D 2015 A QUALITATIVE AND QUANTITATIVE STEREOLOGICAL STUDY OF THE MICROSCOPIC MORPHOLOGY OF THE NERVOUS GANGLIA OF THE BIVALVE PEPPERY FURROW SHELL (SCROBICULARIA PLANA) SUKANLAYA TANTIWISAWARUJI TESE DE DOUTORAMENTO APRESENTADA AO INSTITUTO DE CIÊNCIAS BIOMÉDICAS ABEL SALAZAR DA UNIVERSIDADE DO PORTO EM CIÊNCIAS BIOMÉDICAS SUKANLAYA TANTIWISAWARUJI A qualitative and quantitative stereological study of the microscopic morphology of the nervous ganglia of the bivalve peppery furrow shell (Scrobicularia plana) Tese de candidatura ao grau de Doutor em Ciências Biomédicas submetida ao Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. Orientador – Eduardo Jorge Sousa da Rocha Categoria – Professor Catedrático Afiliação – Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Portugal Coorientador: Uthaiwan Kovitvadhi Categoria – Professor Associado Afiliação – Department of Zoology, Faculty of Science, Kasetsart University, Bangkok, Thailand Coorientador: Maria João Tomé da Rocha Categoria – Professor Auxiliar Afiliação – Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Portugal OBJECTIVES The microscopic morphology of bivalves is still poorly studied, particularly in quantitative terms, and that of peppery furrow shell (Scrobicularia plana) is no exception. With this scenario in mind, the general aim of the Thesis was to enrich the knowledge of the bivalves’ central nervous system, reviewing the state of art and unveiling new qualitative and quantitative cytological and histological data in S. plana, namely using technical approaches that were either never or seldom used in the neuroscience of bivalves. Based on these broad objectives, the specific aims were: To better understand the microscopic anatomy of the central nervous system, by conducting an unprecedented three-dimensional (3D) computer assisted reconstruction of S. plana ganglia, while estimating for the first time their total volumes and surface areas in the 3D models, and the relative volumes of cortex and medulla. Within this scope, we wanted to start studying whether sexual differences exist regarding ganglion size and its internal composition, in view of the key roles of the neurosecretory neurons in governing gonadal maturation, particularly in females. To examine the general histology and cytology of the neural cells in S. plana, registering its main aspects and looking after any still undescribed features, while trying a first identification of potentially neurosecretory neurons — viz. those putatively producing serotonin and dopamine — comparatively across the cerebral, pedal and visceral ganglia, and considering the gonadic sex. To start studying, with design-based stereological methods, theorized structural differences between the various nervous ganglia types, specifically connected with intrinsic features of those elements, like their different body locations and distinct functions, and in view of the gender and gonadal maturation staging (comparing adults with maturing vs. spent gonads). The expectable neural cell targets would be those identified by the previous histological and ultrastructural study. To work further on the possible effects of the animal’s gender in the structure of the central nervous system, and, in parallel, to start exploring the hypothesis that if adult bivalves, including S. plana, continue to grow during its lifespan, then its nervous ganglia may continue to develop with age — at least until senescence, if it happens — and eventually gains in cellularity and other morphofunctional changes plausible occur. Implementing design-based stereological strategies, one aim was thus to search for changes in ganglion size and in number of distinct neural elements. - I - To make the first experimental assay for start testing the hypothesis that if both the nervous ganglia signalling and oestrogens play a modelling role in the bivalves’ gonadal maturation and spawning — and all indicates that such impacts are not equal ways in females and males — then waterborne exposure to xenoestrogens can cause morphofunctional impacts in the central nervous system. This aim makes even more sense if the works proposed for other goals generate evidences supporting the hypothesis that sex may shape the structure of the S. plana central nervous system. Pursuing the cited goals will expand the knowledge of the bivalves’ nervous system, in S. plana especially, and has potential of introducing this species as a valuable model in neuroscience. - II - ABSTRACT This research was carried out on the bivalve peppery furrow shell, Scrobicularia plana, which already well-recognized as one of the important species in environmental monitoring and other types of researches. S. plana has its natural habitat in the intertidal soft sediment