We previously reported that estrogen increased TpH2 expression selectively in the caudal DRN and that TpH2 expression in caudal DRN was inversely associated with anxiety (Hiroi et al., 2006b
). In the present study, we investigated the hypothesis that increased TpH2 in caudal DRN is involved in modulating estrogen’s anxiolytic effects. To this end, we explored the roles of in-vivo
TpH2 knockdown and overexpression selectively in the caudal DRN on the anxiolytic effects of estrogen. Our results suggest that estrogen-induced increases in TpH2 mRNA in the caudal DRN are important to its anxiolytic actions, as these manipulations modulated estrogen’s anxiolytic effects.
We used PMOs, steric-block antisense oligonucleotides to selectively knockdown TpH2 mRNA in caudal DRN; this antisense oligonucleotide strategy for in-vivo
knockdown did not require a transfection reagent in this application (Fujikawa et al., 2005
). It has several advantages over traditional antisense knockdown strategies, including nuclease resistance, high affinity binding, and a coupled fluorescent dye, lissamine, that may enhance cellular uptake by adding polarity to otherwise non-ionic PMOs and allows convenient microscopic localization in tissue (Heasman, 2002
). These factors reduce nonspecific toxicity encountered with transfection reagents or high concentrations of less stable antisense oligonucleotide reagents (Summerton, 1999
, Morcos, 2001
). Knockdown appeared to be very efficient, with over 60% reduction in tryptophan hydroxylase protein as measured by western blot from punches and virtually complete knockdown in neurons exhibiting lissamine fluorescence. The PMO approach is anatomically more precise than chemical inhibitors of tryptophan hydroxylase enzyme activity such as p-chlorophenylalanine because the PMOs are less likely to diffuse away and can be directly detected by fluorescence microscopy. We were able to limit TpH2 knockdown to a select DRN subregion, as shown by decreased tryptophan hydroxylase immunoreactivity restricted to the infused subregion and not in the neighboring areas. Anatomical precision is an advantage because different subregions of DRN provide serotonergic innervation to different forebrain regions that mediate distinct elements of emotional behavior, such as anxiety. Indeed, TpH2 expression is regulated in a region-specific manner by estrogen, and different subregions mediate different effects on emotional behavior (Lowry, 2002
, Hiroi et al., 2006a
, Clark et al., 2007
). Furthermore, this strategy does not lesion the targeted or surrounding non-serotonergic cells as is the case for traditional lesion strategies.
We found that reduction of TpH2 expression selectively in the caudal DRN reversed the anxiolytic effects of estrogen, as measured by decreased time spent in the center of the open field. Ovariectomy increased anxiety, and TpH2 knockdown in these animals did not increase anxiety further. Thus, these results support the hypothesis that induction of TpH2 in the caudal DRN is a key mechanism of how estrogen reduces anxiety. Alternatively, it is also possible that TpH2 knockdown increased the baseline anxiety level, although there was only a slight, non-significant decrease in the time spent in center of the open field with TpH2 knockdown in the OVX group. In any case, this study does not rule out anxiolytic effects of estrogen that are mediated by other brain regions.
In fact, estrogen’s anxiolytic effects are likely mediated by other regions as well, as the two major types of estrogen receptors (ERα and ERβ) have broad distribution throughout the brain. In DRN, ERα is expressed in nonserotonergic neurons, mainly in rostral DRN in rats (Alves et al., 1998
) while ERα is not found in macaque DRN (Bethea et al., 2002
, VanderHorst et al., 2004
, Vanderhorst et al., 2009
); whereas ERβ is expressed in serotonergic neurons of rodents (Lu et al., 1999
, Lu et al., 2001
, Mitra et al., 2003
) and macaque DRN (Gundlah et al., 2000
, Gundlah et al., 2001
, Bethea et al., 2002
), and is clearly expressed in mid and caudal rat DRN (Shughrue et al., 1997
, Lu et al., 2001
). This unique localization of the estrogen receptors may provide a direct mechanism for estrogen-induced increase of TpH2 mRNA in caudal DRN, as ERβ is found on serotonergic neurons in the DRN.
