146

Brain Research, 530 (1990) 146- 152 Elsevier

BRES 16145

Involvement of a protein kinase C-dependent process in long-term potentiation formation in guinea pig superior colliculus slices Hiroshi Tomita, Yuji Shibata, Takashi Sakurai and Yasuhiro Okada Department of Physiology, School of Medicine, Kobe University, Kobe (Japan) (Accepted 10 July 1990) Key words." Superior colliculus slice; Long-term potentiation; Protein kinase C; Phorbol ester; Melittin; Polymyxin B; H-7

Superior collicuius (SC) slices were prepared from guinea pig. Electrical stimulation was applied to the optic layer (OL) of the SC slices and the postsynaptic potential (PSP) was evoked in the superficial grey layer (SGL) of the SC. Tetanic stimulation to the OL evoked long-term potentiation (LTP) in the PSE To investigate the involvement of the protein kinase C (PKC) system on the LTP formation in the SC, the effects of PKC activator (phorbol ester) and PKC inhibitors (polymyxin B, melittin and H-7) on the LTP formation have been studied. Application of phorbol ester to the medium at concentrations between 10-8 and 10-'0 M increased the amplitude of the PSE On the other hand, the presence of phorbol ester at a concentration of 10 -6 M increased the glutamate release from the SGL slices. Tetanic stimulation which could not induce LTP by itself could elicit LTP during application of phorbol esters at the low concentration (3 × 10-12 M). PKC inhibitors such as polymyxin B (10-7 M), melittin (10-8 M) and H-7 (10 -4 M) prevented LTP formation. When H-7 was applied once after LTP was formed, the enhanced PSP reduced to the original level. These results strongly indicate the involvement of PKC system on the LTP formation in the SC slices.

INTRODUCTION In 1973, Bliss and L 0 m o described the p h e n o m e n o n of long-term potentiation (LTP) in the hippocampus which was 'maintained for a long period after tetanic stimulation 7. LTP formation is interpreted as substantial increase in synaptic efficacy and so attracts great interest because of the possibility that the p h e n o m e n o n might underlie some aspect of m e m o r y storage. For this reason, research findings on the formation of LTP in mammalian brain have mainly come from the hippocampus in relation to m e m o r y formation 23'3°'38'39. The LTP phen o m e n o n is not restricted only to the hippocampus 8' 10,12,40, but it has been found in several areas of the mammalian brain, such as the neocortex la, the medial geniculate nucleus 13 and the cerebellum 17. Distinct LTP formation was also found in the superior colliculus (SC) in the guinea pig where the N-methyl-o-aspartate ( N M D A ) receptor may be involved in LTP formation but the reported mode of action of the receptor was quite different from that of the hippocampus 28'29'33. Many accumulative studies on LTP formation 23'38 and learning 3°'34 in the hippocampus have suggested that the protein kinase C (PKC) system is involved. Phorbol esters, activators of PKC, enhance transmitter release 24' 37 and regulate ionic conductance closely related to LTP

formation 5"6'9"11. P K C activity was increased two-fold in membranes and decreased proportionally in the cytosol, 1 h after the occurrence of LTP l, as was also increased after the application of phorbol ester 19. A 47,000 M r protein (F1) which is a substrate for PKC increases after LTP formation 2'2°'21'31. Some studies using PKC inhibitors have indicated that LTP consists of different phases and that P K C inhibitors prevent the maintenance phase of LTP21'35. However, recent studies have shown that PKC inhibitors block induction of LTP in postsynaptic sites and block expression of LTP in presynaptic sites 26'27. Thus, the true action of PKC on the mechanism of LTP in the hippocampus has not been completely clarified. The mechanism of LTP formation and the involvement of PKC in LTP have not been studied in other regions in the brain including SC. Since the functional organization has been well characterized in the SC 14-16, it would be a useful system to study the mechanism of LTP. In the present experiment, the involvement of PKC in the formation of LTP in slices of the SC was studied using activators and inhibitors of PKC such as polymyxin B, melittin and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). MATERIALS AND METHODS Guinea pigs weighing 200-300 g were decapitated and their brains

