- Email: [email protected]
The determination of the extracellular concentration of brain glutamate using quantitative microdialysis
BRAIN RESEARCH ELSEVIER
Brain Research 707 (1996) 131-133
Short communication
The determination of the extracellular concentration of brain glutamate using quantitative microdialysis M. Miele 1, M. Berners, M.G. Boutelle 2, H. Kusakabe 3, M. Fillenz * University Laboratory of Physiology, Oxford OX1 3PT, UK Accepted 24 October 1995
Abstract
Quantitative microdialysis with two enzyme-based assays was used to determine the extracellular concentration of glutamate in the striatum of freely moving rats. From the difference between infused and dialysate glutamate a value of 3.0 + 0.6/.~M for the extracellular glutamate concentration was computed by regression analysis. The in vivo recovery, derived from the slope of the regression line, was 50%. Keywords: Quantitative micmdialysis; Glutamate; Rat striatum; Enzyme assay; Extracellular concentration; Unanesthetized
Glutamate is the main excitatory neurotransmitter [12] and acts on five different classes of receptors [4]. Three of these are ionotropic receptors whose threshold for activation and desensitisation is 0.5-10 /zM [7]. For a full assessment of the extracellular role of glutamate it is therefore important to know its concentration in the extracellular fluid (ECF). Dialysate glutamate concentrations in the literature vary widely with the experimental conditions. The true ECF concentration of glutamate cannot be derived from the in vitro probe recovery since additional factors determine the in vivo recovery [11]. A number of methods have been developed for the determination of the ECF concentration of analytes [5,6,9,10]. In the present study we have used the zero net flux method of L/Snnroth [10] to determine the extracellular concentration of glutamate in the striatum of freely moving rats. Two enzyme-based assays, which both use glutamate oxidase (EC 1.4.3.11) [8] were used. In one
Abbreviations: ECF, extracellular fluid; PPD, poly-(phenylenediamine) * Corresponding author. Fax: (44) (1865) 272469. E-mail: [email protected] 1 Present address: Department of Pharmacology, University of Sassari, 07100 Sassari, Italy. 2 Present address: Department of Chemistry, King's College London, London WC2R, UK. 3Present address: Yamasa Corporation, 10-1 Araoieho 2-chome Choshi, Chiba-ken, 288, Japan. 0006-8993/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 1 3 7 1 - 7
method immobilised enzyme is used in a packed bed and flow injection analysis is carried out at 5 rain intervals [2]; in the other an enzyme-coated tube electrode gives a continuous record of glutamate levels in the dialysate [1]. Male Sprague-Dawley rats, weighing between 180 and 300 g, were anaesthetised using fentanyl/fluanisone and midazolam. A concentric microdialysis probe, 4 mm in length, was implanted in the right striatum. Following surgery the animals were placed in large plastic bowls and maintained in a temperature- and light-controlled environment, with free access to food and water. All procedures were specifically licensed under the Animals (Scientific Procedures) Act 1986. Experiments were carried out 24 h after surgery. The microdialysis probe was perfused with aCSF of the following composition in mM: NaCL 147, KC1 4, CaCI 1.2, MgCI 1. at a flow rate of 2 /~l/min. A series of different glutamate concentrations, chosen to bracket the expected ECF concentration, were perfused in random order. A linear regression through the points obtained by plotting the difference of perfusate and dialysate concentration (AC = Cin - Coat) VS. perfusate concentrations (Cin) yields a slope which represents the recovery. The true ECF concentration is the point at which AC = 0. Two sets of zero-net-flux experiments (4 animals each) were performed using either the tubular electrodes or the packed enzyme bed. When using the tube electrode for the balance experiments the various concentrations of gluta-
132
M. Miele et al. /Brain Research 707 (1996) 131-133
