Effects of serotonin on hilar neurons and granule cell inhibition in the guinea pig hippocampal slice

BRAIN RESEARCH ELSEVIER

Brain Research 633 (1994) 27-32

Research Report

Effects of serotonin on hilar neurons and granule cell inhibition in the guinea pig hippocampal slice Bijan Michael Ghadimi, Wolfgang Jarolimek **, Ulrich Misgeld * I. Physiologisches Institut, Universitiit Heidelberg, Im Neuenheimer Feld 326, D-69120 Heidelberg, FRG (Accepted 10 August 1993)

Abstract

Intracellular recordings in guinea pig hippocampal slices were used to study the effects of serotonin (5-HT) on presumed inhibitory hilar neurons and on postsynaptic inhibition of granule cells. 5-HT applied by the bath hyperpolarized only 50% of the hilar neurons tested but all CA3 neurons and granule cells, presumably by activating a K-conductance. The bath application of 4-aminopyridine (4-AP, 50 /xM) induced burst discharge activity in hilar neurons and giant inhibitory postsynaptic potentials (IPSPs) in granule cells consisting of a C1- and K-component. 5-HT (5-10 p.M) reversibly blocked the K-component of giant IPSPs in granule cells, but not their Cl-component. In the majority of hilar neurons 5-HT increased the frequency of 4-AP induced burst discharges even when hilar neurons were hyperpolarized. Only in a few hilar neurons 5-HT blocked 4-AP induced burst discharges. We conclude that the changes in burst discharge pattern of hilar neurons correspond with the differential effect of 5-HT on C1- and K-mediated inhibition of granule cells. Key words: Serotonin; GABA; Baclofen; 4-Aminopyridine; Burst discharge; Hilar neuron; Granule cell; Hippocampal slice

I. Introduction

Electrophysiological studies in the CA1 and CA3 region of the hippocampus have revealed three major physiological effects of serotonin (5-HT) on pyramidal cells [3,5,7,22,29]. These are a hyperpolarization mediated by the activation of an outward K-current [3,5,7,22], a depolarization mediated either by a decrease in resting K-conductance [3,7] or by an inward current [29], and a reduction of spike frequency accommodation due to a decrease in the Ca-dependent Kcurrent responsible for the afterhyperpolarization that follows burst firing [3,7]. Seven 5-HT-receptors were pharmacologically separated which could mediate the serotonin effects [6,21]. Recent anatomical studies have demonstrated that GABAergic neurons are the main postsynaptic targets of median raphe afferents, which are serotoninergic [8,9]. Therefore, recent interest fo-

* Corresponding author. Fax: (49) (6221) 564561. * * Present address: Dept. of Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 3 ) E 1 1 5 5 - V

cussed on the effects of 5-HT on GABAergic neurons in the hippocampus [19,23,26]. The effects of serotonin on inhibitory neurons were investigated indirectly by recording inhibitory postsynaptic potentials (IPSPs) from CA3 and CA1 pyramidal cells on which GABAergic neurons impinge. It has been shown that 5-HT blocks G A B A n - r e c e p t o r mediated K-IPSPs leaving GABAA-receptor mediated C1IPSPs rather unaffected [19,26]. Other studies revealed an increase in unitary CI-IPSPs following the application of serotonin [23]. The conclusion drawn from these studies was that at least two different response patterns of GABAergic neurons to 5-HT exist. One GABAergic cell type, corresponding to the effects on K-IPSPs, should be inhibited by serotonin, the other cell type should be unaffected or even excited, corresponding to the effects on CI-IPSPs. Therefore, the aim of our study was to investigate directly the effects of serotonin on presumed inhibitory hilar neurons [15] which contribute to inhibition of granule cells. 4-Aminopyridine (4-AP) induces giant IPSPs in granule cells in association with burst discharge activity of hilar neurons [18]. The giant IPSPs consist of a G A B A a - r e c e p t o r mediated Cl-component and a

28

B.M. (;hadtmi et aL / Brain Research 633 (lq94) 27-32

G A B A n - r e c e p t o r mediated K-component. Thus, 4-AP enables to study the activity of presumed inhibitory hilar neurons and the resulting inhibition in granule cells. In the present study we addressed the issue whether there are effects of 5-HT on hilar neurons which can be correlated to the differential effects of 5-HT on postsynaptic inhibition in principal cells of the hippocampus.