Correspondence: Y. Okada, Department of Physiology, School of Medicine, Kobe University, 7-chome, Kusunoki-cho, Chuo-ku, Kobe 650, Japan. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers.B.V. (Biomedical Division)

147 were quickly removed. Tissue blocks of SC were rapidly dissected out and cut sagittaly into slices of between 400 and 600 pm thick with a razor blade. The technical methods to prepare SC slice in detail have been reported elsewhere 3'4'33. Before starting the experiments, the slices were preincubated for a minimum of 30 min in the standard medium (concentration in mM: glucose 10, NaCI 125, KCI 5, KH2PO 2 1.24, MgSO 4 1.3 , CaCI 2 2 and NaHCO 3 26) equilibrated with 95% 0 2 and 5% CO 2 (pH 7.4).

z

pMA ( IO'~°M )

~.

PdlD ( IO"*M )

_~--:

Electrophysiological experiments Each slice was transferred to the recording chamber under a stereomicroscope and submerged in the perfusion medium. The perfusion rate was 8 ml/min and the temperature in the chamber was kept at 35 °C. The evoked postsynaptic field potential (PSP) was recorded from the superficial grey layer (SGL) of the SC using a glass microelectrode (resistance = 1 MD) containing 2 M NaCI. The bipolar silver wire (diameter 100 pm) electrodes were placed in the optic layer (OL) of the SC for stimulation 3. Square pulses of 100/~s in duration were used for stimulation (stimulator, Nihon Koden SEN 7103) which was at a rate of 0.2 Hz. Electrical responses in SGL were recorded with an oscilloscope (Nihon Koden VC10). By raising the strength of stimulation, a maximum amplitude of PSP was first noted and the strength of the stimulation was adjusted to get response which was about 1/3 of the maximum amplitude before the experiment was started. To investigate the effect of PKC activators on LTP formation, stock solutions of phorbol esters, phorbol 12-myristate 13 acetate (PMA) or 4-a-phorbol 12,13didecanoate (PdiD) were prepared at a concentration of 10-2 M by dissolving them in dimethyl sulfoxide (1%). Each solution was diluted in the standard medium just prior to application to the perfusion medium. To test the effect of PKC inhibitors, polymyxin B, melittin or H-7 were dissolved in standard medium to the desired concentration and applied to the perfusion medium. In every experiment, the stable PSP response was recorded at least 30 min before applying tetanic stimulation to induce LTP. Usually tetanic stimulation of 50 Hz and 20 s was applied to the OL through the same electrode for test stimulus because it was found optimum for the induction of LTP in SC 28.

Determination of glutamate release

100 Phorbol ester

21 -,0

-30

0

-,.

,0

20

~0

~/--J~0

Time ( min )

Fig. 1. One of the typical examples showing the time course of the change in the amplitude of PSP in SGL after application of PMA (O--------O) at a concentration of 10- ~° M or PdiD (O O) at 10-1° M. Phorbol ester was applied during the period indicated by the horizontal bar with arrow.

Fig.

2 shows the

amplitude

of the

input-output

slice t r e a t e d

curve

of t h e P S P

with PMA.

PMA

at

c o n c e n t r a t i o n s b e t w e e n 10 -8 M a n d 10 -1° M shifted the i n p u t - o u t p u t c u r v e to t h e left, b u t 10 -11 M P M A did n o t c h a n g e t h e curve. T h e c u r v e o b t a i n e d by t h e a p p l i c a t i o n of P M A at t h e c o n c e n t r a t i o n o f 10 -1° M a c c o r d e d well with that

by P M A

at

10 -8 M.

The

PSP

amplitude

e n h a n c e d by t h e a p p l i c a t i o n o f P M A at t h e c o n c e n t r a t i o n o v e r 10 -1° M was thus h i g h e r by 4 0 % t h a n t h a t o f c o n t r o l slice w h e n t h e s t r e n g t h o f t h e s t i m u l a t i o n was a d j u s t e d to evoke PSP about

1/3 o f m a x i m u m

amplitude

in the

c o n t r o l slice.

