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Relative potency of analogues of excitatory amino acids on hippocampal CA1 neurons
Neuropharrnacology Vol. 22, No. 12A, pp. 1343 1348, 1983
0028-3908/83$3.00+0.00 Pergamon Press Ltd
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RELATIVE POTENCY OF ANALOGUES OF EXCITATORY AMINO ACIDS ON HIPPOCAMPAL CA1 N E U R O N S T. H. LANTHORN* and C. W. COTMAN Department of Psychobiology, University of California, Irvine, CA 92712, U.S.A.
(Accepted 10 April 1983) Summary--The relative potency of analogues of excitatory amino acids to produce depolarization when applied in the apical dendritic field of CA1 cells was studied in the hippocampal slice. The effect of these compounds was measured by recording focal potentials (FPs), the shift in the extracellular d.c. potential produced by the compounds applied. The ability of focal potentials to measure neuronal responses was evaluated. N-methyl-D-aspartate (NMDA)-type agonists were 10-20 times more potent, relative to Lglutamate, than reported from investigations in spinal cord. Quisqualate (QA), + ~-amino-3-hydroxy-5methyl-4-isoazolepropionicacid (AMPA) and kainate (KA) exhibited potencies on CA1 cells similar to those reported for spinal neurons. These data indicate that elements in CA1 cells possess a receptor with an affinity for N-methyl-D-aspartate-type agonists. Some putative antagonists were found to induce negative focal potentials suggestinga direct, excitatory action in this area. Key words: excitatory amino acids, hippocampal slice,CA1 cells, focal potentials.
It has been suggested that glutamate, aspartate or a related compound serves as the transmitter at Schaffer collateral/commissural synapses in area CA1 of the hippocampus (Dudar, 1974; Nadler, White, Vaca, Perry and Cotman, 1980; Schwartzkroin and Andersen, 1975; Storm-Mathisen, 1977; White, Nadler and Cotman, 1979; Wieraszko and Lynch, 1979). However, the type of amino acid receptor used is unknown. Recently, it has been found that quisqualate, N-methyl-D-aspartate, and kainate antagonists (Davies and Watkins, 1979; Evans, Francis, Hunt, Oakes and Watkins, 1979; Watkins, Davies, Evans, Francis and Jones, 1980) are all relatively weak antagonists at Schaffer/ commissural synapses (Koerner and Cotman, 1983) making identification of the receptor uncertain. A large number of agonists exist which might give clues to the structural characteristics of the natural transmitter receptor. In this study, the actions of several transmitter agonists and antagonists on the hippocampal slice were investigated. The effect of these compounds was measured by recording the shift in the extracellular d.c. potential induced by the compounds. This shift in d.c. potential has been investigated in the spinal cord by Flatman and Lambert (1979). They found that excitatory agents induced a negative shift in the extracellular d.c. potential which, except for being opposite in sign, closely corresponded to the intracellularly recorded d.c. shift. Norepinephrine sometimes induced a positive d.c. shift corresponding to its weak hyperpolarizing action. These potentials, *Send correspondence to: Dr Thomas H. Lanthorn, Building 10, Room 5C106, National Institutes of Health, Bethesda, MD 20205, U.S.A.
which Flatman and Lambert called focal potentials (FPs), are probably analogous to synaptic field potentials except that they are induced by substances applied rather than by substances released from nerve endings. Lambert, Flatman and Jahnsen (1981) have also observed focal potentials using the hippocampal slice preparation. The present authors used focal potentials because they are easy to record and appear to measure the direct effect of compounds on neurons. However, since focal potentials have not been used extensively, their ability to reflect changes in membrane polarization was evaluated. METHODS
Slices (350-400/zm thick) were prepared from the hippocampus of male, Sprague-Dawley rats, 45-60 days old. The slices were placed in a chamber previously described (Spencer, Gribkoff, Cotman and Lynch, 1976; White, Nadler and Cotman, 1978) and incubated in a medium containing 124mM NaC1, 3.3raM KC1, 1.2mM KH2PO4, 25.4mM NaHCO3, 2.5raM CaCl2, 2.4raM MgSO4 and 10 mM D-glucose. The slices were superfused with a humidified atmosphere of 95% 02-5% CO2, at a temperature of 31-33°C. Focal potentials and action potentials were recorded with glass pipettes filled with 2 M NaCI and having a resistance of 4-10MO. Direct current (d.c.)-coupled signals were amplified and displayed in a conventional manner. All drugs were dissolved in the same medium as used to support the slices and the solutions were brought to a pH of 7.4 +_ 0.05. Pipettes with their tips broken back to about 50#m were filled with drug solutions and lowered onto the slice until a fluid bridge was created between the two. Application was
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T.H. LANTHORr~and C. W. COTMAN
terminated by raising the pipette from the slice. Drugs were applied for approx. 1 sec unless otherwise noted. To determine the potencies of the amino acid analogues, an ascending series of concentrations of drug (at most three) was applied to one site on each slice. D-~-Aminoadipate (D-AA), t-~-aminopimelate (LAP), and D- and t-~-aminosuberate (D- and L-AS) were gifts from Dr J. C. Watkins. +-Cis-l-amino1,3-dicarboxycyclopentane (ADCP) was a gift from Dr H. McLennan. Quisqualate (QA) was a gift from Dr H. Shinozaki and ibotenate (Ibo) from Dr C. H. Eugster. The _ ~-amino-3-hydroxy-5-methyl-4isoazolepropionic acid (AMPA) was a gift from Dr P. Krogsgaard-Larsen. fl-(p-Chlorophenyl)-GABA (baclofen) was a gift from CIBA-Geigy. All other compounds were obtained from commercial sources. Unless specifically stated, all compounds were applied to the stratum radiatum of CAI very close to a recording electrode from which a subthreshold (for population spike) Schaffer potential of 2.5-4.5mV could be recorded. RESULTS
Focal potentials As focal potentials have not been used extensively some examination of their characteristics in the hippocampus was necessary. Application of experimental saline produced little change (maximum deflection of - 0 . 0 5 m V ) in the d.c. potential unless the tip of the recording electrode was less than 25/~m
..~
(A)
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2mV
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(c)
1
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9
Fig. 1. Focal potentials recorded at different locations in CAI following application of L-glutamate (1 mM) to the stratum lacunosum-moleculare of CA1. Records 1 and 5 were recorded in the stratum lacunosum-molecular near the site of application of L-glutamate. Records 2~4 were recorded at sites 200, 400 and 600/~m from the site of application of L-glutamate towards and past the stratum pyramidale. Records 6-9 were recorded at sites in the stratum lacunosummoleculare 200, 400, 600 and 800pm from the site of L-glutamate's application. into the slice. When the tip was closer to the surface, application of the medium resulted in a larger negative deflection (about 0.1 mV) lasting for around 2 sec. This was probably due to the transient increase in medium around the electrode tip which may provide a current shunt, or to mechanical disturbance of the tip. In order to avoid this artifact, all recordings were made with the electrode tip lowered 75 tim into the slice. L-Glutamate and other depolarizing compounds applied to the surface of the slice produced a negative d.c. potential shift which rapidly returned to the preIB)
5sec |IL
~
3rnv
3
~'
lOsec Fig. 2. Correspondence between intrace]]ularly and extracellulady recorded responses to L-glutamate from 3 cells. ]ntracellular record is the top record of each pair. (A) Small concentration of L-glutamate producing a small intracellular response. (B) Suprathreshold response from a different cell. (C) Three responses from a third cell. Calibrations are the same for all three records. C1, subthreshold response; C2, suprathresho]d response; C3, two applications of L-glutamate showing additive responses in both intrace]lular and extracellular records. Because of slow recording speed, action potentials in B, C2 and C3 show up only as scatter in the records.