2. Materials and methods Transverse hippocampal slices (300-400 izm) were prepared from ether-anesthetized guinea pigs as described before [17]. For recording, slices were transferred to a submersion chamber and fixed between two nylon nets. The small volume of the chamber (0.8 ml) and a flow rate of 2 m l / m i n resulted in complete solution exchange within 40 s. Recordings were done at room temperature. Oxygenated solution (95% O2; 5% C O p for recording contained (in raM): NaC1 130, KCI 2, MgSO 4 1.3, K H 2 P O 4 1.25, CaC12 2.5. NaHCO~ 26, glucose 10, pH 7.35. Intracellular recording electrodes were filled with 3 M KCI (or with 0.6 M K2SO~+0.1 M KCI in a few exceptions) and had resistances between 40-60 M~O. Voltage recordings were done with the Axoclamp 2 amplifier (Axon Instruments). Cell input resistance was determined by the injection of constant current pulses (0.05-0.2 hA, 200-500 ms). All values are given as means_+S.E.M. Hilar neurons had a mean m e m b r a n e potential of - 7 1 _+4 mV and a mean input resistance of 120_+42 MI2, granule cells a mean m e m b r a n e potential of - 72 + 4 mV and a mean input resistance of 93 + 39 M,Q, and CA3 neurons a mean membrane potential of - 6 6 + 3 mV and a mean input resistance of 86_+21 M~Q. Spike amplitude was more than 80 inV. During drug application input resistance was measured 2.5 min after start of the application at the control m e m b r a n e potential. Decrease in resistance was calculated as the percentage change of the control response measured in the same cell before drug application. To prevent the oxidation of serotonin it was dissolved before application with the antioxidant sodium bisulfite (from Sigma). Inorganic salts were of analytical grade (Merck, FRG). GABA, 5-hydroxytryptamine creatinine sulfate, (+)bicuculline and picrotoxin were all from Sigma (FRG), 4-aminopyridine from Merck, Schuchard (FRG), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) from Tocris Neuramin (England). ( - ) Baclofen was kindly provided by Ciba Geigy (Switzerland).

3. Results

3.1. Membrane effects of 5-HT on hilar neurons, CA3 and granule cells First we examined the m e m b r a n e effects of 5-HT on hilar neurons which are presumably inhibitory to granule cells. All hilar neurons were impaled in a zone subjacent to the granule cell layer and shared the electrophysiological characteristics of presumed inhibitory hilar neurons: pronounced afterhyperpolarization following short duration spikes, little accommodation of discharge and outward rectification [15]. In the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 tzM), bicuculline (BIC, 50 # M ) and picro-

