characterization of the distribution of gαo in rat striatal synaptosomes and its colocalization...

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

ABSTRACT Dopaminergic striatal synaptosomes can be detected and isolated with a fluorescence-activated cell sorter (FACS). In the present study, two antigens were detected simultaneously with primary antisera raised in different species and species- specific 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 (Gaol are 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 hydroxylase- containing synaptosomes were detected with a mouse monoclonal antibody to TH and R-phycoerythrin-conjugated secondary antibody. They comprised 15-1 7% 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 non- neuronal tissues (Homburger et al., 1987). Immunohis- tochemical studies have demonstrated that G,, is un- evenly 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- o 1991 WrLEY-Lrss. INC.

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 species- specific 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.

67 COLOCALIZATION OF TYROSINE HYDROXYLASE AND G,,

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 200- 300 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 1 mg protein per ml in HEPES-buffered Krebs-Ringer solution (KRH), pH 7.4, containing 140 mM NaC1, 5 mM KC1,2 mM CaCl,, 1.25 mM MgCl,, 10 mM Hepes, and 10 mM glucose.

Fixation and immunolabeling Synaptosomes were permeabilized and fixed by incu-

bation 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 Labo- ratories) 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 at 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-

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 1 hr 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,, double- labeling 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 (1 or 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). Photomulti- plier 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 1 into fluorescence 2, and vice versa. Synaptosomal fluorescence was analyzed with logarithmic amplifiers set for 4 log

68 M.E. WOLF ET AL.

UNLABELED TH ANTIBODY 11 4

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

decades. The 90" scatter PMT was operated at 400 V. A neutral density filter (ND1 j 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 11/23 based computer (Consort 40; Becton-Dickin- son).

Data analysis Graphic data are presented as either single parame-

ter 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

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.

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 determina- tions 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 communica- tion). 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 to 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.

Gel electrophoresis and Western analysis Sodium dodecyl sulfate-polyacrylamide gel electro-

phoresis (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-

COLOCALIZATION OF TYROSINE HYDROXYLASE AND G,, 69

cific antibody binding sites (Johnson et al., 1984) and reacted with G,, antiserum (1:4,000). The antigen- antibody 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 descrip- tion 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 mito- chondria 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 es- timates 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 immunopre- cipitate a single 39-kDa protein that is specifically [32P]ADP-ribosylated by 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

70 M.E. WOLF ET AL.

3 PREIMMUNE SERUM

I Go ANTISERUM 1

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).


i 7 UNLABELED

<

\

i i

I 1 1 PREIMMUNESERUM 1

FORWARD ANGLE LIGHT SCATTER

Fig. 4. Dual-parameter plots of forward-angle light scatter and fluorescein fluorescence illustrating synaptosomal labeling by preimmune serum and G,, antiserum (see Fig. 1 for details).

Single-parameter histograms illustrating the specificity of synaptosomal labeling by G,, antiserum are shown in Figure 3. Unlabeled synaptosomes (shaded area in each panel) are compared to synaptosomes incubated with preimmune serum (top), G,, antiserum (middle), and G,, antiserum that had been preadsorbed with the synthetic G,, peptide antigen (bottom). The fluorescence intensity of synaptosomes incubated with fluorescein-conjugated secondary antibody alone was identical to that of unlabeled synaptosomes (data not shown). When G,, antiserum was preadsorbed with the synthetic peptide antigen labeling was reduced to the level obtained with preimmune serum. Preadsorption had no effect on preimmune labeling (data not shown). When interpreted in light of the data derived from Western blot analysis, three conclusions were drawn: (1) labeling by G,, antiserum is due to recognition of the appropriate antigen; (2) the difference between labeling by G,, antiserum and preimmune serum is specific to G,,; and (3) as a result of the loss of immunoreactivity upon fixation, not all synaptosomes containing G,, may be detectable by this methodology. In subsequent experiments, specific labeling was defined as the percentage of synaptosomes labeled by G,, antiserum minus the percentage labeled by preimmune serum. On the basis of the maximum positive difference method to quantify specific labeling (Overton, 1988), it was determined that 52.7 k 4.2% of the synaptosomal fraction was specifically labeled by G,, antiserum (n = 3 experiments, 20,000 synaptosomes per group). It is uncertain whether it is appropriate to correct this value for the fact that 20-30% of particles in the synaptosomal fraction are not derived from neurons (see above). Because TH is present only in synaptosomes, this correction was appropriate when quantifying the percentage of synaptosomes labeled by TH. This correction may be less appropriate for G,,, however, since G,, has been found in cultured murine striatal astrocytes (Brabet et al., 19881,

