Topological and histological description of preoptic area and hypothalamus in cardinal tetra Paracheirodon axelrodi ( Characiformes : Characidae )

Topological and histological descriptions of the preoptic area and hypothalamus of the cardinal tetra Paracheirodon axelrodi were performed. Standard histological paraffin sections were used and stained with Nissl technique, and plastic sections for high-resolution optic microscopy (HROM). The preoptic area showed some differences related to the location of the magnocellular preoptic nucleus (PM) and the size of the neurons in this region, as they were the biggest in all the preoptic area. Additionally, within the preoptic area, the different structures that comprise the organum vasculosum of the lamina terminalis (OVLT) were identified and characterized. The hypothalamus could be subdivided in three regions the ventral, the dorsal and the caudal hypothalamic regions neuron morphology, size and staining pattern were similar in all of them.


Introduction
The teleost central nervous system (CNS) plays an important role in the regulation of reproduction.First, it integrates environmental signals received through the sensorial system and internal signals regulated by the hypophysiotropic system.Second, it transmits the integrated signals to endocrine effectors through hypophysal modulation that results in the onset of reproduction-specific behaviours and gonadal maturation (Butler, Hodos, 2005;Cerdá-Reverter, Canosa, 2009).
Pioneer studies revealed similarities between the hypothalamus-hypophysis system of fish and other vertebrates, and its link with the CNS and endocrine system to regulate several vital processes (Kapoor, Khanna, 2004;Pogoda, Hammerschmidt, 2007).However, studies in teleost fish have shown that the regulation of hypophysiary activity is not restricted to the hypothalamus, as it was once believed, but it extends to other brain areas such as the ventral telencephalon and the preoptic area (Kah et al., 1993;Trudeau et al., 1997).The two main areas in the anterior-ventral brain containing neuronal somas whose axons reach the neurohypophysis in teleost fishes are the preoptic area and the hypothalamus (Palay, 1945).
The preoptic area includes different nuclei that are functionally related to the ventricular and tuberal regions of the hypothalamus forming a functional and structural unit (Meek, Nieuwenhuys, 1998).In fish, the hypothalamus is 2 e160145 [2] the biggest part of the diencephalon, located in the ventral brain surface, ventral to the third ventricle.It is connected to the hypophysis through the infundibulum, which contains neuroendocrine fibers, which proceed from preoptic and hypothalamic nuclei (Wullimann et al., 1996;Cerdá-Reverter, Canosa, 2009).For this reason, these two areas preoptic area and hypothalamus are treated as a single complex by endocrinologists when discussing hypophysiotropic control in fish.Secretory neurons of the preoptic area extend their fibers directly to the posterior hypophysis or neurohypophysis (Norris, 2007;Cerdá-Reverter, Canosa, 2009).

Materials and Methods
All procedures described in the following sections take into account the current regulations and protocols for ethic handling and animal care in research (AVMA, 2001;CCAC, 2003).Twelve adult animals were used for the analysis, mean standard length (L S ) ± S.D. = 2.33 ± 0.30 cm, mean total length (L T ) ± S.D. = 2.81 ± 0.39 cm, mean weight ± S.D. = 0.26 ± 0.12 g).All animals were anesthetized with (0.1 g/L) benzocaine and sacrificed by medullar cervical sectioning (Scherck, Moyle, 1990).Whole brain was carefully dissected and processed as follows.
The topology of the main preoptic and hypothalamic nuclei was established based on previously published fish brain atlases from: zebrafish, Danio rerio (Hamilton, 1822) (Wullimann et al., 1996); medaka, Oryzias latipes (Temminck & Schlegel, 1846) (Anken, Bourrat, 1998;Ishikawa et al., 1999) and lubina, Dicentrarchus labrax (Linnaeus, 1758) (Cerdá-Reverter, Canosa, 2009).Additionally, use quantitative and qualitative criteria such as neuron size, neuron shape, staining intensity, cytoplasm distribution pattern, nucleus location, spatial distribution and degree of neuronal grouping per nucleus in the different nuclei identified.In order to show the location of the different nuclei, schematics diagrams were performed indicating the level at which cross sections were obtained, labelled from A to L (Fig. 1).The zero level corresponds to the start of the postoptic commissure (Cpop) and was labelled as E. Each cross section was diagrammed using the "Image J" software (Rasband, 2012) and indicates the distance to the zero level in micrometres (μm).
Morphometrics and statistical analysis.Four nonconsecutive cross section images from the different levels identified were used for the morphometric analysis.Using the "Image J" software (Rasband, 2012), neuronal body area and perimeter were measured and recorded.All data were tested for normality and homoscedasticity with the Kolmogorov-Smirnov test and Levene's test, repectively, for single samples using the SPSS software (SPSS Inc, 2009).Once statistical assumptions were confirmed, one way analysis of variance (ANOVA) was performed followed by a Bonferroni multiple comparisons test (the level of significance was P ˂ 0.05) using the GraphPadPrism, Inc ©2007 software in order to determine significant differences between neuron perimeter and area in the different nuclei identified.(ENv); ventral part of the periventricular pretectal nucleus (PPv); ventral telencephalic area (V); ventral zone of the periventricular hypothalamus (Hv); ventro-lateral nucleus of the semicircular torus (TSvl); ventro-lateral optic tract (VOT); ventro-lateral thalamic nucleus (VL); ventromedial thalamic nucleus (VM).

