Dual effect of serotonin on the dendritic growth of cultured hippocampal neurons: Involvement of 5-HT1A and 5-HT7 receptors

Accepted Manuscript Dual effect of serotonin on the dendritic growth of cultured hippocampal neurons: Involvement of 5-HT1A and 5-HT7 receptors

P.S. Rojas, F. Aguayo, D. Neira, M. Tejos, E. Aliaga, J.P. Muñoz, C.S. Parra, J.L. Fiedler PII: DOI: Reference:

S1044-7431(17)30180-X doi:10.1016/j.mcn.2017.09.009 YMCNE 3232

To appear in:

Molecular and Cellular Neuroscience

Received date: Revised date: Accepted date:

22 May 2017 4 September 2017 29 September 2017

Please cite this article as: P.S. Rojas, F. Aguayo, D. Neira, M. Tejos, E. Aliaga, J.P. Muñoz, C.S. Parra, J.L. Fiedler , Dual effect of serotonin on the dendritic growth of cultured hippocampal neurons: Involvement of 5-HT1A and 5-HT7 receptors. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Ymcne(2017), doi:10.1016/j.mcn.2017.09.009

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ACCEPTED MANUSCRIPT Rojas PS. et al. Molec Cell Neurosci RM

Dual effect of serotonin on the dendritic growth of cultured hippocampal neurons: Involvement of 5-HT1A and 5-HT7 receptors. †Rojas PS2,1, †Aguayo F1., † Neira D1., Tejos M1., Aliaga E3., Muñoz JP1., Parra CS1., and Fiedler JL1*. 1

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Laboratory of Neuroplasticity and Neurogenetics. Faculty of Chemical and Pharmaceutical Sciences. Department of Biochemistry and Molecular Biology. Universidad de Chile, Independencia 8380492, Chile. Present address, Faculty of Medicine, School of Pharmacy, Universidad Andres Bello.

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Department of Kinesiology, Faculty of Health Sciences, Universidad Católica del Maule, Talca, Chile

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Running title: Differential effects of 5-HT on hippocampal neuron morphology

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*Corresponding author: Dr. Jenny L. Fiedler, Laboratory of Neuroplasticity and Neurogenetics. Department of Biochemistry and Molecular Biology. Faculty of Chemical and Pharmaceutical Sciences, Universidad de Chile. Sergio Livingstone 1007, Independencia. Santiago 8380492, Chile. E-mail: [email protected]

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† Authors contributed equally to this study

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ABSTRACT Serotonin acts through its receptors (5-HTRs) to shape brain networks during development and modulates essential functions in mature brain. The 5-HT1AR is mainly located at soma of hippocampal neurons early during brain development and its expression gradually shifts to dendrites during postnatal development. The 5-HT7R expressed early during

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hippocampus development, shows a progressive reduction in its expression postnatally.

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Considering these changes during development, we evaluated in cultured hippocampal

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neurons whether the 5-HT1AR and 5-HT7R change their expression, modulate dendritic growth, and activate signaling pathways such as ERK1/2, AKT/GSK3β and LIMK/cofilin,

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which may sustain dendrite outgrowth by controlling cytoskeleton dynamics.

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We show that mRNA levels of both receptors increase between 2 and 7 DIV; however only protein levels of 5-HT7R increase significantly at 7 DIV. The 5-HT1AR is preferentially distributed in the soma, while 5-HT7R displays a somato-dendritic localization at 7 DIV.

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Through stimulation with 5-HT at 7 DIV during 24 h and using specific antagonists, we

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determined that 5-HT1AR decreases the number of primary and secondary dendrites and restricts the growth of primary dendrites. The activation of 5-HT1AR and 5-HT7R promotes

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the growth of short secondary dendrites and triggers ERK1/2 and AKT phosphorylation through MEK and PI3K activation respectively; without changes in the phosphorylation of

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LIMK and cofilin. We conclude that 5-HT1AR restricts dendritogenesis and outgrowth of primary dendrites, but that both 5-HT1AR and 5-HT7R promote secondary dendrite

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outgrowth. These data support the role of 5-HT in neuronal outgrowth during development and provide insight into cellular basis of neurodevelopmental disorders.

Keywords Serotonin; 5-HT1AR; 5-HT7R; outgrowth; dendrite; hippocampal neurons; ERK1/2; AKT; GSK3β; LIMK; cofilin.

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1. Introduction Serotonin (5-HT) is a chemical mediator that is expressed early during the development of the central nervous system (CNS) and it has been involved in important cognitive processes and human behaviour (Lauder, 1993;Gaspar et al., 2003). Alterations in serotonergic neurotransmission have been linked to the appearance of several neuropathological

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disorders, including anxiety, depressive disorder, eating disorders, and others (Charney and

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Manji, 2004). It has been stressed that 5-HT plays a role during the early development of

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the CNS, which differs from its role in mature brain (Homberg et al., 2013). Several studies support a trophic role for 5-HT during CNS development by regulating cellular

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proliferation, migration, and neuronal differentiation (Lauder, 1993;Gaspar et al., 2003;Cote et al., 2007). Studies show that animals with a diet low in tryptophan (an amino

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acid precursor of 5-HT biosynthesis) show a decrease in dendritic arborisation (GonzalezBurgos et al., 1996). Similarly, the abrogation of 5-HT synthesis using a knock-in mouse

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line in which the tryptophan hydroxylase 2 (Tph2) gene was replaced by the eGFP reporter gene showed severe abnormalities in the formation of serotonergic circuits (Migliarini et

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al., 2013). Furthermore, in vitro studies using hippocampal mouse neurons challenged with 5-HT have shown a reduction in the number of tertiary dendrites (Ferreira et al., 2010),

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indicating that this molecule could act through its receptors to sculpt neuronal morphology. Overall, these studies suggest that alterations of 5-HT levels during brain development

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affect proper neuronal wiring and therefore, may produce long-lasting modifications that

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affect brain functioning in adulthood.

5-HT acts through a large family of serotonin receptors, which are divided into seven subfamilies (5-HT1-5-HT7). Additionally, through alternative processing and editing of transcripts, these subfamilies can generate around 20 additional receptors, with different physiological functions (Mattson et al., 2004). With the exception of the 5-HT3R, which is a ligand-gated ion channel, all serotonergic receptors are G-protein coupled, which positively or negatively regulate adenylate cyclase (AC). These receptors also promote PLC activation and therefore, generate IP3 and diacylglycerol (DAG) (Mattson et al., 2004;Lesch and Waider, 2012). Immunohistochemical studies have shown early embryonic expression 3

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of these receptors, demonstrating a dynamic variation in their levels during pre- and postnatal development (Gaspar et al., 2003). To date, however, the specific contribution of each serotonergic receptor to the regulation of brain development and neuronal morphology has not been determined.

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5-HT1AR is one of the most studied serotoninergic receptors in relation to several

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pathologies (Dutton and Barnes, 2008). Its mRNA is detected in the foetal brain of rodents

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in the E12 stage at the raphe nucleus (Hillion et al., 1993) and in E16 at the hippocampus (Patel and Zhou, 2005), and it is also transiently expressed in hypoglossal motor neurons (Talley et al., 1997) and the cerebellum after birth (Miquel et al., 1994). Furthermore,

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depletion of 5-HT in the early postnatal period (P3) has no effect on the number and length of dendrites in hippocampal granule neurons, but it promotes a reduction of spine density

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(Yan et al., 1997a). This effect is reversed by buspirone, a 5-HT1A agonist (Yan et al., 1997b), suggesting that 5-HT promotes the spinogenesis through the 5-HT1AR. However, in

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vitro studies, using mouse neuroblastoma showed that the activation of 5-HT1AR induces an

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increase in both the number and length of neurites (Fricker et al., 2005).

5-HT7 R, the most recently identified member of the 5-HT receptor family (Volpicelli et al.,

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2014)), is expressed in the hippocampus in early postnatal stages (P2-P6), and its expression decreases during later developmental stages (Kobe et al., 2012). In contrast to

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the 5-HT1AR, the dynamic expression of the 5-HT7R during postnatal development suggests a more restricted role at well-defined developmental stages. In mouse cultured hippocampal

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neurons, specific activation of 5-HT7R promotes neurite elongation (Kvachnina et al., 2005). These evidences suggest that 5-HT1AR and 5-HT7R activation may impact the neuronal morphology. Our recently published study using rat hippocampal primary cultures demonstrated that both 5-HT1AR and 5-HT7R at 2 DIV promote the growth of secondary neurites, with no effect on neuritogenesis (Rojas et al., 2014). These results indicate that 5HT1AR and 5-HT7R have redundant functions at early neuronal stages, in which neuronal polarity is established. Considering that the levels of these receptors change during development, we found it interesting to evaluate the role of these receptors in neuronal

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morphology at a more mature neuronal stage, i.e., when the arborization of dendrites is occurring.

