Distribution of vasoactive intestinal peptide (VIP) immunoreactivity in the rat pallial and subpallial amygdala and colocalization with γ-aminobutyric acid (GABA).

The amygdaloid complex, also known as the amygdala, is a heterogeneous group of distinct nuclear and cortical pallial and subpallial structures. The amygdala plays an important role in several complex functions including emotional behavior and learning. The expression of calcium-binding proteins and peptides in GABAergic neurons located in the pallial and subpallial amygdala is not uniform and is sometimes restricted to specific groups of cells. Vasoactive intestinal polypeptide (VIP) is present in specific subpopulations of GABAergic cells in the amygdala. VIP immunoreactivity has been observed in somatodendritic and axonal profiles of the rat basolateral and central amygdala. However, a comprehensive analysis of the distribution of VIP immunoreactivity in the various pallial and subpallial structures is currently lacking. The present study used immunohistochemical and morphometric techniques to analyze the distribution and the neuronal localization of VIP immunoreactivity in the rat pallial and subpallial amygdala. In the pallial amygdala, VIP-IR neurons are local inhibitory interneurons that presumably directly and indirectly regulate the activity of excitatory pyramidal neurons. In the subpallial amygdala, VIP immunoreactivity is expressed in several inhibitory cell types, presumably acting as projection or local interneurons. The distribution of VIP immunoreactivity is non-homogeneous throughout the different areas of the amygdaloid complex, suggesting a distinct influence of this neuropeptide on local neuronal circuits and, consequently, on the cognitive, emotional, behavioral and endocrine activities mediated by the amygdala.

Renal antifibrotic effect of N-acetyl-seryl-aspartyl-lysyl-proline in diabetic rats.

BACKGROUND AND AIM: Diabetic nephropathy is the main cause of end-stage renal disease. N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), a physiological tetrapeptide hydrolyzed by the angiotensin-converting enzyme (ACE), has antifibrotic effects in the cardiovascular system and in the kidney in experimental models of hypertension, heart failure and renal disease. The aim of the study was to evaluate the effect of Ac-SDKP in diabetic nephropathy and the potential additive effect of Ac-SDKP, when compared to ACE inhibitors alone, on the development of renal fibrosis. METHOD: Diabetes was induced in 28 Sprague-Dawley rats by a single intraperitoneal injection of streptozotocin. Control rats (n = 10) received only buffer solution. An ACE inhibitor (ramipril, 3 mg/kg/day) was administered to 11 diabetic rats. After 2 months, Ac-SDKP (1 mg/kg/day) was administered by osmotic minipumps for 8 weeks to 7 diabetic rats and to 6 diabetic rats treated with ramipril. Osmotic minipumps delivered saline solution in the corresponding sham-treated rats (diabetic rats, n = 10, and ramipril-treated diabetic rats, n = 5). RESULTS: Diabetic rats showed a significant increase in blood glucose level, urinary albumin excretion and renal fibrosis, and a reduction of glomerular nephrin expression with respect to control rats. Ac-SDKP administration significantly reduced renal fibrosis in diabetic rats, without significantly reducing urinary albumin excretion. Ramipril treatment caused a significant decrease in albuminuria and renal fibrosis and restored glomerular nephrin expression. Administration of Ac-SDKP, in addition to ramipril, further reduced renal fibrosis with respect to ramipril alone, while it did not improve the antiproteinuric effect of ramipril. CONCLUSION: Ac-SDKP administration reduces renal fibrosis in diabetic nephropathy. Addition of Ac-SDKP to ACE inhibition therapy improves the reduction of renal fibrosis with respect to ACE inhibition alone, suggesting a beneficial effect of this pharmacological association in diabetic nephropathy.

Connections of the sheep basolateral amygdala: A diffusion tensor imaging study.

