Metabotropic glutamate receptor agonists reduce paired-pulse depression in the dentate gyrus of the rat in vitro

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Neuroscienee Letters 196 (1995) 17-20

NEUBOSCIHC[ LHIERS

Metabotropic glutamate receptor agonists reduce paired-pulse depression in the dentate gyrus of the rat in vitro Ritchie E. Brown*, Klaus G. Reymann Department of Neurophysiology, Federal Institutefor Neurobiology, POB 1860, Brenneckestrasse 6, D-39008, Magdeburg, Germany

Received 14 June 1995; revised versionreceived7 July 1995; accepted7 July 1995

Abstract

We have analyzed the effects of agonists acting at different classes of metabotropic glutamate receptors (mGluRs) on paired pulse depression (PPD) at the medial perforant path/granule cell synapse. Drugs were bath applied and paired pulses delivered at 3-min intervals during control and during drug application. Both 1S,3R-l-aminocyclopentane-l,3-dicarboxylic acid (1S,3R-ACPD, 100/tM), which acts at class I (mGluR1, 5) and class II (mGluR2, 3) mGluRs and L-2-amino-4-phosphobutyric acid (L-AP4, 100/~M) which is specific for class III (mGluR4, 6-8) mGluRs, strongly reduced PPD with an interstimulus interval (ISI) of 40 ms (P < 0.001). The class I specific agonists trans-azetidine-2,4,dicarboxylic acid (t-ADA, 100/tM) and 3,5,dihydroxyphenylglycine (DHPG, 100/~M) did not affect PPD. The relatively specific class II agonists S-3-carboxy-4-hydroxyphenylglycine (3C4HPG) and 2S,3S,4S-acarboxycyclopropyl-glycine (L-CCG-I) did reduce PPD, but only at very high concentrations (500 and 40/tM respectively) with respect to their ECs0 values. These results suggest that two types of mGluRs control PPD at this synapse - a class III mGluR and a class II-like mGluR, which may not correspond to one of the currently cloned receptors. Keywords: mGluR agonists; Paired-pulse depression; ACPD; AP4 receptor; Dentate gyrus; Rat

The most recently discovered group of receptors for the neurotransmitter, L-glutamate are the metabotropic glutamate receptors (mGluRs) [16,22]. These receptors do not directly gate ion channels but instead are linked to a number of intracellular signalling cascades by the action of G proteins. The mGluRs have so far been subdivided into three classes according to pharmacological and biochemical criteria. Class I mGluRs (mGluR1 and 5) are coupled to phospholipase C and are activated most potently by quisqualate; class II mGluRs (mGluR2 and 3) are linked negatively to adenylate cyclase and are activated most potently by 1S,3R-l-aminocyclopentane-l,3dicarboxylic acid (I';,3R-ACPD); class III mGluRs (mGluR4, 6-8) are also negatively linked to adenylate cyclase but are activated preferentially by L-2-amino-4phosphobutyric acid (L.-AP4) (see [19] for review). Glutamatergic synapses exhibit various forms of plasticity which range in duration from milliseconds to days or even months. Many of these plastic processes are de* Corresponding author, Tel: +49 391 6263437; Fax: +49 391 6263438; E-mail: [email protected].

pendent on mGluR activation [2,5,7,9,18]. Paired-pulse phenomena (facilitation and depression) are short-term forms of plasticity operating in the millisecond to second range whereby the second response of a pair of pulses delivered to afferent fibres is either depressed or enhanced. In the dentate gyrus, the medial perforant path input to the granule cells demonstrates paired-pulse depression whilst the lateral perforant path input exhibits paired-pulse facilitation [4,15]. According to current thinking the depression in the medial perforant path is caused by depletion of neurotransmitter [15]. However, it is also possible that glutamate regulates its own release by means of presynaptic autoreceptors. The mGluRs are prominent candidates for these autoreceptors and indeed, recently, it has been shown that mGluR agonists can reduce paired-pulse depression [12]. In this study we have attempted to elucidate which classes of mGluRs are involved in PPD at the medial perforant path/granule cell synapse. Hippocampal slices were prepared from 7-8-week-old, male Wistar rats (Institute breeding stock). Animals were killed by a blow to the neck and rapidly decapitated. The

