The aim of the present study was to investigate the enzymatic activities of TPH (1 and 2) and to assess TPH2 expression and localization in different brain regions of the adult male Sprague-Dawley rat. TPH2 is the rate-limiting enzyme in serotonin synthesis and hence serves as a molecular marker for serotonergic neurons. In general, serotonergic neurons have been thought to originate in the raphe nucleus and to project broadly and diffusely throughout the brain. In the present study, we confirmed the broad dissemination of TPH2, but also identified additional TPH2 neuronal cell body localization to the VTA.
This work demonstrates that enzymatic activity of TPH2 in the VTA was significantly higher than any other brain region we examined in the male rat, except the raphe nucleus. Immunocytochemistry findings confirmed the localization of TPH2 cell bodies within the VTA region. These results were supported by real time PCR findings that showed TPH2 mRNA levels in the VTA at 12% of the raphe nucleus levels (). Indeed, with the exception of the raphe and VTA, very low levels of TPH2 mRNA expression were detected throughout the brain. This may represent previously documented evidence of mRNA in terminal fields (for the related gene, tyrosine hydroxylase) [19
]. It is important to note that, as these studies were confined to use of male subjects, the possibility of gender-based differences remains a topic for future studies.
Previous studies of Deguchi and co-workers investigated TPH activity in the rat brain and compared the raphe nucleus with the rest of the brain stem [6
]. They reported that the raphe nucleus contained seven-fold higher TPH activity compared to the rest of the brain stem, and four-fold higher activity than the hypothalamus; a finding in contrast to their previous publications reporting higher activity in the hypothalamus [4
]. Our findings show that VTA followed the raphe as the region with the second highest amount of TPH2 activity. Moreover, the immunoblot data on TPH2-specific protein levels support these activity findings (). Similarly, Koubi and coworkers [15
] reported that the raphe nucleus expresses the highest levels of TPH activity (presumably TPH2), with lower but comparable levels in the VTA, hippocampus, frontal cortex and substantia nigra; a finding consistent with our own. In the present study, there are significant levels of TPH activity in the SN (), and lower, but detectable, levels of TPH2 protein (, middle panel). Indeed, there is TPH2 mRNA in this region ().
The observations of TPH2-positive neurons in the VTA may provide an area of further research involving interactions between serotonergic and dopaminergic systems within the VTA. Specially, this contributes to the growing understanding of the mesolimbic/mesocortical reward pathway and its role in addictive behaviors. There is evidence that the serotonin system in the VTA plays an inhibitory role on the mesolimbic dopaminergic system [7
]. Roberts and colleagues [22
] showed that the reinforcing efficacy of cocaine can be improved by partial depletion of brain serotonin. Moreover, modulation of serotonin receptor activity will alter both cocaine and MDMA (ecstasy) behavioral effects [8
The presence of somata of neurons containing serotonin, within the VTA, could be an important biological factor for understanding the role of the VTA in depression [24
] and addiction [26
]. Koubi et al. [15
] specifically characterized the VTA in animal models for the treatment of depression. In the rat, electroconvulsive shock (used to treat severe depression in humans) decreased TPH levels in the VTA within 72 hours of the treatment, whereas it increased TPH levels in hippocampus and frontal cortex. In addition, modulation of the serotonergic system in the VTA is a target of cocaine’s actions, as the serotonin transporter plays a very important role in the VTA for reward processes [18
]. In this regard, we have previously reported a cocaine-mediated decrease in raphe TPH activity [29
]. Recent preliminary findings from our laboratory suggest that VTA TPH activity significantly decreases following abstinence from heroin self-administration, while the TPH activity immediately following a period of self-administration remains unchanged (data not shown), suggesting that TPH2 and serotonin may play a role in drug abuse relapse behavior. Therefore, activity of serotonergic cells resident within the VTA, combined with serotonin inputs to the VTA, may play a role in several neuropsychiatric disorders. Characterizing the complexities of TPH2 expression within this brain region will contribute to enhanced understanding of behavioral phenomena controlled by the activity of neurons within the VTA.