Research ArticleDifferential Effects of Extended Exercise and Memantine Treatment on Adult Neurogenesis in Male and Female Rats
Introduction
In the dentate gyrus subregion of the hippocampus, adult-born neurons have enhanced plasticity and unique connectivity relative to older neurons (Snyder and Cameron, 2012, Toni and Schinder, 2015), and they play an important role in memory and emotional behavior (Abrous and Wojtowicz, 2015, Cameron and Glover, 2015). Their functional role has stimulated much research on regulatory factors that could be harnessed, typically to promote neurogenesis, in order to enhance cognitive function or recovery from neurological disorders (Toda et al., 2018). However, most studies have examined neurogenesis regulation over hours, days or weeks. Since most disorders of hippocampal function are chronic, it is important to identify whether neurogenesis can be increased over extended intervals to potentially offset long-term dysfunction (e.g. months in rodents, years in humans).
In humans, reduced hippocampal volume is typically interpreted as a sign of damage and is observed in a number of disorders including depression (McKinnon et al., 2009), schizophrenia (Harrison, 2004), mild cognitive impairment (Yassa et al., 2010), and Alzheimer’s disease (Jack et al., 2000). While the mechanisms underlying hippocampal volume changes are multifaceted, and certainly not wholly reflective of neurogenesis (Schoenfeld et al., 2017), changes in adult neurogenesis could contribute to structural damage as well as recovery. Neurogenesis is difficult to measure in humans, and currently can only be assessed in postmortem tissue. However, a number of known neurogenic treatments (identified in animal studies) are associated with reversal of hippocampal structural deficits in humans. Antidepressant treatment restores dentate gyrus granule cell number in depressed patients (Boldrini et al., 2013, Mahar et al., 2017), possibly by increasing adult neurogenesis (Boldrini et al., 2009). Exercise increases hippocampal volume in healthy individuals (Erickson et al., 2011, Killgore et al., 2013), women with mild cognitive impairment (ten Brinke et al., 2015) and schizophrenic patients (Pajonk et al., 2010), though effects in schizophrenia have been inconsistent (Kim et al., 2018). Moreover, there are sex differences in the prevalence of disorders that impact the hippocampus, with depression (Seedat et al., 2009, Bangasser and Valentino, 2014) and Alzheimer’s disease (Gao et al., 1998) more common in females, and schizophrenia more common in males (Aleman et al., 2003). Thus, it is important to determine which factors can effectively increase adult neurogenesis in males and females, and potentially improve behavioral outcomes.
Neurogenesis is a multistep process whereby precursor cells undergo lineage-directed cell division to produce immature neurons, of which only a fraction are selected to survive and contribute to hippocampal function. Animal models have identified a number of factors that increase neurogenesis, either by promoting precursor proliferation or enhancing immature neuronal survival. For example, factors that can increase proliferation include exercise (Eadie et al., 2005, Kronenberg et al., 2006), antidepressant drugs and electroconvulsive shock (Malberg et al., 2000), synthetic chemicals (Petrik et al., 2012), learning (Dupret et al., 2007) and NMDA receptor antagonists (Cameron et al., 1995, Maekawa et al., 2009). Survival of immature neurons is enhanced by exercise (Snyder et al., 2009b) and learning (Gould et al., 1999, Dupret et al., 2007, Epp et al., 2007). Importantly, regulatory factors can be highly dose- and time-dependent, with some factors increasing or decreasing neurogenesis depending on the conditions (Olariu et al., 2005, Dupret et al., 2007).
