030644921’92 $5.00+ 0.00 0 1991Pergamon Press plc
Vol. 101C. No. 1, pp. 4147, 1992
Camp. Biochem. Phyki.
Printed in Great Britain
PHILANTHOTOXINS BLOCK GLUTAMATERGIC TRANSMISSION IN RAT HIPPOCAMPUS-II. INHIBITION OF SYNAPTIC TRANSMISSION IN THE CA1 REGION N. C.
M. SCHLUTER,* T. PIER* and F. H. LOPES DA SILVA~
*Department of Pharmacology, Mei~rgdreef 1.5,1105 AZ Amsterdam and ~~pa~rnent Kruislaan 318, 1098 SM, Amsterdam, The Netherlands
of Zoology,
(Received 25 March 1991)
Abstract-l. Philanthotoxins decrease the amplitude of the population spike (PS), the field excitatory postsynaptic potential (f-EPSP), and the presynaptic volley (PV), as evoked by Shaffer-collateralcommisural inputs to the CA1 pyramidal cells in the rat hippo~mpus slice. 2. The effects are slow and often not completely reversible. 3. Dideaza-philanthotoxin-12 is, in all experiments, the most active antagonist showing a very poor recovery. 4. Using a twin pulse the percentual decreases of f-EPSP and PV amplitudes are almost identical for the first and second response. However, the first PS is much more affected than the second one, indicating a possible effect on the inhibiting circuit. 5. Philant~otoxins cause a non-competitive inhibition. 6. Besides a possible postsynaptic block and a distinct presynaptic effect (preceding paper) a non-postsynaptic effect (on the PV) is described.
INTRODUCTION
diographical studies showed that delta-philanthotoxin affects transmission by a postsynaptic block of open ~tion~hannels and by a presynaptic reduction of the glutamate uptake in nerve terminals and glial cells (Clark et al., 1982; Piek, 1982; Van Marle et al., 1984, 1986; Schluter et al., 1988; Karst et al., 1991; Karst and Piek, 1991). Synthetic delta-philanthotoxin (Tong, 1988), now called PTX-4.3.3, is chemically identical to the natural toxin and displays the known antagoni~ng effects (Piek et al., 1988; Karst et al., 1991; Karst and Piek, 1991). The present paper presents the results of the effects on the glutamatergic transmission between the Schaffer collaterals and the CA1 pyramidal cells of the rat, of PTX-4.3.3 and eleven of its analogues, together indicated with the collective name philanthotoxins (Fig. 1). The effects were studied in hippocampal slice preparations. Some of the results were published in a preliminary report (Schluter et al., 1988).
Following the discovery that glutamate may function as an excitatory transmitter in the crustacean neuromuscular junction (Florey, 1957) it has been shown that this amino acid plays an important role in the synaptic transmission in various parts of the mammalian central nervous system (Watkins, 1984; Cotman and Iversen, 1987). L-Glutamate is now firmly established as a major excitatory trans~tter substance in the mammalian CNS (McLennan, 1983; Fonnum, 1984; Watkins and Olverman, 1987). Electrophysiological, biochemical and anatomical studies have revealed the existence of at least four receptor subtypes for glutamate (Mayer and Westbrook, 1987). The lack of specific antagonists for some of these receptor subtypes stimulated an extensive search for specific compounds that could function as tools for the analysis of the mechanisms of glutamate transmission. This led to the discovery of the pure synthetic quinoxalinediones that have been reported to be the first potent and selective antagonists active at kainate and quisqualate receptors (Honore et al., 1988). Other important glutamate antagonists are natural compounds isolated from spider and wasp venoms. From these venoms polyamine-like toxins, which are active in glutamatergic transmission systems, have been isolated and chemically characterized (Piek et aZ., 1988; Jackson and Usherwood, 1988; Kawai et al., 1988). Twenty-five years of research on deltaphilanthotoxin, a polyamine isolated from the venom of the digger wasp Philanthus triangulum, demonstrated its antagonistic properties in peripheral glutamatergic and central acetylcholinergic transmission systems of insects (Piek et al., 1988; Piek and Hue, 1989; Piek, 1990). Electrophysiological and autora-
MATERIALS AND
METHODS
Animals and tissue preparation
Female Wistar rats with weights varying from 1.50 to 250g were anaesthetized by diethylether before decapitation. The brain was rapidly removed, divided midsaggitally, and placed in ice cooled Krebs phosphate solution as described in the preceding paper (Schluter et al., 1991). Transverse slices (4OOpm) of both hippocampi were prepared using a hand operated tissue chopper starting about 1.5 mm from the rostra1 {septal) end. By means of a moistened fine brush, the slices were collected and placed in aerated Krebs solution in which they were allowed to equilibrate for at least 1 hr at room temperature. Electrophysiological setup
Hippocampal slices were placed on the stretched gauze bottom of a ring that was fixed in an interface chamber. 41
N. C. M.
42
SCHLUTER
With a constant flow of I-2ml per mitt, aerated Krebs solution at 34°C passed the bottom side of the slice. The upper surface of the slice was prevented from drying out by conducting a constant flow of moistened gas (95% 0, and 5% CO,) over the slice. Two trimel-insulated stainless steel wires with a diameter of 60pm, placed in the stratum radiatum of the CAl-CA2 area, were used to stimulate the Schaffer collateral-commissural inputs to the CA1 pyramidal cells with square wave constant current pulses (0.1 ms in duration and SO-200 PA intensity), applied every 5 or 10 s in a paired-pulse fashion (interstimulus interval: 20 ms). Glass recordine electrodes filled with 3 M NaCl and a resistance of 2-10 MG were placed in the stratum radiatum of the CA1 region for extracellular recording of the presynaptic volley (PV) and the field excitatory post synaptic potential (f-EPSP) or in the stratum pyramidale of the CA1
region for extracellular recording of the population spike (P8). Chemicals: preparation and application
Philanthotoxin-4.3.3 and the eleven structural analogues, listed in Fig. 1, were synthesized by Dr Y. C. Tong (DOW Chemical Research Center, Walnut Creek, California, U.S.A.). Kynurenic acid was obtained from Sigma. All other compounds were from Merck. All chemicals were easily soluble in Krebs buffer solution except dideaza-PTX12, which was first dissolved in bidistilled water (325 PM) using moderate heating at about 50°C and subsequently diluted (1:l) with two times concentrated Krebs solution. Control and experimental fluids were administered by means of a pipette with an inner diameter of about 0.5 mm, which was situated just above the slice near the stimulationrecording area. The pipette was connected to a 10 ml vessel placed about 30 cm above the slice, in order to guarantee a constant gravity-fed flow of 0.2&3ml per min. At the beginning of drug application the aerated Krebs solution in the vessel was replaced by Krebs solution containing the compound to be tested. The washing procedure was started by replacing the content of the vessel for Krebs solution. All compounds were tested at a concentration of 650 PM except dideaza-PTX-12, which was tested at 162.5 PM. PO”7
delta-philanthotoxin,
PTX 43.3
tiH HO~CH2~HCO~NH(CH2)H~CH2)3NH(CH2)3NH2 /
/ 4.3.3
“00.
