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.

Expression of Cannabinoid Receptors in the Trigeminal Ganglion of the Horse.

Cannabinoid receptors are expressed in human and animal trigeminal sensory neurons; however, the expression in the equine trigeminal ganglion is unknown. Ten trigeminal ganglia from five horses were collected post-mortem from an abattoir. The expression of cannabinoid receptors type 1 (CB1R) and type 2 (CB2R), and the cannabinoid-related receptors like transient receptor potential vanilloid type 1 (TRPV1), peroxisome proliferator-activated receptor gamma (PPARÉ£), and G protein-related receptor 55 (GPR55) in the trigeminal ganglia (TG) of the horse were studied, using immunofluorescence on cryosections and formalin-fixed paraffin-embedded (FFPE) sections. Neurons and glial cells were identified using fluorescent Nissl staining NeuroTrace® and an antibody directed against the glial marker glial fibrillary acidic protein (GFAP), respectively. Macrophages were identified by means of an antibody directed against the macrophages/microglia marker ionized calcium-binding adapter molecule 1 (IBA1). The protein expression of CB1R, CB2R, TRPV1, and PPARÉ£ was found in the majority of TG neurons in both cryosections and FFPE sections. The expression of GPR55 immunoreactivity was mainly detectable in FFPE sections, with expression in the majority of sensory neurons. Some receptors were also observed in glial cells (CB2R, TRPV1, PPARγ, and GPR55) and inflammatory cells (PPARγ and GPR55). These results support further investigation of such receptors in disorders of equine trigeminal neuronal excitability.

Endocannabinoid System Receptors at the Hip and Stifle Joints of Middle-Aged Dogs: A Novel Target for the Therapeutic Use of Cannabis sativa Extract in Canine Arthropathies.

The endocannabinoid system (ECS) has emerged as a potential therapeutic target in veterinary medicine due to its involvement in a wide range of physiological processes including pain, inflammation, immune function, and neurological function. Modulation of the ECS receptors has been shown to have anti-inflammatory, analgesic, and immunomodulatory effects in various animal models of disease, including dogs with osteoarthritis. The goal of this study was to identify and compare the cellular expression and distribution of cannabinoid receptor type 1 (CB1R) and type 2 (CB2R) and the cannabinoid-related G protein-coupled receptor 55 (GPR55) on the synovial cells of hip and stifle joints of seven dogs of different breeds without overt signs of osteoarthritis (OA). The synovial membranes of seven hips and seven stifle joints were harvested post mortem. The expression of the CB1R, CB2R, and GPR55 present in the synovial tissues was investigated using qualitative and quantitative immunofluorescence and Western blot (Wb) analysis. Synoviocytes of the stifle and hip joints expressed CB1R, CB2R, and GPR55 immunoreactivity (IR); no significant differences were observed for each different joint. Cannabinoid receptor 2- and GPR55-IR were also expressed by macrophages, neutrophils, and vascular cells. The ECS receptors were widely expressed by the synovial elements of dogs without overt signs of OA. It suggests that the ECS could be a target for the therapeutic use of Cannabis sativa extract in canine arthropathies.

Expression of cannabinoid (CB1 and CB2) and cannabinoid-related receptors (TRPV1, GPR55, and PPARα) in the synovial membrane of the horse metacarpophalangeal joint.

Background: The metacarpophalangeal joint undergoes enormous loading during locomotion and can therefore often become inflamed, potentially resulting in osteoarthritis (OA). There are studies indicating that the endocannabinoid system (ECS) modulates synovium homeostasis, and could be a promising target for OA therapy. Some cannabinoid receptors, which modulate proliferative and secretory responses in joint inflammation, have been functionally identified in human and animal synovial cells. Objective: To characterize the cellular distribution of the cannabinoid receptors 1 (CB1R) and 2 (CB2R), and the cannabinoid-related receptors transient receptor potential vanilloid type 1 (TRPV1), G protein-related receptor 55 (GPR55) and peroxisome proliferator-activated receptor alpha (PPARα) in the synovial membrane of the metacarpophalangeal joint of the horse. Animals: The dorsal synovial membranes of 14 equine metacarpophalangeal joints were collected post-mortem from an abattoir. Materials and methods: The dorsal synovial membranes of 14 equine metacarpophalangeal joints were collected post-mortem from an abattoir. The expression of the CB1R, CB2R, TRPV1, GPR55, and PPARα in synovial tissues was studied using qualitative and quantitative immunofluorescence, and quantitative real-time reverse transcriptase PCR (qRT-PCR). Macrophage-like (MLS) and fibroblast-like (FLS) synoviocytes were identified by means of antibodies directed against IBA1 and vimentin, respectively. Results: Both the mRNA and protein expression of the CB2R, TRPV1, GPR55, and PPARα were found in the synoviocytes and blood vessels of the metacarpophalangeal joints. The synoviocytes expressed the mRNA and protein of the CB1R in some of the horses investigated, but not in all. Conclusions and clinical importance: Given the expression of the CB1R, CB2R, TRPV1, GPR55, and PPARα in the synovial elements of the metacarpophalangeal joint, these findings encouraged the development of new studies supporting the use of molecules acting on these receptors to reduce the inflammation during joint inflammation in the horse.

The use of cannabidiol as a novel treatment for oral stereotypic behaviour (crib-biting) in a horse.

