Preferential release of neuroactive amino acids from the ventrolateral medulla of the rat in vivo as measured by microdialysis

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PREFERENTIAL RELEASE OF NEUROACTIVE AMINO ACIDS FROM THE VENTROLATERAL MEDULLA OF THE RAT IN I/W0 AS MEASURED BY MICRODIALYSIS V. KAPOOR,* D. NAKAHARA,~ R.J. BLOOD and J.P. CHALMERS* *Department of Medicine and Centre for Neuroscience, Flinders Medical Centre, Bedford Park, SA 5042, Austraiia *Department of Psychology, Nagoya University College of Medical Technology, Nagoya, Japan Abstract-The basal overflow of extracellular endogenous amino acids was measured from the ventrolateral medulla of urethane anaesthetized rats in viuo by microdialysis. Inclusion of a mercury salt, p-chloromercuriphenylsulphonic acid, in the dialysate (Krebs’ solution), results in a preferential increase in the overfiow of aspartate, glutamate, glycine and GABA. A smaller increase in the overflow of the glutamate precursor and metabolite, glutamine, was also found. There was no ~gnifi~nt change in the basal extracellular levels of taurine, asparagine, alanine, se&e, omithine or lysine. Inclusion of a speciiic GABA uptake inhibitor, nipecotic acid, in the dialysate results in an immediate, dose dependent increase in the overfiow of GABA, and to a lesser extent, taurine. Since it is likely that mercury salts increase neurotransmitter release by increasing free intracellular calcium ion concentrations, it is suggested that these results provide further evidence for a physiologically relevant neurotransmitter role for aspartate, glutamate, glycine and GABA in the ventrolateral medulla.

The medulla has long been implicated in the tonic and reflex control of blood pressure.* There is accumulat-

ing evidence that amino acid pathways within the medulla play a crucial role in this respect. Amino acid excitatory and inhibitor receptors have been demonstrated within the rostra], vasopressor region of the ventrolateral medulla (VLM)23.24and the caudal vasodepressor region. lo Injections of glutamate (Glu) into the rostra1 VLM increase blood pressure” whereas Glu antagonists and GABAergic agonists produce a fall in blood pressure.” The pressor responses observed during activation of the rostra1 VLM, acting via sympathetic preganglionic neurons in the spinal cord, are attenuated by intrathecal administration of excitatory amino acid antagonists, suggesting that the major descending output from the VLM may also involve amino acid neurotransmitters.23 Recently, a major amino acid pathway from the nucleus tractus solitarius to the VLM has been demonstrated by autoradiography after the accumulation of [3H]-naspartate, a specific excitatory amino acid pathway marker.” Physiolo~cal and neuroanatomical studies do not, in themselves, provide conclusive evidence for a specific neurotransmitter role of amino acids, especially since neuroactive amino acids like Glu and GABA have widespread excitatory and inhibitory actions respectively. In an attempt to gain further evidence for the existence and nature of these amino acid pathways we have measured extracellular leveis Abbrwiuriom: NPA, nipecotic acid; pCMB,pchloromercuri-

benzoate; pCMP, p-chloromercuriphenyl-sulphonate; Tau, taurine; VLM, ventrolateral medulla. I87

of endogenous amino acids with the VLM using Extracellular levels of amino in uivo microdialysis. 2**35 acids are necessarily related to metabolic as well as neurogenic events, hence unlike dopamine and acetylcholine the basai release of neuroactive amino acids, as measured by microdialysis, is not calcium ion or tetrodotoxin sensitive,” and in fact may increase in the absence of calcium ions9q3’(V. Kapoor and J. Chalmers, unpublished observation). Several authors have shown an increased overflow of neuroactive amino acids in the striatum with specific neurotoxins such as kainic acid,‘.” tityustoxinM and veratrine.6 Unfortunately, the mechanism of kainic acid induced effects is controversial7*37 and this effect does not show calcium dependence in viva.’ While the veratrine induced effects on amino acid release are tetrodotoxin sensitive it does cause smaller increases in the overflow of some non-neuroactive amino acids as we1l.6 These studies have also demonstrated reductions in extracellular levels of neuroactive amino acids after selective lesions of presumed amino acid pathways, namely the corticostriatal pathway, providing some evidence for a neurotran~itter function for Gh.F and GABAt in particular. Mercury salts have been shown to increase the release and inhibit the high-affinity uptake of several transmitters in vitro. Inhibition of rH]Glu, [%lGABA and [‘H]monoamine uptake4yt6 and the specific release of Iabelled preloaded dopamine and acetylcholine in rat brain synaptosomes by mercury salts have been reported. ‘.I* The apparent uptake inhibition or increases in overtlow of exogenous prelabelled transmitters may be the net result of increased release, inhibition of uptake or both. Importantly, however,