mate were alternately perfused through the animal and directly through the tubular electrode using a flow switcher. This allowed direct comparisons of Ci, and Co, t without interpolation from calibration curves. Points of zero net flux and recoveries including standard errors were averaged from individual experiments and standard errors calculated using an error propagation formula [13]. The basal striatal dialysate glutamate concentration, using the flow injection method and enzyme bed, was 1.3 + 0.3 /~M (n = 4). This level remained constant provided the animal was quiescent, but fluctuated with activity. Only experiments in which data were collected during a period of quiescence were included. Glutamate concentrations ranging from 0 to 5 /xM were perfused. The resulting changes in dialysate concentration reached a steady level within 10 min. Regression analysis showed the point of zero net flux as 2.6 + 0.5 /xM (n = 4) The in vivo recovery derived from the slope of the curve was 50 _ 1% (Fig. la). In experiments using the tubular enzyme electrode the basal dialysate concentration of glutamate was 1.6 + 0.2 /zM (n = 5) and the point of zero net flux was 3.3 + 0.2 /xM (n = 4) (Fig. lb). The recovery in vivo derived from the slope of the curve was 50 ± 6%. There is no statistically significant difference between the results of the two assay systems. The pooled results gave a figure of 3.0 ___0.6 /xM (n = 8) for extracellular glutamate. Published dialysate glutamate levels, obtained under very different experimental conditions, vary greatly between 0.2 and 2.5/xM [3]. There are no previous measurements of ECF glutamate in unanaesthetised animals. The two published ECF concentrations in anaesthetised animals are 2.9/zM in the rat hippocampus (using the recirculation method) [9], and 4.3 ~ M in the rabbit olfactory bulb (using extrapolation to zero flow) [6]; these are remarkably close to the present values, which suggests that anaesthesia does not affect basal ECF glutamate. The difference between dialysate and ECF concentration represents the in vivo recovery. Whereas values for ECF concentration are method independent, values for in vivo recovery are not. They show variation with flow rate and probe characteristics; indeed it is this variation in in vivo recovery that gives rise to the variation in reported dialysate levels. Zero net flux experiments with glutamate present some special problems. One is the fluctuation of dialysate glutamate levels with the activity of the animal. Another is the fact that infusion of glutamate can sometimes trigger further release of glutamate [14]. Glutamate receptors in the synaptic cleft mediate excitatory transmission. There are also a variety of extrasynaptic receptors on both glia and neurons. The kinetic properties of metabotropic receptors have not been determined. Patch clamp m e a s u r e m e n t s of kinetic parameters of AMPA/kainate receptors suggest that at maintained con-
a' I
0
1
2
I
I
3
4
glutamate (pM) b) A
:i
::L
2-
1
v
m
E
o
,<]
-1-
s
-20
I
I
I
t
1
2
3
4
5
glutamate (I~M) Fig. 1. In vivo zero net flux experiments. The difference between the perfusate and the striatal dialysate concentration is plotted against the perfusate concentration (mean+ S.E.M.). a: experiments measured using the enzyme bed system (n= 4); b: experiments measured using the tubular electrode system(n = 4).
centrations of 3 / z M - 9 / x M glutamate 50% of the receptors would be desensitised [7]. The high value for ECF glutamate determined ion the present study raises the question of the function of the extrasynaptic receptors.
Acknowledgements M.B. is supported by the Dunhill Medical Trust. M.M. acknowledges Grant 95.00853.CT04 from the C.N.R.
References [1] Berners, M.O.M., BouteUe, M.G. and Fillenz, M., On-line measurement of brain glutamate with an enzyme/polymer-coatedtubular electrode, Anal. Chem., 66 (1994) 2017-2021. [2] Boutelle, M.G., Fellows, L.K. and Cook, C., Enzyme packed bed systemfor on-line measurementof glucose, glutamate,and lactate in brain microdialysate, Anal. Chem., 64 (1992) 1790-1794. [3] Fillenz, M., Physiologicalrelease of excitatory amino acids, Behav. Brain Res., in press.
