Overexpression or knockdown of rat tryptophan hyroxylase-2 has opposing effects on anxiety behavior in an estrogen-dependent manner

doi:10.1016/j.neuroscience.2010.12.019

Neuroscience

Volume 176, 10 March 2011, Pages 120-131

https://doi.org/10.1016/j.neuroscience.2010.12.019 Get rights and content

Abstract

Previous studies showed that chronic estrogen treatment increases tryptophan hydroxylase-2 (TpH2) mRNA in the caudal dorsal raphe nucleus (DRN), and this increase was associated with decreased anxiety. The present study explored the interaction of estrogen and targeted, bidirectional manipulation of TpH2 expression in the caudal DRN by knockdown or viral overexpression, to decrease or increase tryptophan hydroxylase expression respectively, on anxiety behavior. Rats were ovariectomized and replaced with empty or estradiol capsules (OVX, OVX/E, respectively). Animals received microinfusions of either antisense TpH2 or control morpholino oligonucleotides into caudal DRN and were later tested in the open field test. A separate group of animals were microinfused with TpH2-GFP or GFP-only herpes simplex viral vectors into caudal DRN and tested in the open field. The bidirectional impact of manipulations on TpH2 expression was confirmed using a combination of quantitative protein and mRNA measurements; TpH2 expression changes were limited to discrete subregions of DRN that were targeted by the manipulations. Estradiol decreased anxiety in all behavioral measures. In the OVX/E group, TpH2 knockdown significantly decreased time spent in the center of the open field, but not in the OVX group, suggesting that TpH2 knockdown reduced the anxiolytic effects of estrogen. Conversely, TpH2 overexpression in the OVX group mimicked the effects of estrogen, as measured by increased time spent in the center of the open field. These results suggest that estrogen and TpH2 in the caudal DRN have a critical interaction in regulating anxiety-like behavior.

Research highlights

▶We examine the role of TpH2 in caudal DRN in estrogen-induced anxiolysis. ▶Estrogen-induced increase in DRN TpH2 mRNA is associated with decreased anxiety. ▶TpH2 overexpression mimics, while knockdown reduces anxiolytic effects of estrogen.

Section snippets

Subjects

A total of 114 female Sprague–Dawley rats between the ages of 60 and 90 days were used for behavioral experiments and 55 male Sprague–Dawley rats around the age of 60–77 days were used for biochemical verification of morpholino antisense oligonucleotide knockdown and virus-mediated gene transfer. Rats were group-housed with three animals per cage on a 12-h light/dark cycle (lights on at 06:00 am), and all behavioral measures were performed during the light period. Animals were acclimated to the

TpH2 antisense PMO infusion decreased TpH protein levels in a discrete subregion of DRN without causing toxicity

PMOs were efficiently taken up by cells without transfection agent as indicated by intense cytoplasmic fluorescence (Fig. 1). There was no histological indication of cytotoxicity and no caspase-3 immunoreactivity was detectable in any of the groups studied (data not shown), suggesting that there was no overt toxicity, including apoptosis, associated with the PMO injections. Scrambled control PMO had no apparent effect on TpH protein levels, as demonstrated by colocalization of PMO label with

Discussion

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., 2006). 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

Conclusion

In summary, the present work highlights the role of the DRN serotonergic system in the regulation of anxiety by estrogen. Estrogen differentially activates TpH2 in select subregions of the DRN (Hiroi et al., 2006), which in turn supplies serotonin innervation to specific brain areas that regulate different aspects of anxiety behavior. The results from our study do not imply that the anxiolytic effect of estrogen is mediated solely through the caudal DRN 5-HT system, thus further studies

Acknowledgments

This work was supported by the National Institute of Mental Health (NIMH) grants MH63303 and MH75279. The authors would like to thank the following for their technical assistance and expert advice: Dr. Michele Kelly and William Brennan for vector packaging, Dr. Susan Ferguson with western blot, and Dr. Catherine Hagan with RT-PCR.