SGL was carefully dissected from SC slice of the guinea pigs. Four or 5 SGL slices were incubated for 10 min in 300 /~1 of the oxygenated medium containing (in mM): KCI 40, NaCI 90, glucose 10, KH2PO 4 1.24, MgSO 4 1.3, CaCI2 2 and NaHCO 3 26 with or without phorbol ester (PMA or PdiD at a concentration of 10-6 M). The temperature of the medium was maintained at 35 °C. Ten min after the incubation, the medium was taken for the measurement of glutamate released from the slices. The slice was saved for determination of the protein content. The glutamate released into the medium was determined using the enzymatic microassay method for glutamate 32. The protein content of the SGL was determined by the method of Lowry et al. 22.

In t h e s t a n d a r d m e d i u m , t e t a n i c s t i m u l a t i o n (50 H z in

:

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RESULTS

D.

Effects of PKC activators To i n v e s t i g a t e the effect o f p h o r b o l esters o n t h e P S P and L T P f o r m a t i o n in S G L , P M A ,

an active f o r m o f

p h o r b o l e s t e r o r P d i D , an i n a c t i v e f o r m o f p h o r b o l e s t e r was a p p l i e d to the p e r f u s i o n m e d i u m . P M A at a c o n c e n t r a t i o n o f 10 -1° M e l e v a t e d t h e a m p l i t u d e o f P S P s to 140% w i t h i n 10 rain, but P d i D at t h e s a m e c o n c e n t r a t i o n failed to e n h a n c e t h e a m p l i t u d e o f P S P (Fig. 1). E l e v a t e d P S P slowly r e t u r n e d to t h e original l e v e l after t h e r e m o v a l o f t h e a g e n t f r o m the m e d i u m ( d a t a n o t shown).

°o~

,

~ Stimulus

~ intensity ( V )

Fig. 2. One o f examples of input-output curve for the PSP in SC slice by application of PMA at concentrations of 10-11 M (& A), 10-l° M (O O) and 10- s M (A A). Line with closed circles (O-----~) indicates the curve from control slice without treatment of PMA. Application of PMA at concentrations between 10-s M and 10-]° M enhanced the amplitude of PSP and shifted the carve to the left. These curves were obtained from one single slice.

148

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Time ( min ) Fig. 3. Effect of phorbol esters on the LTP formation in SC slice. A: the PSP response of the slices treated with PdiD at a concentration of 10-1° M and PMA at 10-l° M and the effect of tetanic stimulation. 1 shows the PSP responses without treatment of phorbol esters, 2 indicates the PSP 45 min after treatment of PdiD at a concentration of 10 z0 M (2b) or PMA at 10-1° M (2c). 3 indicates the PSP 20 min after tetanic stimulation in the presence of phorbol esters (b,c) and in the control slice (a). B shows the time course of the LTP formation during application of PMA at concentrations of 10-11 M (& &), 10 -1° M (A A) and of PdiD at 10-1° M (O O). Line with closed circles (0-----0) indicates the results of the control slice. In each case, pliorbol ester was applied 45 min before the application of tetanic stimulation (50 Hz, 20 s) at the arrow. Each plot indicates the mean of the results from 5-7 slices. Vertical bars shown in each curve indicate the S.E.M. at 4 plots. Two way ANOVAs and Duncan's new multiple range test show significant differences (P < 0.01) between a and c, b and c, and d and c. Among a, b and d, there was no significant difference. f re q u en cy and 20 s in duration) to O L elevated PSPs of

standard m e d i u m could ev o k e LTP in the presence of

SC slices to about 140% in 10 min and induced LTP. LTP

P M A at the concentration of 3 × 10 -]2 M P M A at which

was also induced in the presence of PdiD at the c o n cen t rat i o n of 10-1° M or P M A at the concentration of

the PSP amplitude e v o k e d by test stimulus was not enhanced. In the slice t r eat ed with PdiD at the concentration of 3 x 10 -12 M, LTP could not be induced by