BACKGROUND: A large amount of anatomic data published over the past decade has provided novel insight into the connections of the basolateral amygdala (BLA) in the rat, cat, and monkey. The mammalian (rat, cat, and monkey) BLA has strong connections with the cortex (especially piriform, and frontal cortices), the hippocampal region (especially perirhinal cortex, entorhinal cortex, and subiculum), the thalamus (in particular, the posterior internuclear nucleus and medial geniculate nucleus) and, to some extent, the hypothalamus. An important question remains as to how well the data obtained in rodents and primates can be extrapolated to ruminants. NEW METHOD: To address this issue the connections of the sheep BLA has been determined by Magnetic Resonance Imaging (MRI) and Diffusion Tensor Imaging (DTI, Tractography). RESULTS: Tractography showed ipsilateral connections between the BLA and several areas. COMPARISON WITH EXISTING METHOD(S): Reviews based mainly on description of the results obtained using anterograde and retrograde neuronal tracers. In the present research, we prefer to use a non-invasive technique (DTI). CONCLUSIONS: This report shows the existence of specific amygdaloid connections in the sheep.

Intrinsic innervation of the ileocaecal junction in the horse: preliminary study.

REASON FOR PERFORMING STUDY: In horses, morpho-functional studies related to the enteric nervous system (ENS) controlling the sphincters are lacking. OBJECTIVES: To investigate immunohistochemically the morphology, distribution, density, phenotypes and projections of neurons controlling the ileocaecal junction (ICJ). METHODS: Two young horses were anaesthetised and underwent midline laparotomy. The neuronal retrograde fluorescent tracer Fast Blue (FB) was injected into the wall of the ICJ. A post surgical survival time of 30 days was used. Following euthanasia, the ileum and a small portion of caecum were removed. Cryosections were used to investigate the immunoreactivity (IR) of the neurons innervating the ICJ for choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), substance P (SP), calcitonin gene-related peptide (CGRP) and neurofilament NF200kDa (NF). RESULTS: Ileal FB-labelled neurons innervating the ICJ were located in the myenteric plexus (MP) and submucosal plexus (SMP) up to 48 cm and 28 cm, respectively, from the point of the FB injections. Descending MP and SMP neurons were nitrergic (54 +/- 11% and 68 +/- 4%, respectively), cholinergic (60 +/- 19% and 82 +/- 11%, respectively), NF-IR (54 +/- 9% and 78 +/- 21%, respectively), and SP-IR (about 20% in both the plexuses). CGRP-IR was expressed only by SMP descending neurons (45 +/- 21%). In both the plexuses descending neurons coexpressing nNOS- and ChAT-IR were also observed (25 +/- 11% and 61 +/- 27%, respectively). CONCLUSIONS: The presence of ileal long projecting neurons innervating the ICJ suggests that they are critical for its modulation. Consequently, in bowel diseases in which the resection of the terminal jejunum and proximal ileum are required, it is preferable, whenever possible, to conserve the major portion of the ileum. POTENTIAL RELEVANCE: The knowledge of the phenotype of ENS neurons of the ileum might be helpful for developing pharmaceutical treatment of the ICJ motility disorders.

Mechanoreceptors in the medial and lateral glenohumeral ligaments of the canine shoulder joint.

In order to have a better understanding of the role of ligaments in canine shoulder joint stability, the presence of mechanoreceptors in the medial (MGHL) and lateral (LGHL) glenohumeral ligaments was detected by means of a modified gold chloride stain. Three morphologically distinct mechanoreceptors were identified: Ruffini receptors (type I endings), Pacinian corpuscles (type II endings) and Golgi tendon organ-like receptors (type III endings). These receptors are mainly localized at each end of the ligaments and are prevalently in their glenoid portion. In particular, in the MGHL the highest density was at the cranial arm of the insertion into the scapula. The variety of mechanoreceptors in canine shoulder ligaments might indicate an afferent function in providing the CNS with joint proprioceptive information. Therefore, besides acting as passive mechanical stabilizers, the MGHL and the LGHL may serve as sensory structures, contributing actively to joint stability. Ligamentous injuries which occur in shoulders not only affect mechanical restraint but also alter the proprioceptive input to the CNS by means of disruption of the mechanoreceptors.

Distribution of Calretinin Immunoreactivity in the Lateral Nucleus of the Bottlenose Dolphin (Tursiops truncatus) Amygdala.