Some studies have used overexpression of 5-HT1AR and 5-HT7R in non-neuronal cell lines to evaluate their coupling to several transduction pathways. For instance, the 5-HT1AR has

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a wide spectrum of intracellular transducers, such as AC, potassium and calcium ion

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channels, and -similar to 5-HT7R- is able to activate signaling pathways commonly

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associated with trophic responses, such as ERK and AKT kinases (Rojas and Fiedler, 2016;Wirth et al., 2016). Specifically, ERK and AKT signaling pathways have been related to cytoskeleton reorganization, possibly regulating dendritic arborization (Kim et al.,

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2004;Jaworski et al., 2005;Kumar et al., 2005). In addition, Rho GTPases such as Rac1 induce neurite growth and branching (Govek et al., 2005) through the phosphorylation and

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activation of LIM Kinase 1 (LIMK-1), which mediates actin cytoskeletal reorganization by phosphorylating the actin depolymerization factor cofilin (Endo et al., 2003). Furthermore,

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phosphorylated LIMK induces microtubule stability and actin polymerization in human

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endothelial cells (Gorovoy et al., 2005). Considering these antecedents, both 5-HT1AR and 5-HT7R may modulate dendrite outgrowth through similar or different signal transduction

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pathways depending on the cell phenotype of a particular developmental stage.

We evaluated variations of 5-HT1AR and 5-HT7R levels during the DIV of neuronal

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cultures, their location in neurons and the effect of the activation of these receptors on dendritic outgrowth and signalling pathways related to cytoskeleton dynamics. In contrast

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to the morphological changes observed in immature neurons, we determined that both receptors regulate dendritic morphology. 5-HT1AR inhibits both the formation and growth of primary dendrites, but both 5-HT1AR and 5-HT7R promote the growth of secondary dendrites. Furthermore, we demonstrate a differential distribution of these receptors in the neuron; 5-HT1AR is expressed in the neuronal soma and 5-HT7R has a somato-dendritic distribution. We also found that both receptors induce activation of ERK1/2 and AKT, with no changes in LIMK and cofilin phosphorylation. These modifications may explain, in part, the shared effect of both receptors on dendritic outgrowth.

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2. MATERIALS AND METHODS 2.1 Ethics Statement Pregnant Sprague-Dawley rats were used in these experiments and were obtained from the Faculty of Chemical and Pharmaceutical Sciences, Universidad de Chile. The rats were

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handled according to guidelines outlined and approved by the Ethical Committee of the

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Faculty of Chemical and Pharmaceutical Sciences, Universidad de Chile, and the Science and Technology National Commission (CONICYT), and were in compliance with the

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National Institutes of Health Guide for the care and use of laboratory animals (NIH Publication, 8th Edition, 2011). Pregnant rats on gestational day 18 were euthanised after

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anaesthesia (ketamine 100 mg/kg and xylazine 10 mg/kg i.p.). 2.2 Hippocampal Culture

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Primary hippocampal neurons were cultured as previously described (Rojas et al., 2014). In brief, fetal rat hippocampal tissue (E18) were incubated in Hank’s balanced solution with

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0.05% trypsin (Biological Industries, Beit HaEmek, Israel). Digestion was stopped by

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serum addition and then centrifuged at 235 g for 4 min. The pelleted cells were mechanically dissociated with a Pasteur pipette in DMEM supplemented with 10% foetal

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bovine serum. For immunocytochemistry studies, cells were plated at a density of 80,000 cells/well on 35-mm coverslips pre-coated with poly-D-lysine (0.1 mg/ml). After 2 hr in the

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presence of 5% CO2 at 37°C, DMEM was replaced with an equal volume of neurobasal medium supplemented with 1% v/v B27, 2 mM Gluta-MAX, 1 mM sodium pyruvate, and 1,000 IU/ml penicillin– streptomycin. After 2 days of culture, a mixture of mitosis inhibitor

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5-fluoro-2-deoxyuridine/uridine (Sigma-Aldrich, St. Louis MO, USA) 4.4 µM was added for 24 h and was then replaced by fresh medium. 2.3 Evaluation of 5-HT1AR and 5-HT7R mRNA levels Hippocampal primary cultures (106 cells) were lysed in TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Total RNA (2,500 ng) was reverse transcribed into cDNA by using Superscript II (Invitrogen, Carlsbad, CA) and 250 ng of random primers (Invitrogen, Carlsbad, CA), following the manufacturer’s protocol.

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Quantitative real-time polymerase chain reaction (RT-PCR) experiments were conducted with a specific set of primers to detect 5-HT1AR and 5-HT7R transcript levels, as we previously described (Rojas et al., 2014). The relative gene mRNA level was calculated based on the delta-delta Ct for each mRNA, normalized to β-actin levels, used as a

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housekeeping gene mRNA.

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2.4 Detection of 5-HT1AR and 5-HT7R by indirect immunofluorescence

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Cells grown on coverslips were fixed and permeabilised at DIV 7 as previously described (Rojas et al., 2014). To detect the presence and distribution of receptors in neurons, we

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conducted double immunofluorescence by incubating with an anti-5-HT1AR antibody or an anti-5-HT7R antibody (see Table 1), both in the presence of a 1:1.000 dilution of anti-

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MAP2a (mouse) (M4403, Sigma-Aldrich, St. Louis MI), to detect MAP2a as a marker of dendritic arbor. The antibody against 5HT1AR has been raised against a recombinant

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protein corresponding to amino acids 218–336, mapping near the C-terminus of the human 5-HT1AR (See Table 1). This antibody detects 5-HT1AR immunoreactivity in the cell

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membrane and cytoplasm in a bladder carcinoma cell line and brain tissue (Stamatakis et al., 2015) and recognizes single bands of ~46 kDa (Siddiqui et al., 2006) in bladder

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carcinoma, oligodendrocytes (Fan et al., 2015), amygdalae and cortex from rat (Raftogianni et al., 2012) and human hippocampus (Mizukami et al., 2011). The antibody against 5HT7R

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was generated with a synthetic peptide, corresponding to amino acids 13-28 of rat 5HT7R (see Table 1). This antibody detects 5HT7R immunoreactivity in rat mesenteric vein

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(Mizukami et al., 2011), and recognizes a single band ~ 50 kDa in rat brain extract (Watts et al., 2015).

After 24 h of incubation with primary antibodies at 4°C, the cells were washed with PBS to visualize serotonin receptors, they were incubated with a 1:500 dilution of anti-rabbit Alexa Fluor 488 (Molecular Probes, Eugene, OR), together with a 1:500 dilution of anti-mouse Alexa Fluor 568 (Molecular Probes, Eugene, OR). Nuclei were then stained with 0.5 µg/ml Hoechst 33342 (Molecular Probes. Eugene, OR). Finally, the cells were washed and mounted on glass slides with fluorescence-preserving medium (Dako Mounting Medium; 7

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Dako, Carpinteria, CA). An epifluorescence microscope (Zeiss, Axioscop 20, Alemania) with a 20x objective was used to detect receptors immunolabeling. 2.4 Pharmacological treatments To determine the effect of serotonin receptor activation, 5-HT, 5-HT1AR antagonist WAY-

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100635 (Sigma-Aldrich, St. Louis MO, USA) and 5-HT7R antagonist SB269970 (Tocris

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Bioscience, Ellisville, MO, USA) were used. To determine the effect of 5-HT on neuronal

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morphology of hippocampal primary cultures, cells were cultured for seven days and then stimulated for 24 h (for morphological studies) or 10 min (signalling studies) with 100 nM

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of 5-HT to assess the effect on neuronal morphology or signalling pathways, respectively. We chose the concentration of 100 nM of 5-HT considering that synaptic release of several

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neurotransmitters, including amines, are usually around 100 nM (Trommershauser et al., 2003). Although the estimated synaptic concentration of 5-HT in brain is around the mM

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range (Bunin and Wightman, 1999), during development, the effect of 5-HT may occur distant from its release site; i.e., acting as paracrine transmitter, in which its concentration

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may vary in the nano and micromolar range (Bunin and Wightman, 1998;1999). Furthermore, 5-HT itself displays affinity with most of its receptors in the 10-20 nM range

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(Newman-Tancredi et al., 1997;Leopoldo et al., 2011). Studies conducted with primary culture of hippocampal neurons have also shown a maximal effect of 5-HT 100 nM in both

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ERK phosphorylation and AKT phosphorylation (Errico et al., 2001;Cowen et al., 2005). Furthermore, WAY-100635 and SB-269970, which are the corresponding 5-HT1AR and 5-

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HT7R antagonists, have an affinity in the nM range (Forster et al., 1995;Hagan et al., 2000). Considering that we used 100 nM of 5-HT in our study, it was estimated that a fully competitive antagonist action would be produced around 1 µM. These two antagonists were added 30 min prior to treatments (both at 1 µM). In order to evaluate the contribution of MEK and PI3K activity in the signalling associated with serotonin receptor activation, hippocampal primary cultures were preincubated during 30 min with 20 µM of UO126 (#9938, Cell Signaling Technology, MA. USA) and 20 µM of Ly294002 (#9901 Cell Signaling, MA. USA), respectively.

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2.5 Morphological evaluation of neurons in culture and after treatments To evaluate morphological changes, the cells were permeabilised as described (Rojas et al.,

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2014). incubated with a 1:20,000 dilution of anti-acetylated tubulin antibody (Sigma-

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Aldrich, St Louis MO, U) to detect neurites at DIV 3, followed by incubation with a 1:300

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dilution of anti-mouse Alexa Fluor 568 (Molecular Probes, Oregon, Canada) (Rojas et al., 2014). On the other hand, neurons at DIV8 were permeabilised and incubated with anti-

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MAP2a antibody to detect dendrites, followed by incubation with a 1:300 dilution of antimouse Alexa Fluor 568 (Molecular Probes, Eugene, OR). Coverslips were then rinsed in

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PBS and incubated with Hoechst 33342 (to visualize the nuclei) and with a 1:500 dilution of rhodamine-phalloidin (Invitrogen, Carlsbad, CA) (to label actin filaments), thus allowing

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the visualization of the dendritic growing tip for the determination of neurite and dendrite length. Finally, the cells were mounted with Dako fluorescent mounting media (Dako,

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Carpinteria, CA). Images were obtained with an IX81 disk scanning unit microscope (Olympus, Tokyo, Japan) at a magnification of 40X. Image J software (NIH,

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rsb.info.nih.gov/ij) was used to merge the fluorescent signals from three fluorophores into a single image. Nuclear staining (blue signal) was used to verify that the signal corresponded

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to a single neuron. Actin staining (red signal) was used to observe the growth cone and dendritic growth at the most distal region.

