J. Physiol. (1979), 286, pp. 29-39 With 3 text-figures Printed in Great Britain
29
THE EXCITATION OF MAMMALIAN CENTRAL NEURONES BY AMINO ACIDS
BY J. G. HALL*, T. P. HICKS, H. McLENNAN, T. L. RICHARDSON AND H. V. WHEALt From the Department of Physiology, University of Briti8h Columbia, 2075 Wesbrook Place, Vancouver, B.C., Canada V6T iW5
(Received 22 February 1978) SUMMARY
1. The relative potencies of a number of analogues of L-glutamate as excitants of thalamic neurones in the rat have been compared. The most powerful compounds were kainate, ibotenate and (± )cis-1-amino-1,3-dicarboxycyclopentane. The D- and L-isomers of glutamate and aspartate were also compared. Whereas D-glutamate is approximately one-half as active as the L-form, D-aspartate is more potent than L-aspartate. 2. Computer analysis has indicated that ibotenate and cis-1-amino-1,3-dicarboxycyclopentane have relatively fixed and similar Ca-N, C.-N and Cha-Ch interatomic distances which can also be achieved by glutamate in certain conformations of the molecule, but not by aspartate. 3. Parallel examination of the antagonists glutamate diethylester and D-aaminoadipate has shown that the former preferentially reduces L-glutamate effects while the latter blocks the actions of other amino acid excitants more readily than those of L-glutamate. 4. The evidence is consistent with the hypothesis that at least two populations of neuronal receptors for the excitatory amino acids exist. INTRODUCTION
The dicarboxylic amino acids L-glutamate and L-aspartate which occur naturally in the mammalian central nervous system share two properties. Firstly they are capable of depolarizing and exciting neurones to which they are administered extracellularly (Curtis, Phillis & Watkins, 1960) and the ionic basis for this depolarization closely resembles that induced by stimulation of excitatory synapses (Zieglginsberger & Puil, 1972). Secondly, they are taken up into cells of the nervous system by high affinity mechanisms which are distinct from those used to transport other amino acids (Logan & Snyder, 1972). For these reasons among others there is a widespread belief that the two compounds probably function as mediators of some synaptic * Present address: Department of Pharmacology, Australian National University, Canberra, Australia. t Present address: Neurophysiology Group, University of Southampton, Southampton, England.
J. G. HALL AND OTHERS excitations in the central nervous system of mammals (Johnson, 1972; Curtis & Johnston, 1974; McLennan, 1975). In the original studies by Curtis & Watkins (1960, 1963) the actions of a number of other compounds structurally related to glutamate were examined, from which the conclusion was drawn that the presence of the two acidic groups and the cationic site provided by the amino group were all essential, i.e. that a three-point attachment of the agonist molecule with its receptor was required. Over the years there has been no serious challenge to that view though evidence has recently been advanced to support the concept that three rather than a single molecule may react with the receptor to produce its activation (McLennan & Wheal, 1976a). The questions dealing with the precise conformation of the receptor site, whether or not there exist separate receptors for glutamate and for aspartate, and whether there may be non-specific ' amino acid' receptors, possibly extrasynaptic, in addition to specific synaptic receptors (McLennan, Huffman & Marshall, 1968; McLennan, 1970) remain to be established. The considerable flexibility of the glutamate and aspartate molecules do not permit the first two of these questions to be answered since a minimum distance of 2-6 A between the w-carboxyl and the - NH+ group has been claimed to be possible in both (van Gelder, 1971; Buu, Puil & van Gelder, 1976), although of course the distances will differ when the molecules are extended. However, the fact that in some circumstances a selective antagonism of the excitatory action of the two agonists has been shown (Haldeman & McLennan, 1972; Biscoe, Davies, Dray, Evans, Francis, Martin & Watkins, 1977a) suggests that separate receptors for the two molecules may exist. The use of conformationally restricted analogues of the molecules is more promising (Johnston, Curtis, Davies & McCulloch, 1974; Biscoe, Evans, Headley, Martin & Watkins, 1975), and an examination of the excitatory action of certain compounds correlated with the extent of their molecular restraint should provide valuable information. The final question, whether or not there may be both synaptic and extrasynaptic amino acid receptors mediating neuronal excitation, is more difficult to approach but the use of certain antagonists suggests that indeed two populations exist. Brief accounts of certain of the studies to be reported here have appeared (Hall, McLennan & Wheal 1977; McLennan & Hall, 1978; McLennan & Wheal, 1978). 30
