Ramón Anadón

Distribution of choline acetyltransferase immunoreactivity in the brain of an elasmobranch, the lesser spotted dogfish (Scyliorhinus canicula).

Although the distribution of cholinergic cells is remarkably similar across the vertebrate species, no data are available on more primitive species, such as cartilaginous fishes. To extend the evolutionary analysis of the cholinergic systems, we studied the distribution of cholinergic neurons in the brain and rostral spinal cord of Scyliorhinus canicula by immunocytochemistry using an antibody against the enzyme choline acetyltransferase (ChAT). Western blot analysis of brain extracts of dogfish, sturgeon, trout, and rat showed that this antibody recognized similar bands in the four species. Putative cholinergic neurons were observed in most brain regions, including the telencephalon, diencephalon, cerebellum, and brainstem. In the retrobulbar region and superficial dorsal pallium of the telencephalon, numerous small pallial cells were ChAT‐like immunoreactive. In addition, tufted cells of the olfactory bulb and some cells in the lateral pallium showed faint immunoreactivity. In the preoptic‐hypothalamic region, ChAT‐immunoreactive (ChAT‐ir) cells were found in the preoptic nucleus, the vascular organ of the terminal lamina, and a small population in the caudal tuber. In the epithalamus, the pineal photoreceptors were intensely positive. Many cells of the habenula were faintly ChAT‐ir, but the neuropil of the interpeduncular nucleus showed intense ChAT immunoreactivity. In the pretectal region, ChAT‐ir cells were observed only in the superficial pretectal nucleus. In the brainstem, the somatomotor and branchiomotor nuclei, the octavolateral efferent nucleus, and a cell group just rostral to the Edinger‐Westphal (EW) nucleus contained ChAT‐ir neurons. In addition, the trigeminal mesencephalic nucleus, the nucleus G of the isthmus, some locus coeruleus cells, and some cell populations of the vestibular nuclei and of the electroreceptive nucleus of the octavolateral region exhibited ChAT immunoreactivity. In the reticular areas of the brainstem, the nucleus of the medial longitudinal fascicle, many reticular neurons of the rhombencephalon, and cells of the nucleus of the lateral funiculus were immunoreactive to this antibody. In the cerebellum, Golgi cells of the granule cell layer and some cells of the cerebellar nucleus were also ChAT‐ir. In the rostral spinal cord, ChAT immunoreactivity was observed in cells of the motor column, the dorsal horn, the marginal nucleus (a putative stretch‐receptor organ), and in interstitial cells of the ventral funiculus. These results demonstrate for the first time that cholinergic neurons are distributed widely in the central nervous system of elasmobranchs and that their cholinergic systems have evolved several characteristics that are unique to this group. J. Comp. Neurol. 420:139–170, 2000.

Experimental study of the connections of the telencephalon in the rainbow trout (Oncorhynchus mykiss). II: Dorsal area and preoptic region

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. J. Comp. Neurol. 480:204–233, 2004.

Distribution of choline acetyltransferase (ChAT) immunoreactivity in the brain of the adult trout and tract-tracing observations on the connections of the nuclei of the isthmus.

The distribution of cholinergic neurons and fibers was studied in the brain and rostral spinal cord of the brown trout and rainbow trout by using an antiserum against the enzyme choline acetyltransferase (ChAT). Cholinergic neurons were observed in the ventral telencephalon, preoptic region, habenula, thalamus, hypothalamus, magnocellular superficial pretectal nucleus, optic tectum, isthmus, cranial nerve motor nuclei, and spinal cord. In addition, new cholinergic groups were detected in the vascular organ of the lamina terminalis, the parvocellular and magnocellular parts of the preoptic nucleus, the anterior tuberal nucleus, and a mesencephalic tegmental nucleus. The presence of ChAT in the magnocellular neurosecretory system of trout suggests that acetylcholine is involved in control of hormone release by neurosecretory terminals. In order to characterize the several cholinergic nuclei observed in the isthmus of trout, their projections were studied by application of 1,1`‐dioctadecyl‐3,3,3`,3`‐tetramethylindocarbocyanine perchlorate (DiI) to selected structures of the brain. The secondary gustatory nucleus projected mainly to the lateral hypothalamic lobes, whereas the nucleus isthmi projected to the optic tectum and parvocellular superficial pretectal nucleus, as previously described in other teleost groups. In addition, other isthmic cholinergic nuclei of trout may be homologs of the mesopontine system of mammals. We conclude that the cholinergic systems of teleosts show many primitive features that have been preserved during evolution, together with characteristics exclusive to the group. J. Comp. Neurol. 428:450–474, 2000.