M. Miele et al. /Brain Research 707 (1996) 131-133 [4] Hollman, M. and Heinemann, S., Cloned glutamate receptors, Annu. Rev. Neurosci., 17 (1994) 31-108. [5] Jacobson, I. and Hamberger, A., Veratridine-induced release in vivo and in vitro of amino-acids in the rabbit olfactory-bulb, Brain Res., 299 (1984) 103-112. [6] Jacobson, I., Sanberg, M. and Hamberger, A., Mass transfer in brain dialysis devices--a new method for the estimation of extracellular amino acid concentration, J. Neurosci. Meth., 15 (1985) 263-268. [7] Jonas, P., Glutamate receptors in the central nervous system, Ann. NYAcad. Sci., 707 (1993) 126-135. [8] Kusakabe, H., Midorikawa, Y., Fujishima, T., Kuninaka, A. and Yoshino, H., Purification and properties of a new enzyme, LGlutamtae oxidase, from Streptomyces sp. X-119-6 grown on wheat bran, Agric. Biol. Chem., 47 (1983) 1323-1328. [9] Lerma, J., Herranz, A.S., Herreras, O., Abraira, B. and Rio, R.M.D., In vivo determination of extracellular concentration of amino acids in the rat hippocampus. A method based on brain dialysis and computerized analysis, Brain Res., 384 (1986) 145-155.
133
[10] L6nnroth, P., Jansson, P.-A. and Smith, U., A microdialysis method allowing characterization of intercellular water space in humans, Am. J. Physiol., 253 (1987) E228-E231. [11] Morrison, P.F., Bungay, P.M., Hsiao, J.K., Mefford, I.N., Dykstra, K.H. and Dedrick, R.L., Quantitative Microdialysis. In T.E. Robinson and J.B. Justice Jr. (Eds.), Microdialysis in the Neurosciences, Elsevier, Amsterdam, 1991, pp. 47-80. [12] Orrego, F. and Villanueva, S., The chemical nature of the main central excitatory transmitter: a critical appraisal based upon release studies and synaptic vesicle localization, Neuroscience, 56 (1993) 539-555. [13] Skoog, D.A. and Leary, J.A., Principles of Instrumental Analysis, Sannders College Publishing, Philadelphia, USA, 1992. [14] Young, A., Crouder, J. and Bradford, H., Potentiation by kainate of EAA release in striatum: complementary in vivo and in vitro experiments, J. Neurochem., 50 (1988) 337-345.
Brain Research 707 (1996) 131-133
Short communication
The determination of the extracellular concentration of brain glutamate using quantitative microdialysis M. Miele 1, M. Berners, M.G. Boutelle 2, H. Kusakabe 3, M. Fillenz * University Laboratory of Physiology, Oxford OX1 3PT, UK Accepted 24 October 1995
Abstract
Quantitative microdialysis with two enzyme-based assays was used to determine the extracellular concentration of glutamate in the striatum of freely moving rats. From the difference between infused and dialysate glutamate a value of 3.0 + 0.6/.~M for the extracellular glutamate concentration was computed by regression analysis. The in vivo recovery, derived from the slope of the regression line, was 50%. Keywords: Quantitative micmdialysis; Glutamate; Rat striatum; Enzyme assay; Extracellular concentration; Unanesthetized
Glutamate is the main excitatory neurotransmitter [12] and acts on five different classes of receptors [4]. Three of these are ionotropic receptors whose threshold for activation and desensitisation is 0.5-10 /zM [7]. For a full assessment of the extracellular role of glutamate it is therefore important to know its concentration in the extracellular fluid (ECF). Dialysate glutamate concentrations in the literature vary widely with the experimental conditions. The true ECF concentration of glutamate cannot be derived from the in vitro probe recovery since additional factors determine the in vivo recovery [11]. A number of methods have been developed for the determination of the ECF concentration of analytes [5,6,9,10]. In the present study we have used the zero net flux method of L/Snnroth [10] to determine the extracellular concentration of glutamate in the striatum of freely moving rats. Two enzyme-based assays, which both use glutamate oxidase (EC 1.4.3.11) [8] were used. In one
Abbreviations: ECF, extracellular fluid; PPD, poly-(phenylenediamine) * Corresponding author. Fax: (44) (1865) 272469. E-mail: [email protected] 1 Present address: Department of Pharmacology, University of Sassari, 07100 Sassari, Italy. 2 Present address: Department of Chemistry, King's College London, London WC2R, UK. 3Present address: Yamasa Corporation, 10-1 Araoieho 2-chome Choshi, Chiba-ken, 288, Japan. 0006-8993/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 1 3 7 1 - 7
