The serotonergic neurons derived from rhombomere 2 are localized in the median raphe and project to the dorsal pallium in zebrafish

The serotonergic neurons in the raphe nucleus are implicated in various cognitive functions such as learning and emotion. In vertebrates, the raphe nucleus is divided into the dorsal raphe and the median raphe. In contrast to the abundance of knowledge on the functions of the dorsal raphe, the roles of the serotonergic neurons in the median raphe are relatively unknown. The studies using zebrafish revealed that the median raphe serotonergic neurons receive input from the two distinct pathways from the habenula and the IPN. The use of zebrafish may reveal the function of the Hb-IPN-median raphe pathway. To clarify the functions of the


Introduction
Serotonin, a neuromodulator, is involved in various functions such as learning and emotion (Carhart-Harris and Nutt, 2017;Fernandez et al., 2017).In addition, the dysfunction of serotonergic neurons is thought to be closely related to psychiatric diseases such as depression and obsessive-compulsive disorder (Jacobsen et al., 2012;Lucki, 1998).In vertebrates, serotonergic neurons in the central nervous system originate from the raphe and project axons throughout a wide area of the brain (Lillesaar et al., 2009;Vertes and Linley, 2007).The distribution and gene expression of the raphe serotonergic neurons are conserved across vertebrates, and they are thought to have similar functions (Gaspar and Lillesaar, 2012).
In mammals, depending on their distribution, serotonergic neurons that provide ascending innervation to the forebrain are divided into the dorsal raphe and the median raphe.These subtypes have different neural activities in response to rewards and punishments, as well as distinct functions (Kawai et al., 2022;Li et al., 2016).Animals can accurately understand the rules of the external world and choose the correct action from various options to achieve their goals.This information processing is performed through reinforcement learning by a series of brain regions such as the cerebral cortex and the basal ganglia (Doya, 2007;Maia and Frank, 2011).In reinforcement learning, dorsal raphe serotonergic neurons control the discount rate for future rewards (Miyazaki et al., Abbreviations: IPN, interpeduncular nucleus; vIPN, ventral interpeduncular nucleus; Hb, habenula; vHb, ventral habenula; dHbM, dorsal habenula medial subnucleus; dHbL, dorsal habenula lateral subnucleus; DR, dorsal raphe; MR, median raphe; GFP, green fluorescent protein; Pet1, ETS domain transcription factor Pet-1; OB, olfactory bulb; Tel, telencephalon; CB, cerebellum; OT, optic tectum; HT, hypothalamus; EN, entopeduncular nucleus; PPa, anterior part of the parvocellular preoptic nucleus; 5HT, 5-hydroxytryptamine serotonin.2020, 2012).In contrast, the role of the serotonergic neurons at the median raphe is poorly understood.
To clarify the function of the median raphe serotonergic neurons, it is necessary to distinguish them from the dorsal raphe serotonergic neurons.One method for differentiation is to divide subtypes based on the origin of the rhombomere segment during early development (Jensen et al., 2008).In particular, most median raphe serotonergic neurons originate from rhombomere 2 in mice.This developmental pattern has been used to explore gene expression, and neural circuits, and manipulate neural activity specifically in median raphe serotonergic neurons (Bang et al., 2012;Okaty et al., 2015;Senft et al., 2021;Teissier et al., 2015).However, the functions of the median raphe serotonergic neurons derived from rhombomere 2 in learning, and the brain regions providing input to them, are largely unknown.
Zebrafish can be a powerful model animal to address these questions.Studies using zebrafish have revealed that the median raphe serotonergic neurons receive input from two different pathways.The first pathway gives direct input from the ventral habenula (vHb), which is activated in response to danger prediction during active avoidance learning.Because the vHb is composed mostly of excitatory neurons, this pathway might activate the median raphe serotonergic neurons when aversive stimuli are likely to be received (Amo et al., 2014(Amo et al., , 2010)).The second pathway has indirect input from the dorsal habenular medial subnucleus (dHbM) via the ventral interpeduncular nucleus (vIPN), which is activated in the losers of conspecific social fighting.Most of the dHbM neurons are excitatory, whereas the vIPN, which receives input from the dHbM and projects to the median raphe serotonergic neurons, is composed largely of inhibitory neurons (Agetsuma et al., 2010;Chou et al., 2016;Kinoshita and Okamoto, 2023).Therefore, this pathway is supposed to suppress the median raphe serotonergic neurons in the loser.In other words, these pathways are thought to activate and inhibit the median raphe serotonergic neurons in response to dangerous stimuli such as electric shocks or attacks from the winner.To clarify the function and information processing mechanism of the habenular-interpeduncular-median raphe circuit, it is necessary to elucidate the projection areas and neural activity of the median raphe serotonergic neurons for each subtype.
We hypothesized that the developmental pattern of raphe serotonergic neurons from rhombomeres is conserved in zebrafish.By using this mechanism, we tried to reveal the specific projection areas of the median raphe serotonergic neurons in the forebrain.First, we generated a transgenic line in which specific serotonergic neurons that develop from rhombomere 2 express green fluorescent protein (GFP), and other raphe serotonergic neurons express DsRed.We investigated the localization of GFP and DsRed by immunohistochemistry for this transgenic line and found that GFP signals were observed in the median raphe.This result indicated that the median raphe mainly consists of serotonergic neurons derived from rhombomere 2 and the developmental pattern of serotonergic neurons is conserved in zebrafish.In addition, we investigated the localization of axons and found that many axonal GFP signals were observed in the rostral dorsal pallium.Recently, an experimental system that can measure neural activity by Ca 2+ imaging in the dorsal pallium of zebrafish in virtual reality has been developed.Using this experimental system, it was revealed that there are neural ensembles that encode rules to adapt to the external environment in the dorsal pallium, which is homologous to the mammalian cerebral cortex (Torigoe et al., 2021).In the future, we may be able to measure and manipulate the neural activity of the serotonergic axons by using this experimental system, thereby elucidating the function of the median raphe serotonergic neurons in the formation of these neuronal ensembles.

