NMDA Receptor-dependent and -independent Long-term Depression in the CA1 Region of the Adult Rat Hippocampus In Vitro

Neuropharmacology, Vol. 36, No. 3, pp. 397–399, 1997 Copyright 01997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0028-3908/97 $17.00 + 0.00

Pergamon PII: S0028-3908(96)00015-4

Rapid Communication NMDA Receptor-dependent and -independent Longterm Depression in the CA1 Region of the Adult Rat Hippocampus In Vitro N. KEMP and Z. I. BASHIR* Department ofAnatomy, School of A4edicalSciences, UniversityWallqUniversiQof Bristol, Bristol, BS8 ITD, U.K. (Accepted 15 January 1997) Summary-We have investigateddifferent stimulus parameters in an attempt to induce long-term depression (LTD) in the CA1 region of adult rat hippocampusin vitro. Whereas 900 stimuli delivered at 1 Hz failed to induce LTD, 900 stimuli when delivered as 450 pairs (50 msec inter-stimulus interval) at 1 Hz induced significantand stablefV-methyl-D-aspartate (NMDA)receptor-dependentLTD. However,900 paired stimuli at 1 Hz induced LTD which was only partly blocked by the NMDA receptor antagonist, D-2-amino-5phosphonopentanoate(AP5). ~ 1997 Elsevier Science Ltd. All rights reserved.

Keywords—Long-term depression (LTD), CA1, hippocampus,NMDA, plasticity.

Long-term depression (LTD) is an activity-dependent lasting decrease in synaptic strength (Bear and Abraham, 1996). In the in vitro hippocampus of young (w2 week old) rats the induction of homosynaptic LTD by low frequency stimulation (LFS; 900 stimuli, 1 Hz) has been demonstrated to occur readily (Mulkey and Malenka, 1992). However, whether similar stimulus protocols induce LTD in hippocampal slices from older animals is still a matter of debate and some different methods have now been described to produce LTD in the adult CA1 region (Dudek and Bear, 1992; Bashir and Collingridge, 1994; Kerr and Abraham, 1995; Wagner and Alger, 1995; Staubli and Ji, 1996; Norris et al., 1996).It has been demonstratedthat LTD can be induced in vivo in the CA1 region of adult animals by the use of paired pulse stimulation protocols (Thiels et al., 1994). We have now tested similarprotocolsand show that LTD can be induced in the CA1 region by paired pulse stimulation paradigmq in vitro. Furthermore, the induction of LTD can be either NMDA receptor-dependentor NMDA receptor-independent,depending on the number of paired stimuli delivered. Hippocampal slices were prepared from adult (180– 220 g; *8–15 weeks old) Wistar female rats (B and K *Towhomcorrespondence shouldbe addressed.

Universal, U.K.), as described previously (Bashir and Collingridge,1994). Briefly, animals were anaesthetized with halothane and decapitated. The brain was rapidly removed and placed in ice-cold artificial cerebrospinal fluid (aCSF; bubbled with 95% 0~5% C02), which comprised (mM): NaCl, 124; KC1, 3; NaHC03, 26; NaH2P04, 1.25; CaC12,2; MgS04, 1; D-glucose, 10. Transverseslicesof hippocampus(400 ~m) were cut on a vibroslice (Campden Instruments) and stored in aCSF. After removing the CA3 region, individual slices were placed in a submergedrecordingchamber (28–30”C,flow rate w 2.5 ml/min). Standard techniques were used to record fieid excitatory postsynaptic potentials from the stratum radiatum in response to stimulation (20 ~sec, 3– 10 V) of the Schaffer collateral-eommissural pathway (SCCP). The stimulus intensity was set to evoke responses (between 0.5 and 1 mV) which were roughly 1/3 of the magnitude at which a population spike started to appear. Two separate sets of SCCP fibres were alternately stimulated (each at 0.033 Hz). Evoked responses were monitored on-line and re-analyzed offline using software developedby Dr W. Anderson (Dept of Anatomy, Universityof Bristol, U.K.). Low frequency stimulation (LFS; 900 stimuli, 1 Hz, test intensity) was delivered in an attempt to induce LTD. When a paired pulse paradigm was used, two consecutive stimuli were

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Fig. 1. The induction of LTD in the CA1 region of the in vitro adult rat hippocampus by paired pulse stimulation protocols. (A) Paired pulses (450) result in the inductionof LTD which is reversibly blocked by the NMDA receptor antagonist, AP5. Both the first (.) and the second (0) of the pair of responses exhibited LTD. Each of the first and second of the pair is normalized to its own pre-LFSbaseline level. (B) Paired pulses (900)result in the inductionof LTD in the presence of AP5. (C) In a separate series of experiments, 900 paired pulses in the absence of AP5, induced LTD which was significantly larger than that in the presence of AP5. In both (B) and (C), only the first of the pair of responses is illustrated. The synaptic responsesillustrated are averagesof four consecutiveresponses

andarerepresentative examplestakenfromthetimesindicated. Controland depressedresponses(only first of the pair) are shownsuperimposedon a fastertimebase.Eachpointon the graph (except duringLFS)is the averageof fourconsecutive responses.During LFS, 10 (A) or 20 (B,C) consecutive responsesare averaged.For all experimentsonly the homosynapticpathwayis illustrated.