Results
Preoptic area.The preoptic area in P. axelrodi extends from the medial region of the ventral telencephalon to the beginning of the diencephalon (Fig. 1).It surrounds the anterior diencephalic ventricle (DiV), also known as the third ventricle.Anteriorly, it is limited by the pars dorsalis (Cantd) and pars ventralis (Cantv) of the anterior commissure (Figs.2a-b).Dorsally, it is limited by the dorsal telencephalic area (D), the post commissural nucleus of the ventral telencephalon (Vp), the supra commissural nucleus of the ventral telencephalon (Vs), the ventro-lateral thalamic nucleus (VL) and the ventromedial thalamic nucleus (VM) (Figs.2a-e).Ventrally, it is adjacent to the optic chiasm (CO) and the postoptic commissure (Cpop) 3g).Posteriorly, it borders the ventral zone of the periventricular hypothalamus (Hv) (Fig. 3g).
The magnocellular preoptic nucleus (PM) (Figs.2a-c) is located in the telencephalon, dorsal to the parvocellular preoptic nucleus (PP).It adopts a more diffused disposition towards the caudal region.The parvocellular nucleus is divided in two regions, the anterior part (PPa), located ventrally in the medial telencephalon (Figs.2a-c), and the posterior part (PPp), located ventrally between the caudal telencephalon and the anterior diencephalon 3g).The most ventral margin of the DiV is surrounded by the suprachiasmatic nucleus (SC), situated dorsal to the CO and ventral to the PPp 3g).
Neurons of the PM are spherical and stain moderately with Cresyl Violet acetate and toluidine blue.They are characterized by being dispersed toward the PP dorsolateral region.They have little visible cytoplasm and a spherical central nucleus with one or more nucleoli (Figs.4a-b).Neurons in the PPa and PPp are spherical and stain strongly with Cresyl Violet acetate and toluidine blue.They form compact groups near the ventral PM region.Visible cytoplasm is scarce and the nucleus occupies the center of the cell (Figs.4a-b).In the SC, spherical neurons stain strongly with Cresyl Violet acetate and toluidine blue.They possess little visible cytoplasm and a central nucleus (Fig. 4c).
Hypothalamus.The hypothalamus, part of the diencephalon, is located ventral to the thalamus and caudal to the optic region (Fig. 1).The anteriormost portion is located caudoventrally to the SC and can be easily recognized due to the highly dense cell groups and its position surrounding the paired latero-caudal ventricular recesses (LR).Anteriorly, it is limited by the postoptic commissure (Cpop) and the PPp (Fig. 3g).Dorsally, it neighbours the limitant zone (LZ), the periventricular nucleus of the posterior tuberculum (TPp) and the paraventricular organ (PVO) (Figs.3h-i); ventrally it is limited by the hypophysis (Figs.3k-l).
Several nuclei can be identified in the hypothalamus.They are mostly located towards the ventral diencephalon region along the DiV.Three regions can be clearly distinguishable: the ventral hypothalamic region, the dorsal hypothalamic region and the caudal hypothalamic region.The ventral hypothalamic region corresponds with the ventral zone of the periventricular hypothalamus (Hv), the anterior tuberal nucleus (ATN) and the hypothalamic lateral nucleus (Hl) (Figs.3g-i).The dorsal hypothalamic region forms the paired latero-caudal ventricular recesses (LR) that are surrounded by a cellular mass known as the dorsal zone of the periventricular hypothalamus (Hd) (Figs.3hl).The caudal hypothalamic region corresponds with the caudal zone of the periventricular hypothalamus (Hc) and the posterior recess of the diencephalic ventricle (PR).The Hc is located ventral to the posterior tuberal nucleus (PTN) of the posterior tuberculum (Figs.3j-k).
The ventral and caudal regions cover most of the tuberal hypothalamus (HT).The dorsal region in turn extends laterally to form the hypothalamic inferior lobes (IL) (Fig. 1).The ILs are separated from the HT by a deep ventral furrow.Within the IL there are two other nuclei, the central nucleus of the interior lobe (CIL) (Figs. 3 k-l) and the diffuse inferior lobe nucleus (DIL) (Figs.3i-l).
Ventral hypothalamic region.Neurons in the Hv (Fig. 5a) and Hl (Fig. 5b) are characterized by their spherical shape, little visible cytoplasm, strong Cresyl Violet staining and moderate toluidine blue staining.Cell nuclei are spherical, central, containing a single nucleolus.In the Hv, neurons form a lamina with 7 or more cell layers.Neurons of the Hl form a lamina with 10 or more cell layers in the vicinity of the Hv; whereas in the farthest lateral region this lamina is only 6 to 7 cell layers.Towards the DiV, cylindrically shaped tanycytes (T) can be seen forming a multi-layered structure (Fig. 5a).Neurons in the ATN are spherical, dispersed and strongly stained with Cresyl Violet and toluidine blue.They have little or no visible cytoplasm and are surrounded by amielinic fibers.