2
[email protected]
3
HO(9Pr.3.4.3
PT X 3.4.3
4
HO(9.Pr.3.3.4
PTX~ 3.3.4
5
“00.
PTX
6
HOo.Pr.3.3
7
Pr.4.3.3
PTX
1
Pr.4.3
dehydroxy-PTX-4.33
H.Pr.4.3.3 F3CQ.Pr.4.3.3
10
ZJ.PV4.3.3
11
HOo.Pr.5.3.3 -2
12
HOo.Pr. Lz
4.3
PTX~ 3.3
O.Pr.4.3.3
8 9
methyl-PTX-4.3.3
dephenol-PTX-4.3.3 trifluoromethyl-PTX~4-3
3
indole-PTX-4.3-3 PTX 12
533
dideaza~PTX~l2
Fig. 1. Structure of delta-philanthotoxin, now called philanthotoxin-4.3.3 (PTX-4.3.3) and eleven analogues (from Piek and Hue, 1989). The numbers at the left correspond with those used in Figs 2-5.
et al.
Stimulation and recording protocol
After a 20-30 min preincubation period in the interface chamber, stimulation of the Schaffer collateral fibers was started, followed by a recording of the responses in the CA1 region every 5-10min. For each record, four successive non-filtered responses were collected and averaged using a digital oscilloscope and written on paper with an X-Y plotter. After an initial 30min period during which the averaged response(s) stayed stable, a philanthotoxin analogue or kynurenic acid was applied to the slice. Washing out of the drug was started after the responses reached a steady level (two consecutive recordings did not differ by more than 5%). The last record before wash out was used for estimating the maximum effect of the drug. The recovery value was measured at the moment that there was no further increase in amplitude of the response. Analysis
The amplitudes of the PS were defined as the average of the differences between the negative peak and the preceding and following positive maxima recorded in the stratum pyramidale. The same procedure was applied to measure the PV at the stratum radiatum, where this component is clearly recorded. The f-EPSP was measured as the maximum rising slope of the negative-going potential recorded in the stratum radiatum. The statistical analysis of differences between control and maximal drug effects and between control and recovery were assessed by a (paired) sample t-test (Sokal and Rohlf, 1969). RESULTS Effects
on the population
spike
In a first series of experiments the effects of a series of polyamine antagonists, the philanthotoxins (Fig. l), were studied on the amplitude of the population spike (PS) recorded extracellularly from the
CA1 pyramidal cell layer. The glutamatergic antagonist kynurenic acid was used as a general reference to the new polyamine antagonists. The pyramidal cells were stimulated indirectly by electrical stimulation of the Schaffer collaterals in the stratum radiaturn of the CAl-CAZ region, with current intensities resulting in a submaximal (50-80%) response to the first stimulus of a pair of stimuli. Every 5-10 set a pair of stimuli was given with an interval of 20 msec. The PS were recorded as averages of four individual sweeps. At submaximal intensity, paired pulse stimulation, with an interval of 20 msec, causes facilitation of the generation of the second PS. Figure 2 shows the effects of kynurenic acid and the philanthotoxins on the first and the second PS. Figure 2A shows that both kynurenic acid and the philanthotoxins significantly antagonize the first PS, however to different levels. From the philanthotoxins only trifluoromethyl-PTX-4.3.3 and dideaza-PTX-12 are more active than PTX-4.3.3. The most active one is dideaza-PTX-12. Applied at a concentration of 162.5 PM this antagonist is about as active as kynurenic acid at 650/*M. However, the reversibility of the effect of dideaza-PTX- 12 is much lower. Regarding the second PS (Fig. 2B) significant reduction of the amplitude is only seen with trifluoromethyl-PTX-4.3.3, dideaza-PTX-12 and kynurenic acid. Here again dideaza-PTX-12 shows an inhibition of the PS which is comparable to that of kynurenic acid, the latter applied at a four times higher concentration.
Philanthotoxins
A
0.660
120
43
inhibit Glu transmission mM
0.163
ml ul
100
80
80
40
/
20
,
L
d
0 IJ
K
B
1
2345678
0.650
120
9
10
11
mM
12 0.183
mM
80
80
K
1
234567
8
9
10
11
12
Fig. 2. Effects of kynurenic acid (K) and 12 philanthotoxins (for numbers see Fig. 1) on the amplitude of the first (A) and second (B) population spike, recorded in the hippocampal CA1 region. Solid bars: maximal reduction in amplitude; open bars: recovery. All values are presented as the percentage of control. All antagonists were applied at a concentration of 650pM, except dideaza-PTX-12, which was administered at 162.5 PM. Closed triangles indicate values significantly (p < 0.05) different from control. Open triangles indicate significant difference from PTX-4.3.3 (p < 0.05). Vertical lines indicate SEMvalues (in one direction). (N = 6, except for numbers 6, 9 and 11, where N = 5.)
In order to reach the level of maximal depression of the PS-amplitudes, as well as to get a more or less stable level of recovery, for most antagonists the incubation and washing times generally were in the range of 15-35 min each. However, significantly longer incubations and washing times were needed for dideaza-PTX-12 (Fig. 3). Effects on the f-EPSP Since in the preceding section only two philanthotoxins were significantly more active than PTX-4.3.3, we have studied only the effects of PTX-4.3.3, trifluoromethyl-PTX-4.3.3, dideaza-PTX-12 and kynurenic acid on the slope of the rising phase of the f-EPSP. Figure 4 shows the mean plateau values of the depressions of the first (A) and second (B) f-EPSP caused by 650 PM kynurenic acid, PTX-4.3.3 and trifluoromethyl-PTX-4.3.3, and 162.5 PM of dideazaPTX-12. All compounds cause a significant depression and all effects are reversible. The average of slopes of the f-EPSP recovered from treatment with kynurenic acid is not significantly different from the
control value. The reversibility of the effects of the three philanthotoxins is not complete and as a result their average plateau value is significantly lower than their corresponding control values (Fig. 4). The most incomplete reversibility is seen after blocking the transmission with 162.5 ,uM dideaza-PTX-12. Eflects on the presynaptic volley (PV) Kynurenic acid has no effect on the PV-amplitude (Fig. 5). On the other hand the philanthotoxins significantly depress the PV-amplitude, dideaza-PTX12 again being the most active antagonist. This indicates that with kynurenic acid the input-output ratio significantly increases and the question arises whether this could also be the case for preparations treated with philanthotoxins. Effects on the stimulus-response curves The question whether the input-output relationship of synaptic transmission could increase in the presence of philanthotoxins or not is elucidated by measuring stimulus-response curves for both the
N. C. M.
SCHLUTER et al.
0.850
120
mM
0.183 mM A
100
80
80
40
20
0
1
K
234587
8
9
10
11
12
Fig. 3. Incubation times (solid bars) and washing times (open bars), in minutes, needed for kynurenic acid (K) and the philanthotoxins (for explanation see legend of Fig. 2) to reach a stable level. Triangles indicate values significantly different compared with PTX-4.3.3 (1). Note the remarkable increase in both times for dideaza-PTX-12 (12). Vertical lines indicate SEM (N = 6, except for numbers 6, 9 and 11, where N = 5).
0.860
mM
0.183
mM
80
80
K
(n*7)
1 WI) 0.850
9
(n-9)
mM
. Al 12 b7)
0.183
mM
100 A
80
80
!-__ A
.
