SYNAPSE 9:66-74 (1991)

Characterization of the Distribution of G,, in Rat Striatal Synaptosomes and Its Colocalization With Tyrosine Hydroxylase MARINA E. WOLF, JAMES G. GRANNEMAN, AND GREGORY KAPATOS Center for Cell Biology, Sinai Research Institute, Detroit, Michigan 48235; the Cellular and Clinical Neurobiology Program, Department of Psychiatry, Wayne State University School of Medicine, Detroit, Michigan 48202

KEY WORDS

Flow cytometry, G-proteins, Nigrostriatal dopamine neurons, Signal transduction

Dopaminergic striatal synaptosomes can be detected and isolated with a ABSTRACT fluorescence-activated cell sorter (FACS). In the present study, two antigens were detected simultaneously with primary antisera raised in different species and speciesspecific fluorescent secondary antibodies with different emission spectra. Double-label FACS analysis was used to determine whether tyrosine hydroxylase (TH) and the alpha subunit of Go(Gaolare colocalized in striatal synaptosomes. Rabbit antibodies generated against a synthetic fragment of G, (corresponding to amino acids 22-35) combined with fluorescein-conjugated secondary antibodies were used to detect G,,-containing striatal synaptosomes. Preadsorption of G,, antiserum with the synthetic peptide antigen reduced labeling to the level obtained with preimmune serum. Approximately 6575% of striatal synaptosomes were specifically labeled by G,, antiserum. Tyrosine hydroxylasecontaining synaptosomes were detected with a mouse monoclonal antibody to TH and R-phycoerythrin-conjugated secondary antibody. They comprised 15-17% of total striatal synaptosomes. Double-label studies indicated that at least 50%of TH-containing synaptosomes also contained Gao. These findings suggest that G,, may not be a protein component of all striatal nerve terminals, and provide a basis for a role for G,, in signal transduction within subpopulations of intrinsic and afferent nerve terminals, including those of nigrostriatal dopamine neurons. INTRODUCTION G, is the most abundant guanine nucleotide binding protein (G-protein) in brain (Gierschik et al., 1986). Much lower levels of the a-subunits of Go(Gao)are found in the peripheral nervous system and in certain nonneuronal tissues (Homburger et al., 1987). Immunohistochemical studies have demonstrated that G,, is unevenly distributed in the central nervous system (CNS), with particularly high concentrations in the cortex and other forebrain regions (Gierschik et al., 1986; Worley et al., 1986; Asano et al., 1987). While the precise localization of G,, within the neuron has not been well characterized, G,, is known to be more abundant in neuropil than in cell bodies or white matter, indicating that the protein is concentrated in neuronal processes (Worley et al., 1986; Asano et al., 1987). Although the heterogeneous distribution of G,, within the brain suggests localization to specific populations of neurons, the extent to which G,, is selectively associated with neurons using particular neurotransmitters is unknown. A recent study has shown that G,, mRNA is abundant within the perikarya of monoamine-containing neu-

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rons, including the dopamine (DA) neurons of the nigrostriatal projection (Vincent et al., 1990). In contrast to the abundance of G,, mRNA in these DA neuron perikarya, however, G,, protein within the substantia nigra appears to be concentrated in the terminals of striatonigral projections (Worley et al., 1986). The purpose of this study was to examine G,, immunoreactivity in a preparation of isolated striatal nerve terminals (synaptosomes) that presumably are representative of the neurotransmitter diversity of this brain region and to determine whether dopaminergic synaptosomes, which comprise approximately 15% of this nerve terminal population, contain Ga0.In the present study, two antigens were detected simultaneously with primary antisera raised in different species and speciesspecific fluorescent secondary antibodies with different emission spectra. Double-label fluorescence activated cell sorter (FACS) analysis was used to determine Received September 28,1990;accepted in revised form February 19,1991 Address reprint requests to Dr. Gregory Kapatos, Center for Cell Biology, Sinai Hospital, 6767 West Outer Drive, Detroit, MI 48235.

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whether TH and G,, are colocalized in striatal synaptosomes. Our results demonstrate that G,, is found in 65-75% of striatal synaptosomes and that a t least 50% of TH-containing dopaminergic synaptosomes also contain G,,. These findings suggest a heterogeneity of G,, within subpopulations of nerve terminals derived from intrinsic and afferent striatal neurons, including those originating from nigrostriatal dopamine neurons.