Potency of amino acids in hippocampus
(A)
Table 1. Relative potency of excitatory amino acid analogues 2mV
Compound
5~ec
IB)S
i
O.SmV
f---
Fig. 3. Focal potentials recorded following the application of transmitters to the stratum radiatum of CA1. (A) GABA, (B) 5-hydroxytryptamine, and (C) acetylcholine. Calibrations are the same in B and C. drug baseline after the application was terminated (Fig. 1). On many occasions, the d.c. potential transiently overshot the baseline. The negative deflection was often accompanied by a train of action potentials. Lengthening the period of application prolonged the time before the d.c. potential returned to baseline. Small increases in the duration of application of glutamate also resulted in an increased peak amplitude of the negative deflection, but after 5-10 sec of application a plateau was reached, and was maintained during applications for 30 sec. Except as noted
20
,...-it'--- ~
62 51 36 23 17 3.5 2.4 1.3
/
OAMPA
• L - H CA
r.-.-
note
@-o-glu
• QA
•O -HCA
~--L-g}u
•
•L-AP
~ --L--Cys
;72"
• ~ D - asp
/iY 10-5
./x;-.-,'/// • 10-4
Log
concentration
1
0.75 0.5 0.4 0.35 0.3 0.25
below, drugs could be applied several times to the same site with similar results. The size of the extracellular d.c. shift induced by a particular concentration of an amino acid analogue was correlated with the threshold amplitude of the Schaffer wave in that slice. When the threshold was
mV
"~
215 208 125 80
Potencies were estimated by extrapolating (from Fig. 1) the concentration of each analogue which would produce a 7mV negative focal potential (L-glutamate = I).
OADCP
>,
Potency
ADCP ( _+-cis- l-amino- 1,3-dicarboxycyclopentane) N-methyl-O,L-aspartate Ibotenate Quisqualate AMPA ( +
- ._
5sec
¢c1
1345
.y 10-3
(M)
•
of
Ip~ 10-2
applied
---ii L-glutamine/i O- I
drug
Fig. 4. Amplitude of focal potentials induced by analogues of excitatory amino acids in CA 1 as a function of the concentration of the applied compounds. Points are the mean of at least 4 trials on different slices; SEM was less than + 15% of the mean in all cases. The inset shows examples of the focal potentials produced by application of I 0 mM L-glutamate to three slices. The break in the bottom record was 5 min. Key to abbreviations: ADCP, +_-cis-l-amino-l,3-dicarboxycyclopentane; NMA, N-methyl-o,L-aspartate; QA, quisqualate; AMPA, _+-ct-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; KA, kainate; HCA, homocysteate; AP, ~t-aminopimelate; AS, ~-aminosuberate; glu, glutamate; asp, aspartate; THA, threo-3hydroxy-D,L-aspartate; cys, cysteate; AA, ct-aminoadipate; APP, D,L-2-amino-3-phosphonopropionate; APB, o,L-2-amino-4-phosphonobutyrate; DAPA, ct,e-diaminopimelate; GDEE, L-glutamic acid diethyl ester.
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T.H. LANTHORNand C. W. COTMAN
less than 2 mV, small negative shifts were seen which were often short and followed by large and longlasting positive shifts. In slices exhibiting such low thresholds variable responses were seen with repeated application. It is likely that the low thresholds and unstable responses to drugs were the result of depolarized or otherwise abnormal cells. Such responses were not included in this study. The size and shape of focal potentials in the hippocampal slice changed as the recording electrode was moved away from the site of application. These changes are demonstrated in Fig. 1 using the application of glutamate to the molecular layer of CA1. Records 1 and 5 are from the site of application of glutamate, while the others are from sites in progressive steps of 200 ~m away. When the recording electrode was moved along the main dendritic axis toward, and past, the cell body layer (records 1-4), the shape as well as the amplitude of the focal potential changed. In record 2, the duration of the negative portion was decreased relative to that seen at the site of application of glumate (record 1). At a greater distance from the application site (records 3 and 4), the negative focal potential was replaced by a positive wave with a negative deflection on it. The thickening of the potential record, especially prominent in record 3, was due to extracellularly-recorded action potentials. As the recording electrode was moved perpendicular to the main dendritic axis of CA1 neurons (records 5 9), the shape and time course of the focal potential stayed generally the same. Only the amplutide changed, decreasing with increasing distance from the site of application of glutamate. The changes seen in focal potentials recorded along the main dendritic axis of CA1 were similar to changes in synaptic field potentials observed in a laminar analysis of this area (Andersen, Holmqvist and Voorhoeve, 1966)--changes which reflect neuronal source-sink current flow. Figure 2 illustrates the temporal correspondence between extracellular and intracellular responses to application of glutamate. Records are shown from three CA I cells with resting membrane potentials of - 6 0 and - 7 0 m V , spike amplitudes (from threshold) of 69 to 80 mV and spike durations of 1.7 to 2.2 msec. In all three neurons, the extracellular focal potential closely followed the changes in membrane potential recorded intracellularly. Dendritic application of 7-aminobutyric acid (GABA) evoked a negative focal potential (Fig. 3A). This was consistent with its depolarizing action of CA1 dendrites (Alger and Nicoll, 1979; Andersen, Dingledine, Gjerstad, Langmoen and MosfeldtLarsen, 1980). Also GABA hyperpolarized the soma of CA I neurons, and the application of GABA to the cell body layer resulted in a positive-negative focal potential (data not shown; Lambert et al., 1981). Serotonin, which hyperpolarizes CA1 neurons (Segal, 1980; Jahnsen, 1980), consistently induced positive focal potentials (Fig. 3B). Postive focal
potentials were rarely greater than 2mV. Norepinephrine is reported to hyperpolarize CA1 neurons only slightly (Langmoen, Segal and Andersen, 1981; Segal, 1980). Applied to the apical dendritic field, it sometimes produced positive focal potentials, but most often produced no observable focal potential. Acetylcholine (ACh), applied to the apical dendritic field, evoked a positive focal potential (Fig. 3C). It has been reported that ACh is inhibitory in the dendritic field, probably via presynaptic inhibition (e.g. Valentino and Dingledine, 1981). Hyperpolarization has been reported, but was transient (Bernardo and Prince, 1981).
