Orexin 2 Receptor (OX2R) Protein Distribution Measured by Autoradiography Using Radiolabeled OX2R-Selective Antagonist EMPA in Rodent Brain and Peripheral Tissues

2021 ◽  
Author(s):  
Kayo Mitsukawa ◽  
Haruhide Kimura
Keyword(s):  
Brain Slices ◽  
Brain Regions ◽  
Retrosplenial Cortex ◽  
Biological Functions ◽  
Protein Distribution ◽  
Peripheral Tissues ◽  
Physiological Functions ◽  
Wide Range ◽  
The Central Nervous System

Abstract Orexin, a neuropeptide, performs various physiological functions, including the regulation of emotion, feeding, metabolism, respiration, and sleep/wakefulness, by activating the orexin 1 receptor and orexin 2 receptor (OX2R). Owing to the pivotal role of OX2R in wakefulness and other biological functions, OX2R agonists are being developed. A detailed understanding of OX2R protein distribution is essential for determining the mechanisms of action of OX2R agonists; however, this has been hindered by the lack of selective antibodies. In this study, we first confirmed the OX2R-selective binding of [3H]-EMPA in in vitro autoradiography studies, using brain slices from OX2R knockout mice and their wild-type littermates. Subsequently, OX2R protein distribution in rats was comprehensively assessed in 51 brain regions and 10 peripheral tissues using in vitro autoradiography with [3H]-EMPA. The widespread distribution of OX2R protein, including that in previously unrecognized regions of the retrosplenial cortex and suprachiasmatic nucleus of the hypothalamus, was identified. In contrast, negligible/very low OX2R protein expression was observed in peripheral tissues, suggesting that orexin exerts OX2R-dependent physiological functions primarily through activation of the central nervous system. These data would be useful for understanding the wide range of biological functions of OX2R and the application of OX2R agonists in various disorders.

Physiology, Pharmacology, and Topography of Cholinergic Neocortical Oscillations In Vitro

1997 ◽  
Vol 77 (5) ◽  
pp. 2427-2445 ◽  
Author(s):  
Heath S. Lukatch ◽  
M. Bruce Maciver
Keyword(s):  
Current Source ◽  
Brain Slices ◽  
Receptor Activation ◽  
Brain Regions ◽  
Oscillatory Activity ◽  
Cortical Layers ◽  
Eeg Activity ◽  
Layer 2

Lukatch, Heath S. and M. Bruce MacIver. Physiology, pharmacology, and topography of cholinergic neocortical oscillations in vitro. J. Neurophysiol. 77: 2427–2445, 1997. Rat neocortical brain slices generated rhythmic extracellular field [microelectroencephalogram (micro-EEG)] oscillations at theta frequencies (3–12 Hz) when exposed to pharmacological conditions that mimicked endogenous ascending cholinergic and GABAergic inputs. Use of the specific receptor agonist and antagonist carbachol and bicuculline revealed that simultaneous muscarinic receptor activation and γ-aminobutyric acid-A (GABAA)-mediated disinhibition werenecessary to elicit neocortical oscillations. Rhythmic activity was independent of GABAB receptor activation, but required intact glutamatergic transmission, evidenced by blockade or disruption of oscillations by 6-cyano-7-nitroquinoxaline-2,3-dione and (±)-2-amino-5-phosphonovaleric acid, respectively. Multisite mapping studies showed that oscillations were localized to areas 29d and 18b (Oc2MM) and parts of areas 18a and 17. Peak oscillation amplitudes occurred in layer 2/3, and phase reversals were observed in layers 1 and 5. Current source density analysis revealed large-amplitude current sinks and sources in layers 2/3 and 5, respectively. An initial shift in peak inward current density from layer 1 to layer 2/3 indicated that two processes underlie an initial depolarization followed by oscillatory activity. Laminar transections localized oscillation-generating circuitry to superficial cortical layers and sharp-spike-generating circuitry to deep cortical layers. Whole cell recordings identified three distinct cell types based on response properties during rhythmic micro-EEG activity: oscillation-on (theta-on) and -off (theta-off) neurons, and transiently depolarizing glial cells. Theta-on neurons displayed membrane potential oscillations that increased in amplitude with hyperpolarization (from −30 to −90 mV). This, taken together with a glutamate antagonist-induced depression of rhythmic micro-EEG activity, indicated that cholinergically driven neocortical oscillations require excitatory synaptic transmission. We conclude that under the appropriate pharmacological conditions, neocortical brain slices were capable of producing localized theta frequency oscillations. Experiments examining oscillation physiology, pharmacology, and topography demonstrated that neocortical brain slice oscillations share many similarities with the in vivo and in vitro theta EEG activity recorded in other brain regions.