The amgdaloid complex consists of different nuclei, each with unique cytoarchitectonic, chemoarchitectonic and connectional characteristics. Most of the inputs coming from cortical and subcortical areas enter the amygdala via the lateral nucleus, which makes it the main receiving structure of the complex. The activity of its neurons is coordinated and modulated by different inhibitory, GABAergic-interneurons, which can be classified for their expression of various calcium-binding proteins, as well as by morphological characteristics. This research based on the analysis of the amygdala of three bottlenose dolphins, provides the first description of the topography, cytoarchitecture and distribution of calretinin immunoreactivity of the lateral nucleus. Our observations on the bottlenose dolphin confirmed the general topography of the mammalian amygdala and of the lateral nucleus. Notably, we identified six subdivision of the nucleus, more than those reported until now in the rat, monkey and human lateral nucleus. This could reveal an outstanding capability of integration and elaboration of external stimuli. In addition, we observed a strong presence of CR-immunoreactive (-ir) neurons and fibres. CR-ir neurons were mainly non-pyramidal inhibitory neurons; in particular, 80% of IR-cells were represented by large and small polygonal neurons. In the lateral nucleus of the human amygdala, CR-ir neurons form inhibitory synapses on calbindin-D28k-IR inhibitory interneurons. Since calbindin-D28k-ir interneurons make inhibitory synapses on the pyramidal cells, the final goal of the CR-ir interneurons could be the synchronization of cells activity, thus playing an important role in the control of information flow in the lateral amygdalar nucleus. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:2008-2016, 2017. © 2017 Wiley Periodicals, Inc.

The laminar organization of the motor cortex in monodactylous mammals: a comparative assessment based on horse, chimpanzee, and macaque.

The architecture of the neocortex classically consists of six layers, based on cytological criteria and on the layout of intra/interlaminar connections. Yet, the comparison of cortical cytoarchitectonic features across different species proves overwhelmingly difficult, due to the lack of a reliable model to analyze the connection patterns of neuronal ensembles forming the different layers. We first defined a set of suitable morphometric cell features, obtained in digitized Nissl-stained sections of the motor cortex of the horse, chimpanzee, and crab-eating macaque. We then modeled them using a quite general non-parametric data representation model, showing that the assessment of neuronal cell complexity (i.e., how a given cell differs from its neighbors) can be performed using a suitable measure of statistical dispersion such as the mean absolute deviation-mean absolute deviation (MAD). Along with the non-parametric combination and permutation methodology, application of MAD allowed not only to estimate, but also to compare and rank the motor cortical complexity across different species. As to the instances presented in this paper, we show that the pyramidal layers of the motor cortex of the horse are far more irregular than those of primates. This feature could be related to the different organizations of the motor system in monodactylous mammals.

Localization of the 5-hydroxytryptamine 4 receptor in equine enteric neurons and extrinsic sensory fibers.

BACKGROUND: Serotonin plays a pivotal role in regulating gut motility, visceral sensitivity, and fluid secretion via specific receptors. Among these receptors, 5-HT4 exerts a prominent control on gut motor function. Although the prokinetic effect exerted by 5-HT4 agonists is well known, the cellular sites of 5-HT4 expression remain poorly understood in large mammals, e.g., horses. In this study, we evaluated the distribution of 5-HT4 in the horse intestine and in foals with enteric aganglionosis, reminiscent of human Hirschsprung's disease. METHODS: The intestine and spinal ganglia were obtained from three healthy horses and two foals with hereditary ileocolonic aganglionosis. Tissues were processed for immunohistochemistry using a specific antibody to 5-HT4 and a variety of neuronal markers. Myenteric and submucosal plexus 5-HT4 -immunoreactive (IR) neurons were quantified as relative percentage (mean±SD) to the total number of neurons counted. Furthermore, the density of 5-HT4 -IR nerve fibers was evaluated in the mucosa and tunica muscularis. KEY RESULTS: The 5-HT4 immunoreactivity was localized to large percentages of myenteric neurons ranging from 28±9% (descending colon) to 63±19% (ileum), and submucosal neurons ranging from 54±6% (ileum) to 68±14% (duodenum). The 5-HT4 -immunoreactivity was co-expressed by some substance P-IR (SP-IR) spinal ganglion neurons and extrinsic sensory fibers of aganglionic foals. CONCLUSIONS & INFERENCES: The presence of 5-HT4 in many enteric and extrinsic sensory neurons and nerve fibers provides solid morphological evidence of the cellular sites of 5-HT4 expression in horses. The evidence of SP-IR sensory neurons positive for 5-HT4 suggests its role in visceral sensitivity.

