Elsevier

Neuroscience

Volume 231, 12 February 2013, Pages 125-135
Neuroscience

Chronic d-amphetamine administered from childhood to adulthood dose-dependently increases the survival of new neurons in the hippocampus of male C57BL/6J mice

https://doi.org/10.1016/j.neuroscience.2012.11.028Get rights and content

Abstract

Adderall is widely prescribed for attention deficit hyperactivity disorder (ADHD) though long term neurological effects of the main ingredient d-amphetamine are not well understood. The purpose of this study was to examine effects of clinically prescribed doses of d-amphetamine and one abuse dose administered from childhood to adulthood on adult hippocampal neurogenesis and activation of the granule layer of the dentate gyrus. Beginning in early adolescence (age 28 days) to adulthood (age 71), male C57BL/6J mice were administered twice daily i.p. injections of vehicle, 0.25, 0.5 or 2 mg/kg d-amphetamine. Locomotor activity was measured in home cages by video tracking. At age 53–56, mice received bromodeoxyuridine (BrdU) injections to label dividing cells. Immunohistochemical detection of BrdU, neuronal nuclear protein (NeuN), doublecortin (DCX) and Ki67 was used to measure neurogenesis and cell proliferation at age 71. ΔFosB was measured as an indicator of repeated neuronal activation. An additional cohort of mice was treated similarly except euthanized at age 58 to measure activation of granule neurons from d-amphetamine (by detection of c-Fos) and cell proliferation (Ki67) at a time when the fate of BrdU cells would have been determined in the first cohort. d-Amphetamine dose-dependently increased survival and differentiation of BrdU cells into neurons and increased number of DCX cells without affecting the number of Ki67 cells. Low doses of d-amphetamine decreased c-Fos and ΔFosB in the granule layer. Only the high dose induced substantial locomotor stimulation and sensitization. Results suggest both therapeutic and abuse doses of d-amphetamine increase the number of new neurons in the hippocampus when administered from adolescence to adulthood by increasing survival and differentiation of cells into neurons not by increasing progenitor cell proliferation. Mechanisms for amphetamine-induced neurogenesis are unknown but appear activity independent. Results suggest part of the beneficial effects of therapeutic doses of d-amphetamine for ADHD could be via increased hippocampal neurogenesis.

Highlights

► d-Amphetamine dose-dependently increases adult hippocampal neurogenesis. ► d-Amphetamine enhances survival and differentiation of new hippocampal neurons. ► d-Amphetamine-induced neurogenesis appears to be activity independent.

Introduction

Stimulant medications, such as d-amphetamine are widely prescribed for treatment of attention deficit hyperactivity disorder (ADHD) (Greenhill et al., 2002). Most properly diagnosed individuals require treatment for ADHD starting from childhood through adulthood (Angold et al., 2000). However, few studies have examined the long term neurological effects of d-amphetamine or any other approved medications for ADHD over this time period (Peterson et al., 2008). Experimental data for abuse doses of d-amphetamine suggest it can damage dopamine nerve terminals and induce cell death (Ryan et al., 1990, Cunha-Oliveira et al., 2006, Atianjoh et al., 2008). However, little is known about how lower therapeutic doses administered chronically affect the brain. One measurement of interest is adult hippocampal neurogenesis. New neurons are continuously born in the dentate gyrus of mice and humans and both the proliferation and survival of these cells is strongly influenced by environmental factors such as stress, physical activity, nutrition, drugs and medications (Gould et al., 1991, van Praag et al., 1999, Eisch et al., 2000, Lee et al., 2002a, Lee et al., 2002b, Santarelli et al., 2003). Depending on the environmental factors, the addition or subtraction of new neurons can increase or decrease the total numbers of neurons and volume of the dentate gyrus (Rhodes et al., 2003). Although the functional significance of adult neurogenesis is not known, new neurons in the hippocampus are thought to contribute to cognitive performance, learning and memory (van Praag et al., 1999, Clark et al., 2008, Mustroph et al., 2011).

