The effect of voluntary exercise on adult hippocampal neurogenesis in maternally separated rats

Hardcastle, Natasha Sema

The effect of voluntary exercise on adult hippocampal neurogenesis in maternally separated rats

Master Thesis

2016

Publisher

University of Cape Town

Department

Department of Human Biology

Faculty

Faculty of Health Sciences

Abstract

Maternal separation (MS) has been shown to produce depression-like symptoms in male Sprague Dawley rats. The underlying mechanisms responsible for the development of these depression-like behaviors are unknown. However, a growing body of evidence suggests that a reduction in neurogenesis may be a key-mediating factor. Voluntary wheel running is a form of exercise that increases neurogenesis and decreases depression-like behaviour in rats. However, the exact molecular role of neurogenesis in exercise-induced antidepressant effects still remains unanswered. This requires new tools to explore the interact ion between exercise and neurogenesis in vivo. To this end, the novel mitotic-marker, 5-ethynyl-2'-deoxyuridine (EdU), and Ki-67, an endogenous marker of cell proliferation, was characterised in order to study neurogenesis in an MS rat model of depression. Furthermore, this study aimed to provide insight into the effect of voluntary exercise on cell genesis and survival. To characterise EdU labelling of cells in vivo, male Sprague Dawley rats (Characterisation rats n =13) were injected with 50 mg/kg EdU a s noted in the literature. The optimal time point to inject the EdU label to measure mitotic activity was found to be post-natal day (PND) 60. MS or non-maternal separation (NMS) was conducted from PND 2-14 on experimental rats (n=39). From PND 54 - 74, ex perimental rats were housed in cages with attached running wheels (R) or locked running wheels (NR). All experimental rats were injected with 50 mg/kg EdU on PND 60 and transcardially perfused on PND 74 using Phosphate Buffered Saline (PBS) followed by fre sh 4% paraformaldehyde. Whole brains were then removed from the skull and placed in 4% paraformaldehyde for three hours. The brains were transferred to a 30% sucrose solution, stored in sucrose for 3-5 days and thereafter mounted in optimal cutting mediu m (OCT) and sectioned using a cryostat. Brain sections of 40 μm from 6.96 to 5.52 mm anterior to the inter-aural line were taken as dorsal and 50 μm sections from 3.84 to 2.76 mm were analyzed as ventral. The marker, EdU was detected in rat brains using t he Click-iT EdU Alexa Fluor 488 detection kit. Three molecular marker combinations were used to detect different factors for both dorsal and ventral hippocampi: (1) EdU/GFAP/NeuN, to establish how many EdU labelled cells survive to become neurons or astroc ytes (2) EdU/DCX to determine how many EdU labelled cells that have survived for 14 days are immature neurons and (3) Ki-67/DCX to indicate how many mitotically active cells are immature neurons on PND 74. Brain sections were then scanned using a confocal microscope whereby EdU stained nuclei were manually counted and cell phenotypes identified. The molecular marker combination one and two revealed no differences between treatment groups in the number of EdU-labelled cells in the dorsal and ventral hippoca mpi. However, a significant correlation was found between EdU/GFAP positive cells and EdU/NeuN positive cells in the ventral hippocampus when all treatment groups were pooled (r = 0.82, n=18, p < 0.0001). The third molecular marker combination revealed sig nificant differences in neurogenesis between groups. The MS+R group had fewer dorsal hippocampal Ki-67/DCX cells relative to NMS+R and NMS+R had significantly higher Ki-67/DCX cell count relative to NMS+NR rats. In the ventral hippocampus MS+R rats had few er Ki-67/DCX cells compared to NMS+R rats. The link between neurons and astrocytes in the ventral hippocampi corresponds with reports that an increase in neurons is linked to the presence of astrocytes. However, it may also be due to unavoidable variation in the intensity of the stain. The third molecular marker combination (Ki-67/DCX) revealed the most significant finding of this study. It showed that voluntary wheel running significantly increased the number of Ki-67/DCX co-labelled neurons in the dorsal hippocampus of NMS+R rats relative to NMS+NR which is in agreement with the literature that suggests exercise increases neurogenesis. The literature also reports that stress decreases neurogenesis and interestin gly MS+R rats had a lower cell count than NMS+R rats. This may indicate an interaction between early life stress and exercise-induced neurogenesis. This finding further suggests that MS alters neurogenesis in adult life and attenuates the effect of exercis e on the ventral hippocampus.

Keywords

Neuroscience

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