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At early stage in culture (DIV1-DIV3), neurites do not express microtubule-associated protein 2 (MAP-2) immunoreactivity; nonetheless they can be immunostained with an antibody that recognize stable microtubules (acetylated-tubulin) (Witte et al., 2008). At DIV 3, the primary neurites were defined as any processes, irrespective of its diameter, that extended directly from the neuronal soma, projecting straight forward by decreasing its diameter, and ending in one tip. Its length was determined as the distance between the base and the tip. The longer neurite, which probably differentiates to an axon, was not considered in the analysis. Furthermore, secondary neurites were defined as any diagonal

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branch emerging from a primary neurite and its length corresponded to the distance from the base to its end. At DIV 8, neurons show differentiated axon and dendrites, being the latter immune positive for MAP2a antibody. Primary dendrites were defined as the processes extending directly from the neuronal soma, projecting as a straight extension and ending in one tip. Secondary dendrites were also defined as diagonal branches emanated

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from primary dendrites. Thus, for dendrites, the length between the base and the end of the

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tip was considered.

The numbers and mean lengths of extensions were determined for each individual hippocampal neuron by NeuronJ software. We estimated at DIV 8 the length mean value of

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primary (39.58±1.66 µm) and secondary dendrites (17.11±1.44 µm) and used these criteria to segregate the dendrites according to length: short (≤40 µm) and long (>40 µm) primary

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dendrites and short (≤20 µm) and long (>20 µm) secondary dendrites. The images were renamed randomly as we previously described (Rojas et al., 2014) by using

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BlindTreatment.jar software (Popko et al., 2009), which was generously provided by Dr.

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Lorene Lanier (University of Minnesota). Trace of projections were carried out in NeuronJ and Sholl analysis, conducted with Bonfire 1.0 (Bonnie Firestein Lab Sholl Analysis Software, Rutgers University) software developed for Matlab 7.0 (MathWorks, Natick,

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MA) (Kutzing et al., 2010). NeuronStudio 0.9.92 software (CNIC, Mount Sinai School of

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Medicine) was used to quantify branching points and ending points.

2.6 Protein extraction and western blotting

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Whole cell extracts were obtained as we previously described (Rojas et al., 2011;Rojas et al., 2014). Protein concentrations were measured by using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA), based in the bicinchoninic method (Sapan et al., 1999). Extract samples (15 µg) were resolved on 12% SDS-polyacrylamide gels and were then blotted onto 0.2-µm nitrocellulose (for determination of AKT, ERK1/2, LIMK) or PVDF (for Cofilin determination) membranes. After blocking the membranes in different solutions (Table 1), they were incubated overnight with the appropriate primary antibody diluted in blocking solution. Each Western blot was performed in duplicate. After 10

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rinsing the membranes in TBS-T, blots were incubated with a peroxidase-conjugated antirabbit or anti-mouse secondary antibody (1:10.000, affinity-purified; Thermo Fisher Scientific, Waltham, MA), at room temperature for 2 hr. Membranes were then incubated with an enhanced chemiluminescent (ECL) substrate (Biological Industries, Israel) and exposed to X-ray film (MR-1 Eastman-Kodak, Rochester, NY, USA), or detected by a

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chemiluminescence imager (Syngene, UK). After stripping, membranes were incubated

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overnight in a 1:10.000 dilution of an anti-β-actin antibody (Sigma-Aldrich St. Louis MO,

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USA). Membranes were then incubated with a peroxidase-conjugated anti-rabbit secondary antibody and processed as described above. Band intensities were determined with the UNSCAN-IT program (Silk Scientific, Orem, UT). Data are expressed as the ratio between

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the band intensities of 5-HT1AR or 5-HT7R and that of β-actin.

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2.7 Statistical Analysis

For Sholl analyses, we used two-way ANOVA tests followed by Tukey's post-hoc test. The

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effect of treatments in the number and length of dendrites was analysed by One-way ANOVA, followed by Tukey's multiple comparisons test and in this case, n corresponds to

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the total number of neurons analysed in three independent cultures. Similarly, the statistical significance of differences between densitometric analyses of western blots was assessed

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by either by t-test or one-way analysis of variance followed by Tukey's multiple comparisons test. GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA) was used for

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3. Results

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statistical analyses.

3.1 Morphological differentiation of hippocampal neurons in culture To characterise the morphological maturation of hippocampal neurons, we compared the number, length and complexity of neuronal processes at DIV3 and DIV8. The processes were visualised by immunolabeling with acetylated tubulin (DIV3) or MAP2a (DIV8) and the growing tip was observed by staining actin with rhodamine phalloidin. We used Sholl analysis (Sholl, 1953), which is a morphometric method to measure the arbor complexity

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through the number of intersections between the neurite or dendrite and a series of concentric circles of an increasing radius centered at the cell soma. We compared neurons at DIV3 with DIV 8 (Figure 1A) through the analysis of Sholl curves by two-way ANOVA tests. The analysis revealed a significant main effect of distance from soma (F(9, 68)=177.4, P<0.0001), effect of days in vitro (F(1, 68)=60.82, P< 0.0001), and a significant

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interaction (F(9, 68)= 5.519, P< 0.0001). Tukey’s post-hoc tests indicated that at DIV 8

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there is a significant more complex arbor in the regions more proximal to the cell

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body compared to the DIV3 (P< 0.0001, 10 µm, 20 µm, 30 µm and 40 µm; P< 0.01, 50 µm ; Figure 1A). On the other hand, the variation in arbor complexity

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was not associated with changes in the number (Figure 1B) or average length (Figure 1C) of primary processes. In contrast, increased neuronal complexity that occur

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spontaneously in the culture is fundamentally achieved by a significant raise of almost twice the number (P< 0.001) and the length (P<0.001) of secondary processes (Figure 1B-

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C).

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3.2 Expression and distribution of 5-HT1AR and 5-HT7R in hippocampal neurons in culture

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To explore the role of 5-HT1A and 5-HT7 receptors on morphological development in cultured hippocampal neurons, and considering that the stimulation with 5-HT was to be

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conducted during 24 h from DIV7, we compared 5-HT1AR and 5-HT7R mRNAs levels at DIV2 and DIV7. Quantitative PCR analyses of hippocampal cultures showed at DIV7

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significant raise of almost twice the levels of 5-HT1AR mRNA vs DIV2 (P<0.01, Figure 2A). Furthermore, as shown in Figure 2B, protein levels of this receptor determined by Western blot showed a trend to increase (P= 0.07) at DIV7 vs DIV2. Additionally, 5-HT7R mRNA levels at DIV7 did change almost three folds in comparison to DIV2 (P< 0.001, Figure 2A); variation that correlate with the increased protein levels for the 5-HT7(a)R and 5-HT7(b)R isoforms at DIV7 (P<0.05, Figure 2B). In order to visualize the distribution of these receptors, we used immunofluorescence combined with a dendritic marker (antiMAP2A) and an antibody directed against the third loop of the 5-HT1AR. We observed a predominantly somatic immunoreactivity at DIV8. Using an antibody that recognizes an 12

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extracellular epitope of the 5-HT7R, we observed 5-HT7R immunoreactivity at the soma and also in primary and secondary dendrites at DIV8 (Figure 3). These results demonstrate the differential expression and distribution of both serotonin receptors, suggesting a specific

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function for 5-HT1AR and 5-HT7R in neuronal function.

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3.3 Effect of 5-HT1AR and 5-HT7R on neuronal morphology

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To determine the effect of 5-HT1AR and 5-HT7R on dendrite outgrowth, hippocampal neurons at DIV 7 were pre-incubated for 30 min with 1 µM of 5-HT1AR and 5-HT7R antagonists (WAY-100635 and SB269970 respectively), or the cells were left untreated.