Hypothalamic dorsal region.
Neurons in the Hd are spherical, with little cytoplasm, a large nucleus, and stain strongly with Cresyl Violet and weakly with toluidine blue.They can be found forming groups surrounding the LR (Fig. 5c).Towards the medial LR neurons form 5 cell layers increasing to 7 or more cell layers through the lateral LR.In the lateral LR a single layer of spindle shaped tanycytes (T) strongly stained with toluidine blue can also be observed (Fig. 5c).

Caudal hypothalamic region.
Neurons in the Hc are spherical, with scarce cytoplasm, and a large nucleus.They stain strongly with Cresyl Violet and weakly with toluidine blue (Fig. 5d).They cluster in tight groups when surrounding the posterior recess of the diencephalic ventricle (PR), forming more than 6-cell layers.Towards the dorsal nuclei region, these tight groups branch off into several 5-cell layers areas.Some dispersed tanycytes (T) and glial (Gl) cells can also be observed in this region, both cell types strongly stained with toluidine blue.The Hc region is completely surrounded by amielinic fibers, which in some locations are also visible within the nuclei (Fig. 5d).Neurons of the PTN are spherical with scarce cytoplasm, a big central nucleus and one or two nucleoli.They are found dispersed within the nuclei.Some PTN neurons stain weakly while others stain strongly with both Cresyl Violet and toluidine blue.This region also includes some amielinic fibers and glial cells.