along the Atlantic coast from Europe to Africa. Its nervous system is poorly studied, and thus we aimed here to improve such lack of knowledge. A qualitative study was done to examine the general histology of the nervous ganglia, including the neural cell types at both light and electron microscopy. For light microscopy, animals were measured, anesthetized, dissected and fixed in 10% buffered formalin. They were routinely processed for paraffin embedding, and sectioned for varied purposes along the sagittal plane, using a fully motorized microtome. An immunohistochemical survey was also made for identifying neurons that could contain serotonin and dopamine, as a first step to gather the knowledge about the presence and role of neuroendocrine neurons in S. plana. For transmission electron microscopy, dissected ganglia were fixed in 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide, all buffered, and routinely processed for epoxy embedding. As to serotonergic and dopaminergic cell bodies and neurites, they were identified in all the ganglia, in adults of both sexes and in immature animals (with undefined sex). Both quantitative and qualitative methods were conducted to study three dimensional (3D) features of the ganglia, taking the 3D models to find out differences between ganglia types and if the biometric parameters can be correlated with the ganglionic volumes and surface areas, which was the case for some parameters. Stereology was applied later one, using the Cavalieri’s principle for estimating the ganglion volume and the optical disector-fractionator method for estimating cellularity (numbers of neurons, glia and pigmented cells); namely investigating the influence of the sex and gonad maturation on the quantitative structural parameters. The statistical approach relied on multi-way analysis of variance. In summary, the main findings were as follows: (1) 3D-reconstruction shows that each type of ganglia has a peculiar 3D-shape, and data suggest a slight left-right asymmetry as to the cerebral ganglia shape. Regarding total surfaces, correlations exist for the cerebral and visceral ganglia, but it is the visceral that consistently shows strong positive correlations with each biometric parameter. Despite the differences in volume/surface among ganglia, the volume ratio of cortex vs. medulla is fairly stable (≈ 1.5), suggesting a functional optimum. In this first approach it seemed that no major differences exist between sexes; (2) Histological analysis using light and transmission electron microscopic analysis shows that each ganglion has perineurium (outermost layer), outer cortex, and inner medulla. The neurons (smaller or - III - larger) are typically unipolar, gliocytes are elongated, roundish or triangular, and there are pigmented cells. Generally, glial cells are much smaller than neurons, having higher nucleus to cytoplasm ratio; (3) Unbiased stereology analyses were conducted in three studies. Firstly, for investigating eventual influences of the sex and gonadal maturation status of animals that did not differ in size. Quantitative parameters were estimated in the nervous ganglia and their cells, in males, females, and undifferentiated specimens. Overall, there was a tendency for the ganglionic volume to be greater in females, followed by males, and undifferentiated animals. As for the type of ganglia, the two cerebral ones are similar in size, but the volumes increased significantly towards the pedal ganglia, which is greater than the cerebral and much smaller than the visceral. The size differences between all ganglia types are independent of the gender and of the gonad maturation status at the time. As for the relative volumes (VV) of the cortex and medulla, the cortex is ≈ 60% and the medulla ≈ 40% of the all ganglia. As for the number neural cells, there were no significant differences among gender, but significant difference were found among ganglia types. The visceral ganglion has the highest number of cells (≈ 68000) and the cerebral ganglia have the lowest (≈ 12000). A second stereological study was on hypothesize impacts of age on the nervous ganglia in mature males vs. females. Considering that size is a proxy of age, the animals were split into two-size classes, that we named “Small” (age: 2+ years) and “Big” (age: 3+ years). We disclosed interganglionic, sex- related and size-related significant effects upon the ganglionic volumes, relative volumes of cortex and medulla, and total numbers of neurons, glial cells, and pigmented cells. The effect of size (age) was consistently marked, and statistically significant, with the older specimens having approximately twice as bigger ganglia (regardless of its type and of the animals’ sex), that contained significantly