Effects of Exogenous Hydrogen Sulfide in the Hypothalamic Paraventricular Nucleus on Gastric Function in Rats

Background: Hydrogen sulfide (H2S) is a new type of gas neurotransmitter discovered in recent years. It plays an important role in various physiological activities. The hypothalamus paraventricular nucleus (PVN) is an important nucleus that regulates gastric function. This study aimed to clarify the role of H2S in the paraventricular nucleus of the hypothalamus on the gastric function of rats.Methods: An immunofluorescence histochemistry double-labelling technique was used to determine whether cystathionine-beta-synthase (CBS) and c-Fos neurons are involved in PVN stress. Through microinjection of different concentrations of NaHS, physiological saline (PS), D-2-Amino-5-phosphonovaleric acid (D-AP5), and pyrrolidine dithiocarbamate (PDTC), we observed gastric motility and gastric acid secretion.Results: c-Fos and CBS co-expressed the most positive neurons after 1 h of restraint and immersion, followed by 3 h, and the least was at 0 h. After injection of different concentrations of NaHS into the PVN, gastric motility and gastric acid secretion in rats were significantly inhibited and promoted, respectively (p < 0.01); however, injection of normal saline, D-AP5, and PDTC did not cause any significant change (p > 0.05). The suppressive effect of NaHS on gastrointestinal motility and the promotional effect of NaHS on gastric acid secretion could be prevented by D-AP5, a specific N-methyl-D-aspartic acid (NMDA) receptor antagonist, and PDTC, an NF-κB inhibitor.Conclusion: There are neurons co-expressing CBS and c-Fos in the PVN, and the injection of NaHS into the PVN can inhibit gastric motility and promote gastric acid secretion in rats. This effect may be mediated by NMDA receptors and the NF-κB signalling pathway.

Droplet Degeneration of Hippocampal and Cortical Neurons Signifies the Beginning of Neuritic Plaque Formation

Background: Neuritic plaques contain neural and microglial elements, and amyloid-β protein (Aβ), but their pathogenesis remains unknown. Objective: Elucidate neuritic plaque pathogenesis. Methods: Histochemical visualization of hyperphosphorylated-tau positive (p-tau+) structures, microglia, Aβ, and iron. Results: Disintegration of large projection neurons in human hippocampus and neocortex presents as droplet degeneration: pretangle neurons break up into spheres of numerous p-tau+ droplets of various sizes, which marks the beginning of neuritic plaques. These droplet spheres develop in the absence of colocalized Aβ deposits but once formed become encased in diffuse Aβ with great specificity. In contrast, neurofibrillary tangles often do not colocalize with Aβ. Double-labelling for p-tau and microglia showed a lack of microglial activation or phagocytosis of p-tau+ degeneration droplets but revealed massive upregulation of ferritin in microglia suggesting presence of high levels of free iron. Perl’s Prussian blue produced positive staining of microglia, droplet spheres, and Aβ plaque cores supporting the suggestion that droplet degeneration of pretangle neurons in the hippocampus and cortex represents ferroptosis, which is accompanied by the release of neuronal iron extracellularly. Conclusion: Age-related iron accumulation and ferroptosis in the CNS likely trigger at least two endogenous mechanisms of neuroprotective iron sequestration and chelation, microglial ferritin expression and Aβ deposition, respectively, both contributing to the formation of neuritic plaques. Since neurofibrillary tangles and Aβ deposits colocalize infrequently, tangle formation likely does not involve release of neuronal iron extracellularly. In human brain, targeted deposition of Aβ occurs specifically in response to ongoing ferroptotic droplet degeneration thereby producing neuritic plaques.

