Form, synapses and orientation topography of a new cell type in layer 6 of the cat's primary visual cortex.

Here we report the morpho-functional features of a novel type of deep-layer neuron. The neuron was selected from a large pool of intracellularly labelled cells based on the large cell body, numerous spine-free dendrites with an overall interneuron morphology. However, the axon gave off long-range axons up to 2.8 mm from the parent soma in layers 5/6 before entering the white matter. The boutons were uniformly distributed along the axon without forming distinct clusters. Dendritic length, surface area and volume values were at least 3 times larger than any known cortical neuron types with the exception of giant pyramidal cells of layer 5. Electron microscopy of the boutons revealed that they targeted dendritic spines (78%) and less frequently dendritic shafts (22%). Nearly half of the postsynaptic dendrites were immunopositive to GABA. Superimposing the axonal field on the orientation map obtained with optical imaging showed a preponderance of boutons to cross-orientations (38%) and an equal representation of iso- and oblique orientations (31%). The results suggest an integrating role for the layer 6 stellate neuron which projects to a functionally broad range of neurons in the deep cortical layers and to other cortical and/or subcortical regions.

Application of the mirror technique for block-face scanning electron microscopy.

The mirror technique adapted for electron microscopy allows correlating neuronal structures across the cutting plane of adjoining light microscopic sections which, however, have a limited thickness, typically less than 100 µm (Talapka et al. in Front Neuroanat, 2021, https://doi.org/10.3389/fnana.2021.652422 ). Here, we extend the mirror technique for tissue blocks in the millimeter range and demonstrate compatibility with serial block-face electron microscopy (SBEM). An essential step of the methodological improvement regards the recognition that unbound resin must be removed from the tissue surface to gain visibility of surface structures. To this, the tissue block was placed on absorbent paper during the curing process. In this way, neuronal cell bodies could be unequivocally identified using epi-illumination and confocal microscopy. Thus, the layout of cell bodies which were cut by the sectioning plane can be correlated with the layout of their complementary part in the adjoining section processed for immunohistochemistry. The modified mirror technique obviates the spatial limit in investigating synaptology of neurochemically identified structures such as neuronal processes, dendrites and axons.

Application of the Mirror Technique for Three-Dimensional Electron Microscopy of Neurochemically Identified GABA-ergic Dendrites.

In the nervous system synaptic input arrives chiefly on dendrites and their type and distribution have been assumed pivotal in signal integration. We have developed an immunohistochemistry (IH)-correlated electron microscopy (EM) method - the "mirror" technique - by which synaptic input to entire dendrites of neurochemically identified interneurons (INs) can be mapped due preserving high-fidelity tissue ultrastructure. Hence, this approach allows quantitative assessment of morphometric parameters of synaptic inputs along the whole length of dendrites originating from the parent soma. The method exploits the fact that adjoining sections have truncated or cut cell bodies which appear on the common surfaces in a mirror fashion. In one of the sections the histochemical marker of the GABAergic subtype, calbindin was revealed in cell bodies whereas in the other section the remaining part of the very same cell bodies were subjected to serial section EM to trace and reconstruct the synaptology of entire dendrites. Here, we provide exemplary data on the synaptic coverage of two dendrites belonging to the same calbindin-D28 K immunopositive IN and determine the spatial distribution of asymmetric and symmetric synapses, surface area and volume of the presynaptic boutons, morphometric parameters of synaptic vesicles, and area extent of the active zones.

Regulation of (Pro)Renin Receptor in Renin-Positive Smooth Muscle Cells of Kidney Arterioles in Rats with STZ-Induced Diabetes.

Objective. The nephron (pro)renin receptor may play a pathophysiological role in renal disorders in hypertension or diabetes. The aim of this study was to determine the relationship of (pro)renin receptors and transdifferentiation between the renin-negative and renin-positive SMCs in the afferent arteriole by estimating the distribution of (pro)renin receptors in renin-positive and renin-negative SMCs of the afferent arteriole of kidneys in normal and streptozotocin- (STZ-) induced diabetic rats. Therefore in diabetes the renin granulation of afferent arterioles is different as in normal, the diabetes model for finding the differences to normal in distribution of (pro)renin receptors of afferent arterioles was used. Method. To estimate the number of (pro)renin receptors in arteriolar SMCs a special protocol of immunohistochemistry to stereology was followed. Results. Our results showed that on the surface of renin-positive SMCs the number of (pro)renin receptors was upregulated, while in the cytoplasm of SMCs there was downregulation in comparison to renin-negative SMCs. There is a significant difference between the number of (pro)renin receptors on the surface and in the cytoplasm of renin-positive SMCs in normal rats. These differences in the number of (pro)renin receptors were not present in rats with STZ-induced diabetes. Any other differences in the number of (pro)renin receptors between the STZ-induced diabetic and normal rats were not detected. The tissue level of angiotensin II did not change in the kidneys of STZ-induced diabetic rats. Conclusion. The distribution of (pro)renin receptors in afferent arteriolar SMCs is related to renin granulation of SMCs, but independent of angiotensin II plasma or tissue levels in the kidney.

