Changes in the cerebellar cytoarchitecture of hibernating hedgehog Erinaceus europaeus L. (Mammalia): an immunocytochemical approach

Abstract

Hibernation is an amazing animal strategy to survive when the environmental temperature is very low and food resources are scarce. Successful hibernation requires a variety of complex biological adaptations, in which the brain plays a central regulatory role. Currently, little information is available regarding the morphology and functional activity of specific neurons within the cerebellar cytoarchitecture of hibernating animals. In the present study, we investigated the immunohistochemical expression of essential proteins in the cerebellum of a mammalian hibernator (i.e. hedgehog Erinaceus europaeus L.), focusing on (i) Purkinje neurons, the sole output cells of the cerebellar cortex; (ii) selected neurotransmitters involved in hibernation processes; (iii) intracellular calcium homeostasis, considering that calcium is also an important regulator of neurotransmission mechanisms; and (iv) cytoskeletal proteins, involved in maintenance of neuronal shape and axon calibre. Specifically, we studied in situ immunocytochemical changes during the torpor state of hibernation (November–March) versus the activity phase (April–September). We employed different selected markers, i.e. glutamic acid decarboxylase (GAD67) and postsynaptic glutamate ionotropic receptor GluR2-3, different calcium-binding proteins (i.e. calbindin, parvalbumin and calretinin) and cytoskeletal components (i.e. pNF-H and MAP2). In summary, our data in hibernating animals demonstrated: (i) downregulation of GAD67, indicating loss/changes of synaptic contacts on Purkinje somata and dendrites; (ii) GluR2-3 upregulation in Purkinje neurons, with a drastic decrease of calbindin expression; and (iii) decrease of normal mechanisms regulating intracellular calcium homeostasis. We also found a decrease/modification in the distribution of cytoskeletal proteins, particularly evident for pNF-H. Changes in the functional activity of Purkinje cells were accompanied by some morphological dendrite alterations, signs of degeneration in cell somata and flattened basket pinceaux at the Purkinje axon hillock. These findings confirm that hibernation makes heterothermic animals a valuable model to study physiological adaptations to adverse conditions, and also for understanding cellular and molecular mechanisms aimed at preserving mammalian organs from full degeneration and death.

Keywords

• Hibernating mammals
• cerebellum
• neurotransmission
• calcium homeostasis and cytoskeleton
• immunocytochemistry