Effects of analogues of acidic amino acid To determine the effects of excitatory amino acid agonists and putative antagonists, these compounds were applied to the apical dendritic field of CAl. At least three concentrations of each compound were tested. The dose response relationships observed are portrayed in Fig. 4. N-methyl-D,L-asparate (NMA) and +_-cis-l-amino-l,3-dicarboxycyclopentane (ADCP) were the most potent compounds followed by ibotenate, quisqualate, +~-amino-3hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) and kainate. Homocysteate was less potent and exhibited little stereospecificity. L-~-Aminopimelate and L-~-aminosuberate were two to three times more potent than L-glutamate. D-Glutamate was about equal to L-glutamate. Slightly less potent were L- and D-aspartate, L-cysteate, L-~-aminoadipate and threo3-hydroxy-D, L-asparate. L-Glutamine, which is not an active depolarizing agent, evoked only a 0.1mV negative deflection. Other compounds which were less potent than Lglutamate (produced less than a I mV negative focal potential at a concentration of 10 mM) included D,L~-methylglutamate, methionine sulfoxide, methionine sulfone, methionine sulfoximine, 6aminolevulinic acid, D- and L-cysteine, L-ascorbate, ~-aspartylglycine and ~-aspartyl-/~-alanine. Three compounds exhibited excitatory activity, but were tested at only one concentration. A 10mM solution of D,L-~-methylaspartate evoked a 5.0_+0.4mV negative focal potential (Mn _+ SEM; n = 7), about the same as that of L-glutamate. L-Serine-O-sulfate (10mM) produced a 4.9 _+ 0.4mV (n = l 1) negative focal potential, while D-serine-O-sulfate (10mM) evoked a 9.2_+0.8mV ( n = l l ) negative focal potential. Table 1 summarizes the relative potency of some of these agonists. A direct comparison of the relative potencies of these compounds was made because the slopes of the dose-response relationship were similar. Seven millivolts was chosen as the point of comparison because it was on the rapidly rising (linear) portion of the dose-response function of all of the agonists examined. A number of putative antagonists were also examined (Fig. 4). Baclofen produced a very small
Potency of amino acids in hippocampus focal potential, ct-~-Diaminopimelate (DAPA) and Daminosuberate also evoked only very small negative focal potentials. However, D-e-aminoadipate (D-AA) D,L-2-amino-4-phophonobutyrate (D,L-APB) and especially D,L-2-amino-3-phosphonopropionate (D,LAPP) evoked substantial negative focal potentials when applied at high doses. Focal potentials from D,L-APP were accompanied by action potentials. LGlutamic acid diethyl ester (GDEE), used within 15 min of being dissolved, produced almost no shift in the d.c. potential. However, 4hr later the same solution induced a negative shift similar to that of D,L-APB or D-c~-aminoadipate. This negativity was accompanied by a train of action potentials. DISCUSSION
Focal potentials appear to be a simple and convenient way to observe the direct action of applied compounds. Focal potentials exhibit changes in shape which are consistent with source-sink relationships in CA1 cells. Focal potentials temporally correspond to intracellularly-recorded potential changes. In addition, negative focal potentials are evoked by excitatory, depolarizing compounds like Lglutamate while positive focal potentials are produced by inhibitory, hyperpolarizing compounds like serotonin. Non-active compounds, like L-glutamine, and antagonists, like D-e-aminosuberate, evoke little or no focal potential. It has also been found that focal potentials evoked by excitatory amino acids are blocked by appropriate antagonists, e.g. 2-amino-5phosphonovalerate (APV) blocks focal potentials induced by NMA (Ganong and Cotman, 1982; Ganong, Lanthorn and Cotman, unpublished). As measured by focal potentials, eight amino acid analogues were considerably more potent than Lglutamate. These are -t--cis-l-amino-l,3- dicarboxycyclopentane (ADCP), N-methyl-D, L-aspartate, ibotenate, quisqualate, + c~-amino-3-hydroxy-5methyl-4-isoazolepropionic acid (AMPA), kainate, and D- and L- homocysteate. The three most potent compounds, i.e. ++_-cis-l-amino-l,3-dicarboxycyclopentane, (ADCP), N-methyl-D,L-aspartate (NMA) and ibotentate, are N-methyl-D-aspartate-type agonists (Davies and Watkins, 1979; Evans et al., 1979; McLennan and Lodge, 1979; Watkins et al., 1980). These same eight analogues were also more potent than L-glutamate in the mammalian spinal cord (Watkins, 1978; Watkins et al., 1980; KorgsgaardLarsen, Honore, Hansen, Curtis and Lodge, 1980). Moreover, the potencies, relative to L-glutamate, of quisqualate, _+~t-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid and kainate were similar in CA1 cells and the mammalian spinal cord. However, some compounds have very dissimilar relative potencies in CA1 cells as compared to spinal cord. When applied to the apical dendritic field of CAI, NMA was over 200 times more potent than L-glutamate and ibotehate was 125 times more potent. In contrast, on cat
1347
