Increase in glutamate receptors following repetitive electrical stimulation in hippocampal slices

Life Sciences, Vol. 27, pp. 325-330 Printed in the U.S.A.

Pergamon Press

INCREASE IN GLUTAMATERECEPTORSFOLLOWINGREPETITIVE ELECTRICAL STIMULATION IN HIPPOCAMPALSLICES M. Baudry, M. Oliver, R. Creager, A. Wieraszko* and G. Lynch Department of Psychobiology University of California Irvine, California 92717 and *Department of Biochemistry of Nervous System and Muscle Nencki Institute of Experimental Biology Warsaw, Poland (Received in final form May 27, 1980)

Summary Repetitive electrical stimulation of identified pathways in the hippocampal slice preparation induces long-term potentiation (LTP) of synaptic transmission a~d is accompanied by a long-lasting (up to 30 minutes) increase in L- H-glutamate accumulation by the slices. This increased accumulation of H-glutamate is restricted to the terminal field of the stimulated fibers and does not seem to represent a non-specific increased accumulation of amino acids. In addition, synaptic membranes prepared from stimulated slices exhib i t an increase in the maximal n~mber of the sodium-independent high-affinity binding sites for H-glutamate without changes in their a f f i n i t y . These results suggest that repetitive electrical stimulation e l i c i t s an increased number of glutamate receptors which might be responsible for LTP. Long-term potentiation (LTP) is an extremely persistent increase in synaptic transmission found in the hippocampal formation following brief bursts of high frequency stimulation delivered to its various fiber projections (for a review, see I). Experiments from several laboratories have provided strong evidence that the alterations responsible for LTP are not to be found in the stimulated axons or their target dendrites, indicating that the substratum of the effect is in one or more of the components of the synapses (viz. terminals, spines, etc.) (2,3,4). Glutamate is l i k e l y to be the neurotransmitter in several hippocampal pathways (5) and we have previously shown that the number of Na-independent glutamate binding sites in hippocampal membranes, which exhibit properties of postsynaptic receptors (6), is quite labile and in particular is increased by low concentrations of calcium (7). Since the induction of LTP is dependent upon adequate levels of extracellular calcium (8), these results prompted us to suggest that long-term potentiation might be caused by an increase in glutamate receptors in the stimulated synapses (7). We now report that LTP is accompanied by an increase in H-glutamate binding in hippocampal slices, as well as by an increased number of Hglutamate binding sites in membranes prepared from stimulated slices.

