability is a consequence of the gradual loss of inhibitory function in CA1 during kindling epileptogenesis rE. Paradoxically, the results reported here along with the results of our previous studies lead to the conclusion that hippocampus kindling epileptogenesis and the induced kindled state are associated with an enhanced G A B A exocytosis. Therefore, we hypothesize that the primary changes responsible for a reduced inhibition and thus to a chronic enhanced seizure susceptibility are located at the level of the G A B A receptor complex. This hypothesis is strongly supported by recent electrophysiological evidence 4'8'16.
Acknowledgements. SK&F 89776-A was a gift from Dr. Skidmore (Smith, Kline and French Laboratories). We thank Dr. Wadman, Dr. Ghijsen and Dr. Verhage for their suggestions. We like to thank the CIVO-TNO Institute, Zeist, for their technical support and Hugo Sandman for his excellent technical assistance. This work was supported by the Dutch Committee for Epilepsy Research (CLEOTNO, Grant A-67) and by Medigon (Grant 550-663-024).
REFERENCES 1 Blaustein, M.P. and King, A.C., Influence of membrane potential on the sodium-dependent uptake of gamma-aminobutyric acid presynaptic nerve terminals: experimental observations and theoretical considerations, J. Membr. Biol., 30 (1976) 153-173. 2 Burke, S.P. and Nadler, J.V., Regulation of glutamate and aspartate release from slices of the hippocampal CA1 area: effects of adenosine and baclofen, J. Neurochem., 51 (1988) 1541-1551. 3 Erecinska, M., The neurotransminer amino acid transport systems, Biochem. Pharmacol., 36 (1987) 3547-3555. 4 Gean, P.W,, Shinnick-Gallager, P. and Anderson, A.C., Spon-
5 6 7 8
taneous epileptiform activity and alteration of GABA- and of NMDA-mediated neurotransmission in amygdala neurons kindled in vivo, Brain Research, 494 (1989) 177-181. Geula, C., Jarvie, P.A., Logan, T.C. and Slevin, J.T., Long-term enhancement of K+-evoked release of L-glutamate in entorhinal kindled rats, Brain Research, 442 (1988) 368-372. Goddard, G.V., Mclntyre, P.C. and Leech, C.K., A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol., 25 (1969) 295-330. Gold, B.I. and Roth, R.H., Glutamate decarboxylase activity in striatal slices: characterization of the increase following depolarization, J. Neurochem., 32 (1978) 883-888. Hernandez, T.D., Rosen, J.B. and Gallagher, D.W., Long-term changes in sensitivity to GABA in dorsal raphe neurons
40 following amygdala kindling, Brain Reseach, 517 (1990) 294300. 9 Jarvie, P.A., Logan, T.C., Geula, C. and Slevin, J.T., Entorhinal kindling permanently enhances Ca2+-dependent L-glutamate release in regio inferior of rat hippocampus, Brain Research, 508 (1990) 188-193. 10 Johnston, G.A.R., Stephanson, A.L. and Twitchin, B., Uptake and release of nipecotic acid by rat brain slices, J. Neurochem., 26 (1976) 83-87. 11 Kamphuis, W., Wadman, W.J., Buijs, R.M. and Lopes da Silva, EH., The development of changes in hippocampal gammaaminobutyric acid (GABA) in the rat kindling model of epilepsy: a light microscopic study with GABA-antibodies, Neuroscience, 23 (1987) 433-446. 12 Kamphuis, W., Wadman, W.J. and Lopes da Silva, EH., Changes in local evoked potentials in the rat hippocampus (CA1) during kindling epileptogenesis, Brain Research, 440 (1987) 433-446. 13 Kamphuis, W., Huisman, E., Wadman, W.J. and Lopes da Silva, EH., Decrease in GABA immunoreactivity and alteration of GABA metabolism after kindling in the rat hippocampus, Exp. Brain Res., 74 (1989) 375-386. 14 Kamphuis, W. and Lopes da Silva, EH., The kindling model of epilepsy: the role of GABAergic inhibition, Neurosci. Res. Commun., 6 (1990) 1-10. 15 Kamphuis, W., Huisman, E., Dreijer, A.M.C., Ghijsen, W.E.J.M., Ghijsen, Verhage, M. and Lopes da Silva, EH., Kindling increases the K+-evoked Ca2+-dependent release of endogenous GABA in area CA1 of rat hippocampus, Brain Research, 511 (1990) 63-70. 16 Kamphuis, W., Gorter, J.G. and Lopes da Silva, EH., Longterm decreased sensitivity of hippocampal pyramidal cells to GABA induced by kindling epileptogenesis, Neuroscience, in press. 17 Kapur, J., Michelson, H.B., Buterbaugh, G.E and Lothman, E.W., Evidence for a chronic loss of inhibition in the hippocampus after kindling: electrophysiological studies, Epilepsy Res., 4 (1989) 90-99. 18 Kapur, J., Bennett, J.P., Wooten, G.E and Lothman, E.W., Evidence for a chronic loss of inhibition in the hippocampus after kindling: biochemical studies, Epilepsy Res., 4 (1989) 100-108. 19 Liebowitz, N.R., Pedley, T.A. and Cutler, R.W.P., Release of gamma-aminobutyric acid from hippocampal slices of the rat following generalized seizures induced by daily electrical stimulation of entorhinal cortex, Brain Research, 138 (1978) 369373.
