The central nervous system (CNS) integrates the information from sensory organs and mediates the response to environmental stimuli, whereas the spinal cord controls locomotion independently of the brain (Genten et al. 2009). This is a general pattern in animals, including teleosts (Northcutt and Braford 1980, Nieuwenhuys and Meek 1990, Yamamoto 2008, Senarat et al. 2016), and many studies have demonstrated that the brain morphology has adaptive significance and influences behavior and habits (Kotrschal et al. 1998, Gonzalez-Voyer and Kolm 2010, Park and Bell 2010). For example, the large cerebral hemisphere is associated with a higher degree of learning, sensory integration, and spatial navigation (Gonzalez-Voyer and Kolm 2010, Park and Bell 2010), while a large and optic tectum is associated with good vision and orientation response (Huber et al. 1997, Pollen et al. 2007). Brain morphology investigations therefore elucidate the physiology and behavior of fish.

It is well-known that the reproduction of teleosts is controlled by the hypothalamic-pituitary-gonadal axis (HPG axis) (Nagahama 2000). In this axis, the hypothalamus, a major area of diencephalon, releases hypothalamic hormones, especially gonadotropin releasing hormone (GnRH), to control the synthesis and secretion of the pituitary gonadotropic hormones (gonadotropins, GTHs): GTH I (FSH-like) and GTH II (LH-like). These gonadotropic hormones are essential for gonadal development and maturation as well as stimulation of gametogenesis in several species of fish (King and Millar 1992, Sherwood et al. 1993, Feist and Schreck 1996, Senarat et al. 2019a). There is an increasing interest in locating these reproductive hormones in the brain using immunocytochemistry and immunofluorescence (Naito et al. 1991, Nyuji et al. 2011, Senarat et al. 2019b), which will be useful in the assessment and control of gonadal differentiation of fish (Murata et al. 2012). However, to interpret the results detailed anatomical information on the brain is required. Therefore, the accumulation of neuroanatomical knowledge will be also significant for the development of evidence-based aquaculture.

The Indo-Pacific seahorse Hippocampus barbouri Jordan & Richardson, 1908 (Syngnathidae) is an economically important fish. This fish has been reared at the Phuket Biological Center, Thailand. The next step to broaden the stock of this fish is to increase its sustainable production with appropriate management. Scientific reports on the reproductive biology of this seahorse species is still limited (Nur et al. 2016, Kamnurdnin 2017), and more importantly no neuroanatomical studies have been reported. This study aims to provide the baseline information on the structure and development of the CNS of H. barbouri in captivity. To this end, H. barbouri were subjected to the histological observation from 1 to 35 days after birth (DAB).

Hippocampus barbouri reared in a standard culture system of the PMBC, Thailand, were used for the observation. We collected samples of juvenile (1, 6, 12, 14 and 24 DAB) and adult (35 DAB) stages (n = 3 for each DAB) from October to December, 2017. Kamnurdnin (2017) studied the effects of food on the growth and gonadal development of this fish, and we used the brains of his specimens in this study. Information on the samples are shown in Table 1. All specimens were acclimatized for about 14 days in shaded concrete tanks filled with sea water at 26–28 °C, salinity level of 31–33 ppt, and photoperiod of 12:12 hours light-dark. The fish were fed wild krill twice a day. The experimental protocol was approved by the Animal Care and Use Committee of Faculty of Science in accordance with the guide for the care and use of laboratory animal prepared by Chulalongkorn University (Protocol Review #1623004).

The fish used in the experiment were euthanized by the rapid cooling method (original protocol by Wilson et al. 2009) and then fixed overnight in a solution containing Davidson’s fixative (Dietrich and Krieger 2009) at room temperature. After dissection, the anatomical features of the whole brain were examined from various views (dorsal, longitudinal and ventral), and cross sections of the mid-body (at 35 days) were observed under the SZX12 stereomicroscopy (Olympus, Japan). Photographs were taken with an Olympus DP 11 digital camera. The major anatomical structures were subjected to a morphometric analysis (corpus cerebelli length, corpus cerebelli width, telencephalon width, tectum opticum length, lobus inferior hypothalami length, lobus inferior hypothalami width, cerebellum length, cerebellum width and vagal lobe length) following the standard guideline from Abrahão and Shibatta (2015). All morphometric parameters were measured using an automated cellular image analysis system, Digimizer software, version 3.7.0. Schematic diagrams were drawn using the Adobe Illustrator CS5.

