A Trophic Role for Serotonin in the Development of a Simple Feeding Circuit

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Original Paper

Dev Neurosci 2010;32:217–237 DOI: 10.1159/000304888

A Trophic Role for Serotonin in the Development of a Simple Feeding Circuit Wendi S. Neckameyer

Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, St. Louis, Mo. , USA

posed to 5-HT to augment 5-HT levels during CNS develop-ment display, as mature larvae, a significant decrease in gut fiber branching and total varicosity number, as well as in-creased feeding and a hyposensitivity to the effects of 5-HT. Exposure of embryos unable to synthesize neuronal sero-tonin to 5-HT during late embryogenesis results in rescue of the feeding behavior and abnormalities in the 5-HT gut fiber architecture. These results demonstrate an inverse relation-ship between developmental 5-HT levels and complexity of the fiber architecture projecting to gut tissue, which results in a perturbed feeding pattern. We conclude that 5-HT is tightly regulated during CNS development to direct the nor-mal architecture and mature function of this neural circuit.

Copyright © 2010 S. Karger AG, Basel

Introduction

Neuroactive substances function in the establishment of neural networks before adopting their roles as trans-mitters in the mature central nervous system (CNS) [for review, see Herlenius and Lagercrantz, 2001]. Seminal earlier studies by Lauder and Kater and their colleagues demonstrated that altered levels of specific transmitters during embryogenesis affected the synaptic development of mature neurons [Lauder and Krebs, 1978; Lauder et al.,

Key Words Branching ! Drosophila ! RNAi transgenic lines ! Tryptophan hydroxylase ! Varicosities

Abstract Correct differentiation and positioning of individual synaps-es during development is fundamental to the normal func-tion of neuronal circuits. While classical transmitters such as serotonin (5-HT) play a critical trophic role in neurogenesis in addition to their functions as transmitters in the mature nervous system, this process is not well understood. We used a simple model to assess both development and function of a specific behavioral circuit in the larval stage of the fruit fly (Drosophila melanogaster) . We show that, as in all other spe-cies examined, the neurotransmitter actions of 5-HT depress feeding, and decreased neuronal 5-HT levels increase appe-tite. However, using transgenic tools, we show that constitu-tive knockdown of neuronal 5-HT synthesis to reduce 5-HT levels during central nervous system (CNS) development re-sults in increased branching of the serotonergic fibers pro-jecting to the gut, as well as increased size and number of varicosities along the neurite length. As larvae, these animals display decreased feeding rates relative to controls, and, when given exogenous 5-hydroxytryptophan, feeding is significantly enhanced. Late-stage wild-type embryos ex-

Received: October 14, 2009 Accepted after revision: March 17, 2010 Published online: August 11, 2010

Wendi S. Neckameyer Department of Pharmacological and Physiological Science Saint Louis University School of Medicine 1402 South Grand Boulevard, St. Louis, MO 63104 (USA) Tel. +1 314 977 6346, Fax +1 314 977 6411, E-Mail neckamws @ slu.edu

© 2010 S. Karger AG, Basel0378–5866/10/0323–0217$26.00/0

Accessible online at:www.karger.com/dne

http://dx.doi.org/10.1159%2F000304888

Hieu Nguyen

Neckameyer

Dev Neurosci 2010;32:217–237218

1982; Haydon et al., 1984, 1987; Goldberg and Kater, 1989]. However, behavioral consequences of the develop-mental impairments could not be addressed in these studies. Since perturbations in neurotrophic signaling pathways are believed to underlie the etiology of several depressive disorders [reviewed in Hasler et al., 2004;Sodhi and Sanders-Bush, 2004], as well as autism spec-trum disorders [Pardo and Eberhart, 2007] and the dys-functional brain development observed in Down syn-drome [Whittle et al., 2007], understanding the behav-ioral consequences of impairments in the developmentof neural circuitry is imperative to delineating etiology for these pathologies.

The indolamine serotonin (5-HT) has emerged as one such trophic factor critical for development of synaptic connectivity [see Gaspar et al., 2003], also serving as a neurotransmitter and a neuromodulator in all animal phyla studied [reviewed in Buznikov et al., 2001]. In mammals, 5-HT affects the developing cerebral and pre-frontal cortex [Luo et al., 2003; Janusonis et al., 2004; Beique et al., 2004], and administration of 5-MT, a meth-ylated derivative of 5-HT, affects sprouting of serotoner-gic fibers [Shemer et al., 1991]. Consistent use of selective serotonin reuptake inhibitors as therapeutic targets for depression stimulates hippocampal neurogenesis and dendritic branching [reviewed in Cowan, 2007]. Devel-opmental abnormalities in 5-HT pathways have been im-plicated in autism [Anderson et al., 1990; Chugani, 2002] and schizophrenia [Dean, 2003]. Mice lacking serotonin receptor 5-HT1A function during embryonic and post-natal stages display enhanced anxiety as adults [Gross et al., 2002]; activation of the 5HT1A receptor promotes neurite extension and survival of neuronal cells in cul-ture [Fricker et al., 2005], suggesting that the trophic ac-tions of 5-HT occur, at least in part, via signaling through this receptor. This trophic role for 5-HT appears con-served across evolution. 5-HT acts as a regulatory signal in the development of the antennal lobes during meta-morphosis in the moth [Oland et al., 1995]. Total neurite length is enhanced by 5-HT application to moth neuronal cell populations in culture; in vitro and in vivo, 5-HT in-creases cell excitability and input resistance in antennal neurons after depolarization [Mercer et al., 1995, 1996, 1999]. In Drosophila mutants deficient in dopa decarbox-ylase, the second enzyme in the biosynthetic pathway for both dopamine and 5-HT, peripheral 5-HT fibers inner-vating the midgut display altered branching [Budnik et al., 1989], consistent with later studies which suggest 5-HT is an autoregulator of serotonergic varicosity den-sity [Sykes and Condron, 2005]. Again, these studies,

while demonstrating a critical role for 5-HT in the devel-opment of neural circuitry, did not address the behav-ioral consequences of these changes.

The rate-limiting step in 5-HT synthesis is the hydrox-ylation of tryptophan to 5-hydroxytryptophan (5-HTP), which occurs via the action of tryptophan hydroxylase (Tph); Tph is thus fundamental to supporting serotoner-gic transmission. In Drosophila , as in mammals, there are two discrete Tph enzymes: Drosophila tryptophan-phe-nylalanine hydroxylase (DTPH) is largely nonneuronal, expressed in the periphery, and corresponds to mamma-lian Tph1; Drosophila tryptophan hydroxylase (DTRH)is exclusively neuronal and corresponds to mammalian Tph2 [Walther et al., 2003; Zhang et al., 2004; Coleman and Neckameyer, 2005; Neckameyer et al., 2007]. Bio-chemical regulation of these enzymes is strikingly simi-lar to that of mammals [Coleman and Neckameyer, 2004, 2005].

In the larval stage of Drosophila , there are 84 bilater-ally symmetric interneurons [Valles and White, 1988]; the recurrens nerve, which displays a high degree of 5-HT immunoreactivity, is part of the stomatogastric nervous system and innervates the pharyngeal muscles, the proventriculus (the larval foregut), and the midgut [Budnik et al., 1989]. These fibers originate in the brain and enter the proventriculus as small groups of 2–4 fas-cicles or bundles ‘dotted’ with variscosities along the lengths. They display relatively little branching until reaching the anterior midgut ( fig. 1 ). The cephalopha-ryngeal plates also display intense immunoreactivity against 5-HT, as does the frontal nerve, which directly connects the cephalopharyngeal plates to the brain ( fig. 1 ). The larval mouth hooks (forming the most ante-rior part of the cephalopharyngeal plates) shovel food into the gut at a relatively constant rate, since the animals eat continuously during this stage of development. 5-HT projections from the brain innervate the mouthparts and the digestive tract, strong evidence for a role for 5-HT in the modulation of feeding. This is consistent with the observed role of 5-HT in appetite modulation in numer-ous species, including mammals [Lucki, 1992] and in-sects [e.g. Dacks et al., 2003; Novak and Rowley, 1994; Novak et al., 1995]. While glutamate- and FMRFamide-containing fibers are also present within the proventric-ulus, they are distinct from those that are serotonergic [Budnik et al., 1989]. However, a role for 5-HT in modu-lating appetitive behavior in Drosophila has not yet been formally established.

