Ubiquitous and temperature-dependent neural plasticity in hibernators

Abstract

Hibernating mammals are remarkable for surviving near-freezing brain temperatures and near cessation of neural activity for a week or more at a time. This extreme physiological state is associated with dendritic and synaptic changes in hippocampal neurons. Here, we investigate whether these changes are a ubiquitous phenomenon throughout the brain that is driven by temperature. We iontophoretically injected Lucifer yellow into several types of neurons in fixed slices from hibernating ground squirrels. We analyzed neuronal microstructure from animals at several stages of torpor at two different ambient temperatures, and during the summer. We show that neuronal cell bodies, dendrites, and spines from several cell types in hibernating ground squirrels retract on entry into torpor, change little over the course of several days, and then regrow during the 2 h return to euthermia. Similar structural changes take place in neurons from the hippocampus, cortex, and thalamus, suggesting a global phenomenon. Investigation of neural microstructure from groups of animals hibernating at different ambient temperatures revealed that there is a linear relationship between neural retraction and minimum body temperature. Despite significant temperature-dependent differences in extent of retraction during torpor, recovery reaches the same final values of cell body area, dendritic arbor complexity, and spine density. This study demonstrates large-scale and seemingly ubiquitous neural plasticity in the ground squirrel brain during torpor. It also defines a temperature-driven model of dramatic neural plasticity, which provides a unique opportunity to explore mechanisms of large-scale regrowth in adult mammals, and the effects of remodeling on learning and memory.

Figure 1.

Sample images and Neurolucida reconstructions. A–C, Examples of layer 4 cortical spiny stellate neurons (A), ventral posterior thalamic somatosensory relay neurons (B), and hippocampal CA3 neurons (C) from euthermic (12 h after arousal induction) and torpid (6 d after torpor entrance) ground squirrels. Higher-magnification images from cortical dendritic arbors show dendritic spines. Images are from 100-μm-thick sections, whereas reconstructions also include dendrite branches from adjoining sections. The scale is the same for images and tracings within a cell type. Scale bars, 100 μm.

Figure 2.

Torpor-related changes in neural microstructure. A–C, Structural parameters for cortical spiny stellate cells (A), ventral posterior thalamic relay cells (B), and hippocampal CA3 neurons (C) at specific time points in a torpor bout, as indicated in the body temperature graph below. Parameters shown are mean ± SEM: cell body area (in square micrometers), sum of intersections of the entire dendritic arbor with concentrically drawn circles 10 μm apart starting at the cell body (a modified Scholl analysis), and spine density (number of spines per micrometer of dendrite) on tertiary and quaternary branch segments. Group means comprise data from 25–35 cells from six to seven animals per group. Means from a group of ground squirrels in the summer provide a comparison with winter euthermic values, and are represented by a dashed line through each graph. Statistically significant comparisons (at α = 0.05) are marked with asterisks above bars as follows: *A > B; **A > D; ***E > D. Not all significant comparisons are shown here.

Figure 3.

Time course of neural microstructural change in torpor. A–C, Percentage change of cell body area (in square micrometers) (A), sum of intersections (B), and spine density (number of spines per micrometer of dendrite) (C) from interbout euthermic values. Means + SEM from the cortex, thalamus, and hippocampus are marked with different symbols (see key at bottom). By the end of torpor, all cell types exhibit the same percentage decrease of all three structural parameters. Greatest percentage changes are seen in cell body area. Spine density changes occur most rapidly, with all of the decrease taking place by day 1, and all of the recovery taking place in 2 h.

Figure 4.

Torpor-related changes occur in higher-order branches. A–C, Number and length of dendritic branches by branch order for cortical spiny stellate neurons (A), ventral posterior thalamic relay cells (B), and hippocampal CA3 neurons (C). Apical and basal dendrites are shown separately. Means + SEM are from euthermic (12 h after arousal induction) and torpid (6 d after torpor entrance) animals. Significant comparisons at α = 0.05 are marked with asterisks above bars. For all three cell types, torpor-related changes occur in the higher-order branches.

Figure 5.

Torpor-related changes in thalamic hair-like processes. A, Time course of torpor-related changes in density and length of thalamic hair-like processes (mean + SEM). A body temperature graph from a hibernating ground squirrel is superimposed. B, C, Images of dendrites from the ventral posterior nucleus of the thalamus from animals 12 h after induced arousal (B), and 6 d into a torpor bout (C). The arrowheads denote hair-like processes.

Figure 6.

Linear relationship between body temperature and neural microstructure. A, Sample body temperature graphs from animals hibernating at ambient temperatures of 5 and 15°C. Comparison of neural microstructure was made 3 d into torpor at both temperatures, as indicated in the figure. B–D, Percentage decrease of neural structural parameters as a function of body temperature in euthermia (36°C) and body temperature in torpor at the two temperatures (∼17 and 7°C) (mean + SEM). Neural cell types depicted are cortical layer 4 spiny stellate neurons (B), ventral posterior thalamic somatosensory projection neurons (C), and hippocampal CA3 neurons (D). The interbout euthermic group comprise data from animals hibernating at both temperatures, because there were no statistically significant differences between them.

Figure 7.

Neural structural recovery reaches fixed parameters. Recovery of neural microstructure in animals hibernating at 5 and 15°C (mean + SEM). Group means are from animals 3 d into a torpor bout and 12 h after induced arousal. Neural cell types shown are as follows: cortical layer 4 spiny stellate neurons, ventral posterior thalamic somatosensory projection neurons, and hippocampal CA3 neurons. Neural structural parameters shown are as follows: cell body area (in square micrometers), sum of intersections, and spine density (number of spines per micrometer of dendrite). Despite significant differences in neural microstructure during torpor at the two temperatures (p < 0.05; marked with asterisks), there were no statistically significant differences between recovered structural values.