method immobilised enzyme is used in a packed bed and flow injection analysis is carried out at 5 rain intervals [2]; in the other an enzyme-coated tube electrode gives a continuous record of glutamate levels in the dialysate [1]. Male Sprague-Dawley rats, weighing between 180 and 300 g, were anaesthetised using fentanyl/fluanisone and midazolam. A concentric microdialysis probe, 4 mm in length, was implanted in the right striatum. Following surgery the animals were placed in large plastic bowls and maintained in a temperature- and light-controlled environment, with free access to food and water. All procedures were specifically licensed under the Animals (Scientific Procedures) Act 1986. Experiments were carried out 24 h after surgery. The microdialysis probe was perfused with aCSF of the following composition in mM: NaCL 147, KC1 4, CaCI 1.2, MgCI 1. at a flow rate of 2 /~l/min. A series of different glutamate concentrations, chosen to bracket the expected ECF concentration, were perfused in random order. A linear regression through the points obtained by plotting the difference of perfusate and dialysate concentration (AC = Cin - Coat) VS. perfusate concentrations (Cin) yields a slope which represents the recovery. The true ECF concentration is the point at which AC = 0. Two sets of zero-net-flux experiments (4 animals each) were performed using either the tubular electrodes or the packed enzyme bed. When using the tube electrode for the balance experiments the various concentrations of gluta-
132
M. Miele et al. /Brain Research 707 (1996) 131-133
mate were alternately perfused through the animal and directly through the tubular electrode using a flow switcher. This allowed direct comparisons of Ci, and Co, t without interpolation from calibration curves. Points of zero net flux and recoveries including standard errors were averaged from individual experiments and standard errors calculated using an error propagation formula [13]. The basal striatal dialysate glutamate concentration, using the flow injection method and enzyme bed, was 1.3 + 0.3 /~M (n = 4). This level remained constant provided the animal was quiescent, but fluctuated with activity. Only experiments in which data were collected during a period of quiescence were included. Glutamate concentrations ranging from 0 to 5 /xM were perfused. The resulting changes in dialysate concentration reached a steady level within 10 min. Regression analysis showed the point of zero net flux as 2.6 + 0.5 /xM (n = 4) The in vivo recovery derived from the slope of the curve was 50 _ 1% (Fig. la). In experiments using the tubular enzyme electrode the basal dialysate concentration of glutamate was 1.6 + 0.2 /zM (n = 5) and the point of zero net flux was 3.3 + 0.2 /xM (n = 4) (Fig. lb). The recovery in vivo derived from the slope of the curve was 50 ± 6%. There is no statistically significant difference between the results of the two assay systems. The pooled results gave a figure of 3.0 ___0.6 /xM (n = 8) for extracellular glutamate. Published dialysate glutamate levels, obtained under very different experimental conditions, vary greatly between 0.2 and 2.5/xM [3]. There are no previous measurements of ECF glutamate in unanaesthetised animals. The two published ECF concentrations in anaesthetised animals are 2.9/zM in the rat hippocampus (using the recirculation method) [9], and 4.3 ~ M in the rabbit olfactory bulb (using extrapolation to zero flow) [6]; these are remarkably close to the present values, which suggests that anaesthesia does not affect basal ECF glutamate. The difference between dialysate and ECF concentration represents the in vivo recovery. Whereas values for ECF concentration are method independent, values for in vivo recovery are not. They show variation with flow rate and probe characteristics; indeed it is this variation in in vivo recovery that gives rise to the variation in reported dialysate levels. Zero net flux experiments with glutamate present some special problems. One is the fluctuation of dialysate glutamate levels with the activity of the animal. Another is the fact that infusion of glutamate can sometimes trigger further release of glutamate [14]. Glutamate receptors in the synaptic cleft mediate excitatory transmission. There are also a variety of extrasynaptic receptors on both glia and neurons. The kinetic properties of metabotropic receptors have not been determined. Patch clamp m e a s u r e m e n t s of kinetic parameters of AMPA/kainate receptors suggest that at maintained con-
a' I
0
1
2
I
I
3
4
glutamate (pM) b) A
:i
::L
2-
1
v
m
E
o
,<]
-1-
s
-20
I
I
I
t
1
2
3
4
5
glutamate (I~M) Fig. 1. In vivo zero net flux experiments. The difference between the perfusate and the striatal dialysate concentration is plotted against the perfusate concentration (mean+ S.E.M.). a: experiments measured using the enzyme bed system (n= 4); b: experiments measured using the tubular electrode system(n = 4).