Afferent and efferent connections of the habenula in the rainbow trout (Oncorhynchus mykiss): An indocarbocyanine dye (DiI) study

The habenula is a conserved structure in the brain of vertebrates. With the aim of further understanding of the evolution of the habenular system in vertebrates, we studied the afferent and efferent connections of the habenula of the rainbow trout. Experiments included application of the carbocyanine dye 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (DiI) into the habenula, telencephalon, pineal organ, posterior tubercle, and interpeduncular nucleus (IPN). The results obtained reveal a consistent pattern of habenular connections. Most afferents originate from three nuclei, one extending from the preoptic region to the rostral thalamus (the entopeduncular nucleus), the second located in the region of the hypothalamus‐posterior tubercle and consisting of large bipolar cells (tuberculohabenular nucleus), and the third in the preoptic region (preoptic nucleus). A few large neurons of the locus coeruleus appeared to be labeled in some cases. The trout habenula also receives pineal and parapineal projections. Small labeled glial cells were observed in the thalamus around the fasciculus retroflexus and, sometimes, around the IPN. The most conspicuous efferents coursed in the fasciculus retroflexus to the IPN, the isthmal raphe, and the central gray. The existence of olfactohabenular or habenulotelencephalic projections is discussed.

Calretinin immunoreactivity in the brain of the zebrafish, Danio rerio: distribution and comparison with some neuropeptides and neurotransmitter-synthesizing enzymes. I. Olfactory organ and forebrain.

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. J. Comp. Neurol. 494:435–459, 2006.

Ontogeny of γ‐aminobutyric acid‐immunoreactive neuronal populations in the forebrain and midbrain of the sea lamprey

Although brain organization in lampreys is of great interest for understanding evolution in vertebrates, knowledge of early development is very scarce. Here, the development of the forebrain and midbrain γ‐aminobutyric acid (GABA)‐ergic systems was studied in embryos, prolarvae, and small larvae of the sea lamprey using an anti‐GABA antibody. Ancillary immunochemical markers, such as proliferating cell nuclear antigen (PCNA), calretinin, and serotonin, as well as general staining methods and semithin sections were used to characterize the territories containing GABA‐immunoreactive (GABAir) neurons. Differentiation of GABAir neurons in the diencephalon begins in late embryos, whereas differentiation in the telencephalon and midbrain was delayed to posthatching stages. In lamprey prolarvae, the GABAir populations appear either as compact GABAir cell groups or as neurons interspersed among GABA‐negative cells. In the telencephalon of prolarvae, a band of cerebrospinal fluid‐contacting (CSF‐c) GABAir neurons (septum) was separated from the major GABAir telencephalic band, the striatum (ganglionic eminence) primordium. The striatal primordium appears to give rise to most GABAir neurons observed in the olfactory bulb and striatum of early larval stages. GABAir populations in the dorsal telencephalon appear later, in 15–30‐mm‐long larvae. In the diencephalon, GABAir neurons appear in embryos, and the larval pattern of GABAir populations is recognizable in prolarvae. A small GABAir cluster consisting mainly of CSF‐c neurons was observed in the caudal preoptic area, and a wide band of scattered CSF‐c GABAir neurons extended from the preoptic region to the caudal infundibular recess. A mammillary GABAir population was also distinguished. Two compact GABAir clusters, one consisting of CSF‐c neurons, were observed in the rostral (ventral) thalamus. In the caudal (dorsal) thalamus, a long band extended throughout the ventral tier. The nucleus of the medial longitudinal fascicle contained an early‐appearing GABAir population. The paracommissural pretectum of prolarvae and larvae contained a large group of non‐CSF‐c GABAir neurons, although it was less compact than those of the thalamus, and a further group was found in the dorsal pretectum. In the midbrain of larvae, several groups of GABAir neurons were observed in the dorsal and ventral tegmentum and in the torus semicircularis. The development of GABAergic populations in the lamprey forebrain was similar to that observed in teleosts and in mouse, suggesting that GABA is a very useful marker for understanding evolution of forebrain regions. The possible relation between early GABAergic cell groups and the regions of the prosomeric map of the lamprey forebrain (Pombal and Puelles [ 1999 ] J. Comp. Neurol. 414:391–422) is discussed in view of these results and information obtained with ancillary markers. J. Comp. Neurol. 446:360–376, 2002.