40
20
0 K (n=7)
1 hll)
1 9
(n=9)
.
12 (n-7)
Fig. 4. Effects of kynurenic acid (K), PTX-4.3.3 (l), trifluoromethyl-PTX-4.3.3 (9) and dideaza-PTX-12 (12) on f-EPSP-amplitudes, measured as the slope. The plateau values of depression (solid bars) as well as of the recovery (open bars) are presented as percentages of the control values. Vertical lines indicate SEM-values (in one direction), asterisks indicate values significantly different from control (p < 0.05). Note the similarity of percentual effects between first (A) and second (B) potential, in contrast to that of the PS (Fig. 2).
Philanthotoxins
A
0.660
120 100 aa 60
40
20
a
inhibit Glu transmission
I K
mM
0.163
mM
1
(n=7)
9
1 (n-7)
0.650
12 (n.4)
(w&l)
0.163
mM
mM
80
80
40
20
0 K
1 (n=7)
(n=7)
9
(n=8)
12 (n.4)
Fig. 5. Effects of kynurenic acid (K), PTX-4.3.3 (I), trifluoromethyl-PTX-4.3.3 (9) and dideaza-PTX-12 (12) on the amplitudes of the presynaptic volley (PV). The plateau values of depression (solid bars) as well as of the recovery (shadowed bars) are presented as percentages of the control values. Vertical lines indicate SEM-values (in one direction), asterisks indicate values significantly different from control (p -C0.5). Note the similarity of percentual effects between first (A) and second (B) potential, in contrast to that of the PS (Fig. 2).
f-EPSP (Table 1) and the PV (Table 2). Table 1 shows the effects of kynurenic acid, PTX-4.3.3 and trifluoromethyl PTX-4.3.3 (650 pM) as well as of dideaza-PTX-12 (162.5pM) on the parameters of the stimulus-response curves of the first and second GEPSP in response to a paired pulse stimulation. The
central parts of the curves are considered to be linear and described using linear regression in Table 1 by the equation y = ax + b, in which y is the response in mV/ms and x the stimulus in 10m4A. Hence ‘a’ gives the slope of the curve in V set-’ A-’ and ‘b’ gives the intercept on the y-axis (v set-‘).
Table 1. Effects of four different antagonists: kynurenic acid (K, 650 FM), PTX-4.3.3 (P, 650 pM), trifluoromethyl-PTX-4.3.3 (T, 650 PM) and dideaza-PTX-12 (D, 162.5 pM) on the parameters of the stimulus-response curves of the first and second f-EPSP during paired stimuli (interval 20 msec). The central parts of the curves are considered to be linear and described by the equation y = ax = b in which y is the resoonse in mV/ms and x the stimulus in lo-” A. Values between brackets are SEM a (102V/c/A) Control
Antagonist
Control
Antagonist
(lo-'A) Antarzonist Wash
r-intercept
b (mV/ms) Wash
Wash
Control
First EPSP K (N=4) P(N=7) T(N=7) D (N=5)
147 (61) 195 (52) 221 (74) 140 (52)
73 (28) 115(28) 72 (18) 32(12)
156 (64) 144 (36) 166 (53) 72 (25)
-0.63 (0.23) -0.70 (0.17) -0.94 (0.30) -0.17(0.26)
-0.36(0.13) -0.44 (0.90) -0.36 (0.10) -0.19 (0.06)
-0.71 (0.28) -0.53 (0.11) -0.74 (0.23) -0.44(0.14)
4.3 3.6 4.3 5.5
4.9 3.8 5.0 5.9
4.6 3.7 4.6 6.1
Second EPSP K (N=4) P(N=7) T (N=7) DM=51
212 242 256 180
140 147 103 51
212 170 203 113
-0.84 -0.81 -0.97 -0.84
-0.70 -0.51 -0.48 -0.27
-0.88 (0.35) -0.57(0.15) -0.84 (0.22) -0.63 (0.27)
4.0 3.5 3.8 4.7
5.0 4.5 4.1 5.3
4.1 3.4 4.1 5.6
(84) (79) (79) (76)
(53) (41) (28) (26)
(90) (47) (56) (52)
(0.26) (0.29) (0.30) (0.33)
(0.23) (0.14) (0.13) (0.121
N.C. M. SCHLWTER~~ al.
46
Table 2. Effects of four different antagonists: kynurenic acid and three philanthotoxins on the parameters of the stimulus-response of first and second PV. For further explanation see legend of Table 1
a (mV/d) First PV K (N=4) P(N=3) T(N=6) D (N=3) Second PV K (N=4) P(N=3) T (N=6) D (N=3)
b W) Antagonist
x-intercept Control
Wash
(lOmsA) Antagonist
Control
Antagonist
Wash
101 (24) 64(15) 98 (20) 99 (19)
111(19) 55 (8) 55 (11) 39 (15)
107(17) 55(11) 96(17) 70 (15)
-0.48 -0.24 -0.42 -0.53
(0.10) (0.04) (0.06) (0.16)
-0.55 -0.23 -0.28 -0.25
(0.7) (0.4) (0.6) (0.10)
-0.55 (0.08) -0.23 (0.05) -0.44 (0.07) -0.40(0.10)
4.7 3.8 4.3 5.6
4.9 4.2 5.1 6.4
5.1 4.2 4.6 5.7
107 (14) 54 (6) 54 (12) 30(11)
98 (15) 57 (12) 91 (18) 64(13)
-0.45 -0.26 -0.40 -0.52
(0.08) (0.09) (0.06) (0.16)
-0.53 -0.24 -0.26 -0.18
(0.09) (0.05) (0.06) (0.06)
-0.49 -0.25 -0.41 -0.38
4.8 4.1 4.2 5.4
5.0 4.4 4.8 6.0
5.0 4.4 4.5 5.9
94 64 95 97
(20) (14) (18) (20)
Control
cmws
All antagonists decrease the values for a and b. In all cases the shift of the intercept on the x-axis is not statistically significant (Table 1). The reversibility is complete for kynurenic acid but incomplete for the philanthotoxins. Kynurenic acid does not affect the values for a, b and the x-intercept (Table 2). However, the philanthotoxins tend to affect the a and b values (Table 2). Although not significantly so, dideaza-PTX-12 is again the most active analogue. DISCUSSION
In this study the effects of a group of polyamine toxins called philanthotoxins on the synaptic transmission processes from the Shaffer collateralcommissural inputs to the CA1 pyramidal cells in the rat hippocampus is compared with those of a known glutamatergic antagonist kynurenic acid. The philanthotoxins cause a decrease in amplitude of the population spike (PS), the field excitatory postsynaptic potential (f-EPSP) and amplitude of the presynaptic volley (PV). The first difference between philanthotoxin and kynurenic acid is that the latter affects the first two electrical phenomena but not the amplitude of the PV. The second difference is that the antagonistic effect of kynurenic acid is completely reversible and that the effects of the philanthotoxins are slowly and often also incompletely restored. From the philanthotoxins tested, dideaza-PTX-12 is consistently the most active antagonist, with a restricted recovery. The synaptic transmission was studied using the technique of a twin-pulse, with an interval of 20 ms. The percentual decrease of the f-EPSP and the PV is almost equal in first and second response. However, this is certainly not the case for the population spike. For both kynurenic acid and the philanthotoxins the effects on the first PS is much more pronounced than that on the second PS, indicating a possible effect of the antagonist on the inhibiting circuit. Both kynurenic acid and the philanthotoxins seem to antagonize the transmission in a non-competitive way, since the stimulus-response curves of the f-EPSPs decrease in slope without a significant shift in the x-axis intercept of the linear middle part of the curves. It can be concluded that the three most active philanthotoxins: the natural PTX-4.3.3, as well as trifluoromethyl-PTX-4.3.3 and dideaza-PTX-12, antagonize the PV and the postsynaptic phenomena. The question is whether the PV can be considered to