toxin (P1C, 50 g M ) to reduce fast synaptic transmission bath applied 5-HT produced two different responses in hilar neurons (n = 15). In one group, the membrane potential and input resistance were unchanged (n = 4) or the cells were slightly depolarized up to 2 mV (n = 4) with no significant change in input resistance. In the second group (n = 7), 5-HT (1-10 p.M) induced a concentration dependent hyperpolarization and decrease in input resistance (Figs. IA; 2A1, 2A2). The hyperpolarization was due to an increase in K-conductance as revealed from its reversal potential (Fig. 2B). For comparison the effects of 5-HT on CA3 neurons (n = 11) and granule ceils (n = 6) were examined. All CA3 neurons and granule cells showed a concentration dependent hyperpolarization and decrease in input resistance following the application of 5-HT (1-10 g M ) (Fig. 1B, 1C; 2AI, 2A2). At 10 ~ M 5-HT, the effects on group 2 of hilar neurons appeared to be more pronounced than those on granule cells (Fig. 2AI). However, there was no significant difference in the effects of 5-HT on group 2 of hilar neurons, granule cells and CA3 neurons, as far as m e m b r a n e hyperpolarization and decrease in input resistance were concerned (paired t-test). 3.2. Effects of 5-HT on K-1PSPs in granule cells and on 4-AP induced burst discharges in hilar neurons As reported elsewhere [13,18], 4-aminopyridine (4AP) induced rhythmically occurring giant inhibitory postsynaptic potentials (IPSPs) in granule cells. The giant IPSPs which were unaffected by C N Q X [14] consisted of a GABAA-receptor mediated Cl-component and a GABAB-receptor mediated K-component. When recording with K 2 S O 4 filled electrodes at a m e m b r a n e potential of - 6 5 mV both components were hyperpolarizing (Fig. 3A). However, in the present study KC1 filled electrodes were used as the depolarizing CI-IPSPs could be distinguished from the hyperpolarizing K-IPSPs (Fig. 3B). The GABAA-receptor mediated CI-IPSPs were blocked by BIC (5(I tzM) and PIC (50 ~M). In the presence of these blockers, 4-AP elicited K-IPSPs in granule cells (Fig. 3C) that were larger in amplitude than the hyperpolarizing components of the giant IPSPs induced before the blockade of GABAA-receptors [14]. First, the effects of 5-HT on 4-AP (50 tzM) induced giant IPSPs in granule cells (n = 18) were tested. When 5-HT (5-10 /~M) was applied for a short period (3-5 rain) in the continuous presence of 4-AP all granule cells were hyperpolarized and the K-component of the giant IPSPs was reversibly blocked (Fig. 3D1, 3D2). This blockade persisted when cells were manually clamped back to control membrane potential to show that the effect was independent from membrane potential (Fig. 3D1, 3D2). In contrast to the blockade of

B.M. Ghadimi et al. / Brain Research 633 (1994) 27-32

,

!II

, '

be seen which was eventually due to the blockade of K-IPSPs that were elicited alternating with giant IPSPs (Fig. 3D2) already in control. If 5-HT (5 /~M) was applied together with 4-AP (50/zM), 4-AP elicited only CI-IPSPs, while in control giant IPSPs with CI- and K-components were induced (Fig. 4A). After blockade of GABAA-receptors by PIC (50 /zM) and BIC (50 /zM) ( n = 8), 4-AP (50 /zM) generated rhythmically occurring K-IPSPs. 5-HT (5 /xM) blocked reversibly these K-IPSPs even if the membrane potential was manually clamped back to control membrane potential (Fig. 4B2). In the presence of CNQX (10/xM), 5-HT (5 /zM) showed the same effects on giant IPSPs (n = 4) or on K-IPSPs (n = 4) as without CNQX, excluding a contribution of synaptic excitation to the effects of 5-HT on postsynaptic potentials. The effects of 5-HT on K-IPSPs were concentration dependent. 5-HT (5 /xM) was effective to block the K-IPSPs (Fig. 4B2), whereas 2-3 /zM 5-HT only reduced the amplitude of K-IPSPs (Fig. 4B1). Ci-IPSPs were never blocked, even when 10 /xM 5-HT was applied. Secondly, we tested whether the increase in K-conductance of hilar neurons by 5-HT would suffice to block burst discharges induced by 4-AP in PIC (50 /zM) and BIC (50 /xM) (n = 14). Because CNQX had no effect on the blockade of K-IPSPs, it was not used in these experiments. The 4-AP (50 #M) induced burst discharge frequency ranged from 5-12 bursts per minute. 5-HT (5 /zM) reversibly induced increases in burst frequency by about 91 + 37% in 12 out of the 14 hilar neurons (Fig. 5A) and reduced burst duration in 50% of the cells (see inset Fig. 5A). The apparently excitatory effect of 5-HT (5 /zM) on burst discharge frequency even occurred when hilar neurons were hy-

i!~' 5#M

HN

10#.~.M

-71mV

A

F

.;

ii

"1 r"