Here we focus on two treatments that have been shown to increase neurogenesis in rodents: running (RUN) and memantine (MEM). RUN is likely the most well-studied method for increasing neurogenesis. In addition to increasing proliferation and survival, it also increases the dendritic complexity of newborn neurons, promotes spine formation, and accelerates their functional maturation (van Praag et al., 1999, Redila and Christie, 2006, Piatti et al., 2011, Vivar et al., 2015, Dostes et al., 2016). However, RUN does not increase neurogenesis in socially isolated rats (Stranahan et al., 2006, Leasure and Decker, 2009), in wild mice (Hauser et al., 2009), in mice that run only in the light phase (Holmes et al., 2004), or in animals that run at high intensities (Naylor et al., 2005, Grégoire et al., 2014, So et al., 2017). Furthermore, a number of studies have found that RUN-induced increases in neurogenesis are transient, raising questions about the extent to which it may be used as a strategy for long-term enhanced production of new neurons (Naylor et al., 2005, Kronenberg et al., 2006, Snyder et al., 2009b, Clark et al., 2010). Interestingly, there is evidence that neurogenesis may be sustained for extended durations if RUN amount is restricted (Naylor et al., 2005, Nguemeni et al., 2018). However, since these studies only investigated cells born at a single timepoint in male rats, it remains unclear whether restricted RUN enhances neurogenesis consistently across sexes and over extended periods of time.
MEM is a low-affinity NMDA receptor antagonist that has neuroprotective effects and has been approved as an Alzheimer’s disease treatment (Lipton, 2004). In mice, MEM increases cellular proliferation, the size of the stem cell pool, and the production of new neurons by 2–3-fold (Maekawa et al., 2009, Namba et al., 2009, Akers et al., 2014, Ishikawa et al., 2014). Notably, two other NMDA receptor antagonists, MK-801 and ketamine, have also been found to increase cell proliferation in the dentate gyrus (Cameron et al., 1995, Soumier et al., 2016). While repeat dosing of MEM has been found to broadly increase neurogenesis (Ishikawa et al., 2016), the efficacy of single vs. multiple doses of MEM remain unknown.
Here, we investigated the long-term efficacy of neurogenic treatments in male and female rats. Rats were subjected to RUN, MEM, or alternating blocks of RUN and MEM and multiple immunohistochemical markers (Fig. 1) were used to quantify neurons born at the beginning, middle and end of treatments. While a single MEM injection (sMEM) and continuous RUN (cRUN) only transiently increased neurogenesis, extended treatments were capable of increasing neurogenesis at later timepoints. Neurogenic efficacy depended on sex and treatment: in females, 2 months of interval RUN (iRUN) increased DCX+ cells; in males, DCX+ cells were elevated after 1 month of iRUN followed by 1 month of multiple MEM injections (mMEM). However, thymidine analog labeling revealed that all extended treatments were relatively ineffective at increasing numbers of neurons born in the earlier phases of treatment.
Section snippets
Animals and treatments
All procedures were approved by the Animal Care Committee at the University of British Columbia and conducted in accordance with the Canadian Council on Animal Care guidelines regarding humane and ethical treatment of animals. Experimental Long–Evans rats were generated in the Department of Psychology’s animal facility with a 12-hour light/dark schedule and lights on at 6:00 am. Breeding occurred in large polyurethane cages (47 cm × 37 cm × 21 cm) containing a polycarbonate tube, aspen chip
Short-term continuous running behavior
Rats housed with running wheels ran progressively more over time (Fig. 2B; effect of time: F4,24 = 3.8, P = 0.015). There were no differences in distance run between males and females, but sample sizes were small since rats were pair-housed and we could only determine the distance by the cage rather than per individual rat (but Fig. 2 shows cage distance divided by 2 to facilitate comparisons with later experiments; effect of sex F6,24 = 4.6, P = 0.09).
Five weeks of continuous running transiently increases neurogenesis
Neurogenesis was measured with three
Discussion
Here we examined the effects of two neurogenic treatments, RUN and MEM, on adult neurogenesis in the dentate gyrus of male and female rats. Our general strategy was to immunostain for thymidine analogs to detect treatment effects on cells born long before the experimental endpoint, DCX to detect effects on neurons born in the weeks prior to endpoint, and PCNA to detect effects on cell proliferation at the very end of each experiment. We have two main findings. First, neurogenic effects were
Acknowledgments
This work was supported by an NSERC Discovery Grant (JSS), an NSERC postgraduate scholarship (SPC) and Killam Doctoral Scholarship (SPC).
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