The present results demonstrate that MDMA treatment differentially regulates gene expression of neurotrophins and their receptors in multiple regions of the rat brain (see for summary). In the forebrain, levels of BDNF mRNA were upregulated in several areas at different intervals after MDMA treatment, whereas increases in NT-3 expression were restricted to the prefrontal and anterior cingulate cortices, as well as the dentate gyrus. Both trkB and trkC receptor mRNAs were elevated by MDMA in the PFC only at 24 hours. Increased expression of trkC was found in the entorhinal cortex, whereas trkB mRNA levels were upregulated in the hippocampal CA1 region. The MDMA-induced modulation of neurotrophins and their high-affinity receptors was not limited to cortical and limbic regions. Alterations were also observed in the nigrostriatal system, with expression of BDNF upregulated in the SNpc and trkB in the striatum. Moreover, in addition to the above-noted increases in neurotrophin expression following MDMA administration, several brain regions exhibited decreased hybridization, including the hippocampal CA3 region and dentate gyrus for both BDNF and NT-3, and the locus coeruleus for trkC. Axons projecting from the dorsal raphe nuclei to the forebrain, cortex, striatum, and hippocampus exhibit MDMA-associated morphological changes (O’Hearn et al., 1988
), so it was not entirely unexpected that MDMA would have effects on neurotrophin expression in these regions, but the exact pattern was unknown. Overall, the present data indicate that MDMA-induced modulation of BDNF and trkB mRNA levels are more widespread than previously appreciated (Martínez-Turrillas et al., 2006
). This is also the first study to demonstrate alterations in NT-3 and trkC expression following MDMA exposure.
Summary of MDMA-Induced Neurotrophin Alterations by Brain Region
Martínez-Turrillas et al. (2006)
also examined the effects of MDMA on BDNF in the hippocampus and PFC of adult rats, and some different patterns were observed in that study compared with ours, although only the 24-hour time point overlapped between experiments. For example, increases in BDNF mRNA were noted in the frontal cortex at 24 and 48 hours after a single dose of MDMA (10 mg/kg), with decreases in hippocampal regions CA1, CA3, and the dentate gyrus at 48 hours and 7 days after MDMA (Martínez-Turrillas et al. 2006
). In the current experiment, no decrease in BDNF mRNA in the CA3 region or dentate gyrus at 24 hours was noted (though there were decreases at the 1- and 7-hour time points), and we found that BDNF mRNA in the CA1 region increased at 24 hours following 4 doses of MDMA. No significant increases similar to those observed by Martínez-Turrillas et al. (2006)
in BDNF in the PFC at 24 hours were found.
The differences in BDNF expression between the two studies may be attributable to dosing regimens. Martínez-Turrillas et al. (2006)
administered a single dose of MDMA (10 mg/kg), whereas in the present study MDMA (10 mg/kg) was administered four times with a 2-hour interval between each dose. The current dosing regimen was chosen because it and very similar regimens are widely reported in the literature to induce long-term 5-HT reductions (Shankaran and Gudelsky, 1999
; Morley et al., 2001
; Shankaran et al., 2001
; Clemens et al., 2004
; Bauman et al., 2007
; Bhide et al., 2009
) and, therefore, models similar changes reported in human chronic MDMA users. Nonetheless, both experiments demonstrate that MDMA has significant regionally specific effects on neurotrophins.