GC

"--I

5#M

_

r -1 f----I

,.#.,g,

J

-

r--

10#M

-69mV

B

CA,3

I#M

5#M

==t

~

10p.M

"--t

f--

--I

~

r--

-~

12 m V 1 0 0 ms

Fig. 1. Hyperpolarization and input resistance decrease induced by serotonin (5-HT, 1 - ] 0 / ~ M ) in a hilar neuron (A; HN), a granule cell (B; GC) and a CA3 neuron (C). The perfusate contained throughout bicuculline (BIC, 50 #M), picrotoxin (PIC, 50 /xM) and 6-cyano-7nitroquinoxaline-2,3-dione (CNQX, 10 /zM) to reduce fast synaptic transmission. Horizontal bars indicate the duration of 5-HT perfusion at the respective concentration. The upper traces show the chart record. Downward deflections are e]ectrotonic potentials in response to hyperpolarizing current pulses (0.1 nA, 200 ms). Lower traces show the superimposed voltage responses (middle) and current pulses (bottom) before and during drug application. The calibration in C also applies to A and B. In B hyperpolarizing current pulses applied at shorter time intervals than in A and C. Membrane potential in this and the following figures indicated to the left of the traces.

K-IPSPs by 5-HT, the amplitude and duration of C1IPSPs increased (Fig. 3D3) and they often occurred more frequently (Fig. 3D1). In a few instances, a slight reduction of the frequency of IPSP occurrence could

A1

A2

N rr'

5 o

0.

MEMBRANE POTENTIAL ( m Y )

90

!

Z

o <

B 100

,~E 15

~, z

10

29

'°

-80-/

70 60

-

5o

9

EI r

•

/D /

_oZ "° (u

4o

5

hi

V HN

"

30

•

5

20

o..

10

GC

0 CA3 T

I

s'

lo

SEROTONIN (#M)

Z --

0

• /•

I

1

I

5

- 1 10-

I

10

SEROTONm (#U)

1

r

-0.4

• CONTROL o 5 #M 5-HT

0.0

0.4

CURRENT

(hA)

Fig. 2. A: concentration-response curves for the m e m b r a n e effects of 5-HT on hilar neurons (HN), granule cells (GC) and CA3 neurons. Amplitudes (1) of 5-HT induced hyperpolarization and input resistances (2) during 5-HT effects in percent of control were plotted against 5-HT concentration. Each point represents at least 4 cells. B: I - V curve m e a s u r e d before and during the hyperpolarization induced by 5-HT. Constant current pulses injected had a duration of 200 ms and m e a s u r e m e n t s were taken from the voltage responses near the end of the current pulses. The reversal potential determined from the intersection point was about - 95 mV. All m e a s u r e m e n t s in this figure were obtained in the presence of C N Q X (10/xM), PIC (50 p.M) and BIC (50 ttM).

3(l

B.M. Ghadirni et aL /Brain Research 033 (1994) 27 32

p e r p o l a r i z e d (Fig. 5C). In contrast to the p a t t e r n of the majority of hilar n e u r o n s 5 - H T blocked the burst discharges in two hilar n e u r o n s c o n c u r r i n g with a hyperpolarization (Fig. 5B).

:o~vl

4 -AP

A

B B1 15mY I

1S

C

Ill

lO V I

1sm,n I PI~÷BIC'"

-70mY t

4-AP

D2

+170pA

i

+ 140pA

D1

ls

I

I

•~,/,~..~wJ~ p ~ ~ _ / ~ , ,

I

5#M 5-HT

I ~._.,

Ii

4 -AP 5 ,uM 5

~i

HT

B2

4-AP 2,u,M 5 - H T

/ i

4-AP 5pM

, ~2opA

Ilo~v

5-HT

Fig. 4. Blockade of 4-AP induced K-IPSPs in granule cells by 5-HT. A: in the same cell, 4-AP induced giant IPSPs consisting of depolarizing CI-IPSPs and hyperpolarizing K-IPSPs and, during a second application together with 5-HT, only CI-IPSPs, until 5-HT was washed out. CNQX (10 /xM) present throughout. B: in the presence of CNQX (10 /xM), PIC (50 txM) and BIC (50 #M), 4-AP induced K-IPSPs. 2/xM 5-HT hyperpolarized the granule cell and diminished slightly the 4-AP evoked K-IPSPs (1). In the same granule cell, K-IPSPs were reversibly blocked by 5 ~M 5-HT (2). During the 5-HT induced hyperpolarization, membrane potential was manually clamped back to initial resting potential, to illustrate that the blockade of K-IPSPs was not due to membrane hyperpolarization.

