Indirect potentiation of synaptic transmission by metabotropic glutamate receptors in the rat hippocampal slice

BRAIN RESEARCH Brain Research684 (1995) 165-171

ELSEVIER

Research report

Indirect potentiation of synaptic transmission by metabotropic glutamate receptors in the rat hippocampal slice Dawn R..Collins, Jennifer M. Scollon, David C. Russell, Stephen N. Davies * Department of Biomedical Sciences, Marischal College, University of Aberdeen, Aberdeen, AB9 1AS, UK

Accepted 14 March 1995

Abstract The role that the metabottopic glutamate receptor plays in synaptic transmission is complex due to the multiple subtypes involved, which initiate a number of intracellular mechanisms. Here we have investigated the role of the metabotropic glutamate receptor in the induction of long-term potentiation (LTP). We have shown that, providing the CA3 region remains attached to the slice, it is possible to induce potentiation by bath perfusion of the metabotropic receptor agonist (1S,3R) 1-aminocyclopentane-l,3-dicarboxylic acid (ACPD) alone. The extent of the potentiation observed showed a strong negative correlation with the age of the animal from which the slices were prepared. Perfusion of ACPD was associated with an increase in the excitability of antidromically activated CA3 neurones, the appearance of spontaneous burst firing within the CA3 region, and an increased fibre volley recorded in the CA1 region. Blockade of N-methyl-D-aspartate (NMDA) receptors prevented all these effects. We suggest that the ACPD-induced potentiation of CA1 fEPSPs is an indirect effect caused by spontaneous burst firing and/or increased excitatory drive from CA3 neurones. Keywords: (1S,3R)-l,3-Aminocyclopentane-3-dicarboxylic acid; Metabotropic glutamate receptor; N-Methyl-D-aspartate ; LTP; Hip-

pocampus; CA1-CA3; Synaptic transmission

1. Introduction Long-term potentiation (LTP) describes a form of usedependent synaptic plasticiity that is widely proposed as a possible mechanism underlying memory formation [6]. Experimentally, LTP is commonly induced in the CA1 region of the rat hippocampal slice by applying a brief, high-frequency stimulus (tetanus). It is well established that activation of the N-methyl-o-aspartate (NMDA) subtype of glutamate receptor during the tetanus is vital for the induction of LTP [14], but it is generally found that application of NMDA alone is sufficient to evoke only a short-lasting form of potentiation [14,18]. The involvement of metabotropic glutamate receptors (mGluRs) in the formation of LTP is widely proposed. Antagonism of mGluR subtypes can prevent the induction of LTP [2,5], and it has been shown that application of mGluR agonists can enhance LTP induced by a subthreshold tetanus [19,20]. The question of whether or not activation of mGluRs alone is sufficient to induce LTP is more controversial. Perfusion

* Correspondingauthor. Fax: (1) (124) 273019. 0006-8993/95/$09.50 © 1995 ElsevierScienceB.V. All rights reserved SSD! 0006-8993(95)00410-6

of (1S,3R) 1-aminocyclopentane-l,3-dicarboxylic acid (ACPD, 10 /xM) for 20 min is reported to cause a slowly developing and sustained potentiation of the field excitatory postsynaptic potential (fEPSP) evoked in rat hippocampal slices to which the CA3 region remained attached [7]. In contrast, we found that perfusion of 50/zM ACPD for 5 min had no substantial long-term effect on slices in which the CA3 region was removed [13]. The present study therefore set out to resolve the discrepancy between these two reports. The possible differences between the two protocols include the concentration and period of perfusion of ACPD, the removal of the CA3 region in our experiments, and the age of the rats.