20 Lothman, E.W., Bennett, J.P. and Perlin, J.B., Alterations in neurotransmitter amino acids in hippocampal kindled seizures, Epilepsy Res., 1 (1987) 313-320. 21 Minc-Golomb, D., Levy, Y., Kleinberger, N. and Schramm, M., D-[3H]aspartate release from hippocampal slices studied in a multiwell system: controlling factors and postnatal development of release, Brain Research, 402 (1987) 255-263. 22 McNamara, J.O., Bonhaus, D.W., Shin, C., Crain, B.J., Gellman, R.L. and Giacchino, J.L., The kindling model of epilepsy: a critical review, CRC Neurobiol., 1 (1986) 341-391. 23 Nicholls, D.G., Release of glutamate, aspartate and gammaaminobutyric acid from isolated nerve terminals, J. Neurochem., 52 (1989) 331-341. 24 Nichols, R.A., Sihra, T.S., Czernik, A.J., Nairn, A.C. and Greengard, P., Caicium/calmodulin-dependent protein kinase II increases glutamate and noradrenaline release from synaptosomes, Nature, 343 (1990) 647-651. 25 Pin, J.-P. and Bockaert, J., Two distinct mechanisms, differentially affected by excitatory amino acids, trigger GABA release from fetal mouse striatal neurons in primary culture, J. Neurosci., 9 (1989) 648-656. 26 Pittaluga, A., Asaro, D., PeUegrini, G. and Raiteri, M., Studies on [3H]GABA and endogenous GABA release in rat cerebral cortex suggest the presence of autoreceptors of the GABAB type, Eur. J. Pharmacol., 144 (1987) 45-52. 27 Racine, R.J., Modification of seizure activity by electrical stimulation. II. Motor seizure, Electroencephalogr. Clin. Neurophysiol., 32 (1972) 281-294. 28 Sihra, T.S. and Nicholls, D.G., 4-Aminobutyrate can be released exocytotically from guinea-pig cerebral cortical synaptosomes, J. Neurochem., 49 (1987) 261-267. 29 Verhage, M., Besselsen, E., Lopes da Silva, F.H. and Ghijsen, W.E.J.M., Ca2+-dependent regulation of presynaptic stimulussecretion coupling, J. Neurochem., 53 (1989) 1188-1194. 30 Verhage, M., Besselsen, E., Huisman, E., Kamphuis, W., Lopes da Silva, EH. and Ghijsen, W.E.J.M., Changes in the GABAexocytosis in kindled rats and its relationship to GABA homeostasis, Neuroscience, in press. 31 Waldmeier, P.C., Wicki, P., Feldtrauer, J.J. and Baumann, P.A., The measurement of the release of endogenous GABA from rat brain slices by liquid chromatography with electrochemical detection, Naunyn-Schmiedeberg's Arch. PharmacoL, 337 (1988) 284-288. 32 Yunger, L.M., Fowler, P.J., Zarevics, P. and Setler, EE., Novel inhibitors of gamma-aminobutyric acid (GABA) uptake: anticonvulsant actions in rats and mice, J. Pharmacol. Exp. Ther., 228 (1984) 109-115.
33
BRES 16450
Development of changes in endogenous GABA release during kindling epileptogenesis in rat hippocampus W. Kamphuis, E. Huisman, M.J. Veerman and F.H. Lopes da Silva Department of Experimental Zoology, University of Amsterdam, Amsterdam (The Netherlands) (Accepted 23 October 1990)
Key words: Kindling: 7-Aminobutyric acid; Hippocampus; Neurotransmitter release; ~,-Aminobutyrc acid uptake; Epilepsy; SK&F 89776-A; Glutamate
The calcium-dependent y-aminobutyric acid (GABA) and glutamate release from rat hippocampal CA1 slices, evoked by a 1-min depolarization with 50 mM K +, was investigated in different stages of kindling epileptogenesis. Kindling was induced by tetanic stimulation of the Schaffer collateral]commissural pathway. In agreement with our previous results, we found a significantly increased calcium-dependent GABA release compared to that of implanted controls, in a group of fully kindled animals 1 day after the last seizure and also 25-36 days after the last seizure. In addition, we found that the increase in GABA release was associated with late phases of kindling epileptogenesis since no significant alterations were found in partly kindled animals that had received only 6 kindling stimulations while a significant increase was apparent in animals that had received 14 tetanic stimuli. When the release protocol was carried out in the presence of SK&F 89776-A, a blocker of the GABA uptake carder, an additional amount of GABA was found after depolarization. This additional amount of GABA, reflecting the amount of GABA taken up under conditions without blocker, was in kindled animals not different from controls which demonstrates that a reduced GABA uptake does not account for the observed enhanced release in kindled animals. The calcium-dependent release of glutamate evoked by 1 min of high potassium depolarization was not significantly changed in the kindled groups. Only after prolonged depolarization during 4 subsequent minutes a significant increase in animals of the fully kindled group and at long-term after kindling was observed. The threshold K + concentration for eliciting a calcium-dependent release of GABA and glutamate, was not changed in the kindled animals. The results reported here indicate that the late stages of kindling development and the long-lasting enhanced seizure sensitivity are associated with an enhanced GABA exocytosis, possibly to counteract a long-lasting postsynaptic reduction of GABA receptor sensitivity.