Although we did not examine these rats specifically for neurotransmitter loss, we have previously found that this dosing regimen results in serotonin depletion in the hippocampus, striatum, and prefrontal cortex, as well as dopamine loss in the striatum (Able et al., 2006
; Skelton et al. 2008
). These neurotransmitter alterations may play a role in the changes of neurotrophin expression. Neurotrophins, particularly BDNF, play an important role in synaptic plasticity. For example, an increase in BDNF levels can cause dendritic growth and can result in enhanced synaptic efficacy (Thoenen, 1995
). Plasticity is important for cognition and plays a role in perception, learning, and memory (Black, 1999
). This implies that alterations in neurotrophins within regions important for cognition, learning, and memory, particularly the hippocampus and various cortical regions, may have behavioral consequences (Rattiner et al., 2005
We and others have shown that MDMA exposure produces lasting effects on learning and memory following exposure in adolescent or adult animals (Sprague et al., 2003
; Piper and Meyer, 2004
; Able et al., 2006
; Skelton et al., 2008
). Subregions of the hippocampus play roles in learning and memory processes, including spatial learning and novelty detection (Kesner et al., 2004
). The loss of neurotrophic factors, as observed in this study, may increase the susceptibility of hippocampal neurons to maladaptive plasticity, which over a period of time could contribute to learning and memory changes. The decrease in BDNF may be tied to the increase in corticosterone that was seen in MDMA-treated animals throughout the dosing period. Elevation of corticosterone has been linked to a decrease of BDNF expression in the hippocampus in several animal models (Smith et al., 1995a
; Schaaf et al., 1997
; Song et al., 2006
). It is unclear whether the increases in BDNF, trkB, and NT-3 mRNAs observed in the hippocampal subfields result in increased protein levels that are sufficient to protect these neurons from injury or whether the loss of BDNF expression in the earlier time points results in an environment permissive to the harmful effects of MDMA exposure and the associated increased corticosterone levels.
Why the hippocampus is vulnerable to decreases in BDNF mRNA expression compared with other regions after MDMA exposure is not fully understood. The time points examined were within 24 hours after MDMA exposure, so it is unlikely that the reduction in BDNF expression in the hippocampus is the result of neuronal loss. It seems more likely that increased corticosterone in combination with reduced 5-HT altered BDNF expression acutely. In this regard, activation of 5-HT receptors can result in activation of cyclic adenosine monophosphate response element binding (CREB) activation and stimulation of transcription of the BDNF gene (Mattson et al., 2004
). It is possible that the acute release of 5-HT after MDMA exposure activated 5-HT receptors and caused an upregulation of BDNF mRNA levels as noted in the CA1 region. How this might result in a decrease in BDNF in the CA3 region and dentate gyrus is unknown. Serotonin is depleted in the hippocampus after MDMA exposure (García-Osta et al., 2004
), and reductions may occur more quickly and/or to a greater extent in some subregions relative to others. Regardless of the mechanism, the regionally specific decreases in BDNF expression might explain why some cognitive skills are affected by MDMA, but not others.
Both the PFC and the AC Ctx are important areas in the regulation of stress, memory, and mood, so it is not surprising that MDMA affected both neurotrophins and their receptors in these regions. Serotonin content in forebrain is reduced by MDMA (Green et al., 2003
), and these reductions are long-lasting (Mayerhofer et al., 2001
) but not permanent. In human users, there is evidence of specific effects on executive functions after MDMA use, and these functions are associated with the PFC (Reay et al., 2006
). Both NT-3 and BDNF were increased in the PFC shortly after treatment, whereas their receptors were not upregulated until 24 hours later. We found no significant changes in BDNF mRNA in the AC Ctx after MDMA, although a loss of BDNF in this region after exposure to stress has been reported (Smith et al., 1995c
). Instead, we observed elevations of NT-3 mRNA at the early post-MDMA time points that were not maintained at 24 hours.
