A superfusion system designed to measure release of radiolabeled neurotransmitters on a subsecond time scale

178,8-16

ANALYTICALBIOCHEMISTRY

(1989)

A Superfusion System Designed to Measure Release of Radiolabeled Neurotransmitters on a Subsecond Time Scale Timothy J. Turner, L. Bruce Pearce, and Stanley M. Goldin Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston, Massachusetts 02115

Received

September

7,198E

A new method for subsecond measurement of release of neurotransmitters from nerve terminal preparations (e.g., synaptosomes) in vitro is described. Synaptosomes were prelabeled with [3H]GABA via a Na-dependent GABA uptake system. The prelabeled nerve terminals are retained on small glass fiber filters in a superfusion chamber accessed by three high speed, solenoid-driven valves. Microcomputer-programmed circuitry controls the timing of valve operation. Each valve controls the delivery of a separate solution to the chamber, permitting rapid and independent control of membrane potential, [Ca”],, and drug delivery. The minimal dead volume of the chamber and the relatively high solution flow rate afford time resolution for release of at least 60 ms. This time resolution was necessary to observe the most rapid of at least three components of GABA release. 0 1989 Academic Press, Inc.

lo-ms time scale (5). We have developed a novel rapid superfusion system that also incorporates some of the features of that design and have applied it to study neurosecretion from synaptosomes (6). Our objective was to use superfusion to measure release of preloaded y- [3H] aminobutyric acid ([3H]GABA)1 from synaptosomes on a subsecond time scale, approaching the time scale on which GABA release is thought to occur in mammalian brain (4,7,8). What follows is a detailed description of the superfusion system. We will present a number of parameters that were optimized in order to achieve 60-ms resolution of [3H]GABA release, describe the important factors that make the assay of release reproducible, and suggest ways to further increase the time resolution of the system. MATERIALS

AND

METHODS

Synaptosome Preparation Superfusion is a process in which a solution moves over and around material immobilized on a porous support in order to bathe the material with a solution that may be changed over the course of time. Superfusion has been used to measure the release of neurotransmitters from brain nerve terminal preparations, or synaptosomes, with second to minute time resolution (l-3). However, the time course of neurosecretion, as determined by fast but indirect electrophysiological methods, has been shown to occur on a subsecond time scale for many neurotransmitters (4). The slow time scale used in superfusion experiments has been dictated by physical constraints, such as low flow rates designed to reduce mechanical forces that might disrupt the vesicles and produce artifactual “release.” A rapid superfusion method has been developed to measure cation transport from isolated vesicles mediated by Na/K ATPase on a 8

Synaptosomes were prepared from the brains of male rats (50-200 g, Charles River) using the method of Hajos (9). The 0.8 M sucrose layer was diluted 1:l with basal buffer (in mM: NaCl, 145; KCl, 5; MgClz, 1.2; D-glucose, 10; EGTA-Tris, 1; Hepes-Tris, 10; pH 7.5), pelleted, and resuspended in 1.5 ml basal buffer prior to diluting to the desired protein concentration. For most experiments, synaptosomes were loaded with [3H]GABA (75-110 Ci/ mmol, New England Nuclear) by combining 5-10 &i of the isotope with 100 ~1 of synaptosomes (0.5 mg/ml) and incubating for 10 min. Uptake was stopped by diluting the incubation solution 1:8 with basal buffer and applying the synaptosomes to the filter sandwich. Stimulating 1 Abbreviations used: GABA; y-aminobutyric glycol bis(&aminoethyl ether) NJV’-tetraacetic droxyethyl)-1-piperazine ethanesulfonic acid; external concentration of a substance.

acid, EGTA, ethylene acid; Hepes, 4-(2-hy[ ]i, [ I., internal or

0003-2697/89 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

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A

INLET

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HOUSING

OUTLET TO FILT

FIG. 1. Diagram of the three valve axis of the superfusion chamber. Each accepts the filter sandwich assembly poppet occludes the fluid flow. When to flow outward through the hole in stainless steel.