L lOmV 1mini

5#M 5- HT 4-AP

15mY I ls Fig. 3. A,B,C: 4-aminopyridine (4-AP) induced giant IPSPs in granule cells. A: in a granule cell (membrane potential set to -65 mV) the giant IPSP recorded with a 0.6 M K2SO4+0.1 M KC1 filled electrode was hyperpolarizing. B: at the same membrane potential the giant IPSP in another cell recorded with a 3 M KCI filled electrode consisted of a depolarizing and a hyperpolarizing component. C: biphasic IPSPs recorded in another granule cell. When PIC (50/xM) and BIC (50/xM) were applied during 4-AP application the GABAA-receptor mediated, depolarizing Cl-component disappeared. Only the GABAB-receptor mediated, hyperpolarizing KIPSP remained. D: effects of 5-HT on giant IPSPs induced by 4-AP. DI: 5-HT (5 IxM) hyperpolarized the granule cell and blocked selectively the hyperpolarizing K-IPSP (downward deflection); depolarizing CI-IPSPs (upward deflection) were not reduced. During the 5-HT mediated hyperpolarization, membrane potential was manually clamped back to predrug membrane potential (-70 mV) to show that the effect was independent from membrane potential. Horizontal lines underneath the traces mark the time of drug application. D2: similar recording as in D1 but with higher time resolution. Asterisks mark the giant IPSPs which are shown in D3. Also K-IPSPs which eventually occurred independently from CI-IPSPswere blocked by 5-HT (5 tzM). D3: giant IPSPs from trace D2 shown at high time resolution. During the 5-HT effect, CI-IPSP was enhanced in duration and amplitude. Spikes, which were triggered from the depolarizing component, are truncated in this and all other figures. In this and the following figures, bars on top of the traces indicate the amount and time of current injection required to manually clamp cells to resting membrane potential.

I n contrast to the complex effects of 5 - H T on hilar n e u r o n s was the effect of bath applied ( - ) b a c l o f e n . It has b e e n shown that hilar n e u r o n s are markedly hyperpolarized by ( - ) b a c l o f e n (0.3-0.5 /zM) [16]. W e comp a r e d the effects of ( - ) b a c l o f e n on 4-AP i n d u c e d burst discharges in hilar n e u r o n s a n d giant IPSPs in g r a n u l e cells. All hilar n e u r o n s (n = 5) were hyperpolarized by 10 _+ 1 m V at a ( - ) b a c l o f e n c o n c e n t r a t i o n from 0.3-0.5 ~ M . ( - ) B a c l o f e n in the same c o n c e n t r a tion r a n g e always blocked reversibly hilar n e u r o n burst discharges (Fig. 5C). T h e effects of ( - ) b a c l o f e n on 4-AP i n d u c e d giant IPSPs were e x a m i n e d in n o r m a l solution. ( - ) B a c l o f e n (0.5 # M ) slightly hyperpolarized g r a n u l e cells and blocked giant IPSPs (n = 4), not d i f f e r e n t i a t i n g b e t w e e n CI- a n d K - c o m p o n e n t s , in line with findings previously published [20].

Discussion In d e n t a t e gyrus g r a n u l e cells of g u i n e a pig hipp o c a m p a l slices b a t h applied 5 - H T blocks reversibly 4-AP i n d u c e d K-IPSPs, b u t n o t CI-IPSPs. T h e same effects have b e e n observed in rat CA1 [26] a n d CA3 n e u r o n s [19] a n d in s u b s t a n t i a nigra n e u r o n s [10]. Thus, 5-HT, which is the n e u r o t r a n s m i t t e r of fiber systems originating from the r a p h e nuclei [11], may be capable to t u n e G A B A e r g i c i n h i b i t i o n in various b r a i n areas in such a way that G A B A A - r e c e p t o r m e d i a t e d i n h i b i t i o n is e n h a n c e d a n d G A B A B-receptor m e d i a t e d i n h i b i t i o n is reduced. However, the exact m e c h a n i s m for this 5 - H T effect is unclear. I n addition, it has n o t b e e n