We previously noted that Drosophila mutants lacking DTRH function, which contain very little neuronal sero-

Hieu Nguyen

Hieu Nguyen

Trophic Role for Serotonin in the Development of a Feeding Circuit

Dev Neurosci 2010;32:217–237 219

tonin (although there is no neuronal 5-HT synthesis, a limited amount is taken up from the periphery by the serotonin transporter), display decreased feeding as lar-vae [Neckameyer et al., 2007]. However, in all species ex-amined, acute reduction in 5-HT levels in adult animals increases feeding behavior, and increased 5-HT levels decrease feeding [e.g. Dacks et al., 2003]. Serotonergic cell bodies and some projections can be visualized in the CNS by 16–18 h after fertilized eggs have been laid (ap-proximately 6 h before hatching), before there is a func-tional nervous system. No peripheral fibers appear to be present until 2–4 h before hatching, and serotonergic fi-bers projecting to the gut are not observed until ! 2 h before hatching [Budnik et al., 1989]. We propose that the difference in feeding behavior in DTRH-null Dro-sophila reflects the critical need for 5-HT during neuro-nal development, which can be tested by comparing con-stitutive with temporal knockdown of neuronal DTRH. Our data show that reduction in DTRH levels after the CNS has developed results in increased feeding, with no change in circuit architecture, as expected; but when DTRH synthesis is disrupted during CNS development,

larval feeding is depressed, and serotonergic projections to the gut display increased branching and an increasein the number and size of varicosities along the neurite length. When mature, feeding 5-HT results in an in-creased, rather than decreased, feeding rate. Conversely, when 5-HT synthesis is induced after CNS development, larval feeding is depressed, as expected. Drosophila em-bryos exposed to 5-HT during the last 6 h of embryonic development display increased feeding as mature larvae, with a significant decrease in branching and overall var-icosity number relative to controls, and a hyposensitivity to exogenous 5-HT. When Drosophila embryos carrying a null mutation in DTRH are exposed to 5-HT during the last 6 h of embryogenesis, their feeding behavior is rescued and branching number, area, and size of vari-cosities are significantly decreased. These data demon-strate that neuronal 5-HT levels during development in-versely correlate with the complexity of gut fiber archi-tecture and impairment of the behavior modulated by that neuronal circuitry, and suggests that levels of 5-HT must be tightly regulated for normal development of the behavioral circuit.

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Fig. 1. Serotonergic fibers projecting to the larval gut. Gut tissue was dissected from a third instar larva and immunostained with an antibody raised against 5-HT. a Proventriculus and midgut. a = Proven-triculus (foregut); b = fascicles of 5-HT-immunore active fibers; c = branching of these fibers as they reach the midgut; d = midgut. b , d Feeding circuit. c Close-up of frontal nerve (e) and cephalopharyngeal plates (f). Scale bars = 100 ! m.

Neckameyer

Dev Neurosci 2010;32:217–237220

Methods

Fly Culture Flies were maintained in pint bottles containing standard

agar-cornmeal-yeast food at 25 ° C on a 12-hour light-dark cycle; wandering third instar larvae for immunohistochemical analyses were collected from these bottles. Staged larvae for behavioral studies were collected from a population cage maintained at 25 ° C on a 12-hour light-dark cycle. Females were allowed to lay eggs overnight on apple juice-agar plates, and newly hatched larvae were collected by maintaining plates with newly deposited eggs at 25 ° C for 24 h, and collecting first instars by migration of the an-imals onto yeast paste in the center of the agar plate. Second and early third instar larvae were obtained by allowing first instars to age for 24 or 48 h, respectively. For the 5-HTP studies, second in-star larvae were fed yeast paste or yeast paste plus 10 mg/ml 5-HTP (Sigma, St. Louis, Mo., USA) for 24 h before behavioral analyses (details for this procedure can be found in Neckameyer [1996]).

Fly Strains All strains were maintained as described above. w 1118 – parental strain for the insertional mutation for DTRH

and the RNAi transgenic lines. These flies have white eyes, and are isogenic for the second and third chromosomes. Obtained from the Bloomington stock center.

pBac[PB]CG9122 c01440 – insertional mutation in the DTRH gene generated by Exelixis, Inc., and obtained from the Bloom-ington stock center, first described in Thibault et al. [2004]. We refer to this stock as pBacTRH [Neckameyer et al., 2007].

pP[w +mW.hs = GawB]elav C155 – pan-neuronal Gal4 driver, ob-tained from the Bloomington stock center, which constitutively drives expression in neuronal tissue. This driver initiates expres-sion within neural tissues beginning after embryonic stage 9, peaking within the next few hours, and decreasing afterwards [Lin and Goodman, 1994].

pP[ELAV-GeneSwitch] – inducible pan-neuronal Gal4 driver obtained from Haig Keshishian. Referred to as GSelav. After ex-posure to the induction agent RU486 (mifeprestone), the driver is activated and expression of the upstream activator sequence (UAS) construct is observed within 3 h; peak expression occurs by 22 h, and levels of expression are maintained for the next 24 h [Osterwalder et al., 2001; data not shown]. Exposure of GSelav second instar larvae to RU486 thus results in expression of the transgene only in animals exposed to the ligand, so that unin-duced animals serve as controls.

CS wu – a wild-type Canton S strain established in the labora-tory of Martin Heisenberg in 1978.

Generation of Transgenics Complementary DNA encoding DTRH (CG9122, FBgn0035187)

was subcloned into both the SympUAS vector [Giordano et al., 2002] downstream of the yeast Gal4 UAS (to generate DTRH dsRNA) and a standard pUAS vector (stock No. 1000, Drosophila Genomics Resource Center). These constructs were injected into w 1118 embryos using standard transformation techniques [Robert-son et al., 1988]. The RNAi lines were generated as described in Neckameyer et al. [2007] for DTPH RNAi transgenic flies; the pUAS transgenics were generated using the services of Genetic Ser-vices, Inc. (Cambridge, Mass., USA). Two independent RNAi trans-genic lines were used (TRHE, on chromosome 2, and TRHA, on chromosome 3) to titrate DTRH expression levels, and thus, neu-ronal 5-HT synthesis. Similarly, two independent UAS lines for in-

duction of TRH levels were used (TRH32, on chromosome 2, and TRH27 on chromosome 3). All lines were crossed with either the pan-neuronal Gal4 promoter elav C155 or to the inducible pan-neu-ronal Gene Switch Gal4 transgenic promoter (GSelav) to drive con-stitutive or inducible neuronal expression, respectively, of these transgenes [elav is expressed in all central and peripheral neurons; Robinow and White, 1988; Yao and White, 1994]. The GSelav con-struct contains a human progesterone receptor-ligand-binding do-main, which binds to UAS in the presence of the antiprogestin RU486 to induce expression of UAS fused to the transgene of inter-est. Controlled feeding of RU486 permits discrete manipulation of UAS activation; induction begins approximately 3 h after exposure to RU486, and reaches a peak of expression at 22 h [Roman et al., 2001]. The DTRH sequences used are unique and will not affect expression of any other gene.

Analysis of DTRH Expression in the Transgenic Lines Induction and knockdown of DTRH expression and 5-HT lev-

els were assessed by quantitation of changes in fluorescent inten-sity of specific neurons after visualization with an antibody raised against DTRH [Coleman and Neckameyer, 2005] or against 5-HT (Spring Biosciences, Fremont, Calif., USA). The GSelav driver [Osterwalder et al., 2001] was used to induce temporal expression of the TRH RNAi transgene or the TRH UAS transgene using RU486 (Mifepristone, Sigma). Second instar larvae (carrying one copy each of the GSelav driver and the DTRH transgene) were submerged in either RU486 (6 mg/ml in 80% ethanol, induced) or 80% ethanol (uninduced control) for 2 min, and aged for 24 h (quantitation of neuronal DTRH levels) or for 40–44 h (quantita-tion of neuronal 5-HT levels). Peak expression of the GSelav driv-er occurs approximately 21 h after induction [Osterwalder et al., 2001], and we anticipated that changes in 5-HT levels would occur about 24 h later. The optimum time point for reduced 5-HT levels was estimated based on 24-hour pharmacological inhibition of tyrosine hydroxylase to reduce dopamine levels after exposure to a tyrosine hydroxylase inhibitor [Neckameyer, 1996]. The 5-HT cell pattern in each brain was visualized under fluorescence, and the single pair of neurons in the 8th abdominal neuromere [Valles and White, 1988; Chen and Condron, 2008] was photographed at the same magnification and exposure. These neurons were cho-sen since they were easily identified and quantified. The average density of pixel intensity was sampled from seven regions within each neuron, covering the entire cytoplasmic region (Northern Eclipse, Empix Imaging, North Tonawanda, N.Y., USA). Density was averaged for each singlet pair in larval brains from induced and uninduced tissues treated and assessed in parallel, and statis-tical analyses were performed using Student’s t test.

Levels of constitutive knockdown were assessed by dissecting larval brains from each genotype (elav C155 /w 1118 ; elav C155 /DTRHE, and elav C155 /DTRHA). Each fly thus carried either one copy of the elav Gal4 driver and one copy of the RNAi transgene, or the elav Gal4 driver alone, which served as a control. Quantitation of knockdown was accomplished as described above comparing DTRH immunoreactivity in the neurons in the 8th abdominal neuromere, and all genotypes were assessed in parallel. Statistical analyses were performed using one-way ANOVAs followed by Dunnett’s post-test. Constitutive induction of the DTRH UAS transgenes was assessed by observation of ectopic expression of DTRH in numerous 5-HT and non-5-HT cells within the larval brain (for DTRH levels) and by pixel intensity of the singlet neu-rons as described above (for 5-HT levels).