Calretinin expression in specific neuronal systems in the brain of an advanced teleost, the grey mullet (Chelon labrosus)

The distribution of calretinin (CR) in the brain of an “advanced” teleost, the grey mullet, was studied by using immunoblotting and immunocytochemical techniques. In immunoblots of protein extracts of rat and mullet brains, the CR antibody stained a single band of about 29 kDa. CR immunoreactivity was observed in specific neuronal populations of all brain regions. The primary olfactory system, the optic nerve fibers, and some sensory fibers of other cranial nerves exhibited strong CR immunoreactivity. In the forebrain, the CR‐immunoreactive (CR‐ir) populations were scarce in the telencephalon and hypophysiotrofic hypothalamus, but numerous in many specialized nuclei of the diencephalon (preglomerulosus complex, nucleus glomerulosus, anterior glomerular nucleus, nucleus diffusus) and pretectum (parvocellular and magnocellular superficial pretectal nuclei, central pretectal nucleus), which are related to sensory systems. The two main forebrain bundles, medial and lateral, contained numerous CR‐ir fibers. The midbrain sensory centers (optic tectum and torus semicircularis) exhibited numerous CR‐ir cells and fibers. Likewise, the secondary gustatory nucleus of the isthmus is one of the nuclei exhibiting more intense CR immunoreactivity. Characteristically, the efferent cerebellar system (eurydendroid cells and brachium conjunctivum) and some afferent cerebellar fibers were CR‐ir. In the medulla oblongata, a number of reticular cells, the inferior olive, and the magnocellular octaval nucleus exhibited CR immunoreactivity. CR‐ir motoneurons were also observed in the spinal cord and in the oculomotor nucleus. Together with results obtained in other vertebrates, present results suggest that neural systems using calretinin to maintain intracellular calcium concentration have been rather well conserved during vertebrate evolution. J. Comp. Neurol. 426:81–105, 2000.

An experimental study of the connections of the telencephalon in the rainbow trout (Oncorhynchus mykiss). I: Olfactory bulb and ventral area

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. J. Comp. Neurol. 480:180–203, 2004.

AFFERENT AND EFFERENT CONNECTIONS OF THE PARAPINEAL ORGAN IN LAMPREYS : A TRACT TRACING AND IMMUNOCYTOCHEMICAL STUDY

The neural connections of the parapineal organ of two species of lampreys were studied with the fluorescent dye 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (DiI) and with immunocytochemistry. The lamprey parapineal organ consists of a vesicle and a ganglion that are connected to the left habenula. Labeling experiments included the application of DiI to the parapineal organ, left and right fasciculus retroflexus, left habenula, and the left pretectal region. Afferent parapineal fibers run in the left fasciculus retroflexus to the interpeduncular nucleus. The parapineal fibers of this fascicle arose from parapineal ganglion cells, whereas DiI application to the left habenula labeled both neurons of this ganglion and bipolar cells in the parapineal vesicle. Efferent neurons were observed in the left habenula, and bilaterally in the subhippocampal nucleus and the dorsal pretectum. Labeling with DiI also revealed a hippocampal projection.

Differential bulbar and extrabulbar projections of diverse olfactory receptor neuron populations in the adult zebrafish (Danio rerio).

Immunohistochemical methods were used to characterize the expression of two calcium‐binding proteins, calretinin (CR) and S100, in the olfactory rosette of the adult zebrafish. These proteins are expressed in different sets of sensory neurons, and together represent a large proportion of these cells. Double immunofluorescence for CR and Gαolf protein, and CR immunoelectron microscopy, indicated that most CR‐immunoreactive (ir) cells were ciliary neurons. Differential S100‐ and CR‐ir projections to glomerular fields of the olfactory bulb were also observed, although these projections overlap in some glomeruli. Application of the carbocyanine dye DiI to either S100‐ir or CR‐ir glomerular regions led to labeling of cells mostly similar to S100‐ir and CR‐ir neurons, respectively. Instead, these bulbar regions project to similar telencephalic targets. On the other hand, antibodies against keyhole limpet hemocyanin (KLH)‐stained numerous sensory cells in the olfactory rosette, including cells that were CR‐ and S100‐negative. This antiserum also stained most primary bulbar projections and revealed extrabulbar olfactory primary projections coursing to the ventral area of the telencephalon through the medial olfactory tract. This extrabulbar projection was confirmed by tract‐tracing with DiI. A loose association of this extrabulbar primary olfactory projection and the catecholaminergic populations of the ventral area was also observed with double tyrosine hydroxylase/KLH‐like immunohistochemistry. Comparison between KLH‐like‐ir pathways and the structures revealed by FMRFamide immunohistochemistry (a marker of terminal ganglion cells and fibers) indicated that the KLH‐like‐ir extrabulbar projection was different from the terminal nerve system. The significance of the extrabulbar olfactory projection of zebrafish is discussed. J. Comp. Neurol. 519:247‐276, 2011.