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Previous Article | Next Article
The Journal of Neuroscience, December 1, 2002, 22(23):10251-10266
Number, Density, and Surface/Cytoplasmic Distribution of GABA Transporters at Presynaptic Structures of Knock-In Mice Carrying GABA Transporter Subtype 1-Green Fluorescent Protein Fusions
Chi-Sung Chiu1, Kimmo Jensen4, Irina Sokolova1, Dan Wang3, Ming Li1, Purnima Deshpande1, Norman Davidson1, Istvan Mody4, Michael W. Quick3, Stephen R. Quake2, and Henry A. Lester1 Divisions of 1 Biology and 2 Engineering and Applied Physics, California Institute of Technology, Pasadena, California 91125, 3 Department of Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0021, and 4 Departments of Neurology and Physiology, University of California Los Angeles School of Medicine, Los Angeles, California 90095-1769
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
GABA transporter subtype 1 (GAT1) molecules were counted near GABAergic synapses, to a resolution of ~0.5 µm. Fusions between GAT1 and green fluorescent protein (GFP) were tested in heterologous expression systems, and a construct was selected that shows function, expression level, and trafficking similar to that of wild-type (WT) GAT1. A strain of knock-in mice was constructed that expresses this mGAT1-GFP fusion in place of the WT GAT1 gene. The pattern of fluorescence in brain slices agreed with previous immunocytochemical observations. [3H]GABA uptake, synaptic electrophysiology, and subcellular localization of the mGAT1-GFP construct were also compared with WT mice. Quantitative fluorescence microscopy was used to measure the density of mGAT1-GFP at presynaptic structures in CNS preparations from the knock-in mice. Fluorescence measurements were calibrated with transparent beads and gels that have known GFP densities. Surface biotinylation defined the fraction of transporters on the surface versus those in the nearby cytoplasm. The data show that the presynaptic boutons of GABAergic interneurons in cerebellum and hippocampus have a membrane density of 800-1300 GAT1 molecules per square micrometer, and the axons that connect boutons have a linear density of 640 GAT1 molecules per micrometer. A cerebellar basket cell bouton, a pinceau surrounding a Purkinje cell axon, and a cortical chandelier cell cartridge carry 9000, 7.8 million, and 430,000 GAT1 molecules, respectively; 61-63% of these molecules are on the surface membrane. In cultures from hippocampus, the set of fluorescent cells equals the set of GABAergic interneurons. Knock-in mice carrying GFP fusions of membrane proteins provide quantitative data required for understanding the details of synaptic transmission in living neurons.
Key words: GABA; synapse; transporter; green fluorescent protein; mouse; knock-in
INTRODUCTION
When uptake by the GABA transporter GAT1 is inhibited, evoked IPSCs are prolonged at some synapses, presumably because transmitter molecules remain near receptors and reactivate them (Dingledine and Korn, 1985
; Roepstorff and Lambert, 1992
Understanding the role of GABA uptake in these and other aspects of GABAergic transmission requires knowing key parameters and mechanisms. Quantitative studies of local GABAA receptor number (Nusser et al., 1997
This study was conducted to quantify the number and density of GAT1 near synapses. We constructed a strain of knock-in mice that express a GAT1-green fluorescent protein (GFP) fusion in place of the wild-type (WT) GAT1 gene. We calibrated the fluorescence measurements by exploiting previously described transparent beads that themselves have known GFP densities (Chiu et al., 2001
In addition to timely removal of GABA released spontaneously or by presynaptic impulses, other possible functions of GABA transporters include replenishing the supply of GABA in an inhibitory neuron and releasing GABA in a voltage-dependent but Ca2+-independent mechanism (Lester et al., 1996
MATERIALS AND METHODS
mGAT1 and GFP fusion constructs. GFP37 was fused to the N terminus of mGAT1 (Liu et al., 1992
CGA ATT CTG CAG ATA TCC AGC ACA GTG GCG GCC GCC
R I L Q I S S T V A A A
The WT mGAT1 control and GFP control were subcloned between the HindIII and EcoRI and between the NotI and XbaI sites in pcDNA3.1(+), respectively.
Human embryonic kidney (HEK) 293T cells were plated on 35 mm plates to 50-70% confluence, transfected with the fusion constructs (Effectene, Qiagen), and incubated for an additional 60 hr. For GABA uptake assays, cells were washed twice with Krebs'-Ringer's (KRH) buffer (Ramamoorthy et al., 1998
A lentivirus expression construct was constructed using the three-plasmid expression system as described (Naldini et al., 1996
80°C, thawed once, and applied (15-20 µl) to Madin-Darby canine kidney (MDCK) cells.
Knock-in mouse targeting construct. The mGAT1 cDNA fragment digested with AccI and StuI (position 715-1785 in the coding sequence) was used to synthesize a random primed [
32P]dCTP probe using the protocol from the NEBlot kit (BioLabs). A 129 SVEV Tac FBR mouse spleen genomic library (Lambda FIX II genomic library; Stratagene) was screened. We isolated 26 genomic clones and identified one clone (#6) that carried intron 14 and exon 15 and had >4.0 kb of flanking genomic sequence on both arms.
