Constrained spherical deconvolution on diffusion-weighted images of dolphin brains.

Invasive neuronal tract-tracing is not permitted in very large or endangered animals. This is especially the case in marine mammals like dolphins. Diffusion-weighted imaging of fiber tracts could be an alternative if feasible even in brains that have been fixed in formalin for a long time. This currently is a problem, especially for detecting crossing fibers. We applied a state-of-the-art algorithm of Diffusion-weighted imaging called Constrained Spherical Deconvolution on diffusion data of three fixed brains of bottlenose dolphins using clinical human MRI parameters and were able to identify complex fiber patterns within a voxel. Our findings indicate that in order to maintain the structural integrity of the tissue, short-term post-mortem fixation is necessary. Furthermore, pre-processing steps are essential to remove the classical Diffusion-weighted imaging artifacts from images: however, the algorithm is still able to resolve fiber tracking in regions with various signal intensities. The described imaging technique reveals complex fiber patterns in cetacean brains that have been preserved in formalin for extended periods of time and thus opens a new window into our understanding of cetacean neuroanatomy.

Distribution of calcium-binding proteins immunoreactivity in the bottlenose dolphin entorhinal cortex.

Introduction: The entorhinal cortex has been shown to be involved in high-level cognitive functions in terrestrial mammals. It can be divided into two main areas: the lateral entorhinal area (LEA) and the medial entorhinal area (MEA). Understanding of its structural organization in cetaceans is particularly important given the extensive evidence for their cognitive abilities. The present study describes the cytoarchitectural and immunohistochemical properties of the entorhinal cortex of the bottlenose dolphin (Tursiops truncatus, Montagu, 1821), perhaps the most studied cetacean species and a paradigm for dolphins and other small cetaceans. Methods: Four bottlenose dolphins' entorhinal cortices were processed. To obtain a precise overview of the organization of the entorhinal cortex we used thionin staining to study its laminar and regional organization, and immunoperoxidase technique to investigate the immunohistochemical distribution of three most commonly used calcium-binding proteins (CBPs), calbindin D-28k (CB), calretinin (CR) and parvalbumin (PV). Entorhinal cortex layers thickness were measured, morphological and morphometric analysis for each layer were conducted and statistically compared. Results: Six layers in both the LEA and MEA were identified. The main difference between the LEA and the MEA is observed in layers II and III: the neurons in layer II of the LEA were denser and larger than the neurons in layer II of MEA. In addition, a relatively cell-free zone between layers II and III in LEA, but not in MEA, was observed. The immunohistochemical distribution of the three CBPs, CB, CR and PV were distinct in each layer. The immunostaining pattern of CR, on one side, and CB/PV, on the other side, appeared to be distributed in a complementary manner. PV and CB immunostaining was particularly evident in layers II and III, whereas CR immunoreactive neurons were distributed throughout all layers, especially in layers V and VI. Immunoreactivity was expressed by neurons belonging to different morphological classes: All CBPs were expressed in non-pyramidal neurons, but CB and CR were also found in pyramidal neurons. Discussion: The morphological characteristics of pyramidal and non-pyramidal neurons in the dolphin entorhinal cortex are similar to those described in the entorhinal cortex of other species, including primates and rodents. Interestingly, in primates, rodents, and dolphins, most of the CBP-containing neurons are found in the superficial layers, but the large CR-ir neurons are also abundant in the deep layers. Layers II and III of the entorhinal cortex contain neurons that give rise to the perforant pathway, which conveys most of the cortical information to the hippocampal formation. From the hippocampal formation, reciprocal projections are directed back to the deep layer of the entorhinal cortex, which distributes the information to the neocortex and subcortical area. Our data reveal that in the dolphin entorhinal cortex, the three major CBPs label morphologically heterogeneous groups of neurons that may be involved in the information flow between entorhinal input and output pathways.

Vascularization of the gastrointestinal tract of the bottlenose dolphin (Tursiops truncatus, Montagu 1821).

