The Journal of Neuroscience August 1986, 6(8): 2226-2234 /II Vitro Release and Electrophysiological Effects In Situ of Homocysteic Acid, An Endogenous N-Methyl-(D)-aspartic Acid Agonist, in the Mammalian Striatum Kim Quang DO,* Paul L. Herrling,? Peter Streit,* Waldemar A. Turski,“fsl and Michel Cuenod* *Brain Research Institute, University of Zurich, Zurich, Switzerland, and j-Wander Research Institute, Bern, Switzerland A potassium-induced, calcium-dependent release of endogenous fects of microiontophoretically applied (L)-HCA on membrane homocysteic acid (HCA) from rat striatal slices was demonstrat- potential and cortically evoked EPSPsin cat caudate neurons, ed. A precolumn derivatization high-performance liquid chro- and the pharmacologicalspecificity of (L)-HCA in this structure. matography method was developed that allowed quantitative de- termination of sulfur-containing amino acids at the picomole level. Materials and Methods Intracellular recordings from cat caudate neurons during si- Materials multaneous microiontophoretic application of drugs and electri- cal stimulation of the corticocaudate pathway showed that (L> Release HCA evoked a depolarization pattern similar to that induced 4-N,N-Dimethylamino-azobenzene-4’-isotbiocyanate (DABITC) was ob- by %methyl-(D>aspartic acid (NMDA), and both these depo- tained from Fluka (Buchs, CH) and recrystallized in acetone (Merck, larizations could be selectively inhibited by a specific NMDA Darmstadt, FRG). All other solvents used were ofcommercial analytical antagonist, (D)-Z-amino-7-phosphonoheptanoicacid [(D)-AP-~]. grade from Merck or Fluka. The internal standard (D,L)-2-amino-7- A selective antagonismof (rJ-HCA-induced depolarizations by sulfonoheptanoic acid (AS-7) was a generous gift of Dr. J. C. Watkins. (L)-homocysteic acid [(L)-HCA], (L)-cysteine sulfinic acid [(L)-CSA], (L)- (D>AP-~ was confirmed in quantitative experiments with the cysteic acid [(L)-CA], and veratrine were purchased from Sigma (St. frog hemisectedspinal cord in vitro. Small quantities of ionto- Louis, MO). The Bio-Rad assay kit was used for protein determination. phoretically applied (L)-HCA, but not of quisqualate,potentiat- ed cortically evoked EPSPs in cat caudate neurons. Iontophoresis These observationssuggest that (L)-HCA might be a candi- date as an NMDA-receptor-preferring endogenoustransmitter The following drugs were used in the iontophoretic experiments and ejected as anions (all 0.1 M, except as noted): (D,L)-2-amino-7-phos- in the caudate nucleus. One possible function for such trans- phonoheptanoic acid (AP-7; Wander, CH); (D)-AP-7 (kindly donated mitter systemscould be the enhancementof EPSPs. by J. C. Watkins); (L)-CSA (Sigma); (L)-HCA (Sigma); N-methyl-(D)- aspartic acid, 0.2 M (NMDA, Tocris Chemicals, Buckhurst Hill, UK); Since the first observation that homocysteic acid (HCA) excites and quisqualic acid (QUIS, Sigma). toad spinal neurons (Curtis and Watkins, 196l), this sulfur- All drugs were dissolved in 100 or 200 ~1 of 1 N NaOH and made containing amino acid has been confirmed by many authors as up to 1 ml with distilled water. The pH was adjusted to 8-9 with HCl. one of the most potent neuronal excitants in various parts of All solutions were kept at - 17°C between experiments. the CNS. It has, however, not been shown to be an endogenous substancein the mammalian brain or to be releasedfrom ner- Frog hemisectedspinal cord vous tissue. In order to screen for releasedsubstances, a pre- The compounds used in vitro were from the same commercial sources. column derivatization high-performance liquid chromatogra- After initial solubilization, they were diluted in Ringer’s solution, pH phy method (HPLC) hasbeen developedthat allows quantitative 7.2. determination of HCA at the picomolar level in biological ma- Release terial (Do et al., 1986). We report here on the depolarization- The K+-induced release in rat brain slices was performed as described induced releaseof endogenousHCA from rat brain slice with by Do et al. (1986). Briefly, slices of striatal tissue containing parts of special emphasison striatal tissue, the electrophysiologicalef- caudate-putamen, globus pallidus, and nucleus accumbens were super- fused at a flow rate of 1 ml/min with freshly oxygenated Earl’s bicar- bonate buffered salt solution (Na+, 134 mM; K+, 5 mM; Ca*+, 2 mM; Received Sept. 30, 1985; revised Dec. 26, 1985; accepted Jan. 20, 1986. Mgz+, 1 mM; Cl-, 137.75 mM; HCOS-, 20 mM; H,PO,-, 1.25 mM; SOaz-, We are grateful to Dr. J. C. Watkins for many very helpful discussions and the 1 mM; @)-glucose, 10 mM; gassed with 5% CO,/95% 0,, pH 7.2) at 37°C gift of reference amino acids. We thank Mr. A. Faeh, J. Kuenzli, Ch. Durtschi, (“resting condition”). The eluate was collected as 5 min fractions. The Ms. M. Mattenberger, Ms. F. Meier, E. Schneider, and R. Emch for technical assistance and Dr. H. 0. Kalkman for help with PA, values. This