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Neurons were subsequently stimulated with 100 nM 5-HT for 24 h; i.e., up to DIV 8. Figure 4A shows representative images of control neurons at DIV 8 and after 24 h of

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incubation with 100 nM 5-HT, in the presence or absence of the antagonist (Figure 4A). Dendrites and growing dendritic tip were visualised as described above. Qualitative

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analyses showed that stimulation with 5-HT produced a reduction in the number and length

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of dendrites (Figure 4A). Additionally, this reduction seemed to be prevented by WAY100635, a 5-HT1AR antagonist, but not by SB269970, a 5-HT7R antagonist (Figure 4A). The two-way ANOVA analysis of Sholl curves obtained from control and 5-HT treatments

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either in the presence or absence of WAY-100635 revealed a significant main effect of distance from soma (F(9, 548)=930.4, P<0.0001), effect of treatments (F(3,548)=43.53, P<

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0.0001), and a non-significant interaction (F(27, 548)=1.754, P> 0.2). Tukey’s post-hoc tests revealed that the addition of 5-HT for 24 h significantly reduced the number of

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intersections within a 30-50 µm radius from the soma (Figure 4B). Furthermore, after the blockage of 5-HT1AR with WAY-100635, addition of 5-HT produces a rightward shift of the Sholl plot, compared to control and 5-HT treatment (Figure 4B). A detailed analysis indicated a significant increase in the number of intersections within a 20-70 µm radius from the soma, in comparison to the effect of 5-HT alone, suggesting a complex action of 5-HT through its receptors, along with a repressive action of 5-HT1AR on dendrite complexity. Additionally, a significant difference between control and neurons treated with 5-HT in the presence of WAY-100635 was observed within a 30-50 µm radius from the

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soma, suggesting that this antagonist may produce some effect on neuronal morphology. Furthermore, the two-way ANOVA analysis of Sholl curves obtained from control and 5HT treatment in the presence and absence of SB-269970 revealed a significant main effect of distance from soma (F(9, 478)=704.5, P<0.0001), effect of treatments (F(3,478)=11.29, P< 0.0001), and a non-significant interaction (Figure 4B). Tukey analysis showed that after

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the blockage of 5-HT7R with SB-269970, 5-HT treatment produced a leftward shift of the

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Sholl plot, compared to control, with a significant reduction in the number of intersections

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at 20 µm (Figure 4C). In order to discard the effect of antagonists alone, individual effects of WAY-100635 and SB-269970 on neuron complexity were also evaluated by the Sholl analysis (See Supplementary Material, Figure S4). Two way analysis revealed an effect of

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distance from soma (F(9,134)=398.5, P<0.0001), effect of treatments (F(2,134)=21.91, P=0.026), and a non-significant interaction (F(18,134)= 5.605, P> 0.5). Tukey´s analysis

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indicated that only SB-269970 affects neuronal complexity by producing a reduction in the

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number of intersections at 20 µm (See Supplementary Material, Figure S4).

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To conduct a detailed analysis of the effects of 5-HT1AR and 5-HT7R on neuronal morphology, we measured changes in the number of primary dendrites (Figure 5A). Oneway ANOVA analysis revealed significant differences among treatments (F(3,628)=3.640,

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P=0.0127). Post-hoc Tukey’s analysis showed that stimulation with 5-HT did not promote changes in the number of primary dendrites; however, after blocking 5-HT1AR with

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WAY100-635, the addition of 5-HT for 24 h significantly increased the number of primary dendrites (**P< 0.01; Figure 5A). Interestingly, 5-HT reduces significantly the growth of

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primary dendrites through the activation of 5-HT1AR (Figure 5 B). In order to evaluate whether 5-HT differentially affected the dendrites according their length, we decided to segregate primary dendrites as being short (≤ 40 µm) and long (> 40 µm; see Material and Methods). We observed that treatments significantly affected the number (F(3,628)=3.087; P=0.0267, Figure 5C), but not the length of short primary dendrites (Figure 5D), being this effect insensitive to the 5-HT1AR and 5-HT7R antagonists (Figure 5C and 5D). In contrast, both the number (F(3,628=7.000 P=0.0001) and mean

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length of long primary dendrites (F(3,628)=4.497 P=0.0039) were reduced by 5-HT stimulation (P<0.05, P<0.01; Figure 5C and D respectively) effects only prevented by WAY-100635 (P<0.001, P<0.01; Figure 5C and D respectively). These results indicate that 5-HT1AR specifically reduces the number and length of long primary dendrites.

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Additionally, 5-HT does not change the number and mean length of secondary dendrites

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(Figure 5E and 5F). However, when secondary dendrites were segregated according to their

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length, we observed that short secondary dendrites (≤ 20 µm) did not vary their number under 5-HT exposure (Figure 5G). Nonetheless, their length was affected by treatments (F(3,628)=6.138, P=0.0006); 5-HT increased the length of short dendrites (P<0.01) and this

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effect was sensitive to the pharmacological blockage of 5-HT1AR (P<0.05) and 5-HT7R (P<0.01) (Figure 5H). ANOVA analysis showed that treatments affect the number

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(F(3,628)=3.540; P=0.0145; Figure 5G) but not the mean length (Figure 5H) of long secondary dendrites (>20 µm). Although ANOVA post-hoc analysis did not reveal a

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significant effect of 5-HT, t-test analysis indicated that reduces the number of long

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secondary dendrites. Besides, pharmacological treatment revealed only the participation of 5-HT1AR in this effect (Tukey analysis P<0.01; Figure 5G). These results show that 5-HT 1AR

and 5-HT7R have a similar action promoting short secondary dendrites growth, while

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5-HT1AR has an additional effect restricting both the number of long primary and

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secondary dendrites and the outgrowth of long primary dendrites.

Moreover, individual effects of WAY-100635 and SB-269970 on the number and mean

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dendrite length was evaluated. We did not observe any effect in these parameters (Supplementary Figures S5A and S5B).

3.4 Effect of 5-HT1AR and 5-HT7R on mitogen activated protein kinase activation in hippocampal cultures The mitogen-activated protein kinase (MAPK) pathway, which transduces extracellular signals at the cell membrane into nuclear events, has been shown to be implicated in dendrite outgrowth (Kumar et al., 2005). The representative immunoblot depicts total and 15

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phosphorylated protein levels for each treatment (Figure 6A). By one-way ANOVA analysis (F(3,11)=7.543, P=0.005) and post-analysis, we demonstrated that acute incubation with 100 nM 5-HT (10 min) induces at least a 2-fold increase in ERK1/2 phosphorylation, in comparison to control (P<0.05). This effect was blocked by the 5HT1AR (P<0.05) and 5-HT7R specific antagonists (P<0.05) (Figure 6A). Pre-treatment with

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30 μM UO126, an agent that inhibits MEK1/2 activity, reduced both basal and 5-HT-

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stimulated activation of ERK1/2 (Figure 6B), suggesting that 5-HT activates MEK.

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3.5 Effect of 5-HT1AR and 5-HT7R on AKT-GSK3β signalling in hippocampal cultures PI3K/Akt signalling through the regulation of downstream effectors such as mammalian

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target of rapamycin (mTOR) and glycogen synthase kinase 3β (GSK-3β) has been implicated in neurite outgrowth, including elongation, calibre and branching (Read and

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Gorman, 2009). A representative immunoblot shows that acute treatment with 100 nM 5HT increases the phosphorylation of Akt at Ser473 (Figure 7A). One-way ANOVA test

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revealed differences among groups (F(3,11)=6.448, P=0.0089) and post-test analysis

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revealed a significant enhancement of phospho-AKT levels (p<0.01). Interestingly, both 5HT1AR (p<0.05) and 5-HT7R (p<0.05) antagonists prevented 5-HT-induced AKT phosphorylation (Figure 7A). Pre-treatment with 20 μm LY294002, an agent that inhibits

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PI3K activity, reduced both basal and 5-HT-stimulated activation of Akt (Figure 7B); suggesting that 5-HT activates PI3K. We next evaluated whether 5-HT-induced

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phosphorylation in AKT triggered GSK3β inhibition. We found that 5-HT, along with 5-

7C).

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HT1AR and 5-HT7R, did not modify the phosphorylation state of GSK3β in Ser9 (Figure

3.6 Effect of 5-HT on LIMK and cofilin signalling in hippocampal cultures The Rho/Rac GTPase subfamily plays a key role in regulating axonal and dendritic morphogenesis through actin cytoskeletal reorganization (Luo, 2000). To evaluate the possible participation of serotonin receptors in this pathway, we determined the activation of LIM kinase, which inactivates (by phosphorylation) the actin-depolymerizing factor/cofilin. The Figure 8A shows a representative western and the analysis of treatments 16

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indicated that acute stimulation of hippocampal neurons with 5-HT did not promote any variation in the phosphorylation levels of both proteins (Figure 8B and C). Furthermore, 5HT1AR and 5-HT7R blockage did not evoke any effect (Figure 8).

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4. Discussion

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In primary cultures of hippocampal neurons, we demonstrated that both isoforms of 5-HT7-

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R (a and b) proteins progressively increase their expression during the maturity of the culture, while 5-HT1AR remained relatively constant. By immunofluorescence, we observed

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at DIV 8 that the 5-HT1AR was predominantly expressed at the soma and that the 5-HT7R had a somato-dendritic distribution. Furthermore, we determined that 5-HT1AR reduces the

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number of long primary and secondary dendrites and also restricts the length of long primary dendrites; while both 5-HT1AR and 5-HT7R promote the outgrowth of short

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secondary dendrites (see Figure 9). These results suggest that at the neural stage studied, both receptors influence neuronal morphology, but not in a fully redundant way, which

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could be related to the effects of 5-HT during early postnatal development.