188

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increased spontaneous and evoked (quanta]) release of acetylcholine by mercury salts has been demonstrated at the neuromuscular junction (measured electrophysiologically), along with inhibition of the uptake of calcium ions at intracellular sites like mitochondria.3 More recently, Tachikawa et ~1.~~have demonstrated a calcium dependent exototic release of endogenous catecholamines from cultured adrenal medullary cells in the presence of organic mercury compounds, p-chloromercuribenzoate ( pCMB) and p-chloromercuriphenyl-sulphonate (pCMP). pCMB induced increases in the intracellular calcium ion concentration, the co-release of dopamine-b-hydroxylase but not phenylethanolamine-l-methyltransferase, and the reversal of the catecholamine release with dithiotheritol suggests that the catecholamine release induced by the organic mercuryls was exocytotic and involves the modification of sulphydryl groups.3Z Low doses of methylmercury which are sufficient to inhibit [3H]Glu transport in vitro, do not affect 2-[1,2‘Hldeoxy-o-glucose uptake, suggesting that the Na +, K+-ATPase and hexokinase activity is unaltered and that the effect of mercury ions on the two systems may not be related.’ Taken together these data suggest that the apparent uptake inhibition of neurotransmitters by mercury salts may actually be due to an increased release, secondary to increased intracellular free calcium ion concentrations as a result of increased calcium ion influx,32 impaired intracellular calcium sequestering mechanisms,3.‘6.27 or both. In the present experiments we have used a mercury salt, pCMP, and a specific GABA uptake inhibitor, nipecotic acid (NPA),j2 to try and demonstrate the neuronal release of specific neuroactive amino acids, without a depolarizing stimulus, and to try and establish the presence of high affinity uptake sites for GABA. EXPERIMENTAL PROCEDURES

Male Wistar Kyoto rats (250-300g) were anaesthetixed with urethane (1 g/kg, i.p.) and placed in a small-animal stereotaxic frame. The skull was exposed and a small burr hole drilled above the medulla (coordinates in mm: AP -3.7, L 2.0 from the interaural line according to the atlas of Paxinos and Watson26). A dialysis probe was then stereotaxically lowered to a depth of 9.2 mm below the surface of the cerebellum. The probe was continuously perfused with Krebs’ solution (composition in mM; Na Cl 124; NaHCO, 25; KC1 3.3; KH,PQ, 0.4; M&O, 1.2; CaCI, 2.0; pH = 7.4) at a flow rate of 1 yl/min. After a 90-min equilibration period the samples were collected, at IO-min intervals, directly in auto sampler micro-vials containing 2 ~1 of a 10% sodium axide solution. The first three samples were used to establish the basal amino acid owrflow. All subsequent samples were collected with Krebs’ solution, or with Krebs’ solution containing 0.1 mM pCMP, 5 mM NPA, or 20mM NPA. At the end of the experiment the animals were decapitated and the brains processed for histological verification of probe placement. Dialysisprobe consrrucrion The dialysis probes used in the present study were similar in construction to the ones we have previously described.” The only difference was that silica-coated glass capillary

al.

tubing (lOVS-170/110, Scientific Glass Engineering, Austraha) supported by a 24-gauge stainless steel tube, was used in the construction of the probes. The glass tubing protruded 1mm from the end of the stainless steel supporting tube, and was covered by dialysis tubing (Clarins TE07, Terumo Corp., Japan). The effective length of the probes used was kept at 1mm. Amino acid determination