2. Materials and methods Four-hundred-/xm-thick transverse hippocampal slices were prepared from halothane anaesthetised female Sprague-Dawley rats (University bred), weighing between 130 and 160 g. The brain was removed and placed in ice-cold artificial cerebrospinal fluid (aCSF) containing in mM: NaC1 124, KC1 3, NaHCO 3 26, NaH2PO 4 1.25,

166

Dawn R. Collins et al. /Brain Research 684 (1995) 165-171

3. Results

CaC12 2, MgSO 4 1, D-glucose 10; bubbled with 90% 0 2 / 5% CO 2. The hippocampi were rapidly dissected out and sliced on a McIlwain tissue chopper. The slices were separated and transferred to a constantly perfused interface-type chamber and maintained at 29-31 ° C. For all experiments, in contrast to our previous published work, the CA3 region was present. The Schaffer-collateral commissural fibre pathway was stimulated at a frequency of 0.033 Hz using a bipolar stimulating electrode placed in stratum radiatum at the the fimbrial end of the CA1 region. A 3M NaCl-filled glass electrode (resistance between 2 and 10 MI2) was positioned in the stratum radiatum of the CA1 region to record the evoked fEPSP. The stimulus strength was adjusted to give a 50% maximal response. Following stabilisation of the response, ACPD was applied by addition to the aCSF according to one of two protocols: (i) a concentration of 50 /~M ACPD for 5 min, or (ii) 10 /xM ACPD for 20 min. All drugs were obtained from Tocris Neuramin (now Tocris Cookson). Statistical comparison of pre-and post-treatment fEPSP amplitudes were made using the Student's t-test.

A

Initially, we repeated our previous protocol of perfusion of 50 /zM ACPD for 5 min, but with the CA3 region attached, and this evoked a significant and long-lasting potentiation of the response in 8 out of the 11 slices tested (mean fEPSP amplitude was 135% at 30 min post-application, 171% of control at 90 min post-application, n = 8, P < 0.01. Fig. 1A). We then replicated the work of Bortolotto and Collingridge [7] by perfusing 10 /zM ACPD for 20 min in slices with the CA3 attached. Out of 10 trials 5 slices showed a potentiation which was comparable in time course and magnitude to that described by Bortolotto and Collingridge (mean fEPSP amplitude was 181% at 90 min post-application, n = 5, P < 0.05. Fig. 1B). The remaining trials showed little evidence of an effect (mean fEPSP amplitude was 94% at 90 min post-application, n ffi 5, not significant, data not shown). Regression analysis showed that there was a marked correlation between the weight or age of the rats from

3O(

! ~ A ~

6

I

30

~

j

90

Tm~ (minums)

B

E ~OpMAClm

b

J

3O

Tune (rnbues)

t

~0

=~m~IIIE/~L

I

9O

Fig. 1. A: graph showing the effect of peffusion of 50/.tM ACPD on the mean fEPSP amplitude recorded in the CA1 region. Perfusion of the drug is indicated by the bar and the data shown is pooled for 8 trials. The traces above are taken from a representative slice and are the average of the consecutive responses, taken at (1) control and (2) 90 rain post ACPD application. B: graph showing the effect of 20 rain pcrfusion of 10/.tM ACPD. Data is pooled for 5 trials and the representative traces taken from (1) the control period and (2) 90 rain post-application. For all figures the scale bar represents 10 ms and 2 inV.

Dawn R. Collins et al. /Brain Research 684 (1995) 165-171 A

167

A 2~0

~

200 i

|

~

L

~APS

,oo

0

i

i

i

W~

i

i

i

i

I

~ ar~m.l (Ip'mnmm)

3o

~o

I

0

B ~OC

•)~ A(~ ~MA~

| l,° F

~

ACL~

[__

.g

i / ~I

~

.11 --

i 41

i 42

1 43

l 44

i 4~;

i 46

i 48

47

10(

49 0

A g e o f mLs ( d a y s )

~0

Trae (minutes)

Fig. 2. Graphs depicting the relationship between the weight of the animal (A) or the age of the animal (B) and the amount of potentiation achieved 90 minutes after 5 min pcrfusion of 50/zM ACPD. For both graphs n = 11 and the line represents the linear relationship obtained by regression analysis.