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Levels and cellular distribution of the serotonin receptors 5-HT1A and 5-HT7 5-HT is a neurotransmitter that is detected from the early stages of CNS development

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(Goldstein et al., 1984), where it regulates important events such as cell migration, neuronal connectivity, and cytoarchitecture (Lauder, 1993;Gaspar et al., 2003;Cote et al.,

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2007;Migliarini et al., 2013). Reduction in the levels of 5-HT through pharmacological strategies during embryonic development has been shown to produce long-term effects, such as decreased dendritic complexity (Vitalis et al., 2007). Additionally, deletion of the Tph2 gene produces serotonergic hyper- or hypo-innervation, depending on specific targets in the brain (Migliarini et al., 2013;Mosienko et al., 2014). These evidences strongly suggest that 5-HT is essential in determining neuronal circuitries during the critical period in which neuronal wiring occurs. However, the specific role of the 5-HT receptors during this neuronal wiring process has not been well established. Early expression of 5-HT1AR and 5-HT7R has been detected during embryonic development, suggesting a pivotal role for 17

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both receptors in the formation of the CNS (Hillion et al., 1993;Miquel et al., 1994;Hedlund and Sutcliffe, 2004;Patel and Zhou, 2005;Kobe et al., 2012). We have recently reported that 5-HT1A mRNA and protein are both expressed at an immature stage (DIV 2-3) in cultured hippocampal neurons (Rojas et al., 2014). In the present study, we have demonstrated that 5-HT1AR mRNA increases, and that its protein levels trend to the

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rise between DIV 2-7. These results are consistent with the early expression of 5-HT1AR in

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hippocampal neurons at the E16 stage and the programmed onset of its expression upon

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completion of terminal mitosis has suggested its participation in neuronal migration (Patel and Zhou, 2005). Furthermore, the most rapid rate of growth in apical dendrites of pyramidal neurons occurs during P0 and P10 (Patel and Zhou, 2005), which is coincident

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with the highest expression levels of hippocampal 5-HT1AR (Patel and Zhou, 2005). In whole, these antecedents support a role for the 5-HT1AR in the control of dendritic

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development in the hippocampus. Furthermore, at early postnatal development (P5), the 5HT1AR is located mainly on the soma, but at a more advanced stage of postnatal

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development (P21), is present mainly on the dendrites of stratum radiatum and stratum

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oriens of the hippocampus (Patel and Zhou, 2005). This evidence suggests the existence of some mechanisms that determine somatic or dendritic trafficking of the 5-HT1AR, to a location that probably determines its function and/or the coupling to certain transductional

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cascades. In neurons, Yif1B protein is an adapter that interacts with the C-terminus of 5HT1AR, an interaction which is crucial for recruitment and trafficking toward the distal

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portion of the dendritic tree (Carrel et al., 2008;Al Awabdh et al., 2012). Experiments with transfected hippocampal neurons that overexpressed 5-HT1AR, have permitted its detection

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in somato-dendritic compartments at DIV7, a pattern mediated by Yif1B (Carrel et al., 2008). However, we have determined the cellular distribution of 5-HT1AR at DIV8, detecting immunoreactivity mainly at soma. Nonetheless, in our study, it is plausible that the endogenous levels of 5-HT1AR are not enough to visualize its dendritic targeting. Considering that 5-HT1AR and 5-HT7R are co-expressed in different brain structures, we evaluated the presence of 5-HT7R in primary cultures of hippocampal neurons. We detected an increased expression of 5-HT7R mRNA between DIV 2-7. Moreover, protein levels of the two 5-HT7R isoforms (a and b), derived from splice variants (Heidmann et al., 1997),

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increase between DIV 2 and 7. These results suggest that in vitro, the expression of 5-HT7R isoform proteins occurs in a regulated fashion, depending on the degree of neuronal maturation. In addition, localization of 5-HT7R seems to be developmentally regulated. We visualized the 5-HT7R at DIV8 in the soma and dendrite arbor (i.e., primary and secondary dendrites), a pattern which is similar to that observed when exogenous 5-HT7R is

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transiently expressed in hippocampal cultures obtained from P1-P2 animals (Kvachnina et

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al., 2005).

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Additionally, high levels of 5-HT7R mRNA have been found in the hippocampus at early postnatal stages (P2 and P6), which are reduced in more advanced periods of postnatal development (P12-P90) (Kobe et al., 2012). Concordantly, a similar profile for the 5-HT7R

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protein has been noted in the hippocampus; i.e., high level at P1, followed by a reduction at P7 (Muneoka and Takigawa, 2003). After that, its expression increases again at P15,

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reaching the adult level (Muneoka and Takigawa, 2003). Interestingly, the highest immunoreactivity of 5-HT7R in the CA1 hippocampal area observed at P2 and P6 stages, is

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accompanied by an increase in Gα12 mRNA levels, a heterotrimeric G protein that

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regulates the activity of small monomeric GTPases (Kobe et al., 2012). The Gα12 protein is involved in the regulation of morphology in non-neuronal cell models and primary neuronal cultures (Buhl et al., 1995;Kvachnina et al., 2005). Considering these evidences, the

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variations in 5-HT7R levels observed during the postnatal period may be related to specific

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actions of 5-HT in the hippocampus, mainly in the early stages of brain development. Activation of 5-HT1A and 5-HT7 receptors and their role in dendritic morphology

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Currently, there is little information regarding how serotonin receptors contribute to the regulation of neuronal morphology and the possible signal transduction cascades involved. To conduct an evaluation of 5-HT on neuronal morphology, we decided first to characterise the morphological changes induced by spontaneous neuronal maturation, by contrasting the number of neuronal processes and their length at different time points after plating. When comparing neurons of DIV3 and DIV8, we observed that the genesis of primary processes is reduced; nonetheless the growth and number of secondary extensions are still occurring at more advanced neuronal maturation stages. With this characterization, we evaluated the

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effect of 5-HT for 24 hours on the morphology of cultured hippocampal neurons at DIV 7. Furthermore, using specific antagonists, we dissected the contribution of 5-HT1AR and 5HT7R in a particular morphological effect. We chose this experimental approach considering that 8OH-DPAT- widely used as specific 5-HT1AR agonist- has the ability to bind other serotonin receptors, such as 5-HT7R and 5-HT4R, and also to dopamine

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receptors (Stiedl et al., 2015). According to Sholl analyses, only SB269970 influence the

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neuronal morphology in absence of 5-HT. This antagonist produced a reduction in the

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number of intersection at a 20 µm radius from the soma, with no effect at other distances. The SB-269970 may act as inverse antagonist through its binding to constitutively active 5HT7R, producing a stabilization of them, and thus reducing the activity (negative intrinsic

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activity). Studies in human recombinant 5-HT7R expressed in CHO-K1 cells (Mahe et al., 2004) and in HEK-293 cell line expressing rat 5-HT7R (Sprouse et al., 2004) support this

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notion. Besides, the action of SB-269970 as inverse agonists may be overridden in the presence of 5-HT, showing in this case its action as antagonist (Sprouse et al., 2004).

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Nonetheless, to date there is no information whether basal activity of 5-HT1AR or 5-HT7R

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is observed in non-transfected cell that express endogenous receptor. In the present study we have shown that 5-HT treatment for 24 h reduced the number of

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intersections within a 30-50 µm radius from the soma, suggesting an inhibitory action for this monoamine. Additionally, after blockage of 5-HT1AR with WAY-100635, the addition

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of 5-HT produces a rightward shift of the Sholl plot, compared to control and 5-HT treatments, increasing the number of intersections within a 20-70 µm radius from the soma.

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These data suggest a complex action of 5-HT through its receptors, and part of these effects can be explained by a repressive action of 5-HT1AR on dendrite complexity. Furthermore, the blockage of 5-HT7R did not influence dendrite complexity according to the Sholl analyses. A more detailed analysis of dendritic morphology showed that 5-HT has different effects in dendrites, depending on the type (primary v/s secondary) and degree of dendritic outgrowth (short v/s long), probably mediated by different receptors. For instance, 5-HT increased the number of short primary dendrites (i.e., ≤ 40 µm), an effect that was not related to 5-HT1AR or 5-HT7R. Additionally, as the activity of 5-HT4R and 5-HT3R reduce

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the number of dendrites, the effect we observed is probably related to other serotonin receptors (Kvachnina et al., 2005;Engel et al., 2013;Wirth et al., 2016).

On the other hand, we observed that 5-HT1AR reduces the number of both long primary and secondary dendrites, and reduces the length of long primary dendrites. Paradoxically, 5-

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HT1AR activation promotes an increase in the average length of short secondary dendrites.

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Therefore, the 5-HT1AR has a dual effect on dendritic morphology: it prevents the both the

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formation and growth of long dendrites and encourages the growth of shorter secondary dendrites. The overall effect of 5-HT1AR can be interpreted as a receptor whose activity restricts the density of long dendrites (i.e., either primary or secondary), while promotes the

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growth of short secondary dendrites and in this way, increases the dendritic field. Our results are in agreement with those reported by the group of Ferreira, who suggested that 5-

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HT1AR restricts dendritic growth in hippocampal neurons (Ferreira et al., 2010). By in vivo pharmacological blockade of 5-HT1AR with WAY-100635 during 3-5 weeks of postnatal

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development, there was a significant increase in branch points in the portion of the apical

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dendritic tree, without differences in the most distal and basal dendrites (Ferreira et al., 2010). Importantly, pharmacological inhibition in this study was performed at a developmental time frame when 5-HT7R mRNA levels decrease (Kobe et al., 2012). These

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changes are also consistent with the results obtained in knock-out animals, in which 5HT1AR activation during postnatal weeks 3-5 is required to restrict apical dendritic growth.

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The Ferreira study also showed that in hippocampal neurons from mice (DIV 4-7) incubated during 72 h with 5-HT, branching points of tertiary dendrites selectively decrease

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(Ferreira et al., 2010). However, no pharmacological tool was used to identify the serotonin receptors involved. However, these results are in accordance with ours, regarding the effects of 5-HT on dendritic complexity, which was decreased between 30-50 µm, according to the Sholl analysis.