Odontocetes primarily rely on fish, cephalopods, and crustaceans as their main source of nutrition. In the digestive system, their polygastric complex exhibits similarities to that of their closest terrestrial relatives such as cows, sheep, and giraffes, while the entero-colic tract shares similarities with terrestrial carnivores. The morphology, caliber, and structure of the odontocete intestine are relatively constant, and, since there is no caecum, a distinction between the small and large intestine and their respective subdivisions is difficult. To address this issue, we used the intestinal vascularization pattern, specifically the course and branching of the celiac artery (CA) and the cranial and caudal mesenteric arteries (CrMA and CdMA). A series of pictures and dissections of 10 bottlenose dolphins (Tursiops truncatus) were analyzed. Additionally, we performed a cast by injecting colored polyurethane foam in both arteries and veins to measure the caliber of the arteries and clarify their monopodial or dichotomous branching. Our results showed the presence of multiple duodenal arteries (DAs) detaching from the CA. The CrMA gave origin to multiple jejunal arteries, an ileocolic artery (ICA), and, in six cases, a CdMA. In four specimens, the CdMA directly originated from the abdominal aorta. The ICA gave rise to the mesenteric ileal branches (MIB) and mesenteric anti-ileal branches and the right colic arteries (RCA) and the middle colic arteries. From the CdMA originated the left colic and cranial rectal arteries (LCA and CrRA). The measurements revealed a mixed monopodial and dichotomous branching scheme. The analysis of the arteries and their branching gave us an instrument, based on comparative anatomy, to distinguish between the different intestinal compartments. We used the midpoint of anastomoses between MIB and RCA to indicate the border between the small and the large intestine, and the midpoint of anastomoses between LCA and CrRA, to tell the colon from the rectum. This pattern suggested an elongation of the duodenum and a shortening of the colic tract that is still present in this species. These findings might be related to the crucial need to possess a long duodenal tract to digest prey ingested whole without chewing. A short aboral part is also functional to avoid gas-producing colic fermentation. The rare origin of the CdMA on the CrMA might instead be a consequence of the cranial thrust of the abdominopelvic organs related to the loss of the pelvic girdle that occurred during the evolution of cetaceans.

Report on the brain of the monk seal (Monachus monachus, Hermann, 1779).

The Mediterranean monk seal (Monachus monachus, Hermann, 1779) is an endangered species of pinniped endemic to few areas of the Mediterranean Sea. Extensive hunting and poaching over the last two centuries have rendered it a rare sight, scattered mainly in the Aegean Sea and the western coast of North Africa. In a rare event, a female monk seal calf stranded and died in southern Italy (Brindisi, Puglia). During due necropsy, the brain was extracted and fixed. The present report is the first of a monk seal brain. The features reported are remarkably typical of a true seal brain, with some specific characteristics. The brain cortical circonvolutions, main fissures and the external parts are described, and an EQ was calculated. Overall, this carnivore adapted to aquatic life shares some aspects of its neuroanatomy and physiology with other seemingly distant aquatic mammals.

Interspecies Retinal Diversity and Optic Nerve Anatomy in Odontocetes.

Throughout evolution, odontocete vision has had to readapt to the aquatic environment, which has had far-reaching effects on ocular anatomy and neurology. The most prominent features include the iris with an operculum, a well-developed choroid, the presence of giant ganglion cells in the retina, and the hemispherical shape of the thick eyecup. In the present study, the optic nerve and the retina were comparatively studied in Odontoceti (Cuvier's beaked whale, common bottlenose dolphin, false killer whale, long-finned pilot whale, Risso's dolphin, striped dolphin), the semi-aquatic common hippopotamus, and the fully terrestrial bovine. Cross-sections of the tissue were treated with histological and immunohistochemical techniques. Substantial differences were seen between the odontocetes and the reference species as well as within the cetaceans. The morphological structure of the optic nerve mainly appeared species specific, while the density of retinal ganglion cells was significantly higher in the terrestrial bovine than in the cetaceans. However, some typical characteristics of the cetacean retina were absent: the giant ganglion cells and the high retinal thickness. Immunohistochemical research showed varying degrees of neurofilament 200 expression in the retinal ganglion cells, while calretinin was only expressed in those of the common bottlenose dolphin and bovine.