METHODS
Assessment of the potencies of a number of compounds as neuronal excitants relative to L-
glutamate has been determined upon neurones of the ventrobasal thalami of rats anaesthetized with urethane (1.5 g/kg I.P.). The animals were placed in a stereotaxic frame, calvarium and dura removed and the exposed cortex covered by a pool of warmed liquid paraffin. Electrodes were introduced stereotaxically into the thalamus and records obtained from cells responding with 8-12 msec latency to electrical stimulation of a contralateral hind limb nerve. Confirmation of electrode placements was achieved by the ejection of Pontamine sky blue at the conclusion of the experiment, and subsequent histological verification. The amino acid agonists were administered electrophoretically from seven barrel micropipettes using intensities of ejecting currents which elicited stable, equal and approximately halfmaximal firing frequencies. Not more than three excitants in addition to L-glutamate were tested in any one series, and for inclusion in the results similar values, which were then averaged, were obtained at least twice on each neurone. It was found important also to preserve the order of presentation of the substances and to maintain constant intervals between administrations.
EXCITATORY AMINO ACIDS
31
A typical series of results presented as integrated firing frequencies displayed on a chart recorder is illustrated by Fig. 1. The relative potencies of the compounds are expressed as ratios of the ejecting currents needed to evoke equal rates of firing in comparison with the current required for L-glutamate. The solutions used to fill the electrode barrels were the following: L- and D-glutamate, L- and D-aspartate, L-a-aminoadipate, DL-a-aminopimelate, L-a-aminosuberate, (± )-ci8 and tran8-1 amino-1,3-dicarboxycyclohexane (ADCH) (all 0*5 M, pH 8); DL-homocysteate (DLH), N-methylDL-aspartate (NMA) and (±)-trans-1-amino-1,3-dicarboxycyclopentane (ADCP) (all 0-2 M, pH 8); kainate, ibotenate and (± )-cis-ADCP (0.1 M, 50 or 20 mM in 0- 15 M-NaCl, pH 8); Lglutamic acid diethylester (GDEE) (0-5 M, pH 3.5); D-a-aminoadipate (DAA) (0-2 M, pH 8). All compounds were obtained from commercial sources except the cyclohexane and cyclopentane derivatives which were synthesized and ibotenate and DAA which were received as gifts. 75
0
a-.
GLUT 13
trans-ADCP 8
cis-ADCP 2
30 sec
Fig. 1. Ratemeter records of the integrated firing frequency of a thalamic neurone. The cell was excited by the electrophoretic ejection of L-glutamate (GLUT) and the tranf8- and cis-isomers of ADCP during the periods shown by the horizontal bars and with the indicated currents in nA. Two factors can affect the validity of the relative potencies determined as above: (a) the linearity of the amount of ion ejected with respect both to the intensity of the applied current and the time of application, and (b) the 'transport numbers' of the substances at the tips of the electrodes (Zieglgansberger, Herz & Teschemacher, 1969; Gent, Morgan & Wolstencroft, 1974). Although it was not possible to examine these factors for all of the materials tested since suitably labelled compounds are not available, the behaviour of three ([3H]L-glutamate, [3H]kainate and [14C]L-aspartate) was observed. The labelled solutions were introduced into micropipettes and the quantities ejected into 0- 5 ml. 0-15 M-NaCl assessed by liquid scintillation analysis. Very similar results were obtained with three different micropipette assemblies. The transport number for kainate was also examined using solutions of several different concentrations in the pipette: 0-1 M in water and 20 and 50 mm in 0-15 M-NaCl. Computer models of many of the compounds examined were constructed from crystallographic data, and could be displayed on a quasi-three-dimensional graphics terminal. The program, details of which will appear elsewhere, permitted the various interatomic bonds of the molecules to be rotated individually while the whole molecule was displayed; and a table of bond angles and interatomic distances for any desired configuration could be obtained. Plots of the computed images of L-glutamate in configurations where the C,,-N distances are maximal and minimal, together with the corresponding distances between the presumed three active groups are given in Fig. 2 (see also Table 2).