or rat spinal interneurons N-methyl-D-aspartate was only 7-18 times more potent, NMLA was equipotent, and ibotenate was 3-6 times more potent than L-glutamate. D- and h-Homocysteate were about an order of magnitude more potent in CA1 cells, relative to glutamate, when compared with spinal interneurons. L-~-Aminopimelate and L-~-aminosuberate, the L-isomers of potent N-methyl-D-aspartate-type antagonists, were also about ten times more potent than L-glutamate in CA1 as compared with their potencies in the spinal cord (Evans et al., 1979). Neurons in CA1 are relatively more sensitive to +_-cis-l-amino-l,3-dicarboxycyclopentane, NMA, ibotenate and L-homocysteate, compounds which are susceptible to antagonism by D-~-aminoadipate and other N-methyI-D-aspartate-type antagonists. The greater potency of those agonists which are sensitive to N-methyl-D-aspartate-type antagonists may suggest the presence of N-methyl-D-aspartate-type receptors in the apical dendritic field of CA1. The Schaffer/commissural synapses are a major source of synaptic input to the apical dendrities of CA1 neurons. However, N-methyl-D-aspartate-type antagonists weakly inhibit the Schaffer/commissural evoked response (Koerner and Cotman, 1983). The most effective antagonist of the Schaffer/commissural evoked response was D-c~-aminosuberate, which can reduce responses to kainic acid in addition to blocking N-methyl-D-asparate responses. This suggests that the junctional receptor is not an N-methylD-aspartate receptor. Thus, the site of action of the N-methyl-D-aspartate-type agonists is unknown. In the present study, D, L-APP, D, L-APB and D-~taminoadipate exhibited substantial focal potentials in large concentrations. This suggests that these compounds directly depolarize CA1 neurons in large concentrations, an action which has been taken into account when studying antagonism of CA1 responses by these compounds (Koerner and Cotman, 1983). ~,e-Diaminopimelate and D-~-aminosuberate produced only small negative focal potentials, more consistent with pure antagonism. The kinetics of inhibition of the Schaffer/commissural-evoked response by these agents is consistent with antagonism uncontaminated by depolarizing effects (Koerner and Cotman, 1983). L-Glutamic acid diethylested (GDEE) induced no focal potential when applied within 15 min of being prepared. Four hours later the same solution evoked negative focal potentials and action potentials. This change in activity may have been due to the hydrolysis of GDEE to L-glutamate and is consistent with reports that GDEE is sometimes a useful antagonist in this area, and sometimes not (Spencer et al., 1976; White et al., 1979). Baclofen inhibits responses believed to be mediated by excitatory amino acids, probably through inhibition of their release (Potashner, 1978). Baclofen has been shown to antagonize potently the Shaffer/commissural evoked response (Lanthorn and
1348
T.H. LANTHORNand C.W. COTMAN
C o t m a n , 1981 ; Ault a n d Nadler, 1981; Olpe, Baudry, Fagni and Lynch, 1982). The present results suggest that baclofen does not act as an excitatory depolarizing c o m p o u n d or as a classical hyperpolarizing G A B A - m i m e t i c in suppressing this response.
The authors would like to thank Dr Per Andersen for the use of his facilities and his guidance in securing intracellular recordings. We also wish to thank John Ryan and Alan Ganong for many helpful discussions. We thank Dr J. C. Watkins, H. Shinozaki, H. McLennan, C. H. Eugster and P. Krogsgaard-Larsen, and CIBA-Geigy for their generous gifts of compounds. This work was supported by fellowship MHO8258 to T.L. and grants NSO8957 and NH19691 to C.W.C. Acknowledqements
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Algcr B. E. and Nicoll R. A. (1979) GABA-mediated biphasic inhibitory responses in hippocampus. Nature 281: 315-317. Andcrsen P., Dingledine R., Gjerstad L., Langmoen I. A. and Mosfeldt-Larsen A. (1980) Two different responses of hippocampal pyramidal cells to application of gammaamino butyric acid. J. Physiol. 305: 279-296. Andersen P., Holmqvist B. and Voorhoeve P. E. (1966) Excitatory synapses on hippocampal apical dendrites activated by entorhinal stimulation. Aeta physiol, scand. 66: 461-472. Ault B. and Nadler J. V. (1981) Baclofen: a selective depressant at synapses made by CA3 pyramidal cells in the hippocampal slice. Neurosci. Abs. 7:11 I. Benardo L. S. and Prince D. A. (1981) Acetylcholine induced modulation of hippocampal pyramidal neurons. Brain Res. 211: 227-234. Davies J. and Watkins J. C. (1979) Selective antagonism of amino acid-induced and synaptic excitation in the cat spinal cord. J. Physiol. 297: 621-635. Dudar J. D. (1974) In vitro excitation of hippocampal cell dendrities by glutamic acid. Neuropharmacology 13: 1083-1089. Evans R. H., Francis A. A., Hunt K., Oakes D. J. and Watkins J. C. (1979) Antagonism of excitatory amino acid-induced responses and of synaptic excitation in the isolated spinal cord of the frog. Br. J. Pharmac. 67: 591-603. Flatman J. A. and Lambert J. D. C. (1979) Sustained extracellular potentials in the cat spinal cord during the microiontophoretic application of excitatory amino acids. J. Neurosci. Meth. 1:205 218. Ganong A. H. and Cotman C. W. (1982) Acidic amino acid antagonists of lateral perforant path synaptic transmission: agonist antagonist interactions in the dentate gyrus. Neurosci. Zetl. 34: 195-200. Jahnsen H. (1980) The action of 5-hydroxytryptamine on neuronal membranes and synaptic transmission in area CA1 of the hippocampus in vitro. Brain Res. 197: 83-94. Koerner J. and Cotman C. W. (1983) Response of Schaffer collateral-CAl pyramidal cell synapses of the hippocam-