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Estrogen receptor β activation within dorsal raphe nucleus reverses anxiety-like behavior induced by food restriction in female rats
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Citation Excerpt :
Moreover, it has been reported that estrogen increases TPH2 mRNA, raising 5-HT synthesis. These changes, in specific DRN areas, mainly the caudal part, are closed related to the control of anxiety-like behaviors [15,47,48]. However, no differences were observed in the behavior associated to anxiety responses on the OF paradigm after E2 treatment.
Severe food restriction (FR), as observed in disorders like anorexia nervosa, has been associated to the reduction of estrogen levels, which in turn could lead to anxiety development. Estrogen receptors, mainly ERβ type, are commonly found in the dorsal raphe nucleus (DRN) neurons, an important nucleus related to anxiety modulation and the primary source of serotonin (5-HT) in the brain. Taking together, these findings suggest an involvement of estrogen in anxiety modulation during food restriction, possibly mediated by ERβ activation in serotonergic DRN neurons. Thus, the present study investigated the relationship between food restriction and anxiety-like behavior, and the involvement of DRN and ERβ on the modulation of anxiety-like behaviors in animals subjected to FR. For that, female Fischer rats were grouped in control group, with free access to food, or a FR group, which received 40% of control intake during 14 days. Animals were randomly treated with 17β-estradiol (E2), DPN (ERβ selective agonist), or their respective vehicles, PBS and DMSO. Behavioral tests were performed on Elevated T-Maze (ETM) and Open Field (OF). Our results suggest that FR probably reduced the estrogen levels, since the remained in the non-ovulatory cycle phases, and their uterine weight was lower when compared to control group. The FR rats showed increased inhibitory avoidance latency in theETM indicating that FR is associated with the development of an anxiety-like state. The injections of both E2 and DPN into DRN of FR animals had an anxiolytic effect. Those data suggest thatanxiety-like behavior induced by FR could be mediated by a reduction of ERβ activation in the DRN neurons, probably due to decreased estrogen levels.
Estrogen Receptors Modulation of Anxiety-Like Behavior
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One way in which ERβ may influence anxiety-like behavior through the serotonergic system is by mediating expression of tryptophan hydroxylase, the rate-limiting enzyme required for the synthesis of serotonin. Knockdown of this enzyme within the caudal dorsal raphe nucleus of OVX rats removes the anxiolytic-like effects of estradiol in the open field test (Hiroi, McDevitt, Morcos, Clark, & Neumaier, 2011). Correspondingly, both male and female ERβ-null mice have decreased expression of tryptophan hydroxylase.
Estrogens exert profound effects on the expression of anxiety in humans and rodents; however, the directionality of these effects varies considerably within both clinical and preclinical literature. It is believed that discrepancies regarding the nature of estrogens’ effects on anxiety are attributable to the differential effects of specific estrogen receptor (ER) subtypes. In this chapter we will discuss the relative impact on anxiety and anxiety-like behavior of each of the three main ERs: ERα, which has a generally anxiogenic effect, ERβ, which has a generally anxiolytic effect, and the G-protein-coupled ER known as GPR30, which has been found to both increase and decrease anxiety-like behavior. In addition, we will describe the known mechanisms by which these receptor subtypes exert their influence on emotional responses, focusing on the hypothalamic–pituitary–adrenal axis and the oxytocinergic and serotonergic systems. The impact of estrogens on the expression of anxiety is likely the result of their combined effects on all of these neurobiological systems.
Sex-dependent programming effects of prenatal glucocorticoid treatment on the developing serotonin system and stress-related behaviors in adulthood
2016, Neuroscience
Citation Excerpt :
Previous studies have examined the effects of prenatal stress on TpH2 mRNA in the DRN of mice (Mueller and Bale, 2008); however, no study to date has evaluated the sex-specific effects of prenatal GC overexposure on TpH2 in developing and adult rats. Further, as DRN TpH2 has been demonstrated to have region-specific effects on anxiety-like behaviors (Hiroi et al., 2006a, 2011), unraveling the possible link between prenatal GC overexposure and TpH2 in regulating these behaviors may have important clinical implications. Therefore, in the current study, we examined the effects of prenatal DEX treatment on the development of TpH2 mRNA in the DRN, focusing on the subregions, and on adult anxiety- and depressive- like behaviors.
Prenatal stress and overexposure to glucocorticoids (GC) during development may be associated with an increased susceptibility to a number of diseases in adulthood including neuropsychiatric disorders, such as depression and anxiety. In animal models, prenatal overexposure to GC results in hyper-responsiveness to stress in adulthood, and females appear to be more susceptible than males. Here, we tested the hypothesis that overexposure to GC during fetal development has sex-specific programming effects on the brain, resulting in altered behaviors in adulthood. We examined the effects of dexamethasone (DEX; a synthetic GC) during prenatal life on stress-related behaviors in adulthood and on the tryptophan hydroxylase-2 (TpH2) gene expression in the adult dorsal raphe nucleus (DRN). TpH2 is the rate-limiting enzyme for serotonin (5-HT) synthesis and has been implicated in the etiology of human affective disorders. Timed-pregnant rats were treated with DEX from gestational days 18–22. Male and female offspring were sacrificed on the day of birth (postnatal day 0; P0), P7, and in adulthood (P80–84) and brains were examined for changes in TpH2 mRNA expression. Adult animals were also tested for anxiety- and depressive- like behaviors. In adulthood, prenatal DEX increased anxiety- and depressive- like behaviors selectively in females, as measured by decreased time spent in the center of the open field and increased time spent immobile in the forced swim test, respectively. Prenatal DEX increased TpH2 mRNA selectively in the female caudal DRN at P7, whereas it decreased TpH2 mRNA selectively in the female caudal DRN in adulthood. In animals challenged with restraint stress in adulthood, TpH2 mRNA was significantly lower in rostral DRN of prenatal DEX-treated females compared to vehicle-treated females. These data demonstrated that prenatal overexposure to GC alters the development of TpH2 gene expression and these alterations correlated with lasting behavioral changes found in adult female offspring.
