The distribution of calretinin (CR) in the forebrain and the olfactory system of the adult zebrafish was studied by using immunocytochemical techniques. Previous studies in trout forebrain have indicated that CR-immunoreactive neurons acquire this phenotype rather early in development (Castro et al., J. Comp. Neurol. 467:254-269, 2003). Thus, precise knowledge of CR-expressing neuronal populations in adult zebrafish may help to decipher late stages of forebrain morphogenesis. For analysis of some forebrain nuclei and regions, CR distribution was compared with that of various ancillary markers: choline acetyltransferase, glutamic acid decarboxylase, tyrosine hydroxylase, neuropeptide Y, thyrotropin-releasing hormone, and galanin. The results reveal that calretinin is a specific marker of olfactory receptor neurons and of various neuronal populations distributed throughout the telencephalon and diencephalon. In addition, CR immunocytochemistry revealed characteristic patterns of fibers and neuropil in several telencephalic and diencephalic regions, indicating that it is a useful marker for characterizing a number of neural centers, pathways, and neuronal subpopulations in the zebrafish forebrain. Some ancillary markers also showed a distinctive distribution in pallial and subpallial regions, revealing additional aspects of forebrain organization. Comparison of the distribution of CR observed in the forebrain of zebrafish with that reported in other teleosts revealed a number of similarities and also some interesting differences. This indicates that various neuronal populations have maintained the CR phenotype in widely divergent teleost lines and suggests that CR studies may prove very useful for comparative analysis.

In this study and the accompanying article (Folgueira et al., 2004a), the fluorescent carbocyanine dye 1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was used in fixed tissue to comprehensively analyze the connections of the different regions of the telencephalic lobes and the preoptic region of the rainbow trout. Here, we analyze the connections of the dorsal area (D; pallium) of the telencephalon, and the preoptic region, as well as the telencephalic connections of several structures in the diencephalon and brainstem of juvenile trout. The dorsal plus dorsolateral pallial zone of D (Dd+Dl-d) receives afferents from contralateral Dd+Dl-d, the ventral area of the telencephalon, preoptic nucleus, suprachiasmatic nucleus, medial thalamus, preglomerular complex, anterior and lateral tuberal nuclei, posterior tuberal nucleus, posterior hypothalamic lobe, superior raphe nucleus, and the rhombencephalic central gray and reticular formation, and projects to the central zone of D (Dc), medial thalamus, and some caudomedial hypothalamic regions. The medial zone of D (Dm) maintains reciprocal connections with the preglomerular complex and also receives afferents from the preoptic nucleus, suprachiasmatic nucleus, anterior tuberal nucleus, preglomerular tertiary gustatory nucleus, posterior tubercle, superior raphe nucleus, locus coeruleus, and the rhombencephalic central gray, and reticular formation. Dc receives fibers mainly from Dd+Dl-d, preoptic nucleus, preglomerular complex, and torus semicircularis and projects to several extratelencephalic centers, including the paracommissural nucleus, optic tectum, torus semicircularis, thalamus, preglomerular complex, posterior tubercle nuclei, and inferior hypothalamic lobes. The posterior zone of D (Dp) is mainly connected with the olfactory bulbs, the ventral and supracommissural nuclei of the ventral area (subpallium), the preoptic nucleus, and the preglomerular complex and projects to wide hypothalamic and posterior tubercular regions. The preoptic nucleus projects to the olfactory bulb, to most regions of the telencephalic lobes, and to several diencephalic and brainstem structures. These results reveal complex and specialized connectional patterns in the rainbow trout dorsal telencephalon and preoptic region. Most of these connections have not been described previously in salmonids. These connections indicate that the salmonid telencephalon is involved in multisensorial processing and modulation of brain activity.

The habenulae are bilateral nuclei located in the dorsal diencephalon that are conserved across vertebrates. Here we describe the main afferents to the habenulae in larval and adult zebrafish. We observe afferents from the subpallium, nucleus rostrolateralis, posterior tuberculum, posterior hypothalamic lobe, median raphe; we also see asymmetric afferents from olfactory bulb to the right habenula, and from the parapineal to the left habenula. In addition, we find afferents from a ventrolateral telencephalic nucleus that neurochemical and hodological data identify as the ventral entopeduncular nucleus (vENT), confirming and extending observations of Amo et al. (2014). Fate map and marker studies suggest that vENT originates from the diencephalic prethalamic eminence and extends into the lateral telencephalon from 48 to 120 hour post-fertilization (hpf). No afferents to the habenula were observed from the dorsal entopeduncular nucleus (dENT). Consequently, we confirm that the vENT (and not the dENT) should be considered as the entopeduncular nucleus “proper” in zebrafish. Furthermore, comparison with data in other vertebrates suggests that the vENT is a conserved basal ganglia nucleus, being homologous to the entopeduncular nucleus of mammals (internal segment of the globus pallidus of primates) by both embryonic origin and projections, as previously suggested by Amo et al. (2014).

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The fluorescent carbocyanine dye 1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was used in fixed tissue to comprehensively analyze the connections of the olfactory bulbs and the different regions of the ventral (V) area of the telencephalic lobes (subpallium) of the rainbow trout. With this goal, DiI was applied to the different telencephalic nuclei and zones, as well as to the olfactory nerve, the olfactory bulb, the retina, and to several structures in the diencephalon and brainstem of juvenile trout. The olfactory bulbs maintain reciprocal connections with several regions of the telencephalon [ventral nucleus of V (Vv), supracommissural nucleus (Vs), posterior zone of D (Dp), preoptic nucleus], and also project to the diencephalon (posterior tuberal nucleus, posterior hypothalamic lobe). Vv receives afferents from Vs, the dorsal nucleus of V (Vd), the preoptic nucleus, and from several nuclei in the diencephalon and brainstem (suprachiasmatic nucleus, anterior and lateral tuberal nuclei, preglomerular complex, tertiary gustatory nucleus, posterior tubercle, inferior hypothalamic lobes, thalamus, torus semicircularis, secondary gustatory nucleus, locus coeruleus, superior raphe nucleus, central gray, and reticular formation), and projects to dorsal (pallial) regions and most of the nuclei afferent to Vv. The dorsal nucleus of V (Vd) and Vs mainly project to the dorsal area. In an accompanying article (Folgueira et al., 2004), we present the results of application of DiI to dorsal (pallial) telencephalic regions, as well as of several experiments of tracer application to extratelencephalic regions. The results presented here, together with those of the accompanying article, reveal a complex connectional pattern of the rainbow trout ventral telencephalon, most of these connections having not been described previously in salmonids.