pus to analogues of acidic amino acids. Brain Res. (In Press). Krogsgaard-Larsen P., Honore T., Hansen J. J., Curtis D. R. and Lodge D. (1980) New class of glutamate agonist structurally related to ibotenic acid. Nature 284: 64-66. Lambert J. D. C., Flatman J. A. and Jahnsen H. (1981) Extracellular recordings of amino acid induced potential changes in hippocampal slices. J. Neurosci. Meth. 3: 311-315. Langmoen I. A., Segal M. and Andersen P. (1981) Mechanisms of norepinephrine actions on hippocampal pyramidal cells in vitro. Brain Res. 208:349 362. Lanthorn T. H. and Cotman C. W. (1981) Baclofen selectively inhibits excitatory synaptic transmission in the hippocampus. Brain Res. 225:171 178. McLennan H. and Lodge D. (1979) The antagonism of amino acid-induced excitation of spinal neurones in the cat. Brain Res. 169:83 90. Nadler J. V., White W. F., Vaca K. W., Perry B. W. and Cotman C. W. (1980) Biochemical correlates of transmission mediated by glutamate and aspartate. J. Neurochem. 31:147 155. Olpe H.-R., Baudry M., Fagni L. and Lynch G. (1982) The blocking action of baclofen on excitatory transmission in the rat hippocampal slice. J. Neurosci. 2: 698-703. Potashner S. J. (1978) Baclofen: effects on amino acid release. Can. J. Physiol. Pharmac. 56: 15~154. Schwartzkroin P. A. and Andersen P. (1975) Glutamic acid sensitivity of dendrites in hippocampal slices in vitro. Adv. NeuroL 12:45 51. Segal M. (1980) The action of serotonin in the rat hippocampal slice preparation. J. Physiol. 303: 423-439. Spencer H. J., Gribkoff V. K., Cotman C. W. and Lynch G. S, (1976) GDEE antagonism of iontophoretic excitations in the intact hippocampus and in the hippocampal slice preparation, Brain Res. 105: 471-481. Storm-Mathisen J. (1977) Glutamic acid and excitatory nerve endings; reduction of glutamic acid uptake after axotomy. Brain Res. 120: 379-386. Valentino R. J. and Dingledine R. (1981) Presynaptic inhibitory effect of acetylcholine in the hippocampus. J. Neurosci. !: 784 792, Watkins J. C. (1978) Excitatory amino acids. In: Kainic Acid as a Tool in Neurobiology (McGeer E. G., Olney J. W. and McGeer P. L., Eds), pp. 37-69. Raven Press, New York. Watkins J. C., Davies J., Evans R. H., Francis A. A. and Jones A. W. (1980) Pharmacology of receptors for excitatory amino acids. In: GABA and Glutamate as Transmitters (diChiara G., Ed.), pp. 1 28. Raven Press, New York. White W. F., Nadler J. V. and Cotman C. W. (1978) A perfusion chamber for the study of CNS physiology and pharmacology in vitro. Brain Res. 152:591 596. White W. F., Nadler J. V. and Cotman C. W. (1979) The effect of acidic amino acid antagonists on synaptic transmission in the hippocampal formation in vitro. Brain Res. 164: 177-194. Wieraszko A. and Lynch G. (1979) Stimulation dependent release of possible transmitter substances from hippocampal slices studied with localized perfusion. Brain Res. 160: 372-376.
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RELATIVE POTENCY OF ANALOGUES OF EXCITATORY AMINO ACIDS ON HIPPOCAMPAL CA1 N E U R O N S T. H. LANTHORN* and C. W. COTMAN Department of Psychobiology, University of California, Irvine, CA 92712, U.S.A.
(Accepted 10 April 1983) Summary--The relative potency of analogues of excitatory amino acids to produce depolarization when applied in the apical dendritic field of CA1 cells was studied in the hippocampal slice. The effect of these compounds was measured by recording focal potentials (FPs), the shift in the extracellular d.c. potential produced by the compounds applied. The ability of focal potentials to measure neuronal responses was evaluated. N-methyl-D-aspartate (NMDA)-type agonists were 10-20 times more potent, relative to Lglutamate, than reported from investigations in spinal cord. Quisqualate (QA), + ~-amino-3-hydroxy-5methyl-4-isoazolepropionicacid (AMPA) and kainate (KA) exhibited potencies on CA1 cells similar to those reported for spinal neurons. These data indicate that elements in CA1 cells possess a receptor with an affinity for N-methyl-D-aspartate-type agonists. Some putative antagonists were found to induce negative focal potentials suggestinga direct, excitatory action in this area. Key words: excitatory amino acids, hippocampal slice,CA1 cells, focal potentials.
It has been suggested that glutamate, aspartate or a related compound serves as the transmitter at Schaffer collateral/commissural synapses in area CA1 of the hippocampus (Dudar, 1974; Nadler, White, Vaca, Perry and Cotman, 1980; Schwartzkroin and Andersen, 1975; Storm-Mathisen, 1977; White, Nadler and Cotman, 1979; Wieraszko and Lynch, 1979). However, the type of amino acid receptor used is unknown. Recently, it has been found that quisqualate, N-methyl-D-aspartate, and kainate antagonists (Davies and Watkins, 1979; Evans, Francis, Hunt, Oakes and Watkins, 1979; Watkins, Davies, Evans, Francis and Jones, 1980) are all relatively weak antagonists at Schaffer/ commissural synapses (Koerner and Cotman, 1983) making identification of the receptor uncertain. A large number of agonists exist which might give clues to the structural characteristics of the natural transmitter receptor. In this study, the actions of several transmitter agonists and antagonists on the hippocampal slice were investigated. The effect of these compounds was measured by recording the shift in the extracellular d.c. potential induced by the compounds. This shift in d.c. potential has been investigated in the spinal cord by Flatman and Lambert (1979). They found that excitatory agents induced a negative shift in the extracellular d.c. potential which, except for being opposite in sign, closely corresponded to the intracellularly recorded d.c. shift. Norepinephrine sometimes induced a positive d.c. shift corresponding to its weak hyperpolarizing action. These potentials, *Send correspondence to: Dr Thomas H. Lanthorn, Building 10, Room 5C106, National Institutes of Health, Bethesda, MD 20205, U.S.A.