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18

R.E. Brown, K.G. Reymann / Neuroscience Letters 196 (1995) 17-20

brain was removed and placed into a cold, oxygenated, modified artificial cerebrospinal fluid (ACSF, see below) in which all NaCI was replaced by equimolar sucrose [1,3]. The hippocampi were dissected out, placed on a chopping block with the dentate gyrus facing up and 4 0 0 p m thick, transverse slices cut using a tissue chopper. The slices were transferred to a submerged recording chamber and left to recover for 1 h at 33-34°C in normal A C S F which contained (in mM) NaC1 124, KC1 4.9, KH2PO4 1.2, MgSO4 4, CaC12 4, NaHCO3 25.6, Dglucose 10, picrotoxin 0.05. Following equilibration, a monopolar, lacquer-coated, platinum electrode was placed in the stratum moleculare of the dentate gyrus (approx. 1 5 0 - 2 0 0 # M from the granular layer) to stimulate the medial perforant path input. A glass electrode filled with A C S F was inserted into the molecular layer at the same level as the stimulation electrode to record field excitatory postsynaptic potentials (fEPSPs). The initial slope of the fEPSP (fEPSP SF) was used as the measure of this potential. The medial perforant path input was distinguished by its localization, by the presence of PPD at short and long intervals and by the biexponential decay of the fEPSP [8]. The stimulating voltage was adjusted to elicit a fEPSP of approximately 50% of the maximum and test stimuli (3 bipolar pulses, 10 s interval, 0.1 ms halfwidth) were applied every 3 min to monitor the stability of the responses. Three paired pulses with interpulse interval (ISI) 40 ms or 500 ms were delivered at 3 min intervals before and during drug application, with the degree of PPD being defined as % PPD = ((EPSP SFznd pulse/ EPSP SFls t pulse) × 100). Drugs (obtained from Tocris Table 1 Effects of mGluR agonists on synaptic transmission and paired-pulse depression Drug ACPD (100#M) DHPG (100ktM) t-ADA (100#M) 3C4HPG (lOOflM) 3C4HPG (500#M) L-CCG-I (40 u M ) L-AP4 (100#M)

fEPSP (% control)

% PPD (control)

% PPD (drug)

n

36.6 + 4.1"**

73.5 -+ 1.1

104.0-+3.9***

5

99.6 _+0.7

80.5 _+2.0

77.1 _+2.9

4

97.3 _ 2.6

79.8 _+2.4

79.4 _+2.4

7

93.2 +-3.0

78.6 +_0.8

80.0 _ 0.9

7

60.1 +--11.6"

80.7+ 1.3

105.0+9.7

5

77.2 + 4.5*

81.5 -+ 1.3

91.3 -+ 2.3**

4

63.3 -+ 2.0***

73.1 -+ 1.4

99.1 + 1.9"**

4

The second column from the left shows % fEPSP SF after a 15 min (30 min for L-CCG-I) application of the drug with respect to the baseline period. Columns three and four show % PPD during the control period and during drug application respectively, lnterpulse interval was 40 ms. Asterisks indicate statistical significance using the paired t-test: *P < 0.05, **P < 0.01, ***P < 0.001.

A

etmtrol

B

Control

D

L-AP4

washout

co.~

~o rns

lS,3R-ACPD (100 pM)

lO ms

L-AP4 (100 MM)

10ms

washout (stimulation in~nsity reduced)

10ms

w u h o u t (stimUlation intensity reduced)

°°o L

oooL

10ms

10ms

Fig. 1. Both 1S,3R-ACPD (ACPD) and L-AP4 reduce paired-pulse depression. (A,C) Average data from ACPD (100/~M, n = 5) and LAP4 (100/~M, n =4) experiments respectively. ***P < 0.001. (B,D) Analogue traces from single experiments involving ACPD and L-AP4 respectively. After washout of the drug the stimulation intensity was reduced to make the size of the first pulse in the pair equal to that during drug application (lower traces). As can be seen in these traces and in the average data (compare control and washout columns), pairedpulse depression did not depend on the size of the first pulse. Cookson) were normally bath-applied for 15 min and then washed out. In some cases drugs were applied for 30 min to confirm that the lack of an effect was not due to insufficient time of application or where it was suspected that 15 min was not long enough to achieve a steady-state response. Statistical tests between control values and values during drug application were made using the paired ttest. All values are given as the mean _+ SEM. Bath application of the selective class III agonist LAP4 (ECs0 45 ~ M [ 13]) at a concentration o f 100/~M led to a large and reversible depression of the fEPSP recorded from the middle third of the molecular layer of the dentate gyrus (n = 4, Table 1), as shown previously [13,14]. At the same time, paired pulse depression with an ISI of 4 0 m s was reduced from 73.1 +_ 1.4% in control to 99.1 _+ 1.9% (n = 4, Fig. 1B,D). Similarly, application of the broad spectrum mGluR agonist 1S,3R-ACPD (100ktM), which has been shown to activate class I and class II but not class III mGluRs (ECs0 48 # M at class I