MATERIALS AND METHODS Preparation of synaptosomes Male rats (Hilltop Laboratories, Scottdale, P A 200300 g) were killed by decapitation and the striata were dissected over ice. Striatal synaptosomes were prepared according to a modification of the method of Gray and Whittaker (1962) as described previously (Wolf and Kapatos, 1989a). For fixation and immunolabeling, synaptosomes were resuspended at a concentration of 1mg protein per ml in HEPES-buffered Krebs-Ringer solution (KRH), pH 7.4, containing 140 mM NaC1, 5 mM KC1,2 mMCaCl,, 1.25mM MgCl,, 10mM Hepes, and 10 mM glucose. Fixation and immunolabeling Synaptosomes were permeabilized and fixed by incubation for 30 min with an equal volume of modified Zamboni fluid (Stefanini et al., 1967), yielding final concentrations of 2% paraformaldehyde and 7.5% picric acid. Preliminary studies indicated that this method of fixation results in maximal specific labeling of TH and G,, as compared with other fixation protocols using ethanol, methanol, or paraformaldehyde alone (Wolf et al., 1989~).Synaptosomes were then washed 4 times with modified Dulbecco’s phosphate-buffered saline (DPBS) (Gibco Laboratories, Grand Island, NY) containing 0.5 mM MgCl, and no CaC1,. Tyrosine hydroxylase-containing synaptosomes were identified with a mouse monoclonal antibody of the IgG, class directed against TH purified from the PC12 pheochromocytoma cell line. This antibody selectively labels the 61-kDa TH monomer in Western blots of total synaptosomal protein from fresh or Zamboni-fixed striatal synaptosomes (Wolf and Kapatos, 1989b,c). G,,-containing synaptosomes were identified with antiserum generated against a synthetic fragment of G,, corresponding to amino acids 22-35. The generation and characterization of this antiserum are described in detail elsewhere (Granneman and Kapatos, 1990). Before immunolabeling, synaptosomes were incubated for 3 hr in DPBS containing 10% normal goat serum (NGS) (Gibco Laboratories) to saturate nonspecific antibody binding sites. All subsequent incubations were in DPBS + 10% NGS unless otherwise noted. To detect G,,, permeabilized synaptosome were incubated with antiserum to G,, or preimmune serum a t a dilution of 1:1,000 for 6 hr. Synaptosomes were washed twice (15 min each) by centrifugation and resuspension, and then incubated overnight a t 4°C. The overnight incubation substan-

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tially reduced the level of labeling produced by preimmune serum alone. In the morning, synaptosomes were washed 3 times (5 min each) and then incubated for 1hr at 4°C with a 150 dilution of fluorescein-conjugated F(ab’), goat antirabbit XgG (Jackson ImmunoResearch, Avondale, PA). Synaptosomes were then washed once in DPBS + 10%NGS and twice in DPBS, and resuspended in DPBS prior to FACS analysis. For Th-G,, doublelabeling experiments, a 1:2,000 dilution of anti-TH ascites was added during the overnight wash and a 1:lO dilution of R-phycoerythrin (R-PE)-conjugated goat antimouse IgG (Tago, Inc., Burlingame, CA) was included during the secondary antibody incubation. For experiments in which only TH was analyzed, synaptosomes were incubated for 3 hr in DPBS containing 10% NGS, incubated overnight with TH antibody, washed 3 times, incubated for 1 hr with R-PE-conjugated goat antimouse IgG, and washed 3 times as described above prior to FACS analysis. Control experiments demonstrated that R-PE-conjugated goat antimouse IgG did not recognize G,, rabbit antiserum and that fluorescein-conjugated goat antirabbit IgG did not recognize the mouse monoclonal antibody to TH (data not shown). For preadsorption experiments, a 1:500 dilution of G,, antiserum or preimmune serum was prepared in DPBS containing 100 pglml bovine serum albumin and incubated overnight at 4°C with the synthetic peptide antigen (1or 10 pM). Preadsorbed antiserum or preimmune serum was then added to an equal volume of synaptosomes to achieve a final dilution of 1:lOOO and labeling was carried out as described above. Flow cytometry Flow cytometric analysis was performed with a FACS 440 (Becton-Dickinson, Mountain View, CAI equipped with a 5-W argon ion laser (Spectra Physics, Mountain View, CA) tuned to generate 200 mW with a 488-nm emission line. Tyrosine hydroxylase and G,, were detected using R-PE and fluorescein-conjugated secondary antibodies, respectively. R-PE and fluorescein are both excited by the 488-nm laser line but exhibit different emission spectra. To detect emissions from synaptosomes labeled with fluorescein and/or R-PE, fluorescein and R-PE emissions were separated and reflected to different photomultiplier tubes (PMT) with a dichroic mirror (560 nm). Fluorescein signals (fluorescence 1) were detected after passing through a 530130-nm bandpass filter, and R-PE signals (fluorescence 2) were detected after passing through a 575126-nm bandpass filter (all optics from Becton-Dickinson). Photomultiplier voltages were adjusted so that unlabeled synaptosomes exhibited approximately equal intensities a t both emission wavelengths (fluorescence 1,630-650 V; fluorescence 2, 500-520 V). Compensation circuitry was used to eliminate bleed-over of fluorescence 1into fluorescence 2, and vice versa. Synaptosomal fluorescence was analyzed with logarithmic amplifiers set for 4 log

