Brain Research, 561 (1991) 324-331 (~) 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 0006899391170503

324

BRES 17050

Kindling-induced persistent alterations in the membrane and synaptic properties of CA1 pyramidal neurons Norihito Yamada and David K. Bilkey Department of Psychology and "the Neuroscience Centre, University of Otago, Dunedin (New Zealand) (Accepted 21 May 1991)

Key words: Kindling; Hippocampal CA1 pyramidal cell; N-methyi-D-aspartate receptor; Hippocampal slice, lntracellular recording; Inhibition

Intracellular recordings of CAt pyramidal cells were performed in in vitro hippocampal slices obtained from control and amygdala- or perforant path-kindled rats. Passive membrane properties did not differ between control and kindled cells. Twenty-three percent of kindled cells, however, displayed burst firing with depolarizing current injection, whereas no control cells produced bursts (P < 0.01). Two different types of voltage-dependent alteration of depolarizing postsynaptic potentials (PSPs) were also evident in kindled cells. The majority (26/29) of these cells showed a smaller increase (type 1, n = 18), or a sudden decrease (type 2, n = 8), in PSP amplitude with passive membrane hyperpolarization when compared to controls (P < 0.01). The NMDA antagonist o-APV did not markedly alter the overall slope of the PSP/membrane potential function in either 'type 1' or 'type 2' cells, suggesting that neither behavior was due to a change in the activation characteristics of NMDA receptors. The amplitude of IPSPs was smaller in 'type 1' kindled cells (P < 0.05) than in controls, however, suggesting that the reduced slope of the PSP/membrane function may be accounted for by a change in inhibition.

INTRODUCTION Kindling involves the repetitive electrical or chemical stimulation of the limbic forebrain and induces a longlasting seizure susceptibility in these regions, leading to the development of progressive motor seizures 8'9'33. Since the pharmacobehavioral reports by Peterson et al. 3°'31, a number of studies have demonstrated that the activation of N-methyl-D-aspartate ( N M D A ) receptors, the most well-characterized excitatory amino acid receptor, is an important factor in the kindling phenomenon. Several kinds of N M D A receptor antagonists, for example, suppress the development of kindled seizures, after both systemic application 2'1°'34 or local microinfusion into the brain 4°. While these studies suggest that the activation of N M D A receptors may be an important factor for promoting the induction and propagation of epileptogenesis during kindling, there is less evidence for N M D A receptor involvement in kindling maintenance. Although N M D A antagonists can attenuate fully kindled seizures, more potent effects are usually observed on kindling development 1°,34. Mody et al. have recently shown that the activation characteristics of N M D A receptors are altered in dentate gyrus granule cells recorded in slices obtained from

kindled rats, regardless of time since the last seizure, or of kindling stimulation site 22. This finding suggests that N M D A receptors play a role in the maintenance of the kindled state. It is unclear, however, whether this kindling-induced change is confined to the dentate gyrus or occurs elsewhere in the brain. Several lines of research have suggested that an enhancement of the excitatory system mediated by N M D A receptors 5"7"38 occurs in the hippocampus proper, particularly in the area CA1, following proconvulsive manipulations. Furthermore, some recent biochemical studies provide evidence of postkindling alterations in CA1 N M D A receptors 24"27. Other evidence, obtained from physiological studies, suggests that a reduction of G A B A receptor-mediated inhibition also occurs in this region 1'4'6'12'16'17. Stelzer et al. 37 have indicated that there is a gradual enhancement of N M D A receptor-mediated EPSPs and a reduction of G A B A receptor-mediated IPSPs in CA1 during the in vitro kindling technique. It is unclear what interaction, if any, links these events, as changes in inhibition could alter the depolarization-dependent N M D A receptor characteristics. The present study used intracellular recording techniques to examine the membrane and synaptic properties of CA1 pyramidal cells in in vitro hippocampal slices

Correspondence: D.K. Bilkey, Department of Psychology, University of Otago, P.O. Box 56, Dunedin, New Zealand.