Discussion
Preoptic area.Distribution and location of the different preoptic area nuclei in P. axelrodi is similar to those reported for zebrafish, D. rerio (Wullimann et al., 1996), and goldfish, Carassius auratus (Linnaeus, 1758) (Peter, Gill, 1975;Fryer, Maler, 1981).This is probably associated to the fact that all three species are closely related phylogenetically, belonging to the superorder Ostariophysi, being D. rerio and C. auratus of the Order Cypriniformes and P. axelrodi of the Order Characiformes.Each column represents the mean value and the standard deviation (Perimeter P = 0.16; Area P = 0.12).Different letters above bars denote significant differences, as assessed by the Bonferroni multiple comparisons test.
Similarities in topological distribution of the nuclei between these related species suggest an evolutionary conservation of structure.However, other related species like lubine, D. labrax (Cerdá-Reverter et al., 2001), and the Atlantic salmon, Salmo salar Linnaeus, 1758 (Peter et al., 1991), show some differences, particularly regarding the distribution of the magnocellular preoptic nuclei (PM), which precludes over generalizing the reported pattern.Detailed studies in other species have to be performed in order to fully characterize this brain region in ostariophysians.
As it has been reported in most true teleost fish, in P. axelrodi the preoptic area forms a continuous structure with the hypothalamus.It extends from the medio-posterior telencephalon to the anteriormost diencephalon, disappearing when it reaches the first hypothalamic nuclei (Fryer, Maler, 1981;Nieuwenhuys et al., 1998, Cerdá-Reverter, Canosa, 2009).
In P. axelrodi, the location of the preoptic area nuclei (PM and PP) is similar to that reported for D. rerio (Wullimann et al., 1996), the American catfish, Ictalurus punctatus (Rafinesque, 1818) (Striedter, 1990a), O. latipes (Anken, Bourrat, 1998;Ishikawa et al., 1999) and Anguilla japonica Temminck & Schlegel, 1846 (Mukuda, Ando, 2003).However, when compared with D. labrax, PM distribution is very different.In P. axelrodi PM neurons are confined to the most anterior region of the preoptic area, just dorsal to the PPa.Towards the posterior part of the PPa, PM neurons have a disperse distribution, and when PPp neurons become visible, no neurons are seen in the PM.In contrast, PM neurons in D. labrax are located dorsally in the caudal most regions of both the PPa and PPp; No neurons can be observed in the anterior part of the PPa (Cerdá-Reverter et al., 2001).Alternatively, in S. salar and C. auratus, the PM is located ventrally to the PPa and dorsally to the PPp (Peter, Gill, 1975;Fryer, Maler, 1981;Peter et al., 1991).
Regarding cell size, the PM in P. axelrodi is characterized for having bigger neurons (mean perimeter of 19.66 ± 1.86 µm) than the PPa (mean perimeter of 12.89 ± 0.51 µm) and the PPp (mean perimeter of 13.23 ± 0.35 µm).This is similar to what has been reported for A. japonica, in which PM neurons have a mean perimeter of 20 µm while PP neurons have an average perimeter of 5 µm (Mukuda, Ando, 2003).
In P. axelrodi, the organum vasculosum of the lamina terminalis (OVLT) is located towards the diencephalic ventricular ventral wall, within the preoptic area.The OVLT has been reported in fish (Gómez-Segade et al., 1986;Baile et al., 2008), as well as in other vertebrates (Bosler, 1977;Tsuneki, 1986), and is thought to be comparable to the median eminence of tetrapods.Ultra-structural evidence shows that nervous terminals inside the OVLT contain synaptic vesicles in close contact with fenestrated capillaries, suggesting release of neurohormones directly into the bloodstream.Particularly, terminals containing tyrosine hydroxylase have been observed (Bosler, 1977;Gómez-Segade et al., 1986;Butler, Hodos, 2005).This region also contains high density of GnRH positive fibers in most teleost fish studied thus far (Kah et al., 1993;Gomes et al., 2013).
Tanicytes (T) in the OVLT extend processes towards the ventricle, contacting blood vessels and cerebrospinal fluid.Evidence suggests that T transport hormones and other molecules from cerebrospinal fluid to hypothalamic nuclei for hypothalamic hormones production and regulation.Therefore, cerebrospinal fluid flow serves as a chemical communication pathway, in conjunction with blood flow, for feedback regulation of hypothalamic hormones (Kah et al., 1993;Butler, Hodos, 2005).Tanycytes are also capable of capturing and transfering substances like thyroxin, peroxidase, GnRH, DA and GABA to other cells (Bosler, 1977).Accordingly, the OVLT might be involved in hormonal feed-back circuits directed toward the central nervous system.However, large molecules might be intracellularly transported through the tanycytes as the fenestrations of endothelial cells might also allow passage of intravascular substances in the opposite direction (Nakai, Naito, 1975).
In teleosts, neurons of the dorsal hypothalamic region secrete neuropeptides such as CCK, GRP, SST and neurotransmitters like serotonin in the Hd nuclei (Cerdá-Reverter, Canosa, 2009).In the caudal hypothalamic region, Hc presents similar neuronal morphological features than Hd.Different reports indicate that neurons in the Hc secrete CCK, CRH, Galanin, NPY, SST, TRH and UI, as well as neurotransmitters like DA, GABA, serotonin and noradrenalin (Norris, 2007;Cerdá-Reverter, Canosa, 2009).Regarding the PTN, it has been reported that neurons in this nuclei secrete TRH (Cerdá-Reverter et al., 2001) Tanycytes are observed along all three hypothalamic regions described in P. axelrodi.They surround the diencephalic ventricle and the posterior (PR) and lateral (LR) recesses.It has been reported that tanycytes in the hypothalamic region have long cytoplasmic processes that converge towards the hypophysis, as it could be observed by retrograde labelling using DiI.Those processes colonize the adenohypophyseal tissue and reach secretory cells.Endocytosis and exocytosis can be observed within the processes, adjacent to secretory cells, indicating that ependymary cells can communicate through the intercellular space with cells in the adenohypophysis (Kah et al., 1993;Butler, Hodos, 2005).
In conclusion, the preoptic area in P. axelrodi shows differences in the distribution of the PM relative to other species.However, the other preoptic nuclei and the hypothalamus are similar in composition to those typically described for other teleost species.Our study of the neuroanatomical organization of the preoptic area and the hypothalamus in P. axelrodi provides a useful tool for future morphofunctional studies, such as a detailed inmunohistological characterization in order to identify neurons in these nuclei by their secretion products.Similarly, retrograde and anterograde tracing studies can be useful to determine the origin and destiny of fibers coming into and e160145 [11] leaving from the preoptic area and hypothalamus in order to truly understand the hypophysiotropic pathway.