superfusion device. (A) View from beneath the device, showing three valves arranged around the central solenoid valve controls the flow of a separate buffer solution. The threaded Teflon bushing in the center (see Fig. 2). (B) Cross-sectional view through one of the solenoid valves. In the resting state, the Teflon energized, the solenoid moves toward the center of the coil, forcing the poppet upward; this enables fluids the central axis, into the superfusion chamber. All parts in contact with the fluid are either Teflon or

buffers contained the indicated concentration of KC1 substituted isotonically for NaCl. Where indicated, the [Ca’+] of the buffers used were determined by titration, using a Phillips Ca2+- selective electrode (Moller Glassblowers, Zurich, Switzerland) and Corning 0.1 M calcium molarity standard (Fisher Scientific). All procedures were performed at room temperature. The Superfusion

System

The superfusion system described here has been used to measure cGMP-stimulated release of radiolabeled cations from purified bovine photoreceptor disk preparations (lo), as well as synaptosomal [3H]GABA release (6). It consists of three pressurized fluid reservoirs, a superfusion device (Fig. l), a control system, and a rotating fraction collector constructed from a phonograph turntable. The three stainless-steel fluid reservoirs (Gelman pressure filtration funnels, product No. 4280), containing up to 200 ml of superfusion buffer, are pressurized under N2 to drive the fluid flow. Each reservoir is connected by standard 3.0-mm Teflon tubing (Rainin catalog No. 200-32) to one of the vai:‘q using polypropylene tube end fittings (3 mm, f-28 thread, Rainin No. 200-08). The Superfusion apparatus. Synaptosomes are retained on a “sandwich” of three 5-mm diameter filters.

The filter configuration normally employed (“SC-GF/ F-SC”) is shown in Fig. 2. Synaptosomes are loaded onto the filters in a separate loading chamber (not shown), assembled in one end of a Teflon tube-to-tube connector (General Valve part No. 13-2-062). The loading assembly was composed of a top washer (5 mm od X 3 mm id stainless-steel washer, Rainin part No. 200-08-002), the three-filter sandwich, a 5-mm diameter stainless-steel screen, and a bottom washer. The assembly was secured in one end of the loading chamber with a Teflon nut (General Valve No. 5-1-125). A barbed fitting (General Valve No. 11-12-125) attached to a lo-cm length of 4 in. id. Tygon tubing is secured in the other end of the connector. The solution of synaptosomes is transferred to the Tygon tubing with a g-in. Pastuer pipette, and the solution is pushed through the filter using a 30-ml syringe. The synaptosomes reside on a 3-mm spot on the 5-mm filter sandwich, as defined by the inside diameter of the upper washer in the loading chamber. The superfusion device (Fig. 1) consists of a stainlesssteel housing configured with three solenoid-driven valves (General Valve part No. g-396-901) arranged coaxially around a superfusion chamber. The loaded filter sandwich is transferred to the superfusion chamber and is secured in place with a Teflon outlet fitting (Fig. 2). The outlet fitting differs from the nut used in the loading

10

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c OF/F FILTER

c SUPERFUSATE

I

GFW PREFILTER

5b

SUPPORT SCREEN

PEARCE,

RA POSTFILTER

NOTCH FUR FLulD FLOW CHANNELING

EXIT

I

y

SUPERFUSA~

OUTLET

‘II

FIG. 2. Exploded view of the filter sandwich assembly. The “SCGF/F-SC” filter sandwich containing [3H]GABA-loaded synaptosomes (see text) is shown in an exploded view, with the GF/B prefilter and RA postfilter in place. Scale bar at lower left represents a distance of 2 mm.

chamber in two respects. First, the bore of the fluid path through the nut is reduced by inserting a short length (10 mm) of standard 3.0-mm Teflon tubing into the nut that is flush with the superfusate exit. This reduces the dead volume of the fluid flow path and maintains the effluent in an uninterrupted stream even at low flow rates. Second, the outlet fitting was notched along its perimeter. The notches were necessary to provide a flow path for superfusion fluid to channel around the outside of the filter sandwich. The notches increased the rate of washout of released material and thereby increased the temporal resolution of the system (see below). The superfusion protocol is triggered by the momentary closing of a reed switch as the permanent magnet attached to the turntable swings past. The valves are energized in the desired sequence, resulting in flow of one of three superfusion buffers through the filter sandwich immobilized in the superfusion chamber. The superfusate is channeled through the narrow outlet fitting, exiting as a continuous, uninterrupted stream into the fraction collector. The fraction collector is a circular Lucite platform with 50 circular wells (l&mm diam) juxtaposed on its perimeter designed to accommodate 15 X 45mm glass minivials (Atlantic Biomedical). The platform rotates on a phonograph turntable placed directly beneath the outlet fitting. The effluent streams into the center of the minivials in the fraction collector, which effectively “cuts” the stream into 50 equal segments. At the end of the experiment, the filter is removed from the superfusion chamber, and 1 ml of scintillation cocktail (Hydro-

