BRIEF REPORT |
Correspondence to: Margaret M. Moga, Indiana U. School of Medicine, 135 Holmstedt Hall, ISU, Terre Haute, IN 47809. E-mail: mmoga@medicine.indstate.edu
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Summary |
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We examined the distribution of annexin 6 (ANX6) in rat brain with immunohistochemistry (IHC). Several neuronal cell populations were intensely labeled with the ANX6 monoclonal antibody (MAb), including layer 5 of neocortex, the lateral septum, the lateral hypothalamic area, the red nucleus, and the Purkinje cell layer in cerebellum. Neuronal immunolabeling was localized to the nucleus and the cytosol. Darkly stained ANX6-immunoreactive glia, with the morphology characteristic of astrocytes, were abundant in the hippocampus, substantia nigra reticulata, and cerebellum. Evidence suggests that ANX6 may function in neuronal and glial calcium-dependent processes.
(J Histochem Cytochem 50:12771280, 2002)
Key Words: calcium-binding protein, cerebral cortex, lateral hypothalamus, radial glia, basal forebrain
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Introduction |
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Annexin calcium-binding proteins are part of a large multigene family with at least 12 different annexins expressed in mammalian tissues (
One of the most abundant annexins in the mammalian brain, as determined by biochemical analyses, is annexin 6 (ANX6;
All animal procedures in this study were approved by the Institutional Animal Care and Use Committee (IACUC) at the Indiana University School of Medicine. Twelve adult male SpragueDawley albino rats (Harlan; Indianapolis, IN), weighing 300400 g, were maintained under a 12:12-hr lightdark cycle with food and water freely available. Each animal was deeply anesthetized with sodium pentobarbital (100150 mg/kg, IP) and perfused through the ascending aorta with 150 ml of heparinized saline, followed by 500 ml of 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4. After fixation the brains were removed, allowed to sink overnight in a 30% sucrose solution, and cut into 30-µm sections on a freezing microtome.
A one-in-three series of sections was preincubated for 1 hr in 2% normal donkey serum (NDS), 2% bovine serum albumin (BSA), and 0.3% Triton-X (TX) in 0.01 M PBS, pH 7.4. The sections were incubated with mouse anti-ANX6 (ICN; Aurora, OH), diluted 1:500 in PBSNDSBSATX, with gentle agitation for 24 days at 4C. Next, the sections were rinsed in PBS, incubated in biotinylated SP-conjugated donkey anti-mouse IgG (Jackson; West Grove, PA), diluted 1:200 in PBSNDSTX for 2 hr, rinsed again, and reacted with ABC reagent (Vector; Burlingame, CA) for 2 hr. After several rinses in PBS, the sections were reacted in a solution of 0.035% 3-3' diaminobenzidine tetrahydrochloride (DAB), 2.5% nickel ammonium sulfate, and 0.3% H2O2 in 0.1 M sodium acetate buffer, pH 6.0, for 1 min. The sections were rinsed in PBS and mounted on gelatin-coated slides. The slides were cleared in xylene and coverslipped with Permount (Fisher; Pittsburgh, PA).
The anti-ANX6 monoclonal antibody (MAb) (clone 73; available from both ICN and BD Transduction Laboratories, San Diego, CA) was generated against ANX6 isolated from human placenta and specifically recognizes ANX6 in a variety of tissues (
A subset of neurons in the rat brain showed dense immunolabeling with the ANX6 MAb. In general, neuronal immunolabeling was localized to both the nucleus and the cytosol, with some labeling present in the proximal dendrites and axon. ANX6-IR neurons were numerous in cortical areas, particularly in layer 5 of the neocortex (Fig 1A), entorhinal cortex, pre- and post-subiculum, and layers 26 of lateral occipital cortex, with the exceptions of the insular and piriform cortices, which were largely unstained. Dark-staining neurons were located in several basal forebrain areas, including the diagonal band, the lateral septum, and the magnocellular preoptic nucleus. In the amygdala and striatum, ANX6-IR neurons were most prominently labeled in the olfactory tubercle, the nucleus of the lateral olfactory tract, and the globus pallidus. In the hypothalamus, many ANX6-IR neurons were found in the anteroventral periventricular nucleus, the lateral anterior hypothalamic nucleus, and the ventrolateral portion of the lateral hypothalamic area (Fig 1C). The reticular thalamic nucleus contained the darkest-staining ANX6-IR neurons in the thalamus. In comparison, the caudate putamen and the dorsomedial hypothalamic nucleus showed a relative lack of immunostaining, whereas many other forebrain areas, such as the ventromedial hypothalamic nucleus and the paraventricular thalamic nucleus, contained lightly labeled neurons. In the rostral brainstem, ANX6-IR was particularly robust in neurons located in the cerebellum (Purkinje cells; Fig 1D); lateral vestibular nucleus, red nucleus, oculomotor nucleus, superior colliculus (intermediate and superficial layers), central gray (dorsal and lateral subdivisions), trapezoid nucleus, and lateral substantia nigra.
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ANX6-IR in glial cells was evenly distributed throughout the cell soma and processes. Two types of ANX6-IR glial cells were observed: radial glia and stellate glia. ANX6-IR radial glia were abundant along the brain surface, with many perpendicular processes extending into the optic chiasm, lateral olfactory tract, cerebral peduncle, lateral lemniscus, middle and inferior cerebellar peduncles, spinal tract of the trigeminal nerve, and spinocerebellar and pyramidal tracts. ANX6-IR stellate glia were limited in their distribution to the hippocampus (Fig 1B), cerebellum (Fig 1D), substantia nigra reticulata, internal capsule, and portions of the thalamus. Immunopositive stellate glia displayed a morphology characteristic of astrocytes, with relatively large, rounded cell bodies and long, slender tapering cell processes (Fig 1B).
Our results show that ANX6-IR is expressed in many neuronal cell populations and in a small subset of glial cells in the rat brain.
ANX6-IR was localized in both the cytosol and the nucleus of neurons, but with a nuclear predominance. Previous studies have found that the subcellular distribution of annexin proteins varies by the annexin and, for each annexin, by cell type and with physiological changes in intracellular Ca2+ (
Several cytoskeletal proteins present in the nervous system have been found to interact with ANX6 in a Ca2+- or phospholipid-dependent manner (
Received for publication April 8, 2002; accepted April 17, 2002.
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