The prefrontal cortex of the bottlenose dolphin (Tursiops truncatus Montagu, 1821): a tractography study and comparison with the human.

Cetaceans are well known for their remarkable cognitive abilities including self-recognition, sound imitation and decision making. In other mammals, the prefrontal cortex (PFC) takes a key role in such cognitive feats. In cetaceans, however, a PFC could up to now not be discerned based on its usual topography. Classical in vivo methods like tract tracing are legally not possible to perform in Cetacea, leaving diffusion-weighted imaging (DWI) as the most viable alternative. This is the first investigation focussed on the identification of the cetacean PFC homologue. In our study, we applied the constrained spherical deconvolution (CSD) algorithm on 3 T DWI scans of three formalin-fixed brains of bottlenose dolphins (Tursiops truncatus) and compared the obtained results to human brains, using the same methodology. We first identified fibres related to the medio-dorsal thalamic nuclei (MD) and then seeded the obtained putative PFC in the dolphin as well as the known PFC in humans. Our results outlined the dolphin PFC in areas not previously studied, in the cranio-lateral, ectolateral and opercular gyri, and furthermore demonstrated a similar connectivity pattern between the human and dolphin PFC. The antero-lateral rotation of the PFC, like in other areas, might be the result of the telescoping process which occurred in these animals during evolution.

Consistency and Variation in Doublecortin and Ki67 Antigen Detection in the Brain Tissue of Different Mammals, including Humans.

Recently, a population of "immature" neurons generated prenatally, retaining immaturity for long periods and finally integrating in adult circuits has been described in the cerebral cortex. Moreover, comparative studies revealed differences in occurrence/rate of different forms of neurogenic plasticity across mammals, the "immature" neurons prevailing in gyrencephalic species. To extend experimentation from laboratory mice to large-brained mammals, including humans, it is important to detect cell markers of neurogenic plasticity in brain tissues obtained from different procedures (e.g., post-mortem/intraoperative specimens vs. intracardiac perfusion). This variability overlaps with species-specific differences in antigen distribution or antibody species specificity, making it difficult for proper comparison. In this work, we detect the presence of doublecortin and Ki67 antigen, markers for neuronal immaturity and cell division, in six mammals characterized by widely different brain size. We tested seven commercial antibodies in four selected brain regions known to host immature neurons (paleocortex, neocortex) and newly born neurons (hippocampus, subventricular zone). In selected human brains, we confirmed the specificity of DCX antibody by performing co-staining with fluorescent probe for DCX mRNA. Our results indicate that, in spite of various types of fixations, most differences were due to the use of different antibodies and the existence of real interspecies variation.

Neuroanatomy of the Cetacean Sensory Systems.

Cetaceans have undergone profound sensory adaptations in response to their aquatic environment during evolution. These adaptations are characterised by anatomo-functional changes in the classically defined sensory systems, shaping their neuroanatomy accordingly. This review offers a concise and up-to-date overview of our current understanding of the neuroanatomy associated with cetacean sensory systems. It encompasses a wide spectrum, ranging from the peripheral sensory cells responsible for detecting environmental cues, to the intricate structures within the central nervous system that process and interpret sensory information. Despite considerable progress in this field, numerous knowledge gaps persist, impeding a comprehensive and integrated understanding of their sensory adaptations, and through them, of their sensory perspective. By synthesising recent advances in neuroanatomical research, this review aims to shed light on the intricate sensory alterations that differentiate cetaceans from other mammals and allow them to thrive in the marine environment. Furthermore, it highlights pertinent knowledge gaps and invites future investigations to deepen our understanding of the complex processes in cetacean sensory ecology and anatomy, physiology and pathology in the scope of conservation biology.

Age-related changes in the primary auditory cortex of newborn, adults and aging bottlenose dolphins (Tursiops truncatus) are located in the upper cortical layers.

