Effect of MDMA-Induced Axotomy on the Dorsal Raphe Forebrain Tract in Rats: An In Vivo Manganese-Enhanced Magnetic Resonance Imaging Study

RESEARCH ARTICLE Effect of MDMA-Induced Axotomy on the Dorsal Raphe Forebrain Tract in Rats: An In Vivo Manganese-Enhanced Magnetic Resonance Imaging Study Chuang-Hsin Chiu1,2, Tiing-Yee Siow3, Shao-Ju Weng4, Yi-Hua Hsu5,6, YuahnSieh Huang4, Kang-Wei Chang7, Cheng-Yi Cheng2*, Kuo-Hsing Ma4¤* 1 Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan, 2 Department of Nuclear Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, 3 Department of Medical Imaging and Intervention, Chang Gung Memorial Hospital, Chang Gung University, Kueishan, Taoyuan, Taiwan, 4 Department of Biology and Anatomy, National Defense Medical Center, Taipei, Taiwan, 5 Functional and Micro-Magnetic Resonance Imaging Center, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, 6 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, 7 Institute of Nuclear Energy Research, Taoyaun, Taiwan ¤ Current address: Department of Biology and Anatomy, National Defense Medical Center, Taipei, Taiwan * (KHM); (CYC) OPEN ACCESS Citation: Chiu C-H, Siow T-Y, Weng S-J, Hsu Y-H, Huang Y-S, Chang K-W, et al. (2015) Effect of MDMA-Induced Axotomy on the Dorsal Raphe Forebrain Tract in Rats: An In Vivo ManganeseEnhanced Magnetic Resonance Imaging Study. PLoS ONE 10(9): e0138431. doi:10.1371/journal. pone.0138431 Editor: Ichio Aoki, National Institute of Radiological Sciences, JAPAN Received: January 5, 2015 Accepted: August 30, 2015 Published: September 17, 2015 Copyright: © 2015 Chiu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by the Ministry of Science and Technology of Taiwan (Grant: 99-2314B-016-030-MY3) (http://www.most.gov.tw/) and TriService General Hospital, National Defense Medical Center (Grant: TSGH-C104-179). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Abstract 3,4-Methylenedioxymethamphetamine (MDMA), also known as “Ecstasy”, is a common recreational drug of abuse. Several previous studies have attributed the central serotonergic neurotoxicity of MDMA to distal axotomy, since only fine serotonergic axons ascending from the raphe nucleus are lost without apparent damage to their cell bodies. However, this axotomy has never been visualized directly in vivo. The present study examined the axonal integrity of the efferent projections from the midbrain raphe nucleus after MDMA exposure using in vivo manganese-enhanced magnetic resonance imaging (MEMRI). Rats were injected subcutaneously six times with MDMA (5 mg/kg) or saline once daily. Eight days after the last injection, manganese ions (Mn2+) were injected stereotactically into the raphe nucleus, and a series of MEMRI images was acquired over a period of 38 h to monitor the evolution of Mn2+-induced signal enhancement across the ventral tegmental area, the medial forebrain bundle (MFB), and the striatum. The MDMA-induced loss of serotonin transporters was clearly evidenced by immunohistological staining consistent with the Mn2+-induced signal enhancement observed across the MFB and striatum. MEMRI successfully revealed the disruption of the serotonergic raphe-striatal projections and the variable effect of MDMA on the kinetics of Mn2+ accumulation in the MFB and striatum. Introduction 3,4-Methylenedioxymethamphetamine (MDMA), sold under the street name “Ecstasy”, is an illicit recreational drug of abuse. Numerous animal performed in recent decades have demonstrated the neurotoxic effects of MDMA on central serotonergic systems [1–3]. The long-term PLOS ONE | DOI:10.1371/journal.pone.0138431 September 17, 2015 1 / 16 MEMRI Study in Dorsal Raphe Forebrain Tract with MDMA Treatment Competing Interests: The authors have declared that no competing interests exist. serotonergic damage caused by MDMA was first demonstrated in laboratory animals during the mid-1980s [3–5], when MDMA was shown to produce long-lasting decreases in the number of serotonin uptake sites and in the expression of both serotonin transporters (SERT) and serotonin biomarkers (i.e., 5-hydroxyindoleacetic acid, tryptophan, and tryptophan hydroxylase). The most compelling evidence for MDMA-induced serotonergic damage comes from immunohistological studies that showed the profound loss of fine serotonergic axon terminals throughout the forebrain [6,7]. Serotonergic axons with swollen varicosity and fragmentation were visualized in the cortex, but no aberrant morphological changes in the raphe cell bodies were identified in electron microscopic analyses [8]. These results indicated that MDMA may specifically induce distal axotomy of brain serotonergic neurons. The dorsal raphe nucleus (DRN) consists of the midbrain raphe nuclear complex that is rostrally connected to the basal-ganglia-motor system and caudally connected to the limbic system [9–11]. The DRN typically contains 40–50% of the serotonergic neurons of the brain [12] and is thought to give rise to most of susceptible of serotonergic fibers to MDMA neurotoxicity [13]. Serotonergic fibers originating from the DRN were found to be preferentially damaged following MDMA administration, whereas those originating from the medial raphe nucleus are spared [6,14,15]. Several ascending fiber tracts from the DRN have been identified in the rat central nervous system [16]. The dorsal raphe forebrain tract is a ventral transtegmental efferent pathway coursing through the midbrain forebrain bundle (MFB) and mainly terminating within the striatum [17,18]. It comprises the most prominent anterior projections of the serotonin system from the DRN in the rat brain. There is evidence from light- and electronmicroscopy autoradiography studies that ascending serotoninergic axons not only pass through but also terminate within the ventral tegmental area (VTA) [17]. These long projection pathways extend for 10–11 mm within the rat brain, which makes in vivo investigation of the axonal integrity difficult using traditional neuroscience techniques. Manganese-enhanced magnetic resonance imaging (MEMRI) is an emerging neuroscience investigation technique that enables in vivo visualization of the antegrade connections of a multi-synaptic neuronal pathway [19–22]. The manganese ion (Mn2+) is a paramagnetic metal ion that shortens the T1 relaxation time and enhances the signal in T1-weighted images (T1WIs). The application of MEMRI for tract-tracing exploits the chemical properties of Mn2+ as a calcium ion (Ca2+) analogue, where the induced signal enhancement is contingent upon Ca2+-dependent signaling events [23,24]. When injected intracranially, Mn2+ enters cells via voltage-gated Ca2+ cha (...truncated)