32
J. G. HALL AND OTHERS RESULTS
Tranmport numbers Three electrodes whose barrels contained radioactively labelled solutions of amino acids were used to determine transport numbers. The results from one electrode where L-glutamate was ejected with different intensities of current and for various periods G lu min
Glu-max
"0(9) N(1 0)
Cx-C,,401 A
Cx-C,,, 4*56 A C-, N 2-43 A C^,-N 513 A
CA-N 2 43 A Cs,-N 2 23 A
Fig. 2. Reproductions of computer models of L-glutamate, manipulated so that the
Q),,-N is minimal (Glu-min) or maximal (Glu-max). C. lies between 0 (8) and 0 (9); C. between 0 (1) and 0 (2), and the amino group is indicated by N (10). The correspond-
ing interatomic distances (A) are given. a)
E
I--
C W a)
0)
E
-J
60 Time (mmn)
120
0
125 Current (nA)
250
Fig. 3. The electrophoretic expulsion of [3H]L-glutamate from a pipette (A) as a function of time with constant ejecting current and (B) as a function of current with a constant time of ejection. The 60 min/150 nA point is identical in the two graphs.
of time are illustrated by Fig. 3, and it is clear that there existed an essentially linear relationship between the quantity ejected and those two variables. The transport number for the amino acid in this case was 0-22; and with the same electrode the corresponding values for aspartate and kainate (from a solution of 01 M) were
EXCITATORY AMINO ACIDS 33 0-28 and 0'24 respectively. The value for glutamate is at the lower end of the range reported by Zieglginsberger et al. (1969); more importantly in the present context there is no substantial difference between the values for the three amino acids and thus variations in transport number would appear not to contribute significantly in this instance to changes in the apparent potency of the agonists. Similar results were obtained with the other two electrode assemblies tested and the transport numbers for the three amino acids were approximately equal in every case. An analysis was also made of the effect of concentration of kainate in the electrode on its transport number. When allowance was made for the fact that the diluted solutions were prepared in 0-15 M-NaCl rather than in water and thus a proportion (75 % for 50 mM; 88 % for 20 mM) of the current would have been carried by chloride ions, the transport number for the amino acid remained unaltered. TABLE 1. Potencies of some amino acids as neuronal excitants, relative to the action of L-glutamate (means + S.E. of mean, number of cells tested in parentheses)
Kainate (± )-cius1-amino-1,3-dicarboxycyclopentane Ibotenate N-methyl-DL-aspartate
(± )-tran8-1-amino-1,3-dicarboxycyclopentane DL-Homocysteate L-Glutamate D-Aspartate L-Aspartate D-Glutamate DL-a-Aminopimelate L-a-Aminoadipate L-a-Aminosuberate (± )-ci8-1-amino-1,3-dicarboxycyclohexane (± )-tramsl-amino-1,3-dicarboxycyclohexane
10-62 + 1*70 9 74 + 1P63 7-45 + 1*31 2 84 + 0 54 1 96 ± 0 14 1.84 + 0.42
(12) (20) (18) (11) (17) (17)
1*00 0-95 + 0.08 0 74 + 0 03 0A41+ 0 04 0.23 + 0 04 0415+ 0.02 0.14 + 0 04 0x13 + 0x02 0.02 + 0.02
(17) (24) (21) (16) (24) (8) (14) (5)
Relative potencies Table 1 sets forth the effectiveness of various amino acids as excitants of thalamic neurones relative to the action of L-glutamate. Some of the results are similar to those that have been reported by others (Curtis & Watkins, 1960, 1963; Johnston et al. 1974; Biscoe et al. 1976); however those involving the geometric isomers of ADCH and ADCP are hitherto unreported, and the quantitative assessments here are more firmly based. The implications of the very high potencies of kainate, ibotenate and cis-ADCP will be further discussed below. A consideration of the effects of the stereoisomeric pairs which have been examined is interesting. D-Glutamate was consistently less active than the naturally occurring L-form; but such was not the case for aspartate where D-aspartate was significantly (P < 0.05) more potent than its corresponding stereoisomer. In this context it may be recalled that both D-homocysteate and N-methyl-D-aspartate are similarly much more active than are the L-forms (Curtis & Watkins, 1963). Although L-ac-aminoadipate is a very weak agonist, as has also previously been reported (Curtis & Watkins, 1960), the D-isomer not only showed no excitatory action at all but is in fact an antagonist of amino acid induced excitations (Biscoe et al. 1977a; Biscoe, 2
