The organization of the zebrafish pallium from a hodological perspective

We studied the connections (connectome) of the adult zebrafish pallium using carbocyanine dye tracing and ancillary anatomical methods. The everted zebrafish pallium (dorsal telencephalic area, D) is composed of several major zones (medial, lateral, dorsal, central, anterior, and posterior) distinguishable by their topography, cytoarchitecture, immunohistochemistry, and genoarchitecture. Our comprehensive study reveals poor interconnectivity between these pallial areas, especially between medial (Dm), lateral/dorsal (Dl, Dd), and posterior (Dp) regions. This suggests that the zebrafish pallium has dedicated modules for different neural processes. Pallial connections with extrapallial regions also show compartmental organization. Major extratelencephalic afferents come from preglomerular nuclei (to Dl, Dd, and Dm), posterior tuberal nucleus (to Dm), and lateral recess nucleus (to Dl). The subpallial (ventral, V) zones dorsal Vv, Vd, and Vs, considered homologues of the striatum, amygdala, and pallidum, are mainly afferent to Dl/Dd and Dp. Regarding the efferent pathways, they also appear characteristic of each pallial region. Rostral Dm projects to the dorsal entopeduncular nucleus. Dp is interconnected with the olfactory bulbs. The central region (Dc) defined here receives mainly projections from Dl–Dd and projects toward the pretectum and optic tectum, connections, which help to delimiting Dc. The connectome of the adult pallium revealed here complements extant studies on the neuroanatomical organization of the brain, and may be useful for neurogenetic studies performed during early stages of development. The connectome of the zebrafish pallium was also compared with the pallial connections reported in other teleosts, a large group showing high pallial diversity.

pallium was also compared with the pallial connections reported in other teleosts, a large group showing high pallial diversity.

connectome, Danio rerio, DiI, neural tracing, telencephalon INTRODUCTION
The telencephalon of vertebrates is considered the main high processing center of the brain. It consists of the olfactory bulbs and telencephalic hemispheres, the latter containing two main regions: the pallium and subpallium. In tetrapods as amphibians and mammals, the pallium is organized medio-laterally in main longitudinal zones, namely, the medial pallium or (hippocampus, i.e., dentate gyrus plus Ammon's horn in mammals), the dorsal pallium (DP) or neocortex, the lateral pallium, and the ventral pallium plus its derivative pallial amygdala (Medina & Abellán, 2009;Moreno & González, 2007;Puelles et al., 2000;Watson & Puelles, 2017). This zonal pallial configuration in evaginated telencephalons of tetrapods is apparently different from that of the everted telencephalon that is present in actinopterygians. Everted telencephalons have a single T-shaped ventricle and that extends over most of the pallium, which is reversed and whose ependymal region faces to it and is covered by an extensive choroid tela (Nieuwenhuys, 1963;Nieuwenhuys, 2009aNieuwenhuys, , 2009bNorthcutt & Braford, 1980). The teleost telencephalic hemispheres also consist of dorsal (D) and ventral (V) telencephalic areas homologous to the mammalian pallium and subpallium, respectively. Further subdivisions of the pallium and subpallium in teleosts are most often named based on their topographic position within this everted telencephalon. This is because there are difficulties when establishing homologies between areas of the telencephalon of teleosts and other vertebrates. Thus, in the teleost subpallium, the following areas are generally recognized in most species: the ventral (Vv), dorsal (Vd), central (Vc), lateral (Vl), supracommissural (Vs), and postcommissural (Vp) V nuclei and the entopeduncular nucleus, considered to be field homologues of different subpallial structures of mammals (Ganz et al., 2012;Gerlach & Wullimann, 2021;Maruska et al., 2017;Northcutt & Braford, 1980;Porter & Mueller, 2020). Regarding the teleost pallium (D), several major zones have been recognized in most species, named topographically as lateral (Dl), dorsal (Dd), medial (Dm), central (Dc), and posterior (Dp) zones, plus the nucleus taeniae (nucleus of the lateral olfactory tract) (Meek & Nieuwenhuys, 1998;Nieuwenhuys, 1963Nieuwenhuys, , 2009Nieuwenhuys, , 2011Northcutt, 2006;Northcutt & Braford, 1980;Wullimann & Mueller, 2004;Yamamoto, 2009;Yamamoto & Ito, 2008;Yamamoto et al., 2007). However, cytoarchitectonical identification of pallial areas is often complex and may differ substantially between authors studying the same species (compare maps of goldfish telencephalon in Northcutt, 2006, andYamamoto &Ito, 2008).
These correspondences received some support from functional studies in cyprinids that analyzed fish behavior after lesions into these two pallial areas (Portavella, Vargas, et al., 2002;Portavella, Torres, et al., 2004). Some aspects of "the simple eversion model" have been challenged recently by anatomical and developmental studies that drew a more complex picture of eversion (Folgueira et al., 2012;Mueller & Wullimann, 2009;Yamamoto, 2009;Yamamoto et al., 2007). In this sense, it has been shown that eversion is more complex than a simple medial to lateral out-folding of the neural tube (Folgueira et al., 2012;Furlan et al., 2017). Anyway, the organization of the pallium shows important differences between species and in advanced teleosts, such as acanthopterygians, and the pallium often exhibits much more complex cytoarchitectonical subdivisions without known mammalian correlates (Burmeister et al., 2009;Hagio et al., 2020;Kawaguchi et al., 2019;Maruska et al., 2017).
Deciphering the connectivity of the vertebrate pallium is basic for understanding the functions of its different regions, as well as their role in specific behaviors. A number of experimental studies have been performed in rat and mouse, main mammalian models in neurobiology.