scholarly journals Distribution of the prion protein in sheep terminally affected with BSE following experimental oral transmission

2001 ◽  
Vol 82 (10) ◽  
pp. 2319-2326 ◽  
Author(s):  
J. D. Foster ◽  
D. W. Parnham ◽  
N. Hunter ◽  
M. Bruce
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Experimental Infection ◽  
Oral Route ◽  
Similar Distribution ◽  
Peripheral Tissues ◽  
Oral Transmission ◽  
Wide Range ◽  
Direct Contrast ◽  
The Central Nervous System

This study has examined the distribution of PrPSc in sheep by immunocytochemistry of tissues recovered from terminally affected animals following their experimental infection by the oral route with BSE. Despite a wide range of incubation period lengths, affected sheep showed a similar distribution of high levels of PrPSc throughout the central nervous system. PrPSc was also found in the lymphoid system, including parts of the digestive tract, and some components of the peripheral nervous system. These abundant PrPSc deposits in sheep in regions outside the central nervous system are in direct contrast with cattle infected with BSE, which show barely detectable levels of PrPSc in peripheral tissues. A number of genetically susceptible, challenged animals appear to have survived.

scholarly journals Imaging Subthreshold Voltage Oscillation With Cellular Resolution in the Inferior Olive in vitro

2020 ◽  
Vol 14 ◽  
Author(s):  
Kevin Dorgans ◽  
Bernd Kuhn ◽  
Marylka Yoe Uusisaari
Keyword(s):  
Inferior Olive ◽  
Brain Slices ◽  
Brain Regions ◽  
Mammalian Brain ◽  
Wide Field ◽  
Voltage Imaging ◽  
Subthreshold Oscillations ◽  
Cellular Resolution

Voltage imaging with cellular resolution in mammalian brain slices is still a challenging task. Here, we describe and validate a method for delivery of the voltage-sensitive dye ANNINE-6plus (A6+) into tissue for voltage imaging that results in higher signal-to-noise ratio (SNR) than conventional bath application methods. The not fully dissolved dye was injected into the inferior olive (IO) 0, 1, or 7 days prior to acute slice preparation using stereotactic surgery. We find that the voltage imaging improves after an extended incubation period in vivo in terms of labeled volume, homogeneous neuropil labeling with saliently labeled somata, and SNR. Preparing acute slices 7 days after the dye injection, the SNR is high enough to allow single-trial recording of IO subthreshold oscillations using wide-field (network-level) as well as high-magnification (single-cell level) voltage imaging with a CMOS camera. This method is easily adaptable to other brain regions where genetically-encoded voltage sensors are prohibitively difficult to use and where an ultrafast, pure electrochromic sensor, like A6+, is required. Due to the long-lasting staining demonstrated here, the method can be combined, for example, with deep-brain imaging using implantable GRIN lenses.

scholarly journals NG2 glia are required for maintaining microglia homeostatic state

2019 ◽  
Author(s):  
Yingjun Liu ◽  
Adriano Aguzzi
Keyword(s):  
Brain Aging ◽  
Brain Slices ◽  
List Type ◽  
Growth Factor Signaling ◽  
Highly Efficient ◽  
Pdgf Signaling ◽  
The Central Nervous System ◽  
Homeostatic State