PHY 286
J. G. HALL AND OTHERS 34 Evans, Francis, Martin, Watkins, Davies & Dray, 1977b; Hall et al. 1977; McLennan & Hall, 1978). There is one feature of the excitations elicited by these various compounds which does not appear from the information contained in Table 1, and that is the matter of the time required to achieve the stable levels of firing from which the values in the Table were derived. It had been noted earlier that the excitations of spinal Renshaw cells by kainate, ibotenate and NMA have a very protracted time course in comparison to that elicited by glutamate (Hutchinson, McLennan & Wheal, 1978); a similar phenomenon has been noted with these compounds in the present study and it applied equally to the excitations induced by cis-ADCP, as is shown in Fig. 1. The effect appears not to be due to the fact that very much smaller currents are required to elicit equivalent excitations with the more potent materials, for increasing their ejecting currents to equal that used for L-glutamate yielded intense excitation and subsequent spike inactivation but with a continued lengthy latency of onset. As is true for kainate and NMA however, it is likely that no cellular uptake process for ADCP exists, and this may be a factor in the slow time course of its action (Cox, Headley & Watkins, 1977). The absence of uptake systems may also contribute to the apparently high potency of these compounds. TABLE 2. Interatomic distances of certain amino acid molecules (A). The maximal and minimal permissible distances between the carboxyl carbon atoms (Cat,-) and between the terminal carboxyl carbon and the nitrogen (CQ,-N) have been separately computed.
CarlC
29
THE EXCITATION OF MAMMALIAN CENTRAL NEURONES BY AMINO ACIDS
BY J. G. HALL*, T. P. HICKS, H. McLENNAN, T. L. RICHARDSON AND H. V. WHEALt From the Department of Physiology, University of Briti8h Columbia, 2075 Wesbrook Place, Vancouver, B.C., Canada V6T iW5
(Received 22 February 1978) SUMMARY
1. The relative potencies of a number of analogues of L-glutamate as excitants of thalamic neurones in the rat have been compared. The most powerful compounds were kainate, ibotenate and (± )cis-1-amino-1,3-dicarboxycyclopentane. The D- and L-isomers of glutamate and aspartate were also compared. Whereas D-glutamate is approximately one-half as active as the L-form, D-aspartate is more potent than L-aspartate. 2. Computer analysis has indicated that ibotenate and cis-1-amino-1,3-dicarboxycyclopentane have relatively fixed and similar Ca-N, C.-N and Cha-Ch interatomic distances which can also be achieved by glutamate in certain conformations of the molecule, but not by aspartate. 3. Parallel examination of the antagonists glutamate diethylester and D-aaminoadipate has shown that the former preferentially reduces L-glutamate effects while the latter blocks the actions of other amino acid excitants more readily than those of L-glutamate. 4. The evidence is consistent with the hypothesis that at least two populations of neuronal receptors for the excitatory amino acids exist. INTRODUCTION
The dicarboxylic amino acids L-glutamate and L-aspartate which occur naturally in the mammalian central nervous system share two properties. Firstly they are capable of depolarizing and exciting neurones to which they are administered extracellularly (Curtis, Phillis & Watkins, 1960) and the ionic basis for this depolarization closely resembles that induced by stimulation of excitatory synapses (Zieglginsberger & Puil, 1972). Secondly, they are taken up into cells of the nervous system by high affinity mechanisms which are distinct from those used to transport other amino acids (Logan & Snyder, 1972). For these reasons among others there is a widespread belief that the two compounds probably function as mediators of some synaptic * Present address: Department of Pharmacology, Australian National University, Canberra, Australia. t Present address: Neurophysiology Group, University of Southampton, Southampton, England.
