Immunocytochemical Labeling (immunocytochemical + labeling)

Distribution by Scientific Domains

Selected Abstracts

Adenosine inhibits paraventricular pre-sympathetic neurons through ATP-dependent potassium channels

De-Pei Li
J. Neurochem. (2010) 113, 530,542. Abstract Adenosine produces cardiovascular depressor effects in various brain regions. However, the cellular mechanisms underlying these effects remain unclear. The pre-sympathetic neurons in the hypothalamic paraventricular nucleus (PVN) play an important role in regulating arterial blood pressure and sympathetic outflow through projections to the spinal cord and brainstem. In this study, we performed whole-cell patch-clamp recordings on retrogradely labeled PVN neurons projecting to the intermediolateral cell column of the spinal cord in rats. Adenosine (10,100 ,M) decreased the firing activity in a concentration-dependent manner, with a marked hyperpolarization in 12 of 26 neurons tested. Blockade of A1 receptors with the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine or intracellular dialysis of guanosine 5,- O -(2-thodiphosphate) eliminated the inhibitory effect of adenosine on labeled PVN neurons. Immunocytochemical labeling revealed that A1 receptors were expressed on spinally projecting PVN neurons. Also, blocking ATP-dependent K+ (KATP) channels with 100 ,M glibenclamide or 200 ,M tolbutamide, but not the G protein-coupled inwardly rectifying K+ channels blocker tertiapin-Q, abolished the inhibitory effect of adenosine on the firing activity of PVN neurons. Furthermore, glibenclamide or tolbutamide significantly decreased the adenosine-induced outward currents in labeled neurons. The reversal potential of adenosine-induced currents was close to the K+ equilibrium potential. In addition, adenosine decreased the frequency of both spontaneous and miniature glutamatergic excitatory post-synaptic currents and GABAergic inhibitory post-synaptic currents in labeled neurons, and these effects were also blocked by 8-cyclopentyl-1,3-dipropylxanthine. Collectively, our findings suggest that adenosine inhibits the excitability of PVN pre-sympathetic neurons through A1 receptor-mediated opening of KATP channels. [source]

The glutamatergic nature of TRPV1-expressing neurons in the spinal dorsal horn

Hong-Yi Zhou
Abstract The transient receptor potential vanilloid receptor 1 (TRPV1) is expressed on primary afferent terminals and spinal dorsal horn neurons. However, the neurochemical phenotypes and functions of TRPV1-expressing post-synaptic neurons in the spinal cord are not clear. In this study, we tested the hypothesis that TRPV1-expressing dorsal horn neurons are glutamatergic. Immunocytochemical labeling revealed that TRPV1 and vesicular glutamate transporter-2 were colocalized in dorsal horn neurons and their terminals in the rat spinal cord. Resiniferatoxin (RTX) treatment or dorsal rhizotomy ablated TRPV1-expressing primary afferents but did not affect TRPV1- and vesicular glutamate transporter-2-expressing dorsal horn neurons. Capsaicin significantly increased the frequency of glutamatergic spontaneous excitatory post-synaptic currents and miniature excitatory post-synaptic currents in almost all the lamina II neurons tested in control rats. In RTX-treated or dorsal rhizotomized rats, capsaicin still increased the frequency of spontaneous excitatory post-synaptic currents and miniature excitatory post-synaptic currents in the majority of neurons examined, and this effect was abolished by a TRPV1 blocker or by non-NMDA receptor antagonist. In RTX-treated or in dorsal rhizotomized rats, capsaicin also produced an inward current in a subpopulation of lamina II neurons. However, capsaicin had no effect on GABAergic and glycinergic spontaneous inhibitory post-synaptic currents of lamina II neurons in RTX-treated or dorsal rhizotomized rats. Collectively, our study provides new histological and functional evidence that TRPV1-expressing dorsal horn neurons in the spinal cord are glutamatergic and that they mediate excitatory synaptic transmission. This finding is important to our understanding of the circuitry and phenotypes of intrinsic dorsal horn neurons in the spinal cord. [source]