Introduction: The auditory system of dolphins and whales allows them to dive in dark waters, hunt for prey well below the limit of solar light absorption, and to communicate with their conspecific. These complex behaviors require specific and sufficient functional circuitry in the neocortex, and vicarious learning capacities. Dolphins are also precocious animals that can hold their breath and swim within minutes after birth. However, diving and hunting behaviors are likely not innate and need to be learned. Our hypothesis is that the organization of the auditory cortex of dolphins grows and mature not only in the early phases of life, but also in adults and aging individuals. These changes may be subtle and involve sub-populations of cells specificall linked to some circuits. Methods: In the primary auditory cortex of 11 bottlenose dolphins belonging to three age groups (calves, adults, and old animals), neuronal cell shapes were analyzed separately and by cortical layer using custom computer vision and multivariate statistical analysis, to determine potential minute morphological differences across these age groups. Results: The results show definite changes in interneurons, characterized by round and ellipsoid shapes predominantly located in upper cortical layers. Notably, neonates interneurons exhibited a pattern of being closer together and smaller, developing into a more dispersed and diverse set of shapes in adulthood. Discussion: This trend persisted in older animals, suggesting a continuous development of connections throughout the life of these marine animals. Our findings further support the proposition that thalamic input reach upper layers in cetaceans, at least within a cortical area critical for their survival. Moreover, our results indicate the likelihood of changes in cell populations occurring in adult animals, prompting the need for characterization.

Multimodal Assessment of Bottlenose Dolphin Auditory Nuclei Using 7-Tesla MRI, Immunohistochemistry and Stereology.

The importance of assessing neurochemical processes in the cetacean brain as a tool for monitoring their cognitive health and to indirectly model human neurodegenerative conditions is increasingly evident, although available data are largely semiquantitative. High-resolution MRI for post-mortem brains and stereology allow for quantitative assessments of the cetacean brain. In this study, we scanned two brains of bottlenose dolphins in a 7-Tesla (7T) MR scanner and assessed the connectivity of the inferior colliculi and ventral cochlear nuclei using diffusion tensor imaging (DTI). Serial thick sections were investigated stereologically in one of the dolphins to generate rigorous quantitative estimates of identifiable cell types according to their morphology and expression of molecular markers, yielding reliable cell counts with most coefficients of error <10%. Fibronectin immunoreactivity in the dolphin resembled the pattern in a human chronic traumatic encephalopathy brain, suggesting that neurochemical compensation for insults such as hypoxia may constitute a noxious response in humans, while being physiological in dolphins. These data contribute to a growing body of knowledge on the morphological and neurochemical properties of the dolphin brain and highlight a stereological and neuroimaging workflow that may enable quantitative and translational assessment of pathological processes in the dolphin brain in the future.

The Odontocete Ear Canal-Associated Lymphoid Tissue (ECALT) and Lymph Nodes: Morphological and Pathological Description with Immuno-Phenotypic Characterisation.

A changing marine environment with emerging natural and anthropogenic stressors challenges the marine mammal immune system. The skin and adnexa form a first protective barrier in the immune response, although this is still relatively understudied in cetaceans. The cellular and tissue morphology of the nodular and diffuse lymphoid tissue are not fully charted and the physiological responses are not yet completely understood. The odontocete's external ear canal has a complex relationship with the external environment, with an artificial lumen rendering the inside of the canal a relatively secluded environment. In this work, we studied the odontocete ear canal-associated lymphoid tissue (ECALT) by histo- and immunohistochemistry (HC, IHC) with anti-CD3, anti-CD20, anti-Iba-1, anti-HLA-DR, and anti-vimentin antibodies. The ECALT cellular composition consists mainly of B-lymphocytes with the occasional presence of T-lymphocytes and the dispersed distribution of the macrophages. In cases of activation, the cellular reaction showed a similar pattern with the occasional presence of T-cells, plasma cells, and neutrophils. Nodular lymphoid tissue was generally in line with the description in other odontocetes, although with abundant erythrocytes throughout the entire organ. This study contributes to the understanding of the cellular composition of diffuse and nodular lymphoid tissue in several species of odontocetes, and in association with inflammation of the external ear canal.

Cell cycle alterations due to perfluoroalkyl substances PFOS, PFOA, PFBS, PFBA and the new PFAS C6O4 on bottlenose dolphin (Tursiops truncatus) skin cell.