Recent studies suggest that stimulant drugs decrease adult hippocampal neurogenesis under a variety of conditions in rodent models (Yamaguchi et al., 2004, Lagace et al., 2006, Mandyam et al., 2008, Noonan et al., 2010, Venkatesan et al., 2011, Yuan et al., 2011). However, only a few studies have specifically explored the effect of d-amphetamine on adult hippocampal neurogenesis. A recent study found that 2.5 mg/kg d-amphetamine injections administered for 14 days had no effect on the proliferation or survival of new neurons in the dentate gyrus of adult male Sprague–Dawley rats (Barr et al., 2010). However, in the new neuron survival group, bromodeoxyuridine (BrdU) was administered 2 weeks before amphetamine exposure. At 2 weeks of age, new neurons may already be in the process of integrating into hippocampal circuitry and could be less vulnerable to the amphetamine treatment. To the best of our knowledge, no previous study has explored the effect of chronic administration of d-amphetamine from childhood to adulthood on the survival of new neurons born and surviving in the presence of continuous d-amphetamine administration. Therefore, one of the goals of this study was to measure the effect of two clinically relevant d-amphetamine doses (0.25, and 0.5 mg/kg) and one abuse dose (2 mg/kg) on adult hippocampal neurogenesis.

Given that d-amphetamine at high doses is neurotoxic, (i.e., induces cell death) (Ryan et al., 1990, Atianjoh et al., 2008), one might predict that chronic d-amphetamine exposure would decrease the survival of new neurons. On the other hand, d-amphetamine is a stimulant, and generally increases the activity of neurons in the brain (Rotllant et al., 2010). If the dentate gyrus experiences increased neuronal activation in response to d-amphetamine administration, then the increased activity would be expected to increase the survival of new neurons in an activity-dependent manner (Deisseroth et al., 2004, Clark et al., 2010, Clark et al., 2011). Although many studies have examined patterns of activation in the brain from d-amphetamine using such techniques as immunohistochemical detection of c-Fos and ΔFosB (Johansson et al., 1994, Badiani et al., 1998, Engber et al., 1998, Trinh et al., 2003, Renthal et al., 2008, Rotllant et al., 2010), typically the dentate gyrus is not examined [but see (Trinh et al., 2003) who found increased c-Fos from 2 mg/kg amphetamine in the granule layer]. Acute induction of c-Fos from stimulant drugs is known to decline upon repeated stimulation, whereas ΔFosB accumulates in brain regions such as the striatum (Nestler et al., 2001, Renthal et al., 2008). Hence, another goal of this study was to determine the extent to which the granule layer of the dentate gyrus displays increased c-Fos and ΔFosB accumulation from the chronic d-amphetamine treatments in order to help interpret the results for the neurogenesis measures. Based on the limited literature, it is difficult to predict the outcome of long term administration of therapeutic doses of d-amphetamine on adult hippocampal neurogenesis. If the low doses of d-amphetamine decrease neurogenesis, then that would constitute evidence for neurotoxicity. Alternatively, if the low doses of d-amphetamine increase neurogenesis, then that would support the activity-dependent survival hypothesis. The high dose (2 mg/kg) was expected to decrease neurogenesis as this has been observed for many other drugs of abuse including methamphetamine, cocaine, morphine, heroin, and alcohol (Eisch et al., 2000, Klintsova et al., 2007, Mandyam et al., 2008, Noonan et al., 2010, Venkatesan et al., 2011, Yuan et al., 2011).

Section snippets

Experimental procedures

A total of 60 male C57BL/6J mice from The Jackson Laboratory (Bar Harbor, ME) arrived at our facility at 21 days of age and were placed into cages in groups of 4. The mice were left undisturbed for 1 week except for daily handling to acclimate the mice to the experimenter. At age 28 days (equivalent to late childhood, early adolescence), mice were injected intraperitoneally (i.p.) twice daily at the beginning of the dark cycle and again 3 h later with either saline, 0.25, 0.5, or 2.0 mg/kg

Locomotor activity

Distance traveled 1 h after injections was not significantly different during the first and second tests (separated by three hour intervals) within each day. Therefore, the two tests were averaged to produce a single measurement of distance traveled post injection per individual per day. The repeated measures ANOVA revealed a significant effect of dose (F3,84 = 241.3, P < 0.0001), day (F3,84 = 25.5, P < 0.0001) and the interaction of dose and day (F9,84 = 42.7, P < 0.0001; Fig. 2A). On average across test

Discussion

The main discovery of the study is that chronic d-amphetamine treatment starting in childhood to adulthood increases adult hippocampal neurogenesis in male C57BL/6J mice (Fig. 3). The dose-dependent increase in neurogenesis was supported by three different independent measures of neurogenesis including counts of total numbers of BrdU-labeled cells in the granule layer, the proportion of BrdU cells differentiated as neurons as indicated by co-labeling with NeuN (mature neuron marker), and counts

Acknowledgment

This work was supported by NIH Grants MH 083807 and DA027487.

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