investigation slices were depolarized for a period of 4 min by raising the K+ concen- was supported by grants of the Swiss National Science Foundation (3.455.83 and tration to SO mM or by adding veratrine (33 pdrnl) to the superfusion 3.228.82) and the Dr. Eric Slack-Gyr-Foundation. The HPLC equipment was fluid (“stimulation condition”). The Ca*+ dependence of the K+-induced generously linanced by the Sandoz-Foundation, the “Jubilaeumsstil%mg der release was tested by substituting the superfusion fluid with a modified Schweizerischen Lebensversicherung- und Rentenanstalt,” the “Geigy Jubilae- Earl’s bicarbonate buffered salt solution containing 0.1 mM Ca*+ and umsstiftung,” and the “Hartmann-Mueller-Stifiung” for which we express our 12 mM Mgz+. The superfusates were lyophilized, and the slices were thd-3. collected from the chambers for protein determination. Correspondence should be addressed to Dr. Kim Quang Do, Brain Research Institute, August Fore1 Strasse 1, CH-8029 Zurich, Switzerland. I Present address: Department of Pharmacology, Medical School, Jaczewskiego Preparation of DABTC derivatives 8, 20-090 Lublin, Poland. 4-N,N-Dimethylamino-azobenzene-4’-thiocarbamoyl (DABTC) deriv- Copyright 0 1986 Society for Neuroscience 0270-6474/86/082226-09$02.00/O atives of amino acids in superfusates were prepared by the method of 2226 The Journal of Neuroscience Homocysteic Acid as Endogenous NMDA Agonist 2227 I Stimulation 0.6 0.4 0.2 0 Figure 2. Time course of K+ depolarization-induced release of HCA from striatal slices. Five consecutive 5 min fractions were analyzed and expressed in pmoVmg protein/min; the third fraction corresponded to the 4 min stimulation neriod in 50 mM K+. Column heightsrepresent the mean concentrations (&SEM) of 5 independent experiments; about 1 gm striatal tissue (wet wt) from 4 rats was used in each experiment. Asterisksdenote significant differences between elllux during stimulation or resting conditions: *, p < 0.05, unpaired Student’s t test. , ,,, ,, I',"""'I""""'I tificial respiration and a bilateral pneumothorax and opening of the 0 10 20 30 cistema magna to reduce cardiovascular and respiratory pulsations were Time (min) performed. The skull was exposed and windows (ca. 1.5 x 1.5 cm) opened vertically above the lateral ventricles. The cortical tissue over- Figure 1. Analysis of the K+-induced release from basal ganglia slices lying the caudate was removed by suction until the lateral ventricles in presence of Ca*+. A and B, Chromatograms of DABTC derivatives were reached. Stimulation electrodes were implanted through the opened in superfusates. Perfusion with Earl’s bicarbonate buffer solution con- frontal sinuses in the anterior sigmoid gyms bilaterally. The positions taining 2 mM CaZ+ and 1 mM Mg*+. A, Resting conditions (3.75 mM of the recording electrodes were verified histologically. K+). B, Stimulation conditions (50 mM K+). Chromatographic condi- At the end of surgery, anesthesia was switched to 0.5-1.5% halothane tions were as described in the text. Solvent A was 35 mM acetate buffer, (Halothan, Hoechst, Frankfurt, FRG) in pure oxygen. The body tem- pH 5.5; solvent B, acetonitrile. In this part of the chromatogram, an perature was kept at 37-38°C and end-tidal CO, in the tracheal cammla isocratic elution at 20% of solvent B was used. AS-7 was used as stan- at 3.64.4%. Cell recordings were included in the present study only if dard; in some cases, under stimulation conditions, its peak overlapped mean blood pressure was above 80 mm Hg. partially with that of other compounds; in such cases the integration One channel of the iontophoretic and recording electrode assemblies was made disregarding the overlapping peak. Note the strong increase was filled with a physiological NaCl solution for occasional balance of HCA, while CSA and CA remained unaffected. controls and one channel with the same NaCl solution but at pH 9.8 as an annroximate DH and current control. The intertip distance between recording and ion;ophoretic electrodes was about 50 pm. Chang (198 1) as modified by Do et al. (1986). The lyophilized residue Recording electrodes were filled with 1.6 M potassium citrate adjusted was redissolved in 1 ml water, and 300 ~1 of AS-7 (1 @/ml in water) to DH 7.2 with citric acid. and thev had a resistance of 80-150 Ma was added as internal standard as well as 1.5 ml of DABITC solution measured in the cerebrospinal fluid iust before penetration of the tissue. (0.3 mg/ml in pyridine, freshly prepared). The mixture was heated at The signals from the recording electrode were amplified and displayed 70°C for 90 min, and the excess of DABITC was extracted with heptane- on commerciallv available eauinment and were stored on FM audiotape ethyl acetate (2: 1, vol/vol). The aqueous phase was evaporated to dry- for later analysis. Data were-analyzed by digitizing the membrane po- ness and the residue redissolved in 1 ml aa.