Joiirnai of Neurocheminry, 1977. Vol. 29, pp. 739-741. Pergamon Press. Printed in Great Britain
SHORT COMMUNICATION
Determination of taurine in individual neurones of Aplysia californica (Received 8 February 1977. Accepted 15 April 1977)
ALMOST40 years ago, Sir Henry Dale hypothesized that due to taurine (oida infra). In other experiments reagent neurons can be characterized by the single chemical blanks, taurine standards (604000 pmol), and aliquots of through which they act (DALE,1938). Thus, cholinergic ganglia extracts prepared in 0.1 N HCI were dried and neurons (i.e.those that release ACh) are not at the same time assayed. dopaminergic, serotoninergic, etc. Several recent studies of The method used is similar to that described by ORR the large, readily identifiable neurons of various inverte- et al. (1976). After selective elution from an ion exchange brate preparations have provided direct chemical confir- resin, the fluorescence of taurine produced by the reaction mation of the cell-specific localization of GABA (OTSUKA with fluorescamine was measured. The mixed bed, ion et al., 1967); ACh (MCCAMAN et al., 1973); 5-HT (WEIN- exchange columns were prepared as follows: (a) slurries REICH er al., 1973); dopamine (POWELL & COTTRELL, 1974); of the two resins (AG-1 x 8, 2 W 0 0 mesh, CI- form, and et al., 1975). There is consider- AG-50 x 8,20&400 mesh, H' form) were prepared separand histamtne (WEINREICH able electrophysiological and pharmacological support for ately in water and the fines decanted; (b) a portion of the tranqmitter function of each of the above substances the AG-1 slurry was then transferred to Pasteur pipettes in various invertebrate systems [see review of GERSCHEN- containing a small bolus of glass wool in the tip and FELD (1973) for GABA and ACh; GERSCHENFELD & PAU- allowed to settle to form a bed volume of approx 40 pl (14 PARDIN-TRITSCH (1974), and COTTRELL & MACON(1974) for mm height x 2 mm diameter); (c) a portion of the AG-50 5-HT; BERRY& COTTRELL(1975) for DA; and McCaman slurry was then added on top of the AG-1 to form a settled & McKenna (unpublished observations) and WEINREICH bed volume of about 40pl, giving a total bed volume of (in press) for histamine]. Thus, this neuron-specificity about 80pl. After washing with 1 N HC1 (500~1)followed would appear to be another useful chemical criterion for by several rinses with distilled water, the columns were evaluating the candidacy of putative transmitters. ready to use. They may be regenerated after use by repeatTaurine, a constituent of all animal tissues, is present ing the I N HCI and water washes and stored, covered in high concentration in excitable tissues such as muscle, with water. To the dry samples to be column treated (measurement nerve and brain. Studies of its distribution and neurophysiological actions have suggested that it may act as a of taurine), 4pl 0.01 N HCl were added and, after mixing, neurotransmitter or as a neuromodulator (see recent each solution was pipetted onto a separate column. Each & PASANTES-MOR-of the columns was then washed with 2004 of distilled reviews of BASKINet al., 1976; MANDEL water and the eluent collected in a clean microtube (6 mm ALES, 1976). Using a sensitive and specific microfluorometric procedure (ORRet al., 1976), we have measured I.D. x 70 mm length). The eluent, which contains 94-100% taurine in individual, identifiable neurons from the nervous of the taurine in the tissue extract, was again taken to system of Aplysia californica. Our results show high levels dryness in a vacuum centrifuge. of taurine are present in all neurons studied. The dried samples (column eluents for taurine measurement and portion of original HCI extract for measurement of total fluorescence) were redissolved in 9p1 of METHODS 4 mM-KOH in 80% ethanol. This was followed by the addiProcedures for the isolation of individual Aplysia tion of 3 pl of fluorescamine (Hoffman-LaRoche, Nutley, neurons have been described previously (WEINREICH et al., NJ) (1 mg/ml in acetone) with rapid and immediate mixing. 1973; MCCAMAN et al., 1973). Individual neurons or clus- After 2-10 min at room temperature (21-24"C), 9 p1 aliters were loaded into microtubes and 10 pl 0.1 N HCI were quots were removed and added to 10 x 75mm Pyrex added with mixing. The samples, including blanks and tubes containing 1 ml distilled water. The samples were taurine standard (200 pmol/tube), were held in an ice bath read in a filter fluorometer set for an excitation maximum (24°C) until the dissection was complete (approx 45 min). at 390nm (Corning No. 5970) and a fluorescence maxiAfter centrifugation, duplicate 4 pl aliquots of the super- mum at 475 nm (Corning No. 3385 & No. 9782) as recomnates were removed, transferred to clean microtubes and mended by ORR et al. (1976). We have substituted the 4 m ~ - K o H / 8 0 %ethanol for the taken to dryness in a vacuum centrifuge (Speed Vac Concentrator, Savant Instrument Co.). One set of samples was 0.1~-boratebuffer, pH 9, recommended by ORR et al. assayed directly to obtain a measure of total fluorescence (1976). The addition of fluorescamine/acetone to the former with fluorescamine; the second set was put over the ion solution causes less 'flaring', i.e. creeping of solvent up the exchange columns to obtain a measure of the fluorescence side of the tube, which produced erratic results. The sensi739
'
Short communication
740 OF TABLE1. LEVELS
TAURINE, OTHER AMINO ACIDS AND AMINES IN
Aplysia
NEURONS
Taurine
Aspartate?
