BRAIN RESEARCH

ELSEVIER Brain Research 667 (1994) 115-117

Short communication

LTP in the dentate gyrus is associated with a persistent NMDA receptor-dependent enhancement of synaptosomal glutamate release

Laura Canevari *, Gal Richter-Levin, T.V.P. Bliss Division of Neurophysiology and Neuropharmacology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW71AA, UK

Accepted 4 October 1994

Abstract

LTP was induced unilaterally in the dentate gyrus of the anaesthetized rat by tetanic stimulation of the perforant path. Two hours after tetanization synaptosomes were prepared from control and potentiated sides, and the depolarization-induced release of endogenous amino acid neurotransmitters was compared in the two groups, using 4-aminopyridine, potassium or veratridine as depolarizing agents. Glutamate release was greater in synaptosomes from potentiated than control tissue with all three depolarizing agents, but the highest release from control synaptosomes and the most significant enhancement of release from potentiated synaptosomes was seen with veratridine. The tetanus-induced enhancement of veratridine-induced glutamate release was prevented by pretreatment with MK-801. The results indicate that the induction of LTP in the dentate gyrus is accompanied by a persistent enhancement in the ability of synaptosomes to release glutamate in response to a depolarizing stimulus.

Keywords: Long-term potentiation; MK-801; 4-Aminopyridine; Potassium; Veratridine

A persistent increase in the concentration of gluta- mate in the extracellular compar tment following the induction of LTP has provided one of the principal sources of evidence that presynaptic mechanisms are involved in the maintenance of LTP in the dentate gyrus [1,5]. Corroborative evidence has been obtained from ex vivo studies in which the depolarization-in- duced release of labelled glutamate was measured in slices of dentate gyrus prepared from anaesthetized rats 45 min or more after the unilateral induction of LTP [3,6,9,10]. However, these latter experiments mon- itored exogenous pre-loaded radioactive glutamate which might also label a pool not involved in neuro- transmission [7]. Moreover, the use of the slice prepa- ration left open the question of the pre- or postsynap- tic locus of the increase in release. In the present work, we have refined the evidence from the ex vivo model by demonstrat ing an increase in depolarization-in- duced release of endogenous neurotransmit ter follow- ing the induction of LTP in vivo; moreover, using a synaptosomal preparation, we have provided further

* Corresponding author. Fax: (44) (81) 906-4477.

0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)01172-9

evidence for the presynaptic locus of the enhanced release. We have also verified, using the selective non- competitive N M D A receptor antagonist, methyl-10- 11-dihydro-5-H-dibenzocyclohepten-5,10-imine (MK- 801), that the activation of N M D A receptors is neces- sary for the persistent increase in glutamate release.

Male Sprague-Dawley rats (250-350 g, bred at NIMR) were anaesthetized with urethane (1.2 g /kg , i.p.) and implanted unilaterally with a bipolar stimulat- ing electrode (two insulated and twisted 125 ~ m O nichrome wires; Goodfellow) into the angular bundle (4.0 mm lateral, 8.0 mm posterior to bregma) and a recording electrode (insulated nichrome wire, 125/zm O) into the hilar region of the dentate gyrus (2.5 mm lateral, 4.0 mm posterior to bregma). The position of the electrodes was adjusted to maximize the slope of the positive-going field EPSP; test stimuli (monopolar pulses, duration 50 /xs, intensity adjusted to yield a population spike 20-40% of maximal value) were de- livered at 0.033 Hz throughout the experiment. After the field potential had stabilized, baseline responses were collected for 10 min. LTP was then induced by a tetanus protocol designed to produce maximal potenti- ation (6 trains of 8 pulses at 400 Hz, duration 150 Izs,

116 L. Canecari et aL /Brain Research 067 (1994) 115 117

inter-train interval 4 min, intensity as test stimulus) and potentials recorded for 1 h from the first train of stimuli. Responses were filtered (3 dB points at 1 and 3 kHz), digitized at 10 kHz and stored to disk. The magnitude of LTP was expressed for each animal as the mean percentage increase for the last 10 responses recorded at the end of the first hour after the tetanus, relative to the mean value during the baseline record- ing period. Stimulus timing, data collection and analy- sis were controlled by A/Dvance software (Fine Sci- ence Instruments, Vancouver) on a Macintosh Si with a National Instruments LabNB interface board. Ani- mals were maintained at 37°C by a homeothermic blanket system (Harvard) throughout the experiment; 2 h after the tetanus, animals were decapitated and whole dentate gyri from the non-potentiated and po- tentiated sides were dissected for preparation of synap- tosomes [8]. Synaptosomal suspensions were incubated at 30°C in Krebs-Henseleit-HEPES (with 10 mM glu- cose and 1.2 mM CaCI 2) medium alone or in the presence of the selected depolarizing agent for 15 min; the extrasynaptosomal fluid was then separated from the particulate fraction, deproteinized, neutralized and subjected to amino acid analysis by HPLC-fluorometry ([4], modified); release was calculated as the difference in concentration between basal and depolarized states. Differences between control and potentiated sides (% change) were assessed by t-test. In some animals, MK- 801 (2 mg/kg i.p.) was injected after stabilization of the baseline, and test stimulation and recording contin- ued for 35 min before the tetanus was delivered. Pre- liminary experiments established that release from ve- hicle injected animals was not different from naive animals (results not shown).