Fig. 3. A: graph showing the effect of 5 min pcrfusion of 50/~M ACPD in the presence of 40 ~M AP5 on the amplitude of the fEPSP recorded in the CA1 region. The graph is pooled data for 6 trials and the traces are taken from a representative slice at (1) control and (2) 90 min post-ACPD application. B: graph showing the effect of 5 rain pcrfusion of 50 /~M ACPD 60 min after the above treatment. The pooled data is for 5 slices and the traces taken at (1) control, (2) 60 min after perfusion of 50 /zM ACPD in the presence of 40/~M AP5 and (3) 60 min following perfusion of 50 /zM ACPD alone.

which the slices were prepared, and the magnitude of the

potentiation evoked by perfilsion of ACPD (Fig. 2A and B, respectively). The ability of the slices to potentiate de-

! M

= •

• •

-

".

i W mr ~

• [

qllJll~

I s Ull

qpu~..dlS~21M~

_

i.

6

I

3O

go

I

90

(mmmes) Fig. 4. Graph showing the effect of 5 min pcrfusion of 50 p.M ACPD on the mean population spike amplitude recorded from the CA3 region in response to antidromic stimulation. The data shown is pooled for 4 trials and the traces above are the average of three consecutive responses taken from a representative slice at (1) control and (2) 90 rain post ACPD application.

168

Dawn R. Collins et al. / Brain Research 684 (1995) 165-171

clined dramatically with age ( r = 0.95, n = 11) and body weight (r = 0.88, n = 11) therefore in all subsequent experiments rats in the range 130-150 g (41-46 days old) were used. Bortolotto and Collingridge [7] reported that the potentiation induced by perfusion of ACPD was not affected by blockade of NMDA receptors. We therefore also perfused 50 /~M ACPD for 5 min in the presence of the NMDA antagonist D-2-amino-5-phosphonopentanoate (AP5). AP5 (40 /xM) was perfused for a period from 15 min prior to, until 5 min after, application of ACPD. In none of the 6 trials run was any potentiation observed and instead there was a slight depression of the response (mean fEPSP amplitude was 97% of control at 30 min, not significant; 83% of control 90 rain post-application, P < 0.01, n = 6, Fig. 3A). In 5 of these slices we subsequently attempted to induce potentiation using ACPD alone. Perfusion of 50~M ACPD for 5 min evoked only a depression of the response (mean fEPSP amplitude was 74% 60 rain after application of ACPD alone, n - - 5 , P < 0.05. Fig. 3B). This could either imply that perfusion of ACPD in the presence of AP5 inactivates the induction mechanism, or that this particular batch of slices (all taken from separate rats) were simply not capable of sustaining potentiation in the first place. However, 3 experiments were run in which naive slices, taken from the same animals, were subsequently treated with 5 min perfusion of 5 0 / x M ACPD and all exhibited ACPD induced potentiation. In contrast, ACPD-induced potentiation was blocked by the metabotropic receptor anatgonist (RS)-a-methyl-4-carboxyphenylglycine (MCPG). In three slices MCPG (500 /~M) was perfused from 15 min prior to, until 5 min after, perfusion of ACPD and none of these slices showed any potentiation (data not shown). In order to further characterise the role of the CA3

3

_

_

~ / V**~*.a,**a,.m,.~-

L__ Fig. 5. Traces recordedfrom a single slice showing the effect of 5 min perfusionof 50/xM ACPDon antidromicresponsesrecordedfromwithin the CA3 cell body region. Traces are an average of three consecutive responses taken at (1) control,(2) duringACPD perfusionand (3) 60 rain post-application. region in ACPD induced potentiation, we also performed experiments in which a recording electrode was placed in stratum pyramidale of the CA3 region, while stimulating the Schaffer collateral-commissural fibres as before. Stimulation in normal aCSF at sufficient stimulus intensity evoked a single antidromically activated population spike. In 4 out of 6 trials perfusion of 50/.~M ACPD for 5 rain caused a rapid increase in the amplitude of the population spike (mean population spike amplitude was 140% of control at 30 rain, 176% of control at 90 rain post-application, n -- 4, P < 0.05, Fig. 4) and introduction of up to 5 extra population spikes (Fig. 5). The increase in population spike amplitude and the development of multiple population spikes preceded the time course of potentiation of the fEPSP recorded in the CA1 region in the previous experiments. Perfusion of 50 /.~M ACPD in the presence of 40 /xM AP5 using the same protocol as before prevented the enhancement of the population spike and instead a tran-

300

i

b

I

50pMAQ~O

1

30 (minu~s)

~b

I

90

Fig. 6. Graph showingthe effect of 5 min perfusionof 50 /.LMACPD on the height ofthe presynapticfibre volley. Data is pooled for 3 trials and the representativetraces are taken at (1) controland (2) 90 min post ACPD application.