Protein Variation Associated With Facial Eczema Resistance or Susceptibility in Romney Sheep

<p>The detection of plasma and liver protein markers for facial eczema resistance or susceptibility in Romney sheep was undertaken. A pooling protocol was used to allow rapid comparison of variation between populations. A 2-D PAGE technigue was developed for protein separation. In general, proteins separated by 2-D PAGE were examined on Coomassie blue or silver stained gels. Greater sensitivity was achieved by labelling proteins with radioactive isotopes. Reductive methylation of the free amino groups of proteins with radioactively labelled formaldehyde and sodium cyanoborohydride was used for isotopic labelling of proteins. A double-labelling technique involving 14C and 3H was used to label plasma or liver proteins from facial eczema resistant and susceptible sheep. The labelled proteins were subsequently separated by 2-D PAGE and detected by autoradiography and fluorography. Any detected variation was further analysed for individuals on one-dimensional polyacrylamide gels which allowed more rapid analysis of multiple samples. No significant difference was detected among the liver proteins of resistant and susceptible sheep. However, among the approximately twenty major plasma protein families visualised on 2-D PAGE gels, significant variation between sheep selected for facial eczema resistance or susceptibility occurred at the transferrin locus. Sheep selected for resistance showed a predominance of acidic transferrins while sheep selected for susceptibility contained a basic transferrin in greater abundance. These results were confirmed and their significance was assessed by transferrin phenotyping on one-dimensional polyacrylamide gels. The transferrin A allele was more abundant in sheep selected for resistance while the transferrin D allele showed a greater association with facial eczema susceptibility. The A allele frequency was 0.57 in resistants and 0.05 in susceptibles while the D allele frequency was 0.18 in resistants and 0.68 in susceptibles. The results suggest some separation of transferrin A and D alleles between the animals selected for resistance and susceptibility. The basis of this variation is unknown. It may reflect either a physiological association of transferrin alleles with a character of importance in facial eczema resistance, or it may be a phenomenon unrelated to facial eczema resistance produced as a result of the way in which the facial eczema resistant and susceptible flocks were generated. It is expected that subsequent genetic studies will show whether transferrin phenotype can be used as a marker to select for facial eczema resistance as a means of controlling the disease.</p>

Protein Variation Associated With Facial Eczema Resistance or Susceptibility in Romney Sheep

<p>The detection of plasma and liver protein markers for facial eczema resistance or susceptibility in Romney sheep was undertaken. A pooling protocol was used to allow rapid comparison of variation between populations. A 2-D PAGE technigue was developed for protein separation. In general, proteins separated by 2-D PAGE were examined on Coomassie blue or silver stained gels. Greater sensitivity was achieved by labelling proteins with radioactive isotopes. Reductive methylation of the free amino groups of proteins with radioactively labelled formaldehyde and sodium cyanoborohydride was used for isotopic labelling of proteins. A double-labelling technique involving 14C and 3H was used to label plasma or liver proteins from facial eczema resistant and susceptible sheep. The labelled proteins were subsequently separated by 2-D PAGE and detected by autoradiography and fluorography. Any detected variation was further analysed for individuals on one-dimensional polyacrylamide gels which allowed more rapid analysis of multiple samples. No significant difference was detected among the liver proteins of resistant and susceptible sheep. However, among the approximately twenty major plasma protein families visualised on 2-D PAGE gels, significant variation between sheep selected for facial eczema resistance or susceptibility occurred at the transferrin locus. Sheep selected for resistance showed a predominance of acidic transferrins while sheep selected for susceptibility contained a basic transferrin in greater abundance. These results were confirmed and their significance was assessed by transferrin phenotyping on one-dimensional polyacrylamide gels. The transferrin A allele was more abundant in sheep selected for resistance while the transferrin D allele showed a greater association with facial eczema susceptibility. The A allele frequency was 0.57 in resistants and 0.05 in susceptibles while the D allele frequency was 0.18 in resistants and 0.68 in susceptibles. The results suggest some separation of transferrin A and D alleles between the animals selected for resistance and susceptibility. The basis of this variation is unknown. It may reflect either a physiological association of transferrin alleles with a character of importance in facial eczema resistance, or it may be a phenomenon unrelated to facial eczema resistance produced as a result of the way in which the facial eczema resistant and susceptible flocks were generated. It is expected that subsequent genetic studies will show whether transferrin phenotype can be used as a marker to select for facial eczema resistance as a means of controlling the disease.</p>

