Exercise leads to the re-emergence of the cholinergic/nestin neuronal phenotype within the medial septum/diagonal band and subsequent rescue of both hippocampal ACh efflux and spatial behavior

Abstract

Exercise has been shown to improve cognitive functioning in a range of species, presumably through an increase in neurotrophins throughout the brain, but in particular the hippocampus. The current study assessed the ability of exercise to restore septohippocampal cholinergic functioning in the pyrithiamine-induced thiamine deficiency (PTD) rat model of the amnestic disorder Korsakoff Syndrome. After voluntary wheel running or sedentary control conditions (stationary wheel attached to the home cage), PTD and control rats were behaviorally tested with concurrent in vivo microdialysis, at one of two time points: 24-h or 2-weeks post-exercise. It was found that only after the 2-week adaption period did exercise lead to an interrelated sequence of events in PTD rats that included: (1) restored spatial working memory; (2) rescued behaviorally-stimulated hippocampal acetylcholine efflux; and (3) within the medial septum/diagonal band, the re-emergence of the cholinergic (choline acetyltransferase [ChAT+]) phenotype, with the greatest change occurring in the ChAT+/nestin+ neurons. Furthermore, in control rats, exercise followed by a 2-week adaption period improved hippocampal acetylcholine efflux and increased the number of neurons co-expressing the ChAT and nestin phenotype. These findings demonstrate a novel mechanism by which exercise can modulate the mature cholinergic/nestin neuronal phenotype leading to improved neurotransmitter function as well as enhanced learning and memory.

Keywords: Acetylcholine; BDNF; Diagonal band; Exercise; Medial Septum; NGF; Nestin.

Figure 1. Experimental design

First, all subjects were randomly assigned to either PTD (n=32) or PF (n=32) treatment. Following recovery, all rats had a cannula placed in the ventral hippocampus for later in vivo microdialysis. After surgery, rats were pair-housed and allowed to recover and were then transferred to cages that had either an active running wheel (voluntary exercise [VEx]) or an inactive running wheel (Stationary Controls [Stat]) attached to the home cage for a 2-wk duration. Rats were subsequently tested either 24-hrs or 2-wks later on spontaneous alternation (SA) combined with in vivo microdialysis followed by a novel object recognition (NOR) test. Cellular morphological assessment from the MS/DB was performed in all subjects. In a different cohort of animals, BDNF and NGF levels were obtained 24-hrs or 2-wks after the cessation of VEx and Stat conditions.

Figure 2. Spontaneous alternation behavioral performance (panels A–B) is expressed as mean percent alternation ± S.E.M, while hippocampal acetylcholine (ACh) efflux (panels C–D) is displayed as percent change relative to baseline ± S.E.M

At the 24-hr time point (A), a significant interaction revealed that PTD rats show impaired performance, relative to PF rats, and exercise modestly improved performance, while control PF rats showed no change in spontaneous alternation in response to exercise. Two-weeks post exercise (B), PTD rats that exercised showed a robust recovery in performance and at this time point. Twenty-four hrs post exercise there was a decrease in ACh levels in PTD rats during behavioral testing (M1–M3) compared to PF (C), but no effect of exercise. However, 2-weeks post-exercise, both PTD and PF rats showed an increase in ACh efflux during behavioral testing (D). * indicates a significant effect of Treatment, ^ indicates a significant effect of Exercise, and # indicates a significant Treatment × Exercise interaction

Figure 3. Correlations between cell counts, ACh efflux and behavior

Panel A. At 2-wks post-exercise a positive correlation was found between Nestin+/ChAT+ cell number and hippocampal ACh efflux. This was primary driven by the PTD group. Panel B. There was also a positive correlation between spontaneous alternation performance and hippocampal ACh efflux after the 2-wk restoration period. Triangles indicated PF control data points: filled triangles containing represented the PF VEx group and open triangles represented the PF Stat group. Circles denoted PTD rats: filled circles represented PTD VEx rats and open circles represented PTD Stat rats.

Figure 4. Novel object recognition (NOR) performance obtained from both 24-hrs (A) and 2-wks (B) post exercise

Data are expressed as percent of overall time spent exploring the novel object, where 50% is indicative of chance performance. PTD rats showed no impairments on this task at either time point. However, an overall effect of exercise was observed at the 24-hr time point. ^ indicates a significant effect of Exercise

Figure 5. Changes in hippocampal mBDNF and β-NGF levels at 24-hrs and 2-wks post exercise

In the hippocampus, there was a decrease in mBDNF in PTD rats that was rescued by exercise at both time points (AB). Exercise also increased mBDNF levels in PF rats at both time points. β-NGF levels in the hippocampus (CD) were also decreased by PTD, but increased by exercise in both PTD and PF rats. Numbers within the bars are the actual mean protein values (pg/ml) for the neurotrophins each condition. * indicates a significant effect of Treatment, ^ indicates a significant effect of Exercise, and # indicates a significant Treatment × Exercise interaction

Figure 6. Thalamic damage and hippocampal cannula placement

Intraventricular distance (IVD) data (A) were collapsed across Time point and Exercise conditions, since there were no significant differences among these variables. Group means are reported and plotted for PF (triangles) and PTD (circles) rats, as shown in the legend. Overall, there is a decrease in the IVD in PTD rats, relative to PF control rats. Exercise did not affect IVD. Representative images (B) of the midline thalamus were taken from a PF and PTD rat. A characteristic image (C) of the hippocampal cannula placement for microdialysis. * indicates a significant effect of Treatment.

Figure 7. The overall estimated number of ChAT+ cells within the MS/DB were decreased by PTD treatment at 24-hrs (A) and 2-wks (B) post exercise

Although exercise did not influence total ChAT+ cell numbers 24-hrs post exercise, 2-wks post exercise there was an increase in the number of neurons that expressed the ChAT+ phenotype in both PF and PTD rats. * indicates a significant effect of Treatment, ^ indicates a significant effect of Exercise

Figure 8. Changes in cholinergic phenotype after thiamine deficiency and exercise

Double immunofluorescent staining for ChAT+ and nestin+ cells within the MS/DB of a stationary PTD rat (A) and PTD rat that exercised (B). Detailed representations of the ChAT+ and nestin+ cells and merged phenotypes with in the MS/DB (C). The number of neurons (per mm²) within the MS/DB that have the overall ChAT phenotype, and the subcategories of cholinergic neurons that display the ChAT+/Nestin − phenotype and the Nestin+/ChAT+ phenotype (D). PTD treatment significantly decreased the ChAT+/nestin+ phenotype at both time points. Exercise increased ChAT+/nestin+ phenotype only after the 2-wk adaption period in PTD and PF control rats. The ChAT+/nestin− phenotype was unchanged by pathology or exercise.* indicates a significant effect of Treatment, ^ indicates a significant effect of Exercise