(0.09) (0.08) (0.08) (0.10)
Wash
be a real presynaptic phenomenon. The PV probably is a composite field action potential. The effect of philanthotoxins on this action potential cannot be explained, since they do not affect the resting potential, postpolarization, or spike amplitude in isolated axons of the cockroach (Piek et al., 1984). It is probable that when electrophysiologically measured in the hippocampus, the philanthotoxins have, just as described for the insect neuromuscular junction (Karst et al., 1991; Karst and Piek, 1991), pre- or more generally spoken non-postsynaptic effects, next to the postsynaptic effects described above. At the moment it cannot be decided whether this non-postsynaptic effect of the presynaptic volley is related to the obvious presynaptic inhibition of glutamate uptake, described in the preceding paper (Schluter et al., 1991). The most active anatagonist described here is dideaza-PTX-12. This philanthotoxin analogue is also the most active glutamate uptake inhibitor in the rat hippocampus (Schluter et nl., 1991) and an active inhibitor of the glutamate uptake in nerve endings and glial cells of the locust muscle (Karst et al., 1990). Since in locust muscle dideaza-PTX-12 is only a very weak ion channel blocker, it is suggested that this antagonist also in the rat hippocampus may affect the glutamatergic transmission, mainly at a non-postsynaptic site. Acknowledgements-This
work has been supported by a grant to T.P. from the Foundation for Biological Research
(BION) subsidized by The Netherlands Organization for Scientific Research (NWO). REFERENCES
Clark R. B., Donaldson P. L., Gration K. A. F., Lambert J. J., Piek T., Ramsey R., Spanjer W. and Usherwood P. N. R. (1982) Block of locust muscle glutamate receDtors by S-philaithotoxin occurs after receptor activations. Brain Res. 241, 105-114. Cotman C. W. and Iversen L. L. (1987) Excitatory amino acids in the brain-focus in NMDA receptors. Trends Neurosci. 10, 263-264. Florey E. (1957) Further evidence for the transmitter function of factor I. Nafurwissenschaften 44, 424-425. Fonnum F. (1984) Glutamate: A neurotransmitter in mammalian brain. J. Neurochem. 42, l-11. Honor6 T., Davies S. N., Drejer J., Fletcher E. J., Jacobsen P., Lodge D. and Nielsen F. E. (1988) Quinoxalinediones: Potent competitive non-NMDA glutamate receptor antagonists. Science 241, 701-703. Jackson H. and Usherwood P. N. R. (1988) Spider toxins as tools for dissecting elements of excitatory amino acid transmission. Trends Neurosci. 6, 278-283.
Philanthotoxins
inhibit Glu transmission
Karst H., Piek T. and Van Marle J. (1990) Dissociation of the pre- and postsynaptic inhibiting properties of the glutamatergic transmitter antagonist philanthotoxin (PTX-4.3.3). Neurosci. Res. Comm. I. 69-74. Karst H., Pick T., Van Marle J., Lind A. and Van WeerenKramer J. (1991) Structure-activity relationship of philanthotoxins-I. Pre- and postsynaptic inhibition of the locust neuromuscular transmission. Comp. Biochem. Physiol. 98C, 471477.
Karst H. and Piek T. (1991) Structure-activity relationship of philanthotoxins-II. Effects on the glutamate gated ion channels of the locust muscle fibre membrane. Comp. Biochem. Physiol. 98C, 419-489.
Kawai N., Miwa A., Shimazaki K. and Nakajima T. (1988) Spider toxins as antagonists of glutamate receptors. In Neurotransmifters: Focus on excitatory amino acids. Excerpta Medica Znt. Congress, series 856.
Mayer M. L. and Westbrook G. L. (1987) The physiology of excitatory amino acids in the vertebrate central nervous system. Prog. Neurobiol. 28, 197-216. McLennan H. (1983) Receptors for the excitatory amino acids in the mammalian central nervous system. Prog. Neurobiol. 20, 25 l-27 1. Piek T. (1990) Neurotoxins from venoms of the Hymenoptera-Twenty-five years of research in Amsterdam. Comp. Biochem. Physiol. 96C, 223-293.
Piek T. (1982) Delta-philanthotoxin, a semi-irreversible blocker of ion-channels. Comp. Biochem. Physiol. 72C, 311-315.
Piek T., Hue B., Pelhate M., David J. A., Spanjer W. and Veldsema-Currie R. D. (1984) Effects of the venom of Philanthus rriangulum F. (Hym. Sphecidae) and /I- and &philanthotoxin on axonal excitability and synaptic
transmission
in the cockroach
41 CNS.
Arch.
Insect.
Biochem. Physiol. 1, 291-306.
Piek T., Fokkens R. H., Karst H., Kruk C., Lind A., Van Marle J., Nakajima T., Nibbering N. M. M., Shinozaki H., Spanjer W. and Tong Y. C. (1988) Polyamine-like toxins-a new class of pesticides? In Neurotox ‘88: Molecular Basis of Drug and Pesticide Action (Edited by G. G. Lunt), pp. 61-77. Elsevier, Amsterdam. Piek T. and Hue B. (1989) Philanthotoxins, a new class of neuroactive polyamines, block nicotinic transmission in the insect CNS. Coma. Biochem. Phvsiol. 93C. 403-406. Sokal R. R. and Rohlf F. J. (1969) Biometry. W. H. Freeman, San Francisco. Schluter N. C. M., Lind A., Van Marle J., Van WeerenKramer J. and Piek T. (1988) Inhibition of transmission and reduction of glutamate uptake in rat hippocampus by delta-uhilanthotoxin. Pest. Sci. 24. 273-274. Van Marie J., Piek T., Lind A. and Van Weeren-Kramer J. (1984) Inhibition of the glutamate uptake in the excitatory neuromuscular synapse of the locust by delta-philanthotoxin; a component of the venom of the solitary wasp Philanthus triangulum F. A high resolution autoradiographic study. Comp. Biochem. Physiol. 79C, 213-215. Van Marle J., Piek T., Lind A. and Van Weeren-Kramer J. (1986) Reduction of high affinity glutamate uptake in rat hippocampus by two polyamine-like toxins isolated from the venom of the predatory wasp Philanthus triangulum F. Experientia 42, 157-l 58. Watkins J. C. (1984) Excitatory amino acids and central synaptic tranmission. Trendr Pharmacol. 7, 313-316. Watkins J. C. and Olverman H. J. (1987) Agonists and antagonists for excitatory amino-acid receptors. Trena!s Neurosci. 10, 265-213.