which Flatman and Lambert called focal potentials (FPs), are probably analogous to synaptic field potentials except that they are induced by substances applied rather than by substances released from nerve endings. Lambert, Flatman and Jahnsen (1981) have also observed focal potentials using the hippocampal slice preparation. The present authors used focal potentials because they are easy to record and appear to measure the direct effect of compounds on neurons. However, since focal potentials have not been used extensively, their ability to reflect changes in membrane polarization was evaluated. METHODS
Slices (350-400/zm thick) were prepared from the hippocampus of male, Sprague-Dawley rats, 45-60 days old. The slices were placed in a chamber previously described (Spencer, Gribkoff, Cotman and Lynch, 1976; White, Nadler and Cotman, 1978) and incubated in a medium containing 124mM NaC1, 3.3raM KC1, 1.2mM KH2PO4, 25.4mM NaHCO3, 2.5raM CaCl2, 2.4raM MgSO4 and 10 mM D-glucose. The slices were superfused with a humidified atmosphere of 95% 02-5% CO2, at a temperature of 31-33°C. Focal potentials and action potentials were recorded with glass pipettes filled with 2 M NaCI and having a resistance of 4-10MO. Direct current (d.c.)-coupled signals were amplified and displayed in a conventional manner. All drugs were dissolved in the same medium as used to support the slices and the solutions were brought to a pH of 7.4 +_ 0.05. Pipettes with their tips broken back to about 50#m were filled with drug solutions and lowered onto the slice until a fluid bridge was created between the two. Application was
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T.H. LANTHORr~and C. W. COTMAN
terminated by raising the pipette from the slice. Drugs were applied for approx. 1 sec unless otherwise noted. To determine the potencies of the amino acid analogues, an ascending series of concentrations of drug (at most three) was applied to one site on each slice. D-~-Aminoadipate (D-AA), t-~-aminopimelate (LAP), and D- and t-~-aminosuberate (D- and L-AS) were gifts from Dr J. C. Watkins. +-Cis-l-amino1,3-dicarboxycyclopentane (ADCP) was a gift from Dr H. McLennan. Quisqualate (QA) was a gift from Dr H. Shinozaki and ibotenate (Ibo) from Dr C. H. Eugster. The _ ~-amino-3-hydroxy-5-methyl-4isoazolepropionic acid (AMPA) was a gift from Dr P. Krogsgaard-Larsen. fl-(p-Chlorophenyl)-GABA (baclofen) was a gift from CIBA-Geigy. All other compounds were obtained from commercial sources. Unless specifically stated, all compounds were applied to the stratum radiatum of CAI very close to a recording electrode from which a subthreshold (for population spike) Schaffer potential of 2.5-4.5mV could be recorded. RESULTS
Focal potentials As focal potentials have not been used extensively some examination of their characteristics in the hippocampus was necessary. Application of experimental saline produced little change (maximum deflection of - 0 . 0 5 m V ) in the d.c. potential unless the tip of the recording electrode was less than 25/~m
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(A)
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Fig. 1. Focal potentials recorded at different locations in CAI following application of L-glutamate (1 mM) to the stratum lacunosum-moleculare of CA1. Records 1 and 5 were recorded in the stratum lacunosum-molecular near the site of application of L-glutamate. Records 2~4 were recorded at sites 200, 400 and 600/~m from the site of application of L-glutamate towards and past the stratum pyramidale. Records 6-9 were recorded at sites in the stratum lacunosummoleculare 200, 400, 600 and 800pm from the site of L-glutamate's application. into the slice. When the tip was closer to the surface, application of the medium resulted in a larger negative deflection (about 0.1 mV) lasting for around 2 sec. This was probably due to the transient increase in medium around the electrode tip which may provide a current shunt, or to mechanical disturbance of the tip. In order to avoid this artifact, all recordings were made with the electrode tip lowered 75 tim into the slice. L-Glutamate and other depolarizing compounds applied to the surface of the slice produced a negative d.c. potential shift which rapidly returned to the preIB)
5sec |IL
~
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3
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lOsec Fig. 2. Correspondence between intrace]]ularly and extracellulady recorded responses to L-glutamate from 3 cells. ]ntracellular record is the top record of each pair. (A) Small concentration of L-glutamate producing a small intracellular response. (B) Suprathreshold response from a different cell. (C) Three responses from a third cell. Calibrations are the same for all three records. C1, subthreshold response; C2, suprathresho]d response; C3, two applications of L-glutamate showing additive responses in both intrace]lular and extracellular records. Because of slow recording speed, action potentials in B, C2 and C3 show up only as scatter in the records.
Potency of amino acids in hippocampus
(A)
Table 1. Relative potency of excitatory amino acid analogues 2mV
Compound
5~ec
IB)S
i
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Fig. 3. Focal potentials recorded following the application of transmitters to the stratum radiatum of CA1. (A) GABA, (B) 5-hydroxytryptamine, and (C) acetylcholine. Calibrations are the same in B and C. drug baseline after the application was terminated (Fig. 1). On many occasions, the d.c. potential transiently overshot the baseline. The negative deflection was often accompanied by a train of action potentials. Lengthening the period of application prolonged the time before the d.c. potential returned to baseline. Small increases in the duration of application of glutamate also resulted in an increased peak amplitude of the negative deflection, but after 5-10 sec of application a plateau was reached, and was maintained during applications for 30 sec. Except as noted
20
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62 51 36 23 17 3.5 2.4 1.3
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• QA
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~--L-g}u
•
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~ --L--Cys
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• ~ D - asp
/iY 10-5
./x;-.-,'/// • 10-4
Log
concentration
1
0.75 0.5 0.4 0.35 0.3 0.25
below, drugs could be applied several times to the same site with similar results. The size of the extracellular d.c. shift induced by a particular concentration of an amino acid analogue was correlated with the threshold amplitude of the Schaffer wave in that slice. When the threshold was
mV
"~
215 208 125 80
Potencies were estimated by extrapolating (from Fig. 1) the concentration of each analogue which would produce a 7mV negative focal potential (L-glutamate = I).
