ARTICLE |
Correspondence to: Ida J. LlewellynSmith, Dept. of Medicine, Flinders Medical Centre, Bedford Park, South Australia 5042, Australia..
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Summary |
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Pre-embedding immunocytochemistry for the active form of glutamate decarboxylase (GAD67) and postembedding staining for -aminobutyric acid (GABA) were compared as markers for central GABAergic terminals in the phrenic motor nucleus, in which phrenic motor neurons had been retrogradely labeled with cholera toxin Bhorseradish peroxidase. Nerve terminals with or without GAD67 immunoreactivity were identified in one ultrathin section. GABA was localized with immunogold in an adjacent section after etching and bleaching. GABA labeling density was assessed over 519 GAD67-positive and GAD67-negative nerve terminals in the phrenic motor nucleus. Frequency histograms showed that statistically higher densities of gold particles occurred over most GAD67-positive terminals. However, some GAD67-negative terminals also showed high densities of gold particles, and some GAD67-positive terminals showed low densities. Preabsorption of the anti-GABA antibody with a GABAprotein conjugate, but not with other amino acidprotein conjugates, significantly reduced gold labeling over both GAD67-positive and GAD67-negative terminals. These results show that the presence of GAD67 immunoreactivity correlates strongly with high densities of immunogold labeling for GABA in nerve terminals in the phrenic motor nucleus. Preabsorption controls indicate that authentic GABA was localized in the postembedding labeling procedure. Only a small proportion of intensely GABA-immunoreactive terminals lack GAD67, suggesting that both GAD67 and GABA are reliable markers of GABAergic nerve terminals. (J Histochem Cytochem 46:12611268, 1998)
Key Words: amino acid, immunocytochemistry, motor neuron, spinal cord, ultrastructure
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Introduction |
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GABAergic nerve terminals throughout the central nervous system have been identified using either pre- or postembedding immunogold methods to localize -aminobutyric acid (GABA), or pre-embedding immunocytochemistry to visualize glutamate decarboxylase (GAD), the enzyme required for synthesis of GABA from L-glutamate (
Using similarly processed tissue in this study, we determined whether GAD67-containing nerve terminals labeled by pre-embedding immunocytochemistry showed high densities of gold particles after postembedding immunogold staining for GABA in adjacent etched and bleached ultrathin sections.
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Materials and Methods |
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Retrograde Tracing
The right hemidiaphragms of three male WistarKyoto rats (250350 g) were injected with cholera toxin B-subunit conjugated to horseradish peroxidase (CT-HRP; List Biological Laboratories, Campbell, CA) (
Tissue Processing
After 23 days, the rats were anesthetized with sodium pentobarbital (80 mg/kg IP) and perfused with phosphate-buffered 2.5% glutaraldehyde. Cervical spinal cords were divided in two, postfixed for 6090 min, and cut parasagitally at 50 µm on a Vibratome. The sections were washed in 50% ethanol for 45 min (
Sections were incubated in the following reagents: 10% nonimmune horse serum (NHS) for 30 min, rabbit anti-GAD67 antiserum (Chemicon; Temecula, CA) 1:20,000 in 10% NHS in Tris-PBS for 4872 hr, biotinylated donkey anti-rabbit IgG (Jackson ImmunoResearch, West Grove PA; 1:500 in 1% NHS-TPBS) for 1624 hr, and finally ExtrAvidinhorseradish peroxidase (Sigma, St Louis, MO; 1:1500 in TPBS) for 416 hr. A nickel-intensified diaminobenzidine reaction revealed GAD-immunoreactive nerve fibers (
Stained sections were osmicated, exposed to uranyl acetate, dehydrated, and embedded flat in Durcupan (Sigma). At least two sets of paired serial sections were cut from one tissue block from each of the three animals. Serial ultrathin sections were collected on formvar-coated nickel single-slot grids. Grids were kept in pairs and in serial order. One grid of each pair was stained with Reynold's lead citrate to identify GAD67-immunoreactive nerve terminals. GABA was localized by postembedding immunocytochemistry in the other.
Postembedding Immunocytochemistry
Sections were etched in 2% aqueous periodic acid for 7 min and then bleached for 7 min in 2% aqueous sodium metaperiodate [adapted from
Absorption Controls
Controls for the specificity of GABA immunogold labeling included omitting the primary or secondary antibody and replacing the primary antibody with normal rabbit serum. Overnight preabsorption of the primary antibody with a variety of amino acidhemocyaninglutaraldehyde conjugates was also carried out to test crossreactivity (
Data Analysis
One pair of serial ultrathin sections per block was randomly selected for identification of GAD67-immunoreactive varicosities in the phrenic motor nucleus. In each section postembedding-stained for GABA, nerve terminals in the vicinity of retrogradely labeled phrenic motor neurons were photographed randomly, numbered, located in the adjacent section on the unlabeled grid, and scored for GAD67 immunoreactivity. Terminals that could not be identified in the untreated sections were not analyzed.