PROLACTIN REDUCES HIPPOCAMPAL PARVALBUMIN AND GABAA RECEPTOR EXPRESSION IN FEMALE MICE

Introduction: Parvalbumin (PV)-positive cells are strategic elements of neuronal networks capable of influencing memory and learning processes. However, it is not known whether pituitary hormones may be related to parvalbumin expression in the hippocampus — a part of the limbic system with important functions in learning and memory. Objective: Since previous studies indicate that prolactin (PRL) plays a significant role in hippocampal-dependent learning and synaptic plasticity, we hypothesized that a rise in PRL levels can modify PV expression in the hippocampus. Methods: We employed biochemical, immunohistochemistry and densitometry techniques —as well as a behavioural assay— in a hyperprolactinemia model using subcutaneous osmotic pumps in female mice. Results: PRL treatment via osmotic pump induced an increase in PRL receptor (PRLR) expression in most regions of hippocampus analysed by Western blotting and immunohistochemistry methods. Fluorescent densitometry analysis revealed that PV expression decreases in the same layers in the hippocampus following PRL treatment, while double labelling immunostaining indicated close localization of PV and PRLR in PV-positive interneurons. In addition, we found that PRL induced a reduction in β2/3 subunit of GABAA receptor (GABAAR) expression that was linearly correlated with the reduction in PV expression. This reduction in β2/3 subunit of GABAAR expression was maintained in trained animals in which PRL treatment improved the learning of a spatial memory task. Conclusions: These data show, for the first time, that an increase in PRL level is associated with changes in key constituent elements of inhibitory circuits in the hippocampus and may be of relevance for the alterations in cognitive function reported in hyperprolactinemia.

Immunohistochemical labelling of lower urinary tract afferents in spinal cord v1

This protocol is used for immunohistochemical visualisation of cholera toxin subunit B within afferents innervating the lower urinary tract in cryosections of rat lumbosacral spinal cord. Free-floating sections are processed in a double labelling protocol to distinguish regions of innervation by these afferents. Cholera toxin B antibody [lower urinary tract afferents] Choline acetyltransferase antibody [preganglionic autonomic neurons and motoneurons]

Telocytes and Lymphatics of the Human Colon

Background: Telocytes (TCs) are a peculiar morphological type of stromal cells. They project long and moniliform telopodes, visible on various bidimensional sections. Originally regarded as “interstitial Cajal-like cells”, gastrointestinal TCs were CD34+. Further double-labelling studies found that colon TCs are negative for the expressions of the PDGFR-α and α-SMA. However, the TCs in colon were not distinguished specifically from endothelial cells (ECs), vascular or lymphatic. A combinational approach is important for accurate TC identification. Hence, we designed an immunohistochemical study of human colon to check whether ECs and CD34+ TCs express different markers. Methods: Immunohistochemistry was performed on archived paraffin-embedded samples of human colon (nine cases) for the following markers: CD31, CD34, CD117/c-kit and D2-40 (podoplanin). Results: A distinctive population of CD34+ TCs was found coating the myenteric ganglia. However, also perivascular cells and vascular ECs were CD34+. c-kit expression was equally found in interstitial Cajal cells (ICCs) and perivascular cells. The CD34 TCs did not express c-kit. As they were equally CD31- and D2-40- they were assessed as different from ECs. Conclusions: Testing specific markers of ECs, vascular and lymphatic, in the same tissues in which CD34+ TCs are found, is much more relevant than to identify TCs by transmission electron microscopy alone.

