These results are the first to demonstrate that concentrations of the substituted amphetamine MDMA achieved in vivo can increase the survival of ventral mesencephalic DA neurons in culture in a dose-dependent, and DA-specific manner. The results also demonstrate that MDMA is effective not only against the early wave of cell death in vitro, but also at later intervals, since delayed application of MDMA still significantly increased DA neuron survival. MDMA exposure significantly increased the expression of the slc6a3 gene which encodes for DAT as measured by qPCR. MPH antagonism of DAT in the presence of MDMA attenuated the increased survival of DA neurons suggesting that MDMA’s binding at DAT is a critical first step in this survival effect.
Historically, potential survival promoting factors have often been screened in suboptimal sparse culture conditions that may favor detection of a positive effect at the cost of exaggerating the biological significance of the effect. In the present experiments we specifically selected the microisland cell culture condition, in which cells are maintained at higher density, to more closely approximate cell density in vivo
. Under these same cell culture conditions we have previously demonstrated that glial cell line-derived neurotrophic factor (GDNF) exposure for 96 hours yields a 40% increase in DA neuron survival (Collier et al., 2003
). Similarly, our optimal concentration of MDMA (37.5 μM) produced an approximate 40% increase in DA neuron survival over the same interval suggesting that MDMA is of equivalent efficacy to 50 ng/ml GDNF in this model system.
It is important to emphasize the utility of employing in vivo
MDMA concentrations for these in vitro
studies. The parity between in vivo and in vitro MDMA concentrations can never be perfect. In vivo
MDMA exposure results in compartmentalization and metabolism of the drug that would be difficult to model in vitro
. In addition, whether or not these concentrations are reminiscent of MDMA levels in the fetal brains of humans is unknown. However, this is the first study to establish fetal brain concentrations of MDMA via maternal administration as an externally valid reference point for in vitro
investigations of MDMA. The fact that MDMA concentrations outside the observed in vivo
range either had no effect on DA neuron survival (low doses) or were non-specifically toxic (high doses) suggests that this type of molar approximation has value when creating an in vitro
model. MDMA conditions within the demonstrated in vivo
range increased the survival of DA neurons without increasing the survival of all cells in culture. High concentrations of MDMA (above 100μM) have been shown to be neurotoxic by others and have been used as evidence for its neurotoxic potential during abuse (Capela et al., 2006
). We contend in vivo
estimates of MDMA brain concentrations should be paired with in vitro
culture studies to enable better interpretation and extrapolation of such findings.
Another important consideration for evaluating MDMA’s potential to increase neuronal survival is the variety of insults from which it may provide protection. During the first 48 hr in culture, mesencephalic cells may experience numerous insults including, detachment induced cell death (anoikis), excitotoxicity, oxidative stress and trophic factor withdrawal. In contrast, established mesencephalic cell cultures are likely to experience cell death mainly due to lack of appropriate environmental signals (Sortwell, 2003
). Our results demonstrate that both early and late exposure to MDMA enhances DA neuron survival suggesting that MDMA is potentially protective against a wide range of toxic insults. Survival of DA neurons in the LATE condition was not as robust as in the EARLY or FULL conditions which were similar to each other. However, this may be explained by the inferred significant neuron loss in the LATE condition already occurred during the first 48 hr prior to MDMA application. Thus, there were fewer neurons remaining for MDMA to rescue by the time it was added, hence the blunted survival effect.
We demonstrated that MDMA increased the expression of the slc6a3 gene via qPCR by 3.7 fold after adjusting for increased neuron survival in the cultures. Amphetamine has been shown in some studies to increase surface expression of DAT on DA neurons (Ukairo et al., 2007
) while some suggest the opposite (Sulzer et al., 2005
; Wei et al., 2007
). However, these findings are postulated to be a result of translocation of DAT between the cytosol and the membrane rather than a result of changes in gene expression (Ukairo et al., 2007
). While we need to confirm that these changes in slc6a3 expression translate into greater expression of DAT protein, understanding why MDMA’s occupancy of DAT results in increased gene expression for DAT, and how this enhances DA neuron survival is a key goal of future studies. Future studies to fully elucidate the MDMA-induced alterations in genes associated with DAT trafficking may allow us to connect MDMA’s occupation of DAT with its ability to increase the survival of dopamine neurons.