On the other hand, in mouse hippocampal cultures obtained at P1-P2, 5-HT7R promotes increase in the total length of neurites, without changes in their number (Kvachnina et al., 2005). In agreement, we have previously described that the stimulation of 5-HT7R at DIV 2

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during 24 h promotes the growth of secondary neurites (Rojas et al., 2014). Importantly, this growth promoting effect of 5-HT7R is still preserved in more mature hippocampal neurons and it is redundant with that of 5-HT1AR in short secondary dendrites. In contrast, an additional growth limiting effect of 5-HT1AR appears at more mature neuronal stages (DIV 8). Thus, we can expect that the positive redundant effects of 5-HT1AR and 5-HT7R

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on short secondary dendritic growth are probably mediated by common signalling

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transduction pathways, while the negative effect of 5-HT1AR upon long primary (restricting

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number and outgrowth) and secondary dendrites (restricting their outgrowth) would be mediated by an exclusive pathway.

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From a mechanistic point of view, stimulation of primary cultures of mouse hippocampus at DIV 5 with 5-HT promotes depolymerisation of filamentous actin in the cone growth, an

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effect observed in WT mice and but not in KO mice for 5-HT1AR (Ferreira et al., 2010). Therefore, 5-HT1AR would regulate actin dynamics and restrict dendritic growth and thus,

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modulate neuronal connectivity during a certain period of development (Ferreira et al.,

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2010). Our results regarding the inhibitory action of 5-HT1AR in long primary dendrites, may explain the restrictive effects reported by Ferreira et al. (2010). However, the detailed mechanism has not been described. It is plausible that the activation of 5-HT1AR or 5-HT7R

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mediates modification of actin cytoskeleton dynamics. In this regard, actin depolimerising factor (ADF)/cofilin promotes the severing of actin filaments and actin dynamics by

regulated

by

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accelerating the treadmilling of actin filaments (Carlier et al., 1997). Cofilin is negatively phosphorylation

through

upstream

signalling

pathways

involving

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Rho/Rac/cdc42 small GTPases. Using neuronal cell lines, Cdc42 and Rac1 were reported to induce, while Rho inhibited neurite growth and branching (Carlier et al., 1997). Interestingly, stimulation of 5-HT7R-expressing NIH-3T3 cells with 5-HT has been reported to induce filopodia formation and cellular rounding, through the activation of RhoA and Cdc42 (Kvachnina et al., 2005). Nonetheless, in the present study, we described that neither 5-HT1AR nor 5-HT7R are able to promote activity of transductional pathways related to Rho GTPases/LIMK/cofilin (Maekawa et al., 1999); suggesting that these receptors may control neuronal growth and arbor complexity through other mechanisms. It

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is important to note that in the present study, the activation of both 5-HT1AR and 5-HT7R produced the same effect on secondary dendrite morphology; i.e., they promote the growth of the short secondary dendrites. Along with our previous study (Rojas et al., 2014), these findings demonstrated that both receptors play similar functions along different stages of maturation. Thus, one can assume that the activation of 5-HT7R and 5-HT1AR evokes

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similar transduction signaling pathways that may converge somehow to promote dendrite

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outgrowth. Interestingly, we observed that both 5-HT1AR and 5-HT7R promote ERK and

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AKT phosphorylation, effects that may be related to dendrite outgrowth. Furthermore, activation of ERK1/2 and AKT induced by 5-HT was prevented by MEK and PI3K inhibitors, respectively. Evidences indicate that PI3K activity promotes both the branching

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and growth of dendrites during a stage of development in cultured hippocampal neurons (7–14 DIV) (Jaworski et al., 2005). Additionally, overexpression and loss-of-function

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experiments (LY294002, rapamycin, and/or RNAi) has allowed to assign a pivotal role of PI3K (Cuesto et al., 2011), along with the downstream pathway Akt–mTOR, in the control

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of protein synthesis, which seems to be required for the regulation of dendritic arborization

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(Jaworski et al., 2005). Additionally, an interaction between PI3K–Akt–mTOR and RasMAPK signaling has been described, in which both pathways cooperatively regulate the

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development of dendrite complexity and elongation (Kumar et al., 2005).

More recently, 5-HT7R activation has been described to promote neurite outgrowth in

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cortical and striatal primary cultures through the activation of mTOR, cdc42, Cdk5 and ERK pathways; i.e., pathways that participate in the reorganization of cytoskeletal proteins,

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and that may sustain neurite outgrowth (Speranza et al., 2013;Speranza et al., 2015). Based on these findings, a potential signal transduction pathway has been proposed, in which 5HT7R-induced activation of heterotrimeric Gαs-protein promotes ERK1/2 activity and therefore, impacts microtubule stabilization (Volpicelli et al., 2014). Besides, the differential pattern of 5-HT1AR and 5-HT7R distribution on neurons (i.e., somatic vs somatodendritic location) and the variation in the ratio of 5-HT1AR/5-HT7R may play a role in determining the number of dendrites and growth of dendritic trees. In vivo and in vitro studies have shown that both 5-HT7R and 5-HT1AR could homo- and heterodimerize. As a

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functional consequence of heterodimerization, the activation of the Gαi protein mediated by 5-HT1A decreases, without affecting the activation of the Gαs protein mediated by 5-HT7R (Renner et al., 2012). In the same study, hippocampal neurons expressed 5-HT1AR and 5HT7R and were highly co-localised at the plasma membrane (Kobe et al., 2012). In addition, this study showed that receptor heterodimerization alters the profile of 5-HT1AR

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internalization and that the proportion of receptor heterodimers can vary during

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development, suggesting that there may be heteroregulation of the activity of 5-HT1AR and

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5-HT7R (Kobe et al., 2012). We highlight that there is an increase in the protein levels of 5HT7R and a trend to increase in 5-HT1AR during neuronal maturity in culture. The functional cross-talk mediated by physical interaction between 5-HT1AR and 5-HT7R has

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been recently reviewed as being important in some neuropsychiatric disorders, such as depressive disorder (Naumenko et al., 2014). Thus, changes in the targeting of these

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receptors on neuronal membranes (somatic vs dendritic), together with variations in the ratio of 5-HT1AR/5-HT7R during neuronal maturity, could define the differential effect of

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5-HT on morphology during development. The redundant action of 5-HT1AR and 5-HT7R

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on neuronal morphology could be triggered by each receptor independently or mediated by the physical interaction between these receptors at specific cellular compartments, while the

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negative action of 5-HT1AR on neuronal morphology would be driven independently. Conclusion

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The present study describes the expression of 5-HT1AR and 5-HT7R during neuronal maturation in vitro and their effect on dendritic morphology and signal transduction

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pathways. We showed that both receptors are distributed differently in the neuron. Moreover, we determined that 5-HT1AR inhibits the growth (number and length) of long primary dendrites, suggesting that this receptor may slow down overall dendritic growth in the neuronal stage studied (DIV 8), and explaining why there is no further increase in the number and average length of primary dendrites from DIV 3 to 8. However, both 5-HT1AR and 5-HT7R promote the growth of short secondary dendrites and also promote AKT and ERK activation, an effect that would be related to the increased dendritic complexity at DIV 8. We conclude that in mature stages; i.e., a timing in which neuronal complexity is

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being established, a new inhibitory effect of 5-HT1AR is observed on primary dendrites (Figure 9). Finally, these results contribute to understanding how serotonin acts as a pleiotropic trophic molecule during CNS development, and how its alteration can predispose subjects to acquire future pathologies associated with serotonergic

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neurotransmission.

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Conflict of interest The authors declare that there is no conflict of interest.

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Acknowledgements Authors thank Ana María Avalos for proofreading the article and Nicolas Parra-Fiedler for figure editing.

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This work was supported by FONDECYT [grant N°108-0489 to JL Fiedler], Fondo Central de Investigación, Universidad de Chile [Grant N° ENL025/16 to JL.Fiedler]; Fondo PEEI, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile; Ph.D fellowship from CONICYT [grant N° D-21070424 and AT-24080056 to PS Rojas].

