Perinuclear Zone Research Articles

Pituitary Crooke cell neuroendocrine tumor of adrenocorticotropic hormone differentiation-specific transcription factor lineage: a clinicopathological analysis of six cases

Objective: To investigate the clinicopathological features of Crooke cell tumor of adrenocorticotropic hormone differentiation specific transcription factor (TPIT, also known as transcription factor 19, TBX19) lineage neuroendocrine tumors. Methods: Six cases of Crooke cell tumor diagnosed at the First Affiliated Hospital of University of Science and Technology of China, Hefei, China from October 2019 to October 2023 were collected. The clinical and pathological features of these cases were analyzed. Results: Among the six cases, one was male and five were female, with ages ranging from 26 to 75 years, and an average age of 44 years. All tumors occurred within the sella turcica. Clinical presentations included visual impairment in two cases, menstrual disorders in one case, Cushing's syndrome in one case, headache in one case, and one asymptomatic case discovered during a physical examination. Preoperative serum analyses revealed elevated levels of cortisol and adrenocorticotropic hormones in two cases, elevated cortisol in two cases, elevated adrenocorticotropic hormone in one case, and one case with a mild increase in prolactin due to the pituitary stalk effect. Magnetic resonance imaging revealed uneven enhancement of masses with maximum diameters ranging from 1.7 to 3.2 cm, all identified as macroadenomas. Microscopically, tumor cells exhibited irregular polygonal shapes, solid sheets, or pseudo-papillary arrangements around blood vessels. The cell nuclei were eccentric or centrally located, varying in size, with abundant cytoplasm. Some tumor cells showed perinuclear halo. Immunohistochemistry demonstrated diffuse strong positivity for TPIT in five cases, focal weak positivity for TPIT in one case, diffuse strong positivity for adrenocorticotropic hormone in all cases, and faint staining around the nuclei in a few cells. CK8/18 showed a strong positive ring pattern in more than 50% of tumor cells, focal weak positive expression of p53, and the Ki-67 positive index ranged 1%-5%. Periodic acid-Schiff staining revealed positive cytoplasm and negative perinuclear areas. Conclusions: Crooke cell tumor is a rare type of pituitary neuroendocrine tumors. Its pathological characteristics include a distinctive perinuclear clear zone and immunohistochemical markers, such as CK8/18 exhibiting a ring or halo pattern. This entity represents a high-risk subtype among pituitary neuroendocrine tumors, displaying a high risk of invasion and a propensity for recurrence. Accurate diagnosis is crucial for the postoperative follow-up and multimodal treatment planning.

Investigating mechanisms of diminished GABAergic inhibitory effcacy in the hypothalamic paraventricular nucleus (PVN) of AngII-salt hypertensive mice

The PVN receives glutamatergic excitation from sodium and AngII sensing regions of the forebrain along with dense GABAergic inhibition from the surrounding peri-nuclear zone (PNZ). Studies in various models of hypertension and heart failure report that GABAergic inhibitory tonus is diminished, contributing to the net increase in PVN-driven sympathetic nerve activity that contributes to the pathogenesis of these chronic and prevalent cardiovascular diseases. Here we compared GABAergic inhibition of the PVN in normotensive mice consuming a normal salt (0.4% NaCl) diet (NSD) and hypertensive mice consuming a high salt (4% NaCl) diet (HSD) with systemic infusion of AngII (600 ng/kg/min, s.c.) (AngII-salt HTN). Using gramicidin perforated patch recordings in brain slices, we determined that the GABA equilibrium potential (EGABA) of PVN neurons was significantly depolarized in the AngII-salt HTN group (Δ+34.3 ± 7.5 mV) compared to the NSD group (n=3 cells) (P=0.003), consistent with diminished inhibitory effcacy of GABA and reflecting increased intracellular [Cl−]. Western blot analysis of PVN punches yielded perplexing results, showing increased expression of the Cl− efflux transporter KCC2 (+12%) and diminished expression of the Cl− influx transporter NKCC1 (-38%) — suggestive of compensation for a separate cause of elevated intracellular [Cl−]. Previously, we determined that EGABA can be rapidly and reversibly depolarized (+9 mV) by perfusing slices with high K+ (+10 mM) aCSF in the presence of ionotropic glutamate receptor blockade with CNQX (10 μM) and APV (50 μM). Hence it seems that heightened synaptic GABA release is capable of increased intracellular [Cl−]. Given that the magnitude of the latter effect is small relative to the measured depolarization of EGABA in AngII-salt HTN neurons, increased synaptic GABA release appears inadequate to explain the measured increase of EGABA in AngII-salt HTN. We posit that glial buffering of extracellular Cl− might contribute to the unexpectedly large EGABA depolarization in AngII-salt HTN. Notably, EGABA among PNZ GABAergic neurons was also significantly depolarized in the AngII-salt HTN group (Δ+22.4 ± 8.8 mV, n=3 cells) compared to NSD controls (n=3 cells) (P=0.037). Mechanism(s) supporting this are presently unknown. Of special interest, preliminary data indicate that most GABAergic PNZ neurons have recurrent GABAergic responses that are predicted (based on having depolarized EGABA) to have diminished feedback inhibition in the AngII-salt group. If true, PNZ neurons would be expected to increase synaptic GABA release in the PVN of mice with AngII-salt HTN. What contribution the latter effect makes to the net depolarization of EGABA in PVN neurons is under investigation. NS115072 (GMT). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Glutamate Spillover Dynamically Strengthens Gabaergic Synaptic Inhibition of the Hypothalamic Paraventricular Nucleus.