INTRODUCTION R e p e a t e d focal electrical tetanic stimulation of the brain, once o r twice daily, results in a lengthening of the elicited epileptiform activity and a progression of the b e h a v i o r a l seizure response. This process of epileptogenesis, resulting in a long-lasting e n h a n c e d seizure sensitivity, is called kindling 6'14'22. O n e of the possible neurobiological mechanisms underlying kindling epileptogenesis is an i m p a i r m e n t of y - a m i n o b u t y r i c acid ( G A B A ) m e d i a t e d recurrent inhibition 14. In accordance with this hypothesis, it was shown that kindling leads to a gradual reduction of paired-pulse depression of local e v o k e d field potentials in the C A 1 a r e a of the rat h i p p o c a m p u s 12'17 and to a d e c r e a s e d sensitivity of h i p p o c a m p a l p y r a m i d a l cells to G A B A 16. W i t h i m m u n o c y t o c h e m i c a l techniques, it was investigated w h e t h e r the o b s e r v e d decrease of inhibitory control was caused by a r e d u c e d cellular G A B A content of local interneurons. H o w e v e r , our studies revealed an
e n h a n c e d immunoreactivity of G A B A e r g i c s o m a t a in CA1 during the initial stages of kindling xl and a reduction only at long t e r m after the last seizure in fully kindled animals 13. M o r e o v e r , at the synaptic level evidence was found for an increased c a l c i u m - d e p e n d e n t release of G A B A from CA1 subslices in fully k i n d l e d animals 15. The latter result led us to suggest that an increased exocytosis of G A B A in the initial phase of kindling acquisition m a y l e a d to a r e d u c e d G A B A r e c e p t o r sensitivity and consequently to a r e d u c e d paired-pulse inhibition. In the present r e p o r t we describe a new series of release e x p e r i m e n t s c a r d e d out to clarify the following 3 issues regarding changes in G A B A release. (1) To c o m p r e h e n d the relation b e t w e e n the electrophysiological alterations and the previously o b s e r v e d changes in G A B A release, we studied with an i m p r o v e d technique the release of G A B A at 4 different phases of kindling epileptogenesis. Simultaneously, the release of glutamate, the main excitatory n e u r o t r a n s m i t t e r in C A 1 , was
Correspondence: W. Kamphuis, Department of Experimental Zoology, University of Amsterdam, Kruislaan 320, 1098 SM Amsterdam, The Netherlands.
34
w e c a r r i e d o u t e x p e r i m e n t s in t h e p r e s e n c e o f S K & F
kindled state, evidenced by the occurrence of class 5 generalized tonic-clonic convulsions27, the release was investigated in two subgroups at different intervals after the last elicited convulsion. Fully kindled (FK)-group (n = 6). Kindled animals studied 24 h after the last class 5 seizure, together with 5 rats of the CON-group. Kindled animals of this group experienced a mean of 27 afterdischarges. A mean of 5 generalized tonic-clonic seizures (stage 5) had been elicited. Long-term (LT)-group (n = 7). Kindled animals studied 25-36 days after the last seizure, together with 7 animals of the CON group. A mean of 31 afterdischarges were evoked in these kindled animals. A mean of 5 generalized seizures (stage 5) were elicited.
8 9 7 7 6 - A , a specific b l o c k e r o f t h e G A B A u p t a k e carr i e r 26'31'32. T h i s s e l e c t i v e a n d p o t e n t d r u g h a s t h e a d v a n -
Amino acid release procedures
m e a s u r e d . (2) T h e r e l e a s e m e t h o d u s e d a c t u a l l y d e t e r mines
the
efflux
of
amino
acids
from
slices.
This
corresponds to the net difference between the quantity of a m i n o acid r e l e a s e d f r o m t h e i n t r a c e l l u l a r c o m p a r t m e n t and the quantity removed from the extracellular space by u p t a k e p r o c e s s e s . T h e r e f o r e , a c h a n g e in t h e effiux m a y b e d u e t o a c h a n g e in r e l e a s e a n d / o r u p t a k e p r o c e s s e s . T o be able to differentiate between these two possibilities,
t a g e o v e r n i p e c o t i c acid, p r e v i o u s l y u s e d b y us t o i n h i b i t the
GABA
uptake
m e c h a n i s m 1°'15,
in
that
it is
a
n o n - c o m p e t i t i v e b l o c k e r . S i n c e S K & F is n o t t a k e n u p b y t h e t i s s u e it d o e s n o t d i s t u r b t h e i n t r a c e l l u l a r G A B A homeostasis
and
is w i t h o u t
effect on the
basal and
K + - e v o k e d r e l e a s e o f [ 3 H ] G A B A f r o m s y n a p t o s o m e s 26. (3) T h e e n l a r g e d G A B A
r e l e a s e f o u n d p r e v i o u s l y in fully
kindled animals may be the result of a lowered threshold K ÷ - c o n c e n t r a t i o n f o r eliciting a c a l c i u m - d e p e n d e n t rel e a s e . I n o r d e r to a d d r e s s this p o s s i b i l i t y , t h e t h r e s h o l d was determined GABA
by measuring the calcium-dependent
release over a range of K+-concentrations.