Fig. 4 .
Fig. 4. Cross section photomicrographs of the telencephalon of Paracheirodon axelrodi; a. Delineated area shows neurons of the magnocellular preoptic nucleus (PM).Anterior part of the parvocellular preoptic nucleus (PPa) and diencephalic ventricle (DiV); b.Delineated area shows the anterior part of the organum vasculosum of the lamina terminalis (OVLT).Glial cells (black arrowhead), nerve fibers detail (white arrow), tanycytes (asterisk) and neurons that correspond to magnocellular preoptic nucleus (PM), anterior part of the parvocellular preoptic nucleus (PPa) and diencephalic ventricle (DiV); c.Delineated area shows the posterior part of the parvocellular preoptic nucleus (PPp) and the suprachiasmatic nucleus (SC); d.Delineated area shows posterior part of OVLT.Glial cells (black arrowhead), nerve fibers detail (white arrow), internal capillaries (stars), tanycytes (asterisk) and neurons that correspond to the anterior part of the parvocellular preoptic nucleus (PPa); e. Detail of the most ventral part of the OVLT shows: venule (V), tanycytes (asterisk) and neurons of the anterior part of the parvocellular preoptic nucleus (PPa).Nissl staining (a).Toluidine blue staining (b-e).Bar = 10 μm.

Fig. 5 .
Fig. 5. Cross section photomicrographs of the diencephalon of Paracheirodon axelrodi; a. Photograph showing neurons from the ventral zone of the periventricular hypothalamus (Hv), the diencephalic ventricle (DiV) and tanycytes (asterisk); b.Neurons of the lateral hypothalamic nucleus (Hl); c.Neurons of the dorsal zone of the periventricular hypothalamus (Hd) surrounding the paired latero-caudal ventricular recess (asterisk); d.Neurons of the most ventral part of the caudal zone of the periventricular hypothalamus (Hc) surrounding the posterior recess of the diencephalic ventricle and glial cells (arrows).Toluidine blue staining.Scale bar = 10 μm.

Fig. 6 .
Fig. 6.Morphometry of neurons of preoptic area and hypothalamus.a. Cell soma mean perimeter (P) (µm) and area (A) (µm2) in brain nuclei of the preoptic area of Paracheirodon axelrodi.Each column represents the mean value and standard deviation (P < 0.0001).b.Cell soma mean perimeter (P) (µm) and area (A) (µm2) in brain nuclei of the hypothalamus of Paracheirodon axelrodi.Each column represents the mean value and the standard deviation (Perimeter P = 0.16; Area P = 0.12).Different letters above bars denote significant differences, as assessed by the Bonferroni multiple comparisons test.