Amino acid content of the dialysates was determined by high-performance liquid chromatography with fluorescence detection after automatic precolumn derivatizatian with ortho-phthaldialdehyde. 2i Separation of the amino acid derivatives was achieved using a Spherisorb ODS II column (150 x 4.6 mm) by gradient elution. The mobile phase consisted of a sodium phosphate buffer (50mM, pH 5.8 containing 15% methanol) mixed in a stepwise gradient with methanol containing 2% tetrahydrofuran, starting with 100% buffer. The gradient change was as follows: start 0% methanol: 100% buffer; at 2 min 15:85%; at 22 min 60:40%; at 25min 60:40%. Over the next 10min the gradient returned to initial conditions. The pumps (K25M, ETP Kortec. Austratia) were controlled bv an Keithfev 500 series D/A converter (Keithley DAS, U.S.A.) in conjunction with an IBM XT microcomputer. An AS 2000 automatic injector (ICI instruments, Australia) was used to automate the precolumn derivatization, with an FP-210 spectrofluorometer (Jasco, Japan) as the detector (excitation = 330 nm, emission = 455 nm). Quantitation of the amino acid content of the samples was performed by peak height analysis and comparison with standard solutions. The results are presented as pmol/sample * standard error of the mean (not corrected for recovery). The statistical analysis of the data was performed using an ANOVA followed by Dunnett’s test. Two sets of data were considered to be significantly different when P < 0.05.

RESULTS

In control animals the extracellular concentration of the major amino acids deteetable in the dialysate was stable over the 3.5-h duration of the experiment (Fig. I). The absolute levels of the amino acids found during this study are generally in good agreement with those reported by other workers, in other regions of the brain.33*35In these control animals a manual switch for Krebs’ back to Krebs’ solution was performed during sample 3 to ensure that there was no “switching artifact” in sample 4 due to the accidental introduction of bubbles or a pause in the flow. Effect of p-chloromercuriphenyl-sulphonate cellular amino acid levels

on extra-

In another group of rats, after the equilibration period (90 min) and the collection of the first three samples, the Krebs’ perfusing solution was switched with one containing 0.1 mM pCMP. Inclusion of pCMP led to a specific increase in the extracellular concentration of some of the neuroactive amino acids (Fig. 1). These increases were noted to be specific and gradual, as had been reported in vitro previously,27 reaching maximum levels 40-60 min after the introduction of the pCMP. The greatest increases noted was a five- to six-fold rise in the levels of aspartate (Asp), Glu and GABA. An approximately three-fold

Microdialysis of neuroactive amino acids from the medulla

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10

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Time (minutes) Fig. 1. The effect acids as measured lines and animals the broken lines

of the mercury salt, pCMP, on the extracellular concentrations of endogenous amino by microdialysis in the rat medulla. Control animals (n = 7) are indicated by the solid treated by inclusion of pCMP in the dialysate (marked by the arrows) are indicated by (n = 7). Each sample represents a lO-min collection period. *P < 0.05 vs control, Dunnett’s test.

increase in glycine (Gly) and a two-fold rise in glutamine (Gln) was also found, while there appeared to be a tendency for taurine (Tau) to show an increase, it was not significantly elevated from control values by Dunnett’s test. There was no apparent change in the extracellular levels of asparagine (Asn), alanine (Ala), serine (Ser), omithine (Om) or lysine (Lys) (Fig. 1).

dent but also very rapid, so that a over 70% of the maximal effect was seen within one sampling time, i.e. 10 min. Extracellular levels to Tau were also markedly increased in the presence of NPA, however, the increases in Tau appeared to have a much slower time course. There was no change in the extracellular levels of any of the other amino acids measured (Fig. 2).

Effect of nipecotic acid on extracellular amino acid levels

Inclusion of NPA in the dialysate led to a dramatic, dose dependent increase in the extracellar levels of GABA (Fig. 2). This effect was not only dose depen4

DISCUSSION

Increased overflow of neuroactive amino acids In the present study we have demonstrated specific increases in the overflow in vivo of the neuroactive

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Time (minutes) Fig. 2. The effect of NPA on extracellular concentration of endogenous amino acids measured in microdialysis samples from the rat medulla, collected over a period of 10 min. Control animals (n = 7) are indicated by the solid lines, animals with NPA added to the dialysate (marked by arrows) are indicated by the dotted lines (5 mM NPA, n = 6) or the dashed lines (20 mM NPA, n = 6). *P -z 0.05 vs control, Dunnett’s test.

