Fast and slow depolarizing potentials induced by short pulses of kainic acid in hippocampal neurons

Brain Research, 324 (1984) 279 287

279

Elsevier BRE 1(1444

Fast and Slow Depolarizing Potentials Induced by Short Pulses of Kainic Acid in Hippocampal Neurons SATSUKI SAWADA and CHOSABURO YAMAMOTO

Department ~)[Physiology, Faculo' of Medicine, Kanazawa University, Kanazawa 920 (Japan) (Accepted April 17th, 1984)

Key words: kainic acid - - glutamic acid - - depolarization - - receptor - - hippocampus - - brain slice

Responses of hippocampal neurons to short pulses of ct-kainic acid (KA) were studied intracellularly in thin brain slices of the guinea pig. A KA pulse induced a slow depolarizing potential either with or without a preceding much faster depolarization. Large fast responses were induced only at the center of glutamate-sensitive spots, where short pulses of L~-glutamate (Glu) induced large depolarizations in the impaled neuron. The fast responses resembled Glu-induced depolarizations in time-course, in sensitivity to movement of the tips of amino acid-pipenes, in sensitivity to Glu-antagonists and in reversal potential. The slow response was much more resistant to movement of the amino acid-pipettes and to Glu-antagonists. Mn2+ was without effect on the fast as well as the slow responses. Gluinduced depolarizations super-imposed on the slow response were simply depressed, These results indicate that two different types of receptors are activated by administration of KA, and suggest that the slow response results from a direct action of KA and the fast response is produced as a consequence of either the direct action of KA on the Glu receptors or a calcium-independent release of Glu bv KA.

found that, in addition to the well-known long-lasting

INTRODUCTION

depolarizations, (~-Kainic acid ( K A ) has a potent excitatory action

K A induced fast depolarizations

with brief latencies,

on vertebrate central neurons 2,36 and produces highly selective lesions in the CNS 7-27,28. For these reasons,

MATERIALS AND METHODS

the mode of action of K A has been intensively studied. K A potentiates the depolarizing action of L-glu-

Preparation o f tissues and solutions

tamate (Glu) on the crayfish muscle in which Glu is

Thin transverse sections of the hippocampus were

thought to function as a transmitter 37,~0. In contrast

prepared from the guinea pig as described before 42.

to short, brisk excitations induced by Glu, K A pro-

They were incubated in standard solution at 36 °C for

duces prolonged excitations (depolarizations) in central neurons 4,5,10,31,35,36. Studies with antagonists of

servation ch am b er for recording electrical activity.

at least 40 rain and transferred one by one into an ob-

excitatory amino acids suggest that K A activates spe-

In the chamber, the tissue was continuously super-

cifically a type of receptors ( K A receptors) which are

fused with the standard

or a modified solution

different from receptors activated by quisqualate

(36 °C). Composition of the standard solution was

(Quis)

( N M D A ) ~-25.

(raM): NaC1, 124; KC1, 5; KH2PO a, 1.24; MgSO 4,

Since the action of K A is reduced by removal of ex-

1.3; CaC12, 2.4: N a H C O 3, 26 and glucose, 10. In phosphate-free solution, KH2PO 4 was replaced with

or

by N-methyl-D-aspartate

citatory inputs, a presynaptic action of K A is also postulated 24. In the present experiments, we studied responses of hippocampal neurons to short pulses of KA. We

KCI and Ca 2+ concentration was 1.8 raM. These solutions were saturated with 95% O~ and 5% CO2.

Correspondence: C. Yamamoto, Department of Physiology, Faculty of Medicine, Kanazawa University. Kanazawa 920, Japan. 0006-8993/84/$03.()0 © 1984 Elsevier Science Publishers B.V.

281)

Electrical stimulation and iontophoretic administration of chemicals

were not rectangular in contour but required approximately 100 ms to reach their final value. Therefore. the current intensity of shorter pulses was probably lower than that indicated.