GABA immunoreactivity was assessed on micrographs printed to x37,00040,000. The areas of axon terminals and of mitochondria within them were measured with a digitizing tablet. Gold particles over axoplasmic matrix plus synaptic vesicles and over mitochondria were counted manually. GABA labeling density (gold particles/µm2) was then calculated for axoplasmic matrix plus synaptic vesicles (excluding mitochondria), for mitochondria, and for the whole terminal (axoplasmic matrix, synaptic vesicles, and mitochondria). Labeling over blank resin was determined from gold particle densities over the lumina of at least four blood vessels per sample and ranged from 0.5 to 1.6 gold particles/µm2. Because this labeling was present at such a low level, we did not correct for it in any of our measurements.
Terminals identified as GAD67-positive or GAD67-negative were sorted into groups on the basis of their gold particle labeling density for GABA. Frequency distributions for the densities of immunogold labeling over synaptic vesicles and axoplasmic matrix were constructed for all analyzed terminals from each section because this tissue compartment appeared most likely to reflect the distribution of neurotransmitter GABA.
Statistics
All statistical analyses were carried out according to
Nerve terminals from GABA-labeled sections were sorted into GAD67-positive and GAD67-negative categories. In the four sets of data collected, tests for homogeneity of variance between the GAD67-positive and GAD67-negative groups showed that the variances of these two groups were heterogeneous in almost all cases. Where this occurred, these data were log-transformed before unpaired t-tests were used to compare the density of labeling of the two groups of nerve terminals. The mean densities are given after back-transformation from their log values.
In the preabsorption control experiments, comparisons of the density of gold labeling were made over groups of GAD67-positive and GAD67-negative nerve terminals. The same terminals were identified in adjacent sections. One section of each pair was stained for GABA and the other with anti-GABA antibody that had been preabsorbed overnight with one of the four amino acidhemocyanin conjugates. In these cases, the density of labeling over GAD67-positive or GAD67-negative nerve terminals over the area of synaptic vesicles and axoplasmic matrix was compared with a paired t-test. In several cases, the variances of the densities of gold labeling were heterogeneous, so data were log-transformed before the statistical test was performed.
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Results |
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Four pairs of adjacent ultrathin sections through the phrenic motor nucleus were examined from the three rats that had received injections of CT-HRP into the diaphragm. One section of each pair was untreated so that nerve terminals could be classified as GAD67-positive or GAD67-negative. The other section was stained for GABA after being etched to remove resin and bleached to remove DAB reaction product. An initial sample of 697 GAD67-immunoreactive terminals in the phrenic motor nucleus was photographed; 25% of these (178) made synapses and direct contacts with retrogradely labeled phrenic motor neurons. Five hundred and nineteen of the 697 terminals could be identified in both GABA- and GAD67-labeled sections and were analyzed further for their density of gold labeling and their GAD67 content.
When GAD67-positive and GAD67-negative terminals were assessed for GABA gold labeling in the adjacent section, GAD67-positive nerve terminals generally appeared to have higher levels of GABA labeling than GAD67-negative nerve terminals (Figure 1, Figure 2A, and Figure 2B). Frequency distributions for gold labeling density over synaptic vesicles and axoplasmic matrix were constructed first for all nerve terminals examined in each section (Figure 3A) and then for nerve terminals that had been sorted into GAD67-positive and GAD67-negative classes (Figure 3B). These histograms confirmed that GAD67-positive nerve terminals generally showed higher densities of gold labeling for GABA than GAD67-negative terminals. The presence of two distinct peaks in the overall frequency distribution (Figure 3A) suggested a population of nerve terminals with a low GABA labeling density and a population with a high GABA labeling. However, when frequency distributions were constructed after sorting terminals into GAD67-positive and GAD67-negative classes, the two populations were not as well separated as the overall frequency distribution suggested, i.e., there was some degree of overlap between the labeling densities of GAD67-negative and GAD67-positive nerve terminals (Figure 3B). There were nerve terminals that lacked GAD67-immunoreactivity in the plane of section but showed a high density of GABA labeling. There were also GAD67-positive nerve terminals that displayed low levels of GABA labeling. Nevertheless, in all four pairs of sections from the three animals, unpaired t-tests showed that the density of gold labeling was significantly higher over GAD67-positive nerve terminals than over GAD67-negative nerve terminals (Table 1). This was true regardless of whether the density of gold labeling for GABA was measured over synaptic vesicles and axoplasmic matrix, over mitochondria alone, or over the entire nerve terminal.