Since substituted amphetamines like MDMA primarily act via activation of monoamine transporters, and since MDMA upregulates the expression of slc6a3, it is not a large conceptual leap to hypothesize that the MDMA induced survival effect is mediated by MDMA’s action at the DAT. In order to determine whether another compound with greater affinity for DAT could block the MDMA-induced survival effect, MPH, a competitive DAT inhibitor, was added to the cultures. Unlike MDMA, MPH did not enhance the survival of DA neurons. While both drugs increase extracellular DA levels, MPH inhibits the action of the DAT while amphetamine-like drugs, like MDMA, activate the DAT in the adult brain (Foster et al., 2006
). This suggests that neither occupying the transporter nor increasing extracellular DA is sufficient to produce the observed survival effect. In addition, when MPH levels were reduced in competition studies where MDMA concentrations were maintained, the survival effect returned. This suggests that DAT is being expressed in the cultured cells, and that inhibition of MDMA binding by MPH inhibited the survival effect. These studies provide strong preliminary evidence that MDMA’s ability to increase DA neuron survival is initiated with its activation of DAT, but that occupation of DAT per se is not sufficient to trigger the survival-enhancing effect. Future studies utilizing more specific DAT antagonists, as well as siRNA to silence DAT may more conclusively answer this question.
Interestingly, while MPH had no effect on neuron survival by itself, it did appear to produce a modest, but significant enhancement of neurite outgrowth while yielding a similar level of DA neuron survival as observed in control conditions. One prior study demonstrated a lack of effect of MPH on neuron survival, as well as its ability to successfully block neurotoxins (Ludolph et al., 2006
). However, this previous report did not observe increased neurite density as we have in the current study. The use of stereologic sampling methods to estimate neurite density in the microisland cultures may have increased our sensitivity to observe such differences in neurite density. How MPH enhances neurite outgrowth in DA neurons, and whether specific DAT antagonists, such as GBR-12909, can produce a similar response, warrants further investigation.
These in vitro
studies appear to model some aspects of our prior in vivo
work in which we demonstrated that maternal administration of MDMA increased DA neurite density in prefrontal cortex, striatum and nucleus accumbens in 21 day old rats (Koprich et al., 2003
). In the prior in vivo
study, we hypothesized that MDMA increased DA neuron survival and/or neurite branching in these target structures. Based upon our current in vitro
findings, MDMA augments DA neuron survival but does not produce an increased number of neurites per neuron. This suggests that either the prior finding of increased neurite density is associated with increased DA neurons from source stuctures, or that our in vitro
model does not exactly reproduce the environmental conditions needed to enhance neurite outgrowth. While our in vitro
model establishes that MDMA has a pro-survival effect on DA neurons, these cultures lack both glia and neurons from their target structures. Future studies will examine the role of glia and striatal target cells in a more complex in vitro
model system. The added cell population complexity in the presence of MDMA may provide sufficient homology to produce both increased neuron survival and increased neurite density to better approximate our in vivo
In the current study, prenatal exposure to MDMA resulted in increased DA neuron populations in the SN but not the VTA in brains of rats assessed at P35. Whether this is a result of less apoptotic cell death or increased neurogenesis during development remains to be determined. MDMA has been shown to reduce the survival of newly generated neurons within the adult hippocampus, but not affect the rate of neurogenesis (Hernandez-Rabaza et al., 2006
). While this demonstrates that MDMA can affect the homeostatic balance between neurogenesis in apoptosis in the adult hippocampus, it is unclear whether this occurs in the developing brain. This finding is particularly interesting not only because it provides a parallel in vivo
result that demonstrates greater DA neuron survival as a result of developmental MDMA exposure, but also because it appears to be selective for nigral DA neurons. This provides important data that can be followed up in vitro
to determine if different DA neuron populations show differential responses to the pro-survival effect of MDMA.
While there are many studies that have shown that DAT can be regulated by upstream signals (Ingram and Amara, 2000
), and that the presence of DAT can change neuronal excitability (Ingram et al., 2002
), the downstream result of phosphorylation and endocytosis of DAT has not been well characterized.
It has been postulated that accumulation of cytosolic DA may be responsible for nigral degeneration (Berman et al., 1996
; Caudle et al., 2007
; Hastings et al., 1996a
; Hastings et al., 1996b
; Jenner, 2003
; Kunikowska and Jenner, 2002
; Montine et al., 1997
; Rabinovic et al., 2000
). Amphetamines such as MDMA, act on DAT to reverse the transport of DA out of the cytosolic compartment and into the extracellular space (for a review see (Fleckenstein et al., 2007
)). In the current study, we have demonstrated that MDMA increases expression of slc6a3 which may increase DAT expression. MDMA’s protective action may be a result of these two phenomena which would keep cytosolic DA pools reduced. While this is speculative, future studies examining the generation of reactive oxygen species in culture may help determine the validity of this hypothesis.
This is the first report that MDMA can specifically increase the survival and/or growth of immature DA neurons both in vivo and in vitro. Elucidating the pathway that begins with MDMA induced DAT stimulation, and ends with a DA neuron that can survive in challenging environments such as those employed here in vitro may allow us to better understand how MDMA may reduce normal DA neuron loss that occurs during development.