A bipolar stimulating electrode was inserted into granular layer of the d e n t a t e gyrus. Chemicals were administered iontophoreticaily with triple-barrel pi-

Intracellular recording

pettes, a barrel of which was filled with 5 m M a - K A ( W a k o - J u n y a k u l in 150 m M NaCI (pH 8.0), another pipette with 0.2 M Glu (pH 8.0) and the third with one of the following: t0 m M Quis in 150 m M NaCI (pH 8.0), 0.2 M D-homocysteate ( D H , p H 8.0L 0.2 M 7-D-glutamylglycine ( D G G , pH 7.5~, 0.2 M l + lcis-2,3-piperidine dicarboxylic acid ( P D A , p H 7.5) or 150 mM NaCI. Since KA induced potential changes similar to those induced by Glu Isee below ~. the purity of K A was examined by p a p e r c h r o m a t o graphy. No contamination of Glu or aspartate (ASP1 was detected. The tips of the triple-barrel pipettes were not broken. The resistance of each p i p e t t e was about 90 M Q . A retaining current of 5 - 1 0 n A was continuously passed through each pipette. W h e n amino acids were e j e c t e d rectangular pulses of short duration were applied to the leads connecting an iontophoretic equipment ( N e u r o p h o r e , Medical Systems C o r p . ) with amino acid-filled pipettes. Since the equipment had no facility for capacitance trimming. current pulses actually passed through each pipette A1

Intracellular potentials were r e c o r d e d usually with pipettes of about 0.5 ~ m tip d i a m e t e r filled with 4 M K+-acetate. For m e a s u r e m e n t of reversal potentials of amino acid-induced depolarizations, glass pipettes filled with 3 M Cs+-acetate were used. Cs+ was rejected into i m p a l e d neurons electrophoretically for 2 - 5 min with 0.5 s pulses at 1 Hz. The Cs+-injectton m a r k e d l y b r o a d e n e d action potentials and depolarized neurons by 2 0 - 3 0 mV. U p o n stable impalement of a CA3 neuron with a K ~ or Cs+-acetatefilled microelectrode, a triple-barrel pipette was gently inserted into the stratum r a d i a t u m or the stratum oriens searching for Gtu-sensitive spots, where ejection of Glu induced a large d e p o l a r i z a t i o n in the impaled n e u r o n 43. Potentials were amplified with a D C amplifier and were r e c o r d e d with a J e t - c o r d e r ( N i h o n - K o d e n ) , which was an ink-writer r e s p o n d i n g relativey well to high frequency osciUation (reduction of less than 3 dB at 750 Hzl.

2 extra

aGluII bQ~,~

U-

Q

b 2s

B1

_

2 exlrQ

Fig. 1. Depolarizations induced by short pulses of different amino acids. Traces Ala. AIb and Alc were recorded from a neuron with pulses of 200 ms duration of Glu (11 nA), Quis (53 nA) and KA (172 hA), respectively. The arrow indicates the fast KA response. Traces A2a, A2b and A2c were extracellular potentials taken just outside the cell with a Gtu pulse (200 ms, 31 hA), a Quis pulse (200 ms, 73 nAt and a KA pulse 1100 ms. 292 nAI, respectively. B1 was recorded from another cell with a KA pulse (150 ms. 175nA), The arrow indicates the intermediate response. B2 was taken just outside the cell. Underlines indicate periods of administration of amino acids indicated. In the experiments depicted in Figs l - 4 a n d 7 , tonic hyperpolarizing currents of 1 nA were pas~d through recording pipettes to prevent generation of action potentials.

281 RESULTS

sity of KA-ejecting currents. With increase in dura-

Generation of fast and slow depolarizations by KA

tion of KA pulses, the fast response increased in duration with an eventual loss of the notch between the fast and the slow responses.