Per- and polyfluoroalkyl substances (PFAS) have become ubiquitous environmental contaminants in aquatic ecosystems worldwide. Marine mammals, as top predators, are constantly exposed to several PFAS compounds that accumulate in different tissues. As a proxy to assess cytotoxicity of PFAS in the bottlenose dolphin (Tursiops truncatus), we generated a new immortalized cell line derived from skin samples of bottlenose dolphin. Using high content imaging, we assessed the effects of increasing concentrations of PFOS, PFOA, PFBS, PFBA and C6O4 on cell viability and cell cycle phases. In particular, we classified all cells based on multiple morphometric differences of the nucleus in three populations, named respectively "Normal" (nuclei in G0, S and M phase); "Large" (nuclei showing characteristics of senescence) and "Small" (nuclei with fragmentation and condensed chromatin). Combining this approach with cell cycle analysis we determined which phases of the cell cycle were influenced by PFAS. The results revealed that the presence of PFOS, PFBS and PFBA could increase the number of cells in G0+G1 phase and decrease the number of those in the S phase. Moreover, PFOS and PFBS lowered the fraction of cells in the M phase. Interestingly PFOS, PFBS and PFBA reduced the prevalence of the senescence phenotype ("large" nuclei), suggesting a potential tumorigenic effect. Besides, the presence of PFOS and PFBS correlated also with a significant decrease in the number of "small" nuclei. The C6O4 exposure did not highlighted morphometric alteration or cell cycle modification bottlenose dolphin skin cell nuclei. While the effects of PFAS on cell cycle was clear, no significant change was detected either in term of cell proliferation or of viability. This study fosters the overall knowledge on the cellular effects of perfluoroalkyl substances in marine mammals.

Systematic validation and assessment of immunohistochemical markers for central nervous system pathology in cetaceans, with emphasis on auditory pathways.

Cetacean neuropathology is a developing field that aims to assess structural and neurochemical changes involved in neurodegenerative, infectious and traumatic processes, however markers used previously in cetaceans have rarely undergone systematic validation. This is a prerequisite to investigating the potential damage inflicted on the cetacean auditory system by anthropogenic noise. In order to assess apoptotic, neuroinflammatory and structural aberrations on a protein level, the baseline expression of biomarker proteins has to be characterized, implementing a systematic approach to validate the use of anti-human and anti-laboratory animal antibodies in dolphin tissues. This approach was taken to study 12 different antibodies associated with hypoxic-ischemic, inflammatory, plastic and excitatory-inhibitory changes implicated in acoustic trauma within the ventral cochlear nuclei and inferior colliculi of 20 bottlenose dolphins (Tursiops truncatus). Out of the 12 tested antibodies, pro-apoptotic protease factor 1 (Apaf-1), diacylglycerolkinase-ζ (DGK-ζ), B-cell lymphoma related protein 2 (Bcl-2), amyloid-ß peptide (Aß) and neurofilament 200 (NF200) were validated employing Western blot analyses and immunohistochemistry (IHC). The results of the validation process indicate specific patterns of immunoreactivity that are comparable to those reported in other mammals, thus suggesting a key panel of IHC biomarkers of pathological processes in the cetacean brain. As a consequence, the antibodies tested in this study may constitute a valid tool for supporting existing diagnostic methods in neurological diseases. The approach of systematic validation of IHC markers in cetaceans is proposed as a standard practice, in order for results to be transparent, reliable and comparable.

The orbitofrontal cortex of the sheep. Topography, organization, neurochemistry, digital tensor imaging and comparison with the chimpanzee and human.

Areas dedicated to higher brain functions such as the orbitofrontal cortex (OFC) are thought to be unique to hominidae. The OFC is involved in social behavior, reward and punishment encoding and emotional control. Here, we focused on the putative corresponding area in the sheep to assess its homology to the OFC in humans. We used classical histology in five sheep (Ovis aries) and four chimpanzees (Pan troglodytes) as a six-layered-cortex primate, and Diffusion Tensor Imaging (DTI) in three sheep and five human brains. Nissl's staining exhibited a certain alteration in cortical lamination since no layer IV was found in the sheep. A reduction of the total cortical thickness was also evident together with a reduction of the prevalence of layer one and an increased layer two on the total thickness. Tractography of the sheep OFC, on the other hand, revealed similarities both with human tracts and those described in the literature, as well as a higher number of cortico-cortical fibers connecting the OFC with the visual areas in the right hemisphere. Our results evidenced the presence of the basic components necessary for complex abstract thought in the sheep and a pronounced laterality, often associated with greater efficiency of a certain function, suggested an evolutionary adaptation of this prey species.