Genomic clone #6 was amplified by subcloning into the NotI site in pBluescript. The amplified mGAT1 genomic clone #6 was subcloned into the HindIII site of pBluescriptSK(+) and subsequently subcloned into pKO vector (Lexicon Genetics), which carries the diphtheria toxin gene as a positive selection marker, using the SalI and EcoRI sites from pBluescript. The genomic DNA in the pKO vector was modified by inserting the 12-residue spacer plus GFP coding sequence just 5' to the stop codon of mGAT1 (Exon #15; position 17617) by PCR using three pairs of primers. This construct was confirmed by sequencing. To eliminate restriction sites in the 12-residue spacer between mGAT1 and GFP, the spacer sequence was modified with silent mutations via the PCR primers:
CGC ATT CTC CAA ATC TCA AGC ACC GTA GCC GCA GCC
R I L Q I S S T V A A A
The floxed Neo cassette was inserted between position 17303 and 17311 (intron #14), where AscI sites were created by PCR. The floxed Neo cassette was modified by creating AscI sites on both ends by linkers. An additional 1.6 kb flanking arm at the 3' end of this construct was created by replacing the ApaI/EcoRI sites in the pKO vector with the corresponding mGAT1 genomic DNA sites. The final construct has ~3.8 kb on the 5' arm, -Neo-320 bp-GFP-, and ~4.2 kb on the 3' arm for homologous recombination (Fig. 1A). The 320 bp sequence includes the 34 residues of the coding portion of exon 15.
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Figure 1. Generation and screening of knock-in strains. A, Modification of mGAT1 genomic DNA to generate a targeting plasmid that contains an mGAT1-GFP fusion sequence in an exon and a floxed neomycin selection cassette in an intron. See Materials and Methods for details. B, PCR screening to identify ES cells carrying the mutant gene. A 4.5 kb PCR product is expected. Lanes 1, 3, and 4 represent positive ES cell clones; lane 2 is a negative clone. Lanes 5 and 6 show negative controls with no PCR products from genomic DNA extracted from WT ES cells and from the final pKO plasmid construct shown in A. Lane M shows molecular length standards. C, Generation of the Neo-deleted mGAT1-GFP knock-in mouse. The intron 14-Neo-mGAT1 heterozygotes were mated with DBA mice carrying cre recombinase to eliminate the neomycin selection cassette. D, Exemplar PCR genotyping results. Lanes 1-3 show PCR products for mice that are homozygous for the presence of the GFP fusion, heterozygous, and WT, respectively. Lanes 4-6 represent the screening for mice that are homozygous for the presence of the Neo cassette, heterozygous, and WT, respectively.
Homologous recombination, blastocyst injection, and mouse breeding. J7 embryonic stem (ES) cells from 129S3SvImJ mice were electroporated with the linearized construct and screened with Geneticin (G418; 180 µg/ml). The ES cell clones that survived this screening were further screened by PCR (Long template PCR kit; Roche) using a primer set that anneals to position 13017-43 and the neomycin resistance cassette (Neo): mGAT1 13017-43: 5'-GAC TGG TGG GAG AGG CAG ACT TTG AAC-3'; Neo-cassette 3': 5'-CCA AGT TCT AAT TCC ATC AGA AGC TCC-3'.
The recombined mGAT1 gene showed a 4.5-kb PCR product (Fig. 1B). Positive clones were confirmed by PCR using additional sets of primers. Two clones were injected into the C57 blastocyst. Chimeras that displayed >60% agouti coloring were mated with C57 females to generate heterozygous mice. These mice carry the Neo-cassette in intron 14 and were named intron 14-Neo-mGAT1. The loxP-flanked Neo-cassette was deleted by mating with DBA mice carrying cre recombinase. This deletion leaves the 34 bp of one loxP site plus restriction sites (SwaI-AscI) in the intron, and this Neo-deleted strain was named mGAT1-GFP (Fig. 1C).
To genotype the mice (Fig. 1D), three PCR primers were used for screening the GFP insertion: mGAT1 17399-430: 5'-GAC ATT TGG CTT ACT AGT GAG GAA ACA AGA GC-3'; mGAT1 17830-799: 5'-GCT AAG GGG CCT CTA CGG AAG CCT CCA GAG GC-3'; and GFP37 995-64: 5'-CCA TCT AAT TCA ACA AGA ATT GGG ACA ACT CC-3'.
To confirm deletion of the Neo-cassette, the primers were as follows: mGAT1 16922-54: 5'-CCA TGA GGT TGG CTG GAG GGA GAA TAA TGT AGC-3'; mGAT1 17521-54: 5'-GCA CAA TAT CTT CAC TGG GCT GAA TCA TGA CCT G-3'; and Neo-cassette 3': 5'-CCA AGT TCT AAT TCC ATC AGA AGC TCC-3'.