Vol. 101C. No. 1, pp. 4147, 1992
Camp. Biochem. Phyki.
Printed in Great Britain
PHILANTHOTOXINS BLOCK GLUTAMATERGIC TRANSMISSION IN RAT HIPPOCAMPUS-II. INHIBITION OF SYNAPTIC TRANSMISSION IN THE CA1 REGION N. C.
M. SCHLUTER,* T. PIER* and F. H. LOPES DA SILVA~
*Department of Pharmacology, Mei~rgdreef 1.5,1105 AZ Amsterdam and ~~pa~rnent Kruislaan 318, 1098 SM, Amsterdam, The Netherlands
of Zoology,
(Received 25 March 1991)
Abstract-l. Philanthotoxins decrease the amplitude of the population spike (PS), the field excitatory postsynaptic potential (f-EPSP), and the presynaptic volley (PV), as evoked by Shaffer-collateralcommisural inputs to the CA1 pyramidal cells in the rat hippo~mpus slice. 2. The effects are slow and often not completely reversible. 3. Dideaza-philanthotoxin-12 is, in all experiments, the most active antagonist showing a very poor recovery. 4. Using a twin pulse the percentual decreases of f-EPSP and PV amplitudes are almost identical for the first and second response. However, the first PS is much more affected than the second one, indicating a possible effect on the inhibiting circuit. 5. Philant~otoxins cause a non-competitive inhibition. 6. Besides a possible postsynaptic block and a distinct presynaptic effect (preceding paper) a non-postsynaptic effect (on the PV) is described.
INTRODUCTION
diographical studies showed that delta-philanthotoxin affects transmission by a postsynaptic block of open ~tion~hannels and by a presynaptic reduction of the glutamate uptake in nerve terminals and glial cells (Clark et al., 1982; Piek, 1982; Van Marle et al., 1984, 1986; Schluter et al., 1988; Karst et al., 1991; Karst and Piek, 1991). Synthetic delta-philanthotoxin (Tong, 1988), now called PTX-4.3.3, is chemically identical to the natural toxin and displays the known antagoni~ng effects (Piek et al., 1988; Karst et al., 1991; Karst and Piek, 1991). The present paper presents the results of the effects on the glutamatergic transmission between the Schaffer collaterals and the CA1 pyramidal cells of the rat, of PTX-4.3.3 and eleven of its analogues, together indicated with the collective name philanthotoxins (Fig. 1). The effects were studied in hippocampal slice preparations. Some of the results were published in a preliminary report (Schluter et al., 1988).
Following the discovery that glutamate may function as an excitatory transmitter in the crustacean neuromuscular junction (Florey, 1957) it has been shown that this amino acid plays an important role in the synaptic transmission in various parts of the mammalian central nervous system (Watkins, 1984; Cotman and Iversen, 1987). L-Glutamate is now firmly established as a major excitatory trans~tter substance in the mammalian CNS (McLennan, 1983; Fonnum, 1984; Watkins and Olverman, 1987). Electrophysiological, biochemical and anatomical studies have revealed the existence of at least four receptor subtypes for glutamate (Mayer and Westbrook, 1987). The lack of specific antagonists for some of these receptor subtypes stimulated an extensive search for specific compounds that could function as tools for the analysis of the mechanisms of glutamate transmission. This led to the discovery of the pure synthetic quinoxalinediones that have been reported to be the first potent and selective antagonists active at kainate and quisqualate receptors (Honore et al., 1988). Other important glutamate antagonists are natural compounds isolated from spider and wasp venoms. From these venoms polyamine-like toxins, which are active in glutamatergic transmission systems, have been isolated and chemically characterized (Piek et aZ., 1988; Jackson and Usherwood, 1988; Kawai et al., 1988). Twenty-five years of research on deltaphilanthotoxin, a polyamine isolated from the venom of the digger wasp Philanthus triangulum, demonstrated its antagonistic properties in peripheral glutamatergic and central acetylcholinergic transmission systems of insects (Piek et al., 1988; Piek and Hue, 1989; Piek, 1990). Electrophysiological and autora-
MATERIALS AND
METHODS
Animals and tissue preparation
Female Wistar rats with weights varying from 1.50 to 250g were anaesthetized by diethylether before decapitation. The brain was rapidly removed, divided midsaggitally, and placed in ice cooled Krebs phosphate solution as described in the preceding paper (Schluter et al., 1991). Transverse slices (4OOpm) of both hippocampi were prepared using a hand operated tissue chopper starting about 1.5 mm from the rostra1 {septal) end. By means of a moistened fine brush, the slices were collected and placed in aerated Krebs solution in which they were allowed to equilibrate for at least 1 hr at room temperature. Electrophysiological setup
Hippocampal slices were placed on the stretched gauze bottom of a ring that was fixed in an interface chamber. 41
N. C. M.
42
SCHLUTER
With a constant flow of I-2ml per mitt, aerated Krebs solution at 34°C passed the bottom side of the slice. The upper surface of the slice was prevented from drying out by conducting a constant flow of moistened gas (95% 0, and 5% CO,) over the slice. Two trimel-insulated stainless steel wires with a diameter of 60pm, placed in the stratum radiatum of the CAl-CA2 area, were used to stimulate the Schaffer collateral-commissural inputs to the CA1 pyramidal cells with square wave constant current pulses (0.1 ms in duration and SO-200 PA intensity), applied every 5 or 10 s in a paired-pulse fashion (interstimulus interval: 20 ms). Glass recordine electrodes filled with 3 M NaCl and a resistance of 2-10 MG were placed in the stratum radiatum of the CA1 region for extracellular recording of the presynaptic volley (PV) and the field excitatory post synaptic potential (f-EPSP) or in the stratum pyramidale of the CA1
region for extracellular recording of the population spike (P8). Chemicals: preparation and application
Philanthotoxin-4.3.3 and the eleven structural analogues, listed in Fig. 1, were synthesized by Dr Y. C. Tong (DOW Chemical Research Center, Walnut Creek, California, U.S.A.). Kynurenic acid was obtained from Sigma. All other compounds were from Merck. All chemicals were easily soluble in Krebs buffer solution except dideaza-PTX12, which was first dissolved in bidistilled water (325 PM) using moderate heating at about 50°C and subsequently diluted (1:l) with two times concentrated Krebs solution. Control and experimental fluids were administered by means of a pipette with an inner diameter of about 0.5 mm, which was situated just above the slice near the stimulationrecording area. The pipette was connected to a 10 ml vessel placed about 30 cm above the slice, in order to guarantee a constant gravity-fed flow of 0.2&3ml per min. At the beginning of drug application the aerated Krebs solution in the vessel was replaced by Krebs solution containing the compound to be tested. The washing procedure was started by replacing the content of the vessel for Krebs solution. All compounds were tested at a concentration of 650 PM except dideaza-PTX-12, which was tested at 162.5 PM. PO”7
delta-philanthotoxin,
PTX 43.3
tiH HO~CH2~HCO~NH(CH2)H~CH2)3NH(CH2)3NH2 /
/ 4.3.3
“00.
2
[email protected]
3
HO(9Pr.3.4.3
PT X 3.4.3
4
HO(9.Pr.3.3.4
PTX~ 3.3.4
5
“00.