Dev Neurosci 2010;32:217–237 221

The observed phenotypes are not due to ectopic DTRH ex-pression in non-serotonergic neurons. DTRH is expressed solely within 5-HT neurons; the only homologous genes are DTPH and Drosophila tyrosine hydroxylase, both expressed in DA neurons. However, there is considerable third base wobble, greatly limiting homology at the DNA level, so it is not likely that the dsRNAi DTRH transgene will affect the expression of these genes [Cole-man and Neckameyer, 2005]. Although both the GSelav and elav C155 promoters drive ectopic DTRH expression, by immuno-histochemical analyses, 5-HT expression is limited to 5-HT neu-rons (data not shown). This is likely a consequence of substrate and cofactor requirements.

Immunohistochemistry of Proventricular Tissue The proventriculus and midgut from wandering third instar

larva were dissected in phosphate-buffered saline (PBS), fixed for 1 h (4% EM grade formaldehyde in 1 ! PBS) and washed thor-oughly in PBT (1 ! PBS, 0.1% protease-free bovine serum albu-min, 0.1% Triton X-100). Tissues were incubated in primary anti-serotonin antibody and then washed thoroughly in PBT, followed by incubation in secondary antibody (Alexa Fluor 568 goat anti-mouse or anti-rabbit IgG; 1: 400 dilution; Invitrogen – Molecular Probes, Carlsbad, Calif., USA). After several washes in PBT, the tissues were incubated in 4 m M sodium carbonate, mounted in 4% n-propyl gallate in 20 m M sodium carbonate, and viewed under fluorescence. To enhance 5-HT immunoreactivity, dissected gut tissues were preincubated in 10 –6 M 5-HT for 1 h at room tem-perature before extensive washing and incubation with the pri-mary antibody. Previous studies [Budnik et al., 1989; Sykes and Condron, 2005] have demonstrated that this concentration of ex-ogenous 5-HT does not affect neuronal architecture or varicosity density in immunohistochemical analyses.

Analysis of Neuronal Circuitry Serotonergic fibers projecting to the proventriculus from each

genotype were assessed in at least six independent immunohisto-chemical experiments and were photographed at the same resolu-tion. Quantification of varicosities and branching of fibers were analyzed using Neuroleucida, version 5 and Neuroexplorer (MBF Bioscience, Chicago, Ill., USA). Individual projections were traced within the body of the proventriculus, and varicosity number, branches, and number of large varicosities per 100- ! m length were quantified. Area ( ! m 2 ) per large varicosity was also deter-mined, as was the number of varicosities 5 ! m 2 or greater in di-ameter along the neurite length. A varicosity was defined as a brighter, discrete unit sufficiently enlarged beyond the size of the fiber, and large varicosities were defined as those larger than1 ! m 2 (i.e. larger than the width of the neurite fiber).

Behavioral Paradigms Retraction of the cephalopharyngeal sclerites is a standard

way to assess larval feeding rate, having been used for decades in population evolution studies as a correlate of rapid development [e.g. Sewall et al., 1975; Joshi and Mueller, 1988].

Feeding A single second instar larvae was placed in the center of a 2%

agar-filled Petri dish overlaid with a 2% yeast solution, and the number of mouth hook contractions was counted for 1 min after a 30-second acclimation period [Neckameyer, 1996].

Locomotion Each animal was placed on a 2% agar substrate and allowed to

acclimate for 30 s. The larva was then observed as it crawled over the substrate for a period of 1 min, and each posterior to anterior contractile wave was counted [Neckameyer, 1996]. In general, the same animal was first assessed for locomotor behavior, and then for feeding.

Statistics Statistical analyses were accomplished by one-way ANOVA

using Tukey’s or Dunnett’s post-hoc tests (for constitutive knock-down with elav C155 Gal4) or by Student’s t test (for temporal knockdown with GSelav or for embryonic studies). There were 40 animals from 4–6 independent experiments.

Embryonic Exposure to 5-HT Staged embryos were aged until 16 h after egg laying, dechori-

onated in 50% chlorox (Walmart), washed in embryo wash (0.7% NaCl, 0.05% Triton X-100), rinsed with H 2 O, air dried and then devitellinized by exposure to octane (Sigma Aldrich, electronic grade) for 5 min. Embryos were incubated in 10 –6 M 5-HT in se-rum-free medium (Sf-900 II SFM 1 ! , Gibco) for 4–6 h until hatching, and then placed in yeast paste on an agar plate and kept at room temperature. Animals were analyzed for feeding behavior as second instar larvae; analysis of architecture of the serotonergic fibers projecting to the gut was accomplished using animals aged to third instar.

Results

Neuronal 5-HT Acts as a Transmitter in the Brain to Modulate Feeding in Drosophila While the stomatogastric system is heavily innervated

by 5-HT ( fig. 1 ), and 5-HT is known to modulate feeding in several other species, including insects, formal demon-stration that 5-HT modulates feeding in Drosophila has not been established. We have previously reported that DTRH is solely responsible for neuronal 5-HT synthesis; the peripheral Tph, DTPH, which also encodes a phenyl-alanine hydroxylase activity, is expressed only in dopa-minergic, and not serotonergic, neurons [Neckameyer et al., 2007]. We therefore generated transgenic RNAi lines to knockdown DTRH levels, as well as transgenic UAS lines to induce DTRH expression. We used a GeneSwitch Gal4 pan-neuronal transgenic line to induce expression of both transgenes in second instar larvae; the GeneSwitch construct contains a human progesterone receptor-li-gand-binding domain that binds Gal4 to UAS sequences in the presence of the antiprogestin RU486. Controlled feeding of RU486 permits discrete manipulation of UAS activation [Roman et al., 2001]; since Drosophila do not contain a receptor that binds RU486, there is no expres-sion in its absence. Induction of expression occurs within 3 h and peaks at 22 h, with lowered expression over at least

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Dev Neurosci 2010;32:217–237222

Tukey's multiple comparison testGSelav32 uninduced control vs. GSelav32 uninduced 5-HTPGSelav32 uninduced control vs. GSelav32 RU486 controlGSelav32 uninduced control vs. GSelav32 RU486 5-HTPGSelav32 uninduced 5-HTP vs. GSelav32 RU486 controlGSelav32 uninduced 5-HTP vs. GSelav32 RU486 5-HTPGSelav32 RU486 control vs. GSelav32 RU486 5-HTP

p valuep < 0.001p < 0.001p < 0.001p > 0.05p < 0.001p < 0.001

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GSelav RNAi uninduced controlGSelav RNAi uninduced 5-HTPGSelav RNAi RU486 controlGSelav RNAi RU486 5-HTP

Fig. 2. Neuronal 5-HT acts as a neurotransmitter to modulate feeding behavior in Drosophila larvae. Larval brains were dissect-ed from control (uninduced) or induced (6 mg/ml RU486 for2 min) second instar larvae 40 h after induction using the GeneSwitch elav inducible pan-neuronal driver to express DTRH transgenic constructs in the CNS. The 5-HT cell pattern in each brain was visualized with a monoclonal antibody raised against 5-HT and viewed under fluorescence. The two unpaired neurons in the 8th abdominal neuromere (boxed, a ) were photographed at the same magnification and exposure, and the pixel intensity of the neuronal fluorescence was determined for both knockdown of 5-HT synthesis using a TRH RNAi transgenic construct ( b ) and induction of 5-HT synthesis using a TRH UAS transgenic con-struct ( c ). Uninduced and induced animals were assessed in paral-lel. GS elav TRH RNAi uninduced, n = 37 neurons; GS elav TRH RNAi RU486, n = 46 neurons; GS elav TRH UAS uninduced, n = 43 neurons; GS elav TRH UAS RU486, n = 36 neurons. * p ! 0.05,

* * p ! 0.01, Student’s t tests. d Treatment with 10 mg/ml 5-HTP, the product of the DTRH reaction, decreases feeding, and de-creasing 5-HT levels by reducing DTRH expression increases feeding rate. Animals with reduced neuronal DTRH synthesis when given exogenous 5-HTP display normal feeding. * p ! 0.05, * * p ! 0.01, one-way ANOVA followed by Dunnett’s multiple com-parisons post-test. e Locomotion is unaffected, demonstrating that pharyngeal contractions in the animals are normal. f Exog-enous 5-HTP and induction of neuronal 5-HT synthesis results in decreased feeding. The effect is additive if induced animals are also given 5-HTP. * * * p ! 0.001, one-way ANOVA followed by Tukey’s multiple comparisons post-test. g Locomotion is unaf-fected, demonstrating that pharyngeal contractions in the ani-mals are normal. n = 40 for behavioral analyses, from 4–6 inde-pendent experiments. Lines above the graph depict standard error of the mean.