The zebrafish (Danio rerio) is a model fish species in neurobiology.
A large number of studies in embryos and larvae have revealed the great potential of zebrafish for studying the development of specific neural populations and circuits, as well as their relationship with wellcharacterized behaviors (Antinucci et al., 2019; Kunst et al., 2019;Lal et al., 2018;Miyasaka et al., 2009;Turner et al., 2016;von Trotha et al., 2014). Knowledge of the zonal organization of the adult zebrafish pallium is mainly based on studies using cytoarchitecture and expression patterns of some markers and transgenes (Wullimann et al., 1996;Wullimann & Mueller, 2004;Castro et al., 2006;Mueller et al., 2011;Ganz et al., 2012Ganz et al., , 2014Diotel et al., 2015;Furlan et al., 2017;Lal et al., 2018;Bloch et al., 2020;Porter & Mueller, 2020). With the exception of the olfactory system (Miyasaka et al., 2009Gayoso et al., 2011Gayoso et al., , 2012Kermen et al., 2020), the connectivity of the adult zebrafish pallium has received scarce experimental attention. The aim of the present study is to comprehensively analyze the connections of the pallial zones of the adult zebrafish and contribute to the understanding of brain circuitry in teleosts. Our connectivity results in the adult pallium are complementary to those of single cell studies in larval zebrafish Kunst et al., 2019), and may facilitate comparison with larval connectivity data and also with hodological studies in adult brains of other fishes.

Animals
Forty-four adult zebrafish (Danio rerio) of both sexes, ranging 28-41 mm in length, were used in this study. Fish were kept in aquaria at 28 • C under standard conditions (Aleström et al., 2019).

Carbocyanine dye tracing
Procedures used for carbocyanine dye tracing were similar to those applied in previous studies in zebrafish and other teleosts (Folgueira et al., 2004a(Folgueira et al., , 2004b(Folgueira et al., , 2006Yáñez, Busch, et al., 2009;Yáñez, Souto, et al., 2017 processes. This implies that the dye uptake by an axon can led to both retrograde labeling of the soma and retrograde/anterograde labeling of collaterals and branches (Folgueira et al., 2004a). Thus, connections need to be confirmed by application of the dye to putative targets of a given region.

Complementary material
For the regional analysis of the zebrafish brain and comparison with the topography revealed in DiI experiments, various series of transverse sections stained with general methods (Nissl) or immunohistochemistry against different enzymes and peptides (GAD, ChAT, TH, neuropeptide Y, calretinin, galanin, FMRF, KLH) used in previous studies of our group (Castro et al., 2006;Gayoso et al., 2011;Turner et al., 2016) were at our disposal (for details of methods, see these publications  (Mueller et al., 2011;Ganz et al., 2014;Bloch et al., 2019Bloch et al., , 2020Porter & Mueller, 2020).

Nomenclature used
Unless otherwise stated, the general brain nomenclature used here is based on the atlas of adult zebrafish brain (Wullimann et al., 1996), excepting for the pallium. For convenience, the pallial regions studied here and their names (Dma, Dla, Dm, Dc, Dd, Dl, and Dp) are mainly based in Castro et al. (2006) and Furlan et al. (2017). The correspondence between these pallial regions with some regions proposed in alternative models of zebrafish pallial organization (Mueller et al., 2011;Ganz et al., 2014;Porter & Mueller, 2020) will be addressed appropriately in the text.

Regions of the zebrafish pallium approached experimentally
As mentioned previously, the zones of the zebrafish pallium experimentally accessed are mostly named on those described in Castro et al. (2006), providing also correspondences with the pallial organization proposed by other authors (Porter & Mueller, 2020). The regions of the zebrafish dorsal telencephalic area or pallium (D) were mapped with Nissl staining and calretinin immunohistochemistry (Figures 1   and 2), to assess their locations in order to apply tracers precisely.
The distinguishable pallial regions in which we centered DiI application experiments were the anterior medial and lateral divisions of the anterior pallium (Dma, Dla of Castro et al., 2006), the extensive medial and lateral regions (Dm, Dl), a central region or nucleus of D (Dc; a part of the region named as DP by Porter & Mueller, 2020), a posterior pallium region (Dp; named IOP by Porter & Mueller, 2020) that is located in a caudo-lateral location, and a small dorsal region (Dd) located just lateral to the sulcus ypsiloniformis included in the Dl by Porter and Mueller (2020). Dma is a rostromedial part of the pallium just rostral to Dm from which it can be distinguished by the lack of CR-immunoreactive innervation (Castro et al., 2006;present observations). The remainder Dm can be subdivided into three to four subregions (Dm1, Dm2, Dm3, and Dmp) distinguishable using immunohistochemical methods (Castro et al., 2006; present results, Figure 2). Owing to the difficulty for distinguishing limits between these areas in fresh tissue, for tracer application to Dm, we only considered two regions, the commissural (Dm1 plus Dm2) and caudal (Dm3 plus Dmp), although some subdivisions were distinguishable by their afferents (see below).
We considered Dc as the central region (nucleus) mainly recognizable by its very rich innervation by parvalbumin-immunoreactive fibers and other differential immunochemical signatures (Castro et al., 2006;von Trotha et al., 2014;Furlan et al., 2017;Porter & Mueller, 2020), as well as by differential efferents (present results). In recent proposals, a homonymous Dc is a much wider region extending till the dorsorostral pole of the pallium (DP; Mueller et al., 2011;Ganz et al., 2014;Porter & Mueller, 2020), which includes most of our Dma and Dml and will be discussed below in the light of present experimental results.
Zebrafish Dp (not to be confused with that homonymous region of Porter & Mueller, 2020) is separated from Dl by a lamina of neurons, and shows different immunochemical signatures (Castro et al., 2006) and olfactory connections (Miyasaka et al., 2009;Gayoso et al., 2011Gayoso et al., , 2012present results), as in other teleosts. Although Dp is adjacent to the nucleus of the lateral olfactory tract (nLot, nucleus taeniae) (Porter & Mueller, 2020), this nucleus was not directly accessed in our experiments because of the difficulty to apply DiI here without affecting Dp and the tract. Dd (as used by us) is a region adjacent to and hardly distinguishable from Dl with immunochemical markers but some heterogeneity in distribution of immunochemical markers was noted inside these pallial regions. Examples of the areas accessed in experiments of DiI application to various brain regions are included in the Figure 3.