5-Ethynyl-2,-deoxyuridine labeling detects proliferating cells in the regenerating avian cochlea,

Christina L. Kaiser PhD
Abstract Objectives/Hypothesis: The avian cochlea regenerates hair cells following aminoglycoside treatment through supporting cell proliferation. Immunocytochemical labeling of 5-bromo-2,-deoxyuridine (BrdU), a thymidine analog, is a popular nonradioactive marker for identifying cells in the DNA synthesis (S phase) of the cell cycle. However, it requires harsh treatments to denature double-stranded DNA for the antibody to bind BrdU. We explored a new method using 5-ethynyl-2,-deoxyuridine (EdU) as a thymidine analog and a nonantibody azide/alkyne reaction between EdU and the fluorescent probe. We propose that EdU is as effective as BrdU, but without the requirement for harsh denaturation or the use of antibodies for detection. Study Design: Two-week-old chicks received a single gentamicin injection followed by a single EdU injection 72 hours later. Cochleae were extracted 4,8 hours later, fixed, and processed for fluorescent detection of EdU. Methods: Cochleae were processed for detection of incorporated EdU using the Click-iT Imaging Kit (Invitrogen/Molecular Probes, Carlsbad, CA) and colabeled with Sox2, myosin VI, or myosin VIIa antibodies. Whole-mount cochlear preparations were examined with confocal microscopy. Results: Supporting cells incorporated EdU into their newly synthesized DNA during the 4,8 hours following the EdU injection and were readily detected with little background signal. The intensity and quantity of cells labeled were similar to or better than that seen for BrdU. Conclusions: The EdU method is as effective as BrdU, without requiring harsh denaturation or secondary antibodies to identify proliferating cells. Thus, the nonantibody EdU system allows more flexibility by enabling colabeling with multiple antibodies to other cellular proteins involved in regeneration. Laryngoscope, 2009 [source]

Asator, a tau-tubulin kinase homolog in Drosophila localizes to the mitotic spindle

Hongying Qi
Abstract We have used a yeast two-hybrid interaction assay to identify Asator, a tau-tubulin kinase homolog in Drosophila that interacts directly with the spindle matrix protein Megator. Using immunocytochemical labeling by an Asator-specific mAb as well as by transgenic expression of a GFP-labeled Asator construct, we show that Asator is localized to the cytoplasm during interphase but redistributes to the spindle region during mitosis. Determination of transcript levels using qRT-PCR suggested that Asator is expressed throughout development but at relatively low levels. By P-element excision, we generated a null or strong hypomorphic Asatorexc allele that resulted in complete adult lethality when homozygous, indicating that Asator is an essential gene. That the observed lethality was caused by impaired Asator function was further supported by the partial restoration of viability by transgenic expression of Asator-GFP in the Asatorexc homozygous mutant background. The finding that Asator localizes to the spindle region during mitosis and directly can interact with Megator suggests that its kinase activity may be involved in regulating microtubule dynamics and microtubule spindle function. Developmental Dynamics 238:3248,3256, 2009. 2009 Wiley-Liss, Inc. [source]

Alterations and reversibility in the chromatin, cytoskeleton and development of pig oocytes treated with roscovitine