When short pulses of Glu were ejected at small spots (Glu-sensitive spots) in a dendritic field of a CA3 neuron penetrated with a recording microelectrode, transient depolarizations with steep rising and falling phases were induced (Fig. 1, record Ala) 4~. Pulses of Quis elicited depolarizing potentials of much slower time-course (Fig. 1, record A l b ) . The time-course of depolarizations induced by D H pulses was almost identical with that of Quis responses (records not shown). In about half of the neurons examined, short pulses of KA induced fast depolarizations with short latencies (Fig. 1, record A l c , arrow), which were followed by slow depolarizations declining over 10 s or more, In the remaining neurons, KA pulses produced only the slow responses. These KA responses were elicited usually by KA pulses of 100-200 nA in intensity and of 100-150 ms in duration. The fast KA response was elicited usually at the center of the Glu-sensitive spots, which were activated by Glu pulses of especially low intensities. In some neurons, K A pulses elicited only the slow responses at a Glu-sensitive spot but elicited the fast responses followed by the slow responses at another Glu-sensitive spot. Current pulses through NaClfilled pipettes never produced similar depolarizing potentials. When the recording electrode was withdrawn outside the cell, no upward deflections were observed (Fig. 1, records A 2 a - A 2 c ) . Immediately after the fast KA response and preceding the slow response, another depolarizing deflection of an intermediate time-course appeared in some neurons (Fig. 1, record B1, arrow). Since the intermediate response was observed only occasionally, no systematic experiments were made on this potential. The fast as well as the slow KA responses were induced by KA pulses applied not only to the stratum radiatum but also to the stratum oriens. In the experiments to be reported below, KA was administered to the stratum lucidum, the most proximal portion of the stratum radiatum where mossy fibers made synaptic contact with CA3 neurons. The threshold intensities of KA currents for generation of the fast and the slow responses were almost identical (Fig. 2). The amplitudes of the two responses increased in parallel with increase in inten-

Extension of KA-sensitive spots Movement of the tip of an amino acid pipette from the center of a Glu-sensitive spot resulted in a decrease of amplitudes of Glu responses as well as Quis and KA responses. The fast KA responses were markedly reduced in size by a movement of 5 u m and were almost abolished by a movement of 20 ~tm (Fig. 3). The Glu responses were affected in the same degree or slightly less, and the Quis responses were much less affected by a movement of the amino acid pipette. The slow KA responses were the least sensitive to an advance or withdrawal of the pipette.

5 -

I0~

Zs Fig. 2. Effects of increases in KA pulse intensity. Traces 1,2, 3, 4, and 5 were recorded with KA pulses (200 ms duration, underlined) of 212. 172, I32, 112 and 92 nA, respectively.

282

1o0 % /

tl. . . . . . . jo•/ ' ~ ~1.". . . . "'-. ...

~

Glu KA lost

control

......

C Q

Glu Glu DGG 13nA

KA

2 "6 i-I

DGG

47nA

3 Q

-20

0 ~

20

40 ~m

Fig. 3. Amplitudes of depolarizations induced by pulses (200 ms duration) of amino acids ejected at 0.20 and 40.um from the center of a Glu sensitive spot. Abscissa: distance from the center of a Giu sensitive spot. Ordinate: amplitudes of responses. those at 0/~m being taken as 100%.

W h e n t h e tip o f a K A p i p e t t e w a s p o s i t i o n e d a little remote from the center of a Glu-sensitive spot. theref o r e , e j e c t i o n o f K A i n d u c e d o n l y t h e s l o w K A res p o n s e s . T h e p e a k l a t e n c i e s o f t h e fast a n d s l o w K A r e s p o n s e s i n c r e a s e d w i t h m o v e m e n t o f t h e tips o f K A

4 I 2s

I0 mV

Fig. 4, Effects of DGG on Glu a n d K A responses. Trace 1. control record. Periods at which two Glu pulses (100 ms duration. 27 nA each) and a KA pulse (100 ms duration, 395 nA) were gwen, are indicated with solid lines and a square, respeetivefy. Traces 2 and 3 were recorded during administration of DGG at 13 and 47 nA. respectively. Trace 4 was recorded 120 s after the offset of the DGG current.

b

O control

n

~u

2 *Nn2*

•

3

wash

~A

2,, •

--

a

2s

200ms ~m

i

I

Fig. 5. Effects of Mn 2+. Traces la and lb are control records. Dots~ solid lines and squares indicate times at which a pair ofsupramaximal stimulation of mossy fibers, a pulse of Glu (50 ms duration. 47 nA) and a pulse of KA (50 ms duration. 297 nA) were applied, respectively. Traces 2a and 2b were recorded 10 min after addition of Mn 2÷ at 1.8 mM. Traces 3a and 3b were recorded 6 min after Mn 2~ was removed from the perfusing solution. All records were obtained in the phosphate-free solution containing Ca 2+ at 1:8 mM.