In initial experiments the ability of three different depolarizing agents to induce neurotransmitter amino acid release were evaluated: 4-aminopyridine (AP-4; 5 raM), KC! (40 mM) and veratridine (veratrine; 50/zM). All three agents stimulated the release of glutamate, aspartate and y-aminobutyric acid (GABA). The largest and most consistent effect was obtained with veratri- dine, which led to the release of about 5 times as much of each of the three amino acids as 4-AP and about twice as much as KC1 (Table 1); with a single excep- tion, in every animal (n = 5 for 4-AP, n = 6 for KCI and veratridine), the release of glutamate was greater in synaptosomes from the potentiated side than it was from with the non-potentiated side (the exception was in the 4-AP group). The most significant difference in glutamate release between potentiated and non-poten- tiated side tissue was obtained with veratridine (% increase on the potentiated side: 22.9%, P = 0 . 1 5 ; 18.49%, P = 0.01 and 19.2%, P = 0.0004 for 4-AP, KC1 and veratridine respectively; t-test). In contrast, LTP was not associated with a significant increase in either aspartate or GABA release from the potentiated side

Table 1

Release of glutamate (Glu), aspartate (Asp) and GABA from synap- tosomes from rat dentate gyrus subjected to depolarization by 4- aminopyridine (4-AP, 5 mM), KCI (40 raM) and veratridine (50/xM)

Glu Asp GABA

4-AP 3.65±1.17 0.74±0.18 1.08±0.96 KC1 11.11±3.13 3.83_+0.94 3.40+0.58 Veratridine 19.86+-4.87 8.79± 1.9(I 9.25 + 1.53

Values are the differences ( n m o l / m l / m g protein) between depolar- ized and basal synaptosomes; means +- S.E.M. from n = 5 (4-AP) or n = 6 (KCI, veratridine) experiments.

relative to the non-potentiated side (12.33 +9.75%, P > 0.2 for aspartate, and 6.91 + 12.57%, P > 0.6 for GABA) (Table 2).

To assess the involvement of the NMDA receptor in the tetanus-induced enhancement of release we exam- ined the effect of blocking the induction of synaptic LTP by MK-801. The drug had no effect on baseline EPSP slope (responses before injection of MK801 and 35 min after injection, immediately before the tetanus were: - 0 . 2 + 4.2% and 0.5 + 0.8% of baseline, respec- tively; Fig. 1A). However, synaptic LTP (measured as the % increase in EPSP slope 1 h after the induction of LTP) was significantly reduced by MK-801 (untreated vs. MK-801: P<0.0001 , unpaired t-test, Fig. 1A). MK801 had no effect on release from the control side (14.88 + 0.93 and 15.35 + 0.35 n m o l / m l / m g protein untreated and treated, respectively; P = 0.67). The in- crease in glutamate release observed in untreated ani- mals (14.88 _+ 0.93 to 17.74 _+ 1.13 n m o l / m i n / m g pro- tein P = 0.0008, paired t-test) was completely abol- ished by the drug (15.35 + 0.35 to 15.03 _+ 0.86, P = 0.71 n m o l / m l / m g protein) (Fig. 1B).

These results strengthen the link between NMDA receptor-dependent LTP and presynaptic mechanisms controlling the release of the receptor's endogenous agonist, glutamate. MK-801 reduced LTP most proba-

Table 2 Mean % increase in release of glutamate (Glu), aspartate (Asp) and GABA from synaptosomes prepared from potentiated dentate gyrus compared to release from synaptosomes from the non-potentiated side

Glu Asp GABA

4-AP 22.90 + 12.99 4.99 +- 27.12 7.36 +- 20.7 P = 0.152 P = 0,86 P = 0.73

KCI 18.49+_ 4.48 18.97+_ 9.34 12.32_+19.38 P = 0.009 P = 0,098 P = 0.55

Veratridine 19.20+_ 2.34 12.33_+ 9.75 6.91+-12.57 P = 0.0004 P = 0.22 P = 0.60

Synaptosomes were subjected to depolarization by 4-aminopyridine (4-AP; 5 mM), KCI (40 mM) or veratridine (50 /xM). Values are means+-S.E.M, from n = 6 experiments. P values were obtained from the t-distribution on the null hypothesis of equal release from both sides.