V. KAKXJRet al.

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amino acids namely Asp, Glu, GABA, Gly and Gin, in the presence of a mercury salt, pCMP. The basal extracellular levels of Asn, Ser, Ala, Orn and Lys remained unaltered, while an apparent increase in the levels of Tau did not reach levels of significance. Tau, which may also be released from non-neuronal cells,” has been shown to be released in the presence of increased (exogenous) extracellular GABA.” Such a response during the pCMP evoked release of GABA appears to show much greater variability when compared to the NPA response, perhaps due to the concomitant release of other (excitatory) amino acids; similar results have been found in vitro.*’ Glutamine, the amino acid found in the highest concentration in the extracellular fluid in vivo,33.35has been proposed as both the precursor36 and the main metabolite” of glutamate, and perhaps may subserve both of these roles under different cir~umstan~s. While most releasers of Glu and GABA have been reported to decrease Gln concentrations in the extracellular fluid33.36(Kapoor and Chalmers, unpublished observation), we have demonstrated a variable but significant increase in Gln levels in the presence of pCMP. Since Gln is probably of glial origin,% it may under these circumstances by reflecting primarily glutamines role as the Glu metabolite although any additional effect of pCMP on uptake inhibition has not yet been ruled out. As discussed in the introduction, it is likely that the increased overflow of Asp, Glu, Gly and GABA in the presence of mercury salts is the result of an increased release of these compounds due to raised intracellular calcium ion concentrations subsequent to increased calcium ion uptake and/or impaired calcium sequestering capacity of intracellular compartments.3*2’~32 Further experiments will be necessary to clarify the contribution of uptake and ATPase activity inhibition, if any, to the pCMP induced increases of amino acid release. While this data is suggestive of a neurotransmitter role for Asp, Glu, GABA and Gly in the VLM, it is also possible that some of these amino acids may be the metabolites of the actual transmitter, such as the dipeptide neurotransmitter candidate N-acetyl-aspartyl-glutamate.‘5 GABA

uptake inhibition by nipecotic acid

The results described show that in the presence of NPA the extracellular concentrations of GABA and

Tau were greatly enhanced. The effect of NPA on GABA levels was immediate and concentration dependent, whereas the effect on Tau levels was time dependent and less pronounced. In addition, there was no effect on the extracellular concentrations of Asp, Glu, Gly, Gin, Asn, Ala, Orn or Lys. These effects of NPA are consistent with the known GABA uptake inhibitory actions of NPA’2*31and similar results have been previously reported.% The effect of NPA on Tau overflow, although well documented, remains poorly understood, but it is possible that it is directly or indirectly related to the increase in local GABA concentration.‘9.20 The results do provide evidence for the presence of GABA and high-affinity uptake sites for GABA within the medulla, and demonstrate the usefulness of the dialysis technique in detecting specific changes in the extraceflular leveis of amino acids. Fhys~o~ogical relevance

Physiological studies on blood pressure regulation have demonstrated the importance of Glu, GABA and Gly systems in the medulla,i0~30although the role of Tau remains unclear.*’ In addition to this, glutamate decarboxylase and GA3A-immunoreac~ve nerve terminals,= and high-affinity uptake systems for Glu, GABA and Gly have also been demonstrated in the medulla.2 The electrically evoked, calcium ion and tetrodotoxin sensitive release of Asp, Glu, GABA and Gly from rat medullary slices in vitro has also been reported.‘” More recentiy, as mentioned in the introduction, a major excitatory amino acid pathway from the nucleus tractus solitarus to the VLM has been demonstrated by physiological and autoradiographic techniques.30 COPICLUSlON

With the use of the microdialysis technique in vivo, the results of this study provide further evidence for a physiologically relevant neurotransmitter role for Asp, Glu, GABA and Gly in the rat medulla. The precise origins and projections of amino acid pathways to and from the medulla are the subject of ongoing studies. ,dnow/edgemenrs-The authors are most grateful for the excellent technical assistance of MS A. Piekarz. These studies were supported by grants from the NHMRC and the

Ramaciotti Foundation.