Zebrafish
All protocols were reviewed and approved by the Animal Care and Use Committees of the RIKEN Center for Brain Science.The zebrafish used in this study were reared and bred in a standard environment (Westerfield, 2000).Adult zebrafish were kept in a 7-liter tank, and the temperature of the breeding water was 28.5 • C and was constantly changed under a standard 14-h light/10-h dark cycling.We used Tg  and Et(hoxa2b-SCP1-Ocu.Hbb2:Cre-2A--Cerulean) y571Et , which specifically expresses Cre in rhombomere 2, and was provided by Burgess laboratory (Tabor et al., 2019).The pet1 is ETS-domain transcription factor-encoding gene which is expressed specifically in the raphe serotonergic neurons.By crossing these lines, we generated a double transgenic line of Tg(-3.2pet1:loxP-DsRed--loxP-GFP);y571-cre.
We made maximum-intensity projection images of all sections from a slice, and images were analyzed by ImageJ.

Tyramide signal amplification
Immunohistochemistry using the tyramide signal amplification reaction was performed based on the protocol of Alexa Fluor 488 Tyramide SuperBoost Kit (Thermo Fisher).After fixing and embedding the zebrafish head as described above, we prepared 75 μm coronal slices using a vibratome.Permeabilization was performed with PBS + 0.5% Triton for 15 minutes.After washing with PBS, a quenching treatment was performed with 3% H 2 O 2 .Then, blocking was performed using 10% goat serum, and a primary antibody reaction was performed at 4 • C overnight with an anti-GFP antibody (Roche, 10744900).The next day, after washing with PBS, a secondary antibody reaction was performed overnight at 4 • C with the poly-HRP conjugated secondary antibody from this kit.The next day, after washing with PBS, a tyramide signal amplification reaction was performed.After washing with PBS, the mixture was reacted with PBS containing 4′,6-diamidino-2-phenylindole (DAPI, 0.3 μg/ml) for 2 hours.After mounting samples on glass slides, we observed the fluorescent signals with a Nikon C2+ confocal microscope.