325 o b t a i n e d f r o m k i n d l e d rats. T h e p u r p o s e o f t h e study

TABLE I

was to i n v e s t i g a t e k i n d l i n g - i n d u c e d c h a n g e s in the p r o p -

Membrane properties of CA1 pyramidal cells from kindled and control rats

erties o f t h e s e cells, a n d to a s c e r t a i n w h e t h e r t h e s e c h a n g e s w e r e d u e to a l t e r a t i o n s in an N M D A

receptor-

m e d i a t e d e x c i t a t o r y system.

MATERIALS AND METHODS

The control group includes cells from both sham-operated and naive animals (see text). Action potentials (APs) were generated by depolarizing the cell membrane with 0.5 nA or 0.4 nA (.4) current injection. AP interval was measured between the initial two APs. Values are expressed as mean ± S.E.M.

Kindling preparation Male Sprague-Dawley rats (330-420 g) were chronically implanted with a tripolar recording/stimulating electrode into either the right amygdala (AM) or the right perforant path (PP) under sodium pentobarbital anaesthesia (60 mg/kg i.p.). The stereotaxic coordinates were 0.8 mm posterior to bregma, 5.2 mm lateral to the midline, and 8.0 mm below the dura (incisor bar 5 mm above the interaural line) for the AM, and 4.5 mm lateral to iamda, 2.8 mm below the dura (incisor bar 3 mm below the interaural line) for the PP. A pin electrode, placed on the skull above the frontal sinus, served as a recording indifferent. Kindling stimulation commenced at least one week following surgery. The stimulus current consisted of a 2-s train of biphasic square wave pulses at 100 Hz with a pulse duration of 1 ms. Pulse intensity was at the previously determined afterdischarge threshold which ranged from 50 to 300 /~A. Stimulation was repeated daily for the AM-kindled group and twice daily for the PP-kindled group until a stage 1 motor seizure 33 was observed. Stimulation was then applied once daily until the animals produced at least 5 consecutive stage 4-5 seizures. Shamoperated animals were also implanted with tripolar electrodes into either the right AM or the right PP but were not electrically stimulated.

Slice preparation The animals were decapitated under diethylether anaesthesia from 5 to 7 days after the last stimulation. The brains were removed, and the whole brain was coronally dissected into slices (400 /~m thick) using a vibratome (Campden Instruments). The slicing process was performed in ice-cooled and oxygenated (95% O2/5% CO2) artificial CSF (ACSF), which contained (in mM) 124 NaCI, 3.2 KCI, 1.25 NaH2PO4, 26 NaHCO3, 2 MgSO4, 2 CaC12 and 10 D-Glucose at pH 7.4. Dorsal hippocampal slices were cut from both hemispheres and were incubated for at least one h in a recording interface chamber with continuous perfusion of ACSF (1.0-1.5 ml/ min) and humidified 95% 02•5% CO2 gas. The chamber was maintained at 32 ± 0.2 °C.

n cells RMP (mV) Input resistance (MQ) Time constant (ms) AP threshold (nA) AP latency (ms) "~ Total n APs (ms) n APs in first 20 ms AP interval 1-2 (ms) AP interval 2-3 (ms) AP height (mV) n cells bursting

Kindled

Control

45 68.1 33.5 13.1 0.21 11.9 4.3 1.9 10.5 19.4 86.9 11 +

30 69.9 30.3 13.9 0.23 19.9 3.4 1.3 12.5 28.8 89.6 0

± 0.9 ± 1.5 ± 0.5 - 0.02 ± 1.4" ± 0.2* + 0.1" ± 1.6 --- 2.6 ± 1..6

- 1.2 --- 1.5 ± 0.8 ± 0.02 --- 2.8 -+ 0.2 +- 0.1 ± 1.5 ± 3.5 ± 2.0

*P < 0.05 as compared to control (ANOVA, Tukey's t-test). +P < 0.01 as compared to control (X2 test).

by orthodromie stimulations were obtained at various membrane potentials from 20 mV more negative to 10 mV more positive than RMP, adjusted by continuous current injection. D-2-Amino-5phosphonovaleric acid (D-APV, 30/~M, Sigma) was applied to some slices, using a bath application technique.