GFP calibrations. Brain slices, MDCK cells, GFP-beads, and GFP in polyacrylamide gels were imaged using a Leica SP1 confocal microscope system. The confocal system was warmed up for >2 hr, so that the laser photopower fluctuated less than ±5%, monitored by a photopower meter (ThorLabs Inc.; model S20MM) and by the transmitted light detector in the microscope. During the ~4 months of the quantitative imaging experiments, we monitored the stability of the photomultiplier tube (PMT) periodically by imaging the standardized GFP-beads at a standard photopower. Signals varied by <5%, indicating that the PMT is stable.
Images were taken with a 100× plan apochromatic objective, numerical aperture (NA) = 1.4 (Leica, #506038) using 2× zoom. The pinhole was set at 152 µm, as recommended by the manufacturer. The scan speed was 200 lines per second (slow mode), and the image size was 1024 × 1024 pixels. Each image was scanned with four repeats. The laser power was adjusted using the microscope's acousto-optical tunable filter, so that the fluorescence of a sample fell within the linear range of the detection system.
Bleaching is described by the relation, f(t) = f0e
kIt, where f0 and f(t) are the fluorescence at times zero and t(sec), respectively, k is the cross section for bleaching, measured previously as 4.7 × 10
0.12, so that the average bleaching for a structure within the imaged region was <6%. No formal bleaching corrections were applied.
GFP fluorescence was calibrated using two, well understood His6-GFP preparations. Our primary tool was a series of transparent Ni-NTA beads with known surface densities of His6-GFP (Chiu et al., 2001
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Figure 2. GFP characterization in beads, gels, and tissue samples. A, B, Calibration lines generated for His6-GFP beads (A) and His6-GFP in polyacrylamide gels (B) using the Leica confocal system. The measured slopes for His6-GFP beads are 0.10379 and 0.06944 (counts per pixel per GFP per square micrometer) at 1.15 and 0.77 µW, respectively. The measured slopes for His6-GFP in polyacrylamide gels were 0.02343, 0.01683, and 0.00718 (intensity per pixel per GFP per cubic micrometer) illuminated with 1.11, 0.75, and 0.31 µW, respectively. C, x-z projection of a slice from the cerebellar ML region. Scale bar, 10 µm. D, The profile of the decreased fluorescent intensity along the z-axis, averaged along the x-axis from C.
For GFP in polyacrylamide gels (Fig. 2B), the His6-GFP stock solution was diluted with 50 mM Tris-Cl, pH 8.0, 300 NaCl, 1.5 mg/ml BSA to various concentrations (a final volume of 50 µl) and then mixed with an equal volume of acrylamide solution (38%, 19:1) to generate a homogeneous polyacrylamide gel. The final concentrations of GFP in the gel were 6900, 5500, 4120, 2750, and 1375 GFP per cubic micrometer. The gels were sectioned at 60 µm slices using a vibratome and mounted for imaging. The sectioning and imaging were done within 30 min to prevent GFP diffusion. The sensitivities were proportional to photopower and equaled 0.02343, 0.01683, and 0.00718 (counts per pixel per GFP per cubic micrometer) at photopowers of 1.11, 0.75 and 0.31 µW, respectively (Fig. 2B). When these sensitivities are compared with those obtained with GFP-beads, it may be calculated that the microscope images fluoresce from an effective z-axis thickness of 250 nm, probably because the microscope is gathering fluorescence from a z-axis range somewhat >250 nm, but with an efficiency somewhat less than that for the bead surface.
His6-GFP is stable in polyacrylamide (without SDS). We monitored His6-GFP fluorescent intensity before and after the gel was polymerized using a fluorometer and found no changes (data not shown). This agrees with a previously published paper that characterized GFP in polyacrylamide gels (Dickson et al., 1997
Sample preparation. Mice were anesthetized with halothane (2-bromo-2-chloro-1,1,1-trifluorothane) and perfused with 4% paraformaldehyde in PBS, pH adjusted to 7.6 with Na2HPO4. Brains were dissected and kept in 4% paraformaldehyde for 1 hr in 4°C and then incubated in 30% sucrose in PBS for ~20 hr. The brains were embedded in O.C.T. medium (Tissue-Tek) for either horizontal or sagittal sections and sliced by cryostat at 35 µm. Brain slices were stored in 11 mM NaH2PO4, 20 mM Na2HPO4, 30% ethylene glycol, and 30% glycerol, pH 7.5, at
mGAT1-GFP intensity measurement using CCD camera. Surveys of mGAT1-GFP expression measurement in sagittal brain slices (see Fig. 4B) were performed with an epifluorescence microscope (Nikon Eclipse 300) equipped with a CCD camera using a 2× objective. We chose 14 regions that represent the range of GFP intensity in the brain and gathered five images of each region using a 100× objective (NA 1.4). The averaged fluorescent intensity from each region was normalized to that of the Purkinje cell pinceaux.
Confocal imaging and quantification. For imaging Neo-deleted mice, the laser power was set between 0.75 and 1.1 µW. We estimated that the autofluorescence from a WT brain slice is equivalent to 10-20 GFP per square micrometer (Chiu et al., 2001
Using ImageJ, we selected boutons, axons, pinceaux, and cartridges for quantification from stacks of images (81 nm per step). The GFP volume density of a structure was determined by dividing (calculated average fluorescent intensity)/(slope of the standard curve, intensity per pixel per GFP per cubic micrometer) at similar photopower (Fig. 2B). The total GFP molecules in a structure were calculated by multiplying the volume (total number of voxels) times the measured average density.