Generation of transgenic zebrafish with labeled serotonergic neurons derived from rhombomere 2
In mammals, most of the median raphe serotonergic neurons are known to originate from rhombomere 2 (Fig. 1A; Jensen et al., 2008).We hypothesized that this developmental pattern of raphe serotonergic neurons is conserved in zebrafish.Using this developmental pattern, it may be possible to distinguish the median raphe serotonergic neurons from other subtypes in zebrafish as well as mammalians.We generated a transgenic line in which serotonergic neurons with Cre express GFP, and serotonergic neurons without Cre express DsRed, which is Tg .The pet1 gene is specifically expressed in the raphe serotonergic neurons.Additionally, an enhancer trap line that expresses Cre specifically in rhombomere 2 early in development has been established in zebrafish (Tabor et al., 2018).By crossing these transgenic lines, we generated a new line that expresses GFP specifically in serotonergic neurons derived from rhombomere 2 (Fig. 1B).

Serotonergic neurons derived from rhombomere 2 located in the median raphe serotonergic neurons
We investigated the localization of GFP and DsRed signals using immunohistochemistry for the generated transgenic lines.We also confirmed the location of serotonergic neurons using anti-5HT antibodies.In zebrafish, serotonergic neurons are present from the upper to the caudal areas of the IPN.The subtype present in the upper part of the raphe is the dorsal raphe serotonergic neuron, and the ventral subtype in the caudal part of the raphe is thought to be the median raphe serotonergic neuron.We made coronal and sagittal slices and observed fluorescence in the raphe regions (Fig. 2A, B).In sagittal slices, DsRed signals but no GFP signals were observed in the dorsal raphe serotonergic neurons (Fig. 2C, panels a, a').Weak GFP signals in panel a' were considered the background signals.In contrast, in the coronal and sagittal section of the caudal part of the IPN, DsRed was observed in the dorsal and median raphe serfotonergic neurons, and GFP signals were observed in the median raphe serotonergic neurons (Fig. 2C, panels b,  b', c, and c').The lateral side of the dorsal raphe serotonergic neurons produced positive results for DsRed (Fig. 2C, panels d, d') but were mostly negative for GFP.Most GFP signals were observed in the median raphe serotonergic neurons.These results suggested that in zebrafish, as in mammals, serotonergic neurons derived from rhombomere 2 are localized in the median raphe.In the median raphe nucleus, most serotonergic neurons expressed GFP, and DsRed signal without GFP signal was observed in only a small number of serotonergic neurons (Fig. 2D, panels b'' and c'').The ratios of GFP + / 5HT + neurons are 20/ 33 in the coronal section and 34/63 in the sagittal section.Furthermore, most GFP signals colocalized with 5HT signals (Fig. 2D, panels b''' and c''').The 5HT signals in some GFP-positive neurons were weak.With this immunohistochemistry method, we were not able to detect GFP and DsRed signals in axons in the telencephalic region.

Serotonergic neurons derived from rhombomere 2 project to the anterior side of the dorsal pallium
To identify which brain regions the median raphe serotonergic neurons derived from rhombomere 2 project their axons to, we enhanced the GFP signal using a tyramide signal amplification reaction and confirmed the localization of GFP in the axons.In the anterior side of the telencephalon, many GFP signals were observed in the axons of the dorsal pallium (Fig. 3B, panels a, a', b, b').In the posterior side of the telencephalon, no GFP signals were observed in the axons in the dorsal pallium.In addition, GFP signals were observed in axons in the entopeduncular nucleus (EN) and anterior part of the parvocellular preoptic nucleus (PPa; Fig. 3B, panels c, c′, d, d′).The GFP signals were detected in the median raphe neurons, and few GFP signals were detected in the dorsal raphe (Fig. 3B, panels e, e', f, f').The GFP signals in the regions indicated by white arrows were in axons not in neurons (Fig3B, panel f').Signals in the EN and the PPa may be en passant for axons that project to the dorsal pallium.This result suggested that the median raphe serotonergic neurons derived from rhombomere 2 projected axons to the anterior side of the dorsal pallium.

