Hippocampal Astrocytes in Migrating and Wintering Semipalmated Sandpiper Calidris pusilla

Abstract

Seasonal migratory birds return to the same breeding and wintering grounds year after year, and migratory long-distance shorebirds are good examples of this. These tasks require learning and long-term spatial memory abilities that are integrated into a navigational system for repeatedly locating breeding, wintering, and stopover sites. Previous investigations focused on the neurobiological basis of hippocampal plasticity and numerical estimates of hippocampal neurogenesis in birds but only a few studies investigated potential contributions of glial cells to hippocampal-dependent tasks related to migration. Here we hypothesized that the astrocytes of migrating and wintering birds may exhibit significant morphological and numerical differences connected to the long-distance flight. We used as a model the semipalmated sandpiper Calidris pusilla, that migrates from northern Canada and Alaska to South America. Before the transatlantic non-stop long-distance component of their flight, the birds make a stopover at the Bay of Fundy in Canada. To test our hypothesis, we estimated total numbers and compared the three-dimensional (3-D) morphological features of adult C. pusilla astrocytes captured in the Bay of Fundy (n = 249 cells) with those from birds captured in the coastal region of Bragança, Brazil, during the wintering period (n = 250 cells). Optical fractionator was used to estimate the number of astrocytes and for 3-D reconstructions we used hierarchical cluster analysis. Both morphological phenotypes showed reduced morphological complexity after the long-distance non-stop flight, but the reduction in complexity was much greater in Type I than in Type II astrocytes. Coherently, we also found a significant reduction in the total number of astrocytes after the transatlantic flight. Taken together these findings suggest that the long-distance non-stop flight altered significantly the astrocytes population and that morphologically distinct astrocytes may play different physiological roles during migration.

Stereological Analysis of Early Gene Expression Using Egr-1 Immunolabeling After Spreading Depression in the Rat Somatosensory Cortex

“…As previously described (Carvalho-Paulo et al, 2017), using deep anesthesia, all recorded animals were perfused by a cardiac via with a 0.9% saline solution followed by aldehyde fixative (4% formaldehyde in 0.1 M phosphate buffer, pH 7.2–7.4). The brains were carefully dissected, and the ipsilateral and contralateral sides were separated, post-fixed in 4% formaldehyde in phosphate buffer, and sliced using a Vibratome (Leica VT1000M) in the sagittal plane.…”

“…The protocols applied by the present study were previously used by Carvalho-Paulo et al (2017), with some adaptations related to antibody. Free-floating sections were treated in 0.2 M boric acid (pH 9.0) for antigenic retrieval by incubation in at 70°C for 60 min.…”

“…The reaction was interrupted after cells were detected under the microscope. Sections were rinsed 3 × 5 min each in 0.1 M PBS, mounted on gelatinized slides, dehydrated in an alcohol and xylene series, and mounted on a coverslip with DPX mounting medium (Sigma-Aldrich) (Carvalho-Paulo et al, 2017). Five animals from each experimental group and three animals from the sham groups were used for the stereological analysis of Egr-1 immunohistochemistry.…”

“…The stereological estimations began with delimitation of the region of interest in sagittal sections (Carvalho-Paulo et al, 2017; Gomes et al, 2019). We projected orthogonal lines toward the pia mater of the somatosensory cortex starting from the tips of the hippocampal pyramidal cell layer (Figure 2).…”

“…Within this rectangular area, we counted the immunolabeled cells inside the counting boxes. As detailed by Carvalho-Paulo et al (2017) (Carvalho-Paulo et al, 2017), the thickness of the section was carefully assessed at each counting site using a high-magnification objective, and the microscope’s fine focus was used to determine the immediate defocus above (top of section) and below (bottom of section). As noted previously, because this rectangular contour in the sections can vary from section to section in each animal, we estimated the cell density (cells/μm 3 ).…”