Suspension of mitotic activity in dentate gyrus of the hibernating ground squirrel

Abstract

Neurogenesis occurs in the adult mammalian hippocampus, a region of the brain important for learning and memory. Hibernation in Siberian ground squirrels provides a natural model to study mitosis as the rapid fall in body temperature in 24 h (from 35-36°C to +4-6°C) permits accumulation of mitotic cells at different stages of the cell cycle. Histological methods used to study adult neurogenesis are limited largely to fixed tissue, and the mitotic state elucidated depends on the specific phase of mitosis at the time of day. However, using an immunohistochemical study of doublecortin (DCX) and BrdU-labelled neurons, we demonstrate that the dentate gyrus of the ground squirrel hippocampus contains a population of immature cells which appear to possess mitotic activity. Our data suggest that doublecortin-labelled immature cells exist in a mitotic state and may represent a renewable pool for generation of new neurons within the dentate gyrus.

Figure 1

Comparative analysis of DG images of DCX-immunoperoxidase-labelled cells in S. undulatus in different functional states: (a) normothermic summer animal; (b) normothermic winter animal; (c) 2.5 h provoked arousal; (d) hibernation. Intensive DCX labelling of immature granule cell dendritic tree was revealed in normothermic summer animals (a) in contrast to winter animals (b–d). Solid arrows show DCX-labelled dendritic trees and their retraction in hibernating state. Open arrows show there is significant retraction of mossy fibres in hibernating S. undulatus (d) in comparison to summer animals (a): (e) DCX labelling of neurons in Layer II of entorhinal cortex in summer animal (open arrows). AD: apical dendrite of granule cell; BV: blood vessel; IGCs: immature granule cells; SGZ: subgranular zone of DG.

Figure 2

Light and electron microscopic patterns of dividing immature cells in the DG of hibernating animals. Mitotic phases are seldom found in normothermic animals (not shown here). (a and b) are light microscopic analysis of BrdU-immunoperoxidase-positive cells (arrowheads) in the DG of hibernating ground squirrel after injection of BrdU in interbout animal (on 50 μm thick coronal slices). Note the distribution of BrdU-labelled cells in the SGZ. (b) Higher magnification light micrograph demonstrating granule cells where low temperature has halted division showing clearly different mitotic phases: metaphase and telophase due to hypothermia causing entrance into the hibernation state. (c) Ultrastructural details from an electron micrograph (6000× magnification) of an early metaphase immature granule cell from rat hippocampus, (image adapted from a figure shown in Popov et al. [3]), with an interphase neuron located above the metaphase cell. Both immature interphase and metaphase granule neurons have synapses (interphase neuron-dendritic spine and metaphase one-shaft synapse) which are absent in mature granule cells of hippocampus of either ground squirrel or rat. BV: blood vessel; DG: dentate gyrus; SGZ: subgranular zone; mt: microtubules; and MT: mitochondria.

Figure 3

Light microscopic immunohistochemical demonstration of the BrdU-labelled doublets in DG as a result of apparent mitotic division of cells. Postmitotic BrdU immunoperoxidase-labelled cells in different zones of DG on coronal slices (50 μm thick). (a–c) Localization of postmitotic cells near subgranular zone (SGZ). (d–f) Possible migration of postmitotic cells from SGZ (e) to supragranular zone of DG (d). (f) Because of the thickness of brain slices different BrdU-labelled cells are frequently located at various depths in the 50 μm slice, in (f) (inset) the same cells can be seen at different focal planes. Scale bar = 50 μm.