Statistical analyses The difference between non-operated, sham and kindled groups was analyzed for each membrane parameter using one-way ANOVA (SAS). Post-hoc analysis was performed using Tukey's studentized range test. Mann-Whitney's U-test was applied to compare the ratio obtained by dividing the PSP amplitude observed at various membrane potentials by the PSP amplitude at RMP (PSP/ PSP-rmp). A regression analysis was also performed on the data describing PSP amplitude as a function of membrane potential (Genstat 5).

Intracellular and extracellular recording techniques lntraceilular recording electrodes (50-90 MQ) were pulled from capillary glass (1.0 mm o.d.) on a Model P-77 Brown-Flaming Micropipette Puller (Sutter Instruments) and filled with 4 mM K-acetate. The microelectrodes were bridge-balanced, capacity was compensated by a current injection probe (Dagan, Model 8175) and potentials were amplified through an intracellular recording amplifier (Dagan, Model 8100-1). A tungsten stimulating electrode was placed in CA1 Str. radiatum to activate pyramidal cells by stimulating afferent fibers. Membrane properties and evoked potentials obtained from pyramidal cells were monitored with an oscilloscope and were digitized and stored on a PDPll/34 computer for off-line analysis. Resting membrane potentials (RMPs) were read directly from the oscilloscope. To determine resting input resistance (Rn~), voltage deflections generated by current injections (100 ms, -0.7 to 0.7 nA) were measured. Time constant (To) was calculated from the voltage profile produced by a 100-ms hyperpolarizing current injection of 0.2 nA. Hyperpolarizing or depolarizing pulses of 0.2 nA, with a duration of 100 ms, were injected into cells at various membrane potentials, ranging from 40 mV negative to 20 mV positive to RMP, and slope conductance was obtained by dividing the current by the resulting voltage deflection. Averaged evoked potentials generated

RESULTS

Membrane and synaptic properties o f kindled and control pyramidal cells I n t r a c e l l u l a r r e c o r d i n g s w e r e m a d e in 27 slices obt a i n e d f r o m 17 c o n t r o l animals a n d 39 slices o b t a i n e d f r o m 29 k i n d l e d animals. Forty-five k i n d l e d , 25 s h a m operated control, and 5 naive control CA1 pyramidal cells w e r e r e c o r d e d . T h e d a t a f r o m s h a m c o n t r o l a n d n a i v e c o n t r o l p y r a m i d a l cells w e r e f o u n d n o t to differ significantly ( A N O V A ) w i t h r e s p e c t to any m e m b r a n e p r o p e r t i e s and so w e r e c o l l a p s e d into o n e c o n t r o l g r o u p (n = 30). P h y s i o l o g i c a l p a r a m e t e r s w e r e w i t h i n t h e r a n g e for n o r m a l C A 1 p y r a m i d a l cells, as d e s c r i b e d p r e v i o u s ly 29"35'36. T h e r e w e r e n o significant d i f f e r e n c e s b e t w e e n k i n d l e d a n d c o n t r o l p y r a m i d a l cells in m e m b r a n e p r o p -

326

a

TABLE II Membrane properties of burst firing- and non-burst firing-kindled cells

Action potentials were generated by 0.5 nA or 0.4 nA (,4) depolarizing current injection. Kindled Kindled burst firing non-burst firing

b

RMP (mV) Input resistance (Mf~) Time constant (ms) AP latency (ms) ~ Total n APs (ms) n APs in first 20 ms AP interval 1-2 (ms) AP interval 2-3 (ms) AP height (mV) n cells

67.7 36.0 13.1 10.4 5.3 2.8 5.7 7.9 88.5 11

± 1.4 + 3.1 -+_0.9 -+ 1.4 ± 0.2* ± 0.1" -+ 0.6* ± 1.0" ± 2.6

68.2 32.6 13.1 12.5 3.8 1.6 12.7 25.2 86.2 34

± 1.1 -+ 1.7 + 0.6 -+ 1.9 -+ 0.3 -+ 0.1 ± 2.1 ± 3.3 ± 2.0

*P < 0.05 as compared to non-bursting cells (ANOVA, Tukey's t-test).