PTX
6
HOo.Pr.3.3
7
Pr.4.3.3
PTX
1
Pr.4.3
dehydroxy-PTX-4.33
H.Pr.4.3.3 F3CQ.Pr.4.3.3
10
ZJ.PV4.3.3
11
HOo.Pr.5.3.3 -2
12
HOo.Pr. Lz
4.3
PTX~ 3.3
O.Pr.4.3.3
8 9
methyl-PTX-4.3.3
dephenol-PTX-4.3.3 trifluoromethyl-PTX~4-3
3
indole-PTX-4.3-3 PTX 12
533
dideaza~PTX~l2
Fig. 1. Structure of delta-philanthotoxin, now called philanthotoxin-4.3.3 (PTX-4.3.3) and eleven analogues (from Piek and Hue, 1989). The numbers at the left correspond with those used in Figs 2-5.
et al.
Stimulation and recording protocol
After a 20-30 min preincubation period in the interface chamber, stimulation of the Schaffer collateral fibers was started, followed by a recording of the responses in the CA1 region every 5-10min. For each record, four successive non-filtered responses were collected and averaged using a digital oscilloscope and written on paper with an X-Y plotter. After an initial 30min period during which the averaged response(s) stayed stable, a philanthotoxin analogue or kynurenic acid was applied to the slice. Washing out of the drug was started after the responses reached a steady level (two consecutive recordings did not differ by more than 5%). The last record before wash out was used for estimating the maximum effect of the drug. The recovery value was measured at the moment that there was no further increase in amplitude of the response. Analysis
The amplitudes of the PS were defined as the average of the differences between the negative peak and the preceding and following positive maxima recorded in the stratum pyramidale. The same procedure was applied to measure the PV at the stratum radiatum, where this component is clearly recorded. The f-EPSP was measured as the maximum rising slope of the negative-going potential recorded in the stratum radiatum. The statistical analysis of differences between control and maximal drug effects and between control and recovery were assessed by a (paired) sample t-test (Sokal and Rohlf, 1969). RESULTS Effects
on the population
spike
In a first series of experiments the effects of a series of polyamine antagonists, the philanthotoxins (Fig. l), were studied on the amplitude of the population spike (PS) recorded extracellularly from the
CA1 pyramidal cell layer. The glutamatergic antagonist kynurenic acid was used as a general reference to the new polyamine antagonists. The pyramidal cells were stimulated indirectly by electrical stimulation of the Schaffer collaterals in the stratum radiaturn of the CAl-CAZ region, with current intensities resulting in a submaximal (50-80%) response to the first stimulus of a pair of stimuli. Every 5-10 set a pair of stimuli was given with an interval of 20 msec. The PS were recorded as averages of four individual sweeps. At submaximal intensity, paired pulse stimulation, with an interval of 20 msec, causes facilitation of the generation of the second PS. Figure 2 shows the effects of kynurenic acid and the philanthotoxins on the first and the second PS. Figure 2A shows that both kynurenic acid and the philanthotoxins significantly antagonize the first PS, however to different levels. From the philanthotoxins only trifluoromethyl-PTX-4.3.3 and dideaza-PTX-12 are more active than PTX-4.3.3. The most active one is dideaza-PTX-12. Applied at a concentration of 162.5 PM this antagonist is about as active as kynurenic acid at 650/*M. However, the reversibility of the effect of dideaza-PTX- 12 is much lower. Regarding the second PS (Fig. 2B) significant reduction of the amplitude is only seen with trifluoromethyl-PTX-4.3.3, dideaza-PTX-12 and kynurenic acid. Here again dideaza-PTX-12 shows an inhibition of the PS which is comparable to that of kynurenic acid, the latter applied at a four times higher concentration.
Philanthotoxins
A
0.660
120
43
inhibit Glu transmission mM
0.163
ml ul
100
80
80
40
/
20
,
L
d
0 IJ
K
B
1
2345678
0.650
120
9
10
11
mM
12 0.183
mM
80
80
K
1
234567
8
9
10
11
12
Fig. 2. Effects of kynurenic acid (K) and 12 philanthotoxins (for numbers see Fig. 1) on the amplitude of the first (A) and second (B) population spike, recorded in the hippocampal CA1 region. Solid bars: maximal reduction in amplitude; open bars: recovery. All values are presented as the percentage of control. All antagonists were applied at a concentration of 650pM, except dideaza-PTX-12, which was administered at 162.5 PM. Closed triangles indicate values significantly (p < 0.05) different from control. Open triangles indicate significant difference from PTX-4.3.3 (p < 0.05). Vertical lines indicate SEMvalues (in one direction). (N = 6, except for numbers 6, 9 and 11, where N = 5.)
In order to reach the level of maximal depression of the PS-amplitudes, as well as to get a more or less stable level of recovery, for most antagonists the incubation and washing times generally were in the range of 15-35 min each. However, significantly longer incubations and washing times were needed for dideaza-PTX-12 (Fig. 3). Effects on the f-EPSP Since in the preceding section only two philanthotoxins were significantly more active than PTX-4.3.3, we have studied only the effects of PTX-4.3.3, trifluoromethyl-PTX-4.3.3, dideaza-PTX-12 and kynurenic acid on the slope of the rising phase of the f-EPSP. Figure 4 shows the mean plateau values of the depressions of the first (A) and second (B) f-EPSP caused by 650 PM kynurenic acid, PTX-4.3.3 and trifluoromethyl-PTX-4.3.3, and 162.5 PM of dideazaPTX-12. All compounds cause a significant depression and all effects are reversible. The average of slopes of the f-EPSP recovered from treatment with kynurenic acid is not significantly different from the
control value. The reversibility of the effects of the three philanthotoxins is not complete and as a result their average plateau value is significantly lower than their corresponding control values (Fig. 4). The most incomplete reversibility is seen after blocking the transmission with 162.5 ,uM dideaza-PTX-12. Eflects on the presynaptic volley (PV) Kynurenic acid has no effect on the PV-amplitude (Fig. 5). On the other hand the philanthotoxins significantly depress the PV-amplitude, dideaza-PTX12 again being the most active antagonist. This indicates that with kynurenic acid the input-output ratio significantly increases and the question arises whether this could also be the case for preparations treated with philanthotoxins. Effects on the stimulus-response curves The question whether the input-output relationship of synaptic transmission could increase in the presence of philanthotoxins or not is elucidated by measuring stimulus-response curves for both the
N. C. M.
SCHLUTER et al.
0.850
120
mM
0.183 mM A
100
80
80
40
20
0
1
K
234587
8
9
10
11
12
Fig. 3. Incubation times (solid bars) and washing times (open bars), in minutes, needed for kynurenic acid (K) and the philanthotoxins (for explanation see legend of Fig. 2) to reach a stable level. Triangles indicate values significantly different compared with PTX-4.3.3 (1). Note the remarkable increase in both times for dideaza-PTX-12 (12). Vertical lines indicate SEM (N = 6, except for numbers 6, 9 and 11, where N = 5).
0.860
mM
0.183
mM
80
80
K
(n*7)
1 WI) 0.850
9
(n-9)
mM
. Al 12 b7)
0.183
mM
100 A
80
80
!-__ A
.