The GAT1-GFP surface density was calculated by dividing the surface area (sum of pixels) by the total number of GFP molecules. Heterozygotes and homozygotes gave equally precise data, but counts from the heterozygotes were half that from homozygotes and were multiplied by 2 for density measurements.
Fluorescence intensity decreases beneath the tissue surface along the z-axis with a space constant of ~35 µm (Fig. 2C,D), primarily because of refractive index mismatches and other causes of light-scattering effect that have been described and characterized previously (Hell et al., 1993
Biotinylation. Biotinylation experiments were performed essentially as described (Qian et al., 1997
Dissociated culture. Neuronal cultures were prepared as described (Li et al., 1998
.
Synaptosomal preparation and GABA uptake assay. Mice were anesthetized with halothane, and brains were dissected and collected on ice. The cerebellum (~50 mg) was homogenized in 20× (w/v) medium I (0.32 M sucrose, 0.1 mM EDTA, and 5 mM HEPES, pH 7.5) using a Teflon-glass homogenizer with 16 strokes. Synaptosomes were prepared (Nagy and Delgado-Escueta, 1984
Twenty microliters of the suspension from the P2 particulate fraction were mixed with 280 µl of uptake buffer containing (in mM): 128 NaCl, 2.4 KCl, 3.2 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 10 glucose, 25 HEPES, pH 7.5 (Lu et al., 1998
Translocation treatments. For synaptosomal GABA uptake assays, synaptosomes were pretreated with 50 µM orthovanadate, 100 nM bisindolylmaleimide II, and 0.45 M sucrose at 37°C for 15 min and then transferred to synaptosomal GABA uptake buffer at 4°C. The GABA uptake assay lasted for 1 h at 4°C.
To prepare living brain slices for quantitative imaging and Western blot, mice were anesthetized, and brains were dissected free. Brains were then kept in ice-cold artificial CSF (ACSF) containing (in mM): 126 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, 10 D-glucose, and 3 kynurenic acid, pH 7.3, bubbled with 95% O2 and 5% CO2. The brain was then sliced by vibratome at 120 µm thickness. The slices were incubated at 37°C in 50 µM orthovanadate, 100 nM bisindolylmaleimide II, and 0.45 M sucrose for 15 min. Slices were transferred to ice-cold ACSF. Some slices were subjected to biotinylation and quantitative Western blot as described in the previous section. Other slices were fixed in ice-cold ACSF containing 4% paraformaldehyde for 20 min. After a rinse with ACSF, slices were mounted and subjected to imaging.
Brain slice electrophysiology. Mice (P15-P25) were anesthetized with halothane before decapitation, according to University of California Los Angeles regulations. The brain was placed in ice-cold ACSF bubbled with 95% O2 and 5% CO2. Coronal slices (350 µm thick) were cut with a Leica VT1000S Vibratome and stored in bubbled ACSF for >1 hr.
Whole-cell recordings were made at 32-33°C from CA1 pyramidal cells (Zeiss Axioscope infrared-differential contrast videomicroscopy) with an Axopatch 200B amplifier. Electrodes were pulled (Narishige PP-83; Narishige, Tokyo, Japan) from borosilicate glass and filled with solution containing (in mM): 140 CsCl, 2 MgCl2, 10 HEPES, pH 7.2 with CsOH. Voltage-clamp recordings were made at a Vhold of
RESULTS
cDNA constructs and functional assays
We fused the cDNA for a GFP mutant (GFP37, named for its stability at 37°C) (Grabner et al., 1998
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Figure 3. Testing and selecting the mGAT1-GFP fusion. A, The four constructs, mGAT1-GFP, mGAT1, and GFP in pcDNA3.1(+), and GFP-mGAT1 in pGFP37 were made to test the function of mGAT1-GFP fusions. The spacer sequences (8 residues for GFP-mGAT1, 12 residues for mGAT1-GFP) are shown in red. The C-terminal AYI sequence is shown in blue. B, The GFP-mGAT1 fusion is expressed mostly in the cytoplasm but not the nucleus of HEK cells and shows only slight increases in GABA uptake (data not shown). C, GFP expresses in cytoplasm and in the nucleus. D, The mGAT1-GFP fusion protein expresses on the membrane of HEK cells as well as in the cytoplasm, a typical situation for overexpressed proteins. E, F, Kinetics of GABA uptake by these fusion proteins expressed in HEK cells, tested at 2.5 µM GABA for time dependence (E) and in a 10 min assay for concentration dependence (F). mGAT1-GFP GABA uptake activity ( , green line) was indistinguishable from that of WT mGAT1 (
, black line). On the other hand, HEK cells expressing GFP (
) have GABA uptake activity indistinguishable from noninfected cells. The GFP-mGAT1 fusion showed only slight increases in GABA uptake compared with untransfected cells (data not shown). G, H, Fluorescence on the apical membrane of MDCK cells transfected with a recombinant mGAT1-GFP lentivirus. The face-on view of an image stack (G) and the side view of the same stack (H) are illustrated. I, mGAT1 expressed in a cultured E18 hippocampal cell, infected after 12 d in culture with the recombinant lentivirus and imaged 5 d later. All labeled processes derive from one cell. Scale bars: B-D, 25 µm; G, H, 20 µm; I, 50 µm.