283 pipettes from the center of a Glu-sensitive spot. The

(Fig. 4, record 4). Essentially identical results were

difference in extension of sensitive spots among ami-

obtained in another 10 neurons with D G G or PDA.

no acid responses as shown in Fig. 3 was observed in all of the 6 neurons examined.

Effects off Mn e+

Effects of antagonists of excitatory amino acids"

presynaptic fibers and caused a calcium-dependent

The possibility was examined that K A activated D G G and P D A are relatively specific antagonists

release of neurotransmitters, thereby inducing the

of excitatory amino acids '~,~L33. The fast KA re-

fast or the slow depolarizations in the n e u r o n from which potentials were recorded. For this purpose,

sponses were as sensitive as Glu responses to these blockers, whereas the slow KA responses were more

slices were perfused with the phosphate-free solution

resistant. A representative result is shown in Fig. 4.

and Mn 2+ was added to the solution. Excitatory post-

In this experiment, a pair of Glu pulses induced depo-

synaptic potentials (EPSPs) elicited by supramaximal stimulation of mossy fibers were almost com-

larizations of about 13 mV and a K A pulse elicited a fast response of about 10 mV with a subsequent slow

pletely suppressed at Mn 2+ concentration of 1.8 mM

response in the control (Fig. 4, record 1). During a continuous administration of D G G at low current intensities, the Glu responses and the fast KA re-

(Fig. 5, record 2a, dots), whereas the Glu responses and the fast and slow K A responses were unaffected. Identical results were obtained in 8 neurons.

sponses ~ e r e depressed substantially (Fig. 4, records 2 and 3). Although the slow KA response was usually

Reversal potentials of the KA responses

suppressed by D G G at very high iontophoretic cur-

As reported before 20,-~2, the Glu responses record-

rents (not shown), D G G was sometimes without ef-

ed with K+-acetate-filled electrodes increased in size during tonic hyperpolarizing currents and decreased

fect on the slow responses. All amino acid responses recovered after the offset of the D G G currents

during depolarizing currents. The amplitudes of the

B

A

mV 20

• KA

fast

o KA

slow

o GIu

J r

0 O.

Gio 2

~>,(+--¢.:

O

{; - ,.;-;~ '.~-.a-5<-~

O

o o

o Q,J

0

-50

•

Em

0

|

*20 mV

~,

3.extro

o

0

D 0

n

E - -

2s

20mY

-lO

Fig. 6. Amplitude changes of amino acid responses during tonic depolarization. In A, the amplitudes of responses of a CA3 neuron to pulses ( 1()0 ms duration) of Glu (57 nA) and of KA (399 nA) are plotted against membrane potential. In B, specimen records from another neuron are shown. Traces 1 and 2 show responses to pulses ( 100 ms duration) of Glu (55 nA) and KA ( 195 nA) at membrane potentials of 42 mV and +2 mV, respectively. Trace 3 shows extracellular potentials. In each record, solid lines and squares indicate periods of administration of Glu and KA, respectively.

284

Glu

2 extra Glu

=

KA

÷1 t~A

FiE. 7. Suppression of Glu responses superimposed on a slow K A response. Trace ! was recorded intraceUularly from a neuron with Glu pulses (50 ms duration, 32 n A ) and a K A pulse (150 ms duration, I95 nA). Solid lines and squares indicate periods of administra-