10 -1] M which did not increase the amplitude of PSP by

itself. In the presence of P M A at the concentration of 10 -1° M, the PSP increased by 140% as m e n t i o n e d above

tetanic stimulation at 30 H z for 20 s (Fig. 4),

and the further application of tetanic stimulation in this

Effect of PKC inhibitors

condition did not show further increase in the amplitude of PSP and failed to induce LTP (Fig. 3). We tested the c o o p e r a t i v e effect b e t w e e n tetanic stimulation of low

To test the i n v o l v e m e n t of P K C system on the LTP formation, the effect of P K C inhibitors such as polymyxin B, melittin and H-7 were applied in the perfusion

fr eq u en cy and application of P M A . Tetanic stimulation at the 30 H z for 20 s which could not induce LTP in the

medium. Polymyxin B at a concentration below 10 -7 M, melittin

149 1

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Fig. 4. Cooperative effect of application of low concentration of phorbol ester and tetanic stimulation (30 Hz for 20 s) whose parameter did not evoke LTP in control slice (A A). PMA (0-----------0)at a concentration of 3 × 10-12 M or PdiD (A A) at a concentration of 3 x 10-12 M was applied 30 min before the application of tetanic stimulation. Each plot indicates the mean of the results obtained from 5-7 slices and vertical bars indicate the S.E.M. Two-way ANOVAs and Duncan's new multiple range test show significant differences (P < 0.01) between a and b, and c and b. Between a and c, there was no significant difference. at a concentration below 10 -8 M or H-7 at a concentration below 10 -4 M had no effect on the amplitude of PSP e v o k e d by test stimulus of SC slices. A f t e r the amplitude of PSP was o b s e r v e d to be stable over 30 min after the application of P K C inhibitors, tetanic stimulation was applied. W b e n the concentration of polymyxin B in the perfusion m e d i u m was below 10 -8 M, the tetanic stimulation induced LTP. H o w e v e r polymyxin B at the concentration o v e r 10 -7 M inhibited the LTP without the a p p e a r a n c e of initial increase of PSP after the application of tetanic stimulation (Fig. 5) whereas an increase was o b s e r v e d in the h i p p o c a m p u s 21. Melittin at a concentration of 10 -8 M or H-7 at a concentration of 10 "4 M also inhibited the a p p e a r a n c e of LTP, although neither melittin at a concentration below 10 -9 M nor H-7 at a concentration below 10 -5 M inhibited it (Fig. 6). We further tested the effect of H-7 on the PSP response after LTP was established. Thirty min after the formation of LTP, H-7 was applied to the m e d i u m at a concentration of 10 -4 M. It r e d u c e d the amplitude of PSP enhanced by LTP f o r m a t i o n to the original level o b s e r v e d before tetanus (Fig. 7).

Determination of glutamate release by phorbol ester Tissue slices containing only S G L were p r e p a r e d from SC slices and incubated in m e d i u m containing high concentration of K ÷. G l u t a m a t e content in the m e d i u m released by the t r e a t m e n t of high potassium stimulation was d e t e r m i n e d . In the control m e d i u m (the concentration of K + at 40 m M ) , glutamate was found in the m e d i u m at a concentration of 5.8 nmol/mg protein. H o w e v e r in the m e d i u m with P M A at a concentration of

•zoo ,~

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-

# Tet

T-i -30

, -20

-10

0

, 10

20

30

Time ( rain )