and it has been previously demonstrated by flow cytometry that 5-10% of the particles within the rat striatal synaptosomal preparation are derived from astrocytes (Wolf and Kapatos, 1989a). If one assumes that G,, is only detected in synaptosomes, then these results indicate that 65-75% of striatal synaptosomes contain G,,. Regardless of whether this correction is performed, it is apparent that not all striatal synaptosomes contain detectable levels of G,,.

Dual-parameter plots comparing the fluorescence intensity of unlabeled synaptosomes, synaptosomes incubated with preimmune serum followed by fluorescein secondary antibody, and synaptosomes incubated with G,, antiserum followed by fluorescein secondary antibody are shown in Figure 4. Two things are notable. First, synaptosomes labeled by G,, antiserum are not preferentially located in a particular range of forward angle light scatter. This suggests that G,,-containing synaptosomes are heterogeneous with respect to size, as has been demonstrated previously for TH-containing nerve terminals (Wolf and Kapatos, 198913). Second, synaptosomes that produce the same FALS signal, and are therefore similar in size, exhibit a relatively broad range of fluorescence intensities after labeling with G,, antiserum. This suggests that G,,-containing synaptosomes can differ significantly in their content of G,, and that variations in synaptosomal G,, content are not simply due to differences in the size or surface area of the subcellular particle.

Simultaneous detection of TH and G,, Dual parameter plots of fluorescein fluorescence vs.

R-PE fluorescence were used in the analysis of double- labeling experiments. Representative plots are shown in Figure 5. For each of the 20,000 synaptosomes analyzed in each plot, fluorescein fluorescence and R-PE fluorescence are presented as x- and y-coordinates, respectively, on a grid. Because both colors of fluores-

M.E. WOLF ET AL.

w - w ,

: UNLABELED : PREIMMUNE SERUM

..... --. ................

'i.

r r

I ' : TH/PREIMMUNE

* .

: Go ANTISERUM

-,-

j TH/Go ANTISERUM

FLUORESCENCE 1 (FLUORESCEIN)

Fig. 5. Dual-parameter plots illustrating colocalization of G,, (fluorescence 1) and TH (fluorescence 2). Quadrant I contains synaptosomes immunoreactive for TH only. Quadrant I1 synaptosomes immunoreactive for both TH and 4,. Quadrant I11 contains synaptosomes immunoreactive for neither TH or G,. Quadrant IV contains synaptosomes immunoreactive for G,, only.

cence are presented for each individual synaptosome, it is possible to discriminate between particles containing TH only, G,, only, both TH and G,,, or neither antigen.

To distinguish between unlabeled and labeled synaptosomes, each plot is divided into quadrants (1-IV) with the unlabeled population as a reference. Boundaries were selected such that the vast majority of unlabeled synaptosomes were found in quadrant 111, with less than 0.2% found in either I or IV. Synaptosomes that remain in quadrant I11 after incubation with G,, and TH antibodies contain neither antigen. Synaptosomes that enter quadrant I contain TH only and synaptosomes that enter quadrant IV contain G,, only. Synap- tosomes which enter quadrant I1 contain both antigens because they exhibit increases in both fluorescein and R-PE fluorescence.

Inspection of Figure 5 shows that some synaptosomes clearly contain both G,, and TH (quadrant 111, while some appear to contain only TH (quadrant I). Because the coincidence of two signals is being analyzed, the maximum positive difference method cannot be used for analysis. Specific labeling can be estimated, however,

based on the quadrants shown in Figure 5. Analysis of data obtained from three double-label experiments (20,000 synaptosomes per group) revealed that 26.6 f 7.5% of TH-containing synaptosomes entered quadrant I1 if they were also incubated with preimmune serum (Fig. 5, bottom middle). After incubation with G,, antiserum, 76.8 It_ 2.9% of TH-containing synaptosomes entered quadrant I1 (Fig. 5, bottom right). Subtraction of preimmune labeling demonstrated that 50.2 2 10.2% of TH-containing synaptosomes were specifically labeled by G,, antiserum. However, as noted above, this type of subtraction is based on boundaries defined by the unlabeled synaptosomes rather than the positive difference method. Thus, these data can only provide the minimum percentage of TH-containing synaptosomes which also contain detectable G,, immunoreactivity.