tion of Glu and KA. respectively. Traces 2 were recorded just outside the cell fast and slow K A responses changed in paralled with those of the Glu responses during passage of tonic deor hyperpolarizing currents in all of the 4 neurons examined. No reversal of polarity of these potentials. however, occurred even with depolarizing currents of more than 5 nA. The lack of reversal may be explained by delayed rectification which developed during excessive depolarization. Since Cs+-injection was expected to minimize delayed rectification ~7, reversal potentials were studied in neurons injected with Cs +. In neurons rejected with Cs ÷. tonic hyperpolarizing currents increased the amplitudes of Glu responses and of fast as well as of slow K A responses and depolarizing currents reduced them as in neurons impaled with K+-acetate-filled microelectrodes. At a membrane potential of about 0 mV. these responses reversed in polarity and further increases in intensity of currents resulted in augmentation of reversed responses. Representative data obtained from a Cs+-injected neuron are shown graphically in Fig. 6A and specimen records from another neuron are demonstrated in Fig. 6B. In Fig. 6A, the amplitudes of the slow K A responses were much larger than those of the fast K A responses and of Glu responses before reversal and were proportionately larger after reversal. In each neuron, the 3 types of responses reversed in polarity at a same membrane potential. This was consistently observed in all of the 7 neurons examined. The reversal potentials ranged from - 2 0 to +15 mV with a mean value o f - 4 mV. Interaction between Glu and KA responses K A potentiates both Glu-induced and synaptic excitations in central neurons observed exttacellular1y36. It is of considerable interest, therefore, to examine whether KA potentiates Glu responses observed

intracellularly. In the experiment depicted in Fig. 7. short GIu pulses were applied periodically once every 1,3 s before and after a K A pulse. The Gtu responses superimposed on a slow KA response were reduced in size and gradually recovered with decay of the K A response. Identical results were obtained in 5 neurons. DISCUSSION In this study, we found that KA induced fast as well as slow responses in CA3 neurons. Since the amplitudes of the fast responses increased with intensity of K A currents, the fast responses were not regenerative potentials such as calcium spikes. Suppression of the fast responses by D G G or P D A was not accompanied by a suppresson of the slow responses. This indicates that the slow responses are not causally related to the fast one. and suggests that K A activates two different groups of receptors. K A receptors exist at exceptionally high concentrations in the stratum lucidum where the most of our data were taken1,14,26,41. Generation of two kinds of responses is not characteristic of the terminal zone of the mossy fiber input, however, because they were induced also by K A pulses administered to the stratum oriens which contained no mossy fibers in the guinea pig 16. The time-course of Gtu responses is much faster than that of D H responses. This may be explained by the presence of the high affinity uptake for Gtu 22.3s_ whereas no uptake mechanism has been found for DH. Although Lodge et at. presented the data supporting the presence of uptake mechanisms for Quis 21, the slow time-course o f Quis responses in the present experiments suggests the absence of the high affinity uptake for Quis. Since the uptake for Quis is much less efficient than that for Glu. the former can

285 attain a higher concentration near the p e n e t r a t e d neuron than can the latter, when ejected at r e m o t e sites. Therefore, the sizes of Quis sensitive spots are larger than those of Glu sensitive spots. The presence of the uptake for K A has repeatedly been denied ~-1~. We would expect, therefore, that K A induces prolonged responses, and that the amplitudes of K A responses are less sensitive to a slight movement of the tips of amino acid pipettes than are those of Glu responses. The slow K A responses are in accordance with these predictions. W e think, therefore, that the slow response resulted from the direct action of K A on the neurons from which potentials were recorded. Since the reversal potential of the slow response was identical with that of the Glu response, the K A response is p r o b a b l y generated by the same ionic mechanism for Glu depolarization. The duration of the slow response, however, seems too long to be explained solely by slow diffusion of K A . It is possible that K A activates a biochemical process and induces the slow depolarization. K A has been known to facilitate synthesis of cyclic nucleotides in the cerebellum 34. It remains to be examined which of the high or low affinity binding contributes to the production of the slow depolarization 1,23,41. The slow K A response was unaffected by P D A or D G G at doses sufficient to cause almost complete suppression of Glu responses. This lends support to the hypothesis that Glu and K A receptors are not identical with each o t h e r s,25. The lack of effect of D G G on the K A response is in contradiction with the results obtained extracellularly with multibarrel electrodes that the action of K A is m o r e susceptible to D G G than that of Glu is3-9. W e have no explanation for this discrepancy. Brief Glu-induced depolarizations were not potentiated but were simply suppressed when superimposed on the slow K A response. W e have no data to determine whether the suppression of Glu responses was due to a reduction in input resistance of neurons under observation or due to interaction between K A and Glu receptors. In neuromuscular junction of the crayfish, K A suppresses the amplitude of Glu-induced responses at the synaptic region but potentiates them at the extrasynaptic region 29. In this respect, hippocampal Glu receptors resemble synaptic Glu receptors of the crustacea. In o t h e r respects, however, the properties of these two groups of receptors are differ-