delivered with a 50 msec inter-stimulus interval during baseline recording and during LFS. Any changes in synaptic strength were expressed relative to the normalized pre-LFS baseline. Pooled data is expressed as mean + SEM and significance tested using Student’s ttests. “n” values refer to the number of times an experiment was conducted; each experimentwas carried out on a differentslice taken from a differentanimal.D-2-

amino-5-phosphonopentanoate(AP5; Tocris Neuramin), when used, was applied by perfusion to the bathing medium at a concentrationof 50 pM. As reported previously (Bashir and Collingridge, 1994), low frequency stimulation (900 stimuli, 1 Hz) that is typicallyused to induce LTD, failed to do so in the CA1 region of the adult rat hippocampus in vitro (–4 + 3%, n =6, p >0.05, data not shown). We investigated whether paired pulse stimulation (50 msec inter-stimulus interval) during LFS could induce LTD. During the baseline period, two consecutive stimuli resulted in facilitation (paired pulse facilitation) of the second response of the pair, relative to the first response (facilitation ratio 1.8 t 0.03, n = 7). In these experiments, 450 pairs of stimuli delivered at 1 Hz did not result in LTD in the presence of the NMDA receptor antagonist, AP5 (–4 f 3’%0and –5 t 390, first and second response, respectively; n = 7, p >0.05 for both, Fig. 1A). Following washout of AP5, the same stimulus protocol resulted in the inductionof LTD (17 ~ 4% and 16 ~ 4%; n = 5, p c 0.05, Fig. la). LTD was homosynaptic since there was no effect on the heterosynaptic pathway which did not receive LFS (data not shown). Increasing the number of paired stimuli during LFS (900 pairs) resulted in the induction of LTD in the presence of AP5 (23 t 4% and 18 + 4%, first and second response, respectively; n = 8, p e 0.05, Fig. IB). In a separate series of experiments,the same paired pulse protocol (900 pairs) resulted in the induction of LTD in the absence of AP5 (35 t 3% and 27 + 3; n = 6, p <0.05, Fig. IC). LTD, induced in the absence of AP5, of both of the pair of responses was significantly greater (p< 0.05) than that in the presence of AP5. The induction of LTD, using the standard 900 stimuli at 1 Hz, in the adult hippocampus in vitro has proved somewhatproblematic. Some reports have demonstrated its induction (Dudek and Bear, 1992; Staubli and Ji, 1996)whilst others,under normal conditions,have failed to do so (Bashir and Collingridge, 1994; Wagner and Alger, 1995;Norris et al., 1996).Thiels et al. (1994) and Doy&e et al. (1996) have previously demonstrated the inductionof LTD using a paired pulse paradigm(25 msec paired pulse interval)in adult rat CA1 in vivo. In order to induce LTD, Thiels et al. (1994) used protocols which resulted in paired pulse depression of population spikes and facilitation of field EPSPS, whilst Doy&e et al. (1996)used protocolsresultingin paired pulse depression of field EPSPS. We have now used a paired pulse paradigm, which produced paired pulse facilitation,to induce LTD. Since, in our hands, 900 single stimuli delivered at 1 Hz did not induce LTD, but 450 paired stimuli induced NMDA receptor-dependentLTD, it is likely that the paired pulse paradigm induces LTD because it activates the NMDA receptor system more efficiently than single stimuli. It has previously been suggested that increasing NMDA receptor activation facilitates the induction of LTD (Wagner and Alger, 1995; Kerr and Abraham, 1995).

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Nine hundred paired stimuli resulted in the induction of methyl-11 -aspartame receptorblockade.Proc. Nutn.Acad. Sci. U.S.A. 89: 4363-4367. LTD which was roughly twice the magnitude as that induced by 450 paired stimuli and was only partly KerrD.S. andAbrahamW. C.(1995)Cooperativeinteractions amongafferentsgoverntheinductionof homosynapticlongblocked by AP5. This indicates that both NMDA termdepressionin the hippocampus. Proc. Natn. Acad. Sci. receptor-dependent and NMDA receptor-independent U.S.A. 92: 11637–11641. mechanisms can underlie LTD induced by this means. Acknowledgement—Supported by the Wellcome Trust.

REFERENCES

Bashir Z. I. and Collingridge G. L. (1994) An investigation of depotentiationof long-termpotentiationin the CA1 region of the hippocampus.Exper. Brain Res. 100: 437-443. Bear M. F. and AbrahamW. C. (1996)Long-termdepressionin hippocampus.Ann. Rev. Neurosci. 19: 437-462. Doy&e V., Errington M. L., Laroche S. and Bliss T. V. P. (1996) Low-frequencytrains of paired stimuli induce longterm depression in area CA1 but not in dentate gyrus of the intact rat. Hippocampus 6: 52–57. Dudek S. M. and Bear M. F. (1992) Homosynapticlong-term depression in area CA1 of hippocampus and effects of N-

Mulkey R. M. and Malenka R. C. (1992) Mechanisms ., underlyinginduction of homosynapticlong-term depression in area CA1 of the hippocampus.Neuron 9: 967–975. Norris C. M., Korol D. L. and Foster T. C. (1996) Increased susceptibilityto inductionof long-termdepression and longterm potentiation reversal during aging. J. Neurosci. 16: 5382–5392. Staubli U. V. and Ji Z. X. (1996) The induction of homo- vs heterosynapticLTD in area CA1 of hippocampalslices from adult rats. Brain Res. 714: 169–176. Thiels E., BarrionuevoG. and Berger T. W. (1994) Excitatory stimulation duringpostsynapticinhibition induces long-term depression in hippocampus in vivo. J. Neurophysiol. 72: 3009-3016. Wagner J. J. and Alger B. E. (1995) GABAergic and developmental influenceson homosynapticLTD and depotentiation in rat hippocampus.J. Neurosci. 15: 1577–1586.