AbstractMicroglia play vital roles in the health and diseases of the central nervous system. Loss of microglia homeostatic state is a key feature of brain aging and neurodegeneration. However, the mechanisms underlying the maintenance of distinct microglia states are largely unclear. Here we show that NG2 glia, also known as oligodendrocyte precursor cells, are essential for maintaining the homeostatic microglia state. We developed a highly efficient and selective NG2 glia depletion method using small-molecule inhibitors of platelet-derived growth factor signaling in cultured brain slices. We found that loss of NG2 glia abolished the homeostatic microglia signature without affecting the disease-associated microglia profiles. Similar findings were also observed in vivo by genetically ablating NG2 glia in the adult mouse brain. These data suggest that NG2 glia exert a crucial influence onto microglia cellular states that are relevant to brain aging and neurodegenerative diseases. In addition, our results provide a powerful, convenient and selective tool for the investigation of NG2 glia function.Main pointsPostnatal NG2 glia maintenance obligatorily depends on continuous PDGF signaling.A highly efficient, selective and versatile NG2 glia depletion method is established.Loss of NG2 glia abolishes the homeostatic microglia signature both in vitro and in vivo.

scholarly journals Is the Exposome Involved in Brain Disorders through the Serotoninergic System?

Biomedicines ◽  
2021 ◽  
Vol 9 (10) ◽  
pp. 1351
Author(s):  
Denis Sarrouilhe ◽  
Norah Defamie ◽  
Marc Mesnil
Keyword(s):  
Serotoninergic System ◽  
Vasoactive Agent ◽  
Neurodevelopmental Toxicity ◽  
Wide Range ◽  
Potential Link ◽  
Common Etiology ◽  
The Central Nervous System ◽  
Health And Disease ◽  
Biogenic Monoamine

Serotonin (5-hydroxytryptamine, 5-HT) is a biogenic monoamine acting as a neurotransmitter in the central nervous system (CNS), local mediator in the gut, and vasoactive agent in the blood. It has been linked to a variety of CNS functions and is implicated in many CNS and psychiatric disorders. The high comorbidity between some neuropathies can be partially understood by the fact that these diseases share a common etiology involving the serotoninergic system. In addition to its well-known functions, serotonin has been shown to be a mitogenic factor for a wide range of normal and tumor cells, including glioma cells, in vitro. The developing CNS of fetus and newborn is particularly susceptible to the deleterious effects of neurotoxic substances in our environment, and perinatal exposure could result in the later development of diseases, a hypothesis known as the developmental origin of health and disease. Some of these substances affect the serotoninergic system and could therefore be the source of a silent pandemic of neurodevelopmental toxicity. This review presents the available data that are contributing to the appreciation of the effects of the exposome on the serotoninergic system and their potential link with brain pathologies (neurodevelopmental, neurodegenerative, neurobehavioral disorders, and glioblastoma).

FACTORS AFFECTING THE INCORPORATION OF RADIOACTIVE PHOSPHATE INTO THE PHOSPHOLIPIDS OF SLICES OF CAT BRAIN

1954 ◽  
Vol 32 (1) ◽  
pp. 50-59 ◽  
Author(s):  
K. P. Strickland
Keyword(s):  
Oxygen Consumption ◽  
Inorganic Phosphate ◽  
Brain Slices ◽  
Phospholipid Fraction ◽  
Metabolic Inhibitors ◽  
Factors Affecting ◽  
Wide Range ◽  
Cat Brain

Slices of cat brain respiring in a Krebs–Ringer bicarbonate medium were found to incorporate radioactive inorganic phosphate (P32) into the phospholipid fraction. The addition of glucose or mannose increased the incorporation of P32 into the phospholipids. Fructose caused a small increase, whereas galactose was without effect. Pyruvate and lactate increased the incorporation of P32 into the phospholipids. Succinate, L (+)-glutamate, D (−)-glutamate, α keto-glutarate, citrate, and L-malate failed to support the incorporation.Anaerobic conditions and homogenization of the tissue prevented the in vitro incorporation of P32 into the phospholipids of cat brain. A wide range of metabolic inhibitors (cyanide, azide, malononitrile, chloretone, nembutal, iodoacetate, and fluoride), in concentrations that inhibit the oxygen consumption of brain slices, inhibited the incorporation. The incorporation was also inhibited by 2,4-dinitrophenol in concentrations that do not decrease the oxygen consumption of brain slices.These findings indicated that the incorporation of P32 into the phospholipids of slices of cat brain is a metabolic phenomenon and is dependent upon the maintenance of an adequate phosphorylating mechanism within the slice.