Differential effect of the 17β-aminoestrogens prolame, butolame and pentolame in anxiety and depression models in rats
2016, Progress in Neuro-Psychopharmacology and Biological Psychiatry
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Their effects on anxiety are more controversial even in animal models. Conflicting results seem be linked to the type of estrogen evaluated, but more importantly to the regimen (Hiroi et al., 2011; Kalandakanond-Thongsong et al., 2012). Considering the advantages of ERTs, there continues to be interest in finding new estrogenic molecules with an adequate balance between benefits and risks.
Estrogens of clinical use produce consistent antidepressant- and anxiolytic-like effects in animal models of menopause. Regulation of the hypothalamic–pituitary–adrenal (HPA) or stress axis, has been proposed as a pathway through which estrogens improve affective-like behaviors. Anticoagulant 17β-aminoestrogens (17β-AEs) butolame and pentolame mimic some effects of estradiol (E₂), i.e., on female rodent sexual behavior, with opposite actions on coagulation. However, their psychoactive actions have not been explored. On the basis of similitude with E₂'s effects, we hypothesized that these 17β-AEs would induce anxiolytic- and antidepressant-like effects, which would be reflected in a reduction of activity in the HPA axis. In ovariectomized female rats, chronic treatment with prolame (60 μg/kg), butolame (65 μg/kg) and pentolame (70 μg/kg) reduced anxiety-like behavior in the elevated plus maze (evidenced by an increase in time in open arms, E₂ (40 μg/kg) + 176%; prolame + 201%; butolame, + 237%; and pentolame + 295%, in comparison to the control vehicle group 100%). Pentolame also decreased significantly anxiety-like behavior in the burying behavior test. Prolame and E₂ produced a significantly antidepressant-like action, which was not induced by butolame and pentolame. Behavioral effects of 17β-AEs (and E₂) on anxiety and depression did not follow the same pattern than corticosterone or E₂ levels; they also were associated to changes in locomotor activity, evaluated by the open field test. These results constitute the first evidence of specific and selective actions of butolame and pentolame as anxiolytics for females with a hypoestrogenic condition. Results also confirm the potential of prolame as an antidepressant steroid with equivalent actions to E₂. Psychoactive properties of 17β-AEs in combinations with reduced adverse effects on coagulation, suggest that 17β-AEs may be a good alternative replacement therapy for women with symptoms associated with menopause.
Tryptophan hydroxylase-2: An emerging therapeutic target for stress disorders
2013, Biochemical Pharmacology
Serotonin (5-HT) has been long recognized to modulate the stress response, and dysfunction of 5-HT has been implicated in numerous stress disorders. Accordingly, the 5-HT system has been targeted for the treatment of stress disorders. Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in 5-HT synthesis, and the recent identification of a second, neuron-specific TPH isoform (TPH2) opened up a new area of research. With a decade of extensive investigation, it is now recognized that: (1) TPH2 exhibits a highly flexible gene expression that is modulated by an increasing number of internal and external environmental factors including the biological clock, stressors, endogenous hormones, and antidepressant therapies; and (2) genetically determined TPH2 activity is linked to a growing body of stress-related neuronal correlates and behavioral traits. These findings reveal an active role of TPH2 in the stress response and provide new insights into the long recognized but not yet fully understood 5-HT–stress interaction. As a major modulator of 5-HT neurotransmission and the stress response, TPH2 is of both pathophysiological and pharmacological significance, and is emerging as a new therapeutic target for the treatment of stress disorders. Given that numerous antidepressant therapies influence TPH2 gene expression, TPH2 is already inadvertently targeted for the treatment of stress disorders. With increased understanding of the regulation of TPH2 activity we can now purposely utilize TPH2 as a target to develop new or optimize current therapies, which are expected to greatly improve the prevention and treatment of a wide variety of stress disorders.
Use of vivo-morpholinos for control of protein expression in the adult rat brain
2012, Journal of Neuroscience Methods
Citation Excerpt :
While vivo-morpholinos are superior to non-conjugated morpholinos for achieving suppression following i.v. administration, brain tissue is not significantly accessed following systemic delivery (Li and Morcos, 2008; Morcos et al., 2008; Parra et al., 2011). A few examples of successful knockdown with non-conjugated morpholinos administered directly in the brain have been reported (Hiroi et al., 2011; Oh et al., 2006). However, evidence is not available on the efficacy or toxicity of vivo-morpholinos administered intracranially.
Vivo-morpholinos are commercially available morpholino oligomers with a terminal octa-guanidinium dendrimer for enhanced cell-permeability. Existing evidence from systemically delivered vivo-morpholinos indicate that genetic suppression can last from days to weeks without evidence of cellular toxicity. However, intravenously delivered vivo-morpholinos are ineffective at protein suppression in the brain, and no evidence is available regarding whether intracranially delivered vivo-morpholinos effectively reduce target protein levels, or do so without inducing neurotoxicity. Here we report examples in which in vivo microinjection of antisense vivo-morpholinos directed against three different targets (xCT, GLT1, orexin) in two different brain regions resulted in significant suppression of protein expression without neurotoxicity. Expression was significantly suppressed at six to seven days post-administration, but returned to baseline levels within fourteen days. These results indicate that direct intracranial administration of vivo-morpholinos provides an effective means by which to suppress protein expression in the brain for one to two weeks.