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Kobe, F., Guseva, D., Jensen, T.P., Wirth, A., Renner, U., Hess, D., Muller, M., Medrihan, L., Zhang, W., Zhang, M., Braun, K., Westerholz, S., Herzog, A., Radyushkin, K., El-Kordi, A., Ehrenreich, H., Richter, D.W., Rusakov, D.A., and Ponimaskin, E. 2012. 5-HT7R/G12 signaling regulates neuronal morphology and function in an age-dependent manner. J Neurosci 32, 2915-2930. Kumar, V., Zhang, M.X., Swank, M.W., Kunz, J., and Wu, G.Y. 2005. Regulation of dendritic morphogenesis by Ras-PI3K-Akt-mTOR and Ras-MAPK signaling pathways. J Neurosci 25, 11288-11299. Kutzing, M.K., Langhammer, C.G., Luo, V., Lakdawala, H., and Firestein, B.L. 2010. Automated Sholl analysis of digitized neuronal morphology at multiple scales. J Vis Exp. Kvachnina, E., Liu, G., Dityatev, A., Renner, U., Dumuis, A., Richter, D.W., Dityateva, G., Schachner, M., Voyno-Yasenetskaya, T.A., and Ponimaskin, E.G. 2005. 5-HT7 receptor is coupled to G alpha subunits of heterotrimeric G12-protein to regulate gene transcription and neuronal morphology. J Neurosci 25, 7821-7830. Lauder, J.M. 1993. Neurotransmitters as growth regulatory signals: role of receptors and second messengers. Trends Neurosci 16, 233-240. Leopoldo, M., Lacivita, E., Berardi, F., Perrone, R., and Hedlund, P.B. 2011. Serotonin 5HT7 receptor agents: Structure-activity relationships and potential therapeutic applications in central nervous system disorders. Pharmacol Ther 129, 120-148. Lesch, K.P., and Waider, J. 2012. Serotonin in the modulation of neural plasticity and networks: implications for neurodevelopmental disorders. Neuron 76, 175-191. Luo, L. 2000. Rho GTPases in neuronal morphogenesis. Nat Rev Neurosci 1, 173-180. Maekawa, M., Ishizaki, T., Boku, S., Watanabe, N., Fujita, A., Iwamatsu, A., Obinata, T., Ohashi, K., Mizuno, K., and Narumiya, S. 1999. Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase. Science 285, 895898. Mahe, C., Loetscher, E., Feuerbach, D., Muller, W., Seiler, M.P., and Schoeffter, P. 2004. Differential inverse agonist efficacies of SB-258719, SB-258741 and SB-269970 at human recombinant serotonin 5-HT7 receptors. Eur J Pharmacol 495, 97-102. Mattson, M.P., Maudsley, S., and Martin, B. 2004. BDNF and 5-HT: a dynamic duo in agerelated neuronal plasticity and neurodegenerative disorders. Trends Neurosci 27, 589-594. Migliarini, S., Pacini, G., Pelosi, B., Lunardi, G., and Pasqualetti, M. 2013. Lack of brain serotonin affects postnatal development and serotonergic neuronal circuitry formation. Mol Psychiatry 18, 1106-1118. Miquel, M.C., Kia, H.K., Boni, C., Doucet, E., Daval, G., Matthiessen, L., Hamon, M., and Verge, D. 1994. Postnatal development and localization of 5-HT1A receptor mRNA in rat forebrain and cerebellum. Brain Res Dev Brain Res 80, 149-157. Mizukami, K., Ishikawa, M., Akatsu, H., Abrahamson, E.E., Ikonomovic, M.D., and Asada, T. 2011. An immunohistochemical study of the serotonin 1A receptor in the hippocampus of subjects with Alzheimer's disease. Neuropathology 31, 503-509. Mosienko, V., Matthes, S., Hirth, N., Beis, D., Flinders, M., Bader, M., Hansson, A.C., and Alenina, N. 2014. Adaptive changes in serotonin metabolism preserve normal behavior in mice with reduced TPH2 activity. Neuropharmacology 85, 73-80. 28

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FIGURE LEGENDS

Figure 1. Comparison of the morphological complexity during the culture of

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hippocampal neurons. Neurons were maintained in culture in serum-free medium

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(Neurobasal supplemented with B27) for 3 or 8 days in vitro (DIV). (A) Representative neurons cultured at 3 DIV or 8 DIV. (B) Sholl analysis shows the number of intersections

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of neuronal processes as a function of path length from the soma. Neuronal processes represent (i) neurites at DIV 3 that were immunostained with acetylated tubulin and (ii)

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dendrites at DIV 8 that were immunostained with anti-MAP 2A. F-actin was stained with rhodamine-phalloidin. The number of intersections was determined by the Bonfire

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program. (C) The number and mean length (D) of primary processes (neurites or dendrites) were determined for each individual hippocampal neuron using the NeuronJ software.

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Experiments were conducted three times in triplicate. The n, corresponds to the total number of cell analysed; n=35 for DIV3 and n= 35 for DIV8. **P< 0.01; ***P< 0.001;

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****P< 0.0001.

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Figure 2. Variation of 5-HT1AR and 5-HT7R mRNAs and protein levels of hippocampal neurons from 2 to 7 DIV. (A) Detection of 5-HT1AR and 5-HT7R mRNAs in hippocampal cultures at DIV 2 and 7. The specific mRNAs were detected by real-time qPCR using β-actin as a housekeeping gene and data were normalized to DIV 2 in order to visualize changes during neuron maturity (n=7). (B) Representative western blots showing

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5-HT1AR, 5-HT7R levels, and β-actin in extracts from cultures of DIV 2 and 7. The anti-5-

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HT1AR antibody detected one band of approximately 65 kDa and the anti-5-HT7R antibody

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detected two bands of approximately 50 (5-HT7a) and 45 kDa (5-HT7b). Relative protein levels were analysed by densitometry with β-actin as a loading control. The graph shows variations of 5H1AR, 5-HT7(a)R and 5-HT7(b)R levels during the evolution of the culture

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(DIV 2 n=6 and DIV7 n=7). The data represent mean ± SEM normalized against the levels observed at DIV 2. The data were analysed using one-way ANOVA followed by Tukey’s

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post-hoc test, *P< 0.05; **P< 0.01; *** P< 0.001 in one tailed t-test.

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Figure 3. Distribution of 5-HT1AR and 5-HT7R receptors in cultured hippocampal neurons. Neurons were cultured for eight days, fixed and immunostained with MAP2A (red fluorescence), anti 5-HT1A-R or anti 5-HT7-R (green fluorescence), and with Hoechst dye for nuclear staining (blue fluorescence). An epifluorescence microscope (Zeiss,

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Axioscop 20, Germany) with a 20x objective was used. The bar represents 50 µm.

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Figure 4. Representative images used for morphometric analyses and the effect of 5HT on neuronal morphology of hippocampal primary cultures. On the 7th day in vitro (DIV7), the cells were stimulated for 24 h with 100 nM 5-HT. The antagonists WAY100635 (1 µM) and SB269970 (1 µM) were added 30 min prior to 5-HT. At DIV 8, cells were fixed and immunostained for MAP2a (green fluorescence), stained with rhodamine-

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phalloidin (F-actin marker, red fluorescence) and with Hoechst dye for nuclear staining

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(blue fluorescence). A DSU microscope with a 40x objective was used, and channels were

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merged with the ImageJ program. (A) Representative fluorescence micrographs of untreated neurons (control) or neurons treated with 100 nM 5-HT, 100 nM 5-HT + 1 µM WAY-100635 or 100 nM 5-HT + 1 µM SB269970 are shown. Addition of 5-HT reduced

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the number and length of primary dendrites, effect prevented by the 5-HT1AR antagonist. The bar represents 50 µm. (B) Sholl analysis shows the number of intersections of dendrites

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as a function of path length from the soma. The number of intersections was determined by the Bonfire program. Neurons from independent cultures were analyzed: control (n=280),

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5-HT (n=119), 100 nM 5-HT + 1 µM WAY-100635 (n=152) and 100 nM 5-HT + 1 µM

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SB269970 (n=81). These neurons were processed for various parameters, as shown in subsequent figures. The data represent the mean ± SEM. The insert shows the effect of antagonists alone on neuronal complexity (n=33 for WAY-100635 and n=35 for

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SB269970). The data were analyzed using ANOVA for each condition, followed by the Tukey’s post-hoc test. Post-hoc test: Control vs 5-HT: *P< 0.05; ** P< 0.01; Control vs 5-

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HT + WAY-100635: × P< 0.05 and 5-HT vs 5-HT + WAY-100635: ≠P<0.05, ≠≠P<0.01,

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≠≠≠P<0.001 and ≠≠≠≠P<0.0001.

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Figure 5. 5-HT promotes variations in the number and mean length of primary and secondary dendrites; effects partially prevented by 5-HT1AR and 5-HT7R antagonists. Cultures were incubated for 24 h with 100 nM 5-HT. The antagonists WAY-100635 and SB269970 were added 30 min prior to 5-HT. (A) The number and (B) mean length of primary dendrites were determined for each individual hippocampal neuron using the

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NeuronJ software. Dendrites classified as primary were segregated according to their length

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as ≤ 40 µm and > 40 µm in length. (C) The number and (D) mean length of ≤ 40 µm and >

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40 µm primary dendrites were determined. Data were analysed using ANOVA for each condition, followed by the Bonferroni post-hoc test. (E) The number and (F) mean length of secondary dendrites were determined as described for primary dendrites. Dendrites

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classified as secondary were segregated according to their length as ≤ 20 µm and > 20 µm in length. The number (G) and mean length (H) of ≤ 20 µm and > 20 µm secondary

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dendrites were determined. Neurons from independent cultures were analyzed: control (n=280), 5-HT (n=119), 100 nM 5-HT + 1 µM WAY-100635 (n=152) and 100 nM 5-HT +

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1 µM SB269970 (n=81). The data represent means ± SEM. The effect of treatments on

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each group was analysed using ANOVA, followed by the Tukey´s post-hoc test. *P< 0.05; ** P< 0.01 and ***P<0.001. The effect of 5-HT on the number of secondary dendrites was

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determined by t-test,  P< 0.05.