The hypothalamic paraventricular nucleus (PVN) is strongly inhibited by γ-aminobutyric acid (GABA) from the surrounding peri-nuclear zone (PNZ). Because glutamate mediates fast excitatory transmission and is substrate for GABA synthesis, we tested its capacity to dynamically strengthen GABA inhibition. In PVN slices from male mice, bath glutamate applied during ionotropic glutamate receptor blockade increased PNZ-evoked inhibitory postsynaptic currents (eIPSCs) without affecting GABA-A receptor agonist currents or single-channel conductance, implicating a presynaptic mechanism(s). Consistent with this interpretation, bath glutamate failed to strengthen IPSCs during pharmacological saturation of GABA-A receptors. Presynaptic analyses revealed that glutamate did not affect paired-pulse ratio, peak eIPSC variability, GABA vesicle recycling speed, or readily releasable pool (RRP) size. Notably, glutamate-GABA strengthening (GGS) was unaffected by metabotropic glutamate receptor blockade and graded external Ca2+ when normalized to baseline amplitude. GGS was prevented by pan- but not glial-specific inhibition of glutamate uptake and by inhibition of glutamic acid decarboxylase (GAD), indicating reliance on glutamate uptake by neuronal excitatory amino acid transporter 3 (EAAT3) and enzymatic conversion of glutamate to GABA. EAAT3 immunoreactivity was strongly localized to presumptive PVN GABA terminals. High bath K+ also induced GGS, which was prevented by glutamate vesicle depletion, indicating that synaptic glutamate release strengthens PVN GABA inhibition. GGS suppressed PVN cell firing, indicating its functional significance. In sum, PVN GGS buffers neuronal excitation by apparent "over-filling" of vesicles with GABA synthesized from synaptically released glutamate. We posit that GGS protects against sustained PVN excitation and excitotoxicity while potentially aiding stress adaptation and habituation.

The first arctic strain of Rhizochromulina: Morphology, ultrastructure, and position in the evolutionary tree of the order Rhizochromulinales (Heterokontophyta, Dictyochophyceae)

Rhizochromulina marina is a unicellular amoeboid alga capable of forming flagellate cells; it is a single validly named species in the genus. Besides, there are numerous environmental sequences and undescribed strains designated as Rhizochromulina sp. or R. marina. The biogeography of the genus is understudied: rhizochromulines from the Indian, Southern, and Arctic Oceans are unknown. Here, we present the description of Rhizochromulina sp. B44, which was for the first time isolated from an arctic habitat. Biofilms of this microalga grow at the bottom of a culture vessel, where neighbouring amoeboid cells form associations through a common network of pseudopodia, i.e. meroplasmodia. Pseudopodia branch, anastomose mainly during meroplasmodia formation, and are supported by microtubules that arise from the perinuclear zone. Actin filaments are localized in the cytoplasm and can be revealed only near the bases of pseudopodia. We succeeded in inducing the transformation of amoeboid cells into flagellates using a prolonged agitation of cultures. Morphological and molecular analyses revealed that the studied strain is most closely related to the type strain of R. marina. At the same time, 18S rDNA sequences of early branching-off rhizochromulinids differ significantly from Rhizochromulina sp. B44, suggesting a high divergence at the genus level.

Light disrupts social memory via a retina-to-supraoptic nucleus circuit.

The formation of social memory between individuals of the opposite sex is crucial for expanding mating options or establishing monogamous pair bonding. A specialized neuronal circuit that regulates social memory could enhance an individual's mating opportunities and provide a parallel pathway for computing social behaviors. While the influence of light exposure on various forms of memory, such as fear and object memory, has been studied, its modulation of social recognition memory remains unclear. Here, we demonstrate that acute exposure to light impairs social recognition memory (SRM) in mice. Unlike sound and touch stimuli, light inhibits oxytocin neurons in the supraoptic nucleus (SON) via M1 SON-projecting intrinsically photosensitive retinal ganglion cells (ipRGCs) and GABAergic neurons in the perinuclear zone of the SON (pSON). We further show that optogenetic activation of SON oxytocin neurons using channelrhodopsin is sufficient to enhance SRM performance, even under light conditions. Our findings unveil a dedicated neuronal circuit through which luminance affects SRM, utilizing a non-image-forming visual pathway, distinct from the canonical modulatory role of the oxytocin system.