The C-terminal fusion mGAT1-GFP, which includes a 12-residue spacer between mGAT1 and the GFP, showed clear plasma membrane localization (Fig. 3D) as well as cytoplasmic localization, which is typically found with overexpressed membrane proteins. GABA uptake activity was similar to that of WT mGAT1 (Fig. 3E,F). In Scatchard analysis, the EC50 values for mGAT1 and mGAT1-GFP were 10.9 and 9.3 µM, respectively, and the Vmax values were 3.6 and 3.9 nmol/mg protein per 10 min, respectively.
The N-terminal fusion protein, however, was localized primarily or exclusively in the cytoplasm rather than in the plasma membrane. The transfected cells displayed GABA uptake only slightly higher than untransfected cells and 10-fold less than WT mGAT1 or mGAT1-GFP (data not shown). The lack of nuclear expression suggests that GFP did indeed fuse with mGAT1, forming a protein that fails to pass through the nuclear pore (Fig. 3B).
Thus the C-terminal fusion, mGAT1-GFP, was the appropriate candidate for further study. We constructed a lentivirus expressing mGAT1-GFP and infected MDCK cells in culture. The mGAT1-GFP construct was directed to the apical membrane, as found previously for WT GAT1 expressed in MDCK cells (Fig. 3G,H) (Ahn et al., 1996
mGAT1-GFP knock-in mouse: general pattern of fluorescence
A knock-in mouse strain carrying the mGAT1-GFP fusion was created by homologous recombination to replace the final coding exon of mGAT1 (Fig. 1). There are only two differences between genome of the mGAT1-GFP strain and the WT genome: the coding region of mGAT1 has been extended at the C terminus by the GFP37 sequence, and the adjacent exon retains a single 37 bp loxP site (a vestige of the selection procedure for the embryonic stem cells). Thus the encoded mGAT1-GFP gene matches the cDNA construct that we chose in the experiments of Figure 3. Homozygotes and heterozygotes from the mGAT1-GFP line display normal weight, development, and life span, and, on the basis of anecdotal observations, normal behavior.
Figure 4A presents a fluorescence image giving an overview of the brain of an adult mGAT1-GFP knock-in mouse. The pattern of fluorescence strongly resembles previous immunohistochemical findings: GAT1 is expressed in axons, in synapses, and in astrocytes in many brain regions (Radian et al., 1990
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Figure 4. Overview of fluorescence in mGAT1-GFP mice. A, Montage forming a sagittal section ~1.2 mm from the midline; homozygote. B, Relative mGAT1-GFP fluorescence intensity in various regions. C, D, Cerebellar cortex; comparison of previously published immunohistochemistry (C) (Radian et al., 1990
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Figure 5. Details of mGAT1-GFP fluorescence in cerebellum, including individual boutons. A, In a P9 mGAT1-GFP mouse, mGAT1-GFP expresses in somata of several cell types; however, no obvious axonal expression was observed. B, In a P29 mGAT1-GFP mouse, mGAT1-GFP is clearly expressed in axons and boutons in the molecular layer (ML) and in pinceaux (arrows) surrounding the axonal hillock of Purkinje cells (P). The background stripe-like structures in ML are probably Bergmann glia. C, The P29 WT shows low background fluorescence and no obvious structures. This image was taken under >10-fold higher illumination photopower and has been further brightened digitally more than the other images. D, mGAT1-GFP expresses in pinceaux (arrows) in the hillock of a Purkinje cell (P). E, mGAT1-GFP fluorescence in axons and boutons in ML. Boutons with higher (arrows) and lower (arrowheads) levels of mGAT1-GFP are indicated. F, The diffuse stripe-like structures (arrows) represent the putative Bergmann glia expressing mGAT1-GFP. This structure is most evident in horizontal sections, probably revealing the glial palisades of Altman (Altman and Bayer, 1997
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Figure 6. Details of mGAT1-GFP fluorescence in CA1 region of hippocampus. A, In a heterozygous P9 mGAT1-GFP mouse, fluorescence is observed both in somata of inhibitory interneurons (arrows) and at synapses near and in the pyramidal cell layer. B, In a 3 month postnatal homozygous mouse, fluorescence is observed only in axons and synapses but not in somata. C, A P29 WT mouse shows low background fluorescence and no obvious structures. This image was taken at >10-fold higher illumination photopower and has been further brightened digitally more than the other images. D, High-magnification view of stratum oriens, showing fluorescent axons (arrows) and boutons. The dimmer background fluorescence could be caused by expression in astrocytes. Brain tissues were prepared from homozygote, 60 d postnatal mouse. E, Neuropil (arrows) in pyramidal cell layers. The lack of clear axon and bouton images could be attributable to mGAT1-GFP expression in both astrocytes and axons (Yan et al., 1997
More detailed images show that mGAT1-GFP is expressed primarily on axons and presynaptic boutons of GABAergic inhibitory neurons throughout the brain, again as revealed in previous studies. Figure 4, C and D, compares a previously published immunocytochemical image of cerebellar cortex (Radian et al., 1990