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FORWARD ANGLE LIGHT SCATTER Fig. 1. Dual-parameter contour plots comparing fixed, unlabeled striatal synaptosomes and synaptosome labeled with a monoclonal antibody to TH and R-PE secondary antibody. Analysis was based on 20,000 particles per group. For each particle, forward angle light

scatter and R-PE fluorescence are presented as x- and y-coordinates, respectively, on a grid. The units are arbitrary and intended only to convey relative differences. The relative number of particles at a given location on the grid is indicated by contour lines.

decades. The 90" scatter PMT was operated at 400 V. A neutral density filter (ND1j was used in the detection of forward-angle light scatter. Sheath fluid consisted of 0.9%NaC1. A 50-pm nozzle was used in all experiments. Analysis and sorting were performed at a sample flow rate of approximately 2,000 events per second, and 20,000 events per sample were collected for analysis. The head drive frequency during sorting was approximately 37,500 Hz. List mode data were stored with a PDP 11/23based computer (Consort 40; Becton-Dickinson).

after labeling with G,, antibody. The unshaded portion of this peak represents specific labeling. For both TH and G,,, the percentage of specifically labeled synaptosomes was calculated from data using the maximum positive difference method (Overton, 1988). This method has been shown to provide accurate determinations of the percentages of positive and negative cells in a population even when the percentage of positive cells is less than 10% of total and when there is considerable overlap between the positive and negative cell populations (Overton, 1988; Overton, personal communication). While single parameter histograms are best for quantifying immunoreactivity, dual parameter plots must be used in cases where more than one parameter is important to the analysis. For example, dual parameter plots are the only way t o present double label data because information about two parameters (fluorescein and R-phycoerythrin fluorescence) must be presented simultaneously for each synaptosome in the population in order to identify synaptosomes containing both antigens.

Data analysis Graphic data are presented as either single parameter histograms (Fig. 3) or dual parameter histograms (Figs. 1,4,6).However, calculations of the percentage of synaptosomes exhibiting specific antibody labeling were always determined from single-parameter histograms of fluorescence. In single parameter histograms each level of fluorescence intensity is presented on the x-axis, while the number of synaptosomes exhibiting that level of fluorescence intensity is presented on the y-axis. Specific labeling can then be defined as difference in area between the distribution derived from synaptosomes incubated with primary antisera and the distribution derived from incubation with either control serum or secondary antibody alone. In Figure 3, for example, the shaded peak on the left represents the distribution derived from unlabeled synaptosomes. The right-hand peak represents the distribution obtained

Gel electrophoresis and Western analysis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 1.5-mm-thick 11% polyacrylamide gels by conventional methods (Laemmli, 1970). Proteins were electroblotted to nitrocellulose (Towbin et al., 1979). The Western blot was then incubated in 5 % nonfat dry milk to block nonspe-

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cific antibody binding sites (Johnson et al., 1984) and reacted with G,, antiserum (1:4,000). The antigenantibody complex was visualized enzymatically with goat antirabbit Ig conjugated to alkaline phosphatase (Blake et al., 1984). To generate spot blots, synaptosomes were sorted based on fluorescence or light scatter and collected onto HA-type membrane filters (0.45-pm pore size) (Millipore, Bedford, MA) under vacuum as described previously (Wolf and Kapatos, 1989a). Spot blots were fixed by incubation for 15 min in 10% v/v acetic acid and 25%v/v isopropyl alcohol (Jahn et al., 19841, incubated in 5% nonfat dry milk, and reacted with 0.2 pg/ml of anti-TH IgG. The antigen-antibody complex was detected with goat antirabbit Ig conjugated to alkaline phosphatase. Relative amounts of TH in spot blots were quantified using a Zeineh soft laser scanning densitometer and accompanying software (Biomed Instruments, Fullerton, CAI. Protein concentration was determined by the method of Peterson (1977).