Tracer application to Dm1 plus Dm2 (Dm1/2)
In these experiments, DiI was applied to intermediate precommissural levels of Dm, which include Dm1 and Dm2 regions (Dm1/2). Experiments were performed using three different approaches: (1) from rostral in sectioned brain blocks, (2) from dorsal in whole brains, and (3) from medial after removing the contralateral telencephalic lobe.
The results presented here are mainly based on one experiment of a sectioned brain where DiI diffusion was restricted to the region of interest (Figures 4 and 5). Results of tracer application to Dma and caudal regions of Dm are described in the next sections.
In the telencephalon, tracer application to Dm1/2 labeled numer-

Tracer application to the medial anterior region of D (Dma)
Medial (Dma) and lateral (Dla) anterior regions were distinguished by Castro et al. (2006) in the rostral pallium with immunohistochemical methods. Here, we describe the connections of Dma ( Figure 6).
We observed that connections of Dma with other pallial and subpallial regions were scarce. After applying a minute DiI crystal to the very rostral pole of Dma, we observed densely labeled structures in Dma but not in Dla (Figure 6a), showing a sharp limit between these two areas. This supports the distinction between these two regions by Castro et al. (2006). Dm1 was labeled as a caudal continuation of the Dma neuropil till the level of the rostralmost portion of the sulcus ypsiloniformis, but no labeling was observed in Dm2, Dm3, or posterior Dm (Figures 6b-e). From the densely labeled Dma, bundles of labeled fibers coursed ventrolaterally and caudally, entered the lateral forebrain bundle and were followed to the (dorsal) entopeduncular nucleus, where a few cell bodies were labeled retrogradely. This projection has been previously reported based on NPY expression in zebrafish (see Turner et al., 2016). Some labeled fibers crossed the midline in the anterior commissure, but no cell bodies were labeled in the contralateral side (Figures 6d-f). No labeled fibers or cell bodies

Tracer application to the caudal region of Dm (Dm3/Dmp)
In the pallium, DiI application to the caudal region of Dm (

Tracer application to the lateral zone of the pallium (Dl)
The lateral zone of the zebrafish pallium (Dl) is composed of dorsal (Dld) and ventral (Dlv) regions that can be distinguished using immunochemical markers (Castro et al., 2006). As DiI was applied to unstained brains, we could not distinguish clearly between both regions, so they will be considered together as Dl in this section In extratelencephalic regions, a number of cell bodies were labeled in the lateral and anterior preglomerular nuclei and the lateral recess nucleus (Figures 8h-i and 10c-e), whereas some cell bodies were labeled in the dorsal posterior thalamic nucleus. Occasional small cell bodies, as well as fibers and terminals, were labeled in the habenulae (Figures 8g and 10a). In the rostral rhombencephalon, cell bodies were observed in the raphe caudal to the interpeduncular nucleus (Figures 8k and 10f).

3.6
Tracer application to the dorsal/dorsolateral zone of the pallium (Dd/Dld) As

3.7
Tracer application to the posterior zone of the pallium (Dp) All applications of DiI to Dp were performed in toto, targeting the ventrolateral portion in order to avoid the lateral olfactory tract and nucleus. These experiments led to very intense labeling of mitral cells F I G U R E 7 (a-l) Photomicrographs of transverse sections through the brains of two zebrafish showing labeled cells (arrowheads) and fibers (arrows) after application of DiI to the caudal dorsomedial zone of the pallium (Dmp/Dm3). (a-g) Panoramic views (a, c, g) and details (b, d, e, f) of transverse sections through the telencephalic lobe in an experiment with application of a very small DiI crystal to the area marked with a white star (in g). Note that labeled pallial neurons and fibers in levels away the area of application are mostly restricted to ipsilateral Dm2 (a-d), and a few cells in the subpallium: Vs (e) and entopeduncular nucleus (f). In (c), a few ependymal cells and processes were also labeled (outlined arrows). In (g), too, labeled small fiber bundles extend between Dm and the lateral forebrain bundle through the central region of the pallium. Photographs are ordered from rostral to caudal: (a-d) precommissural, (e-f) commissural, and (g) postcommissural levels. Numbers 1-3 indicate Dm subdivisions. (h-l) Sections of a brain where the area of application of DiI to caudal Dm was more extended showing some ipsilateral neurons labeled in Dld (h), the posterior periventricular preoptic nucleus (i), suprachiasmatic nucleus (j), ventral hypothalamus (k), and medial preglomerular nucleus (l). Ipsilateral side is to the left and the midline is indicated by broken lines or defined by the ventricle (asterisk). Outlined star: lateral recess. For abbreviations, see the list. All photomicrographs are negative images of fluorescent data. Scale bars: 200 µm (a, c, g, k); 100 µm (b, e-f-j, l); 50 µm (d, h-i) F I G U R E 8 (a-k) Schematic drawings of transverse sections of the zebrafish brain showing the distribution of cells and fibers labeled after application of DiI to the lateral zone of the pallium (Dl). The level of sections from rostral to caudal is indicated in a lateral view of the brain. Black circles, retrogradely labeled cells. Small dots and lines, labeled fibers and tracts. Shaded areas in (a-e) represent the areas with dense labeling by the tracer from the point of application of the small DiI crystal (white star in c). Ipsilateral is at the left. Scale bar for sections: 500 µm and, less intensely, fibers in the inner granular layer of the ipsilateral olfactory bulb. A few labeled mitral cells, with their characteristic dendrites branching in the olfactory glomeruli, were also observed in the contralateral olfactory bulb. These observations, with labeling on both sides, demonstrate the bilateral olfactory bulb projection to Dp (Figures 13a and 14a). In the pallium, some cell bodies and fibers Labeled fibers were observed bilaterally in the medial and lateral olfactory tracts, as well as crossing caudally in the anterior commissure ( Figures 13a-d and 14a-b). Small cell bodies were also labeled in the entopeduncular nucleus, close to the lateral olfactory tract and fore-brain bundles, and occasionally in the anterior parvocellular preoptic nucleus (Figures 13d-e and 14c-e).
Most of the extratelencephalic structures labeled after DiI application to Dp were fibers. Rostrally, some of these fibers were observed in the ipsilateral habenula, crossing to the contralateral side through the habenular commissure (Figures 13f and 14g). Some labeled fibers exited the lateral forebrain bundle (lfb) toward the dorsal posterior thalamic nucleus (DP), where occasional periventricular cell bodies were observed, and the lateral preglomerular nucleus (PGl) (Figures 13g   and 14h-j). Numerous labeled fibers from the medial forebrain bundle coursed throughout the lateral hypothalamus to the torus lateralis (TLa), diffuse nucleus of the hypothalamic inferior lobe, the region of the tertiary gustatory nucleus, the nucleus of the lateral recess, and a few entered the hypothalamic PL (Figures 13h-i and 14j-k, m). A few lightly labeled cell bodies were observed lateral to the medial preglomerular nucleus (Figures 13g-h and 14i-j). Occasionally, an ipsilateral labeled fiber bundle was followed from the lateral hypothalamic region directly to the midbrain dorsal tegmental nucleus, which is located rostrally to the lateral NLV (Figures 14k-l). In the NLV, a few faintly labeled fibers and terminals were observed (Figure 14l). In the rostral rhombencephalon, a few cell bodies were labeled bilaterally in the rostral and ventral part of the secondary gustatory/visceral nucleus (Figures 13j and 14n), which likely corresponds to the visceral part of this complex (see Yáñez et al., 2017).

