ARTICLE |
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Summary |
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Analysis of the connectivity between different neuronal cell types is dependent on an appreciation of their dendritic and axonal arborizations. A detailed study of the dendrites and axons of GABAergic neurons has been thwarted by the lack of a suitable technique for enhancing GABA immunoreactivity. This article describes a procedure using tetanus toxin which, when applied to organotypic hippocampal cultures, considerably enhances the immunoreactivity in the dendrites and axons of the GABA- and somatostatin-containing neurons and clearly demonstrates the co-localization of GABA and somatostatin immunoreactivities in the same neuron. Tetanus toxin was applied to the culture medium on Day 14 for a 24-hr period and the cultures were fixed at the end of Day 18. Tetanus toxin-treated cultures (n = 30) or untreated cultures (n = 40) were incubated for either GABA or somatostatin immunoreactivity. Tetanus toxin-treated cultures used for co-localization studies (n = 20) were incubated for both GABA and somatostatin immunoreactivity. (J Histochem Cytochem 46:321326, 1998)
Key Words: GABA, somatostatin, tetanus toxin, hippocampus, organotypic cultures, rat
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Introduction |
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To analyze the connectivity of a particular neuronal cell, it is useful to appreciate the extent of its dendritic and axonal arborizations. Ramon y Cajal (1893) used a technique developed by the Italian histologist Camillo Golgi which, when used in conjunction with intracellular injections of dyes, allows the visualization of extensive dendritic and axonal projections in single neurons.
In the hippocampus, knowledge about the connectivity of the nonpyramidal, presumed GABAergic inhibitory neurons awaited the development of immunocytochemical methods that utilized antisera directed against glutamic acid decarboxylase (GAD) (-aminobutyric acid (GABA) became available and confirmed the observations made by previous workers using GAD (
A method is needed that enhances or amplifies the antigenic signal and that might allow a more thorough examination of the connectivity between GABAergic neurons and other neurons.
Previous work from this laboratory showed that, when tetanus toxin was administered in vivo, a considerably enhanced immunoreactivity was observed in neurons containing somatostatin (
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Materials and Methods |
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Hippocampi were dissected from 8-day-old neonatal rats, placed in Geys medium supplemented with 28 mM glucose, and sliced on a McIlwain tissue chopper set to cut at 400 µm (
The immunocytochemical procedure used to reveal somatostatin and GABA immunoreactivity was a modification of that used previously (
Controls
Specific immunoreactivity for GABA was blocked either by treating cultures (n = 10) with GABA antibody that had been preadsorbed with GABA antigen coupled to polacrylamide beads (
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Results |
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GABA
Control cultures incubated in the presence of the preadsorbed GABA antibody or in the absence of the GABA antibody revealed no staining of GABAergic neurons. In cultures not exposed to tetanus toxin (Figure 1A), only the weakly stained somata of the GABAergic neurons were present.
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Intensely immunoreactive GABAergic neurons were located throughout the septotemporal extent of the hippocampus, in all layers of the hippocampus proper, and in the dentate gyrus. Many neurons in strata oriens, pyramidale, and in the subiculum revealed extensive dendritic and axonal arborizations (Figure 1B and Figure 1C) and an intense terminal staining around the principal cells in stratum pyramidale. A similar GABAergic immunoreactivity was observed in the basket cells of the dentate gyrus, whose dendrites extended deep into the molecular layers and whose axons revealed an intense terminal plexus around the granule cells.
Somatostatin
Control cultures incubated for somatostatin immunoreactivity in the absence of tetanus toxin revealed the characteristic signet-ring staining of their cytoplasm (Figure 2B), and those cultures incubated with preadsorbed antibody revealed no staining.
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After tetanus toxin exposure, the somatostatin cells became intensely labeled and revealed not only increased staining of dendrites but also of axons and terminals. Particularly striking were the somatostatin neurons in stratum oriens (Figure 2C), with axons that extend vertically down through the pyramidal cells into stratum radiatum to terminate in stratum lacunosum moleculare. Somatostatin neurons with long dendrites and axons were also observed in the hilus of the dentate gyrus (Figure 1D). Most of these axons were confined to the hilar area and terminated on somatostatin-negative cells.
Many neurons in stratum oriens were intensely immunoreactive for GABA and somatostatin and were stained a deep blue-black color. In stratum radiatum throughout the hippocampus were isolated neurons, whose somata measured 3035 µm in diameter, which co-localized somatostatin and GABA. The brown DAB reaction product indicative of somatostatin was quite distinct from the blue SG reaction product demonstrating GABA (Figure 1C). The beaded axons of these neurons could be followed into the dentate gyrus. In the dentate gyrus (Figure 1D), as in stratum oriens, many neurons were immunoreactive for either somatostatin or GABA alone. Many hilar cells were somatostatin-immunoreactive but GABA-negative. However, there were neurons in the hilus that co-localized somatostatin and GABA immunoreactivities (Figure 1D).
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Discussion |
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The present study has clearly demonstrated that a single dose of tetanus toxin enhances the immunoreactivity of the dendrites and axons of GABAergic and peptidergic hippocampal neurons in culture.
Although the preparation of the organotypic cultures causes the loss of all extrinsic afferents and results in some reorganization (
It is partly because these cultures maintain a structure and function similar to that seen in vivo and partly because of the large number of slices that can be obtained from each hippocampus that they are increasingly being used as in vitro models to investigate the pathophysiology of amino acid-based neurotransmission in conditions such as hypoxia and epilepsy (
The somatostatin immunoreactive neurons in the hippocampus and dentate gyrus of the present study are similar to those described previously (
The GABAergic neurons that also contained somatostatin and were found in stratum radiatum close to the border with lacunosum moleculare and whose axons could be followed into the dentate gyrus resemble the NPY neurons described previously in organotypic cultures and in vivo (
Indirect evidence from intracellular labeling studies and from immunostaining against receptors associated with somatostatin neurons would confirm that the dendrites of hilar somatostatin neurons remain within the hilus and run parallel with the dentate granule cells (see review by
In summary, tetanus toxin enhances the immunoreactivity of GABAergic dendrites and axons, which should facilitate an analysis of their connectivity and provide a better understanding of their function. Tetanus toxin may also facilitate the localization of other neurotransmitters in alternative culture systems.
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Acknowledgments |
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Supported by Action Research Grant S/P 2796.
I thank Prof Udo Schumacher and Dr Nicholas Best for their valued comments.
Received for publication May 30, 1997; accepted September 24, 1997.
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Literature Cited |
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