In other cerebellar cell types, mGAT1-GFP shows more diffuse fluorescence. For instance, in some sections of the molecular layer, we observed diffuse stripes of fluorescence (Fig. 5B,F); these structures are probably the Bergmann glia (Rattray and Priestley, 1993
In forebrain (including hippocampus), GAT1 is thought to be expressed in GABAergic interneurons, including chandelier, basket, and dendritic inhibitory cells (Freund and Buzsaki, 1996
Figure 6 shows details of fluorescence in hippocampus. Figure 6, A and B, compares GAT1 localization during development. The P9 knock-in mouse shows both synaptic and somatic expression, which agrees with other studies on young mice (Fig. 6A) (Yan et al., 1997
Counting mGAT1 molecules in cerebellum
The data presented thus far indicate that mGAT1-GFP fluorescence occurs as expected from previous studies, with regard to the overall pattern of expression, the cellular localization, and the time course of expression. We therefore proceeded to count mGAT1-GFP molecules, with localization at the resolution of confocal microscopy. These counts were based on two sets of standards. The first standard was a set of transparent beads with calibrated surface densities of GFP. The beads themselves were calibrated on the basis of both single-molecule fluorescence measurements and mass analyses of the GFP preparations used to generate the beads (Chiu et al., 2001
Our most detailed data are from cerebellar molecular layer (ML), where the relatively large size of basket cell and stellate cell boutons is well suited to our methods (Palay and Chan-Palay, 1974
In boutons with higher mGAT1-GFP expression, we observed higher mGAT1-GFP in subregions, ~1.5-2-fold higher (see Fig. 8A). In sections simultaneously immunostained for GABAA receptors and imaged for GFP fluorescence, we have noted that ~90% of the higher-expression boutons are associated with GABAA receptors, suggesting that these are bona fide areas of synaptic contact.
We now describe the 30-50% of boutons with lower fluorescence (Fig. 5E, arrowheads) (also typified by Fig. 8B). The bouton volume is 1.35 ± 0.15 µm3, and surface area is 5.12 ± 0.48 µm2. On the basis of calibrations from polyacrylamide gels, the average total mGAT1-GFP molecules in a bouton is 5070 ± 527, representing a concentration of 3850 ± 126 GFP per cubic micrometer. If all mGAT1-GFP molecules are on the membrane, the surface density is 980 ± 18 mGAT1-GFP per square micrometer (n = 11). Quantifications based on the calibrated GFP-beads yield surface densities ~16% lower, at 820 ± 40 mGAT1-GFP per square micrometer. Thus the lower-intensity class of boutons appear to express mGAT1-GFP at levels 64-75% of that of the higher-intensity class.
The axons connecting the boutons have an apparent diameter of ~0.3 µm (Fig. 5E,F), but this figure is near the limit of resolution and must be considered approximate. We analyzed the linear fluorescence density of axons that ran along the z-axis for a distance >500 nm, or twice the theoretical z-axis resolution, so that adjacent sections were averaged with little distortion from edge effects. (We would prefer axons that run for >1 µm, but the tortuous course may vitiate such a search.) Axons in the higher-intensity class showed fluorescence corresponding to 636 ± 19 mGAT1-GFP molecules per micrometer (n = 4). If one assumes an actual diameter of 0.3 µm, the membrane density of mGAT1-GFP molecules is 677 per square micrometer, close to half that of the boutons. Axons that connect lower-intensity boutons also showed fluorescence at least 30% lower than the high-intensity class.
Each cerebellar pinceau consists of dozens of intertwined basket cell boutons and axons. Pinceaux vary in size and may sometimes associate as linked pairs. We have treated pinceaux as solid objects. The average volume and surface area of a pinceau are 1020 ± 83 µm3 and 470 ± 12 µm2 (n = 8). The total of mGAT1-GFP molecules in a pinceau is ~7.8 × 106, and the average volume density is 7700 ± 443 GFP per cubic micrometer, or 55% higher than the volume density in the high-intensity class of bouton.
We estimate that the density of mGAT1-GFP in Bergmann glia is ~1200-2000 GFP per cubic micrometer. Our observations on GAT1 expression in these cells will be described in a separate publication.
Counting mGAT1 molecules in cortex and hippocampus
GAT1 is expressed on axons and boutons in cortex; boutons have a diameter × length of ~0.8 × ~1.8 µm. The average bouton volume is 1.3 ± 0.1 µm3, and the average surface area is 3.3 ± 0.2 µm2. The volume and surface density of mGAT1-GFP expression are 3120 ± 140 GFP per cubic micrometer and 1180 ± 46 GFP per square micrometer (mean ± SEM; n = 8), respectively. The volume and surface of the measured cortex cartridge are 188 µm3 and 233 µm2, respectively (n = 1). The total GFP is ~4.3 × 105 per cartridge, and the average concentration is ~2400 GFP per cubic micrometer.