Discussion
In this study, we revealed that most of the median raphe serotonergic neurons are derived from rhombomere 2 in zebrafish (Fig. 4A).This developmental pattern is conserved in mammals and teleosts.We also revealed that the median raphe serotonergic neurons derived from rhombomere 2 projected to the anterior side of the dorsal pallium (Fig. 4B).The dorsal pallium are thought to be homologous with the mammalian cerebral cortex, hippocampus, and amygdala (Northcutt and Braford, 1980;Wullimann and Mueller, 2004).Because those serotonergic neurons project to the regions that control learning and behavior, this subtype may play an important role in reinforcement learning.
In mice, the median raphe serotonergic neurons derived from rhombomere 2 project their axons to the olfactory bulb, perlimbocortex, suprachiasmatic nucleus of the hypothalamus (SCN), paraventricular nucleus of the thalamus (PVT), medial and lateral septum, diagonal band, rostral hippocampus, and caudal hippocampus (Bang et al., 2012;Senft et al., 2021).Comparing the published research with the results of this study, it appears that mammals and zebrafish share axonal projections of the median raphe serotonergic neurons derived from rhombomere 2 to evolutionary homologous areas.The median raphe receives input directly from the vHb and indirectly from the dHbM via the vIPN in zebrafish.These neural pathways are thought to activate and inhibit the median raphe serotonergic neurons, respectively, in a dangerous situation.In this study, we also revealed that the median raphe serotonergic neurons derived from rhombomere 2 projects to the rostral dorsal pallium.The results of this study that the median raphe serotonergic neurons may control learning and behavior by transmitting the information from the Hb and the IPN to the dorsal pallium.Recently, an experimental system has been developed that can measure the neural activity in the dorsal pallium of zebrafish during active avoidance learning in virtual reality (Torigoe et al., 2021).Using this system, it was revealed that excitatory neurons in the dorsal pallium, which corresponds to the mammalian cerebral cortex and hippocampus, form neuronal ensembles that encode the rules imposed by the environment.Simultaneous measurement of the neural activity of the serotonergic axons and these ensembles, as well as a manipulation of neural activity of serotonergic axons in the dorsal pallium, may reveal the function of the median raphe serotonergic neurons in the formation of these neuronal ensembles.
In mammals, there are multiple subtypes of median raphe serotonergic neurons depending on the differences in gene expression and neural activity (Luchetti et al., 2020;Ren et al., 2019).Furthermore, a small number of median raphe serotonergic neurons are derived from other rhombomere regions (Jensen et al., 2008).The results of this study

Fig. 1 .
Fig. 1.Developmental pattern of serotonergic neurons derived from rhombomere 2 and schematic diagram of the DNA sequence of transgenic zebrafish.(A) Schematic diagram of rhombomeres during early development in mice and raphe serotonergic neurons in adults (red circles: serotonin neurons) (B) Design of the transgenic lines capable of labeling the median raphe serotonergic neurons derived from rhombomere 2.

Fig. 2 .
Fig. 2. Localization by immunohistochemistry of GFP and DsRed in Tg(-3.2pet1:loxP-DsRed-loxP-GFP);y571-cre.(A, B) The positions of a cross-sections for immunohistochemistry (A, coronal; B, sagittal) (C) Fluorescence results of immunohistochemistry of GFP (Green), DsRed (red), and 5HT (magenta).Panels a, and a' are coronal sections, including the IPN and the dorsal raphe.Panels b and b' are coronal sections, including the dorsal and median raphe.Panels c and c' are sagittal sections including the IPN, the dorsal raphe, and the median raphe.Panels d, and d' are sagittal sections, including the lateral side of the dorsal raphe.(D) Panels b'' and c'' are merged images with GFP (green) and DsRed (red).Panels b''' and c''' are merged images with GFP (green) and 5HT (magenta).The age of zebrafish in this experiment is 4 months.

Fig. 3 .Fig. 4 .
Fig. 3.The median raphe serotonergic neurons derived from rhombomere 2 projected axons to the rostral dorsal pallium.(A) Schematic diagram of the position of the coronal sections.(B) Panel a, and a' are the most anterior section of the telencephalon.Panels b, and b' are sections of the anterior telencephalon.Panels c and c' are sections of the posterior telencephalon.Panels d and d' are the most posterior telencephalon sections.Panels e and e' are sections of the dorsal raphe.Panels f and f' are sections of the median raphe.The white arrows in the panel f' indicate the axonal signals of GFP.The age of zebrafish in this experiment is 15 months.