Fig 1.
Experimental scheme for MDMA treatment, MEMRI and immunohistochemistry.
Fig 2.
Anatomical interrelationships of the assessed regions.
(A) Sagittal view of the midbrain raphe nucleus (RN) and its downstream VTA/IP, MFB, and striatum. (B) Mn2+ injection site on the rat brain atlas and on MEMRI images.
Fig 3.
SERT immunohistochemical staining of rat brain sections.
Immunohistochemical staining of (A) a control rat and (B) a MDMA-treated rat confirmed the degeneration of serotonergic nerve terminals in the midbrain RN and striatum in the MDMA-treated rat, as shown by reduced SERT expression. Scale bar = 50 μm.
Fig 4.
Dynamic MEMRI reveals the temporal and spatial evolution of Mn2+-induced signal enhancements.
The Mn2+-induced signal enhancements along (A) the VTA/IP and (B) the MFB pathway after injecting Mn2+ into the midbrain RN. The series of T1WIs shows the kinetics of Mn2+ uptake in the midbrain RN, accumulation in the VTA/IP, and transport along the MFB pathway.
Fig 5.
Time course of Mn2+-induced rSI reveals the effects of MDMA neurotoxicity.
The midbrain RN downstream including (A) the direct target nuclei (VTA/IP) and (B) the MFB pathway. The temporal evolution of the averaged Mn2+ rSI in the VTA/IP and the MFB pathway of each group is quantitatively expressed as a function of time, and the data are fitted with sigmoid curves (VTA/IP-MDMA: R2 = 0.7359, p<0.0001; VTA/IP-control: R2 = 0.7990, p<0.0001; MFB-MDMA: R2 = 0.6221, p<0.001; MFB-control: R2 = 0.9200, p<0.0001). The Mn2+-induced rSIs are given as mean and S.E.M. values. Blue and red spots correspond to Mn2+-induced rSIs measured from the ROIs in control and MDMA-treated rats, respectively (the corresponding lines are the fitted sigmoid curves). Asterisks indicate the first significant increase in the Mn2+-induced rSI, determined by comparing new images with images acquired 40 min after injecting Mn2+ (* p<0.05). In control rats, the first significant increase in Mn2+-induced rSI occurred at 60 min in the VTA/IP and at 120 min in the MFB. In MDMA-treated rats, the first significant increase in Mn2+-induced rSI occurred at 60 min in the VTA/IP and at 180 min in the MFB. The rSI of the MFB was significantly lower in the MDMA group than in the control group after 210 min (# p<0.05).
Table 1.
rSImax and n coefficient values in VTA/IP and MFB of control and MDMA group, as the derived by fitting of time-rSI curve with sigmoid equation.
Fig 6.
MDMA-induced axotomy of serotonergic neurons in the striatum.
(A) MEMRI showing differences in Mn2+ distribution in the striatum between control and MDMA-treated rats at 38 h after injecting Mn2+. Mn2+-induced signal enhancements were decreased after MDMA treatment. The orange-colored region depicts the area of the striatum, as indicated on the atlas in (B). (C) A quantitative analysis confirmed that Mn2+-induced rSIs in the striatum differed statistically between control and MDMA-treated rats. The increase in Mn2+-induced rSI in MDMA-treated rats was reduced significantly at 34–38 h after injection, compared with controls (*p<0.05). (D) Optical density (OD) ratios of SERT immunoreactivity in the striatum of rat brains from both groups on day 16 after saline or MDMA treatment. Data are presented as mean and S.D. values (** p<0.01). There was a statistically significant reduction in the OD ratio after MDMA treatment.