OADCP
>,
Potency
ADCP ( _+-cis- l-amino- 1,3-dicarboxycyclopentane) N-methyl-O,L-aspartate Ibotenate Quisqualate AMPA ( +
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1345
.y 10-3
(M)
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Fig. 4. Amplitude of focal potentials induced by analogues of excitatory amino acids in CA 1 as a function of the concentration of the applied compounds. Points are the mean of at least 4 trials on different slices; SEM was less than + 15% of the mean in all cases. The inset shows examples of the focal potentials produced by application of I 0 mM L-glutamate to three slices. The break in the bottom record was 5 min. Key to abbreviations: ADCP, +_-cis-l-amino-l,3-dicarboxycyclopentane; NMA, N-methyl-o,L-aspartate; QA, quisqualate; AMPA, _+-ct-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; KA, kainate; HCA, homocysteate; AP, ~t-aminopimelate; AS, ~-aminosuberate; glu, glutamate; asp, aspartate; THA, threo-3hydroxy-D,L-aspartate; cys, cysteate; AA, ct-aminoadipate; APP, D,L-2-amino-3-phosphonopropionate; APB, o,L-2-amino-4-phosphonobutyrate; DAPA, ct,e-diaminopimelate; GDEE, L-glutamic acid diethyl ester.
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T.H. LANTHORNand C. W. COTMAN
less than 2 mV, small negative shifts were seen which were often short and followed by large and longlasting positive shifts. In slices exhibiting such low thresholds variable responses were seen with repeated application. It is likely that the low thresholds and unstable responses to drugs were the result of depolarized or otherwise abnormal cells. Such responses were not included in this study. The size and shape of focal potentials in the hippocampal slice changed as the recording electrode was moved away from the site of application. These changes are demonstrated in Fig. 1 using the application of glutamate to the molecular layer of CA1. Records 1 and 5 are from the site of application of glutamate, while the others are from sites in progressive steps of 200 ~m away. When the recording electrode was moved along the main dendritic axis toward, and past, the cell body layer (records 1-4), the shape as well as the amplitude of the focal potential changed. In record 2, the duration of the negative portion was decreased relative to that seen at the site of application of glumate (record 1). At a greater distance from the application site (records 3 and 4), the negative focal potential was replaced by a positive wave with a negative deflection on it. The thickening of the potential record, especially prominent in record 3, was due to extracellularly-recorded action potentials. As the recording electrode was moved perpendicular to the main dendritic axis of CA1 neurons (records 5 9), the shape and time course of the focal potential stayed generally the same. Only the amplutide changed, decreasing with increasing distance from the site of application of glutamate. The changes seen in focal potentials recorded along the main dendritic axis of CA1 were similar to changes in synaptic field potentials observed in a laminar analysis of this area (Andersen, Holmqvist and Voorhoeve, 1966)--changes which reflect neuronal source-sink current flow. Figure 2 illustrates the temporal correspondence between extracellular and intracellular responses to application of glutamate. Records are shown from three CA I cells with resting membrane potentials of - 6 0 and - 7 0 m V , spike amplitudes (from threshold) of 69 to 80 mV and spike durations of 1.7 to 2.2 msec. In all three neurons, the extracellular focal potential closely followed the changes in membrane potential recorded intracellularly. Dendritic application of 7-aminobutyric acid (GABA) evoked a negative focal potential (Fig. 3A). This was consistent with its depolarizing action of CA1 dendrites (Alger and Nicoll, 1979; Andersen, Dingledine, Gjerstad, Langmoen and MosfeldtLarsen, 1980). Also GABA hyperpolarized the soma of CA I neurons, and the application of GABA to the cell body layer resulted in a positive-negative focal potential (data not shown; Lambert et al., 1981). Serotonin, which hyperpolarizes CA1 neurons (Segal, 1980; Jahnsen, 1980), consistently induced positive focal potentials (Fig. 3B). Postive focal
potentials were rarely greater than 2mV. Norepinephrine is reported to hyperpolarize CA1 neurons only slightly (Langmoen, Segal and Andersen, 1981; Segal, 1980). Applied to the apical dendritic field, it sometimes produced positive focal potentials, but most often produced no observable focal potential. Acetylcholine (ACh), applied to the apical dendritic field, evoked a positive focal potential (Fig. 3C). It has been reported that ACh is inhibitory in the dendritic field, probably via presynaptic inhibition (e.g. Valentino and Dingledine, 1981). Hyperpolarization has been reported, but was transient (Bernardo and Prince, 1981).
Effects of analogues of acidic amino acid To determine the effects of excitatory amino acid agonists and putative antagonists, these compounds were applied to the apical dendritic field of CAl. At least three concentrations of each compound were tested. The dose response relationships observed are portrayed in Fig. 4. N-methyl-D,L-asparate (NMA) and +_-cis-l-amino-l,3-dicarboxycyclopentane (ADCP) were the most potent compounds followed by ibotenate, quisqualate, +~-amino-3hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) and kainate. Homocysteate was less potent and exhibited little stereospecificity. L-~-Aminopimelate and L-~-aminosuberate were two to three times more potent than L-glutamate. D-Glutamate was about equal to L-glutamate. Slightly less potent were L- and D-aspartate, L-cysteate, L-~-aminoadipate and threo3-hydroxy-D, L-asparate. L-Glutamine, which is not an active depolarizing agent, evoked only a 0.1mV negative deflection. Other compounds which were less potent than Lglutamate (produced less than a I mV negative focal potential at a concentration of 10 mM) included D,L~-methylglutamate, methionine sulfoxide, methionine sulfone, methionine sulfoximine, 6aminolevulinic acid, D- and L-cysteine, L-ascorbate, ~-aspartylglycine and ~-aspartyl-/~-alanine. Three compounds exhibited excitatory activity, but were tested at only one concentration. A 10mM solution of D,L-~-methylaspartate evoked a 5.0_+0.4mV negative focal potential (Mn _+ SEM; n = 7), about the same as that of L-glutamate. L-Serine-O-sulfate (10mM) produced a 4.9 _+ 0.4mV (n = l 1) negative focal potential, while D-serine-O-sulfate (10mM) evoked a 9.2_+0.8mV ( n = l l ) negative focal potential. Table 1 summarizes the relative potency of some of these agonists. A direct comparison of the relative potencies of these compounds was made because the slopes of the dose-response relationship were similar. Seven millivolts was chosen as the point of comparison because it was on the rapidly rising (linear) portion of the dose-response function of all of the agonists examined. A number of putative antagonists were also examined (Fig. 4). Baclofen produced a very small