Dawn R. Collins et a l . / Brain Research 684 (1995) 165-171

sient depression of the population spike was observed during perfusion, but no significant long-term effects were obtained (mean population spike amplitude was 105% at 30 min, 88% of control at 90 min post-application, n -- 4, not significant, data not ,;hown). In 3 trials subsequent application of ACPD alone', led only to a marginal depression of the response (data not shown). In the same slices we looked for the occurrence of spontaneous burst firing by monitoring the amplifier output on a chart recorder. While recording in the CA3 region, we observed spontaneous burst firing in 5 out of 6 trials. Bursting was found to occur maximally during perfusion and for at least 30 min after perfusion, with sporadic but prolonged periods of bursting throughout the remainder of the recording period. In most of our recordings the presynaptic fibre volley is either very small or non-existant. To establish if the changes in fEPSP amplitude were accompanied by changes in the fibre volley we therefore performed some experiments where we recorded in the CA1 region, but positioned the stimulating electrode in closer proximity to the recording electrode. We have previously found that this technique

6

5 ¢m

4

3 f~

,J

2 1

0

|

0

!

i

i

|

5

10 15 20 Stimultus Strength (V)

5

10 15 20 Stimulus Strength (V)

25

.N

o

0

'

0

I

,

I

,

I

i

I

25

Fig. 7. Input-outputcurves showingthe effect of perfusionof ACPD on the relationship between stimulus strength and fEPSP height (upper panel) or fibre volley amplitude (lower panel). Open symbols indicate control responsesand closed symbolsindicate responsesrecorded90 rain after perfusionof ACPD (50 /xM for 5 rain).

169

evokes a much bigger recordable fibre volley. In 3 out of 4 trials 5 min perfusion of 50 /zM ACPD elicited a slowly increasing and long-lasting potentiation of the fibre volley, comparable to fEPSP potentiation (mean fibre volley amplitude was 125% at 30 min and 144% of control at 90 min post-application, n = 3, P < 0.05, Fig. 6). Fig. 7 shows the input-output curve generated from a single slice plotting the amplitude of the fibre volley and the fEPSP against stimulus strength for control responses, and 90 min after perfusion of ACPD.

4. Discussion

The first finding from this study is confirmation that the presence of the CA3 region is necessary for ACPD induced synaptic potentiation [7]. Both the time course and the magnitude of potentiation were comparable using either of the two concentrations of ACPD suggesting that selective actions on specific mGluR subtypes are not involved. We have, however, also demonstrated that the effect of ACPD shows a marked dependence on the age of the animals from which the slices were prepared. This may be caused by alterations in the mGluR subtypes present during the early postnatal weeks [8-10,15,16], which are also reflected in age-dependent changes in agonist efficacy [21]. According to the data of Catania et al. [10], the loss of ACPD induced potentiation correlates most closely with the loss of mGluR3 and mGluR5 subtypes, thereby implicating these in the synaptic potentiation. The age or weight of the animals used in experiments are often not specified in the methods, but this may be the reason why ACPD induced potentiation has not been commonly observed in most other published reports [3,4,13,19,20]. Although we have been able to replicate the ACPD induced potentiation reported by Bortolotto and Collingridge [7], there are two important discrepancies. The first is the sensitivity of the ACPD induced potentiation to AP5. It is hard to prove convincingly that AP5 blocks the effect when not all the the control slices showed potentiation, and the problem is compounded by the fact that subsequent perfusion of ACPD alone did not induce potentiation. However, it is very unlikely that all 6 slices, prepared from separate rats, would have been unable to express potentiation, especially since on 3 occasions perfusion of ACPD later in the day onto naive slices prepared from the same rat consistently induced potentiation. We therefore tend towards the alternative conclusion that perfusion of ACPD in the presence of AP5 turned off the LTP induction mechanism thereafter. The second important discrepancy concerns whether the effect of ACPD on the CA1 fEPSP is a direct potentiation of synaptic transmission, or whether it is an indirect effect on the presynaptic CA3 neurones. Both removal of the CA3 region, and blockade of NMDA receptors, prevented