J. G. HALL AND OTHERS excitations in the central nervous system of mammals (Johnson, 1972; Curtis & Johnston, 1974; McLennan, 1975). In the original studies by Curtis & Watkins (1960, 1963) the actions of a number of other compounds structurally related to glutamate were examined, from which the conclusion was drawn that the presence of the two acidic groups and the cationic site provided by the amino group were all essential, i.e. that a three-point attachment of the agonist molecule with its receptor was required. Over the years there has been no serious challenge to that view though evidence has recently been advanced to support the concept that three rather than a single molecule may react with the receptor to produce its activation (McLennan & Wheal, 1976a). The questions dealing with the precise conformation of the receptor site, whether or not there exist separate receptors for glutamate and for aspartate, and whether there may be non-specific ' amino acid' receptors, possibly extrasynaptic, in addition to specific synaptic receptors (McLennan, Huffman & Marshall, 1968; McLennan, 1970) remain to be established. The considerable flexibility of the glutamate and aspartate molecules do not permit the first two of these questions to be answered since a minimum distance of 2-6 A between the w-carboxyl and the - NH+ group has been claimed to be possible in both (van Gelder, 1971; Buu, Puil & van Gelder, 1976), although of course the distances will differ when the molecules are extended. However, the fact that in some circumstances a selective antagonism of the excitatory action of the two agonists has been shown (Haldeman & McLennan, 1972; Biscoe, Davies, Dray, Evans, Francis, Martin & Watkins, 1977a) suggests that separate receptors for the two molecules may exist. The use of conformationally restricted analogues of the molecules is more promising (Johnston, Curtis, Davies & McCulloch, 1974; Biscoe, Evans, Headley, Martin & Watkins, 1975), and an examination of the excitatory action of certain compounds correlated with the extent of their molecular restraint should provide valuable information. The final question, whether or not there may be both synaptic and extrasynaptic amino acid receptors mediating neuronal excitation, is more difficult to approach but the use of certain antagonists suggests that indeed two populations exist. Brief accounts of certain of the studies to be reported here have appeared (Hall, McLennan & Wheal 1977; McLennan & Hall, 1978; McLennan & Wheal, 1978). 30
METHODS
Assessment of the potencies of a number of compounds as neuronal excitants relative to L-
glutamate has been determined upon neurones of the ventrobasal thalami of rats anaesthetized with urethane (1.5 g/kg I.P.). The animals were placed in a stereotaxic frame, calvarium and dura removed and the exposed cortex covered by a pool of warmed liquid paraffin. Electrodes were introduced stereotaxically into the thalamus and records obtained from cells responding with 8-12 msec latency to electrical stimulation of a contralateral hind limb nerve. Confirmation of electrode placements was achieved by the ejection of Pontamine sky blue at the conclusion of the experiment, and subsequent histological verification. The amino acid agonists were administered electrophoretically from seven barrel micropipettes using intensities of ejecting currents which elicited stable, equal and approximately halfmaximal firing frequencies. Not more than three excitants in addition to L-glutamate were tested in any one series, and for inclusion in the results similar values, which were then averaged, were obtained at least twice on each neurone. It was found important also to preserve the order of presentation of the substances and to maintain constant intervals between administrations.
EXCITATORY AMINO ACIDS
31
A typical series of results presented as integrated firing frequencies displayed on a chart recorder is illustrated by Fig. 1. The relative potencies of the compounds are expressed as ratios of the ejecting currents needed to evoke equal rates of firing in comparison with the current required for L-glutamate. The solutions used to fill the electrode barrels were the following: L- and D-glutamate, L- and D-aspartate, L-a-aminoadipate, DL-a-aminopimelate, L-a-aminosuberate, (± )-ci8 and tran8-1 amino-1,3-dicarboxycyclohexane (ADCH) (all 0*5 M, pH 8); DL-homocysteate (DLH), N-methylDL-aspartate (NMA) and (±)-trans-1-amino-1,3-dicarboxycyclopentane (ADCP) (all 0-2 M, pH 8); kainate, ibotenate and (± )-cis-ADCP (0.1 M, 50 or 20 mM in 0- 15 M-NaCl, pH 8); Lglutamic acid diethylester (GDEE) (0-5 M, pH 3.5); D-a-aminoadipate (DAA) (0-2 M, pH 8). All compounds were obtained from commercial sources except the cyclohexane and cyclopentane derivatives which were synthesized and ibotenate and DAA which were received as gifts. 75
0
a-.