Fig. 5. Effect of polymyxin B on the LTP formation in the SGL slice. Upper inserted figure shows one of the typical examples showing the inhibitory action of polymyxin B on the LTP formation. (a) indicates the PSP of control slice and in (b), polymyxin B at a concentration of 10-7 M was applied to the medium. 1 shows the potentials before application of tetanic stimulation and 2 indicates that 15 min after tetanic stimulation. Figure at the bottom shows the effect of PMB at concentrations of 10-s M (b) and 10 -7 M (C) on the formation of LTP. (a) indicates the control slice. At the arrow (T), tetanic stimulation was applied to the optic layer. Each point indicates the average of the result of 5-7 slices and vertical bars show the S.E.M. Two-way ANOVAs and Duncan's new multiple range test show that only polymyxin B (10-7 M) was significantly different from control values (P < 0.01). M, released g l u t a m a t e increased to 12.5 nmol/mg protein, whereas P d i D did not enhance the glutamate release (Fig. 8). 10 -6

DISCUSSION The e v o k e d potential was r e c o r d e d in the superficial grey layer of SC slices in response to electrical stimulation of the optic layer. T h e p o t e n t i a l was r e v e a l e d to be postsynaptic in nature 4'33. Tetanic stimulation o f O L increased the a m p l i t u d e o f PSP by 4 0 - 5 0 % and e v o k e d distinct LTP in the SC slice. A p p l i c a t i o n o f P M A , the active form of p h o r b o l ester, at a concentration of 10- l ° M e n h a n c e d the a m p l i t u d e of PSP to 140% of original level (Fig. 1). This increase in the a m p l i t u d e by P M A appears to be indistinguishable from the LTP e v o k e d by tetanic stimulation in the h i p p o c a m p u s 25. A f t e r maxim u m synaptic e n h a n c e m e n t by P M A , LTP could no longer be elicited by further tetanic stimulation to O L . A s m e n t i o n e d in M e t h o d s for o b s e r v a t i o n of the LTP formation, the stimulation strength to O L was a d j u s t e d to e v o k e the PSP whose a m p l i t u d e was 1/3 of the m a x i m u m response. W i t h tetanic stimulation, the amplitude increased to 140% of original level forming LTP

6O

150

A % 150

'O

a

;

= Control

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.=

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c

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"= Mellttln ( 10 ~ M )

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&

100

Tel -30

-20

;

-10

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20

I

~I

so

Time ( min )

B

% 150 a :

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'D

d '~

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c H.7(104M)

E

.E

100

Tet

T_I -30

' -20

-10

0

' 10

20

30

II

J 60

Time ( min )

Fig. 6. Effects of melittin (A) and H-7 (B) on the formation of LTP in SC slice. Melittin at concentrations of l 0 -9 M (b) and 10-s M (c) or H-7 at concentrations of 10-5 , (d) and 10-~ M (e) did not give any influence on the PSP amplitude evoked by test stimulus: Tetanic stimulation was applied at ( 1' ). (a) indicates the results of the control slice. Each point indicates the average of the results from 5-7 slices. Vertical bars show S.E.M. Two-way ANOVAs and Duncan's new multiple range test show that only melittin (10-s M) and H-7 (10-4 M) were significant different from control values (P < 0.01).

(Fig. 3A). This increase a g r e e d well with the enhancem e n t of a m p l i t u d e with the application of P M A when the stimulus strength was a d j u s t e d to e v o k e about 1/3 of m a x i m u m a m p l i t u d e in control slice (see d a s h e d vertical line in Fig. 2). This indicates that application of P M A caused an increase in responses similar to that e v o k e d by tetanic stimulation and thus tetanic stimulation in addition to P M A t r e a t m e n t failed to elicit further increase in the PSP amplitude. It is interesting to note, however, that

in the presence of P M A at the concentration of 3 x 1 0 - 1 2 M, LTP could be induced by tetanic stimulation of 30 Hz for 20 s at which LTP could not be o b s e r v e d in the standard m e d i u m without the p h o r b o l ester. Physiological and biochemical studies in combination with d e n e r v a t i o n studies r e v e a l e d that the PSP r e c o r d e d in S G L was e v o k e d by the activation of retino-tectal p a t h w a y which could be glutamatergic 29'36. In the present study, P M A e n h a n c e d the K + - s t i m u l a t e d release of

151

%

150

a ;

;Control

[

T

m

o

#

! 101 . o

o

¢

'