Fig. 1. Non-burst (a) and burst (b) firing generated by 0.5 nA depolarizing current injection recorded from two kindled CAI pyramidal cells. Note the underlying slow depolarizing hump and small slow spikes occurring during burst firing. RMP and RIN of these cells were -72 mV, 32.3 Mfl (a) and -68 mV, 29.3 Mr2 (b), respectively. Calibration: 20 mV, 20 ms.

erties such as RMP, RIN, Tc and action potential amplitude (Table I). Twenty-three percent (11/45) of the kindled cells, however, p r o d u c e d a burst-type firing pattern, characterized by multiple fast action potentials superimposed on a slow depolarizing hump, when activated by a 0.5 n A depolarizing intracellular current injection (Fig. l b ) . None of the control cells displayed bursting behavior (Fig. l a ) ( P < 0.01, Z2 test). Burst firing was reflected in the shorter action potential latency, greater total numb e r of action potentials, and greater n u m b e r of action potentials in the 20 ms following stimulus onset of kindled cells ( P < 0.05, Tukey's test) when action potentials were generated by intracellular injections of 0.4 or 0.5 n A depolarizing current (Table I). W h e n characteristics of burst firing-kindled cells and non-burst firing-kindled cells were c o m p a r e d , there were significant differences in the n u m b e r of action potentials and interaction potential intervals ( P < 0.05, Tukey's test. Table II). The action potential firing p a r a m e t e r s of non-burst firing-kindled cells a p p e a r e d to be similar to those of control cells (Tables I and II). These results suggested that burst firing cells m a d e the primary contribution to the changes in action potential p a r a m e t e r s of all kindled cells. There were no significant differences

in the values of RMP, R~N, Tc and action potential amplitude between burst firing- and non-burst firing-kindled cells ( A N O V A ) . Passive m e m b r a n e properties were also c o m p a r e d for kindled cells from ipsilateral and contralateral hippocampus, however, there were no significant differences between the two groups ( A N O V A , Table IlI). To d e t e r m i n e whether there were voltage-dependent alterations in m e m b r a n e conductance, we measured slope conductance at various m e m b r a n e potentials in control (n = 5) and kindled cells (n = 9). In both groups, enhanced conductance (an average increase of 15% in controls and 33% in kindled cells) was seen when hyperpolarizing current pulses (20 mV) were used to determine slope conductance (Fig. 2). This finding is in agreement with the results of a previous study 32, however, there was not a significant difference ( M a n n - W h i t ney's U-test) between control and kindled cells. Stimulation of Str. r a d i a t u m e v o k e d o r t h o d r o m i c depolarizing PSPs in CA1 p y r a m i d a l cells. The average am-

TABLE III Basic membrane properties of ipsilateral and contralateral kindled cells

RMP (mV) Input resistance (Mr2) Time constant (ms) AP height (mV) n cells

lpsilateral kindled

Contralateral kindled

68.2 33.5 13.1 86.3 39

67.0 33.4 13.1 90.4 6

-+ 1.0 ± 1.7 ± 0.6 ± 1.8

-+ 1.7 ± 3.0 -+ 0.7 _+ 2.7

The two groups were not significantly different from each other (ANOVA).

327

(j.~

0.06 -

control J kindled

plitudes of depolarizing PSPs at R M P were identical in both groups (5.9 +- 0.5 mV, control and 5.9 -+ 0.5 mY, kindled, m e a n +- S.E.M.). PSPs were also obtained at various m e m b r a n e potentials held by continuous injec-

=

tion of depolarizing or hyperpolarizing current. As expected, in control pyramidal cells, the amplitude of de-

0.05.

oe..

polarizing PSPs increased as the cell was hyperpolarized (Fig. 3a). The function describing this voltage-dependent alteration was linear when the ratio of PSP amplitude/

"o

~" 00

0.04"