Dev Neurosci 2010;32:217–237 223

the next 24 h [Osterwalder et al., 2001; data not shown]. Exposure of GSelav second instar larvae to RU486 thus results in expression of the transgene only in animals ex-posed to the ligand, so that the uninduced animals serve as age- and environment-matched controls.

Although elav directs expression within all neural tis-sue, DTRH expression is limited to 5-HT neurons. When analyzing knockdown using a DTRH RNAi, the homol-ogy between DTRH and its sister enzymes ( Drosophila tyrosine hydroxylase and DTPH) at the DNA level is suf-ficiently limited that the DTRH RNAi and UAS con-structs will not affect expression of these genes [Necka-meyer and White, 1992; Neckameyer et al., 2007]. Thus, no other cells were directly affected, permitting con-trolled and specific manipulation of neuronal 5-HT syn-thesis in staged larvae. When analyzing induction of DTRH using a UAS transgene, while DTRH protein was ectopically observed in the CNS, the 5-HT pattern was unchanged except for intensity, implying that all the nec-essary factors for 5-HT synthesis (substrate, cofactor) were only available in 5-HT neurons.

Changes in DTRH expression were assessed by quan-titation of changes in fluorescent intensity in specific 5-HT neurons after visualization with the DTRH anti-body (data not shown) as well as with an antibody raised against 5-HT ( fig. 2 ). DTRH protein is undetectable in brain by Western immunoblot analysis, since it is ex-pressed in a limited subset of neurons within the CNS [Neckameyer et al., 2007], and a decrease in neuronal Tph activity levels would not be observed with these trans-genes, since DTPH Tph activity would not be affected and would obscure results from enzymatic assays of brain tis-sues. The intense 5-HT expression within the ring gland, which is physically associated with the brain lobes, could obscure HPLC or ELISA analyses of 5-HT levels in dis-sected larval brains. We thus chose to quantitate DTRH and 5-HT levels within easily identifiable 5-HT larval neurons. The two unpaired, bilaterally symmetric neu-rons in the 8th abdomere ( fig. 2 a) were assessed in third instar larval brains from uninduced and induced GS elav /DTRH RNAi ( fig. 2 b) and Gselav/DTRH UAS ( fig. 2 c) animals (each carrying a single copy of the inducibleGS elav Gal4 driver and a single copy of the TRH trans-gene) treated in parallel (quantitation occurred 24 h after induction for DRTH and 44 h after induction for 5-HT levels). Fluorescence from these neurons was not affected by the fluorescence from the dense serotonergic neuropil found within the brain lobes and ventral ganglia. The other neurons within the ventral ganglion are bilaterally symmetrical doublets, and often partially overlap, which

affects the fluorescence intensity. The relative fluores-cence of each neuron, photographed at the same exposure and magnification, was determined by quantitating the relative intensity of a circular sampling region placed at seven different locations and covering the cytoplasm of each cell. This number was averaged to give a score for each neuron. Both DTRH (uninduced, n = 14 neurons; induced, n = 22 neurons; p = 0.0002, Student’s t test, data not shown) and 5-HT levels ( fig. 2 b; p ! 0.05) were re-duced when expression of the DTRH RNAi transgene was induced in larvae during the second instar, demon-strating that reduction in DTRH levels resulted in dimin-ished 5-HT synthesis. Similarly, induction of DTRH ex-pression resulted in increased 5-HT fluorescence ( fig. 2 c; p ! 0.01); relative levels of DTRH could not be assessed since the protein is ectopically expressed in the brain (see fig. 7a).

We then examined these animals for changes in feed-ing rate by feeding 5-HTP (the product of the DTRH re-action) to increase 5-HT levels (this approach has been used successfully to increase neuronal 5-HT levels [e.g. Kaplan et al., 2008]), and by inducing knockdown of the RNAi construct to decrease DTRH levels and thus 5-HT synthesis ( fig. 2 d). In mammals, increased brain sero-tonin levels decrease appetite, and lesioning of 5-HT neu-rons increases feeding behavior [see Blundell, 1986, for review]. Since Drosophila larvae at this developmental stage (second to third instar) engage full-time in feeding activity in the presence of food, the continuous contrac-tion of the mouth hooks is equivalent to the feeding rate [Sewall et al., 1975], in anticipation of the enormous en-ergy demands for metamorphosis. Also, in contrast to adults, there is no gender difference in feeding rate [de Miranda and Eggleston, 1988].

As expected, when the animal was placed in a yeast solution, feeding 5-HTP decreased its feeding rate (mea-sured by the number of mouth hook contractions), and a reduction in neuronal 5-HT synthesis increased feeding rate. Animals with reduced neuronal DTRH levels fed 5-HTP displayed a feeding rate indistinguishable from normal. To establish that the effect of 5-HTP was not due to a generalized physiological impairment, we also as-sayed locomotor behavior, which is stable in second and third instar larvae [Sewall et al., 1975]. Foraging larvae use their mouth hooks to dig into the substrate, which may facilitate pulling the larvae forward by muscular body wall contractions. These same muscles are used by the animal to shovel food into the larval pharynx. Chang-es in 5-HT levels either by feeding 5-HTP or by reducing DTRH levels had no effect on locomotion ( fig. 2 e).

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Similarly, feeding 5-HTP to uninduced GS elav /TRH UAS animals reduced appetite; induction of neuronal 5-HT synthesis also reduced feeding, and induced ani-mals given 5-HTP displayed an additive effect ( fig. 2 f). Again, motor behavior was normal ( fig. 2 g), demonstrat-ing that changes in neuronal 5-HT specifically affected appetitive behavior. Thus, 5-HT acts as a neurotransmit-ter to modulate feeding in Drosophila , as it does in all other species tested.

Constitutively Reduced Neuronal Serotonin Levels throughout Development Result in Perturbed Larval Feeding Behavior To address the trophic rather than transmitter role for

5-HT in the modulation of feeding rate, we used a con-stitutive pan-neuronal driver (elav C15 5 Gal4) to perturb

DTRH expression during neuronal development. It was not feasible to reduce 5-HT levels during the last 6 h of embryonic development via pharmacological inhibition of DTRH or via neuronal induction of the DTRH trans-gene, since neither approach would have resulted in re-duced 5-HT levels during the critical time frame. We thus chose to use the pan-neuronal elav driver to reduce DTRH levels, and thus 5-HT synthesis, beginning from stage 9 of embryogenesis. Since the elav C155 Gal4 driver is strongest during late embryogenesis, and displays re-duced expression in the larval CNS, use of this driver al-lowed us to knockdown DTRH levels during embryo-genesis, with a lesser reduction in expression during the larval stages. We identified two independent insertions of the same dsRNAi transgene (DTRHE, on chromo-some 2, and DTRHA, on chromosome 3), and quantified

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Fig. 3. Constitutive reduction in neuronal 5-HT results in de-pressed feeding behavior and abnormal responses to exogenous 5-HT. Two independent RNAi transgenic lines (DTRHE, inserted on chromosome 2, and DTRHA, on chromosome 3) were used to titrate constitutive knockdown of DTRH levels throughout CNS development. Larval brains were dissected from each genotype (elav C155 /w 1118 , n = 9; elav C155 /DTRHE, n = 10, and elav C155 /DTRHA, n = 14). The 5-HT cell pattern in each brain was visual-ized with a polyclonal antibody raised against DTRH [Coleman and Neckameyer, 2005] and viewed under fluorescence. a The pixel intensity of the DTRH immunofluorescence of the two un-paired neurons in the 8th abdominal neuromere was determined to ascertain the extent of knockdown of DTRH. All genotypes

were assessed in parallel. * p ! 0.05; * * p ! 0.01, one-way ANOVAs followed by Dunnett’s multiple comparisons post-test. The ani-mals were then assayed for feeding ( b ) and locomotor behavior ( c ). An additional control, DTRH/w 1118 , was included in these studies. Feeding was decreased in DTRH knockdown animals, and loco-motion was unaffected. d Feeding rate was dramatically increased in elav C155 /DTRHA larvae given 10 mg/ml 5-HTP for 24 h. e Con-sistent with the results obtained with the DTRH knockdown lines, the DTRH-null pBacTRH mutation was similarly assessed for feeding rate and compared with the parental w 1118 controls.n = 40 for each genotype and condition. Lines above the graph depict standard error of the mean. * * p ! 0.01; * * * p ! 0.001, one-way ANOVAs followed by Tukey’s post-test.