Immunohistochemical localization of alpha(1a)-adrenoreceptors in muscle spindles of rabbit masseter muscle.

The expression of alpha(1a)-adrenoreceptors (alpha(1a)-ARs) within the muscle spindles of rabbit masseter muscle was investigated. The alpha(1a)-ARs were detected by immunohistochemical fluorescent method and examined along the entire length of 109 cross serially sectioned spindles. The sympathetic fibers were visualized by the immunofluorescent labeling of the noradrenaline synthesizing enzymes tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). In order to recognize the intrafusal muscle fiber types, antibodies for different myosin heavy chain isoforms (MyHCI) were used. TH and DBH immunolabeled nerve fibers have been observed within the capsule lamellar layers, in the periaxial fluid space and close to intrafusal muscle fibers. The alpha(1a)-ARs were detected on the smooth muscle cells of the blood vessels coursing in the muscle and in the capsule lamellar layers or within the periaxial fluid space of the spindles. Moreover, at the polar regions of a high percentage (88.1%) of muscle spindles a strong alpha(1a)-ARs immunoreactivity was present on the intrafusal muscle fibers. In double immunostained sections for alpha(1a)-ARs and MyHCI it was evidenced that both bag, and nuclear chain fibers express alpha(1a)-ARs. The receptors that we have detected by immunofluorescence may support a direct control by adrenergic fibers on muscle spindle.

Central distribution of nociceptive intradental afferent nerve fibers in the rat.

The central distribution of intradental afferent nerve fibers was investigated by combining electron microscopic observations with a selective method for inducing degeneration of the A delta- and C-type afferent fibers. Degenerating terminals were found on the proprioceptive mesencephalic trigeminal neurons and on dendrites in the neuropil of the trigeminal motor nucleus after application of capsaicin to the rat's lower incisor tooth pulp. The results give anatomical evidence of new sites of central projection of intradental A delta- and C-type fibers whereby the nociceptive information from the tooth pulp can affect jaw muscle activity.

Distribution of α-transducin and α-gustducin immunoreactive cells in the chicken (Gallus domesticus) gastrointestinal tract.

The expression and distribution patterns of the taste signaling molecules, α-gustducin (Gαgust) and α-transducin (Gαtran) G-protein subunits, were studied in the gastrointestinal tract of the chicken (Gallus domesticus) using the immunohistochemical method. Gαgust and Gαtran immunoreactive (-IR) cells were observed in the mucosal layer of all examined segments, except the esophagus, crop, and the saccus cranialis of the gizzard. The highest numbers of Gαgust and Gαtran-IR cells were found in the proventriculus glands and along the villi of the pyloric, duodenum, and rectal mucosa. Gαgust and Gαtran-IR cells located in the villi of the jejunum, ileum, and cloaca were much less numerous, while only a few Gαgust and Gαtran-IR cells were detected in the mucosa of the proventriculus and cecum. In the crypts, IR cells were observed in the small and large intestine as well as in the cloaca. Gαgust and Gαtran-IR cells displayed elongated ("bottle-" or "pear-like") or rounded shape. The demonstration of Gαgust and Gαtran expression provides evidence for taste receptor mediated mucosal chemosensitivity in the chicken gastrointestinal tract.

The recent European isolate (08P178) of equine arteritis virus causes inflammation but not arteritis in experimentally infected ponies.