The Heart of the Killer Whale: Description of a Plastinated Specimen and Review of the Available Literature.

The killer whale (Orcinus orca, Linnaeus, 1958) is the largest extant delphinid. Despite its worldwide distribution in the wild and in dolphinariums, its anatomy remains relatively poorly described. In the present study, we describe the detailed morphology of a plastinated killer whale heart. The gross description of the arteries and veins reaching the organ and its coronary vessels are reported. Additional endoscopy and CT (computed tomography) scanning were performed to provide extensive measurements of its parts. In many aspects, the killer whale heart conformed to other delphinid heart descriptions, including position, relative size and shape and specific features such as extensive papillary muscles, trabecular endocardium and trabecula septomarginalis. These characteristics are representative of the delphinid family, suggesting that its functions and capacities are similar to that of other, smaller, dolphins and help understand the conditions in which these predators exert their remarkable physical performance necessary for their survival.

The primary visual cortex of Cetartiodactyls: organization, cytoarchitectonics and comparison with perissodactyls and primates.

Cetartiodactyls include terrestrial and marine species, all generally endowed with a comparatively lateral position of their eyes and a relatively limited binocular field of vision. To this day, our understanding of the visual system in mammals beyond the few studied animal models remains limited. In the present study, we examined the primary visual cortex of Cetartiodactyls that live on land (sheep, Père David deer, giraffe); in the sea (bottlenose dolphin, Risso's dolphin, long-finned pilot whale, Cuvier's beaked whale, sperm whale and fin whale); or in an amphibious environment (hippopotamus). We also sampled and studied the visual cortex of the horse (a closely related perissodactyl) and two primates (chimpanzee and pig-tailed macaque) for comparison. Our histochemical and immunohistochemical results indicate that the visual cortex of Cetartiodactyls is characterized by a peculiar organization, structure, and complexity of the cortical column. We noted a general lesser lamination compared to simians, with diminished density, and an apparent simplification of the intra- and extra-columnar connections. The presence and distribution of calcium-binding proteins indicated a notable absence of parvalbumin in water species and a strong reduction of layer 4, usually enlarged in the striated cortex, seemingly replaced by a more diffuse distribution in neighboring layers. Consequently, thalamo-cortical inputs are apparently directed to the higher layers of the column. Computer analyses and statistical evaluation of the data confirmed the results and indicated a substantial correlation between eye placement and cortical structure, with a markedly segregated pattern in cetaceans compared to other mammals. Furthermore, cetacean species showed several types of cortical lamination which may reflect differences in function, possibly related to depth of foraging and consequent progressive disappearance of light, and increased importance of echolocation.

Anatomical organization of the lateral cervical nucleus in Artiodactyls.

The presence of the lateral cervical nucleus (LCN) in different mammals, including humans, has been established in a number of anatomical research works. The LCN receives its afferent inputs from the spinocervical tract, and conveys this somatosensory information to the various brain areas, especially the thalamus. In the present study, the organization of the calf and pig LCN was examined through the use of thionine staining and immunohistochemical methods combined with morphometrical analyses. Specifically, the localization of calbindin-D28k (CB-D28k) and neuronal nitric oxide synthase (nNOS) in the LCN was investigated using the immunoperoxidase method. Calf and pig LCN appear as a clearly defined column of gray matter located in the three cranial segments of the cervical spinal cord. Thionine staining shows that polygonal neurons represent the main cell type in both species. The calf and pig LCN contained CB-D28k-immunoreactive (IR) neurons of varying sizes. Large neurons are probably involved in the generation of the cervicothalamic pathway. Small CB-D28k-IR neurons, on the other hand, could act as local interneurons. The immunoreactivity for nNOS was found to be mainly located in thin neuronal processes that could represent the terminal axonal portion of nNOS-IR found in laminae III e IV. This evidence suggests that nitric oxide (NO) could modulate the synaptic activity of the glutamatergic spinocervical tracts. These findings suggest that the LCN of Artiodactyls might play an important role in the transmission of somatosensory information from the spinal cord to the higher centers of the brain.