0024-3205/300325-06502.00/0 Copyright (c) 1980 Pergamon Press Ltd

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Methods Rat hippocampal slices were prepared as previously described (9). B r i e f l y , hippocampi were quickly removed following decapitation and cut into 0.5 mm slices on a Mcllwain tissue chopper. Slices were placed on nets in a chamber which contained Krebs-Ringer bicarbonate buffer consisting of: NaCl, 121 ~ ; KCI, 5 ~4, KHpP04, 1.25n~I; MgSO~, 1.3 mM; CaClp, 3.1 mM; NaHCO:, 25.7 mM and G~ucose, ~0 ~4, oxygenated w~th a mixture o~ 0 :CO2 (95:5) ~nd kept at 33-35vC. Usually adjacent slices were matched int~ pa~rs, one serving as the experimental slice the other as the control. Stimulating electrodes were placed at opposite ends of the s. radiatum in order to activate two populations of Schaffer collateral-commissural (S-C) fibers of the regio superior while a recording micropipette was placed between them in dendritic zones innervated by both projections. After ascertaining that typical synaptic potentials were obtained in response to single pulse stimulation of each electrode, brief high frequency trains (3-4 trains at 100 sec- for I sec. each) were delivered to both pathways with the duration and voltage of the pulses increased so as to activate the maximum possible number of fibers. This stimulation paradigm t y p i c a l l y increased the slope of the control dendrit i c response by 40-120% when tests with single pulses were carried out 5-30 minutes after the train (for stimulation parameters see ref. 4). At various times after the stimulation, s~ices were removed in pairs from the chamber and incubated for I0 minutes at 37 C in 0.5 ml ~f freshly oxygenated medium in the presence of v a r i o ~ radioactive1~ompounds ( H-~lutamate, H-aspartate, New England Nuclear, C-tyrbsine, C-aspartate, H-GABA, Amersham). The incubation was stopped by pouring the slices on a paper f i l t e r under vacuum and washing of the slice with 5 ml of the incubation medium at room temperature. The slice was then homogenized in 0.3 ml of d i s t i l l e d water by sonication and aliquots were used to determine total r a d i o a c t i v i t y and protein content. Under these conditions TLC chromatography indicate~ that more than 90% of the accumulated r a d i o a c t i v i t y migrated l i k e authentic H-glutamate in the following solvent system: butanol: acetic acid: water (25:4:10). In order to measure characteristics of h i g h - a f f i n i t y 3H-glutamate binding sites in hippocampal membranes, several slices (control or potentiated) were pooled together and homogenized in 2 ml of cold 0.32 M sucrose. Membranes were prepared and immediately assayed for the binding of various concentrations of L- H-glutamate in the absence of sodium as previously described (6,7). The saturation curves were analyzed with a MINC-11 computer following the method of Parker and Waud (I0). Results Althou~h no s i g n i f i c a n t changes were detected when the slices were incubated with H-glutamate immediately after the stimulation, a sizable increase ("potentiated" versus control; +20% ± 4,% mean ± S.E.M. of 34 experiments) was found in the slices removed 7 minutes after the high frequency trains. A similar difference between potentiated and control slices (+18% ± 4%, mean ± S.E.M. of 18 experiments) was also evident 30 minutes after the stimulation (Fig. i ) . Since the terminal fields of the stimulated pathways are mainl~ localized to the regio superior we tested for changes in the accumulation of H-glutamate, following r e p e t i t i v e stimulation, in both the regio superior and the dentate gyrus. In similar experiments, we also measured ~he accumulation of L-~-aspartate and in some cases, simultaneously that of H-glutamate3and L- C-aspartate. As shown in Table I, the increased accumulation of H-glutamate was restricted to the regio superior with no s i g n i f i c a n t effect in the

Vol. 27, No. 4, 1980

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Glutamate Receptors and LTP

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TIME AFTER STIMULATION(mln)

FIGURE 1

At various times ~fter r e p e t i t i v e electric~l stimulation, the slices were incubated with L- H-glutamate (50 nM) and H-glutamate accumulation was ~easured as described under Methods. Results were calculated in pmoles of H-glutamate accumulated per mg of protein. The amount of H-glutamate in stimulated slices was expressed as percentage of that accumulated in the matched, non-stimulated control slices. The hatched area represents the S.E.M. of the control slices. Mean ± S.EoM. of 16-34 experiments (*p
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percentage of labelled amino acid was localized to i n t r a c e l l u l a r compartments. Glutamate uptake is Na-dependent ( I I ) and we used this to make an estimate of the amount of uptake exhibited by the slices. Incubation in sodium-free medium decreased glutamate accumulation by about 30% (2.60 ± 0.06 pmol/mg protein versus 3.47 ± 0.17 pmol/mg protein; mean ± S.E.M. of 10 experiments); t h i s r e l a t i v e l ~ low level of uptake probably r e f l e c t s the fact that the concentration of H-glutamate used in the assay is well below the Km of the uptake system (12). Further evidence that the whole s l i c e assay does detect s i g n i f i c a n t levels of binding comes from experiments in which the commissural afferents were destroyed by unilateral hippocampal a s p i r a t i o n ; under these circumstances h i g h - a f f i n i t y uptake into homogenates of the intact hippocampus was reduced by 30% but there was no reduction in accumulation into slices (Michel Baudry, unpublished r e s u l t s ) . Recently we reported the existence in hippocampal membranes of Na-independent L- H-glutamate binding sites which e x h i b i t several characteristics expected of postsynaptic glutamate receptors (6 and Baudry and Lynch, submitted). Therefore we measured H-glutamate binding in membranes3Prepared from control and stimulated slices and found that the binding of H-glutamate at a concentration of i00 nM was s i g n i f i c a n t l y increased by 45% (0.72 ± 0.16 pmol/mg protein in control vs. 1.04 ± 0.12 pmol/mg protein in stimulated, TABLE I Changes in Accumulation of 3H-Glutamate and 3H-Aspartate in Hippocampal Slices Following LTD Regio Superior