170

Dawn R. Collins et al. /Brain Research 684 (1995) 165-171

the ACPD-induced potentiation. We have considered two mechanisms which might reconcile these observations. (i) ACPD has a number of actions on cortical neurones including potentiation of NMDA receptor mediated responses [1,17] and blockade of K ÷ conductances [11]. Such an effect on the reciprocally connected CA3 neurones could lead to burst firing which would effectively create a 'physiological tetanus' which would be expected to potentiate, in an AP5 sensitive manner, any synapses that those neurones innervated. Comparable ACPD induced burst firing has previously been demonstrated in the dorsolateral septal nucleus [22,23]. (ii) Alternatively, ACPD might cause an increased excitability of the stimulated CA3 neurones such that the recorded CA1 neurones are subject to greater excitatory drive which would be evident as an increased presynaptic fibre volley. Our experiments have shown evidence for both of these mechansisms, ie spontaneous burst firing and increased CA3 excitability. Perfusion of ACPD increased the amplitude and number of antidromically activated population spikes in the CA3 region, induced spontaneous burst firing in the CA3 region, and also increased the amplitude of the presynaptic fibre volley recorded in the CA1 region. Either, or both, of these mechanisms may therefore contribute to the potentiation of the fEPSP. It is notable that all of these effects were blocked by AP5 and therefore presumably rely at some stage on the activation of NMDA receptors. During review of this manuscript comparable results were reported from another laboratory [12] which indicated changes in fibre volley amplitude and appearance of 'paroxysmal' activity induced by perfusion of ACPD. Therefore, it would appear that in our preparation, ACPD induced potentiaton of the CA1 fEPSP is an indirect effect relying on modulation of excitability in the CA3 region to either induce spontaneous burst firing and/or increase the number of presynaptic neurones activated by the electrical stimulus. Both these mechanisms rely on functioning NMDA receptors. The indirect nature of the slowly developing potentiation caused by perfusion of ACPD alone suggests that it is not mechanistically comparable to the maintenance phase of LTP. We conclude that activation of metabotropic glutamate receptors alone is insufficient to evoke synaptic potentiation in the CA1 region of the hippocampal slice. Acknowledgements This work was supported by the Medical Research Council and the Wellcome Trust. References [1] Aniksztejn, L., Bregestovski, P. and Ben-Ari, Y., Selective activation of quisqualate metabotropic receptor potentiates NMDA but not AMPA responses, Eur. J. Pharmacol., 205 (1991) 327-328.