Nitrergic and Substance P Immunoreactive Neurons in the Enteric Nervous System of the Bottlenose Dolphin (Tursiops truncatus) Intestine.

Compared with other mammals, the digestive system of cetaceans presents some remarkable anatomical and physiological differences. However, the neurochemical features of the enteric nervous system (ENS) in these animals have only been described in part. The present study gives a description of the nitrergic and selected peptidergic systems in the myenteric plexus (MP) and submucosal plexus (SMP) of the intestine of the bottlenose dolphin (Tursiops truncatus). The distribution and morphology of neurons immunoreactive (IR) for the neuronal nitric oxide synthase (nNOS) and Substance P (SP) were immunohistochemically studied in formalin-fixed specimens from the healthy intestine of three animals, and the data were compared with those described in the literature on other mammals (human and non-human). In bottlenose dolphins, the percentages of nitrergic neurons (expressed as median and interquartile range-IQR) were 28% (IQR = 19-29) in the MP and 1% (IQR = 0-2) in the SMP, while the percentages of SP-IR neurons were 31% (IQR = 22-37) in the MP and 41% (IQR = 24-63) in the SMP. Although morphological features of nNOS- and SP-IR neurons were similar to those reported in other mammals, we found some noticeable differences in the percentages of enteric neurons. In fact, we detected a lower proportion of nNOS-IR neurons in the SMP and a higher proportion of SP-IR neurons in the MP compared to other mammals. To the best of the authors' knowledge, this study represents the first description and quantification of nNOS-IR neurons and the first quantification of SP-IR neurons in the intestine of a cetacean species. As nNOS and SP are important mediators of intestinal functions and the nitrergic population is an important target for many neuroenteropathies, data obtained from a healthy intestine provide a necessary basis to further investigate and understand possible functional differences and motor intestinal dysfunctions/alterations in these special mammals.

Topographical and structural characterization of the V1-V2 transition zone in the visual cortex of the long-finned pilot whale Globicephala melas (Traill, 1809).

The visual system of cetaceans is at best poorly understood. With a handful of electrophysiological studies and a limited number of histological preparations from well-preserved specimen, the investigation of the principles underlying the cortical organization in cetaceans remains a challenge. In the course of our current investigation, we identified the transition from V2 to V1 in the long-finned pilot whale Globicephala melas, only recognizable through immunocytochemistry, and a similar if not homologue transition in the sheep Ovis aries. Our results emphasize the importance of differential pattern recognition in which the application of different markers uncovers a diversity in a delphinid's cortex, formerly widely considered as uniform and archetypal. In fact, the evidence that we present suggests the existence of relatively unacknowledged areas beyond the well-known sensory territories in cetaceans.

The claustrum of the sheep and its connections to the visual cortex.

The present study analyses the organization and selected neurochemical features of the claustrum and visual cortex of the sheep, based on the patterns of calcium-binding proteins expression. Connections of the claustrum with the visual cortex have been studied by tractography. Parvalbumin-immunoreactive (PV-ir) and Calbindin-immunoreactive (CB-ir) cell bodies increased along the rostro-caudal axis of the nucleus. Calretinin (CR)-labeled somata were few and evenly distributed along the rostro-caudal axis. PV and CB distribution in the visual cortex was characterized by larger round and multipolar cells for PV, and more bitufted neurons for CB. The staining pattern for PV was the opposite of that of CR, which showed densely stained but rare cell bodies. Tractography shows the existence of connections with the caudal visual cortex. However, we detected no contralateral projection in the visuo-claustral interconnections. Since sheep and goats have laterally placed eyes and a limited binocular vision, the absence of contralateral projections could be of prime importance if confirmed by other studies, to rule out the role of the claustrum in stereopsis.