MATERIALS AND METHODS
Implantation and kindling Male Wistar rats (200-250 g) were used in this study. Chronic indwelling electrodes were placed in CA1 area of the left dorsal hippocampus to enable kindling stimulations to this area. The details of this procedure were described previously 12. In short: under pentobarbital anesthesia, two bundles of stainless steel wire electrodes were implanted. The stimulation bundle was placed in the Schaffer-collaterai/commissural fiber pathway and the recording bundle was placed in stratum radiatum of CA1. Local evoked field potentials, elicited by test stimuli, were used to optimize the position of the electrodes. Electrodes were fixed in position with dental cement and placed in a connector plug. After at least 2 weeks of recovery, field potentials were recorded in the freely moving rat. The stimulation electrode that elicited the optimal evoked field potential in response to single pulse stimulation of the Schaffer collateral/commissural fiber pathway was selected and used to deliver kindling stimulations. A group of 49 implanted animals was divided into a nonstimulated control group (n = 18) and a group (n = 31) that received, twice daily, kindling stimulations at an intensity suprathreshold for the induction of an afterdischarge (200-300/zA, 50 Hz, 1 s). The animals of the control group (CON) were handled throughout the experimental period in a way comparable to the kindled rats but did not receive tetanic stimulations. The release of CA1 subslices was studied in the following groups of stimulated animals. 6AD-group (n = 9). Animals that had received 6 kindling stimulations. The release was studied 24 h after the last stimulation. All kindling stimulated animals showed at this early phase of the kindling process stage 1-2 behavioral seizures 27. Together with this group, 4 animals of the CON group were studied. 14AD-group (n = 9). Animals that had received 14 kindling stimulations were studied 24 h after the last stimulation. All stimulated animals in this group showed stage 2-3 seizures. Two control animals were studied together with the stimulated animals. When the remaining stimulated animals had acquired the fully
Rats were anesthetized with ether, decapitated and the brains were rapidly removed from the skull. Both hippocampi were dissected on an ice-cooled plate. From each hippocampus, 12 slices were cut along the septotemporal axis. Slices obtained from the ipsilateral and contralateral hemisphere in respect to the kindled side were collected in 95% 0 2 - 5 % CO 2 gassed Krebs-Ringer buffer at room temperature. Composition of the buffer in raM: NaCI 124, KCI 3.5, CaCI 2 2.0, MgSO 4 2.0, NaH2PO 4 1.25, NaHCO 3 26.0, D-glucose 10.0 with pH 7.4. The slices were divided at random in 4 sets of 6 slices. The CA1 area was dissected from the hippocampal slice to study specifically the release from this area 2'9'15. The CA1 subslices were placed in transfer chambers that were made from small tubes provided with a fine meshed nylon gauze bottom. At t = 0 min, the transfer chambers with the subslices laying on the meshed bottom were placed in a large reservoir filled with Krebs-Ringer buffer. All media in the release protocol were kept at a constant temperature of 34.4 °C. After a recovery period of 15 min (t = 0-15 min), the chambers were transferred to the wells of a 24-well tissue culture plate filled with 1000 #1 medium. From that moment onward, the chambers were transferred every 5 rain to the next row of wells filled with fresh medium. This method, based on a multiwell system described by Minc-Golomb 2a, allows a change over of the tissue from one medium to the next in a stepwise manner. To obtain an optimal exchange between the medium and the slices during the incubation, the submerged slices were gently shaken in the transfer chamber. Three different protocols were employed in this study; The standard protocol, the SK&F protocol and the potassium protocol. A schematic representation of the protocols is given in Fig. 1. Standard protocol. The basal release in normal Krebs-Ringer medium was determined from t = 40-45 min. The exocytotic release of G A B A and glutamate was stimulated by raising the extracellular K ÷ concentration from 3.5 to 50 mM. A first sample of the stimulated release was taken from the first minute (t = 45-46 min). Then the slices were transferred to the next well, also containing 50 mM K ÷, for a prolonged stimulation of 4 min, yielding a second sample (t = 46-50; see Fig. la). This design provides information both on the release during a short initial period of depolarization and on effects of a prolonged depolarization, comparable with the protocol used in our previous study (5 min incubation with 50 mM K+). SK&F protocol. Identical to the standard protocol but 10 #M SK&F 89976-A was added to all media from t = 35 min onward. Potassium protocol. Identical to the standard protocol but at t = 45 rain the K ÷ concentration was increased to 15 mM, followed after 1 min by a transfer to medium with 20 mM K ÷. Thereafter, the K ÷ concentration was increased with steps of 5 mM K ÷ to 50 mM K + (see Fig. lb). For each of above-mentioned release protocols two transfer chambers were used. In the first, the protocol was carried out with 2 mM Ca 2 present in all media to determine the total release. In the second, Ca 2 was replaced by 2 mM EGTA in all media from t = 35 min onward, to study the calcium-independent release. Since from one rat 4 transfer chambers could be filled, only two of the 3 protocols could be carried out for one particular animal. After completion of the protocol, the medium of the relevant
35
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53
Time (min)
Fig. 1. a: schematic representation of the standard and the SK&F release protocol. Basal release was determined from t = 40-45 min in medium containing 3.5 mM K +. Thereafter, the tissue was transferred to medium containing 50 mM K +. To study the evoked amino acid in the first minute of stimulation, a sample was taken from 45-46 min (1'). After transfer to fresh medium also containing 50 mM K ÷, a sample was taken from 46--50 min (2'-5') to study the release after prolonged depolarization. The SK&F protocol was identical to the standard protocol but 10/~M SK&F was present in all media from t = 35 onward, b: schematic representation of the potassium protocol. Slices of CA1 were kept in normal medium until t = 45 min, then the tissue was transferred to 15 mM K ÷ . Thereafter, the K ÷ concentration was increased every minute with steps of 5 mM K ÷ to 50 mM K ÷.