L. Caneuari et al. / Brain Research 667 (1994) 115-117 117

02, (n

..Q

"6

v

t~ 0.0

co o_

n uJ

A

100-

80"

60'

4 0

2 0

0

-20

o untreated

2 0 4 0 6 0 8'0

Time (min)

460

20 ¸

B .k [ ] control • LTP

¢9 t~ ~- 15

c~ E

t~ E I O E~ t~ E

(.9 5

0 Untreated MK-801

Fig. 1. The NMDA receptor antagonist MK-801 significantly reduces the induction of LTP, and the tetanus-induced increase in glutamate release. A: effect of treatment with MK-801 (2 mg/kg i.p.) on the EPSP slope. The filled arrow indicates the injection of the drug, and open arrows indicate trains of tetanic stimulation. B: glutamate release (nmol /ml /mg protein) from synaptosomes stimulated by veratridine (50 /xM) in untreated animals and in animals injected with MK-801. Values are means 5-S.E.M. from n = 5 (untreated) and n = 6 (MK-801) experiments. * P = 0.0008 potentiated vs. control sides, paired t-test.

bly by affecting postsynaptic NMDA receptors since it had no effect on baseline responses. The data are thus also consistent with the notion that a retrograde mes- senger is required to carry information back to recently

activated presynaptic terminals to activate mechanisms leading to a persistent increase in transmitter release [1,2].

This work was supported by grants from EEC (L.C.) and HFSP (G.R.-L.).

[1] Bliss, T.V.P., Douglas, R.M., Errington, M.L. and Lynch, M.A., Correlation between long-term potentiation and release of en- dogenous amino acids from dentate gyrus of anesthetized rats, J. Physiol., 377 (1986) 391-408.

[2] Bliss, T.V.P., Errington, M.L., Fazeli, S., Li, Y.-G., Murphy, K.P.S.J. and Williams, J., The role of intercellular messengers in long-term potentiation. In P. Andersen, O. Hvalby, O. Paulsen and B. H6kfelt (Eds.), Memory Concepts - 1993; Basic and Clinical Aspects, Elsevier, Amsterdam, 1993, pp. 163-172.

[3] Bliss, T.V.P., Errington, M.L., Laroche, S. and Lynch, M.A., Increase in K+-stimulated, caa÷-dependent release of [3H]glutamate from rat dentate gyrus three days after induction of long-term potentiation, Neurosci. Lett., 83 (1987) 107-112.

[4] Canevari, L., Vieira, R., Aldegunde, M. and Dagani, F., High performance liquid chromatographic separation with electro- chemical detection of amino acids focusing on neurochemical application, Anal. Biochem., 205 (1992) 137-142.

[5] Dolphin, A.C., Errington, M.L. and Bliss, T.V.P., Long-term potentiation of the perforant path in vivo is associated with increased glutamate release, Nature, 297 (1982) 496-498.

[6] Errington, M.L., Lynch, M. A. and Bliss, T.V.P., Long-term potentiation in the dentate gyrus: induction and increased gluta- mate release are blocked by D-(-)-aminophosphonovalerate, Neuroscience, 20 (1987) 279-284.

[7] Ferkany, J.W. and Coyle, J.T., Evoked release of aspartate and glutamate: disparities between prelabelling and direct measure- ment, Brain Res., 278 (1983) 279-282.

[8] Lynch, M.A. and Voss, K.L., Arachidonic acid increases inositol phospholipid metabolism and glutamate release in synapto- somes prepared from hippocampa[ tissue, J. Neurochem., 55 (1990) 215-221.

[9] Lynch, M.A., Errington, M.L. and Bliss, T.V.P., Long-term potentiation of synaptic transmission in the dentate gyrus: in- creased release of [14C]glutamate without increase in receptor binding, Neurosci. Left., 62 (1985) 123-129.

[10] Lynch, M.A., Errington, M.L. and Bliss, T.V.P., The increase in [3H]glutamate release associated with long-term potentiation in the dentate gyrus is blocked by commissural stimulation, Neu- rosci. Lett., 103 (1989) 191-196.