In the last two decades, outbreaks of equine viral arteritis (EVA) have been reported in Europe, but little is known about these European isolates of equine arteritis virus (EAV). EAV European strain (08P178, EU-1 clade) isolated from one of these recent outbreaks is able to cause clinical signs on experimental infection. The aim of the present study was to investigate the microscopical lesions induced by this isolate after experimental infection of ponies. Animals were killed at 3, 7, 14 and 28 days post infection (dpi). At 3 dpi, lesions were essentially restricted to the respiratory tract and intestines and were characterized by mild multifocal epithelial degeneration and associated mononuclear cell infiltration. Lesions were more severe at 7 dpi and by 14 dpi, respiratory lesions were even more severe and lymphoplasmacytic infiltrates extended to other organs. At 28 dpi, lesions were still present in the viscera. In all specimens the most prominent histological change was intraepithelial, subepithelial and perivascular lymphoplasmacytic infiltration, ranging from mild and multifocal to extensive and diffuse. No signs of arterial damage such as infarcts, haemorrhages or necrosis were found. In conclusion, infection of naïve animals with the European 08P178 strain of EAV is associated with inflammation, but not arteritis.

Urinary exosomes and diabetic nephropathy: a proteomic approach.

Urinary exosomes (UE) are nanovesicles released by every epithelial cell facing the urinary space and they are considered a promising source of molecular markers for renal dysfunction and structural injury. Exosomal proteomics has emerged as a powerful tool for understanding the molecular composition of exosomes and has potential to accelerate biomarker discovery. We employed this strategy in the study of diabetic nephropathy (DN) and the consequent end stage renal disease, which represent the dramatic evolution of diabetes, often leading the patients to dialysis or kidney transplantation. The identification of DN biomarkers is likely to help monitoring the disease onset and progression. A label free LC-MS/MS approach was applied to investigate the alteration of the proteome of urinary exosomes isolated from the Zucker diabetic fatty rats (ZDF), as a model of type 2 DN. We collected 24 hour urine samples from 7 ZDF and from 7 control rats at different ages (6, 12 and 20 weeks old) to monitor the development of DN. Exosomes were isolated by ultracentrifugation and their purity assessed by immunoblotting for known exosomal markers. Exosomal proteins from urine samples of 20 week old rats were pooled and analyzed by nLC-ESI-UHR-QToF-MS/MS after pre-filtration and tryptic digestion, leading to the identification and label free quantification of 286 proteins. Subcellular localization and molecular functions were assigned to each protein by UniprotKB, showing that the majority of identified proteins were membrane-associated or cytoplasmic and involved in transport, signalling and cellular adhesion, typical functions of exosomal proteins. We further validated label free mass spectrometry results by immunoblotting, as exemplified by: Xaa-Pro dipeptidase, Major Urinary Protein 1 and Neprilysin, which resulted increased, decreased and not different, respectively, in exosomes isolated from diabetic urine samples compared to controls, by both techniques. In conclusion we show the potential of exosome proteomics for DN biomarker discovery.

Characterization of spinal ganglion neurons in horse (Equus caballus). A morphometric, neurochemical and tracing study.

Spinal ganglion (SG) neurons have been widely described in rodents, and classified according to various criteria. On the basis of such studies, many features of rodent SG neurons have become benchmarks to classify these cells. However, these traits cannot be confirmed in all other species. In the present study, horse SG neurons were morphometrically and neurochemically characterized by detecting the neuronal markers calcitonin gene-related peptide (CGRP), substance P (SP), neuronal nitric oxide synthase (nNOS) and isolectin B4 (IB4) from Griffonia simplicifolia. Moreover, spinal cord staining and tracer studies were also performed injecting Fast Blue tracer in the ileo-cecal junction. The statistical analysis of the histograms related to the cross sectional area of dark and light SG neurons confirmed the presence of the categories of small and large neurons. The staining methods employed yielded the following results: (1) in all triple staining experiments performed, most SG stained neurons were triple-labeled; (2) SP-IR neurons showed the largest percentages of co-localization with the other markers studied; (3) CGRP-IR and IB4-labeled neurons were the SG neurons showing the largest percentages of single staining; (4) nNOS-IR neurons were more represented in horse SGs than in those from rodents; (5) IB4 was widely co-localized with both CGRP and SP. Retrograde tracer investigation combined with neurochemical evaluation showed that in horse, contrarily to rodents, IB4-labeled neurons are widely involved in visceral innervations. The results obtained from the observations of serial stained sections and from a critical analysis of triple-labeling experiments allowed us to conclude that (1) most stained SG neurons co-expressed IB4-nNOS-CGRP-SP neuronal markers, (2) IB4 is not indicated as a marker of non-peptidergic neurons in the horse, (3) horse IB4-labeled neurons are widely involved in visceral sensation, (4) differently from rodents, horse IB4-, CGRP- and SP-labeled fibers share the same spinal cord level terminations.