Tracer application to the central zone of the pallium (Dc)
In this hodological study, we considered Dc ( Some labeled fibers also entered the rostral optic tectum (Figure 15c).
Somas were labeled bilaterally in the preglomerular complex (PGl and PGm) and ipsilaterally in the posterior (PTN) and periventricular (TPp) tuberal nuclei. Labeled fibers were also observed in the F I G U R E 1 1 (a-k) Schematic drawings of transverse sections of the zebrafish brain showing structures labeled after application of a DiI crystals centered in the dorsal zone of pallium (Dd) at precommissural levels. In (a-f), note that tracer diffusion (gray areas) also extends to neighboring ipsilateral pallial regions (Dl and Dc), and also to the contralateral lobe. Note also ipsilateral labeling in Vd. In (g-k), labeled extratelencephalic structures, mostly ipsilateral, are represented. Sections are arranged from rostral to caudal and the levels of the sections are indicated in the figurine of the lateral view of the brain. Black circles, retrogradely labeled cells. Small dots and lines, labeled fibers. Shaded areas in (a-e) represent the high-or less-dense labeled regions from the application point (white star in d). Scale bar for sections: 500 µm caudomedial region of the ipsilateral dorsal periventricular hypothalamic nucleus and bilaterally in the dorsal region of the hypothalamic PL (Figures 15d-f). Some cell bodies and many fibers were also labeled bilaterally in the medial mesencephalic tegmentum, where some fibers decussate at the level of the oculomotor nucleus, and in the lateral valvular nucleus, the superior raphe and the secondary gustatory/visceral nucleus (Figures 15g-i). Finally, a small nucleus was labeled in the dorsomedial rhombencephalic tegmentum at the level of entrance of the trigeminal nerve ( Figure 15j): The nucleus appears to be the dorsal portion of superior raphe nucleus.

Pallial connections revealed by retrograde tracing from extrapallial areas
Direct application of DiI to the various pallial regions, as reported above, revealed the presence of afferent perikarya in other pallial and extrapallial regions, as well as labeled fibers. As most of the pal-lium regions of zebrafish were accessed directly from the ventricular surface, which is away the main telencephalic tracts, the labeled perikarya represent bona fide afferent neurons to these regions, However, the fibers labeled in other brain regions might not be originated in neurons of the approached pallial region, but from neurons in other centers that send collaterals both to the problem pallium region and to the putative target. This is possible because the free diffusion of DiI along cell membranes (indistinctly in retrograde and anterograde directions) characteristic of this small lipophilic molecule. A way to demonstrate that fibers are actually efferent of the problem center is to apply DiI to the putative target in order to reveal the presence of labeled perikarya in that pallial center. Here, we report the pallial neuronal populations and fibers labeled after DiI application to various extratelencephalic regions that showed labeled neurons or fibers after DiI application to pallial zones. These experiments included application of DiI to the anterior and lateral preglomerular nuclei, the TLa, the lateral and medial regions of the hypothalamic inferior lobes, the PTN, the optic tectum, and the olfactory bulb (Figures 16-19). In the case of the olfactory bulb, the goal of these injections was helping to F I G U R E 1 2 (a-j) Photomicrographs of transverse sections of zebrafish brain showing intra-(a-g) and extratelencephalic (h-j) structures labeled in an experiment of application of DiI to Dd (open arrow in c-e). Correspondences with schemes of Figure 11 are indicated. Black arrowheads: cell bodies. Arrowheads: fibers and fiber bundles. (a-g) Sections through rostral (a-b), precommissural (c), commissural (d-f), and postcommissural (g) telencephalic levels, showing ipsilateral (at left) and contralateral (at right) labeling of pallial areas well delimited mainly in Dd, Dld, Dc, and Dma, and also in the subpallial Vd. Dm and Dp are mostly free of labeling. In €, note a detail of Vd showing retrogradely labeled cells (black arrowheads). (h-j) Sections through the ipsilateral hypothalamus showing dense labeling of the lateral preglomerular nucleus (h) and bundles of labeled fibers (black arrows) coursing from the lateral forebrain bundle (see g) and reaching caudally the hypothalamic posterior lobe (j), as well as occasional labeled cells (black arrowheads) dorsal to the PGm, Hv, and periventricular TPp (i) (correspondence of (h-j) with levels depicted in Figure 11i-j is partial). Numbers 1-3 indicate Dm subdivisions. Asterisks: ventricle. Stars: inferior lobe recess. All photomicrographs are negative images of fluorescent data. For abbreviations, see the list. Scale bars: 200 µm (b-d, g); 100 µm (a, f, h-j); 50 µm (e) delimit Dp anatomically, facilitating thus the approach to this region.
Other zebrafish extratelencephalic brain regions analyzed by us with tracing experiments that did not show connections with the pallium were not considered (see Yáñez et al., 2009;Yáñez et al., 2017;Yáñez et al., 2018).