Behaviour is the response of living things to their environment and external stimulation, and is one of the parameters to be observed when assessing animal welfare. Any alteration from the conditions found in nature can lead to the occurrence of some specific behaviours, called stereotypies which are characterised as repetitive, consistent patterns of behaviour usually defined as having no apparent ultimate or proximal functions. It has been reported that once stabled or subjected to stressful activities, horses have more susceptibility of developing behavioural disturbances; therefore, behavioural disorders in horses are a strong indicator of poor welfare. Cannabis spp.-derived molecules have been studied under different medical conditions; the therapeutic potentials of phytocannabinoids are related to the effects of delta-9-tetrahydrocannabinol, cannabidiol (CBD), and other compounds. Cannabidiol has many activities within the central nervous system, such as anxiolytic, antidepressant, antipsychotic, anticonvulsant, and anti-inflammatory activities. Some studies have recently shown the potential and successful therapeutic use of phytocannabinoids in veterinary medicine. This clinical case report described a 22-year-old mare suffering from chronic crib-biting and wind-sucking, and the successful outcome of four weeks-therapy with CBD. This is the first report of the successful therapeutic use of phytocannabinoids in equine behavioural disorders.

Cannabinoid receptors in the inflammatory cells of canine atopic dermatitis.

Background: Atopic dermatitis (AD) is one of the most common cutaneous inflammatory and pruritic diseases in dogs. Considering its multifactorial nature, AD can be a challenging disease to manage, and the therapeutic strategy must often be multimodal. In recent years, research has been moving toward the use of natural products which have beneficial effects on inflammation and itching, and no side effects. Cannabinoid receptors have been demonstrated to be expressed in healthy and diseased skin; therefore, one of the potential alternative therapeutic targets for investigating AD is the endocannabinoid system (ECS). Objective: To immunohistochemically investigate the expression of the cannabinoid receptor type 2 (CB2R), and the cannabinoid-related receptors G protein-coupled receptor 55 (GPR55), transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1) in mast cells (MCs), macrophages, dendritic cells (DCs), T cells, and neutrophils of the skin of dogs with AD. Animals: Samples of skin tissues were collected from eight dogs with AD (AD-dogs). Materials and methods: The immunofluorescent stained cryosections of the skins of 8 dogs with AD having antibodies against CB2R, GPR55, TRPV1, TRPA1 were semiquantitatively evaluated. The inflammatory cells were identified using antibodies against tryptase (mast cells), ionized calcium binding adaptor molecule 1 (IBA1) (macrophages/DCs), CD3 (T cells), and calprotectin (neutrophils). The proportions of MCs, macrophages/DCs, T cells, and neutrophils expressing CB2R, GPR55, TRPV1 and TRPA1 were evaluated. Results: The cells of the inflammatory infiltrate showed immunoreactivity (IR) for all or for some of the cannabinoid and cannabinoid-related receptors studied. In particular, MCs and macrophages/DCs showed CB2R-, GPR55-, TRPA1-, and TRPV1-IR; T cells showed CB2R-, GPR55- and TRPA1-IR, and neutrophils expressed GPR55-IR. Co-localization studies indicated that CB2R-IR was co-expressed with TRPV1-, TRPA1-, and GPR55-IR in different cellular elements of the dermis of the AD-dogs. Conclusions and clinical importance: Cannabinoid receptor 2, and cannabinoid-related receptors GPR55, TRPV1 and TRPA1 were widely expressed in the inflammatory infiltrate of the AD-dogs. Based on the present findings, the ECS could be considered to be a potential therapeutic target for dogs with AD, and may mitigate itch and inflammation.

Immunohistochemical Distribution of Serotonin Transporter (SERT) in the Optic Lobe of the Honeybee, Apis mellifera.

Visual information is processed in the optic lobes, which consist of three retinotopic neuropils. These are the lamina, the medulla and the lobula. Biogenic amines play a crucial role in the control of insect responsiveness, and serotonin is clearly related to aggressiveness in invertebrates. Previous studies suggest that serotonin modulates aggression-related behaviours, possibly via alterations in optic lobe activity. The aim of this investigation was to immunohistochemically localize the distribution of serotonin transporter (SERT) in the optic lobe of moderate, docile and aggressive worker honeybees. SERT-immunoreactive fibres showed a wide distribution in the lamina, medulla and lobula; interestingly, the highest percentage of SERT immunoreactivity was observed across all the visual neuropils of the docile group. Although future research is needed to determine the relationship between the distribution of serotonin fibres in the honeybee brain and aggressive behaviours, our immunohistochemical study provides an anatomical basis supporting the role of serotonin in aggressive behaviour in the honeybee.

Ex vivo ultrasonographic and histological morphometry of small intestinal wall layers in horses.

Ultrasonographic morphometry of wall layers is commonly used in veterinary patients with suspected small intestinal disease, however published studies comparing this method with histopathology in horses are limited. This prospective, methods comparison study compared the qualitative and quantitative characteristics of small intestinal wall layers using ex vivo high-frequency ultrasound versus histopathology in a sample of 16 horses. Transverse section images of duodenum, distal jejunum, and ileum were acquired with a high-frequency linear transducer (7-15 MHz). Transverse histological cryosections were obtained at the same level. Appearance and measurements of the intestinal wall layers were assessed on the ultrasonographic and histological images. High-frequency scanning with the probe in close contact with the serosal surface of the equine intestinal wall allowed a clear and detailed definition of wall layers. A hyperechoic line was consistently detected within the tunica muscularis in all the intestinal tracts, corresponding histologically to the interface between its longitudinal and circular muscle layers. The overall trend of the values for wall layers thickness was comparable between ex vivo ultrasonography and histology. However, a poor agreement was found between the two methods for all layers. The ultrasonographic measurements were thicker compared to histological measurements, with the exception of the total wall and the muscular layer thicknesses. These layers were thinner on ultrasonography in the duodenum and in all the intestinal segments, respectively. Findings from the current study can be used as background for future ultrasonographic investigations of small intestinal diseases in horses.

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.