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the relative intensity of DTRH expression in these ani-mals using the approach described above to demonstrate that a single copy of the driver and the RNAi construct significantly decreased DTRH levels, and DTRHA re-sulted in a greater knockdown of DTRH levels than did DTRHE, thus permitting ‘titration’ of the decrease in DTRH expression and 5-HT synthesis ( fig. 3 a). Progeny from parental animals (w 1118 , the strain used for genera-tion of these transgenics, and the elav Gal4 driver) were used as controls. Decreased levels of DTRH knockdown directly correlated with reduced feeding relative to con-trols (w 1118 /DTRHA and elav C155 /w 1118 ; fig. 3 b) and gen-eral motor activity was unaffected ( fig. 3 c), demonstrat-ing the effect was specific to feeding behavior and notthe result of a generalized impaired physiology. While consistent with our published observations for the null DTRH mutation, pBacTRH [Neckameyer et al., 2007], these findings are contrary to what was observed for knockdown of the DTRHA transgene using the induc-ible pan-neuronal promoter, suggesting a critical role for 5-HT during CNS development in the maturation of this neural circuitry.

We predicted, since feeding exogenous 5-HTP de-creases appetite, and since appetite is also depressed in animals with developmentally reduced 5-HT levels, that exposing these animals to 5-HTP would significantly further impair feeding rate. Surprisingly, we observed that feeding 5-HTP to elav C15 5 /DTRHA ( fig. 3 d) and pBacTRH animals ( fig. 3 e) resulted in a highly increased appetite; locomotor control was unaffected (data not shown). These results clearly demonstrate that although 5-HT still acts to modulate feeding at this circuit, its ac-tions are impaired as a consequence of reduced neuronal 5-HT levels during development.

Titration of Neuronal 5-HT Synthesis during CNS Development Affects the Architecture of 5-HT Fibers Projecting to the Proventriculus Using Drosophila mutants deficient in dopa decarbox-

ylase, and thus unable to synthesize either dopamine or serotonin, Budnik et al. [1989] noted that these animals displayed increased branching of serotonergic fibers pro-jecting to the midgut, as well as increased numbers of varicosities; however, these studies could not distinguish whether this phenotype was a consequence of altered se-rotonin or altered dopamine levels, or both, and whether the perturbed fibers were sensitive to these factors acting neuronally or peripherally. We thus ascertained whether manipulating neuronal DTRH levels to titrate 5-HT lev-els throughout CNS development would correlate with

this phenotype, as it did with feeding rate. This was ac-complished by quantitating the numbers of branch points, as well as the size and number of varicosities, along the lengths of the serotonergic fibers projecting to the foregut. We limited our analyses to the fibers within the center of the proventriculus, since they are easily vi-sualized and, in control animals, display little branching; the earlier studies with dopa decarboxylase mutants as-sessed the branching of these fibers in the midgut, which normally displays greater branching. It is not possible to quantitate changes in branching and varicosity number within the frontal nerve and recurrens nerve, since they are tightly bundled, and single fibers cannot be adequate-ly resolved, nor can individual varicosities be distin-guished (see fig. 1 ).

We analyzed gut tissues from the two transgenic RNAi lines driven by the pan-neuronal elav promoter, as well as from the pBacTRH mutation. The pBacTRH mutation re-sults in barely detectable levels of 5-HT within the CNS; although the mutation does not express DTRH protein, there is a small amount of 5-HT taken up via the sero-tonin transporter into the nervous system [Neckameyer et al., 2007]. The w 1118 allele was used in the generation of the elav Gal4 driver [Lin and Goodman, 1994] as well as the pBacTRH mutation, but they still differ in their ge-netic backgrounds. In addition, w 1118 animals are blind, which impacted their behavioral analyses (w 1118 animals move and feed at a reduced rate relative to elav Gal4/w 1118 , data not shown; see also Mackenzie et al. [1999]). There-fore, the mutation and the transgenic knockdown lines were not directly compared.

We observed a correlative increase in arborization with decreasing levels of neuronal 5-HT in the DTRH transgenic knockdown animals relative to controls ( fig. 4 ). While there was some overlap in branching phe-notype, animals with constitutively decreased levels of neuronal 5-HT showed greater complexity of arboriza-tion.

We also assessed the total number of varicosities per unit length (0.1 mm), defining a varicosity as a brightly staining, discrete vesicle containing 5-HT dotting the neurite length, with an area about the width of the neurite fiber, which is less than 1 ! m 2 . Large varicosities were defined as those enlarged beyond the width of the neurite (area greater than 1 ! m 2 ). Ultrastructural analysis of similar varicosities in the CNS neuropil suggests they are presynaptic terminals that release 5-HT [Sykes and Con-dron, 2005]. In general, the number of total varicosities increased in animals with perturbed DTRH levels ( fig. 5 ). While large varicosities could be observed in the gut pro-

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Dev Neurosci 2010;32:217–237226

jections of control animals, animals with constitutively decreased neuronal 5-HT levels displayed a far greater number of these vesicles; some even in the range of 25–50 ! m 2 ( fig. 5 a). While the pBacTRH mutant did not dis-play the greatest increase in varicosity number ( fig. 5 b), gut projections from the pBacTRH mutant appeared to have a significantly greater number of larger varicosities ( 1 5 ! m 2 ; fig. 5 c). Closer observation of the larger vari-

cosities revealed that they were composed of numerous smaller ones ( fig. 5 d), suggesting that, with decreasing amounts of neuronal 5-HT during development, they sufficiently increase in number and then fuse. Thus, while pBacTRH gut fibers may appear to have fewer dis-crete varicosities relative to the neuronal TRH knock-downs, the significantly larger vesicles along the lengths of the gut fibers may contain a far greater number of

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Fig. 4. Decreasing amounts of neuronal serotonin during critical developmental windows inversely correlate with the degree of branching of serotonergic fibers projecting to the proventriculus. The mouthparts, brain and foregut were dissected from third in-star larvae and immunostained with an anti-serotonin antibody. The samples were viewed under fluorescence and photographed at the same magnification. a Representative examples of increased arborization in animals with constitutively decreased neuronal 5-HT levels. Scale bar = 20 ! m. b Quantitation of arborization. Branch points for projecting fibers were quantitated using the Neuroleucida analysis program. elav C155 /w 1118 , 50 projecting fi-bers from 40 independent gut dissections; DTRHA/w 1118 , 53 pro-

jecting fibers from 45 independent gut dissections (controls); elav C155 /TRHE, 45 projecting fibers from 34 independent gut dis-sections; elav C155 /TRHA, 54 projecting fibers from 39 indepen-dent gut dissections; w 1118 and pBacTRH, 18 and 19 projecting fibers from 17 and 20 independent gut dissections, respectively. Lines above the graph depict standard error of the mean. Signifi-cance was assessed by one-way ANOVAs followed by Dunnett’s post-tests (elav C155 /w 1118 , DTRHA/w 1118 , elav C155 /TRHE, elav C155 /TRHA) or Student’s t tests (w 1118 and pBacTRH). * p ! 0.01;** p ! 0.001. These groups were not analyzed together because they did not share the same genetic background.


Dev Neurosci 2010;32:217–237 227

smaller, discrete varicosities whose membranes have fused to form the large ones, consistent with a correlative increase in varicosity number with decreasing develop-mental neuronal 5-HT levels.

To demonstrate that these abnormalities were the re-sult of perturbations in developmental neuronal 5-HT levels, and not a consequence of altered neuronal 5-HT transmission, we analyzed the architecture of serotoner-

gic fibers projecting to the proventriculus in GSelav/TRHA RNAi animals, inducing neuronal expression of the RNAi transgene during second instar, and analyzing the animals during early third instar (40–44 h after in-duction, when 5-HT levels have been significantly re-duced; fig. 6 ). No differences were observed in arboriza-tion of fibers ( fig. 6 a), varicosity number ( fig. 6 b), number of large varicosities ( fig. 6 c), or in varicosity area ( fig. 6 d).

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gut dissections; elav C155 /TRHE, 46 projecting fibers from 34 in-dependent gut dissections; elav C155 /TRHA, 58 projecting fibers from 39 independent gut dissections; w 1118 and pBacTRH, 23 and 24 projecting fibers from 17 and 20 independent gut dissections, respectively. Lines above the graph depict standard error of the mean. Significance was assessed by one-way ANOVAs followed by Tukey’s post-tests (elav C155 /w 1118 , DTRHA/w 1118 , elav C155 /TRHE, elav C155 /TRHA) or Student’s t tests (w 1118 and pBacTRH). * * p ! 0.01; * * * p ! 0.001. These groups were not analyzed together because they did not share the same genetic background. d Examples of less dense varicosities (from pBacTRH gut fibers), which appear to contain multiple smaller varicosities. Scale bar = 10 ! m.

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Fig. 6. Reduction in neuronal 5-HT after the CNS has matured does not affect the architecture of serotonergic fibers project-ing to the proventriculus. Tissues were dis-sected from control (uninduced) or in-duced (6 mg/ml RU486 for 2 min) GS elav /TRHA larvae 40–44 h after induction. a Branch number. b Varicosity number. c Number of large varicosities per 0.1 mm. d Varicosity area. Control, 30 projecting fibers from 24 independent gut dissec-tions; RU486, 28 projecting fibers from 21 independent gut dissections. Lines above the graph depict standard error of the mean. Significance was assessed by Stu-dent’s t tests.