Following MDMA exposure, BDNF expression was elevated at 7 hours in the parietal cortex. Loss of 5-HT cell bodies has been described in this region following MDMA exposure (Schmued, 2003
). In addition, the parietal cortex sends projections to the entorhinal cortex, which has an important function in egocentric learning (Parron and Save, 2004
). We have previously reported egocentric deficits in the Cincinnati water maze after treatment with MDMA (Able et al., 2006
; Skelton et al., 2008
). However, these deficits are seen at longer intervals after MDMA treatment and cannot be directly attributed to the BDNF expression changes seen here. The entorhinal cortex also plays an important role in transmitting sensory information to the hippocampus, as well as receiving output from hippocampal subfields (Kerr et al., 2007
). BDNF mRNA levels were upregulated at all three time points, and trkC receptor mRNA was elevated at 1 hour in the entorhinal cortex. Both BDNF and NT-3 can be retrogradely transported by neurons in both the hippocampus and the entorhinal cortex (DiStefano et al., 1992
Levels of BDNF mRNA were increased at all three time points in the piriform cortex. This region is part of the olfactory system, and extends projections to the medial PFC. Olfactory perception is dependent on learning and memory, and the piriform cortex plays an important part in object recognition and discrimination of odors (Wilson and Stevenson, 2003
; Keller et al., 2005
). Expression of BDNF mRNA increases in the piriform cortex after exposure to stressors (Nibuya et al., 1995
; Katoh-Semba et al., 1999
), and it has been suggested that such increases serve to protect neurons (Nibuya et al., 1995
), but may not always be fully effective (Schmidt-Kastner et al., 1996
). Upregulation of BDNF mRNA is also observed in the piriform and entorhinal cortices during the formation of recognition memory (Broad et al., 2002
), and there are reports that MDMA interferes with novel object recognition learning (Morley et al., 2001
; Piper and Meyer, 2004
; but see Able et al., 2006
Neurotrophin expression levels were also altered in regions outside cortical and limbic regions. We observed MDMA-induced changes within the striatum, with trkB mRNA expression exhibiting a substantial increase at the 1- and 7-hour time points. This striatal trkB upregulation paralleled the similarly large increase in BDNF expression in the cerebral cortex (PFC, frontal and parietal cortices; see ) at the same early post-MDMA time points. Because BDNF produced in the cortex is anterogradely transported to the striatum (Altar et al., 1997
), such temporal correspondence may suggest MDMA-induced enhanced BDNF/trkB trophic communication in corticostriatal systems, at least early on following drug administration. In the SNpc, levels of BDNF mRNA were increased by 24 hours post-MDMA. BDNF and NT-3, as well as their receptors trkB and trkC, are synthesized by dopaminergic neurons in the SNpc (Seroogy et al., 1994
; Numan and Seroogy, 1999
). Nigral BDNF is also anterogradely transported to the striatum where it activates trkB receptors (Altar et al., 1994
). The delayed elevation of nigral BDNF mRNA (at the 24-hour time point) relative to the early striatal trkB mRNA increase (at the 1-and 7-hour time points) is less consistent with MDMA-induced augmented nigrostriatal neurotrophin signaling because the striatal trkB levels have already returned to control levels by 24 hours post-treatment. Finally, we observed no changes in TH mRNA within the SNpc at any post-MDMA time point. It should be noted that upregulation of trkB mRNA in the striatum has also been seen following administration of the catecholamine-specific neurotoxin 6-hydroxydopamine (Numan and Seroogy, 1997
), probably in response to the loss of dopaminergic afferent innervation. The present results raise the possibility that loss of 5-HT afferent innervation similarly induces a compensatory elevation of trkB expression in the striatum.
In the LC, a region that contains noradrenergic neurons, chronic stress causes an upregulation in NT-3 and TH mRNA expression (Smith et al., 1995b
), whereas BDNF mRNA is decreased (Hemmerle et al., 2007
). The LC was the only other area besides the hippocampus to demonstrate a decrease in mRNA expression after MDMA exposure, in this case in trkC mRNA at 7 hours. However, there were no changes in either NT-3 or BDNF mRNAs.
In conclusion, MDMA has selective regional effects on BDNF and NT-3 mRNAs. Now that we have established that there are changes in expression after MDMA treatment in regions associated with cognition, memory, and egocentric learning, future studies assessing longer intervals will be necessary to elucidate the relationship between the behavioral changes and neurotrophin expression.