DISCUSSION Some G-proteins exhibit strong tissue specificity. For

example, transducin is found only in rod photoreceptor cells of vertebrate retina (Grunwald et al., 1986). Rela-


tively little is known, however, about the extent to which particular G-proteins in brain are selectively localized to particular types of neurons. Although the heterogeneous distribution of G,, within the brain suggests localization to specific populations of neurons, little is known to what extent G,, is selectively associated with particular neurotransmitter phenotypes. Within the basal ganglia, G,, protein appears to be concentrated in the striatal projections to the dorsome- dial region of the substantia nigra zona reticulata (Worley et al., 1986). These striatonigral neurons are believed to contain the neurotransmitter y-aminobu- tyric acid (GABA) and a tachykinin peptide (Gerfen et al., 1990). In contrast to this immunohistochemical localization of G,, protein within the substantia nigra, a recent study has shown that G,, mRNA is concentrated within the perikarya of the DA-containing neurons of the zona compacta (Vincent et al., 1990). This differen- tial cellular localization of G,, mRNA and protein dem- onstrates the importance of localizing G,, protein to specific populations of neurotransmitter-defined nerve terminals. The purpose of the present study was to examine the distribution of G,, within a striatal synaptosomal fraction and, more specifically, to determine whether G,, is present in synaptosomes derived from the nerve terminals of nigrostriatal DA neurons.

In agreement with previous results from our labora- tory and results obtained with other methods (Hokfelt, 1968; Hokfelt and Ungerstedt, 1969; Iversen and Schon, 1973), the present findings confirmed that approximately 15-17% of striatal synaptosomes contain TH and are therefore dopaminergic. G,, was found to be present in approximately 65-75% of total striatal synaptosomes and in a t least 50% of TH-containing synaptosomes. Although G,, has been reported to be a major protein component of the growing nerve terminal (Strittmatter et al., 1990), this is the first study to localize G,, to the adult nerve terminal region.

Heterogeneity of G-protein content has previously been reported for midbrain DA neurons. Gaia immunoreactivity has been selectively localized to neurons containing cholecystokinin (CCK) or both CCK and TH and, in particular, to CCK- and TH-positive cell bodies in the substantia nigra and ventral tegmental area (Cortes et al., 1988). However, while coexistence of Gaia and CCK immunoreactivity was always observed, some TH- positive neurons in the ventral tegmental area ap- peared to lack GaiS. Because the vast majority of DA nerve terminals in the striatum are derived from the DA cell group located in the zona compacta of the substantia nigra, our results indicate that up to 50% of these neurons may either contain no G,, or levels of this protein which are not detectable by our methodology. An evaluation of whether the presence of G,, within nigrostriatal DA neurons correlates with the existence of a peptide co-neurotransmitter, as is the case for GaiS in the ventral tegmentum, awaits further study using the techniques presented here.

There are many functions that G-proteins may sub- serve in DA perikarya and nerve terminals. For example, pertussis toxin treatment has been shown to block the inhibitory effects of DA autoreceptor stimulation on the activity of DA neurons in rat substantia nigra (Innis and Aghajanian, 1987) and on the synthesis of DA in rat striatum (Bean et al., 1988), suggesting a role for G- proteins in transducing the effects of both somatodendritic and nerve terminal DA autoreceptors. The inhibitory effect of somatodendritic autoreceptor stimulation on DA cell activity may be due to an autoreceptor- mediated increase in potassium conductance in DA neurons of the substantia nigra (Lacey et al., 1987). An increase in potassium conductance may also be involved in the autoreceptor-mediated inhibition of DA release from DA nerve terminals in the striatum (Bowyer and Weiner, 1989). Both G,, and Gaia are candidates for the autoreceptor-coupled G-protein, since both are pertussis toxin substrates and both have been shown to acti- vate potassium channels (VanDongen et al., 1988; Co- dina et al., 1988). It is relevant to this report that not all midbrain DA neurons appear to possess functional DA autoreceptors (Chiodo et al., 19841, but this distinction may correlate with the level of G,, protein rather than the presence of the DA autoreceptor itself.