GLUT 13
trans-ADCP 8
cis-ADCP 2
30 sec
Fig. 1. Ratemeter records of the integrated firing frequency of a thalamic neurone. The cell was excited by the electrophoretic ejection of L-glutamate (GLUT) and the tranf8- and cis-isomers of ADCP during the periods shown by the horizontal bars and with the indicated currents in nA. Two factors can affect the validity of the relative potencies determined as above: (a) the linearity of the amount of ion ejected with respect both to the intensity of the applied current and the time of application, and (b) the 'transport numbers' of the substances at the tips of the electrodes (Zieglgansberger, Herz & Teschemacher, 1969; Gent, Morgan & Wolstencroft, 1974). Although it was not possible to examine these factors for all of the materials tested since suitably labelled compounds are not available, the behaviour of three ([3H]L-glutamate, [3H]kainate and [14C]L-aspartate) was observed. The labelled solutions were introduced into micropipettes and the quantities ejected into 0- 5 ml. 0-15 M-NaCl assessed by liquid scintillation analysis. Very similar results were obtained with three different micropipette assemblies. The transport number for kainate was also examined using solutions of several different concentrations in the pipette: 0-1 M in water and 20 and 50 mm in 0-15 M-NaCl. Computer models of many of the compounds examined were constructed from crystallographic data, and could be displayed on a quasi-three-dimensional graphics terminal. The program, details of which will appear elsewhere, permitted the various interatomic bonds of the molecules to be rotated individually while the whole molecule was displayed; and a table of bond angles and interatomic distances for any desired configuration could be obtained. Plots of the computed images of L-glutamate in configurations where the C,,-N distances are maximal and minimal, together with the corresponding distances between the presumed three active groups are given in Fig. 2 (see also Table 2).
32
J. G. HALL AND OTHERS RESULTS
Tranmport numbers Three electrodes whose barrels contained radioactively labelled solutions of amino acids were used to determine transport numbers. The results from one electrode where L-glutamate was ejected with different intensities of current and for various periods G lu min
Glu-max
"0(9) N(1 0)
Cx-C,,401 A
Cx-C,,, 4*56 A C-, N 2-43 A C^,-N 513 A
CA-N 2 43 A Cs,-N 2 23 A
Fig. 2. Reproductions of computer models of L-glutamate, manipulated so that the
Q),,-N is minimal (Glu-min) or maximal (Glu-max). C. lies between 0 (8) and 0 (9); C. between 0 (1) and 0 (2), and the amino group is indicated by N (10). The correspond-
ing interatomic distances (A) are given. a)
E
I--
C W a)
0)
E
-J
60 Time (mmn)
120
0
125 Current (nA)
250
Fig. 3. The electrophoretic expulsion of [3H]L-glutamate from a pipette (A) as a function of time with constant ejecting current and (B) as a function of current with a constant time of ejection. The 60 min/150 nA point is identical in the two graphs.