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¹: Present address: Department of Basic Medical Sciences, University of Arizona College of Medicine in Phoenix, Phoenix, AZ 85004, USA.

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Cellular and Molecular NeuroscienceResearch PaperOverexpression or knockdown of rat tryptophan hyroxylase-2 has opposing effects on anxiety behavior in an estrogen-dependent manner

Abstract

Research highlights

Section snippets

Subjects

TpH2 antisense PMO infusion decreased TpH protein levels in a discrete subregion of DRN without causing toxicity

Discussion

Conclusion

Acknowledgments

Neuropharmacology

Psychoneuroendocrinology

Front Neuroendocrinol

Front Neuroendocrinol

Neuroscience

Pharmacol Biochem Behav

Physiol Behav

Neuroscience

Neuropharmacology

Lancet

Brain Res Mol Brain Res

Brain Res Mol Brain Res

Dev Biol

Biol Psychiatry

Behav Brain Res

Neuroscience

Physiol Behav

Pharmacol Biochem Behav

Brain Res

Brain Res

Physiol Behav

Psychiatr Clin North Am

Behav Brain Res

Behav Brain Res

Biol Psychiatry

Brain Res Mol Brain Res

Biol Psychiatry

Biochim Biophys Acta

Brain Res Mol Brain Res

Neuroscience

Neuroscience

Biochem Pharmacol

Cellular and Molecular Neuroscience
Research Paper
Overexpression or knockdown of rat tryptophan hyroxylase-2 has opposing effects on anxiety behavior in an estrogen-dependent manner