In summary, as determined by flow cytometric techniques, 65-75% of striatal synaptosomes contain G,, and at least 50% of TH-positive synaptosomes, derived primarily from nigrostriatal DA neurons, also contain G,,. These findings suggest that G,, may not be a protein component of all nerve terminals, and provide a basis for a role for G,, in signal transduction within subpopulations of striatal nerve terminals derived from intrinsic and afferent neurons, including those of nigrostriatal dopamine neurons.

ACKNOWLEDGMENTS We thank Vickie Kemski for excellent technical assis-

tance. This work was supported by U.S.P.H.S. grants MH 43758, NS 08413, NS 26801, and DK 37006.

REFERENCES Asano, T., Semba, R., Ogasawara, N., and Kato, K. (1987) Highly

sensitive immunoassay for the (Y subunit of the GTP-binding protein Go and its regional distribution in bovine brain. J. Neurochem.,

Bean, A.J., Shepard, P.D., Bunney, B.S., Nestler, E. J., and Roth, R.H. (1988) The effects of pertussis toxin on autoreceptor-mediated inhibition of dopamine synthesis in the rat striatum. Mol. Pharmacol., 34:715-718.

Blake, M.S., Johnston, K.H., RussellJones, G.J., and Gotschlich, E.C. (1984) A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on western blots. Anal. Biochem.,

Bowyer, J.F., and Weiner, N. (1989) K' channel and adenylate cyclase involvement in regulation of Ca2+-evoked release of L3H1dopamine from synaptosomes. J. Pharmacol. Exp. Ther., 248:514520.

Brabet, P., Dumuis, A,, Sebben, M., Pantaloni, C., Bockaert, J., and Homburger, V. (1988) Immunocytochemical localization of the guanine nucleotide-binding protein Go in primary cultures of neuronal and glial cells. J. Neurosci., 8:701-708.

Chiodo, L.A., Bannon, M.J., Grace, A.A., Roth, R.H., and Bunney, B.S. (1984) Evidence for the absence of impulse-regulating somatodendritic and synthesis-modulating autoreceptors on subpopulations of mesocortical dopamine neurons. Neurosciences, 12:l-16.

48~1617-1623.

136:175-179.

74 M.E. WOLF ET AL.

Codina, J., Olate, J., Abramowitz, J., Mattera, R., Cook, R.G., and Birnbaumer, L. (1988) ai-3 cDNA encodes the (1 subunit of G,, the stimulatory G protein of receptor-regulated K' channels. J. Biol. Chem., 263:6746-6750.

Cortes, R., Hokfelt, T., Schalling, M., Goldstein, M., Goldsmith, P., Spiegel, A., Unson, C., and Walsh, J. (1988) Antiserum raised against residues 159-168 of the guanine nucleotide-binding protein Gi,, reacts with ependymal cells and some neurons in the rat brain containing cholecystokinin- or cholecystokinin- and tyrosine 3-hydroxylase-like immunoreactivities. Proc. Natl. Acad. Sci. U.S.A.,

Gabrion, J., Brabet, P., Nguyen Than Dao, B., Homburger, V., Dumuis, A,, Sebben, M., Rouot, B., and Bockaert, J . (1989) Ultrastructural localization of the GTP-binding protein Go in neurons. Cell. Signal- ling, 1:107-123.

&&en, C.R., Engber, T.M., Mahan, L.C., Susel, Z., Chase, T.N., Monsma, F.J., and Sibley, D.R. (1990) D1 and D2 dopamine receptor- regulated gene expression of striatonigral and striatopallidal neurons. Science, 250:1429-1432.

Gierschik, P., Milligan, G., Pines, M., Goldsmith, P., Codina, J., Klee, W., and Spiegel, A. (1986) Use of specific antibodies to quantitate the guanine nucleotide-binding protein Go in brain. Proc. Natl. Acad. Sci.

Granneman, J.G., and Kapatos, G. (1990) Developmental expression of Go in neuronal cultures from rat mesencephalon and hypothalamus. J . Neurochem., 54:1995-2001.

Gray, E.G., and Whittaker, V.P. (1962) The isolation of nerve endings from brain: A electron-microscopic study of cell fragments derived by homogenization and centrifugation. J . Anat., 96:79-88.