Effects of intravenous infusions of vasopressin and angiotensin II on central and peripheral noradrenergic function in conscious rabbits

1987 ◽  
Vol 65 (5) ◽  
pp. 765-772 ◽  
Author(s):  
K. P. Patel ◽  
C. A. Whiteis ◽  
D. D. Lund ◽  
P. G. Schmid
Keyword(s):  
Angiotensin Ii ◽  
Low Dose ◽  
Cardiovascular Effects ◽  
Brain Regions ◽  
Locus Ceruleus ◽  
Baroreflex Control ◽  
Peripheral Tissues ◽  
Norepinephrine Turnover ◽  
The Central Nervous System ◽  
Noradrenergic Function

Vasopressin (AVP) and angiotensin II (AII) are proposed to exert part of their cardiovascular effects via different actions within the central nervous system. These peptides are also known to alter central noradrenergic function. In the present study we determined the effects of these peptides administered intravenously on norepinephrine (NE) turnover in discrete brain regions thought to be involved in the regulation of circulation, and simultaneously, in various peripheral tissues. An index of NE turnover was determined by measuring the decline in tissue NE concentration 75 min after administration of α-methyl tyrosine (240 mg∙kg−1∙min−1, i.p.). During NE synthesis blockade, five separate groups of rabbits were infused intravenously (1 h) with either saline, AVP (4 and 16 mU∙kg−1∙min−1), AII (0.1 μg∙kg−1∙min−1), or phenylephrine (PE) (5 μg∙kg−1∙min−1). The low dose of AVP produced an increased index of NE turnover in the median preoptic area and the paraventricular nucleus, and concomitantly, a decreased index of NE turnover in kidney and skeletal muscle. In contrast, AII produced an increased index of NE turnover in the locus ceruleus and the intestine. Neither the infusion of vehicle nor the infusion of phenylephrine, which increased arterial pressure comparable to AVP and AII, produced detectable changes in indices of central and peripheral norepinephrine turnover. A higher dose of AVP produced a different pattern of changes in NE turnover than the low dose. These results demonstrate that intravenous infusion of the low dose of AVP produced changes in noradrenergic function in specific central areas known to be involved in autonomic outflow. Concomitant with the central changes, there were selective changes in peripheral noradrenergic function. AII produced changes in both central and peripheral noradrenergic function that differed from the effects of AVP. These desperate actions might account for the previously observed differences in effects of AVP and AII on baroreflex control of the circulation.

scholarly journals Reference-based annotation of single cell transcriptomes identifies a profibrotic macrophage niche after tissue injury

2018 ◽  
Author(s):  
Dvir Aran ◽  
Agnieszka P. Looney ◽  
Leqian Liu ◽  
Valerie Fong ◽  
Austin Hsu ◽  
...  
Keyword(s):  
Single Cell ◽  
Lung Fibrosis ◽  
Tissue Injury ◽  
Granular Cell ◽  
Cellular Heterogeneity ◽  
Trophic Effect ◽  
Peripheral Tissues ◽  
Wide Range

AbstractMyeloid cells localize to peripheral tissues in a wide range of pathologic contexts. However, appreciation of distinct myeloid subtypes has been limited by the signal averaging inherent to bulk sequencing approaches. Here we applied single-cell RNA sequencing (scRNA-seq) to map cellular heterogeneity in lung fibrosis induced by bleomycin injury in mice. We first developed a computational framework that enables unbiased, granular cell-type annotation of scRNA-seq. This approach identified a macrophage subpopulation that was specific to injured lung and notable for high expression of Cx3cr1+ and MHCII genes. We found that these macrophages, which bear a gene expression profile consistent with monocytic origin, progressively acquire alveolar macrophage identity and localize to sites of fibroblast accumulation. Probing their functional role, in vitro studies showed a trophic effect of these cells on fibroblast activation, and ablation of Cx3cr1-expressing cells suppressed fibrosis in vivo. We also found by gene set analysis and immunofluorescence that markers of these macrophages were upregulated in samples from patients with lung fibrosis compared with healthy controls. Taken together, our results uncover a specific pathologic subgroup of macrophages with markers that could enable their therapeutic targeting for fibrosis.