Effect of dietary cereal type, crude protein and butyrate supplementation on metabolic parameters of broilers.

This study investigates the metabolic effects of maize- or wheat-based diets with normal (NP) and lowered (LP) dietary crude protein level [the latter supplemented with limiting amino acids and sodium (n-)butyrate at 1.5 g/kg diet] at different phases of broiler fattening. Blood samples of Ross 308 broilers were tested at the age of 1, 3 and 6 weeks. Total protein (TP) concentration increased in wheat-based and decreased in LP groups in week 3, while butyrate reduced albumin/TP ratio in week 1. Uric acid level was elevated by wheat-based diet in week 1 and by wheat-based diet and butyrate in week 3, but decreased in LP groups in weeks 3 and 6. Aspartate aminotransferase activity was increased by wheat-based diet in week 3, and creatine kinase activity was intensified by LP in weeks 3 and 6. Blood glucose level decreased in wheat-based groups in week 3; however, triglyceride concentration was augmented in the same groups in week 3. No change of glucagon-like peptide 1, glucose-dependent insulinotropic polypeptide and insulin concentration was observed. In conclusion, an age-dependent responsiveness of broilers to dietary factors was found, dietary cereal type was a potent modulator of metabolism, and a low crude protein diet supplemented with limiting amino acids might have a beneficial impact on the growth of chickens.

Elevated levels of macromolecular damage are correlated with increased nitric oxide synthase expression in erythrocytes isolated from twin neonates.

Pregnancy is a state associated with an enhanced metabolism and demand for O2 , which may lead to the overproduction of reactive oxygen species (ROS) and hence to oxidative stress. An elevated ROS level may result in delayed development and a low birth weight. The aim of this study was to reveal the consequences of multiple pregnancies on the redox status of neonatal human red blood cells (RBCs) and evaluate the role of endothelial nitric oxide synthase (NOS3) - expressing RBCs in the generation of oxidative stress. The study presents evidence of higher levels of production of hydrogen peroxide, peroxynitrite and nitrate content in the RBCs of twin neonates, clearly reflected by an elevated level of protein and lipid damages. This phenotype appears to be a consequence of multiple pregnancies, regardless of the level of maturity or the birth weight of the twins. Besides the higher level of ROS, there was a general decrease in the expression of genes coding for antioxidants. The first data are presented on NOS3-expressing neonatal human RBCs. The number of RBCs producing NOS3 was more than twice as high in twin neonates compared to singletons, with no correlation to maturity.

Structural and molecular features of intestinal strictures in rats with Crohn's-like disease.

AIM: To develop a new rat model we wanted to gain a better understanding of stricture formation in Crohn's disease (CD). METHODS: Chronic colitis was induced locally by the administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS). The relapsing inflammation characteristic to CD was mimicked by repeated TNBS treatments. Animals were randomly divided into control, once, twice and three times TNBS-treated groups. Control animals received an enema of saline. Tissue samples were taken from the strictured colonic segments and also adjacent proximally and distally to its 60, 90 or 120 d after the last TNBS or saline administrations. The frequency and macroscopic extent of the strictures were measured on digital photographs. The structural features of strictured gut wall were studied by light- and electron microscopy. Inflammation related alterations in TGF-beta 2 and 3, matrix metalloproteinases 9 (MMP9) and TIMP1 mRNA and protein expression were determined by quantitative real-time PCR and western blot analysis. The quantitative distribution of caspase 9 was determined by post-embedding immunohistochemistry. RESULTS: Intestinal strictures first appeared 60 d after TNBS treatments and the frequency of them increased up to day 120. From day 90 an intact lamina epithelialis, reversible thickening of lamina muscularis mucosae and irreversible thickening of the muscularis externa were demonstrated in the strictured colonic segments. Nevertheless the morphological signs of apoptosis were frequently seen and excess extracellular matrix deposition was recorded between smooth muscle cells (SMCs). Enhanced caspase 9 expression on day 90 in the SMCs and on day 120 also in myenteric neurons indicated the induction of apoptosis. The mRNA expression profile of TGF-betas after repeated TNBS doses was characteristic to CD, TGF-beta 2, but not TGF-beta 3 was up-regulated. Overexpression of MMP9 and down-regulation of TIMP1 were demonstrated. The progressive increase in the amount of MMP9 protein in the strictures was also obvious between days 90 and 120 but TIMP1 protein was practically undetectable at this time. CONCLUSION: These findings indicate that aligned structural and molecular changes in the gut wall rather than neuronal cell death play the primary role in stricture formation.