Influence of a high-carbohydrate high-fat diet on age-related changes in the myocardium in rats

The increase in cardiovascular diseases and metabolic disorders associated with longevity actualizes the study of the effect of high-calorie diets on heart aging.Aim: the experimental study of the effect of a high-carbohydrate hig -fat diet on the myocardium in young and old age.Material and Methods. A morphological study of the myocardium was carried out in four groups of male Wistar rats: group 1 - 150-day-old animals were kept on a standard diet; 2nd - 150 days, kept at a high-carbohydrate high-fat diet (HCHFD) for 90 days (from 60 days of age); 3rd - 540 days old, kept on a standard diet; 4th - 540 days old, kept at HCHFD for 90 days (from450 days of age). ELISA method in blood serum was used to determine the concentration of fibronectin, transforming growth factor beta-1, connective tissue growth factor.Results. In groups 2-4, leukostasis, focal lympho-monocytic infiltration of the myocardial stroma, an increase in the number of myocardial cells with karyopyknosis and edema of the perinuclear zone of the sarcoplasm, contracture disorders, and an increase in the specific volume of the connective tissue of the stroma were detected. The defeat of myocardial cells and fibrosis were most pronounced in group 4. HCHFD increased the concentration of fibronectin in animals in both age groups with predominance in group 4, caused a tendency to increase the content of transforming growth factor beta-1, connective tissue growth factor in blood serum. Thus, HCHFD accelerates and enhances age-related changes in the white rat myocardium.

Sortilin knock-down alters the expression and distribution of cathepsin D and prosaposin and up-regulates the cation-dependent mannose-6-phosphate receptor in rat epididymal cells

The selective transport to lysosomes can be mediated by either mannose-6-phosphate receptors (CD-MPR and CI-MPR) or sortilin. In mammalian epididymis, some lysosomal proteins are secreted into the lumen through unknown mechanisms. To investigate the underlying mechanisms of lysosomal protein transport in epididymal cells we studied the expression and distribution of cathepsin D (CatD) and prosaposin (PSAP) in a sortilin knocked down RCE-1 epididymal cell line (RCE-1 KD) in comparison with non-transfected RCE-1 cells. In RCE-1 cells, CatD was found in the perinuclear zone and co-localize with sortilin, whereas in RCE-1 KD cells, the expression, distribution and processing of the enzyme were altered. In turn, PSAP accumulated intracellularly upon sortilin knock-down and redistributed from LAMP-1-positive compartment to a perinuclear location, remaining co-localized with CatD. Interestingly, the sortilin knock-down induced CD-MPR overexpression and a redistribution of the receptor from the perinuclear zone to a dispersed cytoplasmic location, accompanied by an increased co-localization with CatD. The increase in CD-MPR could result from a compensatory response for the proper delivery of CatD to lysosomes in epididymal cells. The intracellular pathway taken by lysosomal proteins could be an approach for addressing further studies to understand the mechanism of exocytosis and therefore the role of these proteins in the epididymis.

Effect of Viral Infection on the Ultrastructural Organization of Black Currant Leaf Tissue Cells

Effect of Viral Infection on the Ultrastructural Organization of Black Currant Leaf Tissue Cells

The objectivation of the concept of war in the Hryhir Tyutyunyk’s story "Klymko"

The article identifies and characterizes the lexical-semantic means of objectifying the concept of war in Hryhir Tyutyunnyk’s story "Klymko", traces the semantic connections of the nuclear, peri-nuclear and peripheral zones of the concept of war with the components of emotional-evaluative lexical-semantic zones. The concept of war in the story has lexical means of objectification, the totality of which represents the plan of expression of the corresponding lexical-semantic field, centered around the dominant represented by the name of this concept. Lexical units are concentrated around the name of the concept, which reflect the dynamics of events, their perception by the character. Lexemes of the perinuclear zone are filled with specific functional and stylistic dimensions, they undergo contextual actualization of their associative and emotive-evaluative semantic plans. The lexical-semantic field of the concept testifies to the subjective vision, understanding, evaluation and reproduction of the events of the war in the story. Features of objectification of the concept are related to the age of the main character, his emotional states and reactions. The specificity of the objectification of the concept of war in Hryhir Tyutyunnyk’s story "Klymko" can be seen in its versatile characteristics, in connections with concepts belonging to emotive-evaluative lexical-semantic zones. The analysis of the contexts of the story based on the lexical-semantic field of the concept of war proves that the author objectifies the features of representatives of the Ukrainian mentality, contrasting them with the brutality of the invaders. The specificity of the objectification of the concept of war in Hryhir Tyutyunnyk’s story "Klymko" can be seen in its versatile characteristics, in connections with concepts belonging to emotive-evaluative lexical-semantic zones. Humanity knows numerous military operations, battles in which millions of people participated, in which it was involved and which cannot be erased from the memory of every person, regardless of his nationality, state, age, gender, etc. Unfortunately, Ukrainians are forced to experience this terrible phenomenon even today, in the 21st century.