steps was collected (700 #1) from the wells, an internal reference amino acid (homoserine) was added and the samples were stored frozen in the presence 0.1% TCA. The wet weight of the tissue in each chamber was determined and the tissue was frozen for later determination of the total amino acid content. For this purpose material was homogenized at 4 °C in aqua dest + 0.1% Triton X-100 (15 mg tissue/100 gl). A sample from this homogenate was diluted 1:10 in methanol 100% and the amino acid concentration was determined. H P L C amino acid analysis Amino acid analysis was carried out with an isocratic high performance liquid chromatography (HPLC) system using a reversed-phase column and a fluorometrical detection. Details of this method were given previously 14"28. Peak height amplitudes from baseline were measured. Calibration was carried out by means of GABA and glutamate standards with a concentration of 1.0/~M and the internal standard present in each release sample. Release under basal and stimulated conditions was calculated as pmol GABA/glutamate expressed per minute per mg tissue. RESULTS
C O N group. Release pattern o f G A B A
aspartate, glutamate, glutamine, serine, glycine, alanine, taurine and G A B A
from hippocampal subslices. A n
increase of the KCI concentration to 50 m M , e q u i m o l a r reduction
of NaCI, induced
with
an enhanced
release of aspartate, glutamate and G A B A while the of glutamine
groups of control animals studied at different times along with the 4 kindled groups. Th e controls were, therefore, pooled into a single control g r o u p for comparison with the e x p e r i m e n t a l groups. Th e basal G A B A release from subslices of CA1 region of the C O N group in normal m e d i u m was 0.3 + 0.1 p m o l / m i n / m g tissue ( m ean + S . E . M . , Table I). This basal release was c a l c i u m - i n d e p e n d e n t because it was not significantly changed when Ca 2÷ in the m e d i u m was
decreased.
In
the
absence
of
Mean G A B A release (pmol/mg tissue +_S. E. M.) 0]:6 CA1 subslices of the control group (CON) under basal and under stimulated conditions of the Standard protocol and of the S K & F protocol The evoked release is given for the first (1"/50K) and the subsequent 4 minutes (2"-5"/50K) of depolarization and is separated into the calcium-independent and the calcium-dependent release. All release values are expressed as pmol/min/mg tissue.
extracellular Ca 2÷, the e n h a n c e m e n t of glutamate and
Standard (n = 16)
G A B A was strongly reduced. It is important to note that
1 750K
we use in this report the term release to describe the effiux of amino acids; that is the quantity of amino acids released by the CAI subslices into the incubation media. In the present report the main emphasis will be on the release of G A B A
in controls
No significant differences w e r e f o u n d b e t w e e n the
TABLE I
T h e amino acid analysis revealed a basal release of
release
observed in the kindled groups is c o m p a r e d to that of the
since only a few kindling related
changes in the glutamate release w e r e found. First, the release characteristics o f G A B A in the control group for the 3 protocols are presented. Second, the release
SK&F (n = 12) 2"--5 750K 1 750K
2"--5 750K
Basal release 0.3+0.1 0.9+0.1"** Stimulated release CA 2+ independent 5.6+0.3 2.5+0.2 3.3+0.3* 3.3+0.2** CA 2÷ dependent fraction 13.4+0.5 14.9+0.6 41.7+1.5"** 30.0+0.8*** *P ~< 0.01, **P ~< 0.005, ***P < 0.001; Student's t-test with paired comparisons (n = 21) between standard and SK&F protocol of the same animal.
36
25"
& •
14AD+FK (n=5) LT (n=4) CON(n=8)
strated a small increase of the c a l c i u m - d e p e n d e n t stimulated release during p r o l o n g e d stimulation (Table I). In contrast, the calcium-independent stimulated release was reduced by a factor 0.45 ( P < 0.001, p a i r e d Student's t-test, Table I) during p r o l o n g e d depolarization. Inhibition of G A B A u p t a k e by 10 /~M S K & F was studied in 12 control animals. T h e results are p r e s e n t e d in Table I. The addition of S K & F d e a r l y e n h a n c e d the detected a m o u n t of G A B A u n d e r basal conditions. During stimulation, the presence of S K & F increased the detected a m o u n t of G A B A from the c a l c i u m - d e p e n d e n t release during the first minute of high K +, a p p r o x i m a t e l y 3-fold. During the following 4 min, the d e t e c t e d a m o u n t of c a l c i u m - d e p e n d e n t stimulated G A B A release was about 2-fold of that found without u p t a k e blocker. The calcium-independent fraction b e c a m e significantly smaller in the presence of S K & F during the first minute of high K +, but was slightly increased in the following 4 min. S K & F had no detectable effect on the release of glutamate (data not shown). Stepwise elevation of the K ÷ level by 5 m M every minute resulted after a threshold concentration in a gradual increase of the c a l c i u m - d e p e n d e n t stimulated G A B A release and reached a p l a t e a u at 45 m M K ÷ of 11-12 pmol/min/mg tissue issue (Fig. 2). In each control animal studied (n = 8), the threshold concentration of K + that e v o k e d a significant c a l c i u m - d e p e n d e n t fraction, was between 25 and 30 mM.