Upregulation of the L-type Calcium Channel in Renin-Positive Smooth Muscle Cells of Arterioles in the Kidneys of Rats with Streptozotocin-Induced Diabetes.

OBJECTIVE: To estimate the number of L-type calcium (Ca2+) channels in the afferent and efferent arteriolar renin-positive and renin-negative smooth muscle cells and endothelial cells of the kidney arterioles in order to seek a relationship between the cell functions and the number of ion channels in the cell membranes. STUDY DESIGN: In diabetes the main source of renin/prorenin is the tubules, while the renin granulation of the afferent arterioles is increased relative to the normal. The release of renin is related to the Ca2+ levels of the intracellular and extracellular spaces of the afferent arterioles. The L-type calcium channel blockers have a stimulatory effect on the renin system. The immunohistochemical signal of the antibody against the L-type Ca2+ channels was estimated stereologically. RESULTS: In the diabetic kidney the relative number of L-type Ca2+ channels on the surface of the renin-positive cells of the afferent arterioles was significantly increased. In the normal kidney there was a difference in the relative number of L-type Ca2+ channels on the surface of the endothelial cells between the renin-positive and renin-negative profiles of the afferent arterioles. CONCLUSION: The increment in the number of Ca2+ channels on the renin-positive cell surface in the diabetic kidney could be related to the high renin granularity of the afferent arterioles. In the normal kidney the differences in the number of L-type Ca2+ channels on the endothelial cell surface may play a role in the higher permeability of the renin-positive part of the afferent arterioles.

Alleviated mucosal and neuronal damage in a rat model of Crohn's disease.

AIM: To establish a rat model suitable to investigate the repetitive relapsing inflammations (RRI) characteristic to Crohn's disease. METHODS: Colitis was induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS). RRI were mimicked by repeating administrations of TNBS. Tissue samples were taken from control, once, twice and three times treated rats from the inflamed and adjacent non-inflamed colonic segments at different timepoints during the acute intestinal inflammation. The means of the ulcerated area were measured to evaluate the macroscopic mucosal damage. The density of myenteric neurons was determined on whole mounts by HuC/HuD immunohistochemistry. Heme oxygenase-1 (HO-1) expression was evaluated by molecular biological techniques. RESULTS: TNBS-treated rats displayed severe colitis, but the mortality was negligible, and an increase of body weight was characteristic throughout the experimental period. The widespread loss of myenteric neurons, and marked but transient HO-1 up-regulation were demonstrated after the first TNBS administration. After repeated doses the length of the recovery time and extent of the ulcerous colonic segments were markedly decreased, and the neuronal loss was on a smaller scale and was limited to the inflamed area. HO-1 mRNA level was notably greater than after a single dose and overexpression was sustained throughout the timepoints examined. Nevertheless, the HO-1 protein up-regulation after the second TNBS treatment proved to be transient. Following the third treatment HO-1 protein expression could not be detected. CONCLUSION: Experimentally provoked RRI may exert a protective preconditioning effect against the mucosal and neuronal damage. The persistent up-regulation of HO-1 mRNA expression may correlate with this.

Regionally distinct alterations in the composition of the gut microbiota in rats with streptozotocin-induced diabetes.