RESULTS Detection of TH Dopamine-containing striatal synaptosomes can be selectively detected and isolated with TH immunolabeling and FACS (Wolf and Kapatos, 1989b). The same approach was used in the present study, except that the secondary antibody was conjugated to R-PE rather than to fluorescein. Specific detection of TH-positive nerve terminals is illustrated in the form of dual parameter histograms of fluorescence versus forward-angle light scatter; each analysis is based on analysis of 20,000 synaptosomes (Fig. 1) (see under Methods for description of data analysis). Forward angle light scatter, a measure of particle size, is shown on the x-axis, while relative fluorescence intensity is shown on the y-axis. A box is drawn in each plot to illustrate the level of fluorescence exceeded by only 0.2% of unlabeled synaptosomes. Following immunolabeling, a subpopulation of particles, corresponding to TH-containing synaptosomes, became sufficiently immunofluorescent to enter this window. Synaptosomes specifically labeled with the TH antibody were found to comprise 12.1 2 0.6% of the synaptosomal fraction (n = 8 experiments, 20,000 synaptosomes per group). When corrected for the fact that 20-30% of the particles in the synaptosomal fraction represent nonsynaptosomal elements (free mitochondria or particles derived from glial cells) (see Wolf and Kapatos, 1989a), this yields an estimate of 15-17% for the percentage of striatal synaptosomes that are dopaminergic. This estimate concurs with previous estimates based upon different techniques (Hokfelt, 1968; Hokfelt and Ungerstedt, 1969; Iversen and Schon, 1973) and with results obtained with the same monoclonal antibody to TH and fluorescein-conjugated secondary antibody (Wolf and Kapatos, 1989b). To corroborate further the flow cytometric identifica-

Fig. 2. Western analysis of proteins from fresh synaptosomes (lane A) and synaptosomes fixed with Zamboni solution (lane B). In both lanes, G,, antiserum recognizes a 39-kDa protein band. Each lane contained 60 kg of protein.

tion of DA synaptosomes, immunoblot analysis of TH protein content was used to verify that synaptosomes labeled by TH antibody and R-PE are actually enriched in TH. FACS was used to collect a equal number of synaptosomes labeled by TH antibody and R-PE secondary antibody, and unlabeled synaptosomes. The relative TH content of the synaptosomal spots was then compared with Western blot techniques. In agreement with an earlier study (Wolf and Kapatos, 1989b), a 5.9 i 0.3 fold (n = 3 experiments) enrichment of TH immunoreactivity was found for synaptosomes which were collected based on labeling by TH antibody and R-PE.

Detection of G,, The G,, antiserum used in these studies has been shown to recognize on Western blots and immunoprecipitate a single 39-kDa protein that is specifically [32P]ADP-ribosylatedby pertussis toxin. The antiserum did not recognize 41-kDa pertussis toxin substrates from brain or liver, or 40-kDa pertussis toxin substrate from lung or liver (Granneman and Kapatos, 1990). It was important, however, to demonstrate that permeabilization and fixation of synaptosomes with Zamboni solution did not interfere with the specificity of the antiserum. Figure 2 shows a Western blot of equal

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FLUORESCENCE 1 (FLUORESCEIN) Fig. 3. Single-parameter histograms comparing synaptosomal labeling by preimmune serum, G,, antiserum, and G,, antiserum preadsorbed with the synthetic peptide antigen. The histogram obtained with unlabeled synaptosomes is shown in each panel for comparison (shaded area). Preadsorption of G,, antiserum with the peptide reduced labeling to the level obtained with preimmune serum.

amounts of synaptosomal protein obtained from fresh synaptosomes (lane A) and Zamboni-fixed synaptosomes (lane B) probed with G,, antiserum. The antiserum recognized a 39-kDa band in both lanes, indicating that specificity for G,, is maintained after Zamboni fixation. However, fixation decreased the relative amount of G,, immunoreactivity by approximately 50% (lane B). Loss of G,, from synaptosomes during the penneabilization and wash steps could account for this decrease in immunoreactivity. This seems unlikely,

however, since we have shown that most TH (82 2 7%), a soluble protein, is retained during the process of synaptosome permeabilization (Wolf et al., 1989~).A more likely explanation is that the affinity of G,, antiserum for G,, was decreased by Zamboni fixation, as it is well known that fixation can have profound effects on antigenicity, especially when the epitope recognized is a relatively short peptide sequence (Kerr et al., 1988). Preimmune serum did not recognize any protein band on Western blot (data not shown).

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Characterization of the distribution of G alpha o in rat striatal synaptosomes and its colocalization with tyrosine hydroxylase.

Dopaminergic striatal synaptosomes can be detected and isolated with a fluorescence-activated cell sorter (FACS). In the present study, two antigens w...
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