REFERENCES 1. Baba A., Fisherman J. S. and Cooper J. R. (1979) Actions of sulphydryl agents on cholinergic mechanisms in synaptosomes. Biochem. Pharmac. Z$ 1879-1883. 2. Bennett J. P., Logan W. J. and Snyder S. H. (1973) Amino acids as central nervous system transmitters: the influence of ions, amino acid anaiogue, and ontogeny on transport systems for L-glutamic and L-aspartic acids and glycine into central nervous synaptosomes of the rat. J. ~e~~~ern. 21, 15334550. 3. Binah O., Meiri U. and Rahamimoff H. (1978) The effects of HgCl, and mersalyl on mechanisms regulating int~~iiular calcium and transmitter release. Eur. J. Phurmac. 51, 453-457. 4. Bondy S. C., Anderson C. L., Harrington M. E. and Prasad K. N. (1979) The effect of or8anic and inorganic lead and mercury on neurotransmitter high-affinity transport and release mechanism. Emdr. Res. 19, 102-l 11. 5. Brookes N. (1988) Specificity and reversibility of the inhibition by HgCI, of glutamate transport in astrocyte cultures. J. Neurochem. 50, 1117-1122.

Microdialysis of neuroactive amino acids from the medulla

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6. Butcher S. P. and Hamberger A. (1987) In nioo studies on the extracellular and veratrine releasable, pooh of endogenous amino acids in the rat striatum: effects ofcorticostriatal deafferentation and kainic acid lesion. J. Neurochem. 48,713-721. 7. Butcher S. P., Lazarewicz J. W. and Hamberger A. (1987) In viuo microdialysis studies on the effect of decortication and excitotoxic lesions on kainic acid-induced calcium fluxes, and endogenous amino acid release, in the rat striatum. J. Neurochem. 49, 1355-1360. 8. Chalmers J. P. (1975) Brain amines and models of experimental hypertension. Circulation Res. 36, 469-480. 9. Girault J. A., Barbeito L., Spampinato U., Gozlan H., Glowinski J and Besson M.-J. (1986) In uiuo release ofendogenous amino acids from the rat striatum: further evidence for a role of glutamate and aspartate in corticostrial neurotransmission. .I. Neurochem. 47, 98-106. 10. Gordon F. J. (1987) Aortic baroreceptor reflexes are mediated by NMDA receptors in caudal ventrolateral medulla. Am. J. Physiol. 252, R628-R633. 1I. lnoue A., Takahashi H., Lee L.-C., Sasaki S., Takeda K., Yoshimura M., Nakagawa M. and ljichi H. (1986) Hypotensive response to centrally administered taurine in DOCA-salt hypertensive and spontaneously hypertensive rats. Jap. Circul. J. 50, 1215-1223. 12. Johnson G. A. R., Krogsgaard-Larsen P., Stephanson A. L. and Twitchin B. (1976) Inhibition of the uptake of GABA and related amino acids in rat brain slices by the optical isomers of nipecotic acid. J. Neurochem. 26, 1029-1032. 13. Kapoor V. and Chalmers J. P. (I 987) A simple, sensitive method for the determination of extracellular catecholamines in the rat hypothalamus using in oiuo dialysis. J. Neurosci. Mesh. 19, 173-182. 14. Kihara M., Misu Y. and Kubo T. (1989) Release by electrical stimulation of endogenous glutamate, y-aminobutyric acid, and other amino acids from slices of the rat medulla oblongata. J. Neurochem. 52, 261-267. 15. Keller K. J., Zaczek R. and Coyle J. T. (1984) N-Acetyl-aspartyl-glutamate: regional levels in rat brain and the effects of brain lesions as determined by a new HPLC method. J. Neurochem. 43, 1136-l 142. 16. Komulainen H. and Tuomisto J. (1981) Interface of methylmercury with monoamine uptake and release in rat brain synaptosomes. Acra pharmacol. ro.ricol. 48, 214-222. 17. Kubo T. and Kihara M. (1987) Studies on GABAergic mechanisms responsible for cardiovascular regulation in the rostra1 ventrolateral medulla of the rat. Archs inr. Pharmacodyn. Thk. 285, 277-287. 18. Lehmann A. (1987) Evidence for a direct action of N-methylaspartate on non-neuronal cells. Brain Res. 411, 95-101. 19. Lerma J.. Herranz A. S.. Herreras 0.. Munoz D.. Solis J. M.. de1 Rio R. M. and Deealdo J. M. R. (1985) v-Aminobutvric * acid greatly increases the in oiuo extracellular taurine in the rat hippocampus. i Neurochem. k$ 983-986. 20. Lerma J., Herreras 0.. Herranz A. S., Munoz D. and de1 Rio R. M. (1984) In uiuo effects of nipecotic acids on levels of extracellular GABA and taurine, and hippocampal excitability. Neuropharmacology 23, 595-598. 21. Lindorth P. and Mopper K. (1979) High performance liquid chromatographic determination of subpicomole amounts of amino acids by precolumn derivatization with o-phthaldialdehyde. Analyt. Chem. 51, 1667-1674. 22. Meeley M. P.. Ruggiero D. A., lshituka T. and Reis D. J. (1985) Intrinsic y-aminobutyric acid neurons in the nucleus of the solitary tract and the rostra1 ventrolateral of the rat: an immunohistochemical and biochemical study. Neurosci. LXII.