I

H-7

Tet

o'

I

~o

e'o

I 9O

Time ( min )

Fig. 7. Effect of H-7 applied after the formation of LTP in SC slice. (a) shows the formation of LTP in control slice (6 slices). In (b), H-7 at a concentration of 10-4 M once after the LTP was formed. Horizontal thick bars indicate the period of the application of H-7. Each plot indicates the average of the results from 5-7 slices. Vertical bars show S.E.M. Two-way ANOVAs and Duncan's new

multiple range test show significant differences (P < 0.01) among (a)-(b). glutamate in the isolated SGL-slice (Fig. 8). This enhancement of the glutamate release can be attributed to the release from nerve endings 24, although in this experiment calcium-free medium was not used. As we have not measured the endogeneous release of glutamate after tetanic stimulation and during formation of LTP in

15 A

O.

"6 E C

_e .= ¢0

E _= o

5

Ill m

o

O

o

ta 'O

a.

,¢ ft.

Fig. 8. Endogeneous glutamate release from superficial grey layer in SC after 10 min exposure to 40 mM K ÷. Application of PMA at a concentration of 104 M increased endogeneous glutamate release about 2-fold, however, PdiD at a concentration of 10-6 M had no effect on the K+-stimulated glutamate release. Each horizontal histogram shows the mean of the results obtained from 6 slices of SGL and the horizontal bars indicate S.E.M. One-way ANOVAs and Duncan's new multiple range test between control and PdiD treated slices and between control and PMA treated slice show significant difference (P < 0.01).

SC, it is difficult to conclude that PKC-dependent process is related to LTP formation through modifying the releasing mechanism of the transmitter. Although the site of action of phorbol ester in the hippocampus has not been well characterized24--26whether it is presynaptic or postsynaptic, the result in the present experiment showing the enhancement of PSP amplitude by phorbol ester together with the enhancement of K÷-stimulated release of glutamate from SGL slice indicate the possibility that PKCdependent process may be related to the LTP formation. To test the effect of PKC inhibitors on the LTP formation, polymyxin B, melittin or H-7 were applied to the medium. Polymyxin B (10-7 M), melittin (10-s M) or H - 7 (10 -4 M ) prevented the formation of LTP although application of the compounds at these concentrations did not influence on the neural transmission evoked by test stimulus. The concentrations of each compound are similar or even less to those used for testing the effect in the hippocampus 21'35. The mode of the inhibitory action of these compounds on the LTP formation differed between SC and hippocampus. It was reported that in the CA1 area of hippocampus, PKC inhibitors did not depress the initial increase of the amplitude of the synaptic potential after tetanic stimulation but inhibited the maintenance phase of LTP21'35. In the SC, neither the initial phase of increase in synaptic potential nor the maintenance of LTP could not be observed after tetanic stimulation in the presence of PKC inhibitors. If the initial induction phase and later maintenance phase of LTP are mediated by different mechanisms, then the PKC inhibitors may block the initial phase of the induction of LTP in the SC whereas in CA1 neuron in the hippocampus PKC may act mainly on the persistence mechanism of LTP. It is interesting to note that 30 min after the formation of LTP, application of H-7 reduced the enhanced amplitude to the initial level (Fig. 7). In our previous study 29'33, the LTP formation in SC was found t o be mediated by the NMDA receptor, a subtype of glutamate receptors and the application of 2-amino-5-phosphonovaleric acid (APV) as a specific antagonist for NMDA receptor prevented the formation of LTP. However once the LTP was formed, the application of APV failed to reduce the enhanced amplitude of PSP by LTP. This discrepancy in the action of H-7 and APV indicates that the NMDA receptor-dependent process and PKC-dependent one in the LTP formation are different to each other and that the PKC-dependent process is related to the expression rather than the induction of LTP formation. Although it has been clearly shown in this experiment that a PKC-dependent process is related to the formation of LTP in SC slice, further studies on its involvement in pre- or postsynaptic events are necessary.

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