Regional Variation of Gap Junctional Connections in the Mammalian Inner Retina

The retinas of many species show regional specialisations that are evident in the differences in the processing of visual input from different parts of the visual field. Regional specialisation is thought to reflect an adaptation to the natural visual environment, optical constraints, and lifestyle of the species. Yet, little is known about regional differences in synaptic circuitry. Here, we were interested in the topographical distribution of connexin-36 (Cx36), the major constituent of electrical synapses in the retina. We compared the retinas of mice, rats, and cats to include species with different patterns of regional specialisations in the analysis. First, we used the density of Prox1-immunoreactive amacrine cells as a marker of any regional specialisation, with higher cell density signifying more central regions. Double-labelling experiments showed that Prox1 is expressed in AII amacrine cells in all three species. Interestingly, large Cx36 plaques were attached to about 8–10% of Prox1-positive amacrine cell somata, suggesting the strong electrical coupling of pairs or small clusters of cell bodies. When analysing the regional changes in the volumetric density of Cx36-immunoreactive plaques, we found a tight correlation with the density of Prox1-expressing amacrine cells in the ON, but not in the OFF sublamina in all three species. The results suggest that the relative contribution of electrical synapses to the ON- and OFF-pathways of the retina changes with retinal location, which may contribute to functional ON/OFF asymmetries across the visual field.

Mapping the vocal circuitry of Alston's singing mouse with pseudorabies virus

Vocalizations, like many social displays, are often elaborate, rhythmically structured behaviors that are modulated by a complex combination of cues. Vocal motor patterns require close coordination of neural circuits governing the muscles of the larynx, jaw, and respiratory system. In the elaborate vocalization of Alstons singing mouse (Scotinomys teguina), for example, each note of its rapid, frequency-modulated trill is accompanied by equally rapid modulation of breath and gape. To elucidate the neural circuitry underlying this behavior, we introduced the polysynaptic retrograde neuronal tracer pseudorabies virus (PRV) into the cricothyroid and digastricus muscles, which control frequency modulation and jaw opening respectively. Each virus singly labels ipsilateral motoneurons (nucleus ambiguous for cricothyroid, and motor trigeminal nucleus for digastricus). We find that the two isogenic viruses heavily and bilaterally co-label neurons in the gigantocellular reticular formation, a putative central pattern generator. The viruses also show strong co-labeling in compartments of the midbrain including the ventrolateral periaqueductal grey and the parabrachial nucleus, two structures strongly implicated in vocalizations. In the forebrain, regions important to social cognition and energy balance both exhibit extensive co-labeling. This includes the paraventricular and arcuate nuclei of the hypothalamus, the lateral hypothalamus, preoptic area, extended amygdala, central amygdala, and the bed nucleus of the stria terminalis. Finally, we find doubly labeled neurons in M1 motor cortex previously described as laryngeal, as well as in the prelimbic cortex, which indicate these cortical regions play a role in vocal production. Although we observe some novel patterns of double-labelling, the progress of both viruses is broadly consistent with vertebrate-general patterns of vocal circuitry, as well as with circuit models derived from primate literature.

Diabetes Affects the Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP)-Like Immunoreactive Enteric Neurons in the Porcine Digestive Tract

Diabetic gastroenteropathy is a common complication, which develops in patients with long-term diabetes. The pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide known for its cytoprotective properties and plays an important role in neuronal development, neuromodulation and neuroprotection. The present study was designed to elucidate, for the first time, the impact of prolonged hyperglycaemia conditions on a population of PACAP-like immunoreactive neurons in selected parts of the porcine gastrointestinal tract. The experiment was conducted on 10 juvenile female pigs assigned to two experimental groups: The DM group (pigs with streptozocin-induced diabetes) and the C group (control pigs). Diabetes conditions were induced by a single intravenous injection of streptozocin. Six weeks after the induction of diabetes, all animals were euthanised and further collected, and fixed fragments of the stomach, duodenum, jejunum, ileum and descending colon were processed using the routine double-labelling immunofluorescence technique. Streptozotocin-induced hyperglycaemia caused a significant increase in the population of PACAP-containing enteric neurons in the porcine stomach, small intestines and descending colon. The recorded changes may result from the direct toxic effect of hyperglycaemia on the ENS neurons, oxidative stress or inflammatory conditions accompanying hyperglycaemia and suggest that PACAP is involved in regulatory processes of the GIT function in the course of diabetes.