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Fig. 7. Induction of DTRH levels during CNS development does not result in increased 5-HT levels. a–d Immunohistochemical analyses of third larval instar CNS tissues from animals carrying a UAS TRH transgenic construct expressed in the CNS and visu-alized with an antibody raised against DTRH. a GSelavTRH UAS32, uninduced. As expected, the pattern is restricted to 5-HT neurons. b GSelavTRH UAS32, induced 6 mg/ml RU486 for 2 min as third instar larvae. DTRH is expressed ectopically in all neu-rons, demonstrating the protein is induced. Brains were dissected 24 h after induction. c The same transgene driven by elav C155 Gal4. d An independent UAS TRH (TRH UAS27) on a separate chro-

mosome, also driven by elav C155 Gal4. Both transgenes are ex-pressed ectopically, as expected. Scale bar = 50 ! m. e In contrast to expressing the DTRH UAS after the larval CNS has matured ( fig. 2 ), there is no increase in 5-HT levels, measured by the pixel intensity of the two unpaired neurons in the 8th abdominal neu-romere in brains from newly hatched first larval instars visualized with an antibody raised against 5-HT. elav C155 /w 1118 , n = 16; elav C155 /UAS27, n = 20, and elav C155 /UAS32, n = 8. Significance was assessed by one-way ANOVAs followed by Tukey’s post-tests. Lines above the graph depict standard error of the mean.


Dev Neurosci 2010;32:217–237 229

5-HT Synthesis Is Tightly Regulated during CNS Development To determine whether increased 5-HT levels above a

normal threshold also affected development of the sero-tonergic feeding circuit, we attempted to use the elav C155 driver to express a TRH UAS transgene in the develop-ing CNS. When DTRH expression was induced in sec-ond larval instars using the GSelav driver, DTRH was expressed in cells throughout the larval CNS ( fig. 7 a, b), which results in increased neuronal 5-HT levels (see fig. 2 c). 5-HT was observed only in 5-HT neurons (data not shown), suggesting that although DTRH may be ex-pressed in dopamine neurons, regulatory mechanisms must be in place to ensure that only the correct trans-mitter is synthesized (tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, is a sister en-zyme that shares many biochemical features, including an absolute requirement for tetrahydrobiopterin as co-factor). When the constitutive pan neuronal elav Gal4 driver was used to induce the DTRH UAS transgenes, DTRH was also expressed ectopically ( fig. 7 c, d). How-ever, unlike the inducible promoter, this induction did not result in increased neuronal 5-HT levels in late-stage (second and third instar) larvae (data not shown). Since it was possible that DTRH synthesis and 5-HT levels might be ‘normalized’ over time, the CNSs of newly-hatched larvae were examined for increased 5-HT levels. However, even at this early stage, no increase in neuro-nal 5-HT was observed ( fig. 7 e). Since the enzyme activ-

ity is limited by cofactor and substrate availability, it is likely that these factors preclude increased synthesis during this developmental period, even though DTRH levels are induced. These data suggest that regulatory mechanisms are in place to not only restrict 5-HT ex-pression only to 5-HT neurons, but that the developing CNS is highly sensitive to perturbations above normal 5-HT threshold levels.

Increased Levels of 5-HT during DevelopmentResult in Increased Feeding and Decreased GutFiber Complexity Since constitutive induction of neuronal 5-HT during

CNS development was not feasible, staged wild-type (CS wu ) embryos were aged until 16 h after egg laying (when projections from serotonergic cell bodies are first observed), and directly exposed to 10 –6 M 5-HT until hatching ( fig. 8 ). In contrast to what was observed for constitutive neuronal induction of transgenic animals carrying a DTRH RNAi transgene, the feeding rate of these animals, when assessed as second instar larvae, was significantly increased ( fig. 8 a); locomotor behavior was normal ( fig. 8 b). Larvae exposed to 5-HT during the last 6 h of embryogenesis were also capable of reducing their feeding rate when given exogenous 5-HTP as second in-star larvae, although not to wild-type levels, suggesting a hyposensitivity to the neurotransmitter actions of sero-tonin ( fig. 8 c). In addition, branching of gut fibers was significantly decreased ( fig. 9 a), the opposite of that ob-

Tukey’s multiple comparison testCSwu-control-control vs. CSwu-control-5-HTPCSwu-control-control vs. CSwu-5-HT-controlCSwu-control-control vs. CSwu-5-HT-5-HTPCSwu-control-5-HTP vs. CSwu-5-HT-controlCSwu-control-5-HTP vs. CSwu-5-HT-5-HTPCSwu-5-HT-control vs. CSwu-5-HT-5-HTP

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Fig. 8. Embryos exposed to 5-HT during the last 6 h of development display in-creased feeding as larvae. Wild-type (CS wu ) embryos were collected for 2 h, aged for14 h, dechorionated, devitellinized, and ex-posed to 10 –6 M 5-HT in serum-free media until hatching. Control embryos were pro-cessed in parallel but not exposed to 5-HT. Treated embryos displayed a significant in-crease in feeding rate ( a ) with no change in locomotion ( b ). * * * p ! 0.001, Student’s t tests. c The effect of exogenous 5-HT was determined by feeding 10 mg/ml 5-HTP for 24 h before assaying for feeding. Al-though exogenous 5-HTP decreases feed-ing rate, the response is not as robust as it is for controls. Statistical analyses accom-plished by one-way ANOVAs followed by Tukey’s multiple comparisons post-test(n = 40). Lines above the graph depict stan-dard error of the mean.

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Dev Neurosci 2010;32:217–237230

served when neuronal 5-HT levels were decreased during development. While the total number of varicosities along the fiber lengths was unchanged ( fig. 9 b), the num-ber of large ( 1 1 ! m 2 ) varicosities was significantly de-creased relative to controls ( fig. 9 c). Since the larger vari-cosities appear to be composed of multiple fused smaller varicosities, this would suggest an overall decrease in var-icosity number, the converse of that observed with re-duced neuronal developmental 5-HT levels. Thus, devel-opmental levels of neuronal 5-HT inversely correlate with the amount of branching of serotonergic fibers projecting to the gut, as well as varicosity number along the fiber lengths. In addition, too little neuronal 5-HT during de-velopment results in animals with decreased feeding, while excess 5-HT during development results in in-

creased feeding relative to controls, demonstrating that the effects of developmental 5-HT on fiber architecture and behavioral output of the mature circuit can be titrat-ed, and suggesting that there is a concentration threshold above and below which the integrity of the circuit is com-promised.

Feeding and Gut Fiber Architecture Abnormalities in DTRH Null Mutants Are Rescued by Exposure to 5-HT during the Last 6 Hours of Embryogenesis pBacTRH embryos were aged until 16 h after egg lay-

ing and directly exposed to 10 –6 M 5-HT until hatching, when they were placed in yeast paste without additional exogenous 5-HT. When assessed as second instar larvae, their feeding behavior was indistinguishable from that of

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Fig. 9. Embryos exposed to 5-HT during the last 6 h of develop-ment display decreased branching of 5-HT fibers projecting to the gut, as well as fewer overall numbers of serotonergic varicosities. Wild-type (CS wu ) embryos were collected for 2 h, aged for 14 h, dechorionated, devitellinized, and exposed to 10 –6 M 5-HT in se-rum-free media until hatching. Control embryos were processed in parallel but not exposed to 5-HT. a Treated embryos displayed decreased branching of gut fibers relative to controls. While the

number of individual varicosities was similar ( b ), the number of large varicosities was also significantly less ( c ). There were 29 pro-jections from 25 guts from 6 independent experiments for control and 36 projections from 30 guts from 5 independent experiments. * p ! 0.05, * * * p ! 0.001, Student’s t tests. Lines above the graph depict standard error of the mean. Representative 5-HT immu-nostaining of control ( d ) and treated ( e ) embryos. Scale bar = 50 ! m.

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Dev Neurosci 2010;32:217–237 231

Tukey’s multiple comparison testpBacTRH –5-HT –5-HTP vs. pBacTRH –5-HT +5-HTPpBacTRH –5-HT –5-HTP vs. pBacTRH +5-HT –5-HTPpBacTRH –5-HT –5-HTP vs. pBacTRH +5-HT +5-HTPpBacTRH –5-HT +5-HTP vs. pBacTRH +5-HT –5-HTPpBacTRH –5-HT +5-HTP vs. pBacTRH +5-HT +5-HTPpBacTRH +5-HT –5-HTP vs. pBacTRH +5-HT +5-HTP

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Fig. 10. Embryos lacking DTRH function exposed to 5-HT for the last 6 h of embryonic development display normal feeding behav-ior. pBacTRH embryos, which are null for DTRH function and contain barely detectable levels of neuronal 5-HT, were collected for 2 h, aged for 14 h, dechorionated, devitellinized, and exposed to 10 –6 M 5-HT in serum-free media until hatching. Control em-bryos were processed in parallel but not exposed to 5-HT. a Treat-ed embryos displayed a significant increase in feeding rate relative

to pBacTRH animals that were not exposed to any treatment as well as to pBacTRH animals exposed only to serum-free medium; treated animals were indistinguishable from the w 1118 parental controls. b Treatment with 5-HT during the last 6 h of embryo-genesis also rescues the abnormal feeding response to the actions of exogenous 5-HTP. * * * p ! 0.001, one-way ANOVAs followed by Tukey’s multiple comparisons post-test (n = 40). Lines above the graph depict standard error of the mean.