AND

GOLDIN

fluor, National Diagnostics, Manville, NJ) is added to each vial. The amount of radioactivity in each fraction is determined by liquid scintillation counting. The amount of release (expressed as a percentage of total) is calculated as the quotient of radioactivity per fraction, divided by the sum of all radioactivity released plus radioactivity remaining on the filter at the end of the experiment. The amount of radioactivity remaining on the filter at the end of the experiment is generally >95% of the total, indicating that the amount radiolabel released per pulse is a small fraction of the total pool. Electronic control system. Each valve of the superfusion device is independently controlled by an Apple IIe computer equipped with an Applied Engineering digitalto-analog converter (items in brackets { } refer to Fig. 3). The computer (1) uses its own microprocessor clock to time the superfusion protocol. The phonograph turntable { 10) employed as the fraction collector includes a magnetic reed switch { 121 (Hamlin type MRG-DT-175, Newark Electronics) triggered by a permanent magnet { ll} (Hamlin H33-606) attached to the turntable platter {lo}. The closing of the switch provides the computer with a reference signal as to the rotational position of the turntable. The magnetic reed switch and a manual switch { 13) are both electrically connected across an analog-to-digital (A/D) input port { 14) to the computer. A 9-V battery { 15) and a 1000-B resistor (16) are con-

C-XL I,, l-l p ,s’3 F/

12

'5.

B.

a

b

c

FIG. 3. (A) Schematic of the computer-controlled, three-valve superfusion system. (B) Computer protocol for superfusion of synaptosomes to measure rates of neurotransmitter release (see text for details).

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netted in series with switches (121 and { 13) to apply an input signal to A/D port { 14) when either switch closes. The solid-state relays {5,6,7} (Model 6321, Crydom Inc., El Segundo, CA), which derive operating power from a 5-V power supply (91, are activated by control signals produced by the computer at the digital-to-analog (D/A) output port (8). The valves are energized for a precise duration by the action of the relays, supplying the necessary current from a separate 12-V power supply (17). The (+) output of power supply { 17) is connected to the common terminal of each valve. The computer is programmed to allow the operator to select the parameters of the superfusion collection cycles in a menu-driven, step-by-step fashion. Referring to Fig. 3B, in step {a}, the operator enters the total duration of the superfusion collection cycle. The superfusate is collected as discrete fractions (see below). In step {b}, the operator enters the parameters of operation for each valve, i.e., the sequence and duration of the opening of valve { 2)) with reference to the closure of the magnetic reed switch during turntable rotations. In steps {c} and {d}, the operator enters the sequence and duration of opening of valves {3 and 4f during the collection cycle. The parameters entered in steps {a-d} may then be stored on a floppy disk of the computer {e}. The parameters may also be graphed and printed { f}. The operator then initiates {g} the collection cycle protocol programmed in steps {a-d}. RESULTS

Retaining the vesicles. A primary concern in designing a superfusion system for synaptosomes is the filter sandwich used to immobilize them in the superfusion chamber. The ideal filter support would retain a large amount of vesicles without clogging, allow a high flow rate through the loaded filter, and have sufficient mechanical stability to prevent the vesicles from being damaged or dislodged during superfusion. We tested the following filters with regard to the desired characteristics: Millipore (cellulose ester membrane) SS (3.0-pm pore size) and HA (0.45 pm), Whatman (glass fiber) GF/ F (0.7 pm) and a combination of GF/A (1.6 pm), GF/C (1.2 pm), and GF/B (1.0 pm). The filters were superfused using N2 pressure (10 psi) with a solution of synaptosomes at a protein concentration of 20 pg/ml. The effluent was collected in 10-s fractions, the volume of each fraction was measured to determine flow rate, and the protein content of each fraction was determined by a TCA-precipitation method (11). This assay tested the ability of the various filters to retain synaptosomal protein and to maintain an adequate flow rate. The HA filters were quickly eliminated from consideration due to unacceptably low flow rates. The remaining three candidates had satisfactory flow rates, but the SS filters retained only about half as much protein as the glass fiber

