Investigating Neotenic and Metamorphic Axolotl Brain Complexity: A Stereological and Immunohistochemical Perspective

Abstract

The ability of certain tetrapods, such as amphibians, to regenerate complex structures, such as organs or limbs, is well-established, though this capacity varies significantly across species, with humans exhibiting limited regenerative potential. Ependymoglia cells in the ventricular region of the brain are known to exhibit proliferative properties during homeostasis and damage and to perform stem cell functions. This study investigated changes occurring in neurons and glia in the central nervous system following metamorphosis in axolotls. Morphological alterations in brain tissue, newly formed neurons, and cellular organizations in different brain regions were assessed using stereological and immunohistochemical methods, as well as light and electron microscopy. Interestingly, we observe no statistically significant difference in total neuron numbers in the telencephalon region between neotenic and metamorphic axolotls. However, the proliferation index and the numbers of cells expressing NeuN were significantly higher in metamorphic axolotls. Furthermore, structural changes in neuronal nuclei and myelin sheath organization were determined at the light and electron microscopic levels post-metamorphosis. Ultrastructural analyses revealed a change in chromatin organization from euchromatic to heterochromatic in neurons after metamorphosis, and morphological changes were also demonstrated in myelinated nerve fibers in the telencephalon. Additionally, mucopolysaccharide-containing secretory sacs were also identified on the apical surfaces of a subgroup of ependymoglia cells located in the lateral ventricle wall. Overall, this study sheds useful light on the intricate changes occurring in the central nervous system during metamorphosis in axolotls and provides valuable insights into the mechanisms underlying these processes.

Keywords: RRID:AGSC_100A; axolotl; ependymoglia; metamorphosis; stereology; telencephalon.

FIGURE 1

(a,b,d,e) Cresyl violet staining brains of neo and met axolotls. (c) Physical fractionator cell count results. (f) Total cell volume results of Ki‐67 positive cells. (g–m) Ki‐67 immunohistochemistry staining of the telencephalon regions of neo and met axolotl brains. There are Ki‐67‐expressing neurons (arrow) at different distances from the ventricle. (l, m) Magnifications of (k). Arrow: Ki‐67 positive cell, *: artifact, Cp: choroid plexus, Dp: dorsal pallium, Lp: lateral pallium, Mp: medial pallium, Olf B: olfactory bulb, S: septum, S: striatumVt: ventricle.

FIGURE 2

(a–h) NeuN immunohistochemistry staining of the telencephalon region of the Neo and Met axolotl brain. NeuN‐expressing cells (arrow) are located at different distances from the ventricular wall. (ı) Total cell volume results of NeuN positive cells. Arrow: NeuN positive cell, arrowhead: NeuN negative cell. Dp: Dorsal pallium, Lp: lateral pallium, Mp: medial pallium, S: Septum, S: Striatum, Vt: ventricle.

FIGURE 3

Schematic view of anterior, midpiece, and posterior portions of the telencephalon. (a–p) GFAP immunohistochemistry staining of the telencephalon regions of Neo and Met axolotl brains. GFAP‐expressing cells (arrow) and their extensions (arrowhead) located in the ventricular wall are shown. (m) Magnifications of (l). (p) GFAP‐expressing cell extensions can be seen surrounding the blood vessel. Dp: Dorsal pallium, Lp: lateral pallium, Mp: medial pallium, S: septum, S: striatum, Vt: ventricle.

FIGURE 4

(a–f) MBP immunohistochemistry staining of the telencephalon region of the Neo and Met axolotl brain. MBP‐expressing nerve fibers could not be detected in the telencephalon. However, blood cells (dashed circles) were found to express MBP. (g, h, k, l) MBP immunohistochemistry staining of diencephalon regions of Neo and Met axolotl brains. MBP‐expressing nerve fibers are seen in the dashed circle. (j, m) When serial twin sections taken from the diencephalon region are examined, cells containing red granules (*) organized in groups are seen. Dp: dorsal pallium, Lp: lateral pallium, Ob: olfactory bulb, V: blood vessel.

FIGURE 5

(a) Neo neurons (N) are seen. (b) A microglia‐like cell (Mg) surrounded by neuropil (c) oligodendroglia (Og) producing a myelin sheath is seen. (d–f) Morphologically changed neurons (N) are seen in the metamorphic axolotl. (g–i) Transmission electron microscopic images taken from the diencephalon region of the neotenic axolotl brain. (h) Black granular structures in the regions of group‐organized neurons facing the pial surface. (i) The structures marked in red in the cell cytoplasm maybe neuromelanin. (j–o) Transmission electron microscopic images taken from the telencephalon region of the neotenic axolotl brain. A nerve fiber (Ax) with a compact (j, k, m, n) and non‐compact (l, o) myelin sheath is examined (white arrowhead: soma, black arrowhead: impaired myelin structure, arrow: myelinated nerve; the dashed rectangle in g is magnified in h and i).

FIGURE 6

Light (a–h), transmission (j–m), and scanning (n–p) electron microscopic images of the secretion product (arrowhead, Sp) located in the apical part of the ependymoglia cells arranged around the ventricle in the telencephalon region of the Neo and Met axolotl brain. The crystalloid structure of the secretory granule was seen. (a–f) Sacs were stained with alcian blue (arrowhead). (g, h) Semi‐thin section of ependymoglia cells and sacs within different cells shown with arrowhead. (j, k, l) Secretory products (arrowhead, Sp) at the apical surface of ependymoglia cells were seen. (m) At higher magnification of j, red arrows showed the basal body of the cilium of the cell (o). Higher magnification of n, secretion product on the ventricle wall was seen, and the cilium of the cell was shown with a white arrow. (p) Secretion products protruding towards the ventricle are seen on the apical surfaces of some ependymoglia cells; cilia (white arrow) and microvilli (green arrow) are located at the apical surface. Nc: nucleus, Sp: secretion product, Vt: ventricle.

FIGURE 7

Scanning electron microscope images of the telencephalon of the neotenic axolotl brain. (a) Ependymoglia cells with long cytoplasmic extensions are seen (green). (b) Ependymoglia cells with primary cilium are seen. (d) Ventral telencephalon. (e, g) higher magnification white dashed rectangular in d. (c, f, h) There are many apical surface specializations (arrowheads) on the apical parts of the cells on the surface of the wall facing the ventricle; some cells do not have any cilia (*) (g, f) A structure that maybe a secretory sac is seen between the cilia (red dashed circle).