31

B.M. Ghadimi et al. /Brain Research 633 (1994) 27-32

in hilar neurons [18]. Because burst discharges persisted even if hilar neurons were hyperpolarized, the blockade of GABAB-inhibition in granule cells cannot be correlated to hilar neurons which were either hyperpolarized or not hyperpolarized. However, the effects of the GABAa-agonist baclofen are likely to be explained by the induction of a K-conductance increase in hilar neurons because 4-AP induced burst discharges in hilar neurons and all IPSPs in granule cells disappeared at the same time. To our opinion two possibilities remain to correlate the effects of 5-HT on hilar neurons and granule cell inhibition: 1) The shortening in burst duration in about 50% of hilar neurons could result in the disappearance of K-IPSPs, but not of CI-IPSPs, because activation of GABAB-receptors might require a prolonged activation of inhibitory synapses. 2) The small group of hilar neurons in which all burst discharges were blocked could represent the hilar cell type that generates KIPSPs mediated through GABAa-receptors , while all other cells are inhibitory neurons generating GABA Areceptor mediated Cl-IPSPs. While we cannot exclude the first possibility, there are some hints in favour of the second possibility: 1) The shortening of burst discharges by 5-HT occurred in about 50% of the hilar neurons studied. This would indicate that about 50 % of so called "fast spiking" hilar neurons [24] would generate GABAB-inhibition. More likely, it was suggested that only a few inhibitory neurons generate GABAa-inhibition [25]. Interestingly also, not all but only a subset of hilar neurons receive a strong serotoninergic input [8,9]. 2) It has been suggested [18,20,28]

shown that endogenous 5-HT has comparable effects under physiological conditions. Because the postsynaptic effects of exogenously applied G A B A on GABA~-receptors were not blocked by 5-HT, it was concluded that 5-HT either reduces the release of G A B A from certain presynaptic terminals [10] or inhibits predominantly GABAergic neurons which generate GABAa-inhibition [19,26]. We believe that a direct interaction of 5-HT and G A B A by converging on the same K-channel [2] is unlikely because K-IPSPs were blocked before the peak of the 5-HT induced hyperpolarization was reached. Thus, the observed 5-HT effects on GABAergic inhibition seem to support the suggestion [18,26,28] that different subsets of neurons generate G A B A A- and GABAa-inhibition. The present study restricted itself to the issue of whether two response patterns of presumed inhibitory neurons exist which could fit to the different effects of 5-HT on Cl-and K-inhibition. There were indeed two types of responses of hilar neurons. One group was hyperpolarized by 5-HT. The effect may be mediated by 5-HTtA-receptors [3,5,7]. Little membrane effects were observed in the other group. In particular, we did not see a significant depolarization which could underlie an excitation. In the CA1 region such an excitation of inhibitory neurons was anticipated, because unitary IPSPs were strongly enhanced by 5-HT via 5-HT3-receptors [23]. Indeed, CA1 pyramidal cells are excited by 5-HT through activation of 5-HTtc-receptors if 5HTtn-receptors are blocked [4]. In 4-AP, the occurrence of giant IPSPs in granule cells is correlated to the occurrence of burst discharges

5/~M 5 - H T

5/~M 5 - H T

A

B

-70mY

-S5mV I10mV lrnin

100ms

I! !Itb!

i i: !~.., i ~ i ~ i ' ~• ~i

-75mV

ii~~ ,!~,.~i~. "!::: . ~ . . ~ i

'~ '

5/~M 5 - H T

~'~' ~

2rain

IO/~M 5 - H T

. ~ . ; . ~ .• ~'~-~

:"

0.5/~M Boc

Fig. 5. Effects of 5-HT on 4-AP induced burst discharges in hilar neurons. Recordings were obtained in picrotoxin (PIC, 50 p.M), bicuculline (BIC, 50/.~M) and 4-AP (50 p.M). A: 5-HT (5/zM) increased the burst discharge frequency in a hilar neuron. Recovery was obtained within 5 min. Additionally to burst frequency increase 5-HT reversibly reduced burst duration as seen in the traces obtained with high time resolution shown below the chart recording. B: in another hilar neuron burst discharges were blocked during hyperpolarization of the cell by 5-HT. C: despite hyperpolarizing the cell, 5-HT did not block burst discharges while they were blocked in the same cell during a hyperpolarization induced by ( - )baclofen (Bac).