Galectin-8 induces mitochondrial fission and redistribution to the perinuclear zone associated with epithelial–mesenchymal transition in MDCK cells

Galectin-8 induces mitochondrial fission and redistribution to the perinuclear zone associated with epithelial–mesenchymal transition in MDCK cells

Microscopic focus on ependymal cells of the spinal cord of the one-humped camel (Camelus dromedarius): Histological, immunohistochemical, and transmission microscopic study.

The current study was designed to give a complete microscopic description of the ependymal cells of the one-humped camel (Camelus dromedarius) using histological, immunohistochemical, and transmission microscopic descriptions of the ependymal cells of the fresh 35 spinal cord samples immediately after their slaughtering. In our findings, the central canal of the spinal cord was lined by multilayered stratified cuboidal or columnar ependymal cells. The ependymal cells had an irregular striated border at their free surface. The ependymal cells do not exhibit a basement membrane. The simple oval nucleus was occupied a large part of the cell with spherical mitochondria. The apical surface of the ependymal cells possesses long cilia; each cilium was bounded by an evagination of the luminal plasma membrane. Some ependymal cells had minute finger-like projections on their luminal plasma membrane. In the perinuclear zone of ependymal cells, many cristiform mitochondria, free ribosomes, and Golgi complexes usually occur. Vacuoles with homogenous and clear fluid were observed. The lateral surface of the adjacent ependymal cells exhibits several tight junctions represented by zonulae occludens and adherens. There were many desmosomes between the neighboring ependymal cells. A perinuclear whorl of filaments fills the lateral part of these ependymal cells. The ependymal cells revealed a clear immunohistochemical reaction with proliferating cell nuclear antigen and nestin stain. There were no obvious differences between the different segments of the spinal cord. Our data concluded that the ependymal cells display clear differences in anatomy as well as ultrastructure that may reflect their distinct functional activity.

Species-specific differences of the avian oesophagus: Histological and Ultrastructural study.

The oesophagus is a muscular tube comprised of cervical and thoracic regions. Several studies have clarified the histological structure of the oesophagus. However, its histoarchitecture in relation to variable dietary habits of each species is still unclear. In the current study, 21 pigeons, cattle egrets and ducks, n=7, each was used. Macroscopically, the oesophagus of cattle egrets either the cervical or thoracic parts was the longest among the pigeons and ducks. Histologically, the oesophagus comprised of four distinct tunicae: mucosa, propria submucosa, musculosa and adventitia or serosa. A great structural variation in these layers among the three investigated species was recorded. In the cervical oesophagus of pigeons, the superficial squamous cells showed perinuclear halo zone, the propria submucosa was characteristically lacked any gland. Moreover, its musculosa was very thick. On the other hand, the intraepithelial glands were characteristically distributed along the whole length of the cattle egret's oesophagus. Interestingly, the cervical esophagus of the ducks showed submucosal associated lymphatic tissue; diffuse and nodular Ultrastructurally, the oesophageal glands showed secretory granules of variable electron densities, electron-lucent in the pigeons and ducks and electron-dense in the cattle egrets.

Diminished inhibitory efficacy of GABA on PVN neurons in AngII‐salt hypertension reflects a KCC2‐independent increase of intracellular Cl‐ concentration