In hippocampus, the neuropil in the pyramidal cell layer showed the highest levels of fluorescence, followed by stratum oriens (Fig. 6B). Presynaptic boutons of inhibitory neurons have only slightly greater diameter than do axons and similar fluorescence levels; therefore, in many cases the identification of a bouton is tentative. We estimate that a bouton in stratum oriens has dimensions of ~0.4-0.5 × 1.2 µm, slightly larger than previous measurements (i.e., 0.3 × 0.9 µm) (Somogyi et al., 1983
Surface/cytoplasmic partitioning of mGAT1-GFP in WT and mGAT1-GFP mice
The mGAT1-GFP construct was selected because it expresses, functions, and sorts like WT mGAT1 in heterologous expression systems (Fig. 3). The comparisons between mGAT1-GFP fluorescence and previous immunocytochemistry also show a good agreement with regard to localization in the animals (Fig. 4). Nonetheless, we found differences between the functional expression level of functional GAT1 in mGAT1-GFP knock-in animals versus WT animals. We assayed crude synaptosome preparations from knock-in and WT mice for [3H]GABA uptake (Fig. 7A). In Scatchard analyses, the EC50 values for wild type, heterozygotes, and homozygotes were 9.41, 11.95, and 11.47 µM, respectively, reaffirming that the mGAT1-GFP fusion construct functions like the WT. However, the Vmax values of the three genotypes were 6.2, 5.2, and 2.2 (nanomoles per milligram of protein per 5 min), respectively, consistent with the presence of approximately one-third as many functional mGAT1-GFP molecules on the plasma membrane as compared with WT (n = 3 for each genotype) (Fig. 7A). In a total of six sets of synaptosome preparations each of the three genotypes (three experiments with complete concentrations series and three with a single [3H]GABA concentration), the homozygote and heterozygote knock-in mice displayed 32 ± 5% and 76 ± 7% (mean ± SEM) of the WT activity, respectively.
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Figure 7. GABA uptake and biotinylation assays for mGAT1-GFP partitioning. A, Scatchard plot of [3H]GABA uptake for all three mGAT1-GFP genotypes (mean ± SEM; n = 3). Inset shows image of green fluorescent synaptosomes. B, Manipulation of membrane/cytoplasmic partitioning, assayed by [3H]GABA uptake. Data show NO-711-sensitive [3H]GABA uptake, measured over a time course of 1 hr at 4°C. Synaptosomes were preincubated for 10 min with control solution or subjected to "translocation treatment" with orthovanadate (50 µM), bisindolylmaleimide II (100 nM) to inactivate PKC, and 0.45 M sucrose, all at 4°C. C, Manipulation of membrane/cytoplasmic partitioning, assayed by surface biotinylation of cerebellar slices. Lanes 1-4, Tissue from mGAT1-GFP mice, probed with anti-GFP antibody. Lanes 5-8, Tissue from WT mice, probed with anti-GAT1 antibody. Lanes 1, 2, 5, and 6 were also probed with anti-actin antibody. D, Quantitation of immunostaining for mGAT1-GFP and mGAT1 in lanes 1-8. y-axis shows percentage of total staining (intracellular + extracellular) in the pair of lanes denoted by similar patterns. Lane pairs 1 + 3, 2 + 4, 5 + 7, and 6 + 8 add up to 100%. Data are mean ± SEM from three experiments like that of C. E, Fluorescence intensity in single boutons, untreated or subjected to translocation treatment.
Because of this unexpected threefold decrease in surface transporter expression in mGAT1-GFP mice, we performed additional manipulations to ensure that fluorescence measurements were made on mice with mGAT1-GFP molecules that were all translocated to the cell surface (Corey et al., 1994
We performed surface biotinylation on cerebellar slices to confirm and explain these manipulations as well as to estimate the proportion of surface versus cytoplasmic transporters in WT mice (Fig. 7C) (Beckman et al., 1999
We then performed additional fluorescence imaging experiments on boutons in cerebellar slices, the structures that we consider the most amenable to quantitative data. There was no significant difference (<3%) between the average mGAT1 fluorescence of individual untreated boutons (n = 35 boutons) and those that were subjected to the translocation treatment (n = 33 boutons) (Fig. 7E). These data show that the parameters of Table 1 may be interpreted as the maximal GAT1 density that can be translocated to the surface membrane.
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Table 1. Counts of mGAT1-GFP molecules in presynaptic structures of inhibitory interneurons Many images of synaptic boutons (Figs. 5E, 8) show slightly elevated fluorescence intensity in the intracellular compartment. We asked whether this apparently intracellular fluorescence corresponds to the nonbiotinylated mGAT1. We compared the spatial profile of fluorescence of the mGAT1-GFP on boutons from the cerebellar molecular layer (Fig. 8A,B) with His6-GFP on the surface of latex beads 1.5 µm in diameter (Fig. 8C). The beads also appeared to have a "tail" of internal fluorescence similar to that of the boutons, and this tail did not change after translocation treatment (Fig. 8D). We have not systematically explored the cause of this fluorescence spread, but both the boutons and the beads have elevated refractive indices relative to the external solution, and this refractive index difference may distort the optical signals slightly. Regardless of the source of this optical effect, we conclude that our optical measurements do not resolve a component of mGAT1-GFP fluorescence away from the surface membrane. The data suggest that the cytoplasmic fraction of cellular mGAT1 remains within a few hundred nanometers of the surface membrane and cannot be distinguished from the fluorescence of surface-membrane mGAT1.