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Control experiments confirmed that the heavy labeling over GAD67-immunoreactive terminals was likely to be authentic GABA immunoreactivity. Overnight preabsorption of the primary antibody with increasing concentrations of GABAhemocyanin resulted in a graded decrease in labeling density over both GAD67-positive and GAD67-negative nerve terminals (Figure 2C and Figure 2D), with the highest concentration (1:100) virtually abolishing gold labeling. This reduction in gold particle density was statistically significant (paired t-test p<0.001; Table 2). In contrast, incubation of the anti-GABA antiserum with glutamate, glycine, or glutaminehemocyanin conjugates did not significantly alter gold labeling density (Table 2), although the density of labeling over both GAD-positive and GAD-negative nerve terminals showed a decrease with all conjugates tested. When the GABAglutaraldehydehemocyanin conjugate diluted 1:2500 was substituted for primary antibody or when primary antibody was omitted, no gold labeling was observed. Substitution of normal rabbit serum for the primary antibody resulted in very sparse labeling, with densities similar to that seen in sections incubated with GABAhemocyanin-preabsorbed primary antibody.
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Discussion |
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Our correlation of the densities of GABA labeling with the occurrence of GAD67 immunoreactivity in adjacent sections of nerve terminals in the phrenic motor nucleus and of terminals synapsing on retrogradely labeled phrenic motor neurons has demonstrated that GAD67-positive nerve terminals have a statistically higher density of GABA gold labeling than GAD67-negative terminals. Although some GAD67-positive terminals showed low levels of GABA labeling and some GAD67-negative terminals showed high levels, the overall correspondence between the presence of immunoreactivity for GABA and GAD67 suggests that both are useful markers of GABAergic terminals.
In this study, GAD67-positive nerve terminals showed statistically higher GABA labeling over synaptic vesicles and axoplasmic matrix, over mitochondria, and over the whole nerve terminal, than was seen over GAD67-negative nerve terminals. This difference between GAD67-positive and GAD67-negative nerve terminals was apparent in frequency distribution histograms based on gold particle density. Nevertheless, all of the frequency distributions for GABA labeling densities over the GAD67-negative and GAD67-positive groups of terminals showed some degree of overlap. Some GAD67-positive terminals showed low levels of GABA labeling and some GAD67-negative terminals showed levels of GABA labeling that were as high as those seen in GAD67-positive terminals. GAD67 terminals were marked by peroxidase reaction product, the presence of which unequivocally identifies them as possessing the active form of the enzyme necessary for GABA synthesis. However, the absence of GAD67 immunoreactivity from a nerve terminal, particularly from a single ultrathin section through a nerve terminal, does not prove that the terminal cannot synthesize GABA. Because the primary antibody used here recognizes GAD67 and not GAD65 (
Only one previous study has directly examined GABA immunogold labeling over GAD-positive and GAD-negative nerve terminals (
The data from our control experiments indicate that we have localized authentic GABA immunoreactivity in both GAD67-positive and GAD67-negative nerve terminals. First, there was no gold labeling over any tissue structures in the absence of primary antibody, and gold labeling was minimal in experiments in which the primary antibody was replaced with normal rabbit serum. We also showed concentration-dependent reductions in the density of gold labeling when the primary antibody to GABA was preabsorbed with increasing concentrations of a GABAprotein conjugate. This anti-GABA antibody has been used in our previous studies on amino acid content of synaptic inputs to rat sympathetic preganglionic neurons, and to bulbospinal neurons of the rostral ventrolateral medulla, in which we showed that terminals that contained high densities of immunogold labeling for GABA contained low levels of GLU labeling, and vice versa (
In this study, the presence of peroxidase reaction product in GAD67-labeled nerve terminals did not appear to cause any significant quenching of immunogold labeling for GABA, as has been reported in other studies (e.g.,
In summary, our postembedding immunogold studies have shown that, in the phrenic motor nucleus, GAD67-immunoreactive nerve terminals, including many terminals that synapse with phrenic motor neurons, are more heavily stained for GABA than terminals that lack GAD67 immunoreactivity. Our results indicate that both immunoreactivity for GABA and immunoreactivity for GAD67 are good markers for central GABAergic terminals.
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Footnotes |
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1 Present address: Dept. of Anatomy and Cell Biology, The University of Melbourne, Parkville, VIC 3052, Australia.
2 Present address: Dept. of Neurosurgery, Royal North Shore Hospital, St Leonards NSW 2065, Australia.
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Acknowledgments |
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Supported by grants from the National Health and Medical Research Council of Australia, the National Heart Foundation of Australia, and the National Sudden Infant Death Syndrome Research Council of Australia. SMM was supported by a Post-doctoral Research Fellowship from the National SIDS Research Council.
We would like to thank Carolyn Martin for expert technical assistance.
Received for publication April 3, 1998; accepted July 13, 1998.
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