The hypothalamic PVN receives glutamatergic excitation from sodium and AngII sensing regions of the forebrain along with dense GABAergic inhibition from the surrounding peri-nuclear zone (PNZ). Previously, we found that synaptic glutamate release in PVN enhances GABA inhibition, but whether forebrain inputs can do so in the setting of hypertension (HTN) is unknown. Studies indicate that a high salt diet alone does not raise mean arterial pressure (MAP), while AngII treatment raises MAP in proportion to the level of salt intake. This suggests that whereas excitatory inputs to PVN driven by high salt intake alone cannot overcome PVN GABA inhibition, AngII driven inputs can. The notion that GABA inhibition of PVN is strengthened forebrain inputs in AngII-salt HTN is contrary to a prior report that GABA inhibition in AngII-salt HTN is reduced. To reconcile these seemingly disparate findings, we compared mechanisms regulating PVN GABA inhibition in mice consuming a normal salt (0.4% NaCl) diet (NSD), a high salt (4% NaCl) diet (HSD) or a HSD with AngII (600 ng/kg/min, s.c.) infusion (AngII-salt HTN). Using gramicidin perforated patch recordings, we determined that the GABA equilibrium potential (EGABA) of PVN neurons was not different between NSD (-67.2 ± 6.3 mV, n=3 cells) and HSD (-53.7 ± 15.6 mV, n=4 cells) mice, but was significantly depolarized in mice with AngII-salt HTN (-32.9 ± 8.8 mV, n=6 cells)(P=0.0033). This indicates that in AngII-salt HTN the inhibitory effect of GABA on PVN neurons decreases as [Cl-]i increases. Normally, [Cl-]i is held low by activities of KCC2 and NKCC1, which promote neuronal Cl- efflux and influx, respectively. In brain slices, KCC2 inhibition (VU0463271, 10 µM) rapidly depolarized PVN EGABA, consistent with blunting of GABA inhibition. Accordingly, PVN inhibition of KCC2 (VU0463271, 100 pmol, 25 nL) in vivo increased renal SNA, suggesting GABA was switched from an inhibitory to an excitatory transmitter. Consistent with this, PVN GABA-AR activation (muscimol, 50 pmol, 25 nL) during KCC2 inhibition increased renal SNA. Thus, KCC2 appears critical for maintaining low [Cl-]i and a hyperpolarized EGABA in PVN. In light of this, it would seem likely that elevated PVN [Cl-]i in AngII-salt HTN reflects a relative loss of KCC2 Cl- efflux. To investigate this, western blot analysis of PVN punches was performed and revealed that expression of KCC2 and NKCC1 was increased and decreased, respectively, both in mice consuming a HSD alone and mice with AngII-salt HTN. These changes were unexpected as they are predicted to drive [Cl-]i low, not high. Therefore, expression changes appear aimed at buffering elevated [Cl-]i caused by an alternative mechanism, possibly excess Cl- influx through GABA-AR, reflecting increased, not decreased, GABA release in AngII-salt HTN. Similar expression changes in HSD mice suggests that high salt intake generates a permissive state of Cl- ionic plasticity that blunts PVN GABA inhibition and facilitates AngII-salt HTN.

Heterogeneity of nanomechanical properties of the human umbilical vein endothelial cell surface

Endothelial cells, due to heterogeneity in the cell structure, can potentially form an inhomogeneous on structural and mechanical properties of the inner layer of the capillaries. Using quantitative nanomechanical mapping mode of atomic force microscopy, the parameters of the structural, elastic, and adhesive properties of the cell surface for living and glutaraldehyde-fixed human umbilical vein endothelial cells were studied. A significant difference in the studied parameters for three cell surface zones (peripheral, perinuclear, and nuclear zones) was established. The perinuclear zone appeared to be the softest zone of the endothelial cell surface. The heterogeneity of the endothelial cell mechanical properties at the nanoscale level can be an important mechanism in regulating the endothelium functions in blood vessels.

ПАРАДИГМАТИЧНІ ЗВ’ЯЗКИ КОНЦЕПТУАЛЬНОЇ ОДИНИЦІ ПЕРЕТВОРЕННЯ

In the article the paradigmatic relations of the name of the TRANSFORMATION concept are defined and characterized on the basis of lexicographic works and text realizations. Synonymous with the conceptual unit of TRANSFORMATION lexemes objectify the phenomenon of transformation in the Ukrainian language, differentiating its expression by a set of conceptual features, such as: specificity / abstractness, spontaneity / purposefulness, procedurality and effectiveness, including various dimensions of transformations (in particular, ontological, social, individual). Antonymous relations that verbalize the phenomenon of transformation form groups of antonyms in the Ukrainian language, corresponding to three types of opposition: express the gradual qualitative opposite and the opposite of coordinated concepts, the opposite direction of action or feature, complementary opposite, i.e. the opposite of concepts that complement each other. Since the TRANSFORMATION concept has a certain number of conceptual features, the hierarchy can be as complete as possible on many features. According to the logical-conceptual criterion, the following types of ordering the conceptual features of the TRANSFORMATION concept are distinguished: 1) superordination (from a hierarchically higher notion) and subordination (from a hierarchically lower notion): transformation ↔ metamorphosis ↔ metempsychosis; 2) coordination (between notions that are simultaneously and directly attributed to the general superordinative notion) and diagonal relations (between opposite subordinate notions): transformation ↔ evolution / revolution; modification ↔ permutation / replacement / truncation / addition. The paradigmatic relations of the concept name, as well as the syntagmatic relations, form the perinuclear zone of the TRANSFORMATION concept field. Placement in this part of the field occurs in the direction of movement from the core to the periphery, which hierarchizes synonyms (ideographic, stylistic, contextual) to the word-name of the concept and antonyms.