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Figure 6: Effect of 5-HT1AR and 5-HT7R on phosphorylation of ERK1/2. Hippocampal neurons were (A) preincubated during 30 min with the antagonists WAY-100635 and SB269970 and then incubated with 100nM 5-HT during 10 min. (B) In order to evaluate the involvement of MEK in 5-HT actions, cells were preincubated during 30 min with 30 μm UO126, an agent that inhibits MEK1/2 activity and then incubated for additional 10

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min alone or in the presence of 100 nM 5-HT. Cell extracts were obtained and analysed by

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immunoblotting with antibody to phospho-ERK1/2. Membranes were then stripped and

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incubated with antibody to total ERK1 and ERK2 (ERK1/2). Net intensities of phosphoproteins relative to their total were determined as described in methods. Immunoblots are representative of different experiments (n=3-4) performed in duplicate. The data represent

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means ± SEM. The effect of treatments was analysed using ANOVA, followed by the

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Tukey´s post-hoc test. *P<0.05; **P<0.01; ***P<0.001 and ****P<0.0001.

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Figure 7: Effect of 5-HT1AR and 5-HT7R on phosphorylation of AKT and GSK3β. (A) Hippocampal neurons were incubated for 10 min with 100 nM 5-HT. The antagonists WAY-100635 and SB269970 were added 30 min prior to 5-HT and the phosphorylation of phospho-Ser473 AKT (p-AKT) was determined. (B) Cultures were pre-treated during 30 min with 20 μM LY294002, an agent that inhibits the PI3K activity. Cultures were additionally

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incubated during 10min with and without 100 nM 5-HT and levels of p-AKT were

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determined. (C) Effect of 100 nM 5-HT and WAY-100635 and SB269970 on phospho-Ser9

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GSK3β levels. Cell extracts were obtained and analysed by immunoblotting with antibody to p-GSK3 β. Membranes were then stripped and incubated with antibody to total AKT or GSK3β. Net intensities of phospho-proteins relative to their total were determined as

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described in methods. Immunoblots are representative of different experiments (n=4) performed in duplicate. The data represent means ± SEM. The effect of treatments was

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analysed using ANOVA, followed by the Tukey´s post-hoc test. *P<0.05; **P<0.01;

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***P<0.001; ****P<0.0001.

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Figure 8: Effect of 5-HT1AR and 5-HT7R on LIMK/cofilin pathway activities. Hippocampal neurons were incubated for 10 min with 100 nM 5-HT. The antagonists WAY-100635 and SB269970 were added 30 min prior to 5-HT. Cells extracts were obtained and analysed by immunoblotting with antibody to phospho-LIMK (p-LIMK) or phospho-Cofilin (p-Cofilin) levels. After stripping, membranes were incubated with

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antibodies that recognize total Cofilin or β-actin. Net intensities of phospho-proteins

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relative to their total or to β-actin was determined as described in methods. Immunoblots

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are representative of different experiments (n=5-7) performed in duplicate. The data

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represent means ± SEM.

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Figure 9: Summary of 5-HT1AR and 5-HT7R actions on neuronal morphology.

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The 5-HT1AR has a dual effect on dendritic morphology: it prevents the both the formation and growth of long dendrites and encourages the growth of shorter secondary dendrites. The 5-HT1AR restricts the density of long dendrites (i.e., either primary or secondary), while promoting the growth of short secondary dendrites and in this way, increases the dendritic field. Similarly to 5-HT1AR, 5-HT7R activity produces an increase in the average length of the short secondary dendrites. The similar effect of 5-HT1AR and 5-HT7R on secondary dendrite can be produced by each receptor independently or mediated by the physical interaction between these receptors at specific cellular compartments.

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Supplementary Material Figure S4. Effect of the antagonists WAY-100635 (1 µM) and SB269970 (1 µM) on neuronal morphology of hippocampal primary cultures. On the 7th day in vitro (DIV7), the cells were incubated for 24 h with WAY-100635 (1 µM) and SB269970 (1 µM). At DIV 8, cells were fixed and immunostained for MAP2a, stained with rhodamine-phalloidin.

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A DSU microscope with a 40x objective was used, and channels were merged with the

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ImageJ program. Neurons from independent cultures were analyzed: control (n=44), 1 µM

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WAY-100635 (n=45) and 1 µM SB269970 (n=45). Sholl analysis shows the number of intersections of dendrites as a function of path length from the soma. The number of

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intersections was determined by the Bonfire program. The two-way ANOVA analysis of Sholl curves revealed a significant main effect of distance from soma (F(9,134)=2390,

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P<0.0001), effect of treatments (F(2,134)=21.91, P=0.026), and a non-significant interaction between treatments and distance from soma (F(18,134)= 5.605, P> 0.5). Tukey’s post-hoc tests only found difference among control and SB269970 at 20 µm (P<

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0.05).

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Figure S5. Effect of WAY-100635 and SB269970 in the number and length of dendrites. (A) Effect of WAY-100635 and SB269970 in the number and length of dendrites. The number and mean length of primary dendrites were determined for each individual hippocampal neuron using the NeuronJ software. Dendrites classified as primary were segregated according to their length as ≤ 40 µm and > 40 µm in length. The number and

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mean length of ≤ 40 µm and > 40 µm primary dendrites were determined. (B) The number

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and mean length of secondary dendrites were determined as described for primary

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dendrites. Dendrites classified as secondary were segregated according to their length as ≤ 20 µm and > 20 µm in length. The number and mean length of ≤ 20 µm and > 20 µm secondary dendrites were determined. Neurons from independent cultures were analyzed:

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control (n=81), 1 µM WAY-100635 (n=36) and 1 µM SB269970 (n=49). The data represent means ± SEM. The effect of treatments on each group was analysed using

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ANOVA, followed by the Tukey´s post-hoc test.

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Table 1. Primary Antibodies and blocking conditions Description of Immunogen

Source, Host Species, Cat. #, Clone or Lot#, RRID

Concentration used

β-Actin

Synthetic actin N-terminal aa.20-33

Sigma-Aldrich, rabbit polyclonal, Cat# A5316, RRID:AB_476743

2.68 µg/mL

AKT

Amino acids 345-480 of Akt1 of human origin.

Santa Cruz Biotechnology, polyclonal Cat# sc-8312, RRID:AB_671714, clone H136

0.4 µg/mL

pSer473-AKT

Synthetic phosphopeptide corresponding to residues surrounding Ser473 of Mouse Akt

Cell Signaling Technology Cat# 9271S, RRID:AB_32982

0.4 µg/mL

pSer3-Cofilin

Synthetic phosphopeptide coupled to KLH corresponding to surrounding residues Ser3 of human cofilin

Cell Signaling Technology, rabbit polyclonal, Cat# 3311S, RRID:AB_330238)

0.2 µg/mL

Cofilin

N-terminal 13-22 amino acids of human cofilin1

Cytoskeleton, rabbit polyclonal, Cat# ACFL02-A, RRID:AB_10708808

0.1 µg/mL

Santa cruz, rabbit polyclonal, Cat# SC-94, RRID:AB_2140110

0.2 µg/mL

Cell Signaling Technology, rabbit monoclonal, Clone 197G2 Cat# 4377S, RRID:AB_331775

0,7 µg/mL

3% nonfat milkPBS

Santa Cruz Biotechnology, mouse monoclonal antibody Cat# sc-7291, RRID:AB_2279451

0.2 µg/mL

3% nonfat milkTBS 0.1% Tween-

pThr202/Tyr204ERK

GSK3β

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Epitope mapping between subdomain XI of ERK1-encoded MAPKinase p44 of rat origin Synthetic phosphopeptide corresponding to residues surrounding pThr202/Tyr204 of human p44/42 MAPK Amino acids 1-420 representing full length glycogen synthase kinase3β of Xenopus origin

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Primary antibody blocking solution 3% nonfat milkTBS 0.1% Tween20 3% nonfat milkTBS 0.1% Tween20 3% nonfat milkTBS 0.1% Tween20 2% BSATBS 0.1% Tween20 3% nonfat milkTBS 0.1% Tween20 3% nonfat milkPBS

ACCEPTED MANUSCRIPT Rojas PS. et al. Molec Cell Neurosci RM

20 Short amino acid sequence containing Ser 9 phosphorylated GSK-3β of human origin

Santa Cruz Biotechnology Cat# sc-11757-R, RRID:AB_653246

5HT1AR

Synthetic peptide including amino acids 218-336 of 5HT1AR of human origin

5HT7R

Synthetic peptide: YGHLRSFLLPEVGRGL, corresponding to amino acids 13-28 of Rat 5HT7 Receptor

Abcam, rabbit polyclonal Cat# Ab13898; RRID:AB_300724

pThr508-LIMK

Synthetic peptide derived from human LIMK around the phosphorylation site of Thr505

Sigma-Aldrich, rabbit polyclonal, Cat# 4504460, RRID: AB_2491619

0.4 µg/mL

0.8 µg/mL

2 µg/mL

3% nonfat milkTBS 0.1% Tween20 3% nonfat milkTBS 0.1% Tween20 3% nonfat milkTBS 0.1% Tween20 3% nonfat milkTBS 0.1% Tween20

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Santa Cruz Biotechnology, rabbit polyclonal Cat# sc10801, clone H-119; RRID:AB_2119583

0.2 µg/mL

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pSer9-GSK3β

The protein levels of 5-HT7R are increased in hippocampal neurons during DIV 5-HT reduces significantly the growth of primary dendrites through the activation of 5HT1AR 5-HT1AR and 5-HT7R have a similar action by promoting short secondary dendrites growth 5-HT1AR and 5-HT7R trigger activation of ERK and AKT which are important signaling molecules involved in dendrite outgrowth. 5-HT1AR and 5-HT7R activation do not promote phosphorylation and inactivation of actinremodeling protein cofilin

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