Glutamatel (pmol/pg protein)
Glycines
ACh(/
5-HTI
-
-
t0.2
SHORT COMMUNICATION
Determination of taurine in individual neurones of Aplysia californica (Received 8 February 1977. Accepted 15 April 1977)
ALMOST40 years ago, Sir Henry Dale hypothesized that due to taurine (oida infra). In other experiments reagent neurons can be characterized by the single chemical blanks, taurine standards (604000 pmol), and aliquots of through which they act (DALE,1938). Thus, cholinergic ganglia extracts prepared in 0.1 N HCI were dried and neurons (i.e.those that release ACh) are not at the same time assayed. dopaminergic, serotoninergic, etc. Several recent studies of The method used is similar to that described by ORR the large, readily identifiable neurons of various inverte- et al. (1976). After selective elution from an ion exchange brate preparations have provided direct chemical confir- resin, the fluorescence of taurine produced by the reaction mation of the cell-specific localization of GABA (OTSUKA with fluorescamine was measured. The mixed bed, ion et al., 1967); ACh (MCCAMAN et al., 1973); 5-HT (WEIN- exchange columns were prepared as follows: (a) slurries REICH er al., 1973); dopamine (POWELL & COTTRELL, 1974); of the two resins (AG-1 x 8, 2 W 0 0 mesh, CI- form, and et al., 1975). There is consider- AG-50 x 8,20&400 mesh, H' form) were prepared separand histamtne (WEINREICH able electrophysiological and pharmacological support for ately in water and the fines decanted; (b) a portion of the tranqmitter function of each of the above substances the AG-1 slurry was then transferred to Pasteur pipettes in various invertebrate systems [see review of GERSCHEN- containing a small bolus of glass wool in the tip and FELD (1973) for GABA and ACh; GERSCHENFELD & PAU- allowed to settle to form a bed volume of approx 40 pl (14 PARDIN-TRITSCH (1974), and COTTRELL & MACON(1974) for mm height x 2 mm diameter); (c) a portion of the AG-50 5-HT; BERRY& COTTRELL(1975) for DA; and McCaman slurry was then added on top of the AG-1 to form a settled & McKenna (unpublished observations) and WEINREICH bed volume of about 40pl, giving a total bed volume of (in press) for histamine]. Thus, this neuron-specificity about 80pl. After washing with 1 N HC1 (500~1)followed would appear to be another useful chemical criterion for by several rinses with distilled water, the columns were evaluating the candidacy of putative transmitters. ready to use. They may be regenerated after use by repeatTaurine, a constituent of all animal tissues, is present ing the I N HCI and water washes and stored, covered in high concentration in excitable tissues such as muscle, with water. To the dry samples to be column treated (measurement nerve and brain. Studies of its distribution and neurophysiological actions have suggested that it may act as a of taurine), 4pl 0.01 N HCl were added and, after mixing, neurotransmitter or as a neuromodulator (see recent each solution was pipetted onto a separate column. Each & PASANTES-MOR-of the columns was then washed with 2004 of distilled reviews of BASKINet al., 1976; MANDEL water and the eluent collected in a clean microtube (6 mm ALES, 1976). Using a sensitive and specific microfluorometric procedure (ORRet al., 1976), we have measured I.D. x 70 mm length). The eluent, which contains 94-100% taurine in individual, identifiable neurons from the nervous of the taurine in the tissue extract, was again taken to system of Aplysia californica. Our results show high levels dryness in a vacuum centrifuge. of taurine are present in all neurons studied. The dried samples (column eluents for taurine measurement and portion of original HCI extract for measurement of total fluorescence) were redissolved in 9p1 of METHODS 4 mM-KOH in 80% ethanol. This was followed by the addiProcedures for the isolation of individual Aplysia tion of 3 pl of fluorescamine (Hoffman-LaRoche, Nutley, neurons have been described previously (WEINREICH et al., NJ) (1 mg/ml in acetone) with rapid and immediate mixing. 1973; MCCAMAN et al., 1973). Individual neurons or clus- After 2-10 min at room temperature (21-24"C), 9 p1 aliters were loaded into microtubes and 10 pl 0.1 N HCI were quots were removed and added to 10 x 75mm Pyrex added with mixing. The samples, including blanks and tubes containing 1 ml distilled water. The samples were taurine standard (200 pmol/tube), were held in an ice bath read in a filter fluorometer set for an excitation maximum (24°C) until the dissection was complete (approx 45 min). at 390nm (Corning No. 5970) and a fluorescence maxiAfter centrifugation, duplicate 4 pl aliquots of the super- mum at 475 nm (Corning No. 3385 & No. 9782) as recomnates were removed, transferred to clean microtubes and mended by ORR et al. (1976). We have substituted the 4 m ~ - K o H / 8 0 %ethanol for the taken to dryness in a vacuum centrifuge (Speed Vac Concentrator, Savant Instrument Co.). One set of samples was 0.1~-boratebuffer, pH 9, recommended by ORR et al. assayed directly to obtain a measure of total fluorescence (1976). The addition of fluorescamine/acetone to the former with fluorescamine; the second set was put over the ion solution causes less 'flaring', i.e. creeping of solvent up the exchange columns to obtain a measure of the fluorescence side of the tube, which produced erratic results. The sensi739
'
Short communication
740 OF TABLE1. LEVELS
TAURINE, OTHER AMINO ACIDS AND AMINES IN
Aplysia
NEURONS
Taurine
Aspartate?
Glutamatel (pmol/pg protein)
Glycines
ACh(/
5-HTI
-
-
t0.2