PSP amplitude at R M P was plotted as a function of

0.03

-lOO

-9o

-;o

Vm (mY) Fig. 2. Slope conductance at various membrane potentials in a control (open circle) and a kindled cell (closed square). Both cells showed anomalous inward current with membrane hyperpolarization. In this figure, the kindled cell showed a greater change in conductance from -75 to -90 mV, although the change of conductance averaged across all cells tested was not significantly different to controls (see text). Values at each point represent the mean obtained from 4 samples. RMP was -70 mV for both cells.

m e m b r a n e potential (PSP/V m plot), (Fig. 4). Sixteen out of 20 control cells showed an increase in PSP amplitude of greater than 40% when hyperpolarized by up to 20 mY. W h e n the m e m b r a n e was depolarized, hyperpolarizing IPSPs were observed. The amplitude of these IPSPs increased as the membrane was further depolarized (Fig. 3b). In pyramidal cells from the hemisphere ipsilateral to the kindling stimulation site, however, the amplitude of depolarizing PSPs showed little increase as the membrane was hyperpolarized (Fig. 3c). Twenty-six out of 29 kindled pyramidal cells ipsilateral to the kindling site in which PSPs were recorded showed less than a 40% increase in PSP amplitude with m e m b r a n e hyperpolariza-

"type r' kindled

control

"type 2" kindled

3

a C

=

e

f i

I

! !

Fig. 3. a: orthodromically-evoked postsynaptic potentials (PSPs) in a control CA1 cell. Since the RMP of this cell was -76 mV, the PSP was purely depolarizing (1). The amplitude of the PSP gradually increased with membrane hyperpolarizations of 10 mV (2) and 20 mV (3). b: PSPs from another control cell. This cell showed a large hyperpolarizing fast IPSP at --63 mV (1), which gradually decreased as the membrane potential was shifted negatively by 5 mV (2) and 10 mV (3). RMP was -68 mV. c: 'type 1' kindled cells showed a smaller increase in the amplitude of depolarizing PSPs with hyperpolarization; (1) at RMP (-74 mV), (2) with membrane hyperpolarizations of 10 mV and (3) 20 mV. d: another 'type 1' kindled cell showing fast IPSPs with membrane depolarizations of 10 mV (1) and 5 mV (2). The IPSP was not obvious at RMP (3, -70 mV). Note the difference in the amplitude of IPSP at around -60 mV, comparing control (b-l, left) and this cell (1). e: an example of PSPs in a 'type 2' kindled cell. The depolarizing PSP showed an amplitude reduction with membrane hyperpolarizations of 10 mV (2) and 20 mV (3). f: the size and shape of the depolarizing PSP recovered in the cell shown in e when membrane potential was returned to RMP. Each trace is an average of 8 consecutive stimulations. Calibration: 2 mV, 10 ms.

328 tion. Eight cells from this subpopulation of kindled ipsilateral cells showed a sudden drop-off (greater than 20% decrease as compared with baseline) in depolarizing PSP amplitude when the membrane was hyperpolarized by about 10-15 mV (Fig. 3e). Since the amplitude of the depolarizing PSPs recovered when the membrane was brought back to RMP (Fig. 3f), it is unlikely that the change in the size of PSP was due to an irreversible deterioration of cell membranes produced by the application of a hyperpolarizing current injection. The former type of kindled cells were named 'type 1' kindled cells and the latter, 'type 2'. For this grouping, cells that showed a rapid and consistent reduction in PSP at hyperpolarized membrane potentials were classified as 'type 2' (n = 9) and the remaining kindled ipsilateral cells were classified as 'type 1' cells (n -- 30). 'Type 1' cells included 3 cells that showed increases of more than 40% in PSP amplitudes at hyperpolarized membrane potentials (within the normal range). Passive membrane properties were not significantly different between 'type 1' and 'type 2' kindled cells (ANOVA, Table IVa). The difference in the PSP ratio between 'type 1' and control

I~

kindled,"type1" kindled,"type2" kindled,contralateral

13.