To examine the CNS structure, all brain regions, including the spinal cord of all samples (1, 6, 12, 14, 24 and 35 DAB), were processed using a standard histological technique (Presnell and Schreibman 2013, Suvarna et al. 2013). The paraffin blocks were crossly and longitudinally sectioned at a thickness of 4 µm and stained with Harris’s hematoxylin and eosin (H&E). All histological sections were examined for the CNS structure, whereas brain development was assessed by comparing images from 1 to 35 DAB taken by the TE750-Ua camera (Leica, Heidelberg, Germany).

Studies in the field of evolutionary neuroscience have shown that the physiological and reproductive behaviors of a species are reflected in the structure of the CNS (Kotrschal et al. 1998, Nieuwenhuys et al. 1998, Gonzalez-Voyer et al. 2016, Tsuboi et al. 2017). We show that the brain of H. barbouri contains a large optic tectum, whereas the size of the cerebellum is not abnormal. Such structural characteristics have been associated with a high-level sensory integration and spatial navigation (Gonzalez-Voyer and Kolm 2010, Park and Bell 2010), visual-tactile information and orienting response (Huber et al. 1997, Pollen et al. 2007). It is also associated with feeding on fast-moving prey (Kotrschal et al. 1998) because the optic tectum is involved in multisensory integrations of visual signals with sensory information from other modalities (Davis and Northcutt 1983, Bodznick 1991, Meek and Nieuwenhuys 1998). Although no information is available on the feeding ecology of this species during the first month of development, our finding probably reflects the structurally complex habitat where H. barbouri catches prey. This may be a unique characteristic for syngnathid fish since they commonly have the longest snout and consume highly mobile prey, such as mysids, shrimps and fish (Kotrschal et al. 1998, Kendrick and Hyndes 2005, Garamszegi et al. 2005, de Lussanet and Muller 2007, Van Wassenbergh et al. 2011, MacLean et al. 2014, Lefebvre et al. 2016, Tsuboi et al. 2017).

According to the histological observation, the brain of H. barbouri is subdivided into five regions: telencephalon, mesencephalon, diencephalon, myelencephalon and metencephalon, as generally observed in other teleosts (Northcutt and Braford 1980, Nieuwenhuys and Meek 1990, Genten et al. 2009, Yamamoto 2008, Senarat et al. 2016). The optic tectum of H. barbouri consisted of five layers, including the stratum marginale, stratum opticum, stratum album central, stratum griseum central, and stratum periventriculae. This result is not in line with previous observations of six layers in the optic tectum of Rastrelliger brachysoma (Bleeker, 1851) (Senarat et al. 2016) and other cyprinids as Cyprinalla lulrensis (Baird & Girard, 1853), Notropis bairdi Hubbs & Ortenburger, 1929 and Notropis amabilis (Girard, 1856) (Huber and Rylander 1991. Immunohistochemical observations, for example targeting the estrogen receptor and neurotrophin Trk receptor expression, will be needed to further test this feature.

An increase in the amount of blood vessel in the saccus vasculosus of H. barbouri happened at 14 DAB. As reported by Nakane et al. (2013), the saccus vasculosus is a complex organ and functions as a seasonal sensor by recognizing the photoperiods. Dammerman (1910) claimed that capillaries are responsible for nutritive substances that significantly affect the function of the saccus epithelium. On the other hand, Purkinje cell differentiation has been described for Danio rerio (Hamilton, 1822) (Hamling et al. 2015), but no studies have addressed this issue for Syngnathidae. We showed that Purkinje cells differentiate as early as 6 DAB in H. barbouri and continue to multiply until 35 DAB. This may be necessary to coordinate movements of various body and swimming motions in the larval fish (Kimmel et al. 1995, Hamling et al. 2015). To investigate possible connectivity the cerebellum function and Purkinje cells, further studies using a variety of different labeling techniques will be needed.

In conclusion, our new description of the CNS and brain development in H. barbouri provides a foundation for neurobiology and the potential structural basis of the ecology of this seahorse. In particular, the largest optic tectum implies a great capacity for learning and the propensity to feed on fast-moving prey. Another important finding in this study is that the increase in blood vessels in the optic tectum and the saccus vasculosus, as well as the development of the Purkinje cell layer. Since these structures develop at 14 DAB, we speculate that appropriate behavior responses will be observed around this time in H. Barbouri, but this hypothesis should be confirmed by future chronology studies on the feeding behavior of this fish.