32

B.M. Ghadbni et al. /Brain Research 033 (1994) 27-32

that separate neuronal populations generate either GABA A- or GABAB-receptor mediated inhibition because C[-IPSPs and K-IPSPs could occur independently of each other as also seen in Fig. 3D2 in this paper. In this case only the small group of neurons in which burst discharges were blocked by 5-HT is generating K-IPSPs. 3) The duration of CI-IPSPs in 5-HT increased which is not supporting the idea that, because of a shortening in burst duration, only a small amount of GABA is released. However, we cannot exclude the possibility that, after blockade of K-IPSPs by 5-HT, the depolarizing component was prolonged by the activation of N-methyl-d-aspartate receptors. Therefore, we believe that the correlation with GABAa-inhibition is likely to be through the small group of hilar neurons in which burst discharges were indeed blocked by 5-HT. In the dentate hilus 21 different cell types have been classified anatomically [1]. For the sake of simplicity these cells were lumped into two groups [12]: spiny and aspiny or sparsely spiny cells. Roughly, this classification may correspond to excitatory (e.g. mossy cells [27]) and inhibitory neurons. If so, aspiny neurons should be further divided into those generating GABAA-inhibition and those generating GABAB-inhibition which are differentially modulated by 5-HT. The exact cell types and receptors involved remain to be determined. Acknowledgements. The authors highly appreciate the technical help of Andrea Lewen and Heidi Bittler. The authors thank Dr. Hector Lopez for reading the manuscript and Dr. H. Brunner for helpful discussions. Supported by the Deutsche Forschungsgemeinschaft (SFB 317; B/13).

References [1] Amaral, D.G., A golgi study of cell types in the hilar region of the hippocampus in the rat, J. Comp. Neur., 182 (1978) 851-914. [2] Andrade, R., Malenka, R.C. and Nicoll, R.A., A G protein couples serotonin and GABA n receptors to the same channels in hippocampus, Science, 234 (1986) 1261-1265. [3] Andrade, R. and Nicoll, R.A., Pharmacologically distinct actions of serotonin on single pyramidal neurones of the rat hippocampus recorded in vitro, J. PhysioL, 394 (1987) 99-124. [4] Beck, S.G., 5-Hydroxytryptamine increases excitability of CA1 hippocampal pyramidal cells, Synapse, l0 (1992) 334-340. [5] Beck, S.G. and Choi, K.C., 5-Hydroxytryptamine hyperpolarizes CA3 hippocampal pyramidal cells through an increase in potassium conductance, Neurosci. Lett., 133 (1991) 93-96. [6] Cbaput, Y., Araneda, R.C. and Andrade, R., Pharmacological and functional analysis of a novel serotonin receptor in the rat hippocampus, Eur. J. Pharmacol., 182 (1990) 441-456. [7] Colino, A. and Halliwell, J.V., Differential modulation of three separate K-conductanees in hippocampal CA1 neurons by serotonin, Nature, 328 (1987) 73-77. [8] Freund, T.F., Gulyfis, A.I., Acsfisy, L., G6rcs, T. and T6th, K., Serotonergie control of the hippocampus via local inhibitory interneurons, Proc. Natl. Acad. Sci. USA, 87 (1990) 8501-8505. [9] Halasy, K., Miettinen, R., Szabat, E. and Freund, T.F., GABAergic interneurons are the major postsynaptic targets of