ent. For instance, the former is sensitive to D H whereas the latter is not 39. A possible explanation for the origin of the fast K A response is to assume that K A activates the Glu receptors directly and gives the fast K A response. This explanation would fit the observations that the fast K A responses rose as fast as Glu responses and that the fast K A response and Glu response reversed in polarity at the same depolarized levels and both of them were blocked by D G G and P D A . The falling phase of the fast response, however, was very fast and its amplitude decreased m a r k e d l y with a slight m o v e m e n t of the tips of K A - e j e c t i n g pipettes. In view of the absence of the uptake for KA6,~s, these features of the fast K A response cannot easily be explained on the assumption that the fast response resuited from a direct action of K A on the neurons under observation. Therefore, we tentatively propose an alternative explanation that K A , at relatively high concentrations, causes a rapid release of Glu (or Asp) from presynaptic boutons in a calcium-independent manner. W e assume that during and just after a K A pulse, K A concentration in a small space surrounding the tip of the K A pipette is sufficiently high to liberate Glu from the boutons, and that the liberated Glu diffuses to the neuron under observation to induce the fast response (Fig. 8). Also K A diffuses to

Fig. 8. A scheme for explaining generation of the fast and slow KA responses. 1, pipette for KA ejection: 2, a bouton en passant near the tip of the KA pipette; 3, the neuron from which potentials are being recorded,

286 the n e u r o n directly from the tip of the pipette to induce the slow response. All our observations can be explained by this hypothesis. With increases m the distance between the n e u r o n and the site of K A ejec-

EGTA (ethyleneglycol-bis-([3-ammoethyl

ether)N.

N'-tetraacetic acid). F r o m this observation. Ferkany and his associates concluded that the KA-induced Glu release was calcium-dependent 12~l~. However.

tion. the a m o u n t of Glu arriving at the n e u r o n is pro-

the complete lack of calcium ions ~s so detrimental

gressively reduced because of the uptake on the path. Thus. the fast K A response can be much more sensi-

to brain tissues that action potentials of presynaptic fibers are irreversibly depressed30. I~ remains to be ex-

tive to a m o v e m e n t of the tip of K A pipette than is the

amined whether the K A - i n d u c e d (ilu release is sup-

slow response. According to this hypothesis, it ~s natural that fast K A responses reversed in polarity at the

pressed under a more mild condition as used in the

reversal potential of Glu responses, and that the for-

present experiments. This is the first physiological study demonstrating

mer was as sensitive as the latter to P D A and D G G . Our hypothesis is in accordance with the previous

that K A activates, either directl~ or indirectly, two

finding that the action of K A is reduced by removal

der observation, and suggesting possible participation of a Glu release in KA-induced excitation. In view of the complicated mode ol KA excitation, re-

of excitatory inputs, suggesting that K A produces an effect on presynaptic etements 24. In our hypothesis, we postulate that the KA-induced Glu release is calcium-independent, because

different populations of receptors on the neurons un-

suits of pharmacological experiments on K A receptors must be interpreted with caution ,~,'

Mn 2+ had no effect on the fast response. Haycock et al.~5 and Katz et al.~9 reported calcium-independent release of amino acids from brain slices and synapto-

ACKNOWLEDGEMENTS

somes by high K + concentrations and by electrical

We thank Dr. Satoru Kato in N e u r o i n f o r m a t i o n Research Institute. Kanazawa University for allow-

stimulation. Kato found a calcium-independent release of 7-aminobutyric acid from the carp retimt (personal communtcation). Ferkany and his associates found a release of Glu and Asp by K A from

ing us to cite his unpublished finding. W e also t h a n k Dr. H Shinozaki for the generous gift of Quis. This work was supported by a grant from the Ministry of

brain slices12,13. The release of Glu and Asp is signifi-

Education of Japan.

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