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ositi

es >

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ngth

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pBacTRH controlpBacTRH +5-HT

0

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Fig. 11. Exposure of embryos lacking DTRH function to 5-HT for the last 6 h of embryonic development decreases the complexity of the serotonergic gut fibers projecting to the proventriculus. pBacTRH embryos were collected for 2 h, aged for 14 h, dechori-onated, devitellinized, and exposed to 10 –6 M 5-HT in serum-free media until hatching. Control embryos were processed in parallel but not exposed to 5-HT. a Treated embryos displayed decreased

branching of gut fibers relative to controls. The number of large varicosities ( b ) and the average varicosity area ( c ) were also sig-nificantly less. d The total number of individual varicosities was similar. There were 38 projections from 27 guts from 8 indepen-dent experiments for control, and 35 projections from 23 guts from 9 independent experiments. * * * p ! 0.001, Student’s t tests. Lines above the graph depict standard error of the mean.

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the parental w 1118 ( fig. 10 a), and the hypersensitivity to exogenous 5-HTP was also rescued ( fig. 10 b). Consistent with the changes in feeding behavior, branching of the proventricular gut fibers was decreased ( fig. 11 a), as was the varicosity area ( fig. 11 b) and number of extremely large varicosities ( fig. 11 c). The varicosity number was unchanged ( fig. 11 d), but since the larger varicosities ap-pear to be composed of numerous small varicosities, a decrease in the number of large varicosities, and in vari-cosity area, would suggest that the overall number ofvaricosities has been reduced, as expected. These results provide confirmation that it is the developmental actions of serotonin that affect the architecture and function of the 5-HT feeding circuit in the DTRH knockdown and mutant animals.

Discussion

Neurotransmitters can function as growth factors in developing neuronal and nonneuronal tissues before adopting their roles as signaling molecules in the mature nervous system, since stimulation of their receptors, also present, can activate downstream intracellular signaling cascades. Thus, the machinery is in place for neurotrans-mitters to exert pleiotropic effects in both development and function of nervous tissue [reviewed in Weiss et al., 1998]. 5-HT is present not only in the most primitive an-imal, the sponge [Salmoun et al., 2002], but it acts as a trophic factor to stimulate the development of specialized feeding cells in juvenile hydra [McCauley, 1997]. Here, we present data to show that 5-HT acts as a trophic factor in the development of a specific neural circuit in Drosophila , as well as acting as a neurotransmitter to modulate the behavior arising from the mature circuit.

Serotonergic Feeding Circuit in Drosophila The cephalopharyngeal plates are strongly 5-HT-im-

munoreactive; a continuous serotonergic nerve fiber con-nects the plates to the brain and then the brain to the proventriculus (the larval foregut) [Budnik et al., 1989]. The latter part of this nerve, the recurrens nerve, fascicu-lates in the proventriculus into individual fibers that are dotted with serotonergic varicosities along the lengths; additional fibers branch off when the fibers reach the midgut. This defines the larval serotonergic feedingcircuit, which is an integral part of the stomatogastric pattern generator. Although both glutaminergic and FRMFamide fibers innervate the foregut, they are dis-tinct from the 5-HT circuitry, and do not change their

architecture as a consequence of impaired 5-HT levels [Budnik et al., 1989], although presumably all contribute to the generation of proventricular muscular contrac-tions.

The control condition used when exposing larvae to exogenous 5-HTP differed from that used when assessing basal feeding rate, although the same yeast source was used. The minor differences in control conditions slight-ly affected responses when comparing the two different conditions, demonstrating that even small changes in the ‘normal’ environment may contribute to the variability observed in the behavioral outcomes. We have previous-ly observed this for adult behavioral paradigms [Necka-meyer and Matsuo, 2008], as have others, not only in Dro-sophila [e.g. Martin et al., 1999], but in mice as well [Wahlsten et al., 2006]. Therefore, although we observed the same changes in behaviors (i.e. decrease or increase in feeding rate) in different experiments, the actual num-ber of mouth hook contractions between control condi-tions may differ, depending upon the environment.

Serotonin modulates feeding behavior in all species tested; rodent studies have revealed the involvement of 5-HT 1 and 5-HT 2 receptor agonists in promoting the hy-pophagic response to 5-HT [De Vry and Schreiber, 2000]. Consistent with these studies, we have shown that in-creased neuronal 5-HT levels in the mature larva de-crease feeding rate, and reduced 5-HT levels within the CNS increase feeding. However, since 5-HT is also known to act as a trophic factor in the development of neural cir-cuitry, and, given the extensive innervation of the mouth-parts and gut by 5-HT, we asked whether the trophic ef-fects of 5-HT could impair the development, and thus the mature function, of this feeding circuit.

Serotonin Is Required during Development of the Feeding Circuit for the Normal Architecture of 5-HT Projections from the Brain to the Gut Using transgenic, genetic and pharmacological tools

to constitutively titrate DTRH and 5-HT levels during CNS development, we have shown that developmental 5-HT levels inversely correlate with the degree of arbori-zation and of varicosity number and size along the length of neurites projecting to the foregut from the CNS. Our results are consistent with the seminal work of Kater and colleagues, who demonstrated that application of 5-HT to cultured Helisoma B19 buccal ganglion neurons re-pressed neurite outgrowth, with secondary effects on synaptogenesis [Haydon et al., 1984; Goldberg and Kater, 1989; reviewed in Goldberg, 1998]. Similarly, treatment of Helisoma embryos with a Tph inhibitor affected the de-

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velopment of early differentiating serotonergic neurons (ENC1s) by increasing the arborization of a neurite pro-jecting from these neurons to the foot primordium, which occurred through the actions of 5-HT autoreceptors [Diefenbach et al., 1995]. Later studies demonstrated that 5-HT induced a calcium influx-dependent inward sodi-um current via the actions of cyclic-nucleotide-gated channels [reviewed in Goldberg, 1998]. 5-HT has been shown to induce growth cone collapse and inhibit neurite outgrowth in Helisoma by depolymerizing F-actin, a crit-ical cytoskeletal component of growth cones [Torreano et al., 2005]. While these studies demonstrated localized ac-tions of 5-HT directly on growth cone activity in isolated neurons, further studies have shown that 5-HT can have more widespread effects on developing neuronal popula-tions [Goldberg, 1998]. In addition, the Helisoma C1 ce-rebral ganglion, which extends through the buccal gan-glia to target tissues required for the feeding response, may release 5-HT nonsynaptically to bind to 5-HT recep-tors on the B19 neuron and elsewhere [Goldberg, 1998]. Thus, the actions of serotonin in the developing nervous system may be specific for a given cell type and target tis-sue, and at a given developmental window. Development of the serotonergic innervation of target tissues for feed-ing in Drosophila is similar to that of the snail in that in-nervation of the buccal mass occurs during late embryo-genesis, with increasing numbers of 5-HT-immunoreac-tive fibers in muscle during the early juvenile stages [Goldberg and Kater, 1989; Budnik et al., 1989; Balog and Elekes, 2008]. In both systems, depletion of 5-HT during this critical developmental time resulted in increased ar-borization of 5-HT fibers projecting to the target tissues. However, these analyses were limited to components of the feeding circuit; while gross development was unaf-fected, and simple behavior (locomotion) was normal in our studies, other circuitry could have been differentially affected.

Earlier studies suggested that 5-HT acts to inhibit neu-rite outgrowth in actively elongating fibers, but it can also reinitiate growth in fibers whose outgrowth has been ar-rested; the former occurs in both embryonic and mature neurons, but the latter only occurs in embryonic neurons [Goldberg et al., 1991]. In addition, the local milieu must be important, since individual neurites projecting from the same neuron can respond differently to 5-HT ex-posure [Goldberg et al., 1991]. Depletion of serotonin in the developing neocortex results in depressed dendritic growth of cortical interneurons [Durig and Hornung, 2000], and 5-HT applications to moth antennal lobe neu-rons cultured in vitro increases branching of these cells

[Kim et al., 2005], the converse of what we have observed for serotonergic fibers projecting from the Drosophila brain to the foregut. Thus, the actions of 5-HT during the developing CNS can be both inhibitory and stimulatory. While the actions of 5-HT induce different signaling pathways in these developing circuits, so that the effect of 5-HT application or reduction may have opposing conse-quences for the neuronal architecture, 5-HT still acts as a trophic factor to initiate these changes.