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Figure 8. Single-bouton images and point-spread function (A, B). Projections are of confocal stacks in cerebellar ML, 1-1.5 µm thick. Scale bar, 1 µm. A, Boutons with higher total mGAT1-GFP expression show subregions of higher fluorescence. B, Boutons with lower fluorescence levels show more evenly distributed fluorescence. C, Single confocal image of an individual latex bead coated with His6-GFP. Scale bar, 1 µm. D, Profiles of GFP fluorescence in single confocal images of the beads (black), boutons from slices subjected to translocation treatment (red), and boutons from untreated slices (blue). Mean ± SEM; n = 16 each.
mGAT1-GFP mice display normal GABAergic transmission
We measured spontaneous IPSCs in whole-cell patch-clamp experiments on CA1 hippocampal pyramidal cells in slices from the brains of WT and mGAT1-GFP mice (Fig. 9A). There were only minor changes in average frequency, amplitude, and waveform of these events between WT and mGAT1-GFP mice. We also tested the effects of blocking GAT1 with NO-711 and found few differences between the effects on WT and mGAT1 mice.
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Figure 9. Electrophysiology of GABAergic transmission to CA1 pyramidal neurons in hippocampal slices from WT and mGAT1-GFP mice. A, Spontaneous GABAA-mediated IPSCs in Cl
In previous experiments on CA1 hippocampal pyramidal cells, pharmacological inhibition of GAT1 also produced tonic GABAA currents, corresponding to a steady GABA concentration estimated at ~1 µM (Frahm et al., 2001
Cultured neurons make inhibitory synapses if and only if they are fluorescent
Figure 10, A and B, shows micrographs of dissociated cultures from mGAT1-GFP knock-in embryo hippocampus, cultured at E16 and studied after 16 d in culture. Of the cultured neurons, 10-20% are fluorescent. mGAT1-GFP is expressed throughout these cells, similar to our observations of early postnatal mice (Fig. 10A,B). Although the fluorescence in mice became restricted to axons and synapses after the third postnatal week (Figs. 5A,B, 6A,B), the pattern of whole-cell fluorescence persisted after >29 d in culture, suggesting that the details of localization differ between cells in dissociated culture and in vivo. A previous report shows that GAT1 immunocytochemistry persists in the soma and dendrites of cultured rat hippocampal neurons, ruling out major effects of the GFP moiety in this case (Pietrini et al., 1994
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Figure 10. Electrophysiological measurements show that inhibitory interneurons fluoresce in hippocampal dissociated culture. A, B, Cultured hippocampal neurons were imaged with 10× (A) and 20× (B) objectives. The images show the fluorescent neurons under epifluorescence (left) and all neurons under transmitted light (right). The merged images are shown in the center panels. There are 16-18 fluorescent neurons in A and 5 (of which the 2 most obvious are marked with arrows) in B. Scale bars: A, 100 µm; B, 50 µm. C, D, Exemplar waveforms of voltage-clamp currents recorded from nonfluorescent postsynaptic cells studied at various holding potentials. C, Records during stimulation of a fluorescent presynaptic cell. D, Another cell; records during stimulation of a nonfluorescent presynaptic cell. E, The axon of a dissociated hippocampal interneuron forms synaptic boutons on nearby excitatory neurons. Scale bar, 20 µm. Left panel, Fluorescence only; arrows point to boutons that make contact on a nonfluorescent soma. Right panel, Fluorescence overlaid on Nomarski image of the culture. In the right-hand panel, the pointer identifies a glial cell expressing GAT1 (arrowhead).
The major goal of the culture experiments was to determine whether the set of fluorescent neurons is identical to the set of GABAergic interneurons. Neurons of homozygous or heterozygous mGAT1-GFP mice were cultured for 8-14 d and then screened, in paired whole-cell patch-clamp recordings, for the presence of synaptic connections (Fig. 10C,D). Thirty-nine fluorescent cells were stimulated in this series of experiments. Thirty-four of the fluorescent neurons induced postsynaptic currents in the second, nonfluorescent neuron of a cell pair (Fig. 10C). In all cases the reversal potential of this current, when corrected for the liquid junction potential, was approximately
In 30 tested pairs of nonfluorescent cells, there were 36 synapses. In all cases the reversal potential of the postsynaptic current induced by stimulation of nonfluorescent cells was more positive than
Thus the set of fluorescent neurons in hippocampal cultures equals the set of GABAergic interneurons. Fluorescent neurons formed boutons on nearby nonfluorescent neurons (Fig. 10E) as well as on themselves, and these boutons are probably some of the inhibitory synapses.
We make an additional, more technical point. The fluorescent interneurons were detected readily in a standard inverted microscope equipped with an