Grunwald, G.B., Gierschik, P., Nirenberg, M., and Spiegel, A. (1986) Detection of a-transducin in retinal rods but not cones. Science, 231:85&859.

Hokfelt, T. (1968) In vitro studies on central and peripheral monoamine neurons at the ultrastructural level. Z. Zellerforsch., 91:l-74.

Hokfelt, T., and Ungerstedt, U. (1969) Electron and fluorescence microscopical studies on the nucleus caudatus putamen of the rat after unilateral lesions of ascending nigro-neostriatal dopamine neurons. Acta Physiol. Scand., 76:415-426.

Homburger, V., Brabet, P., Audigier, Y., Pantaloni, C., Bockaert, J . , and Rouot, B. (1987) Immunological localization of the GTP-binding protein G, in different tissues of vertebrates and invertebrates. Mol. Pharmacol., 31:313-319.

Innis, R.B., and Aghajanian, G.K. (1987) Pertussis toxin blocks autoreceptor-mediated inhibition of dopaminergic neurons in rat substantia nigra. Brain Res., 411:139-143.

Iversen, L.L., and Schon, F.E. (1973) The use of autoradiographic techniques for the identification and mapping of transmitter-specific neurones in CNS. In: New Concepts in Neurotransmitter Regula- tion. A.J. Mandell, ed. Plenum Press, New York, pp. 153-193.

85:9351-9355.

u.s.A., 83:2258-2262.

Johnson, D.A., Gautsch, J.W., Sportsman, J.R., andElder, J.H. (1984) Improved technique utilizing nonfat dried milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene Anal. Technol., 1:3-8.

Kerr, R.S.C., Haye, A.H., and Bartlett, P.F. (1988) A quantitative assessment b y flow cytometry of the preservation of the Thy-1 antigen by various fixation procedures. Neurosci Lett., 87:250-265.

Lacey, M.G., Mercuri, N.B., and North, R.A. (1987) Dopamine acts on D, receptors to increase potassium conductance in neurones of the rat substantia nigra zona compacta. J. Physiol. (Lond.), 392:397- 416.

Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of Bacteriophage T4. Nature, 227:68C-685.

Lehman, J. , and Langer, S.Z. (1983) The striatal cholinergic interneu- ron: Synaptic target of dopaminergic terminals? Neuroscience, 10:1105-1120.

Overton, W.R. (1988) Modified histogram subtraction technique for analysis of flow cytometry data. Cytometry, 9:619-626.

Peterson, G.L. (1977) A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochcbm., 83:34&356.

Strittmatter, S.M., Valenzuela, D., Kennedy, T.E., Neer, E.J., and Fishman, M.C. Go is a major growth cone protein subject to regulation by GAP-43. Nature, 344:836841.

Towbin, H., Staehlein, T., and Gordon, J. (1979) Electrophoretic trans- fer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. U.S.A., 76:4350-4354.

Van Dongen, A.M.J., Codina, J., Olate, J., Mattera, R., Joho, R., Birnbaumer, L., and Brown, A.M. (1988) Newly identified brain potassium channels gated by the guanine nucleotide binding protein Go. Science, 2421433-1437.

Vincent, S.R., Hope, B.T., Drinnan, S.L., and Reiner, P.B. (1990) G protein mRNA expression in immunohistochemically identified dopaminergic and noradrenergic neurons in the rat brain. Synapse, 6:23-32.

Wolf, M.E., and Kapatos, G. (1989a) Flow cytometric analysis of rat striatal nerve terminals. J . Neurosci., 9:94-105.

Wolf, M.E., and Kapatos, G. (198913) Flow cytometric analysis and isolation of permeabilized dopamine nerve terminals from rat striatum. J. Neurosci., 9:106-114.

Wolf, M.E., Zigmond, M.J., and Kapatos, G. (1989~) Tyrosine hydroxylase content of residual striatal dopamine nerve terminals following 6-hydroxydopamine administration: A flow cytometric study. J. Neurochem., 53:879-885.

Worley, P.F., Baraban, J.M., Van Dop, C., Neer, E. J., and Snyder, S.H. (1986) Go, a guanine nucleotide-binding protein: Immunohistochem- ical localization in rat brain resembles distribution of second mes- senger systems. Proc. Natl. Acad. Sci. U.S.A., 83:45614565.

characterization of the distribution of gαo in rat striatal synaptosomes and its colocalization...

Documents