Potency of amino acids in hippocampus focal potential, ct-~-Diaminopimelate (DAPA) and Daminosuberate also evoked only very small negative focal potentials. However, D-e-aminoadipate (D-AA) D,L-2-amino-4-phophonobutyrate (D,L-APB) and especially D,L-2-amino-3-phosphonopropionate (D,LAPP) evoked substantial negative focal potentials when applied at high doses. Focal potentials from D,L-APP were accompanied by action potentials. LGlutamic acid diethyl ester (GDEE), used within 15 min of being dissolved, produced almost no shift in the d.c. potential. However, 4hr later the same solution induced a negative shift similar to that of D,L-APB or D-c~-aminoadipate. This negativity was accompanied by a train of action potentials. DISCUSSION
Focal potentials appear to be a simple and convenient way to observe the direct action of applied compounds. Focal potentials exhibit changes in shape which are consistent with source-sink relationships in CA1 cells. Focal potentials temporally correspond to intracellularly-recorded potential changes. In addition, negative focal potentials are evoked by excitatory, depolarizing compounds like Lglutamate while positive focal potentials are produced by inhibitory, hyperpolarizing compounds like serotonin. Non-active compounds, like L-glutamine, and antagonists, like D-e-aminosuberate, evoke little or no focal potential. It has also been found that focal potentials evoked by excitatory amino acids are blocked by appropriate antagonists, e.g. 2-amino-5phosphonovalerate (APV) blocks focal potentials induced by NMA (Ganong and Cotman, 1982; Ganong, Lanthorn and Cotman, unpublished). As measured by focal potentials, eight amino acid analogues were considerably more potent than Lglutamate. These are -t--cis-l-amino-l,3- dicarboxycyclopentane (ADCP), N-methyl-D, L-aspartate, ibotenate, quisqualate, + c~-amino-3-hydroxy-5methyl-4-isoazolepropionic acid (AMPA), kainate, and D- and L- homocysteate. The three most potent compounds, i.e. ++_-cis-l-amino-l,3-dicarboxycyclopentane, (ADCP), N-methyl-D,L-aspartate (NMA) and ibotentate, are N-methyl-D-aspartate-type agonists (Davies and Watkins, 1979; Evans et al., 1979; McLennan and Lodge, 1979; Watkins et al., 1980). These same eight analogues were also more potent than L-glutamate in the mammalian spinal cord (Watkins, 1978; Watkins et al., 1980; KorgsgaardLarsen, Honore, Hansen, Curtis and Lodge, 1980). Moreover, the potencies, relative to L-glutamate, of quisqualate, _+~t-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid and kainate were similar in CA1 cells and the mammalian spinal cord. However, some compounds have very dissimilar relative potencies in CA1 cells as compared to spinal cord. When applied to the apical dendritic field of CAI, NMA was over 200 times more potent than L-glutamate and ibotehate was 125 times more potent. In contrast, on cat
1347
or rat spinal interneurons N-methyl-D-aspartate was only 7-18 times more potent, NMLA was equipotent, and ibotenate was 3-6 times more potent than L-glutamate. D- and h-Homocysteate were about an order of magnitude more potent in CA1 cells, relative to glutamate, when compared with spinal interneurons. L-~-Aminopimelate and L-~-aminosuberate, the L-isomers of potent N-methyl-D-aspartate-type antagonists, were also about ten times more potent than L-glutamate in CA1 as compared with their potencies in the spinal cord (Evans et al., 1979). Neurons in CA1 are relatively more sensitive to +_-cis-l-amino-l,3-dicarboxycyclopentane, NMA, ibotenate and L-homocysteate, compounds which are susceptible to antagonism by D-~-aminoadipate and other N-methyI-D-aspartate-type antagonists. The greater potency of those agonists which are sensitive to N-methyl-D-aspartate-type antagonists may suggest the presence of N-methyl-D-aspartate-type receptors in the apical dendritic field of CA1. The Schaffer/commissural synapses are a major source of synaptic input to the apical dendrities of CA1 neurons. However, N-methyl-D-aspartate-type antagonists weakly inhibit the Schaffer/commissural evoked response (Koerner and Cotman, 1983). The most effective antagonist of the Schaffer/commissural evoked response was D-c~-aminosuberate, which can reduce responses to kainic acid in addition to blocking N-methyl-D-asparate responses. This suggests that the junctional receptor is not an N-methylD-aspartate receptor. Thus, the site of action of the N-methyl-D-aspartate-type agonists is unknown. In the present study, D, L-APP, D, L-APB and D-~taminoadipate exhibited substantial focal potentials in large concentrations. This suggests that these compounds directly depolarize CA1 neurons in large concentrations, an action which has been taken into account when studying antagonism of CA1 responses by these compounds (Koerner and Cotman, 1983). ~,e-Diaminopimelate and D-~-aminosuberate produced only small negative focal potentials, more consistent with pure antagonism. The kinetics of inhibition of the Schaffer/commissural-evoked response by these agents is consistent with antagonism uncontaminated by depolarizing effects (Koerner and Cotman, 1983). L-Glutamic acid diethylested (GDEE) induced no focal potential when applied within 15 min of being prepared. Four hours later the same solution evoked negative focal potentials and action potentials. This change in activity may have been due to the hydrolysis of GDEE to L-glutamate and is consistent with reports that GDEE is sometimes a useful antagonist in this area, and sometimes not (Spencer et al., 1976; White et al., 1979). Baclofen inhibits responses believed to be mediated by excitatory amino acids, probably through inhibition of their release (Potashner, 1978). Baclofen has been shown to antagonize potently the Shaffer/commissural evoked response (Lanthorn and
1348
T.H. LANTHORNand C.W. COTMAN
C o t m a n , 1981 ; Ault a n d Nadler, 1981; Olpe, Baudry, Fagni and Lynch, 1982). The present results suggest that baclofen does not act as an excitatory depolarizing c o m p o u n d or as a classical hyperpolarizing G A B A - m i m e t i c in suppressing this response.
The authors would like to thank Dr Per Andersen for the use of his facilities and his guidance in securing intracellular recordings. We also wish to thank John Ryan and Alan Ganong for many helpful discussions. We thank Dr J. C. Watkins, H. Shinozaki, H. McLennan, C. H. Eugster and P. Krogsgaard-Larsen, and CIBA-Geigy for their generous gifts of compounds. This work was supported by fellowship MHO8258 to T.L. and grants NSO8957 and NH19691 to C.W.C. Acknowledqements
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