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1.
Figure 5

Figure 5. From: 3,4-Methylenedioxy-N-methamphetamine (Ecstasy) Promotes the Survival of Fetal Dopamine Neurons in Culture.

Representative image of TH+ cultures after 4 DIV in the absence (A) or presence (B) of 37.5 μM MDMA. Scale bar in A = 20 μm.

Jack W. Lipton, et al. Neuropharmacology. ;55(5):851-859.
2.
Figure 10

Figure 10. From: 3,4-Methylenedioxy-N-methamphetamine (Ecstasy) Promotes the Survival of Fetal Dopamine Neurons in Culture.

Representative images of the TH+ neurons of the SN of P35 rats exposed to (A) vehicle (B) or MDMA from E14-20. Scale bar in A = 100 μm.

Jack W. Lipton, et al. Neuropharmacology. ;55(5):851-859.
3.
Figure 2

Figure 2. From: 3,4-Methylenedioxy-N-methamphetamine (Ecstasy) Promotes the Survival of Fetal Dopamine Neurons in Culture.

Example of the counting frame used to count both neurites and neurons in cover-slip mounted cultures from experiment 6 using the Stereo Investigator software. Any neuronal cell bodies within the counting frame our touching the green line were included while those outside the frame or touching the red line were excluded. Any neurite that crossed the black lines (Merz radius) within the counting frame was counted. If the same neurite crossed a black line more than once, each instance was counted.

Jack W. Lipton, et al. Neuropharmacology. ;55(5):851-859.
4.
Figure 9

Figure 9. From: 3,4-Methylenedioxy-N-methamphetamine (Ecstasy) Promotes the Survival of Fetal Dopamine Neurons in Culture.

Stereologic estimates of TH+ neuron populations of the SN and the VTA in animals exposed to MDMA prenatally from E14-20 and examined at postnatal day 35. The population of TH+ neurons in the SN but not the VTA were significantly increased as a result of prenatal MDMA exposure. (*= p<0.05 vs. control; graphs represent a merging of 3 repeated experiments)

Jack W. Lipton, et al. Neuropharmacology. ;55(5):851-859.
5.
Figure 7

Figure 7. From: 3,4-Methylenedioxy-N-methamphetamine (Ecstasy) Promotes the Survival of Fetal Dopamine Neurons in Culture.

A saturating level of MPH (2uM) alone in culture did not alter TH+ neuron survival but it completely attenuated the MDMA induced survival effect. As MPH levels were reduced in increments of 1:10, the MDMA survival effect returned when MPH was reduced to 0.002 μM. This implies that MPH was able to outcompete MDMA at the DAT at higher concentrations and block the survival effect of MDMA at the DAT. (Post-hoc comparisons: *= p<0.05; graphs represent a merging of 3 repeated experiments)

Jack W. Lipton, et al. Neuropharmacology. ;55(5):851-859.
6.
Figure 3

Figure 3. From: 3,4-Methylenedioxy-N-methamphetamine (Ecstasy) Promotes the Survival of Fetal Dopamine Neurons in Culture.

The concentration of MDMA as a function of time in fetal brain after a 15 mg/kg s.c. injection of MDMA on E18 to the dam. The dam had been injected previously from E14 to E17 with 15 mg/kg MDMA (b.i.d). The dashed line represents the concentration of MDMA used in culture studies. Each time point represents three fetuses from a separate dam. These data demonstrate that the concentration of MDMA used for in vitro culture studies are within the physiologic range of a fetus exposed to a 15 mg/kg injection of MDMA to a drug-experienced dam.

Jack W. Lipton, et al. Neuropharmacology. ;55(5):851-859.
7.
Figure 6

Figure 6. From: 3,4-Methylenedioxy-N-methamphetamine (Ecstasy) Promotes the Survival of Fetal Dopamine Neurons in Culture.

TH+ neuron survival after 4 days in vitro (DIV) when cultures were exposed to culture media only (CTRL), MDMA during the first 2 DIV (EARLY), MDMA during the last 2 DIV (LATE)or MDMA for the full 4 DIV (FULL). All MDMA conditions were significantly greater than the CTRL conditions. The EARLY and MDMA conditions were significantly better than the LATE condition at improving TH+ neuron survival, although the LATE condition was better than CTRL. (Post-hoc comparisons: *= p<0.05; graphs represent a merging of 3 repeated experiments)

Jack W. Lipton, et al. Neuropharmacology. ;55(5):851-859.
8.
Figure 8

Figure 8. From: 3,4-Methylenedioxy-N-methamphetamine (Ecstasy) Promotes the Survival of Fetal Dopamine Neurons in Culture.

Examination of neuron survival and neurite density in control cultures or cultures exposed to MDMA, MPH or MDMA+MPH. At first glance it appears the MDMA increases both TH+ neuron survival and neurite outgrowth in culture (A). However when the data for each well was calculated as a ratio of neurites per neuron, this finding was not evident (B). This suggests that in vitro exposure to MDMA increases TH+ neuron survival but does not increase the density of neurites expressed by each neuron. Interestingly, MPH appears to increase TH+ neurite density independent of neuron number. (*= p<0.05 vs. control; graphs represent a merging of 3 repeated experiments)

Jack W. Lipton, et al. Neuropharmacology. ;55(5):851-859.
9.
Figure 1

Figure 1. From: 3,4-Methylenedioxy-N-methamphetamine (Ecstasy) Promotes the Survival of Fetal Dopamine Neurons in Culture.

Schematic diagram illustrating the microdissection technique to harvest ventral mesencephalic tissue. 1. Incisions are made at the rostral and caudal borders of the mesencephalic flexure to excise the mesencephalon. 2. The dorsal segment of the mesencephalon (tectum, //////) is removed and discarded. 3. The remaining ventral tegmental tissue is laid flat exposing the floor of the 4th ventricle. Incisions of appropriate length are made utilizing the isthmus and sulcus limitans as landmarks. 4. The isolated ventral mesencephalic tissue piece is collected and processed to single cell suspension. MF = mesencephalic flexure, PF = pontine flexure, DIEN = diencephalon, TELEN = telencephalon, MES = mesencephalon,----------- = incision line.

Jack W. Lipton, et al. Neuropharmacology. ;55(5):851-859.
10.
Figure 4

Figure 4. From: 3,4-Methylenedioxy-N-methamphetamine (Ecstasy) Promotes the Survival of Fetal Dopamine Neurons in Culture.

Dose response curve for E14 midrain neurons over a 96 hr MDMA exposure in vitro. A: MDMA significantly enhanced TH+ neuron survival when incubated within a physiologically relevant range based upon our in vivo model. General toxicity was observed in concentrations higher than 75 μM. The greatest survival occurred in the 37.5 μM MDMA condition. B: MTT Assay: MDMA had no effect on general cell viability until 750 μM was applied which resulted in a significant reduction in cell survival. Together, these data demonstrate that MDMA’s ability to enhance cell survival is specific to DA neurons. (Graphs represent a merging of 4 repeated experiments; *= p<0.05 vs. control)

Jack W. Lipton, et al. Neuropharmacology. ;55(5):851-859.

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