Infl uence of coal-rock dust on the risk of developing morphological disorders of the cardiovascular system

Cardiovascular pathology prevails in the structure of somatic diseases of miners. As a complication of pneumoconiosis, the development of a chronic pulmonary heart in workers against the background of pulmonary hypertension and congestive heart failure is considered. Data from clinical studies also indicate remodeling of the blood vessel wall, the formation of atherosclerosis, endothelial dysfunction and others. There are practically no in-depth comprehensive studies on the contribution of industrial dust to the development and course of this pathology. To implement this task, the simulation of occupational diseases in animals helps to trace the dynamics of the pathological process from the moment of exposure to the body of the production factor.The aim of the study is to study the infl uence of coal-rock dust on the risk of developing morphological disorders of the cardiovascular system in an experiment.Experiments were conducted on white non-linear male rats that were exposed to inhaled coal-rock dust of the “gas-fat” brand. For histological study in animals aft er 1, 3, 6, 9 and 12 weeks of exposure to the damaging factor, fragments of the brain, lungs, heart, liver and kidneys were taken. The quantitative content of matrix metalloproteinases, their inhibitor and vascular endothelial growth factor in blood plasma was studied using enzyme immunoassay.Morphological changes in the heart muscle and blood vessels are a systemic response of the body to prolonged inhalation of coal-rock dust. Violations develop from the moment of exposure to the damaging factor, so they can be considered as one of the manifestations of pneumoconiosis, and not its late complication. In the early stages of the experiment (1-3 weeks), the development of diff use protein dystrophy of cardiomyocytes with pronounced thickening of some fi bers due to cell hypertrophy is noted in the heart muscle of experimental animals. Increasing the timing of seed leads to the progression of pathological changes, increased apoptotic activity of cells and the accumulation of lipofuscin granules in the perinuclear zone, as well as the formation of atrophic damage in muscle tissue. By the 12th week of the experiment, diff use cardiosclerosis and pronounced dystrophy of cardiomyocytes with fragmentation and myocytolysis of individual sections of muscle fi bers develop in the heart muscle. Long-term inhalation of coal-rock dust causes progressive development in the wall of blood vessels, regardless of their organ affi liation, of the same type of pathological changes in the form of endotheliosis, diff use hypertrophy of the smooth muscle component of the medial layer and perivascular fi brosis against the background of pronounced microcirculatory disorders.The obtained results indicate the need for pre-nosological diagnostics of cardiovascular diseases and their prevention among miners working in harmful working conditions.

Glutamate strengthening of GABAergic inhibition in the hypothalamic PVN: Dependence on EAAT3‐mediated Glu uptake into local GABA terminals

In unstressed animals, pre‐sympathetic neurons (PSNs) of the hypothalamic paraventricular nucleus (PVN) are held in a state of near discharge quiescence by the dominance of GABAergic inhibition. In brain slice studies we found that the amplitude of PSN GABAergic inhibitory postsynaptic currents (IPSCs) is increased by elevated ECF L‐glutamate (Glu). This Glu‐GABA strengthening (GGS) phenomenon, originally described in the hippocampus, can be elicited in PVN PSNs both by exposure to bath Glu (100 μM) or high K+ (+10 mM) for 10 min. PVN GGS is (1) mediated by a presynaptic mechanism that increases unitary quantal size (more GABA molecules/vesicle), (2) is unaffected by inhibition of glial GLT1‐mediated Glu uptake, and (3) is prevented by global inhibition of Glu uptake. Collectively, these findings indicate that PVN GGS depends on Glu uptake by the neuronal Glu transporter EAAT3. Importantly, bath Glu elicits a delayed GABA‐AR‐mediated suppression of PSN firing, indicating that the Glu‐induced increase of IPSC amplitude (i.e., GGS) is sufficient to regulate PSN discharge quiescence. Moreover, in vivo studies indicate that PVN Glu enhances the increase of renal SNA caused by local blockade of GABA‐AR, consistent with elevated Glu increasing functional GABAergic inhibitory tone in the PVN. Here we performed PVN immunohistochemistry and found EAAT3 localized diffusely within somatodendritic compartments and densely in puncta resembling synaptic terminals. EAAT3+ puncta co‐localized with GAD65 punctate staining, consistent with EAAT3 expression on local GABAergic synaptic terminals. To test dependence of GGS on EAAT3 uptake of Glu specifically by PVN GABA terminals, we used a CRISPR/Cas9 gene editing strategy in which spCas9 was expressed in a Cre‐dependent manner amongst vGAT+ (GABAergic) neurons. To knockdown (KD) EAAT3 specifically amongst GABAergic inputs to PVN, AAV1‐CAG‐EAAT3:sgRNA‐GFP was nanoinjected (400 nl) into the surrounding peri‐nuclear zone (PNZ). Western blot analysis of tissue punches (Cntrl: n=10, CRISPR EAAT3: n=12) revealed a nearly 40% loss of EAAT3 in PVN. Given that EAAT3 amongst non‐GABAergic (i.e., glutamatergic) PVN neurons were not targeted by our CRISPR/Cas9 strategy, loss of EAAT3 specifically amongst PVN‐projecting GABAergic PNZ neurons would likely have been much greater than ~40%. Crucially, strengthening of PNZ evoked GABAergic IPSCs by bath Glu was effectively abolished (before: −238 ± 10 pA, after: −226 ± 12 pA; n=3) by CRISPR EAAT3 KD amongst PNZ GABAergic neurons. Results indicate that EAAT3‐mediated Glu uptake into PVN GABA terminals actively defends and restores PSN quiescence following acute glutamatergic excitation.Support or Funding InformationNIH HL088052 (GMT)