20" 15"
<[ 10 O
S 0 ~'5 ~o ~'s 3'o 3's 4'o ,,'s s o
(mM)
potassium concentration
Fig. 2. Potassium protocol; mean calcium-dependent fraction of GABA release (_+ S.E.M.) as a function of stepwise increased extracellular potassium concentration. Release is expressed as pmol GABA per minute per mg tissue. The animals of the 14AD (n = 2) and the FK groups (n = 3) were pooled. No animals of the 6AD group were studied with this protocol. The increases in the 14AD + FK group were significant for potassium concentrations greater or equal than 30 mM and for the LT group from 35 mM on (P <~ 0.05, Student's t-test).
r e p l a c e d by E G T A . In contrast, the release of G A B A , e v o k e d by 50 m M K +, was of p r e d o m i n a n t calciumd e p e n d e n t nature. To d e t e r m i n e the calcium-dependent stimulated release, the calcium-independent stimulated release d e t e r m i n e d u n d e r E G T A conditions, was subtracted from the total release d e t e r m i n e d in the presence of Ca 2÷. The protocols were designed in such a way that the release during the first minute of depolarization could be c o m p a r e d with the release e v o k e d during p r o l o n g e d stimulation in the following 4 min (Fig. 1). This demon-
Retained G A B A content in control and kindled groups The m e a n r e t a i n e d content of the CA1 slices of the C O N - g r o u p after the standard p r o t o c o l was 1084 + 38 p m o l G A B A p e r mg tissue (-- 100%). In comparison to
lO-a
35tb
08-
30
~,
25 2o
0.4-
T
-t-
~-
'~ E "" ~ 15 ~
T
~
j
-~ ~o
02
0.0
: "
T
CON 6AD 14AD FK
I
LT
.
.
.
. CON 6AD 14AD FK
LT
Fig. 3. Standard protocol, a: mean basal GABA release (_+ S.E.M.) of 6 CA1 subslices of the CON group and of the kindled groups. Release is expressed as pmol GABA per min per mg tissue. The mean release of the pooled animals of the 6AD, 14AD and FK groups (0.38 + 0.04) was larger than that of controls (P ~<0.031). b: mean calcium-dependent fraction of the stimulated GABA release under 50 mM K + conditions. The release is given for the first (1"/50K) and the subsequent 4 min (2"-5"/50K) of depolarization. Values are expressed as pmol GABA + S.E.M. per min per mg tissue. **P ~< 0.004, ***P < 0.001; Student's t-test, comparison between kindled and control animals. Number of observations: CON, n = 16; 6AD, n = 9; 14AD, n = 9; FK, n = 4; LT, n = 5.
37 35
!
E= =o
30 o~ E~ ¢1 "~U 25
o<
~o t-o
basal 1'/50K + SK&F 2'-5750K + SK&F
10 5
0
CC~
6AD
14AD
FK
LT
Fig. 4. S K & F protocol. Additionally detected amount of G A B A in comparison to the standard protocol for the CON and the kindled groups under basal and stimulated conditions. The excess G A B A obtained in the presence of S K & F 89776-A was calculated by subtracting the mean release in the standard protocol from the release, under similar conditions, in the presence of 10/~M S K & E The amounts for stimulated conditions arc given for the first (]'/50K) and the subsequent 4 rain (2"-5"/50K) of depolarization. Values are expressed as mean _+ S.E.M. per rain per mg tissue. Number of observations: CON, n = ]2; 6AD, n = 9; 14AD, n =
5;FK, n = 4;LT, n = 5. this control value, a significant increase was found in the 6AD (116%, P ~< 0.009, Student's t-test), the 14AD (123%, P ~< 0.001) and the FK group (121%, P ~< 0.008). No significant differences were found between these groups of kindled animals. The retained content of the LT group did not differ from that of the controls.
Development of GABA release in kindled groups The mean basal release from the control and the kindled groups is illustrated in Fig. 3a. As in the CON group, in all kindled groups the mean basal G A B A release was not different from the basal release when Ca 2+ in the medium was replaced by EGTA. The mean basal release of all the kindled groups studied 24 h after the last seizure was larger than the basal release of controls (Fig. 3a). However, the detected amount of G A B A in the basal release samples was close to the detection limit of the H P L C and this caused a large relative variation in the measurements. When the kindled group 6AD, 14AD and FK were pooled, we found a significant increase of the basal release (P ~< 0.031, Student's t-test). The values of the stimulated calcium-dependent G A B A release are presented in Fig. 3b. Comparison with the CON group ( = 100%) showed a highly significant increase in the 14AD (128%), the FK (199%) and the LT (146%) groups in the first minute of depolarization. These differences were also found in the following 4 rain of depolarization. In contrast to the release of the other kindled groups, the evoked G A B A release from the 6AD group gave no indication of an increase. It is important to emphasize that the animals of the 6AD
group showed clear signs of kindling acquisition; the animals responded with stage 1-2 behavioral seizures to the last tetanic stimulation and the duration of the afterdischarge became gradually longer from 21 + 4 s (mean + S.E.M., n --- 9) at session 1 to 33 + 2 s at session 6 (P -<-< 0.001, paired comparison). Moreover, field potential recordings indicated a reduction of paired-pulse depression at this phase of kindling development (data not shown) 12. The calcium-independent stimulated release in the kindled groups was not significantly different from the CON group.