Preglomerular complex
Application of DiI to the lateral preglomerular nucleus (PGl), also affecting the attached anterior preglomerular nucleus (PGa), led to labeling abundant fibers in the ipsilateral pallium, mainly at precom-missural and commissural levels of Dl, Dd, Dm, and Dc ( Figure 16). Retrogradely labeled cells were also noted in the pallium, and these cells were mostly observed in a deep (away the ventricle) ventral zone of Dm at commissural and postcommissural levels (Figures 16d-g and 19b-c).
A few cell bodies were also labeled in Dd (not shown), where also a number of varicose fibers could be observed coming from the lateral forebrain bundle. In addition, we observed a few faintly labeled cell bodies in dorsal Dp (Figures 16i-j), which may represent cells that project to the TLa and/or the diffuse nucleus and whose axons could get labeled as they traversed the region of the lateral preglomerular nucleus (see the next paragraph).  (Figures 17a-b).
More caudally, only a few cells were labeled in Dm (Dm1) at commissural levels (Figures 17e-f) and some cells at the ventral border of medial Dp (Figure 17g). These latter cells belong to Dp that was not assessed.

Anatomical regions of the zebrafish pallium
Recent molecular studies of the zebrafish telencephalon are introducing new views regarding the regional organization of the zebrafish pallium, especially attending the characterization of rostral Dm, Dc, Dd, and Dl. Establishment of connections of neuronal groups is the result of the expression of a large number of regulatory factors, proteins, signals, and receptors directing the birth and progressive specification of neuron types, guiding the axon growth, selective search for one or several types of target neurons, and establishment of functional synapses (Kwan et al., 2012). Thus, the patterns of connections reflect the differential characteristics of various neuronal populations, and reveal the areal /topographical organization of neural centers, even though they appear rather homogeneous. Following this idea, connectional results presented here may reveal hidden properties of pallial territories and thus be useful for assessing the organization of pallium in zebrafish.
As suggested previously using immunohistochemical methods (Castro et al., 2006), present results support the notion that major pallium regions (Dm, Dl) are not homogeneous rostrocaudally, because rostral parts are distinguishable from more caudal portions by connections. Castro et al. (2006) showed that the lateral part of the rostral pallium (Dla) can be distinguished from more caudal Dl and Dc based on the distribution of different markers, among others the calciumbinding protein calretinin. Parvalbumin (PV) expression also allows distinguishing in Dl a rostral region that lacks PV from more caudal regions with intense PV-positive cells (figure S4-A1-2 in Aoki et al., 2013, Supplemental information). Rostrocaudal differences in these main areas have been reported in the trout (Castro et al., 2003;Folgueira et al., 2004b) and goldfish (Northcutt, 2006). Recent studies named a wide region that comprises to the entire rostral pole of the zebrafish pallium as "Dc" (Mueller et al., 2011;Porter & Mueller, 2020), which includes our Dma and Dla and more caudal territories. The use of the name Dc in this sense causes homonymy confusion with the traditional definition of Dc in studies of different teleosts as a large-celled central region (Nieuwenhuys, 1963), as used here. Moreover, the anterior pallium differs from the rest of the pallium, and may be divided into medial and lateral divisions (Dma and Dla) (Castro et al., 2006). ter interpreted in terms done by Castro et al. (2006).
New neurogenetic data indicate the progressive generation of the zebrafish pallium in a temporal and medio-lateral zonal sequence (Dirian et al., 2014;Furlan et al., 2017). Results of these studies suggest that the deepest pallial neurons (possibly our Dc neurons among others) are generated early during development from the pallial ventricular zone, forming the core of the pallium, and then the ventricular zone gives rise to neurons (Dma, Dm, Dla, Dl, Dd, and Dp?) that surround the Dc core, as proposed by early models (see Nieuwenhuys & Meek, 1990). If the new neurogenetic ideas on the zebrafish pallium organization (Dirian et al., 2014;Furlan et al., 2017) also apply to advanced teleosts that exhibit much more complex pallia need to be investigated.
For further discussion on the organization and connections of Dc, see Section 4.5.
Results in zebrafish and other species indicate a high complexity in the pallial subdivisions of teleosts. For instance, the existence of neurochemical differences between rostral Dm and more caudal parts of the pallium has also been reported in trout (Castro et al., 2003) and goldfish (Northcutt, 2006). In goldfish, Northcutt's (2006) cytoarchitectonic study proposed a more complex subdivision of main pallial areas, which is in contrast with the subdivision of the pallium in the same species done by Yamamoto and Ito (2008), more similar to the zebrafish "new model" of Mueller et al. (2011). Complex rostral to caudal subdivision of the main pallial areas is outstanding in some acanthopterygians, both in medial and lateral regions (see Burmeister et al., 2009;Dewan & Tricas, 2014;Maruska et al., 2017;Hagio et al., 2020).