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types. The GF/F and the GF/A,C,B combination were comparable in terms of flow rate and protein retention, but the combination is 10 times thicker than GF/F. Thus, the GF/F filter was chosen on the basis of high flow rate and protein retention, with relatively small volume. A disadvantage of using GF/F filters is that they tend to disintegrate, probably due to fluid shear forces produced by high flow rates. To add structural support to the filter, a Millipore cellulose ester SC filter (8.0 pm) was placed both above and below the GF/F filter to form a sandwich. The SC filters did not significantly alter the protein retention or flow rate of the GF/F filter. Loading the vesicles. The next factor of importance was to determine the amount of synaptosome-associated radioactivity we could retain on the 3-mm diameter spot where the synaptosomes are applied to the filter sandwich (Fig. 4). Synaptosomes (100 pg in 100 ~1) were incubated with 50 nM [3H]GABA (0.5 &i, 110 Ci/mmol), and a time course of [3H] GABA uptake was observed by filtering the synaptosomes at various intervals and determining the amount of radioactivity retained on the filters. Under these conditions, total uptake of [3H]GABA reached a level of 14 pmol/mg at 10 min. A parallel experiment was conducted in a low Na+ and Clbuffer, using N-methyl glucamine SO, to replace NaCl, to estimate nonspecific association of [3H]GABA with the loaded filter, since an inwardly directed gradient for both Na+ and Cl- is required to concentrate [3H]GABA in nerve terminals (12). Uptake in N-methyl glucamine SO, was less than 0.3% of the total uptake. Nipecotic acid, a selective blocker of GABA uptake (13), completely blocked Na+- and Cl-dependent [3H]GABA uptake, with a Ki value of 6 pM as derived from Hill plot xintercepts (not shown). The internal volume of the synaptosomes was determined to be 3 pl/mg, based on the inulin-excluded 3Hz0 space. At a level of 14 pmol/mg, the minimum 3H-labeled [GABA]i was 4.6 pM. Thus, [3H]GABA is concentrated by synaptosomes at least loo-fold, assuming uniform distribution of [3H]GABA within the inulin-excluded water space, and higher concentrations would be expected for cytosolic subcompartments such as secretory vesicles. The uptake experiment was repeated at a higher [GABA],, using 0.90 pM [3H]GABA or 21 pM [‘“Cl GAB A (not shown). Synaptosomes concentrated GABA to a level inversely proportional to [GABA],, with the radiolabel concentrated ‘IO-fold at 0.90 pM ([GABA]i = 65 PM), and 50-fold at 21 pM ([GABA]i = 1.1 mM). Using a 3H-labeled [GABA], of 0.9 pM, a 50-pg sample of synaptosomes contains about 1 &i of radiolabel. This is sufficient radioactivity to measure basal rates of synaptosomal [3H]GABA release at 15-ms intervals when radioactivity is counted for 10 min per fraction at 30 to 40% efficiency.

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o A 0

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4 0

2

4

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a

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20

TIME (min)

FIG. 4. The time course of [3H]GABA uptake. Synaptosomes (100 temperature for the indicated interval. [3H]GABA uptake was halted The filters were washed with two 4-ml portions of basal buffer, placed liquid scintillation counting. The time course of [‘H]GABA uptake in shown.