40
20
0 K (n=7)
1 hll)
1 9
(n=9)
.
12 (n-7)
Fig. 4. Effects of kynurenic acid (K), PTX-4.3.3 (l), trifluoromethyl-PTX-4.3.3 (9) and dideaza-PTX-12 (12) on f-EPSP-amplitudes, measured as the slope. The plateau values of depression (solid bars) as well as of the recovery (open bars) are presented as percentages of the control values. Vertical lines indicate SEM-values (in one direction), asterisks indicate values significantly different from control (p < 0.05). Note the similarity of percentual effects between first (A) and second (B) potential, in contrast to that of the PS (Fig. 2).
Philanthotoxins
A
0.660
120 100 aa 60
40
20
a
inhibit Glu transmission
I K
mM
0.163
mM
1
(n=7)
9
1 (n-7)
0.650
12 (n.4)
(w&l)
0.163
mM
mM
80
80
40
20
0 K
1 (n=7)
(n=7)
9
(n=8)
12 (n.4)
Fig. 5. Effects of kynurenic acid (K), PTX-4.3.3 (I), trifluoromethyl-PTX-4.3.3 (9) and dideaza-PTX-12 (12) on the amplitudes of the presynaptic volley (PV). The plateau values of depression (solid bars) as well as of the recovery (shadowed bars) are presented as percentages of the control values. Vertical lines indicate SEM-values (in one direction), asterisks indicate values significantly different from control (p -C0.5). Note the similarity of percentual effects between first (A) and second (B) potential, in contrast to that of the PS (Fig. 2).
f-EPSP (Table 1) and the PV (Table 2). Table 1 shows the effects of kynurenic acid, PTX-4.3.3 and trifluoromethyl PTX-4.3.3 (650 pM) as well as of dideaza-PTX-12 (162.5pM) on the parameters of the stimulus-response curves of the first and second GEPSP in response to a paired pulse stimulation. The
central parts of the curves are considered to be linear and described using linear regression in Table 1 by the equation y = ax + b, in which y is the response in mV/ms and x the stimulus in 10m4A. Hence ‘a’ gives the slope of the curve in V set-’ A-’ and ‘b’ gives the intercept on the y-axis (v set-‘).
Table 1. Effects of four different antagonists: kynurenic acid (K, 650 FM), PTX-4.3.3 (P, 650 pM), trifluoromethyl-PTX-4.3.3 (T, 650 PM) and dideaza-PTX-12 (D, 162.5 pM) on the parameters of the stimulus-response curves of the first and second f-EPSP during paired stimuli (interval 20 msec). The central parts of the curves are considered to be linear and described by the equation y = ax = b in which y is the resoonse in mV/ms and x the stimulus in lo-” A. Values between brackets are SEM a (102V/c/A) Control
Antagonist
Control
Antagonist
(lo-'A) Antarzonist Wash
r-intercept
b (mV/ms) Wash
Wash
Control
First EPSP K (N=4) P(N=7) T(N=7) D (N=5)
147 (61) 195 (52) 221 (74) 140 (52)
73 (28) 115(28) 72 (18) 32(12)
156 (64) 144 (36) 166 (53) 72 (25)
-0.63 (0.23) -0.70 (0.17) -0.94 (0.30) -0.17(0.26)
-0.36(0.13) -0.44 (0.90) -0.36 (0.10) -0.19 (0.06)
-0.71 (0.28) -0.53 (0.11) -0.74 (0.23) -0.44(0.14)
4.3 3.6 4.3 5.5
4.9 3.8 5.0 5.9
4.6 3.7 4.6 6.1
Second EPSP K (N=4) P(N=7) T (N=7) DM=51
212 242 256 180
140 147 103 51
212 170 203 113
-0.84 -0.81 -0.97 -0.84
-0.70 -0.51 -0.48 -0.27
-0.88 (0.35) -0.57(0.15) -0.84 (0.22) -0.63 (0.27)
4.0 3.5 3.8 4.7
5.0 4.5 4.1 5.3
4.1 3.4 4.1 5.6
(84) (79) (79) (76)
(53) (41) (28) (26)
(90) (47) (56) (52)
(0.26) (0.29) (0.30) (0.33)
(0.23) (0.14) (0.13) (0.121
N.C. M. SCHLWTER~~ al.
46
Table 2. Effects of four different antagonists: kynurenic acid and three philanthotoxins on the parameters of the stimulus-response of first and second PV. For further explanation see legend of Table 1
a (mV/d) First PV K (N=4) P(N=3) T(N=6) D (N=3) Second PV K (N=4) P(N=3) T (N=6) D (N=3)
b W) Antagonist
x-intercept Control
Wash
(lOmsA) Antagonist
Control
Antagonist
Wash
101 (24) 64(15) 98 (20) 99 (19)
111(19) 55 (8) 55 (11) 39 (15)
107(17) 55(11) 96(17) 70 (15)
-0.48 -0.24 -0.42 -0.53
(0.10) (0.04) (0.06) (0.16)
-0.55 -0.23 -0.28 -0.25
(0.7) (0.4) (0.6) (0.10)
-0.55 (0.08) -0.23 (0.05) -0.44 (0.07) -0.40(0.10)
4.7 3.8 4.3 5.6
4.9 4.2 5.1 6.4
5.1 4.2 4.6 5.7
107 (14) 54 (6) 54 (12) 30(11)
98 (15) 57 (12) 91 (18) 64(13)
-0.45 -0.26 -0.40 -0.52
(0.08) (0.09) (0.06) (0.16)
-0.53 -0.24 -0.26 -0.18
(0.09) (0.05) (0.06) (0.06)
-0.49 -0.25 -0.41 -0.38
4.8 4.1 4.2 5.4
5.0 4.4 4.8 6.0
5.0 4.4 4.5 5.9
94 64 95 97
(20) (14) (18) (20)
Control
cmws
All antagonists decrease the values for a and b. In all cases the shift of the intercept on the x-axis is not statistically significant (Table 1). The reversibility is complete for kynurenic acid but incomplete for the philanthotoxins. Kynurenic acid does not affect the values for a, b and the x-intercept (Table 2). However, the philanthotoxins tend to affect the a and b values (Table 2). Although not significantly so, dideaza-PTX-12 is again the most active analogue. DISCUSSION
In this study the effects of a group of polyamine toxins called philanthotoxins on the synaptic transmission processes from the Shaffer collateralcommissural inputs to the CA1 pyramidal cells in the rat hippocampus is compared with those of a known glutamatergic antagonist kynurenic acid. The philanthotoxins cause a decrease in amplitude of the population spike (PS), the field excitatory postsynaptic potential (f-EPSP) and amplitude of the presynaptic volley (PV). The first difference between philanthotoxin and kynurenic acid is that the latter affects the first two electrical phenomena but not the amplitude of the PV. The second difference is that the antagonistic effect of kynurenic acid is completely reversible and that the effects of the philanthotoxins are slowly and often also incompletely restored. From the philanthotoxins tested, dideaza-PTX-12 is consistently the most active antagonist, with a restricted recovery. The synaptic transmission was studied using the technique of a twin-pulse, with an interval of 20 ms. The percentual decrease of the f-EPSP and the PV is almost equal in first and second response. However, this is certainly not the case for the population spike. For both kynurenic acid and the philanthotoxins the effects on the first PS is much more pronounced than that on the second PS, indicating a possible effect of the antagonist on the inhibiting circuit. Both kynurenic acid and the philanthotoxins seem to antagonize the transmission in a non-competitive way, since the stimulus-response curves of the f-EPSPs decrease in slope without a significant shift in the x-axis intercept of the linear middle part of the curves. It can be concluded that the three most active philanthotoxins: the natural PTX-4.3.3, as well as trifluoromethyl-PTX-4.3.3 and dideaza-PTX-12, antagonize the PV and the postsynaptic phenomena. The question is whether the PV can be considered to