58, 83-89.

Mills E., Minson J., Drolet G. and Chalmers J. (1990) Effect of intrathecal amino acid receptor antagonists on basal blood pressure and pressor responses to brain stem stimulation in normotensive and hvoertensive rats. J. cardiouasc. _. Pharmbc. (in press).. 24. Mills E.. Minson J., Pilowsky P. and Chalmers J. (1988) N-Methyl-o-aspartate receptors in the spinal cord mediate pressor responses to stimulation of the RVLM in rat. Clin exa. Pharm. Phvsiol. 15. 147-155. 25. Minson J. -B., Chalmers J. P., Caon A. C. and Renaud B. (1987) Separate-areas of the rat medulla oblongata with populations of serotonin- and adrenaline-containing neurons alter blood pressure after L-glutamate stimulation. J. aufon. 23.

new.

Syst.

19, 39-50.

Paxinos G. and Watson C. (1986) The Rat Brain in Stereotuxic Coordinates. Academic Press, Australia. 27. Reynolds J. N. and Racz W. J. (1987) Effects of methylmercury on the spontaneous and potassium-evoked release of endogenous amino acids from mouse cerebehar slices. Can. J. Physiol. Pharmac. 65, 791-798. 28. Sandberg M. and Lindstrom S. (1983) Amino acids in the dorsal lateral geniculate nucleus of the cat-collection in uiuo.

26.

J. neurosci. 29.

Merh.

9, 65-74.

Segovia J.. Tossman U.. Herreras-Marschitz M., Garcia-Munoz M. and Ungentedt U. (1986) y-Aminobutyric release in the globus pallidus in uiuo after a 6-hydroxydopamine lesion in the substantia nigra of the rat. Neurosci.

acid Left.

70, 364-368. 30.

Somogyi P., Minson J. B., Morilak D., Llewellyn-Smith I., Mcllhinney J. R. A. and Chalmers J. (1989) Evidence for an excitatory amino acid pathway in the brain stem and for its involvement in cardiovascular control. Brain Res. 496, 401-407.

31. 32. 33. 34. 35.

Szerb J. C. (1982) Effect of nipecotic acid, a y-aminobutyric acid uptake inhibitor, on the turnover and release of y-aminobutyric acid in rat cortical slices. J. Neurochem. 39, 850-858. Tachikawa E., Takahashi S. and Kashimoto T. (1989) p-Chloromercuribenzoate causes Ca*+-dependent exocytotic catecholamine secretion from cultured bovine adrenal medullary cells. J. Neurochem. 53, 19-26. Tossman U., Jonsson G. and Ungerstedt U. (1986) Regional distribution and extracellular levels of amino acids in the rat central nervous system. Acra physiol. stand. 127, 533-545. Waniewski R. A. and Martin D. L. (1986) Exogenous glutamate is metabolised to glutamine and exported by rat primary astrocyte cultures. J. Neurochem. 47, 304-313. Westerink B. H. C., Hofsteede H. M., Damsma G. and de Vries J. B. (1988) The significance of extracellular calcium for the release of dopamine, acetylcholine and amino acids in conscious rats, evaluated by brain microdialysis. Naunyn-Schmiedeberg’s

36. 37.

Arch.

Pharmac.

337, 373-378.

Young A. M. J. and Bradford H. F. (1986) Excitatory amino acid neurotransmitters in the corticostriate pathway: studies using intracerebral microdialysis in uiuo. J. Neurochem. 47, 1399-1404. Young A. M. 1.. Crowder J. M. and Bradford H. F. (1988) Potentiation by kainate of excitatory amino acid release in striatum: complementary in ciao and in uirro experiments. J. Neurochem. 50, 337-345. (Accepted

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