median raphe afterents in the rat dentate gyrus. Eur..I. Neu rosci., 4 (1992) 144-153. [10] Johnson, S.W., Mercuri, N.B. and North, R.A., 5-ttydroxy tryptamine IB receptors block the GABAI~ synaptic potential in rat dopamine neurons, J. Neurosci., 12 (1992) 2000-2006. [11] K6hler, C. and Steinbusch, H., Identification of serotonin and non-serotonin-containing neurons of the mid-brain raphe projecting to the entorhinal area and the hippocampal formation. A combined immunohistochemical and fluorescent retrograde tracing study in the rat brain, Neuroscience. 7 (1982) 951-975. [12] Livsey, C.T. and Vicini, S., Slower spontaneous excitatory postsynaptic currents in spiny versus aspiny hilar neurons, Neuron, 8 (1992) 745-755. [13] Misgeld, U., Bijak, M. and Brunner, H., Granule cell inhibition and the activity of hilar neurons. In C.E. Ribak, C.M. Gall and I. Mody (Eds.), The dentate gyrus and its roh, in seizure, Elsevier, 1992, pp. 113 118. [14] Misgeld, U., Bijak, M., Brunner, H. and Dembowsky, K., K-dependent inhibition in the dentate-CA3 network of guinea pig hippocampal slices, J. Neurophysiol., 68 (1992) 1548-1557. [15] Misgeld, U. and Frotscher, M., Postsynaptic-GABAergic inhibition of non-pyramidal neurons in the guinea-pig hippocampus, Neuroscience, 19 (1986) 193-206. [16] Misgeld, U., Mfiller, W. and Brunner, H., Effects of ( - )baclofen on inhibitory neurons in guinea pig hippocampal slice. Pfliigers Arch., 414 (1989) 139-144. [17] Misgeld, U., Sarvey, J.M. and Klee, M.R., Heterosynaptic postactivation potentiation in hippocampal CA3 neurons: longterm changes of the postsynaptic potentials, Exp. Brain Res., 37 (1979) 217-229. [18] Miiller, W. and Misgeld, U., Inhibitory role of dentate hilus neurons in guinea pig hippocampal slice, Z Neurophysiol., 64 (1990) 46-56. [19] Oleskevich, S. and Lacaille, J.C., Reduction of GABA u inhibitory postsynaptic potentials by serotonin via presynaptic and postsynaptic mechanisms in CA3 pyramidal cells of rat hippocampus in vitro, Synapse, 12 (1992) 173-188. [20] Otis, T.S. and Mody, I., Differential activation of GABA A and GABAI~ receptors by spontaneously released transmitters, .L Neurophysiol.. 67 (1992) 227-235. [21] Peroutka, S.J., 5-Hydroxytryptamine receptor subtypes, Ann. Rev. Neurosei., 11 (1988) 45-60. [22] Ropert, N., Inhibitory action of serotonin in CA1 hippocampal neurons in vitro, Neuroscience, 26 (1988) 69-81. [23] Ropert, N. and Guy, N., Serotonin facilitates GABAergic transmission in the CA1 region of rat hippocampus in vitro, J. Physiol., 441 (1991) 121-136. [24] Scharfman, H.E., Kunkel, D.D. and Schwartzkroin, P., Synaptic connections of dentate granule cells and hilar neurons: results of paired intracellular recordings and intracellular horseradish peroxidase injections, Neuroscience, 37 (1990) 693-707. [25] Segal, M., Repetitive inhibitory postsynaptic potentials evoked by 4-aminopyridine in hippocampal neurons in vitro, Brain Res., 414 (1987) 285-293. [26] Segal, M., Serotonin attenuates a slow inhibitory postsynaptic potential in rat hippocampal neurons, Neuroscience, 36 (1990) 631-641. [27] Storm-Mathisen, J., Leknes, A.K., Bore, A.T., Vaaland, J.L., Edminson, P., Haug, F.-M.S. and Ottersen, O.P., First visualization of glutamate and GABA in neurones by immunocytochemistry, Nature, 301 (1983) 517-520. [28] Sugita, S., Johnson, S.W. and North, R.A., Synaptic input to GABA A and GABA u receptors originate from discrete afferent neurons, Neurosci. Lett., 134 (1992)207-211. [29] Yakel, J.L. and Jackson, M.B., 5-HT 3 receptors mediate rapid responses in cultured hippocampus and a clonal cell line, Neuron, 1 (1988)615-621.