The aim of this study was to map the microbiota distribution along the gut and establish whether colon/faecal samples from diabetic rats adequately reflect the diabetic alterations in the microbiome. Streptozotocin-treated rats were used to model type 1 diabetes mellitus (T1D). Segments of the duodenum, ileum and colon were dissected, and the microbiome of the lumen material was analysed by using next-generation DNA sequencing, from phylum to genus level. The intestinal luminal contents were compared between diabetic, insulin-treated diabetic and healthy control rats. No significant differences in bacterial composition were found in the luminal contents from the duodenum of the experimental animal groups, whereas distinct patterns were seen in the ileum and colon, depending on the history of the luminal samples. Ileal samples from diabetic rats exhibited particularly striking alterations, while the richness and diversity obscured some of the modifications in the colon. Characteristic rearrangements in microbiome composition and diversity were detected after insulin treatment, though the normal gut flora was not restored. The Proteobacteria displayed more pronounced shifts than those of the predominant phyla (Firmicutes and Bacteroidetes) in the rat model of T1D. Diabetes and insulin replacement affect the composition of the gut microbiota in different, gut region-specific manners. The luminal samples from the ileum appear more suitable for diagnostic purposes than the colon/faeces. The Proteobacteria should be at the focus of diagnosis and potential therapy. Klebsiella are recommended as biomarkers of T1D.

Gut region-specific rearrangement of the cellular and subcellular compartments of nitric oxide synthase isoforms after chronic ethanol consumption in rats.

We recently provided evidence of cell-type-specific differences in the subcellular distributions of the three nitric oxide synthase (NOS) isoforms in the myenteric neurons, enteric smooth muscle cells and the capillary endothelium of the rat duodenum. We hypothesized that the presence of three NOS isoforms in the same type of cells with differences in subcellular compartmentalization might reflect a functional plasticity. Therefore, investigation of the possible rearrangement of cellular and subcellular NOS compartments in different gut segments following chronic ethanol treatment was the aim of this study. Rats were randomly divided into two groups and received water or 20% ethanol solution, preceded by short periods of adaptation with 10% and 15% ethanol. After 8 weeks, segments of duodenum, ileum and colon of the control and the alcohol-treated rats were processed for post-embedding immunohistochemistry and RT-PCR. The quantitative differences in the numbers of gold particles indicative of the different NOSs and their relative mRNA levels between the two experimental groups varied greatly, depending on the gut segment, and also on the cellular and subcellular compartments investigated. The chronic ethanol administration had the opposite effect on the quantitative distribution of the neuronal and endothelial NOS labelling gold particles in the different cellular compartments and resulted in subcellular rearrangement of NOS labels along the gastrointestinal tract. The intestinal region-specific rearrangement of the cellular and subcellular NOS compartments may possibly result in functional plasticity and help to maintain the optimum NO level under pathological conditions.

Heterogeneous downregulation of angiotensin II AT1-A and AT1-B receptors in arterioles in STZ-induced diabetic rat kidneys.

INTRODUCTION: The renin granulation of kidney arterioles is enhanced in diabetes despite the fact that the level of angiotensin II in the diabetic kidney is elevated. Therefore, the number of angiotensin II AT1-A and AT1-B receptors in afferent and efferent arteriole's renin-positive and renin-negative smooth muscle cells (SMC) was estimated. METHOD: Immunohistochemistry at the electron microscopic level was combined with 3D stereological sampling techniques. RESULTS: In diabetes the enhanced downregulation of AT1-B receptors in the renin-positive than in the renin-negative SMCs in both arterioles was resulted: the significant difference in the number of AT1 (AT1-A + AT1-B) receptors between the two types of SMCs in the normal rats was further increased in diabetes and in contrast with the significant difference observed between the afferent and efferent arterioles in the normal animals, there was no such difference in diabetes. CONCLUSIONS: The enhanced downregulation of the AT1-B receptors in the renin-negative SMCs in the efferent arterioles demonstrates that the regulation of the glomerular filtration rate by the pre- and postglomerular arterioles is changed in diabetes. The enhanced downregulation of the AT1-B receptors in the renin-positive SMCs in the arterioles may result in an enhanced level of renin granulation in the arterioles.

Spatiotemporal expression pattern of DsRedT3/CCK gene construct during postnatal development of myenteric plexus in transgenic mice.