The medial zone of the zebrafish pallium (Dm) is organized in territories with differential connections
The zebrafish Dm is considered to be topologically similar to the cortical amygdala of mammals (Porter & Mueller, 2020), despite its topographically medial location, which results from the eversion process. Our results indicate the existence of regional differences within Dm with regard to its connections, despite that all individual zones could not be accessed separately by technical reasons. In general, Dm exhibits abundant internal connections. Dm1 shows reciprocal connections with Dma, and projects to Dm3 and probably Dm2. Thus, Dm1/2 and Dm3 appear connected, whereas no direct connection between Dma and Dm3 was observed. However, connections of zebrafish Dm subregions with other pallial regions are rather limited. Only the applications of DiI to Dm3 labeled some neurons in a lateral region of Dl.
Thus, zebrafish Dm, excepting its caudal part, appears largely independent from other pallial regions.
From a comparative point of view, the connections of zebrafish Dm with other pallial zones resemble those reported in goldfish (Northcutt, 2006) and Gymnotus carapo , but not those reported in the rainbow trout (Folgueira et al., 2004b) and Sebasticus marmoratus (Murakami et al., 1983). Although similar, Dlcaudal Dm connections are much more limited in zebrafish than in goldfish (Northcutt, 2006). In the rainbow trout, most intrapallial connections of Dm were restricted to other Dm subregions (Folgueira et al., 2004b), and no connections with Dl were observed. The intrapallial pattern of connections of zebrafish Dm also differs from the abundant interconnections between Dm and Dc reported in S. marmoratus (Murakami et al., 1983). Finally, in the mormyrid Gnathonemus petersii, tracer application in the auditory and lateral line related areas of Dm led to labeling pallial neurons only in Dm (von der Emde & Prechtl, 1999).
With regard to their connections with extrapallial territories, the zebrafish Dma, Dm1/2 and caudal Dm3/Dmp, show remarkable differences between them. In general, Dma connections with extrapallial nuclei are scant, showing mainly projections to the (dorsal) entopeduncular nucleus, from which it also appears to receive fibers (Turner et al., 2016). Zebrafish Dma shows more restricted connections than those reported for rostral Dm (Dmr) in goldfish (Northcutt, 2006), suggesting that the zebrafish Dma approached by us may not correspond in extension to goldfish Dmr. In goldfish and carp, Yamamoto and Ito (2008) reported afferents and neurons labeled from the preglomerular complex to Dm mostly at its commissural levels, but not rostrally. Compared to Dma, intermediate (Dm1/2) and caudal (Dm3) regions of Dm show more broad connections. Present results show that Dm1/2 receives abundant innervation from the lateral and anterior preglomerular nuclei and the PTN, and projects fibers to the thalamus, pretectum, and the hypothalamic lobes. Among afferent to the precommissural and commissural region of Dm are notable the fibers from the preglomerular complex that form a terminal field in parallel to the ependymal surface. As shown in toto in the brain of a transgenic zebrafish line, this terminal field originating from a preglomerular population expressing GFP does not extend along all Dm, and fibers probably are collaterals of the projection of these neurons on Dl (see below) (Bloch et al., 2020). A study in transgenic lines of zebrafish has identified a neuronal population in Dm (120A-Dm neurons) that projects to the hypothalamus and is essential for fear conditioning (Lal et al., 2018). These results indicate that afferents to Dm3/Dmp mostly differ from those to Dm1/2. From a comparative point of view, the pattern of Dm efferents observed in zebrafish seems more restricted than that reported for the caudal Dm of goldfish (Northcutt, 2006). This suggests that the region approached by us in zebrafish is only equivalent to a part of Northcutt's caudal Dm in goldfish. Comparison with other teleosts indicates that Dm connections with preglomerular nuclei and the tertiary gustatory nucleus are conserved. That is the case for the rainbow trout, for instance, in which Dm is reciprocally connected with the preglomerular nuclei and receives afferents from the tertiary gustatory nucleus (Folgueira et al., 2003(Folgueira et al., , 2005. In S. marmoratus (Murakami et al., 1983), there are connections between Dm and the acousticolateral preglomerular nuclei, as well as direct projections from the Dm and Dd to the acoustic region of the torus semicircularis.
In goldfish, tracer injections in the tertiary gustatory nucleus labeled fibers and many neurons in dorsal Dm (Kato et al., 2012). Reciprocal connections between the preglomerular complex and division 2 of Dm, but not from rostral Dm, have been reported in Gymnotus (Corrêa & Hoffmann, 1999;, and between dorsal Dm and the auditory preglomerular nucleus in the carp and goldfish (Yamamoto & Ito, 2005a, 2005b, 2008. In Gnathonemus, the auditory/lateral line ventral preglomerular nucleus projects on Dm (von der Emde & Prechtl, 1999). In addition, a few afferent fibers to Dm originate from the commissural nucleus of Cajal in carp (Uezono et al., 2015), but not in tilapia or zebrafish (Yoshimoto & Yamamoto, 2010;Yáñez et al., 2017;present results). Together, these observations reveal

The lateral zone of the zebrafish pallium (Dl)
In zebrafish, the lateral zone of the pallium (Dl) comprises a wide region dorsal and rostral to Dp, lateral to Dc and the sulcus ypsiloniformis (Figure 1). This region is not homogeneous from rostral to caudal, as noted by the differential innervation by NPY-immunoreactive fibers (very dense in the dorsal part of precommissural Dl; Castro et al., 2006), the differential expression of the cannabinoid receptor 1 (cb1) in rostral Dl (Lam et al., 2006) and the central-caudal distribution of parvalbuminimmunoreactive neurons in Dl (see supplemental figure S4 in Aoki et al., 2013). These data allow distinguishing roughly anterior, middle, and posterior regions in Dl, with the middle one consisting of a dorsal and a ventral part (Castro et al., 2006). Moreover, dorsal Dl appears to be continuous with Dd in zebrafish, which makes it difficult to distinguish between these two regions.  (Rink & Wullimann, 2004), which suggests that connections may be reciprocal. In goldfish and carp, Dl receives afferents from Vd and Vs (Northcutt, 2006;Yamamoto & Ito, 2008), and in trout, Vd, Vv, and Vs are also afferent to pallial areas (Folgueira et al., 2004a;2004b). These subpallial regions (Vv, Vd, Vs) may correspond to the mammalian pallidum, striatum, and subpallial amygdala, as proposed on the base of a variety of molecular markers (Ganz et al., 2012;Porter & Mueller, 2020). However, discussion about possible homologies in detail with mammals is out of our focus and the reader is referred to these molecular studies. The interhemispheric interconnections of Dl via the anterior commissure may be topographically organized, as observed in trout (Folgueira et al., 2004b), leading probably to topographical contralateral regulation of activity as reported for contralateral projections of the olfactory bulbs (Kermen et al., 2020). Moreover, the connectivity observed between Dl and subpallial centers may be related with the roles of Dl in learning and spatial memory reported in goldfish (Portavella et al., 2002;Ocaña et al., 2017;Rodríguez-Expósito et al., 2017). Experimental evidence after ablation of Dlv in goldfish indicates that this zone of the pallium is necessary for development of allocentric, relational spatial learning and memory, but not for egocentric, nonrelational strategies for orientation (Rodríguez et al., 2002;Rodríguez-Expósito et al., 2017).

Regarding its intrapallial connections, DiI application to
Based on these results, it has been proposed that the goldfish Dlv is functionally comparable to the hippocampus of mammals. Whether this is the case for zebrafish, who is phylogenetically close to goldfish, needs to be further investigated.
With regard to the extratelencephalic afferents to zebrafish Dl, the most important comes from the lateral and anterior preglomerular nuclei. This Dl afferent was very clearly demonstrated using a transgenic zebrafish line in which the lateral preglomerular neurons express GFP (Bloch et al., 2020); interestingly, this projection only covers the middle region of Dl, and simultaneously projecting on Dm, as indicated above, providing main inputs to two well-separated pallial regions.
This input was proposed to be part of an ascending visual pathway retina-tectum-preglomerular complex-pallium (Bloch et al., 2020). A recent neurogenetic study using the same transgenic line suggested that many preglomerular neurons originate in the embryonic mesencephalon near the midbrain-hindbrain boundary (Bloch et al., 2019).
This teleost visual circuit appears to be very unlike the visual circuits of amniotan vertebrates, indicating convergent evolution. How this Dl circuitry compares with that of the mammalian hippocampus is unclear.