E

d. a,

ffl

The effect of D-APV on kindled pyramidal cells We examined 5 control and 6 kindled cells to investigate whether 'type 1' or 'type 2' cell behavior was due to a change in the activation characteristics of NMDA receptors. Fig. 5 shows the effect of bath application of

control

2.0"

L

cells was statistically significant at the points o f - 1 0 , -15 and -20 mV negative to RMP (P < 0.05, Mann-Whitney's U-test, Fig. 4). A regression analysis performed on the PSP/Vm plot also indicated that there was a significant difference in the slope of the P S P / V m plot between the control and the two kindled groups (P < 0.01). Pyramidal cells recorded from the hippocampus contralateral to the kindled site showed an increase in PSP amplitude with membrane hyperpolarization similar to that of control cells (Fig. 4). The amplitude of the fast IPSP at depolarized membrane potentials (around -60 mV, Table IVb) was also significantly smaller in 'type 1' kindled cells than in control cells (ANOVA, P < 0.05, Fig. 3d, Table IVb), although 'type 2' and contralateral kindled cells were not significantly different from controls. The amplitude of the depolarizing PSP at RMP was not significantly different when the kindled and control group were compared (ANOVA, Table IVb).

TABLE IV

Membrane properties of "type 1' and "type 2' kindled cells (see text), two groups that were not significantly different from each other (ANOVA) (a) and mean (+- S.E.M.) amplitude of depolarizing PSPs at RMP, and IPSPs at membrane potential shown in the table (b)

(a)

1,0 '

L

0o0

"30

i

"20

"I0

0

i

i

10

20

aVm (mY) Fig. 4. Mean (+- S.E.M.) ratio of the amplitude of depolarizing PSPs at various membrane potentials divided by that at RMP (PSP/V m plot). The ipsilateral kindled cells ('type 1', closed circle) displayed a function with a lower slope than control cells (open circle). The slope of 'type 1' kindled cells appeared to be somewhat further reduced as the membrane was hyperpolarized by more than 15 mV. Some kindled cells showed a drop-off in PSP/V m plot at between - 5 and -10 mV membrane hyperpolarization ('type 2', closed square). Contralateral kindled cells (closed triangle) had a similar slope to that of control cells. *indicates statistically significant (P < 0.05, Mann-Whitney's U-test) as compared to control. There were significant differences in the slope of the plot between control and 'type 1' (P < 0.01) and between control and 'type 2' kindled cells (P < 0.01).

RMP (mV) Input resistance (Mff~) Time constant (ms) A P height (mV) n cells

'Type 1' kindled

'Type 2' kindled

67.5 31.2 14.1 88.4 19

66.6 33.7 12.2 85.8 7

-+ -+ -+ -+

1.6 2.2 1.0 1.8

-+ -+ -+ -+

2.3 4.2 1.5 3.4

(b)

'Type 1' kindled Depolarizing (PSP) amplitude (mV) 6.3 - 0.5

'Type 2" kindled

4.5 --_ 1.0

Contralateral

6.9 -+ 1.2

Control

5.9 +-- 0.5

Fast IPSP amplitude(mV) 2.1 -+ 0.3* 3.1 -+ 1.3 3.2 -+ 0.7 4.8 -+ 0.8 Membrane potential at which IPSP was measured (mV) 60.1 -+ 1.0 59.0 +- 2.7 60.0 -+ 0.7 61.3 -+ 1.5 *, P < 0.05 as compared to control (ANOVA, Tukey's t-test).

329

a

DISCUSSION

control

Before APV Wash

1.8' Q. 1.6 1.4 (n 1.2 Q. 1.0 (n Q. 0.8 0.6 -30

E

"10

~Vm (mV)

"type

b Q,

-20

I"

0

1'0

kindled

'~

1,3 " 1.2"

Before APV Wash

U~ Q. 1.1 Q. 1.0 m O. 0.9" 0.8

-lo

-30

C

~-

o

lO

"type 2" kindled 1.4.

Q"

AVm (mY)

~ - - - ~ - -

Before

APV I

1.2 1.0'

eL m Q.