(0.09) (0.08) (0.08) (0.10)
Wash
be a real presynaptic phenomenon. The PV probably is a composite field action potential. The effect of philanthotoxins on this action potential cannot be explained, since they do not affect the resting potential, postpolarization, or spike amplitude in isolated axons of the cockroach (Piek et al., 1984). It is probable that when electrophysiologically measured in the hippocampus, the philanthotoxins have, just as described for the insect neuromuscular junction (Karst et al., 1991; Karst and Piek, 1991), pre- or more generally spoken non-postsynaptic effects, next to the postsynaptic effects described above. At the moment it cannot be decided whether this non-postsynaptic effect of the presynaptic volley is related to the obvious presynaptic inhibition of glutamate uptake, described in the preceding paper (Schluter et al., 1991). The most active anatagonist described here is dideaza-PTX-12. This philanthotoxin analogue is also the most active glutamate uptake inhibitor in the rat hippocampus (Schluter et nl., 1991) and an active inhibitor of the glutamate uptake in nerve endings and glial cells of the locust muscle (Karst et al., 1990). Since in locust muscle dideaza-PTX-12 is only a very weak ion channel blocker, it is suggested that this antagonist also in the rat hippocampus may affect the glutamatergic transmission, mainly at a non-postsynaptic site. Acknowledgements-This
work has been supported by a grant to T.P. from the Foundation for Biological Research
(BION) subsidized by The Netherlands Organization for Scientific Research (NWO). REFERENCES
Clark R. B., Donaldson P. L., Gration K. A. F., Lambert J. J., Piek T., Ramsey R., Spanjer W. and Usherwood P. N. R. (1982) Block of locust muscle glutamate receDtors by S-philaithotoxin occurs after receptor activations. Brain Res. 241, 105-114. Cotman C. W. and Iversen L. L. (1987) Excitatory amino acids in the brain-focus in NMDA receptors. Trends Neurosci. 10, 263-264. Florey E. (1957) Further evidence for the transmitter function of factor I. Nafurwissenschaften 44, 424-425. Fonnum F. (1984) Glutamate: A neurotransmitter in mammalian brain. J. Neurochem. 42, l-11. Honor6 T., Davies S. N., Drejer J., Fletcher E. J., Jacobsen P., Lodge D. and Nielsen F. E. (1988) Quinoxalinediones: Potent competitive non-NMDA glutamate receptor antagonists. Science 241, 701-703. Jackson H. and Usherwood P. N. R. (1988) Spider toxins as tools for dissecting elements of excitatory amino acid transmission. Trends Neurosci. 6, 278-283.
Philanthotoxins
inhibit Glu transmission
Karst H., Piek T. and Van Marle J. (1990) Dissociation of the pre- and postsynaptic inhibiting properties of the glutamatergic transmitter antagonist philanthotoxin (PTX-4.3.3). Neurosci. Res. Comm. I. 69-74. Karst H., Pick T., Van Marle J., Lind A. and Van WeerenKramer J. (1991) Structure-activity relationship of philanthotoxins-I. Pre- and postsynaptic inhibition of the locust neuromuscular transmission. Comp. Biochem. Physiol. 98C, 471477.
Karst H. and Piek T. (1991) Structure-activity relationship of philanthotoxins-II. Effects on the glutamate gated ion channels of the locust muscle fibre membrane. Comp. Biochem. Physiol. 98C, 419-489.
Kawai N., Miwa A., Shimazaki K. and Nakajima T. (1988) Spider toxins as antagonists of glutamate receptors. In Neurotransmifters: Focus on excitatory amino acids. Excerpta Medica Znt. Congress, series 856.
Mayer M. L. and Westbrook G. L. (1987) The physiology of excitatory amino acids in the vertebrate central nervous system. Prog. Neurobiol. 28, 197-216. McLennan H. (1983) Receptors for the excitatory amino acids in the mammalian central nervous system. Prog. Neurobiol. 20, 25 l-27 1. Piek T. (1990) Neurotoxins from venoms of the Hymenoptera-Twenty-five years of research in Amsterdam. Comp. Biochem. Physiol. 96C, 223-293.
Piek T. (1982) Delta-philanthotoxin, a semi-irreversible blocker of ion-channels. Comp. Biochem. Physiol. 72C, 311-315.
Piek T., Hue B., Pelhate M., David J. A., Spanjer W. and Veldsema-Currie R. D. (1984) Effects of the venom of Philanthus rriangulum F. (Hym. Sphecidae) and /I- and &philanthotoxin on axonal excitability and synaptic
transmission
in the cockroach
41 CNS.
Arch.
Insect.
Biochem. Physiol. 1, 291-306.
Piek T., Fokkens R. H., Karst H., Kruk C., Lind A., Van Marle J., Nakajima T., Nibbering N. M. M., Shinozaki H., Spanjer W. and Tong Y. C. (1988) Polyamine-like toxins-a new class of pesticides? In Neurotox ‘88: Molecular Basis of Drug and Pesticide Action (Edited by G. G. Lunt), pp. 61-77. Elsevier, Amsterdam. Piek T. and Hue B. (1989) Philanthotoxins, a new class of neuroactive polyamines, block nicotinic transmission in the insect CNS. Coma. Biochem. Phvsiol. 93C. 403-406. Sokal R. R. and Rohlf F. J. (1969) Biometry. W. H. Freeman, San Francisco. Schluter N. C. M., Lind A., Van Marle J., Van WeerenKramer J. and Piek T. (1988) Inhibition of transmission and reduction of glutamate uptake in rat hippocampus by delta-uhilanthotoxin. Pest. Sci. 24. 273-274. Van Marie J., Piek T., Lind A. and Van Weeren-Kramer J. (1984) Inhibition of the glutamate uptake in the excitatory neuromuscular synapse of the locust by delta-philanthotoxin; a component of the venom of the solitary wasp Philanthus triangulum F. A high resolution autoradiographic study. Comp. Biochem. Physiol. 79C, 213-215. Van Marle J., Piek T., Lind A. and Van Weeren-Kramer J. (1986) Reduction of high affinity glutamate uptake in rat hippocampus by two polyamine-like toxins isolated from the venom of the predatory wasp Philanthus triangulum F. Experientia 42, 157-l 58. Watkins J. C. (1984) Excitatory amino acids and central synaptic tranmission. Trendr Pharmacol. 7, 313-316. Watkins J. C. and Olverman H. J. (1987) Agonists and antagonists for excitatory amino-acid receptors. Trena!s Neurosci. 10, 265-213.