pg in 100 by adding in a vial, N-methyl

As shown in Fig. 5A, Reducing the switching artifact. a surge of radioactivity in the effluent was observed that coincides with the switching of valves necessary to change the fluid composition from the basal buffer to a stimulating buffer. This surge was also observed when switching from the stimulating buffer back to the basal buffer at the end of the depolarizing pulse or when switching between identical basal buffers. We hypothesized that this surge was due to turbulence and shear forces generated when flow through the superfusion chamber was perturbed by valve switching. Therefore, we measured the switching surge by recording a switch between identical basal buffers as a function of superfusion pressure. Decreasing the superfusion pressure from 110 to 38 psi reduced this artifact fivefold, while cutting flow rate by only 50% to 2 ml/s. The twofold reduction in flow rate represents a twofold decrease of the Reynolds number for the inlet pipe to the superfusion chamber (where fluid flow is the highest) from about 3400 to 1700. The critical Reynolds number is 2300; the flow must be laminar when the Reynolds number is less than 2300. While the flow may be turbulent at values greater than 2300, laminar flow may occur at values up to 40,000 (14). The lower flow rate thus may reduce the Reynolds number below the critical level for transition between laminar and turbulent flow in the inlet pipe, since a correlation was observed between the reduced flow rates and a reduced level of switching surge. Thus, we have sacrificed some of the temporal resolution of the system by

~1) were incubated with [3H]GABA (0.5 &i, 50 nM) at room 4 ml of basal buffer and filtering over Whatman GF/C filters. and extracted overnight ip 3 ml of scintillation cocktail before glucamine SO1, and in the presence of 30 pM nipecotate, is also

decreasing flow rate to improve the mechanical stability of the filter sandwich. The switching surge was further reduced by adding a “prefilter” in the superfusion chamber flow path directly above the filter sandwich containing the synaptosomes. Again, a variety of filters and multifilter combinations were tested to determine which would produce the optimal combination of reduced switching surge and maximal flow rate, with an additional constraint of minimizing the volume of the superfusion chamber. The best filter for this task was the GF/B filter. In addition, we found that the addition of a Millipore cellulose ester RA “postfilter” (l-pm pore size) directly below the filter sandwich and above the stainless-steel screen was helpful in reducing the switching surge even further. The RA postfilter is probably acting as a “net,” retaining synaptosomal particles that become dislodged during the switching event. Once again, we sacrificed temporal resolution by increasing the volume of the superfusion chamber in exchange for improved stability of the filter sandwich. The combination of reduced flow rates, a GF/ B prefilter, and a RA postfilter nearly eliminates the switching surge, reducing it to no more than twice the level of the release baseline. The remaining switching surge is a convenient marker for the start or the end of a pulse. Because there is an uncertainty as to the exact instant the switch occurs during the superfusion experiment of plus or minus one fraction, the switching surge (roughly analogous to an electrophysiological “stimulus

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FIG. 5. Reducing the switching artifact. Synaptosomes (50 rg) were loaded with [3H]GABA for 10 min, applied to the filter sandwich, and placed in the superfusion chamber. The standard superfusion protocol was to wash the synaptosomes with basal buffer for 10 s to remove external [3H]GABA from the filter sandwich. The fraction collector (33 rpm, 1.8 s per revolution) was started, and the superfusate was collected in the following sequence: basal buffer (0.65 s), stimulating buffer (0.75 s), and basal buffer (0.40 s). The synaptosomes were superfused at 110 psi using the filter sandwich alone (A) or at 38 psi along with GF/B prefilters and RA postfilters (B). The basal buffer was switched to the following stimulating buffers: (i) 110 mM K+/pCa 2.6; (ii) 110 mM K+/pCa 8; or (iii) 5 mM K+/pCa 2.6.

artifact”) is routinely used to confirm that ,release data from different experiments are synchronized. Calibrating the system. It is important to determine how rapidly solutions within the superfusion chamber can be changed under conditions of flow through the loaded system, in order to determine the upper limit of time resolution of the system. Biological release processes that occur more rapidly than the solutions can be exchanged within the superfusion chamber will tend to be masked. The rate of exchange of solutions within the

superfusion chamber was estimated by observing the rate of washin and washout of a buffer containing 0.5 &i/ml 45Ca2+ (Fig. 6). The rate of exchange of 45Ca2+ using this method can be described as a first-order process, with a time constant of 55 ms. Similar results were obtained using 3Hz0 as a calibration marker. The upper limit of time resolution we can observe using this system in the current configuration is 50-60 ms. Further modifications may be able to improve the temporal resolution to lo-20 ms.