Uptake of GABA in kindled animals A part of the G A B A released from the terminals is taken up by the active carder mechanism before it can reach the incubation medium in which the slices are kept. We estimated the amount taken up, by blocking the carder with SK&F and calculating the difference between the mean release found in the standard protocol and the release found in the presence of 10 /~M SK&E This additionally detected amount of G A B A under SK&F conditions, reflects the activity of the G A B A uptake carrier. The additionally detected amount was for the kindled groups under basal conditions not different from controls (Fig. 4). No significant, kindling related, change in the additionally detected amount of G A B A under high K ÷ conditions was encountered (Fig. 4). The small SK&F-induced decrease of the calciumindependent fraction during the first minute of high K ÷ in controls (Table I), was also found in the kindled groups. This decrease did not differ between kindled and control animals. Potassium threshold for calcium-dependent GABA release in kindled animals The results of the potassium protocol for the kindled groups are presented in Fig. 2. The threshold concentration for eliciting a calcium-dependent G A B A release was between 25 and 30 mM for all investigated kindled animals and is therefore not different from the threshold observed in control animals. The G A B A release of the kindled animals was larger compared to controls at 30 mM K ÷ and at all following K ÷ steps. The relative increase in kindled animals was about the same for the different K + concentrations. This increase in comparison to the CON group was significant from 30 mM onward for the pooled animals of the 14AD and the FK groups (n = 5) and from 35 mM onward for the LT group. Glutamate release During prolonged depolarization a significant increase of the mean calcium-dependent glutamate release, was
38 found in the FK and LT groups as compared to the CON group (CON, 21.9 + 1.3; vs FK, 30.8 + 1.6; P ~< 0.002, Student's t-test; and CON vs LT, 34.6 + 2.7; P ~< 0.044, one-tailed). In contrast, during the first minute, the increase measured in the mean calcium-dependent fraction of the FK (23.0 + 2.7) and the LT (25.6 _+ 2.5) groups was not statistically different from controls (20.3 + 1.8). With the potassium protocol the threshold for eliciting a calcium-dependent glutamate release was established between 30 and 40 mM KCI. Similar thresholds were found for kindled animals. DISCUSSION In the present study the effects of Schaffer collateral/ commissural pathway kindling on the release of the amino acid neurotransmitters G A B A and glutamate in CAI area were investigated. Our main finding was that the calcium-dependent, high K÷-evoked G A B A release was significantly enhanced in animals that had received 14 kindling stimulations, in fully kindled animals 24 h and 25-36 days after the last generalized seizure. The observed increase was found in the first minute of evoked release. Prolonged stimulation with high K ÷ showed also an enhanced release confirming our previous observations 15. In animals that had received 6 stimulations no increase was observed despite the fact that these animals showed clear manifestations of kindling epileptogenesis. From the observations reported here, follows the conclusion that kindling stimulations in CAI lead to a gradual increase in calcium-dependent stimulated G A B A release in the same region with an onset of this change between session 6 and session 12. The K÷-evoked, calcium-dependent net efflux of G A B A and glutamate from CA1 subslices in vitro, termed in this report release, is generally considered to provide information on the calcium-dependent exocytosis from synaptic vesicles in the terminals triggered by the high K÷-induced depolarization 2'23'31. It is important to note that in the intact system exocytotic release is likely to occur in milliseconds while our measurements are made on a time scale of 1 min. Notwithstanding this fact, the results reported here indicate that the late phases of kindling epileptogenesis are associated with a long-lasting enhanced exocytotic release of GABA. The retained total tissue content of G A B A was enlarged in the 6AD, the 14AD and the FK groups probably underlying the enhanced basal release from the cytosolic compartment via a calcium-independent mechanism. This alteration was not found in the LT group, suggesting that this change is seizure associated and different from the changes underlying the enhanced G A B A exocytosis 7.