Considerations about the dorsal zone of the pallium (Dd)
As indicated above, the dorsal zone of the zebrafish pallium (Dd) extends laterally to Dm, between the sulcus ypsiloniformis and dorsal Dl (Figure 1). The Dd and dorsal Dl zones are so poorly differentiated cytoarchitectonically in the adult zebrafish that can be considered as a single area judging from our tracing results. In electric fishes as Gymnotus and Apteronotus, immunohistochemical markers such as the kinase CaMKIIa clearly distinguish between both pallial zones . In zebrafish, despite the difficulty to differentiate between Dd and Dl, this domain is far from being homogeneous rostrocaudally with regard to the expression of gene markers or fiber innervation. For instance, numerous Dd+Dl neurons express eomesa rostral to the anterior commissure but not caudal to it (Ganz et al., 2014). In middle and caudal levels, Dd+Dl shows numerous parvalbumin positive perikarya, but not in rostral levels (Dla). A small zone of Dd at the level of the anterior commissure shows calretinin-immunoreactive cells, which are lacking in rostral and caudal levels of Dd or in Dl (Castro et al., 2006). Likewise, notable regional density differences in innervation by NPY-positive fibers were reported among rostral and caudal Dl and Dc (Castro et al., 2006). Thus, these differences in expression patterns and innervation reveal a more complex area than suggested by cytoarchitectonic analysis. While with respect to cytoarchitectonical aspects, the Dd and Dl areas may be largely viewed as a continuum, functional differentiation would be based on the information processed in their different parts.

4.5
The central zone of the zebrafish pallium (Dc) Although the central zone of the teleost pallium (Dc) is considered a heterogeneous region in most teleost species, it was generally identified cytoarchitectonically by the presence of large neurons in low to moderate density (Northcutt & Braford, 1980;Nieuwenhuys, 1998 Prox1-immunoreactive fibers (see figure 5B in Ganz et al., 2014); these fibers appear to originate from PV-positive neurons of Dl, which is in agreement with our tracing results, revealing neurochemical signatures of this afferent system to our Dc. In contrast with neighbor regions, this almond-shaped Dc neuropil is poorly innervated by GADimmunoreactive fibers (Castro et al., 2006). Together, these results in zebrafish indicate that Dc is distinguishable from the other pallial regions pallial by its specific neuronal population, connections, and immunohistochemistry. We conclude that this zebrafish central nucleus is the pallial area giving rise to a pallial dorsal efferent pathway in zebrafish, relaying information mainly from dorsal Dl-Dd and projecting toward the pretectum (Yáñez et al., 2018) and rostral optic tectum (present results). In electric fish, similar Dc neurons were also characterized neurochemically as glutamatergic cells that receive diffuse projections from Dl and project to the tectum; they have been compared with layer 5 and layer 6 neurons of the mammalian pallium that project to the superior colliculus, the optic tectum homologue .
Hodological data show that the zebrafish Dc (as characterized here) is clearly distinct by its pattern of connections from the other pallial areas, including those rostrodorsal pallial regions included in "Dc" by Mueller et al. (2011). A cytoarchitectonically distinct Dc central zone well separated from Dl and rostral pallium was recognized in the telencephalon of various teleosts (Murakami et al., 1983;Harvey-Girard et al., 2012;Elliott et al., 2017). Hodologically, Dc neurons project to the optic tectum in goldfish (Grober & Sharma, 1981) and the goby Acanthogobius flavimanus (Hagio et al., 2018;Hagio et al., 2021), which is similar to that shown in zebrafish. However, in the goby, tectal afferent neurons are located in a central part (Dcm) adjacent to Dm within a large Dc. In trout, a small but well-defined Dc, similar to that shown in zebrafish, was demonstrated by retrograde labeling from the paracommissural pretectum (Folgueira et al., 2004b). In the carp, large Dc neurons and occasional small neurons in Dm were also labeled from the ipsilateral auditory torus semicircularis (Echteler, 1984). A central region (Dc) receiving rich innervation from Dl was reported in goldfish (Northcutt, 2006). In the electric fish Gymnotus, a conspicuous Dc is richly innervated with fibers from Dl , as noted in zebrafish. In Sebastiscus, Dc is an anatomically very well-defined central area that maintains connections with other pallial areas and gives rise to extensive projections to various brain regions (Murakami et al., 1983).