GABAergic synaptic inhibition in the hypothalamic paraventricular nucleus (PVN) is strengthened by acute activation of glutamatergic inputs from the median preoptic nucleus

Pre‐sympathetic neurons (PSNs) in the hypothalamic PVN are normally maintained in a state of near discharge quiescence reflecting the resting dominance of GABAergic inhibition. Our studies indicate that synaptic inhibition is actively maintained in the PVN, in part, by EAAT3‐mediated uptake of L‐glutamate (Glu) into local GABAergic synaptic terminals arising from the surrounding peri‐nuclear zone (PNZ). Notably, this Glu‐GABA strengthening (GGS) mechanism relies on apparent increases both in GABA synthesis and packaging into synaptic vesicles. Studies have so far demonstrated that PVN GGS can be elicited either by exposure of brain slices to bath Glu (100 μM) or high K+ (+10 mM). Studies performed in vivo indicate that Glu nanoinjection into the PVN increases GABAergic inhibitory control of renal sympathetic nerve activity (SNA). To investigate whether endogenous synaptic Glu release can drive PVN GGS, glutamatergic EPSCs were evoked in PVN PSNs from the median preoptic nucleus (MnPO). To accomplish this, we created an angled horizontal slice preparation containing the MnPO, PVN and PNZ. Single pulse (0.5 ms, 0.5 to 1.0 mA) electrical stimulation of MnPO evoked amongst PVN PSNs (n=2) mostly unitary EPSCs (ampl.: −139 ± 5 pA, tau: 3.27 ± 0.01). In slices prepared from vGAT‐ChR2(H134R) expressing mice, GABAergic IPSCs were evoked by blue light stimulation (20 ms, 0.4 mW, 0.067 Hz) of PNZ GABA neurons before and after glutamatergic EPSCs were electrically evoked from MnPO (1 Hz, 10 min). The amplitude of photostimulated GABAergic IPSCs at baseline (−152 ± 47 pA) was increased (−220 ± 83 pA) within ~3 minutes following MnPO stimulation (n=2). PNZ eIPSC amplitude was not increased (before: −89.5 ± 9.7 pA, after: −86.5 ± 10.3, pA) when MnPO stimulation was performed in the presence of the pan‐specific Glu uptake inhibitor TBOA (100 μM). Findings indicate that excitatory inputs to PVN from the forebrain lamina terminalis can acutely strengthen GABAergic inhibition through an EAAT3 uptake‐dependent mechanism consistent with PVN GGS. Grading of PVN synaptic inhibition in pace with excitation could help prevent excitotoxic injury of PSNs during prolonged physiological challenges (e.g., water deprivation) and in chronic diseases (e.g., neurogenic hypertension) accompanied by persistently elevated SNA.Support or Funding InformationNIH HL088052 (GMT)

Effects of water deprivation on inhibitory GABAergic synaptic transmission in the hypothalamic paraventricular nucleus (PVN)