Cholecystokinin (CCK) is an early marker of both neuronal and endocrine cell lineages in the developing gastrointestinal tract. To determine the quantitative properties and the spatial distribution of the CCK-expressing myenteric neurones in early postnatal life, a transgenic mouse strain with a CCK promoter-driven red fluorescent protein (DsRedT3/CCK) was established. The cell-specific expression of DsRedT3/CCK was validated by in situ hybridization with a CCK antisense riboprobe and by in situ hybridization coupled with immunohistochemistry involving a monoclonal antibody to CCK. A gradual increase in the DsRedT3/CCK-expressing enteric neurones with clear regional differences was documented from birth until the suckling to weaning transition, in parallel with the period of rapid intestinal growth and functional maturation. To evaluate the proportion of myenteric neurones in which DsRedT3/CCK transgene expression was colocalized with the enteric neuronal marker peripherin, immunofluorescence techniques were applied. All DsRedT3/CCK neurones were peripherin-immunoreactive and the proportion of DsRedT3/CCK-expressing myenteric neurones in the duodenum was the highest after the third week of life, when the number of peripherin-immunoreactive myenteric neurones in this region had decreased. Nearly all of the DsRedT3/CCK-expressing neurones also expressed 5-hydroxytryptophan (5-HT). Thus, by utilizing a new transgenic mouse strain, we have demonstrated a small number of CCK-expressing myenteric neurones with a developmentally regulated spatiotemporal distribution. The coexistence of CCK and 5-HT in the majority of these neurones suggests their possible regulatory role in feeding at the suckling to weaning transition.

Gut region-specific diabetic damage to the capillary endothelium adjacent to the myenteric plexus.

OBJECTIVE: Damage in the capillaries supplying the MP has been proposed as a critical factor in the development of diabetic enteric neuropathy. We therefore investigated connections between STZ-induced diabetes and the BM morphology, the size of caveolar compartments, the width of TJs, the transport of albumin, and the quantitative features of Cav-1 and eNOS expression in these microvessels. METHODS: Gut segments from diabetic rats were compared with those from insulin-treated diabetics and those from controls. The effects of diabetes on the BM, the caveolar compartments, and the TJs were evaluated morphometrically. The quantitative features of the albumin transport were investigated by postembedding immunohistochemistry. The diabetes-related changes in Cav-1 and eNOS expression were assessed by postembedding immunohistochemistry and molecular method. RESULTS: Thickening of the BM, enlargement of the caveolar compartments, opening of the junctions, enhanced transport of albumin, and overexpression of Cav-1 and eNOS were documented in diabetic animals. Insulin replacement in certain gut segments prevented the development of these alterations. CONCLUSIONS: These data provide morphological, functional, and molecular evidence that the endothelial cells in capillaries adjacent to the MP is a target of diabetic damage in a regional manner.

Subcellular distribution of nitric oxide synthase isoforms in the rat duodenum.

AIM: To study the cell-type specific subcellular distribution of the three isoforms of nitric oxide synthase (NOS) in the rat duodenum. METHODS: Postembedding immunoelectronmicroscopy was performed, in which primary antibodies for neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), were visualized with protein A-gold-conjugated secondary antibodies. Stained ultrathin sections were examined and photographed with a Philips CM10 electron microscope equipped with a MEGAVIEW II camera. The specificity of the immunoreaction in all cases was assessed by omitting the primary antibodies in the labeling protocol and incubating the sections only in the protein A-gold conjugated secondary antibodies. RESULTS: Postembedding immunoelectronmicroscopy revealed the presence of nNOS, eNOS, and iNOS immunoreactivity in the myenteric neurons, the enteric smooth muscle cells, and the endothelium of capillaries running in the vicinity of the myenteric plexus of the rat duodenum. The cell type-specific distributions of the immunogold particles labeling the three different NOS isozymes were revealed. In the control experiments, in which the primary antiserum was omitted, virtually no postembedding gold particles were observed. CONCLUSION: This postembedding immunoelectronmicroscopic study provided the first evidence of cell-type-specific differences in the subcellular distributions of NOS isoforms.

Spatial pattern analysis of nitrergic neurons in the myenteric plexus of the duodenum of different mammalian species.

Nitrergic myenteric neurons are especially susceptible to the development of neuropathy in functional gastrointestinal disorders. Investigations of the similarities and dissimilarities in the organization of nitrergic neurons in the various mammalian species are therefore important in an effort to determine the extent to which the results obtained in different animal models can be generalized. In the present work, the density and the spatial organization of the nitrergic neurons in the myenteric plexus of the duodenum were investigated in 7 mammalian species. After nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry, the Plexus Pattern Analysis software (PPAs) was applied to count the nuclei of nitrergic neurons, calculate the proportions of the areas covered by the plexus and perform randomization analysis. All 7 species exhibited a large population of nitrergic myenteric neurons, with densities in the range 12-56 cells/mm 2 . The distribution patterns of these neurons differed markedly in the different species, however, the rat was the only species in which the nitrergic neurons appeared to be randomly distributed. The PPAs in conjunction with NADPH-d histochemistry proved to be a simple and fast tool with which to reveal similarities and dissimilarities in the spatial arrangement of the nitrergic neurons in the different species.