14

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0.15

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flME(eec)

FIG. 6.

Calibrating the system. The exchange of solutions within the superfusion chamber was monitored by measuring the rate of appearance and disappearance of &Ca’+ in the effluent. The chamber was superfused with basal buffer for 150 ms, at which time the buffer was switched to 300 ms, the superfusate was switched back to the basal buffer for the final 330 an identical buffer with 0.5 &i/ml 46Ca2+. After an additional ms. The amount of “Ca*+ in each 15 ms fraction was normalized to the plateau level (time between 210 and 300 ms) by dividing the radioactivity in each fraction by the plateau level. The surge observed at 300 ms was not observed when 3H20 was used as a calibration marker and may be an artifact due to 45Ca2+ binding to the glass fibers. The time constant of the washout of “Ca’+ was calculated by linear regression analysis of the logarithm of the relative amount of radioactivity per fraction versus time. The regression line of the semilog plot was linear over >95% of the time course, with a correlation coefficient (r2) of 0.94, and a time constant (-l/slope) of 55 ms.

Once the [3H]GABA release is kinetically complex. superfusion system had been optimized with respect to the switching artifact, the K+-stimulated release of [3H]GABA from synaptosomes was measured again, using buffers containing either 100 nM or 2.4 mM Ca2+ (Fig. 5B). Synaptosomes (50 pg/sample) were superfused at 38 psi, resulting in a flow rate of 1.5 ml/s, and fractions were collected at 33 rpm (36 ms/fraction). Switching from 5 mM K+, pCa 8 to 5 mM K+, pCa 2.6 did not alter the rate of [3H]GABA efflux. Elevating [KC11 to 110 mM at pCa 8 resulted in an increase in the rate of [3H]GABA efflux to 2-3 times the basal rate, which persisted as long as the synaptosomes were depolarized. At pCa 2.6, there was a rapid, transient increase in the rate of GABA release to 8-12 times the basal rate, which decayed within 200 ms to a level that was initially 4-6 times the basal rate and decayed slowly over the course of 5-10 s (not shown). Thus [3H] GABA release is composed of at least three components, a Ca-independent component, a transient Ca-dependent component (phasic component) that lasts less than 200 ms, and a slowly decaying Ca-dependent component (tonic component) that lasts 5-10 s. The amount of [3H]GABA released per 36-ms fraction ranged between a basal level of 80-200 cpm to a peak of 600-1500 cpm observed at 110 mM K+, pCa 2.6. These results are described in greater detail elsewhere (6).

The [ Ca”], during preincubation affects Ca-dependent [3HJGABA release. A direct relationship between

[Ca2’], and [Ca2+]i of synaptosomes has been shown using the Ca2+-sensitive dye fura- (16). Thus, an issue of concern is whether synaptosomes are able to maintain the submicromolar [Ca2+]i observed in intact neurons, after prolonged exposure to physiological [Ca2+], . Therefore, Ca-dependent [3H]GABA release was measured as a function of [Ca2’], in the preincubation buffer over the range of 10 nM to 3 mM (Fig. 7). Synaptosomes were resuspended in a basal buffer containing 1 mM EGTA and the indicated free [Ca2+] (pCa). The synaptosomeswere preincubated 5-30 min at room temperature in this buffer prior to the lo-min [3H]GABA loading period conducted in the same buffer. [3H]GABA release was measured under the same conditions regardless of the preincubation conditions; the synaptosomes were washed with basal buffer (5 mM K+, pCa 8) for 10 s and superfused with the basal buffer (5 mM K+, pCa 8) and stimulating buffer (110 mM K+, pCa 2.6 or 8.0) using the standard protocol (see legend to Fig. 5). Varying the [Ca2’], in the preincubation buffer between 10 nM and 3.0 mM had no significant effect on the amount of synaptosomal [3H]GABA uptake. However, we observed an inverse relationship between [Ca2+le in the preincubation buffer and the magnitude of Ca-dependent release; release was greatest at 10 nM [Ca2+],, was reduced by roughly one half between 3 and 100 PM, and was greatly attenuated at 3 mM. While both the phasic and the tonic components of Ca-dependent release were affected, the