Presympathetic neurons (PSNs) of the hypothalamic PVN mostly lack basal firing in unstressed animals due to the dominance of GABAergic inhibition, which our findings indicate is actively promoted and defended by the coupling of synaptic glutamate (Glu) release to heightened synthesis and packaging of GABA into synaptic vesicles. Our findings indicate that the latter process, referred to as Glu‐GABA strengthening (GGS), depends on EAAT3‐mediated uptake of Glu into PVN GABA terminals. To investigate the impact of PVN GGS on PSN responses to short‐term exposure to a physiological challenge that increases glutamatergic excitation of the PVN, male C57Bl/6 mice were either euhydrated (EUH) or water deprived (WD) for 48 h. Western blot analysis revealed significantly increased (P<0.05) PVN EAAT3 in WD (132 ± 1.2%) compared to EUH (100 ± 9.5 %) mice (n=18/group). Together with our previous report of reduced PVN expression of the glial Glu transporter GLT‐1 (EAAT2) in WD rats, the present findings suggest that Glu clearance in the PVN is shift during dehydration to favor uptake by neuronal EAAT3. If true, this could indicate that PVN GGS becomes enhanced during dehydration. To explore this further, we determined in brain slices from WD mice (n=4) that the amplitude of evoked IPSCs at baseline (−397 ± 102 pA) was unaffected by bath exposure to Glu (100 μM, 10 min) (−352 ± 89 pA), indicating either that dehydration disrupts PVN GGS or enhances it nearly to its maximum. To distinguish between these possibilities, we generated a cumulative eIPSC amplitude curve from 1000 stimuli delivered at a frequency of 5 Hz to the GABAergic peri‐nuclear zone (PNZ) that surrounds the PVN. The slope (‐pA/stimulus) of this relation in WD mice early during the stimulus train (stimuli 1–100) was similar to that of mice lacking GGS due to EAAT3 knockdown (KD) in PNZ GABA neurons (WD: 186 ± 0.9, EAAT3 KD: 180 ± 0.9). By contrast, the late phase slope (stimuli 901–1000) was 3 time higher for PSNs from WD mice than from the PNZ EAAT3 KD group (WD: 52 ± 0.2, EAAT3 KD: 17 ± 0.1). The latter indicates that eIPSCs in the WD group are resistant to rundown/depression and suggests that PVN GGS is likely near maximally enhanced, not disrupted, during dehydration. We posit that enhanced PVN GGS may be critical for preserving synaptic homeostasis and preventing excessive PSN activation and sympathoexcitation during short‐term dehydration stress.Support or Funding InformationNIH HL088052 (GMT)

Kisspeptin Regulation of Oxytocin Neuron Activity in Late Pregnancy

Oxytocin is secreted from the posterior pituitary gland by oxytocin neurons to trigger uterine contractions during birth. We have recently shown that the oxytocin neuron firing rate increases in response to intracerebroventricular (ICV) kisspeptin only in late‐pregnant rats. Furthermore, the kisspeptin projection from the periventricular nucleus of the hypothalamus (PeN) to the perinuclear zone (PNZ) surrounding the supraoptic nucleus (SON) increases in late pregnancy. This study aimed to determine the site and mechanism of central kisspeptin activation of oxytocin neuron activity in late pregnant rats. First, in vivo extracellular single unit recordings were made from SON neurons in urethane‐anaesthetised non‐pregnant and late‐pregnant rats during microdialysis application of kisspeptin (100 μM in the dialysate) into the SON. Intra‐SON kisspeptin consistently increased the firing rate of oxytocin neurons in late‐pregnant rats but not in non‐pregnant rats. Two‐way repeated measures (RM) ANOVA revealed a significant interaction between REPRODUCTIVE STATUS and TIME (F(6,88) = 10.31, p < 0.001); post hoc all pairwise Holm‐Sidak tests revealed a significant increase in firing rate in late‐pregnant rats at 30 (p < 0.05), 40, 50 and 60 minutes (all p < 0.001 versus pre‐kisspeptin), and a significantly higher firing rate than in time‐matched recordings from non‐pregnant rats at 50 and 60 minutes (p < 0.05).Then, to examine whether excitatory effects of central kisspeptin emerges directly within the SON and paraventricular nucleus (PVN) containing oxytocin neurons, we performed single label diaminobenzidine (DAB) immunostaining against phosphorylated extracellular regulated kinase (pERK), a second messenger activated by kisspeptin. ICV cannulae were implanted on day 13 or 14‐pregnant and non‐pregnant rats and one week later, day 21 pregnant and non‐pregnant rats were perfused 15 min after ICV administration of kisspeptin (2 μg/2 μl) or vehicle under pentobarbital anaesthesia. ICV kispeptin injection did not alter pERK expression in the SON or PVN. Two‐way RM ANOVA revealed a significant effect of REPRODUCTIVE STATUS (F(1,28) = 4.78, p = 0.03) but no significant effect of TREATMENT (kisspeptin/vehicle) (F(1,28) = 0.04, p = 0.82) and no REPRODUCTIVE STATUS X TREATMENT interaction (F(1,28) = 0.81, p = 0.37 ) in the SON. There was no significant effect of REPRODUCTIVE STATUS (F(1,27) = 0.93, p = 0.34, two‐way RM ANOVA) or TREATMENT (F(1,27) = 0.93, p = 0.34) and no REPRODUCTIVE STATUS X TREATMENT interaction (F(1,27) = 0.29, p = 0.59) in the PVN. Similarly, there was no significant effect of REPRODUCTIVE STATUS (F(1,27) = 2.28, p = 0.14, two‐way RM ANOVA) or TREATMENT (F(1,27) = 0.28, p = 0.59) and no REPRODUCTIVE STATUS X TREATMENT interaction (F(1,27) = 0.07, p = 0.78) in the magnocellular division of PVN. Hence, it appears that kisspeptin excites oxytocin neurons at the end of pregnancy by a presynaptic action at local nerve terminals to stimulate peripheral oxytocin release and so might contribute to oxytocin neuron activation for parturition.