0.8"

0.8

-30

.

-20

.

.

-10

AVm (mY)

.

0

10

Fig. 5. a: the bath application of 30/~M D-APV produced little effect on the slope of the PSP/V m plot in control cells. R M P of this control cell was - 6 0 mV. b, c: D-APV did not have a significant

effect on the slope of PSP/Vm plot in 'type 1' (b) and 'type 2' (c) kindled cells. APV did not affect any of the 5 other 'type 1' kindled cells. RMP: -69 mV (b), -72 mV (c), respectively. Values at each point represent the means of 8 PSPs.

30/zM D-APV on depolarizing PSP amplitude in 'type 1' and 'type 2' kindled and control cells. D-APV slightly reduced the peak amplitude of depolarizing PSPs and slightly reduced the slope of the PSP/V m plot in control cells (n -- 5, Fig. 5a), but appeared to have no effect in 'type 1' kindled cells (n = 5). D-APV was only applied to one 'type 2' cell and in this case it did not abolish the drop-off phenomenon, although the point of drop-off shifted leftward, occurring at a more negative membrane potential (Fig. 5b,c).

In the present study, substantial differences were observed in the membrane and synaptic properties of control and kindled cells. Twenty-three percent of kindled cells displayed burst firing when activated by intracellular injection of depolarizing current, whereas control cells did not generate burst responses. This is the first report of such a kindling-induced change in region CA1 of hippocampus. Although one explanation for the large proportion of burst firing cells observed in kindled slices is that these cells were not physiologically 'healthy', the typical passive membrane properties used for measurement of 'cell health', such as RMP, RzN, Tc and action potential amplitude, did not differ between burst firing-kindled, nonburst firing-kindled and control cells, being close to those of previous reports 29'35'36. A number of other factors could explain the change in burst responses. There are indications that a voltagegated Ca 2÷ influx causes the slow depolarizing hump 41 underlying burst firing. Kindled cells might, thereby, acquire enhanced Ca 2+ influx, although systematic investigation using drugs such as Ca 2+ antagonists or tetraethylammonium would be necessary to clarify this hypothesis. A further possibility is that, in kindled ceils, there may be some loss of the CI- channel-mediated recurrent inhibition which is normally activated after pyramidal cell firing. In a previous study, it was demonstrated that a small population of naive CA1 pyramidal cells generated burst responses 2°. The lack of burst firing cells in the control slices of our study may be due to recording position within CA12° or to differences in ACSF ionic concentrations 2°,35. Our data demonstrate that there are two subpopulations of ipsilateral kindled cells, differentiated according to the characteristics of the slope of the function relating depolarizing PSP amplitude to membrane potential. 'Type 1' cells show a reduction of this slope compared to control cells whereas the slope for 'type 2' cells falls off rapidly at hyperpolarized membrane potentials of between -5 and -10 mV negative to RMP. In contrast, all the kindled cells from the contralateral hippocampus showed an increase in depolarizing PSP amplitude with membrane hyperpolarization, with a slope that was similar to that of control cells. The kindling-induced changes in PSPs did not appear to be due to a voltage-dependent alteration of intrinsic membrane properties since the magnitude of the anomalously rectifying inward current was not significantly different between kindled and control cells. Moreover, several studies have indicated that even a large anomalous