centrations of 3 / z M - 9 / x M glutamate 50% of the receptors would be desensitised [7]. The high value for ECF glutamate determined ion the present study raises the question of the function of the extrasynaptic receptors.
Acknowledgements M.B. is supported by the Dunhill Medical Trust. M.M. acknowledges Grant 95.00853.CT04 from the C.N.R.
References [1] Berners, M.O.M., BouteUe, M.G. and Fillenz, M., On-line measurement of brain glutamate with an enzyme/polymer-coatedtubular electrode, Anal. Chem., 66 (1994) 2017-2021. [2] Boutelle, M.G., Fellows, L.K. and Cook, C., Enzyme packed bed systemfor on-line measurementof glucose, glutamate,and lactate in brain microdialysate, Anal. Chem., 64 (1992) 1790-1794. [3] Fillenz, M., Physiologicalrelease of excitatory amino acids, Behav. Brain Res., in press.
M. Miele et al. /Brain Research 707 (1996) 131-133 [4] Hollman, M. and Heinemann, S., Cloned glutamate receptors, Annu. Rev. Neurosci., 17 (1994) 31-108. [5] Jacobson, I. and Hamberger, A., Veratridine-induced release in vivo and in vitro of amino-acids in the rabbit olfactory-bulb, Brain Res., 299 (1984) 103-112. [6] Jacobson, I., Sanberg, M. and Hamberger, A., Mass transfer in brain dialysis devices--a new method for the estimation of extracellular amino acid concentration, J. Neurosci. Meth., 15 (1985) 263-268. [7] Jonas, P., Glutamate receptors in the central nervous system, Ann. NYAcad. Sci., 707 (1993) 126-135. [8] Kusakabe, H., Midorikawa, Y., Fujishima, T., Kuninaka, A. and Yoshino, H., Purification and properties of a new enzyme, LGlutamtae oxidase, from Streptomyces sp. X-119-6 grown on wheat bran, Agric. Biol. Chem., 47 (1983) 1323-1328. [9] Lerma, J., Herranz, A.S., Herreras, O., Abraira, B. and Rio, R.M.D., In vivo determination of extracellular concentration of amino acids in the rat hippocampus. A method based on brain dialysis and computerized analysis, Brain Res., 384 (1986) 145-155.
133
[10] L6nnroth, P., Jansson, P.-A. and Smith, U., A microdialysis method allowing characterization of intercellular water space in humans, Am. J. Physiol., 253 (1987) E228-E231. [11] Morrison, P.F., Bungay, P.M., Hsiao, J.K., Mefford, I.N., Dykstra, K.H. and Dedrick, R.L., Quantitative Microdialysis. In T.E. Robinson and J.B. Justice Jr. (Eds.), Microdialysis in the Neurosciences, Elsevier, Amsterdam, 1991, pp. 47-80. [12] Orrego, F. and Villanueva, S., The chemical nature of the main central excitatory transmitter: a critical appraisal based upon release studies and synaptic vesicle localization, Neuroscience, 56 (1993) 539-555. [13] Skoog, D.A. and Leary, J.A., Principles of Instrumental Analysis, Sannders College Publishing, Philadelphia, USA, 1992. [14] Young, A., Crouder, J. and Bradford, H., Potentiation by kainate of EAA release in striatum: complementary in vivo and in vitro experiments, J. Neurochem., 50 (1988) 337-345.