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The [Cax+], during preincuhation affects the the indicated [Cax+],, collected on the filter sandwich, superfusion protocol used was identical to that described K+, pCa 8 or 2.6. The difference between evoked release used in the loading buffer. Data shown are the averages

magnitude of [3H]GABA release. The synaptosomes were loaded in a buffer containing and superfused with basal buffer (5 mM K+, pCa 8) for 10 s prior to superfusion. The in Fig. 5. Release was evoked by switching to a superfusion buffer containing 110 mM at pCa 8 and 2.6 (Ca-dependent release) is plotted as a function of time for each [Ca”], of four independent experiments.

tonic component was more sensitive to preincubation in elevated [Ca2+lethan the phasic component. This is consistent with the observation that evoked release mediated by the tonic component is more sensitive to [Ca2’], than the phasic component (6). These results suggest that the Ca-dependent GABA release process not only requires Ca2+ entry during depolarization, but is also regulated by Ca2+ in the resting state, since maximal release is evoked at 2.4 mM [Ca2’], from samples that are maintained in submicromolar [Ca2+], . The synaptosomes may be deficient in their ability to maintain the internal [Ca2+] at levels optimal for evoked release of GABA (16). Alternatively, physiological [Ca2+] may exert an inhibitory influence on nerve terminals, maintaining evoked release at a relatively low level. Introducing EGTA into the buffer reduces the [Ca2+];, relieving the inhibitory influence of [Ca2’],. In either case, it is clear that Ca-dependent release of GABA is regulated by the resting [Ca”+]i in synaptosomes. DISCUSSION

To achieve subsecond temporal resolution of the superfusion system, it was necessary to optimize a number of opposing factors. The time resolution of the system improves as the flow rate increases and the “dead volume” of the superfusion chamber decreases, and hence the ratio of flow rate to dead volume must be increased to improve the temporal resolution. An additional constraint that effects both the flow rate and the dead volume is that there must be a sufficient amount of

radioactive material retained in the superfusion chamber to obtain a signal in the effluent that is readily measured. Increasing the flow rate increases the pressure gradient across the region where fluids are flowing. This increases the likelihood of mechanical damage to the porous support, especially when the fluid flow is perturbed by the switch between different valves in the superfusion device. This superfusion system has a number of advantages over other superfusion systems reported previously. The use of glass fiber filters as a support for the synaptosomes represents a distinct advantage over the use of membrane filters (5), as the glass fiber filters have at least an order of magnitude higher loading capacity. The use of a multivalve superfusion chamber allows rapid and independent control of membrane potential, [Ca2+], and drug delivery. The computer-programmable circuitry permits the investigator to control the stimulation of the nerve terminals in analogy to electrophysiological experiments. The 60-ms time resolution obtained using this system represents an increase of at least fourfold over the fastest biochemical measurement of synaptosomal neurotransmitter release previously reported (2); these investigators collected fractions at 0.25-s intervals, but the rate of exchange of solutions within the superfusion chamber was not determined. The phasic component of GABA release lasts less than 200 ms, and hence the 60ms time resolution is necessary to resolve this component. Further improvements in the temporal character-

TURNER,

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istics of the superfusion system should allow us to measure the actual rates of decay of the phasic component. An implicit advantage of rapid superfusion is that potential neuromodulators released into the environment are washed away during superfusion, preventing the buildup of such compounds during the efflux experiment. This gives the investigator the ability to control the local concentration of agents that may be involved in regulating neurosecretory activity. ACKNOWLEDGMENTS The authors thank the following project. Mr. Gerald Toohey of General ufactured the three-valve superfusion cations. Dr. Roger Calhoon of Cetus gramming the superfusion protocol. tragadda Subbarao provided helpful

people who contributed to this Valve Corp. designed and mandevice according to our specifiCorp. wrote the software for proDrs. Kathleen Sweadner and Kacomments on the manuscript.

M., and Levi, G. (1978) Reu. Neurosci. 3,77-130. C. W., Haycock, J. W., and White, W. F. (1976)

iol. 264,475-505.

3. Minnema,

D., and Michaelson,

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