R.E. Brown, K.G. Reymann I Neuroscience Letters 196 (1995) 17-20

and 6/zM at class II [7',1]) depressed the fEPSP (Table 1), and reduced PPD (ISI 40 ms) from 73.5 _+ 1.1% in control, to 103.9 _+3.9% (Fig. 1A,C). The reductions in PPD were not due to the smaller size of the first pulse per se, since reduction of the stimulus intensity to give a fEPSP of the same size as during drug application did not affect the amount of PPD obtained (Fig. 1). None of the drugs tested in this study affected PPD with an ISI of 500 ms (data not shown). Since 1S,3R-ACPD activates both class I and class II mGluRs, in the next series of experiments we used more specific activators of cilass I and class II mGluRs to try to determine which of these two classes were responsible for the effect of 1S,3R-ACPD. Application of the class I specific agonists 3,5-dihydroxyphenylglycine (DHPG, ECs0 28/~M [21]) or trans-azetidine-2,4,dicarboxylic acid [6] (tADA, ECs0 32/tM at mGluR5 and 190/~M at mGluRla (J.-P. Pin, personal communication)), both at a concentration of 100/tM, had no effect on baseline synaptic transmission, and did not affect PPD (see Table 1). DHPG (70/.tM) did, however, strongly reduce accommodation of firing (data not shown). Therefore, the class I mGluRs do not appear to mediate the effects of 1S,3R-ACPD on PPD. Application of the class II agonist S-3-carboxy-4-hydroxyphenylglycine (3C4HPG, ECs0 70/tM at class II, IC5o 500/xM at class I i(10]) at a concentration of 100/~M had no effect on either baseline or on PPD but at a higher concentration of 5 0 0 ~ M the first pulse was depressed to 60.1_ 11.6% of control and PPD was reduced from 80.4_ 1.3% in control to 101.7 _+9.7%. This effect did not quite reach significance (P = 0.087) due to the large standard error, although in every case (n = 5) PPD was reduced compared to ,:ontrol. The reason for the large variability between slices is at present unclear. Another, more potent class II ~tgonist, 2S,3S,4S-ct-carboxycyclopropyl-glycine (L-CCG-I, ECs0 0.3/zM at class II and 5 0 ~ M at class I and III [11]) did not affect PPD at 10 or 20/~M (data not shown) but did slightly reduce PPD at 40/tM (n = 4, Table 1). However, at this concentration, this drug also activates class III mGluRs and so may be activating the same receptors as L-AP4. Our data demonstrate that a class III as well as a class II-like mGluR is involved in PPD at the medial perforant path synapse. Class I rnGluRs appear to play no role in this process. Among the four class III mGluRs that have been cloned so far (mGluR4, 6-8), mGluR6 is only expressed in the retina [ 17]. The other three receptors, however, cannot be excluded from mediating the effects of LAP4 in this region. In comparison with the lateral perforant path, the effects of L-AP4 on synaptic transmission in the medial pathway are quite small, indicating perhaps, that a lower affinity receptor (mGluR7) [20] is preferentially activated. Alternatively, it may be that only a small proportion of the synapses have AP4 receptors. Indeed, the effects of L-AP4 vcere very fast to wash in, and quickly reached a plateau, which may indicate the pres-

19

ence of the same high affinity receptor, as in the lateral path, but more sparsely distributed. The results obtained with the class I and class II agonists are more difficult to interpret. 1S,3R-ACPD activates class I and class II mGluRs, as well as activating a number of biochemical mechanisms which have so far not been linked to any of the cloned mGluRs [19]. Activation of either mGluR1 or 5 is unlikely to be responsible for its effects since we found that two class I agonists did not affect synaptic transmission or PPD. This is consistent with normal PPD in m G l u R l a - mutant mice [5]. In addition, mGluR2 and 3 may also not be responsible for the effects of 1S,3R-ACPD since 1S,3R-ACPD was much more effective than the class II agonists, 3C4HPG and LCCG-I, than a comparison of their respective ECs0 values would predict. Furthermore, the effect of L-CCG-I may rather have been mediated by an action at a class III mGluR. However, this conclusion depends on the validity of the ECso values we have quoted for the class II agonists, which were obtained in expression systems and not in hippocampal slices. Thus, the mGluR mediating the effects of 1S,3R-ACPD is at present unclear. One possibility is that it corresponds to the recently described mGluR which is positively coupled to adenylyl cyclase [23]. We found that mGluR agonists only affected PPD when the interval between the pulses was short, in agreement with the findings of Kahle and Cotman [12]. Therefore, there appear to be two phases of PPD, one which operates over a short time period and is regulated by presynaptic mGluRs and a longer phase which may depend on depletion of transmitter available for release. In conclusion we have found that 2 types of mGluRs control PPD at this synapse - a class III mGluR and a class II-like mGluR. The fact that two receptors seem to be involved in the regulation of the early phase of PPD suggests that this phase is tightly regulated; the elucidation of the differential role of the two receptor classes in this process will have to await further study. The authors wish to thank Dr. Detlef Balschun for critical reading of this manuscript. This research was supported by the Bundesministerium fiir Bildung, Wissenschaft, Forschung und Technologie (BMFT, BEO 21/0316912 A) and the European Biomed 1 program (BMH1-CT93-1033) to K.G.R. [1] Aghajanian,G.K. and Rasmussen, K., Intracellularstudies in the facial nucleus illustrating a simple new method for obtaining viable motoneuronsin adult rat brain slices, Synapse,3 (1989) 331338. [2] Bashir, Z.I.. Bortolotto, Z.A., Davies, C.H., Berretta, N, Irving, A.J., Seal. A.J., Henley, J.M., Jane, D.E., Watkins, J.C. and Collingridge, G.L., Induction of LTP in the hippocampusneeds synaptic activation of glutamate metabotropicreceptors, Nature, 363 (1993) 347-350. [3] Buhl, E.H., Han, Z.-S., l..6dnczi, Z., Stezhka, V.V., Karnup, S.V., and Somogyi, P., Physiologicalproperties of anatomically identi-

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