4.6
The posterior zone of the zebrafish pallium (Dp) The posterior zone of the pallium (Dp) of teleosts and other ray-finned fishes is the main target of mitral cells of the olfactory bulbs (Nikonov et al., 2005;Huesa et al., 2006). Our results expand our knowledge regarding the connectivity of this telencephalic area in zebrafish, showing its extratelencephalic connections, mainly with preglomerular nuclei and hypothalamus. Present results confirm projections of mitral cells to Dp in zebrafish shown by DiI labeling (Gayoso et al., 2011(Gayoso et al., , 2012) and single-cell labeling (Miyasaka et al., 2009. Single-cell labeling shows that most mitral cells project their axons to Dp bilaterally , and the contralateral projections are specifically organized (Kermen et al., 2020). Tract-tracing experiments further reveal that the olfactory bulb-Dp connection is reciprocal, as indicated by the numerous neurons labeled in Dp after DiI applica-tion into the olfactory bulb (Gayoso et al., 2011(Gayoso et al., , 2012 and the abundance of fiber labeled from Dp in the inner granular layer of the bulb present results). Similar results obtained with neural tracing methods were reported in different teleosts, as S. marmoratus, rainbow trout, and goldfish (Murakami et al., 1983;Folgueira et al., 2004a;Northcutt, 2006) and in a chondrostean (Huesa et al., 2000(Huesa et al., , 2006. In the goldfish, a ventral region of Dl (Dl-v), rostral to Dp, also maintains reciprocal connections with the olfactory bulb (Northcutt, 2006). This Dl-v olfactory region was also observed in zebrafish, appearing as a rostral extension of Dp. Recent molecular studies in adult zebrafish identify Dl-v with a rostral part of the nucleus of the lateral olfactory tract (nLot; Porter & Mueller, 2020). For details of differential connections in zebrafish of the different olfactory bulb glomerular fields and the subpallium, the reader is directed to studies of Gayoso et al. (2011Gayoso et al. ( , 2012. With regard to intrapallial connections of Dp, only the application of DiI to Dl led to labeling of some fibers and cell bodies in Dp. These results, showing connections between Dl and Dp, differ from those reported in other species, such as the rainbow trout (Folgueira et al., 2004b), S. marmoratus (Murakami et al., 1983), or Gymnotus . In the rainbow trout, Dp was interconnected with Dm but not with Dl-Dd (Folgueira et al., 2004b). Similarly, in S. marmoratus, numerous Dp neurons project to ventral Dm but not to Dl, whereas dorsal Dm projects to Dp (Murakami et al., 1983). In Gymnotus, Dl and Dp are not interconnected and their limit is very sharp after tracing experiments from Dl . In goldfish, application of HRP to the caudal Dm, Dl, or Dl-v produced labeling of neurons and fibers in Dp (Northcutt, 2006). This pattern observed in goldfish seems less specialized than that in zebrafish, also a cyprinid, and in other teleosts studied.
With regard to the subpallial connections of Dp, our experiments in zebrafish labeled numerous cell bodies ipsilaterally in the dorsal region of Vv (Vv-d) and, bilaterally, in the supracommissural nucleus (Vs), with fibers crossing in the anterior commissure. The Vs and Vv neurons, as for most subpallial nuclei, are GABAergic (Mueller & Guo, 2009). Accordingly, these nuclei may contribute to the abundance of GADimmunopositive (inhibitory) fibers observed in Dp (Castro et al., 2006).
In the rainbow trout, Dp receives projections from Vv and projects to the Vd (Folgueira et al., 2004a(Folgueira et al., , 2004b. Moreover, interconnections between Dp and Vv were reported in S. marmoratus (Murakami et al., 1983).
Regarding extratelencephalic connections of zebrafish Dp, one of the most consistent extratelencephalic pathway labeled after DiI application to Dp was that to the PTN. As most mitral cell axons project to both Dp and the posterior tubercle in zebrafish , the fibers in the posterior tubercle we observed after DiI application to Dp probably represent, in fact, mitral cell axons labeled retrogradeanterogradely from Dp because DiI diffuses freely along cell membranes. Results of DiI application to the posterior tubercle ruled out direct projections from Dp to the PTN, because only an occasional neuron was labeled in Dp. Likewise, labeled fibers observed in the habenulae after DiI application to Dp also appear to be en-passant labeled mitral cell fibers , as DiI tracing from the habenula did not reveal labeled cell bodies in Dp (Turner et al., 2016).
However, Dp neurons were retrogradely labeled from the TLa and the lateral region of the hypothalamic lobes, which also appear to be targets of ascending gustatory and visceral pathways (Yáñez et al., 2017), indicating convergence of chemical information from olfactory and taste pathways on these areas. On the other hand, extratelencephalic afferents to Dp (from the anterior and medial preglomerular nuclei, PTN, and secondary gustatory/visceral nucleus) appear very scant in zebrafish by comparison with the numerous afferent sources to Dp reported in the rainbow trout (Folgueira et al., 2004b). In this latter species, two subregions (dorsal and ventral) can be identified within Dp based on connection patterns (Folgueira et al., 2002;Folgueira et al., 2004aFolgueira et al., , 2004b. Thus, it seems that there are important differences between this two species regarding Dp organization and circuitry.

Other pallial afferents
The pallium of teleosts receives diffuse projections from various regulatory systems, as revealed by the presence of fibers immunoreactive to various peptides and neurotransmitters using immunohistochemical methods (Batten et al., 1990;Kaslin & Panula, 2001). The cells of origin of these fibers are difficult to label with DiI because the extensive axonal branching characteristic of these cells limits the amount of tracer diffusing to cell perikarya, which at most appear faintly or very faintly labeled. In any case, the DiI-labeled cells from the pallium observed in the zebrafish raphe (labeled from Dl and Dd) may be part of the serotonergic system, and those labeled in the hypothalamic lateral or posterior recess region (from Dl) may be part of the histaminergic system (Kaslin & Panula, 2001;Xavier et al., 2017), which send numerous fibers to rostral pallial regions. Further analysis and discussion of these and other regulatory systems are away from our present purposes.

CONCLUSIONS
We present the results of the first comprehensive study of connections of the adult zebrafish pallium with tracing methods. This study reveals a wide intrazonal connectivity in pallial regions that is in contrasts with the poor interzonal connections observed, especially between medial (Dm), dorsolateral (Dl, Dd), and posterior (Dp) regions. This suggests that the zebrafish pallium has a modular organization (Figure 19), with dedicated modules for different neural processes. Pallial afferents also show compartmental organization. Most extratelencephalic afferent fibers come from preglomerular nuclei, whereas subpallial afferents to Dl and Dp originate from Vd, Vs, or dorsal Vv, regions that may be comparable with the mammalian striatum, amygdala, or pallidum (Wullimann & Mueller, 2004;Porter & Mueller, 2020). Moreover, output pathways of the pallium appear characteristic of different regions.
Interestingly, zebrafish Dc neurons characterized here originate the main pallial output toward the pretectum and optic tectum. In electric fish, these neurons were also characterized neurochemically as glutamatergic cells that project to the tectum and have been compared with layer 5 and 6 neurons of the mammalian pallium that project to the superior colliculus . Finally, we hope that the connections of the adult zebrafish pallium revealed in our experiments may be useful for neurogenetic studies of individual pallial neurons or the roles of different pallial zones in the zebrafish behavior.

CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest. All authors had access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

DATA SHARING AND AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

AUTHOR CONTRIBUTIONS
Study concept and design: JY and RA. Acquisition of data: JY, IL.
Analysis and interpretation of data: JY, MF, and RA. Drafting of the manuscript: JY, MF, and RA.

FUNDING
Funding for open access charge: Universidade da Coruña / CISUG.

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1002/cne.25268.