Jyh-Cherng Ju
Abstract Germinal vesicle (GV) breakdown in mammalian oocytes is regulated by the activation of maturation promoting factor (MPF). We investigated a specific cdc2 kinase inhibitor, roscovitine, to maintain pig oocytes in the GV stage. Cumulus-oocyte complexes (COCs) were aspirated from slaughterhouse ovaries and cultured for 44 hr in NCSU#23 medium containing different levels of roscovitine (0, 10, 20, 30, 40, 50 ,M in Experiment 1 and 0, 40, 60, 80, 100, 120 ,M in Experiment 2). The COCs were cultured for another 44 hr after removal of the chemical. Twenty oocytes in each group were fixed at 44 hr for immunocytochemical labeling of the cytoskeleton and the rest (,20/group) were fixed at the end of 88 hr after culture. Results showed that the inhibition of the oocyte in the GV stage was not effective when 10,50 ,M (Experiment 1) of roscovitine were used (19,34%). When oocytes were released from the inhibitor, similar proportions (70,83%) of oocytes were observed in the MII or advanced stages among treatments. However, when higher concentrations of roscovitine were used (Experiment 2), significantly greater inhibitory effect was observed at the levels of 80,120 ,M with 83,91% oocytes being blocked in the GV stage when compared to the control (9%) and the 40,60 ,M (27,43%) groups (P,<,0.05). Although 15,21% of the oocytes showed abnormal MII morphology with aberrant meiotic spindles and/or formation of cytoplasmic microtubules, a substantial number of oocytes resumed meiosis and reached MII stage at 44 hr after removal of this chemical. In Experiment 3, different concentrations of roscovitine (0, 20, 40, and 80 ,M) were tested to examine the length of intervals (0, 11, 22, 33, and 44 hr) for an effective inhibition. Results showed that the inhibitory effect was significantly more prominent at 22 hr than that at 33 and 44 hr after roscovitine treatment in all treatment groups (P,<,0.05). This study demonstrated that roscovitine-treated oocytes resumed meiosis after removal of the inhibitor. This could provide flexibility for studying porcine oocyte development and embryo cloning and may have application in other species. Mol. Reprod. Dev. 64: 482,491, 2003. 2003 Wiley-Liss, Inc. [source]

ORIGINAL RESEARCH,BASIC SCIENCE: Neuroanatomical Evidence for a Role of Central Melanocortin-4 Receptors and Oxytocin in the Efferent Control of the Rodent Clitoris and Vagina

Helene Gelez PhD
ABSTRACT Introduction., The clitoris and the vagina are the main peripheral anatomical structures involved in physiological changes related to sexual arousal and orgasm. Their efferent control and, more particularly, the neurochemical phenotype of these descending neuronal pathways remain largely uncharacterized. Aim., To examine if brain neurons involved in the efferent control of the clitoris and the vagina possess melanocortin-4 receptor (MC4-R) and/or contain oxytocin (OT). Methods., Neurons involved in the efferent control of the vagina and clitoris were identified following visualization of pseudorabies virus (PRV) retrograde tracing. PRV was injected into the vagina and clitoris in adult rats in estrous. On the fifth day postinjection, animals were humanely sacrificed, and brains were removed and sectioned, and processed for PRV visualization. The neurochemical phenotype of PRV-positive neurons was identified using double or triple immunocytochemical labeling against PRV, MC4-R, and OT. Double and triple labeling were quantified using confocal laser scanning microscopy. Main Outcome Measure., Neuroanatomical brain distribution, number and percentage of double-labeled PRV/MC4-R and PRV-/OT-positive neurons, and triple PRV-/MC4-R-/OT-labeled neurons. Results., The majority of PRV immunopositive neurons which also expressed immunoreactivity for MC4-R were located in the paraventricular and arcuate nuclei of the hypothalamus. The majority of PRV positive neurons which were immunoreactive (IR) for OT were located in the paraventricular nucleus (PVN), medial preoptic area (MPOA), and lateral hypothalamus. PRV positive neurons were more likely to be IR for MC4-R than for OT. Scattered triple-labeled PRV/MC4-R/OT neurons were detected in the MPOA and the PVN. Conclusion., These data strongly suggest that MC4-R and, to a less extent, OT are involved in the efferent neuronal control of the clitoris and vagina, and consequently facilitate our understanding of how the melanocortinergic pathway regulates female sexual function. Gelez H, Poirier S, Facchinetti P, Allers KA, Wayman C, Alexandre L, and Giuliano F. Neuroanatomical evidence for a role of central melanocortin-4 receptors and oxytocin in the efferent control of the rodent clitoris and vagina. J Sex Med 2010;7:2056,2067. [source]