The presence of G A B A and glutamate/aspartate reuptake mechanisms in the tissue, in addition to technical limitations which only allow the detection of the 'overflow' of amino acid neurotransmitters from the slices, leads to an underestimation of the actual amount of neurotransmitter exocytotically released into the extracellular space. It is possible that the observed enhanced release of G A B A from the slices may be accounted for by a decreased uptake. This possibility is not excluded by the fact that the rise in K ÷ and the simultaneous lowering of Na ÷, to depolarize the tissue, leads to a partial suppression of Na÷-dependent G A B A uptake 1'3. This possibility was checked by blocking the G A B A uptake carrier with SK&F 89776-A 26'32. Our observations on the effects of SK&F on G A B A release are in agreement with the finding that SK&F enhanced the amount of G A B A detected after electrical or high potassium stimulated release of endogenous G A B A from cortical brain slices 31. In addition, we also found that SK&F decreased the calcium-independent, K÷-evoked, G A B A release during the first minute of depolarization which may be related to an inhibition of the reversal of the uptake carrier during depolarization 25' 28. The amount of G A B A additionally detected due to SK&F presence, representing the amount of G A B A taken up in the standard protocol, was unchanged in the kindled animals. Thus, the changes in G A B A release found in kindled animals are not different in the presence of the G A B A uptake blocker, indicating that the kindling related enhanced release originates from a calciumdependent pool likely from exocytotic origin. This interpretation is further supported by our recent release experiments using purified synaptosomal suspensions where re-uptake is negligible. The K÷-evoked calciumdependent G A B A release in the first minute of stimulation was enhanced in synaptosomes prepared from kindled animals 3°. The threshold K+-concentration for the calcium-dependent G A B A release was found to be between 25 and 30 mM KC12. In kindled animals this threshold was unaltered while the release was enhanced over the whole range of investigated K÷-concentrations, suggesting no clear change in the threshold level of depolarization that is needed to evoke exocytotic release in kindled animals. Concerning the release of glutamate, we found an enhanced glutamate release in the FK and LT groups. However, this change was clearly dependent upon a prolonged depolarization since the release during the first minute of stimulation was not significantly increased. An increased glutamate release from hippocampal slices, 1 month after the last seizure in entorhinal kindled rats, was demonstrated by Geula et al. 5 and Jarvie et al. 8 who used in their studies a long depolarization of 5 min with
39 55 mM KC1. The presence of a glutamate uptake mechanism 3, that may mask possible changes occurring in glutamate exocytosis during kindling epileptogenesis, prevents definite conclusions on basis of the present data. Data available in the literature are in agreement with the notion of an enhanced G A B A release in kindled animals. An increased K÷-stimulated (40 rain with 60 mM KCI) release of G A B A from hippocampal slices of entorhinal kindled rats, 24 h after the last convulsion, was demonstrated by Liebowitz et al. 19. Jarvie et al. 9 studied in entorhinal cortex kindled rats, 1 month after the last seizure, the K+-evoked (5 min with 55 mM KCI), calcium-dependent release of amino acids from 3 hippocampal regions. The mean G A B A release from one ipsiand contralateral side was enhanced by approximately 70% in CA1 and in CA3, but due to a large variability in their measurements these increases were not statistically significant 9. In contrast to our results, a decreased total G A B A level and a significant long-lasting decrease of the K÷-stimulated (30 min with 60 mM KCI), calcium-dependent G A B A release was found in hippocampal slices obtained from animals that were stimulated with a large number of rather long tetanic stimulations applied with a short interstimulus interval 18'2°. The electrical stimulation protocol in these studies is clearly different from the classical kindling paradigm used by us 6 and the differences with the results of experiments described in the present report can probably be attributed to the differences in electrical stimulation paradigm. At present it is not clear which mechanism is underlying the enhanced G A B A exocytosis. On the one hand, the changes may be located in the cascade of processes between the stimulus and the release of neurotransmitter; for instance they may be due to an enhanced calcium influx or an enhanced CaE÷/calmodulin-dependent protein kinase II activity24. On the other hand, structural changes resulting in a larger number of GABAergic terminals due to sprouting process may also contribute to the observed changes. The latter would also explain why the reduced number of GABA-immunoreactive somata present in CA1 at long-term after kindling is associated
with an enhanced release of G A B A 13. The fact that no alterations occur in the 6AD group merits special attention. At this phase of kindling an attenuation of paired-pulse depression in CA1 is already clearly present 12. On the basis of the results of our previous release study in fully kindled animals, we suggested that an enhanced G A B A exocytosis could lead to a G A B A receptor desensitization resulting in a reduced paired-pulse inhibition and in a decreased sensitivity of hippocampal pyramidal cells to G A B A 15'16. On the basis of the present results, we now have to conclude that the enhanced G A B A exocytosis cannot be responsible for the observed electrophysiological changes. It appears that the presynaptic changes are manifested only after the changes in the field potentials become evident which indicates that the enhanced G A B A exocytosis may be a compensatory response of the cells to counteract the decreased stability caused by a reduced postsynaptic GABAergic function. On the basis of field potential alterations in the kindled focus we put forward the suggestion that the long-lasting kindling induced hyperexcitability is a consequence of the gradual loss of inhibitory function in CA1 during kindling epileptogenesis rE. Paradoxically, the results reported here along with the results of our previous studies lead to the conclusion that hippocampus kindling epileptogenesis and the induced kindled state are associated with an enhanced G A B A exocytosis. Therefore, we hypothesize that the primary changes responsible for a reduced inhibition and thus to a chronic enhanced seizure susceptibility are located at the level of the G A B A receptor complex. This hypothesis is strongly supported by recent electrophysiological evidence 4'8'16.
Acknowledgements. SK&F 89776-A was a gift from Dr. Skidmore (Smith, Kline and French Laboratories). We thank Dr. Wadman, Dr. Ghijsen and Dr. Verhage for their suggestions. We like to thank the CIVO-TNO Institute, Zeist, for their technical support and Hugo Sandman for his excellent technical assistance. This work was supported by the Dutch Committee for Epilepsy Research (CLEOTNO, Grant A-67) and by Medigon (Grant 550-663-024).
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