A recent report showing that the ERβ agonist, DPN, is anxiolytic and increases TpH2 mRNA in the caudal DRN (Donner and Handa, 2009
) corroborates the idea that estrogen-induced induction of TpH2 may be mediated via ERβ. However, the authors of this study also showed that while a systemic treatment with DPN decreased anxiety-like behaviors, direct application of DPN in the DRN did not alter anxiety-like behaviors in either the open field test or elevated plus maze, albeit the increased expression of TpH2 in the caudal DRN. There are procedural differences between this previous study and our present report that may shed light into understanding these seemingly disparate results. First, the duration of the hormone treatment at the time of behavioral testing in the previous study was notably shorter than the present study (5 days vs 14 days, respectively). As dose and duration of hormone treatment has been shown to be a critical factor in determining the direction of change in serotonin gene expression (Sumner et al., 1999
, Smith et al., 2004
), as well as anxiety and other behaviors (Diaz-Veliz et al., 1991
, Diaz-Veliz et al., 1999
, Estrada-Camarena et al., 2003
, Wide et al., 2004
), longer hormone treatment may be necessary to effectively induce anxiolytic effects. Moreover, the pellet implantation of the hormones in the previous study was reported to spread over all of rostrocaudal and medial-lateral subregions of the DRN, whereas our present study selectively targeted caudal DRN for TpH2 manipulation. As subdivisions of the DRN may have opposing (Hiroi et al., 2006b
) and undoubtedly other complex effects on anxiety, diffusion of estrogens into all DRN subdivisions most likely have other unknown effects on the DRN as a whole that counteract the anxiolytic effects of elevated TpH2 mRNA specifically in caudal DRN. Nevertheless, these results gives credence to the argument that although serotonin activity in the caudal DRN plays a critical role in modulating estrogen’s anxiolytic effects, estrogen also has complex interactions with multiple brain systems to regulate specific aspects of anxiety behavior. Although the present study focused on the bidirectional effects of TpH2 manipulation as a strategy rather than multiple behavioral measures to investigate the role of caudal DRN in modulating the anxiolytic effects of estrogen, careful examination of the effects of estrogen in multiple brain regions and its impact on anxiety behavior is warranted.
Conversely, we found that TpH2 overexpression in the caudal DRN in OVX animals was anxiolytic, similar to estrogen’s anxiolytic effects. This result supports the hypothesis that estrogen-induced increase of TpH2 mRNA in the caudal DRN is important for its anxiolytic effects. However, increasing TpH2 mRNA expression in the caudal DRN in OVX/E group had the opposite effect; in this case TpH2 overexpression may be mildly anxiogenic when estrogen is present. There are at least two possible interpretations of these results. First, there may be a bimodal effect of TpH2 expression in caudal DRN with a U-shaped relationship between TpH2 and anxiety (). In other words, increasing TpH2 activity from low to moderate levels is associated with reduced anxiety; but higher levels of TpH2 activity could cause increased anxiety. A bimodal relationship may partly explain the seemingly ambiguous literature surrounding 5-HT and anxiety. At first glance, it may seem inconsistent to find that increased 5-HT tone in the caudal DRN can be anxiolytic, since in male rats, increased 5-HT activity in the caudal DRN is usually thought to be anxiogenic (Lowry et al., 2000
, Hammack et al., 2002
). However, males appear to have higher serotonin synthetic capacity than females (Nishizawa et al., 1997
). Perhaps, OVX/E rats with TpH2 overexpression (who presumably have higher TpH2 levels than OVX rats with TpH2 overexpression) have similar 5-HT levels seen in the male rats, thereby resulting in anxiogenic behavior.
Figure 7 Theoretical serotonin-anxiety interaction. Our data suggest a U-shaped relationship between serotonin and anxiety. Since TpH2 knockdown was anxiogenic in OVX/E rats (red arrow) while TpH2 overexpression was anxiolytic in OVX rats (green arrow), it suggests (more ...)
Second, these bimodal effects indicate that there may be a differential effect of the level of synthetic capacity of serotonin in low versus high estrogenic states. Although the mechanism underlying TpH2 overexpression is distinct from selective serotonin reuptake inhibitors (SSRIs), a class of antidepressant commonly used to treat anxiety, interesting comparisons are possible. SSRIs successfully treat some symptoms of depression associated with menopause, yet there is evidence that an incomplete response can be augmented by estrogen treatment (Zanardi et al., 2007
). It is possible that anxiolytic effects of SSRIs are mediated via increased serotonin activity, and that estrogen may facilitate the antianxiety effects of SSRIs by increasing TpH2 expression and restoring serotonin synthetic capacity.
TpH2 overexpression in a low estrogenic state may restore higher levels of serotonin synthesis in caudal DRN neurons and their projections to forebrain; however, further increases in TpH2 in the estrogen treated group, who presumably already have higher TpH2 expression in caudal DRN (Hiroi et al., 2006b
) has no further benefit for anxiety, or could even be anxiogenic. This excess serotonin activity may lead to a host of other symptoms similar to that seen in “serotonin syndrome” in overdoses of drugs that increases serotonin activity. It would be interesting to examine other physiological characteristics associated with serotonin syndrome, such as increased heart rate, overresponsive reflexes, pupillary dilation and myoclonus in these animals for comparison in future studies. It is also possible that there is a ceiling effect of TpH2 expression, and that estrogen may also have other effects on serotonin neurons, such as modulating enzyme activity or autoreceptor expression (Rubinow et al., 1998
, Birzniece et al., 2001
, Robichaud and Debonnel, 2005
, Hiroi and Neumaier, 2009
), as well as on other nonserotonergic systems, as estrogen receptors have a diverse distribution in the brain.