Dentate Gyrus

3H-glutamate (11)

+25% ~ 10%*

+6,,~ oi z 8%N.S.

3H-aspartate (11)

+22% ± 8%*

-7% ± 7%N'S"

3H-glutamate/14C-Aspartate

2% ~ 2%

-2% ± 2%

Hippocampal slices were prepared and e l e c t r i c a l l y stimulated as described in Methods. Seven minutes following r e p e t i t i v e ~ l e c t r i c a l stimulation t~ey were transferred into incubation tubes c~ntaining ~H-glutamate (5~4nM) or Haspartate (125 nM) or a mixture of H-glutamate (50 riM) and C-aspartate (200 nM). A f t e r a I0 minute incubation they were placed on a paper f i l t e r and the dentate gyrus was dissected free. Both regions were then processed as described in Methods. Results were calculated as pmol/mg protein and were expressed as percent increase over the nonstimulated, matched control s l i c e s . Number of experiments in parentheses (*p
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TABLE 11 Characteristics of 3H-glutamate Binding in Membranes Prepared from Control or Stimulated Slices Bmax

Kd

Hill

(pmol/mg prot.)

(nM)

Coefficient

Control

2.57 ± 0.26

288 ± 73

1.18 ± 0.07

Stimulated

3.37 ± 0.39*

299 ± 74N'S"

0.97 ± 0.08 N'S"

(+30%) Hippocampal slices were prepared and e l e c t r i c a l l y stimulated as described in Methods. Seven minutes following electrical stimulation they were transferred to cold 0.32M sucrose. 3-4 slices were pooled together and homogenized in 2 ml of 0.32M sucrose and hippocampal membranes were ~repared and immediately assayed for the binding of various concentrations of H-glutamate. Mean ± S.E.M. of 7 experiments (*p
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The above results suggest an explanation for the remarkably stable synapt i c f a c i l i t a t i o n which occurs after r e p e t i t i v e stimulation in hippocampus. The changes in glutamate binding appear within minutes of the stimulation t r a i n and are quite prominent 30 minutes l a t e r . The biochemical effects thus exhibit the temporal parameters required for the substrates of LTP. With regard to the mechanisms by which the stimulation produces i t s effects on glutamate binding, the number of sodium-independent glutamate binding sites in hippocampal membranes is markedly and i r r e v e r s i b l y increased by modest levels of calcium (10-100 ~M) (7). Furthermore, low temperatures, reducing, and alkylating agents, as well as a number of protease i n h i b i t o r s , all i n h i b i t basal glutamate binding as well as the effects of calcium (15). These results suggest that a calcium sensitive thiol-protease is present in hippocampal membranes, the a c t i v i t y of which "uncovers" binding sites which otherwise would be inaccessible to glutamate. Possibly then, the calcium influx into the dendrites (16,17) reaches s u f f i c i e n t magnitude during r e p e t i t i v e stimulation, that the postulated calcium-sensitive protease is activated and additional receptor sites become exposed. This could occur in a matter of minutes (or less) and could provide an increase in synaptic transmission for the l i f e of the additional receptors. Ac knowl edgments This work was supported by NIMH grant MH-19793 and a Career Development Award, NS-00043 to G.L. We wish to thank Darlene Fujimoto-Thompson for typing the manuscript. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. II. 12. 13. 14. 15. 16. 17.

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