330 rectifier has little effect on PSP or EPSP amplitude 11,15. The behavior of PSPs in kindled cells is thus more likely to be due to changes in synaptic events. Since it has been demonstrated that an N M D A receptor-mediated postsynaptic component of synaptic transmission is enhanced in dentate gyrus after in vivo kindling 22 or in CA1 after kindling-like stimuli applied in vitro 37, we investigated the possibility that the changes in PSPs might be due to alterations in the N M D A receptor activation characteristics. As Martin et al. TM have suggested, if there was an abatement of the Mg 2+ regulation of the N M D A receptor-coupled ion channel, we could expect a somewhat similar change in PSPs to that of 'type 1' or 'type 2' cells, due to an increase in EPSP amplitude as the cell was depolarized. The effect of the N M D A antagonist, D-APV, on the slope of the PSP/V m plot of type 1 kindled cells, however, was minimal. Although D-APV, at a concentration which had previously been shown to block N M D A receptors 25, reduced the depolarizing PSP amplitude at RMP by 17%, this decrease was not significantly different to that seen in control cells (12%). The hyperpolarization-associated reduction in PSP was also left intact in the one type 2 cell that was tested with APV. This result suggests that the changes in PSP/V m function observed in both type 1 and type 2 cells are probably not attributable to an alteration of N M D A receptors. This finding does not, however, discount the role of N M D A receptors in kindling, as they may play a differential role in the developmental and maintenance phase of kindling. This hypothesis may account for the relative impotence of N M D A receptor antagonists on established kindled seizures. An alternative mechanism underlying changes in 'type 1' cells is related to levels of inhibition. A considerable reduction in amplitude of the fast IPSP was observed in 'type 1' cells, suggesting that kindling produces a reduction in orthodromically evoked, G A B A A receptor/Clionophore-mediated postsynaptic inhibition 26. Most of the pyramidal cells recorded in this study had a RMP which was either very near, or more negative than, the reversal potential of C1-. Since fast IPSPs are depolarizing at these and more negative membrane potentials, a reduction in the IPSP would result in a reduction in the PSP recorded in a hyperpolarized cell. Furthermore, since with cell hyperpolarization the amplitude of the EPSP would increase more slowly than that of the depolarizing IPSP due to the difference in the IPSP/EPSP reversal potentials, the slope of the PSP/V m plot would also be reduced. This would explain why the PSP/Vm slope decline occurs at membrane potentials of more than about 10 mV negative to RMP, where the influence of the depolarizing fast IPSP on the total depolarizing PSP amplitude becomes greater. In accord with the

above hypothesis, a preliminary study from our laboratory suggests that a substantial reduction in the slope of the PSP/Vm plot can be obtained in control cells when fast IPSPs are abolished by bath application of 50/~M picrotoxin. Our observation that kindling reduces the amplitude of IPSPs in CA1 pyramidal cells does not agree with a previous report 29. The difference may arise from the variations in the kindled sites or the interval between the last kindled seizure and the slice experiment. The time course between kindling and analysis in the previous study is not clearly specified, but it may be shorter ('at least 24 hours') than ours (5-7 days). There is evidence that the potency of GABAergic inhibition is temporarily enhanced before gradually decreasing after kindling of both the hippocampus 13'14"17"19'28 and the amygdala 23. The time course may, therefore, be an important factor to consider when investigating the role of the G A B A system in kindling 3. There is no clear explanation for the mechanism underlying 'type 2' cells. Since there was a very early negative shift of the potentials after the stimulus artifact, there is still a possibility that 'type 2' characteristics are the result of technical artifact, such as a sudden transient DC shift of the amplifier. The fact that we found no 'type 2' cells in control slices and also obtained both 'type 1' and 'type 2' cells in the same slice with the same electrode, however, would suggest that this is not a valid explanation. Furthermore, the 'bridge balance' of the recording amplifier was checked carefully during cell penetration, eliminating the possibility that this effect could have been caused by a transient change in electrode resistance. The 'type 2' effect could be obtained if kindling enabled a certain type of synaptically activated, voltage-gated, rectifying K + channel in the proximal dendrite or soma. At present, however, no such current has been described. Blockade of K + channels and/or a voltage-clamp study of these cells might be helpful for understanding this phenomenon. In conclusion, the kindling-induced loss of inhibition in CA1 pyramidal cells coupled with the onset of burst firing may be an important mechanism for the propagation of seizure activity through this brain region. This effect is particularly interesting considering the recent hypothesis that area CA1 is an important site for triggering rhythmic seizure discharges in the seizure-sensitive hippocampus 21"39. At present we do not understand what factors are critical for the onset of this effect or at what point during the kindling procedure it develops, however further investigation should clarify these points.

Acknowledgement. This research was supported by the New Zealand Neurological Foundation.

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