of time are illustrated by Fig. 3, and it is clear that there existed an essentially linear relationship between the quantity ejected and those two variables. The transport number for the amino acid in this case was 0-22; and with the same electrode the corresponding values for aspartate and kainate (from a solution of 01 M) were
EXCITATORY AMINO ACIDS 33 0-28 and 0'24 respectively. The value for glutamate is at the lower end of the range reported by Zieglginsberger et al. (1969); more importantly in the present context there is no substantial difference between the values for the three amino acids and thus variations in transport number would appear not to contribute significantly in this instance to changes in the apparent potency of the agonists. Similar results were obtained with the other two electrode assemblies tested and the transport numbers for the three amino acids were approximately equal in every case. An analysis was also made of the effect of concentration of kainate in the electrode on its transport number. When allowance was made for the fact that the diluted solutions were prepared in 0-15 M-NaCl rather than in water and thus a proportion (75 % for 50 mM; 88 % for 20 mM) of the current would have been carried by chloride ions, the transport number for the amino acid remained unaltered. TABLE 1. Potencies of some amino acids as neuronal excitants, relative to the action of L-glutamate (means + S.E. of mean, number of cells tested in parentheses)
Kainate (± )-cius1-amino-1,3-dicarboxycyclopentane Ibotenate N-methyl-DL-aspartate
(± )-tran8-1-amino-1,3-dicarboxycyclopentane DL-Homocysteate L-Glutamate D-Aspartate L-Aspartate D-Glutamate DL-a-Aminopimelate L-a-Aminoadipate L-a-Aminosuberate (± )-ci8-1-amino-1,3-dicarboxycyclohexane (± )-tramsl-amino-1,3-dicarboxycyclohexane
10-62 + 1*70 9 74 + 1P63 7-45 + 1*31 2 84 + 0 54 1 96 ± 0 14 1.84 + 0.42
(12) (20) (18) (11) (17) (17)
1*00 0-95 + 0.08 0 74 + 0 03 0A41+ 0 04 0.23 + 0 04 0415+ 0.02 0.14 + 0 04 0x13 + 0x02 0.02 + 0.02
(17) (24) (21) (16) (24) (8) (14) (5)
Relative potencies Table 1 sets forth the effectiveness of various amino acids as excitants of thalamic neurones relative to the action of L-glutamate. Some of the results are similar to those that have been reported by others (Curtis & Watkins, 1960, 1963; Johnston et al. 1974; Biscoe et al. 1976); however those involving the geometric isomers of ADCH and ADCP are hitherto unreported, and the quantitative assessments here are more firmly based. The implications of the very high potencies of kainate, ibotenate and cis-ADCP will be further discussed below. A consideration of the effects of the stereoisomeric pairs which have been examined is interesting. D-Glutamate was consistently less active than the naturally occurring L-form; but such was not the case for aspartate where D-aspartate was significantly (P < 0.05) more potent than its corresponding stereoisomer. In this context it may be recalled that both D-homocysteate and N-methyl-D-aspartate are similarly much more active than are the L-forms (Curtis & Watkins, 1963). Although L-ac-aminoadipate is a very weak agonist, as has also previously been reported (Curtis & Watkins, 1960), the D-isomer not only showed no excitatory action at all but is in fact an antagonist of amino acid induced excitations (Biscoe et al. 1977a; Biscoe, 2
PHY 286
J. G. HALL AND OTHERS 34 Evans, Francis, Martin, Watkins, Davies & Dray, 1977b; Hall et al. 1977; McLennan & Hall, 1978). There is one feature of the excitations elicited by these various compounds which does not appear from the information contained in Table 1, and that is the matter of the time required to achieve the stable levels of firing from which the values in the Table were derived. It had been noted earlier that the excitations of spinal Renshaw cells by kainate, ibotenate and NMA have a very protracted time course in comparison to that elicited by glutamate (Hutchinson, McLennan & Wheal, 1978); a similar phenomenon has been noted with these compounds in the present study and it applied equally to the excitations induced by cis-ADCP, as is shown in Fig. 1. The effect appears not to be due to the fact that very much smaller currents are required to elicit equivalent excitations with the more potent materials, for increasing their ejecting currents to equal that used for L-glutamate yielded intense excitation and subsequent spike inactivation but with a continued lengthy latency of onset. As is true for kainate and NMA however, it is likely that no cellular uptake process for ADCP exists, and this may be a factor in the slow time course of its action (Cox, Headley & Watkins, 1977). The absence of uptake systems may also contribute to the apparently high potency of these compounds. TABLE 2. Interatomic distances of certain amino acid molecules (A). The maximal and minimal permissible distances between the carboxyl carbon atoms (Cat,-) and between the terminal carboxyl carbon and the nitrogen (CQ,-N) have been separately computed.
CarlC