scholarly journals A fluorescent sensor for spatiotemporally resolved endocannabinoid dynamics in vitro and in vivo

2020 ◽  
Author(s):  
Ao Dong ◽  
Kaikai He ◽  
Barna Dudok ◽  
Jordan S Farrell ◽  
Wuqiang Guan ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Basolateral Amygdala ◽  
Fluorescent Sensor ◽  
Brain Slices ◽  
High Specificity ◽  
Cultured Neurons ◽  
Spatiotemporal Resolution ◽  
Wide Range

Endocannabinoids (eCBs) are retrograde neuromodulators that play an important role in a wide range of physiological processes; however, the release and in vivo dynamics of eCBs remain largely unknown, due in part to a lack of suitable probes capable of detecting eCBs with sufficient spatiotemporal resolution. Here, we developed a new eCB sensor called GRABeCB2.0. This genetically encoded sensor consists of the human CB1 cannabinoid receptor fused to circular-permutated EGFP, providing cell membrane trafficking, second-resolution kinetics, high specificity for eCBs, and a robust fluorescence response at physiological eCB concentrations. Using the GRABeCB2.0 sensor, we monitored evoked changes in eCB dynamics in both cultured neurons and acute brain slices. Interestingly, in cultured neurons we also observed spontaneous compartmental eCB transients that spanned a distance of approximately 11 μm, suggesting constrained, localized eCB signaling. Moreover, by expressing GRABeCB2.0 in the mouse brain, we readily observed foot shock-elicited and running-triggered eCB transients in the basolateral amygdala and hippocampus, respectively. Lastly, we used GRABeCB2.0 in a mouse seizure model and observed a spreading wave of eCB release that followed a Ca2+ wave through the hippocampus. Thus, GRABeCB2.0 is a robust new probe for measuring the dynamics of eCB release under both physiological and pathological conditions.

scholarly journals The ryanodine receptor/calcium channel genes are widely and differentially expressed in murine brain and peripheral tissues.

1995 ◽  
Vol 128 (5) ◽  
pp. 893-904 ◽  
Author(s):  
G Giannini ◽  
A Conti ◽  
S Mammarella ◽  
M Scrobogna ◽  
V Sorrentino
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Intracellular Calcium ◽  
Ryanodine Receptor ◽  
Calcium Release ◽  
Ryanodine Receptors ◽  
Calcium Release Channels ◽  
Peripheral Tissues ◽  
The Central Nervous System

Ryanodine receptors (RyRs) are intracellular calcium release channels that participate in controlling cytosolic calcium levels. At variance with the probably ubiquitous inositol 1,4,5-trisphosphate-operated calcium channels (1,4,5-trisphosphate receptors), RyRs have been mainly regarded as the calcium release channels controlling skeletal and cardiac muscle contraction. Increasing evidence has recently suggested that RyRs may be more widely expressed, but this has never been extensively examined. Therefore, we cloned three cDNAs corresponding to murine RyR homologues to carry a comprehensive analysis of their expression in murine tissues. Here, we report that the three genes are expressed in almost all tissues analyzed, where tissue-specific patterns of expression were observed. In the uterus and vas deferens, expression of RyR3 was localized to the smooth muscle component of these organs. In the testis, expression of RyR1 and RyR3 was detected in germ cells. RyR mRNAs were also detected in in vitro-cultured cell lines. RyR1, RyR2, and RyR3 mRNA were detected in the cerebrum and in the cerebellum. In situ analysis revealed a cell type-specific pattern of expression in the different regions of the central nervous system. The differential expression of the three ryanodine receptor genes in the central nervous system was also confirmed using specific antibodies against the respective proteins. This widespread pattern of expression suggests that RyRs may participate in the regulation of intracellular calcium homeostasis in a range of cells wider than previously recognized.

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