Sympathetic innervation of the ileocecal junction in horses.

The distribution and chemical phenotypes of sympathetic and dorsal root ganglion (DRG) neurons innervating the equine ileocecal junction (ICJ) were studied by combining retrograde tracing and immunohistochemistry. Immunoreactivity (IR) for tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), neuronal nitric oxide synthase (nNOS), calcitonin gene-related peptide (CGRP), substance P (SP), and neuropeptide Y (NPY) was investigated. Sympathetic neurons projecting to the ICJ were distributed within the celiac (CG), cranial mesenteric (CranMG), and caudal mesenteric (CaudMG) ganglia, as well as in the last ganglia of the thoracic sympathetic chain and in the splanchnic ganglia. In the CG and CranMG 91 +/- 8% and 93 +/- 12% of the neurons innervating the ICJ expressed TH- and DBH-IR, respectively. In the CaudMG 90 +/- 15% and 94 +/- 5% of ICJ innervating neurons were TH- and DBH-IR, respectively. Sympathetic (TH-IR) fibers innervated the myenteric and submucosal ganglia, ileal blood vessels, and the muscle layers. They were more concentrated at the ICJ level and were also seen encircling myenteric plexus (MP) and submucosal plexus (SMP) descending neurons that were retrogradely labeled from the ICJ. Among the few retrogradely labeled DRG neurons, nNOS-, CGRP-, and SP-IR nerve cells were observed. Dense networks of CGRP-, nNOS-, and SP-IR varicosities were seen around retrogradely labeled prevertebral ganglia neurons. The CGRP-IR fibers are probably the endings of neurons projecting from the intestine to the prevertebral ganglia. These findings indicate that this crucial region of the intestinal tract is strongly influenced by the sympathetic system and that sensory information of visceral origin influences the sympathetic control of the ICJ.

Angiotensin II increases tissue-specific inhibitor of metalloproteinase-2 expression in rat aortic smooth muscle cells in vivo: evidence of a pressure-independent effect.

1. Angiotensin (Ang) II plays a major role in vascular remodelling. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in the tissue remodelling processes. The aim of the present study was to investigate whether AngII modulates TIMP-2 expression in rat aortic smooth muscle cells in vivo. 2. Angiotensin II (200 ng/kg per min, s.c.) or AngII + losartan (10 mg/kg per day, s.c.) or normal saline was administered continuously by osmotic minipumps to Sprague-Dawley rats for 1 week. In addition, the effect of endogenous AngII on TIMP-2 expression was evaluated in renovascular hypertensive rats (two kidney, one clip (2K1C) and one kidney, one clip (1K1C) models). Control rats (sham 2K1C and sham 1K1C rats) underwent sham-clipping of the left renal artery. At the end of the treatment, plasma renin activity was measured by radioimmunoassay, aortic TIMP-2 mRNA expression was evaluated by real-time polymerase chain reaction and/or northern blotting and protein expression was evaluated by immunohistochemistry. Systolic blood pressure (SBP) was measured twice a week by the tail-cuff method. 3. Exogenous AngII administration produced the expected increase in SBP (P = 0.02) compared with the control saline-treated group. The increase in SBP was abolished in AngII + losartan-treated rats. Administration of AngII caused a significant increase in TIMP-2 expression (P = 0.01) in rat aortic smooth muscle cells that was abolished in AngII + losartan-treated rats. In renovascular hypertensive rats, SBP was higher (P < 0.0001) in 2K1C and 1K1C rats compared with the corresponding sham-operated rats. Plasma renin activity was higher (P < 0.01) in 2K1C rats compared with the other groups. The expression of TIMP-2 was significantly (P < 0.05) increased only in 2K1C rats. 4. Our in vivo data demonstrate that exogenous and endogenous AngII increases TIMP-2 expression in rat aortic smooth muscle cells. This effect is not dependent on the AngII-induced increase in blood pressure and is mediated by angiotensin AT1 receptors.