[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 Collingndge, G.L., Induction of LTP in the hippocampus needs synaptic activationof glutamate metabotropic receptors, Nature, 363 (1993) 347-350. [3] Baskys, A. and Malenka, R.C., Trans-ACPD depresses synaptic transmission in the hippocampus, Eur. J. PharmacoL, 193 (1991) 131-132. [4] Behnisch, T. and Reymann, K.G., Co-activation of metabotropic glutamate and N-methyl-D-aspartate receptors is involved in mechanisms of long-term potentiation maintenance in rat hippocampal CA1 neurons, Neuroscience, 54 (1993) 37-48. [5] Behnisch, T., Fjodorow, K. and Reymann, K.G., L-2-Amino-3-phosphonopropionate blocks late synaptic long-term potentiation, NeuroReport, 2 (1991) 386-388. [6] Bliss, T.V.P. and Collingfidge, G.L., A synaptic model of memory: long-term potentiation in the hippocampus, Nature, 361 (1993) 31-39. [7] Bortolotto, Z.A. and Collingridge, G.L., Characterisation of LTP induced by the activation of glutamate metabotropic receptors in area CA1 of the hippocampus, Neuropharmacology, 32 (1993) 1-9. [8] Boss, V., Desai, M.A., Smith, T.S. and Conn, P.J., Trans-ACPD-induced phosphoinositide hydrolysis and modulation of hippocampal pyramidal cell excitability do not undergo parallel developmental regulation, Brain Res., 594 (1992) 181-188. [9] Casabona, G., Genazzani, A.A., Di Stefano, M., Sortino, M.A. and Nicolletti, F., Developmental changes in the modulation of cyclic AMP formation by the metabotropic glutamate receptor agonist 1S,3R-aminocyclopentane-l,3-dicarboxylic acid in brain slices, J. Neurochem., 59 (1992) 1161-1163. [10] Catania, M.V., Landwehrmeyer, G.B., Testa, C.M., Standaert, D.G., Penney, J.B. and Young, A.B., Metabotropic glutamate receptors are differentially regulated during development, Neuroscience, 61 (1994) 481-495. [11] Charpak, S., Giih~viler, B.H., Do, K.Q. and Kn~pfel, T., Potassium conductances in hippocampal neurons blocked by excitatory aminoacid transmitters, Nature, 347 (1990) 765-767. [12] Chinestra, P., Diabara, D., Urban, N.N., Barrionuevo, G. and BenAft, Y., Major differences between long-term potentiation and ACPD-induced slow onset potentiation in hippocampus, Neurosci. Left., 182 (1994) 177-180. [13] Collins, D.R. and Davies, S.N., Potentiation of synaptic transmission in the rat hippocampal slice by exogenous L-glutamate and selective L-glutamate receptor subtype agonists, Neuropharmacology, 33 (1994) 1055-1063. [14] Collingridge, G.L., Kehl, S.J. and McLennan, H., Excitatory amino acids in synaptic transmission in the Schaffer collateral-commissural pathway of the rat hippocampus, J. Physiol., 334 (1983) 33-46. [15] Desai, M.A., Smith, T.S. and Corm, P.J., Multiple metabotropic glutamate receptors regulate hippocampal function, Synapse, 12 (1992) 206-213. [16] East, S.J. and Garthwaite, J., Actions of a metabotropic glutamate receptor agonist in immature and adult rat cerebellum, Eur. J. Pharmacol., 219 (1992) 395-400. [17] Harvey, J., Frenguelli, B.G., Sunter, D.C., Watldns, J.C. and Collingridge, G.L., The actions of 1S,3R-ACPD, a glutamate metabotropic receptor agonist, in area CA1 of rat hippocampus, Br. J. Pharmacol., 104 (1991) 75P. [18] Kaner, J.A., Malenka, R.C. and Nicoll, R.A., NMDA application potentiates synaptic transmission in the hippocampus, Nature, 334 (1988) 250-252. [19] McGuinness, N., Anwyl, R. and Rowan, M., Trans-ACPD enhances long-term potentiation in the hippocampus, Eur. J. Pharmacol., 197 (1991) 231-232. [20] Otani, S. and Ben-Ari, Y., Metabotropic receptor-mediated long-term potentiation in rat hippocampal slices, Eur. J. PharmacoL, 205 (1991) 325-326.

Dawn R. Collins et al. / Brain Research 684 (1995) 165-171

[21] Schoepp, D.D., Johnson, B.G., True, R.A. and Monn, J.A., Comparison of (1S,3R)-l-aminocyclopentane-l,3-dicarboxylic acid (1S,3R-ACPD)-and 1R,3S-ACPD-stimulated brain phosphoinositide hydrolysis, Eur. J. Pharmacol., 207 (1991) 351-353. [22] Zheng, F. and Gallagher, J.P., Trans-ACPD (trans-D,L-1-Amino1,3-Cyclopentanedicarboxylic Acid) elicited oscillation of membrane potentials in rat dorsolateral septal nucleus neurons recorded intracellularly in vitro, Neurosci. Lett., 125 (1991) 147-150.

171

[23] Zheng, F., Lonart, G., Johnson, K.M. and Gallagher, J.P., (1S,3R)1-Aminocyclopentane-l,3-dicarboxylic acid (1S,3R-ACPD) induces burst firing via an inositol-l,4,5-triphosphate-independent pathway at rat dorsolateral septal nucleus, Neuropharmacology, 33 (1994) 97-102.