5-HT also acts to spatially regulate varicosity forma-tion along the neurite length. The diameter of normal serotonergic varicosities in the neuropil of the Drosophila distal abdominal ganglia are approximately 0.5–1.0 ! m in size [Sykes and Condron, 2005], comparable to the ma-jority of varicosities observed in the serotonergic projec-tions to the gut in our control animals. Application of exogenous 5-HT to intact Drosophila larval ventral nerve cords in culture resulted in a reversible loss of serotoner-gic varicosities in second and third instar larvae [Sykes and Condron, 2005], consistent with our observations that reduced 5-HT levels increase varicosity size and number and increased 5-HT levels reduce them. Since we did not observe any change in varicosity number or size when comparing control and induced GS elav TRH RNAi animals ( fig. 6 ), the effect of reduced 5-HT levels on var-icosity density in animals with constitutively reduced neuronal 5-HT levels must have occurred prior to second instar (2 days after hatching). Assessment of reduced 5-HT synthesis during more narrowly defined develop-mental windows will also clarify whether this effect rep-resents an ongoing process or whether there is a more limited developmental window.

Peripheral fibers begin to project from serotonergic neurons 2–4 h before hatching, and the fibers projecting to the gut are not observed for another 2 h [Budnik et al., 1989]. In addition, these fibers must grow with the en-larging proventriculus, which increases in size through-out the larval molts. Since knockdown (using RNAi) and knockout (via the pBacTRH mutation) of DTRH oc-curred across this developmental window, and since em-bryonic exposure to exogenous 5-HT also occurred over this same time span, it is also possible the changes in ar-borization reflected temporal sensitivity to 5-HT levels. It is conceivable that perturbations in regional 5-HT con-centrations at specific times direct growth of the fascicu-lating fibers to limit arborization until they enter the midgut, which would occur shortly before hatching.

Expression levels of the serotonin transporter might be upregulated to counteract limited neuronal 5-HT syn-thesis by having increased reuptake from the periphery.

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Hieu Nguyen

How so?

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Dev Neurosci 2010;32:217–237234

5-HT transporter mRNAs are first observed in stage 15 embryos, before 5-HT can be detected in serotonergic neurons [Demchyshyn et al., 1994]. However, in the DTRH null mutant pBacTRH, 5-HT levels in the brain are barely detectable [Neckameyer et al., 2007]; since all neuronal 5-HT must occur via reuptake from circulating 5-HT, it is therefore unlikely 5-HT transporter expres-sion is significantly enhanced. While DTRH levels can be upregulated during early CNS development, this does not result in increased levels of 5-HT. While it is also possible that expression of the 5-HT transporter is downregulat-ed, it is not clear that such downregulation would have any significant effect on neuronal 5-HT levels, sinceneuronal and peripheral 5-HT synthesis are distinct pathways [Neckameyer et al., 2007]. In addition, when exposure to enhanced 5-HT is limited to late embryo-genesis ( fig. 9 ), the larvae still display aberrant feeding and neurite architecture of fibers projecting to the fore-gut, suggesting that the last 6 h of embryogenesis provide a critical developmental window; similar treatment of pBacTRH embryos rescues both the behavioral and gut architecture abnormalities ( fig. 10 , 11 ).

It is possible that constitutive knockdown of DTRH synthesis could affect expression of the any of the four serotonin receptors to compensate for limited 5-HT lev-els, since all the receptors are expressed in the CNS dur-ing embryonic stage 16–17 [Saudou et al., 1992; Colas et al., 1995]. However, it is not yet known which of these re-ceptors are involved in the development of the serotoner-gic innervation of the foregut. Since 5-HT signaling must be propagated via the actions of 5-HT receptors, it is con-ceivable that, depending upon the temporal expression of a given receptor, the actions of 5-HT may act to both in-duce and repress neurite extension and arborization.

Changes in Gut Fiber Architecture Correlate with Impairments in Feeding Behavior, the Functional Output of the Mature Circuit The architectural changes in this 5-HT circuit as a con-

sequence of changes in 5-HT levels during development also correlate with impaired feeding response in the ma-ture larva. The precise causative mechanisms for the be-havioral aberrations remain to be elucidated. While re-duced developmental 5-HT results in an increase in the number of varicosities, and the formation of large vesicles that likely arise from the fusion of numerous smaller ves-icles, their contribution to the behavioral phenotype is unknown. The small vesicles along the neurite length are likely to be synaptic varicosities that contain release sites for serotonin. However, increased varicosity number and

size correlates with decreased DTRH levels, and thus 5-HT synthesis. It is possible that a stimulus resulting in release of 5-HT from these vesicles can surpass the normal concentration threshold and functionally affect down-stream signaling pathways. The increased arborization and abnormal neurite structures may also result in local release of 5-HT that surpasses a threshold. Manipulation of Helisoma embryos to transiently depress extracellular 5-HT levels increased synaptic connectivity; enhanced dye coupling between neurons was observed, which cor-related with the increase in neurite outgrowth [Goldberg and Kater, 1989]. Both large and small 5-HT varicosities have been identified along axonal processes in rat raphe 5-HT neurons; the larger varicosities comprise less than 10% of the total vesicle population, and may play a differ-ent functional role [Benzekhroufa et al., 2009]. These may be analogous to the fine 5-HT fibers, with small varicosi-ties, and beaded 5-HT fibers, with larger varicosities, found in the retrohippocampal areas in the rabbit; these fibers are spatial distinct and are thus also likely to serve different functions [Bjarkam et al., 2005].

While feeding in animals with constitutively reduced neuronal 5-HT is depressed, their response to exogenous 5-HTP as mature larvae suggests a hypersensitivity to its neurotransmitter actions. Conversely, embryos exposed to 5-HTP during development of the circuitry are hypo-sensitive to 5-HTP when mature. Therefore, sensitivity of the feeding circuit to the neurotransmitter actions of 5-HT also appears to correlate with the degree of arbori-zation and varicosity size and number.

It is also possible that perturbations in developing 5-HT levels may affect either the frontal or recurrens nerves; however, these nerve fibers are highly 5-HT im-munoreactive and tightly aligned, making quantitation of changes in the fibers difficult to assess. Similarly, the cephalopharnygeal plates are intensely 5-HT immunore-active (see fig. 1 ). When placed on an agar substrate, lar-vae will insert their mouth hooks into the agar with each forward muscular contraction, and as the animal moves, the tracks left by the mouth hook indentations are clearly visible. Thus, locomotor behavior on an agar substrate not only acts as a control to demonstrate that the animals are not generally physiologically impaired, but damage to the frontal nerve and cephalopharyngeal plates as a con-sequence of perturbed developmental 5-HT levels should have affected this aspect of locomotor behavior, which it did not. Constitutive reduction in 5-HT synthesis had no effect on locomotion, neither did temporal up- or down-regulation of 5-HT levels, or exposure of embryos to ex-ogenous 5-HT during the last 6 h of embryogenesis. Since

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the recurrens nerve is merely an extension of the frontal nerve, it is reasonable to assume that any alterations in the frontal nerve would also occur in the recurrens nerve. Thus, while we cannot rule out that the frontal nerve, re-currens nerve, or the cephalopharyngeal plates do not have an effect on the changes in feeding, since serotonin does not appear to alter mouth hook contractions except when the animal is placed in a food source, it is reason-able to assume that any changes as a consequence of per-turbations in developmental neuronal 5-HT levels are likely limited to the proventricular and midgut tissues.

In summary, neuronal developmental 5-HT levels in-versely correlate with the amount of branching, and the number and size of varicosities of 5-HT fibers projecting from the brain to the gut. Developmental neuronal 5-HT levels directly correlate with changes in feeding behavior in mature larvae and an inappropriate feeding response to exogenous 5-HT. These data demonstrate that 5-HT acts as a trophic factor in CNS development, with direct effects on neuronal architecture, and impairment of the behavior modulated by that neuronal circuitry. Many converging lines of evidence now suggest that abnormal levels of 5-HT

during CNS development result in permanent alterations in neuronal circuitry and may explain the antidepressant actions of selective serotonin reuptake inhibitors in neu-rogenically active regions of the adult brain. Inhibition of 5-HT synthesis increases the rate of proliferation of sero-tonergic neurons in the dentate gyrus [reviewed in Djava-dian, 2004]; chronic fluoxetine treatment enhances den-dritic arborization of newborn hippocampal granule cells [Wang et al., 2008] and increases hippocampal pyramidal neuron dendritic spine density [Hajszan et al., 2005]. Thus, understanding the trophic actions of serotonin on developing neurons in the immature and mature CNS will provide critical insight into not only a fundamental ques-tion in developmental neuroscience, but into the thera-peutic actions of antidepressant treatments.

Acknowledgements

This work has been supported by NSF Grant No. 0616062 to W.S.N. We would like to acknowledge Dr. W. Michael Panneton for the generous use of microscopes and analysis programs, and Parag Bhatt for technical assistance.

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A Trophic Role for Serotonin in the Development of a Simple Feeding Circuit

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