Dual Immunocytochemistry Research Articles

Transmembrane and coiled-coil 2 associates with Alzheimer's disease pathology in the human brain.

Transmembrane and coiled-coil 2 (TMCC2) is a human orthologue of the Drosophila gene dementin, mutant alleles of which cause neurodegeneration with features of Alzheimer's disease (AD). TMCC2 and Dementin further have an evolutionarily conserved interaction with the amyloid protein precursor (APP), a protein central to AD pathogenesis. To investigate if human TMCC2 might also participate in mechanisms of neurodegeneration, we examined TMCC2 expression in late onset AD human brain and age-matched controls, familial AD cases bearing a mutation in APP Val717, and Down syndrome AD. Consistent with previous observations of complex formation between TMCC2 and APP in the rat brain, the dual immunocytochemistry of control human temporal cortex showed highly similar distributions of TMCC2 and APP. In late onset AD cases stratified by APOE genotype, TMCC2 immunoreactivity was associated with dense core senile plaques and adjacent neuronal dystrophies, but not with Aβ surrounding the core, diffuse Aβ plaques or tauopathy. In Down syndrome AD, we observed in addition TMCC2-immunoreactive and methoxy-X04-positive pathological features that were morphologically distinct from those seen in the late onset and familial AD cases, suggesting enhanced pathological alteration of TMCC2 in Down syndrome AD. At the protein level, western blots of human brain extracts revealed that human brain-derived TMCC2 exists as at least three isoforms, the relative abundance of which varied between the temporal gyrus and cerebellum and was influenced by APOE and/or dementia status. Our findings thus implicate human TMCC2 in AD via its interactions with APP, its association with dense core plaques, as well as its alteration in Down syndrome AD.

Calcium released by osteoclastic resorption stimulates autocrine/paracrine activities in local osteogenic cells to promote coupled bone formation.

A major cause of osteoporosis is impaired coupled bone formation. Mechanistically, both osteoclast-derived and bone-derived growth factors have been previously implicated. Here, we hypothesize that the release of bone calcium during osteoclastic bone resorption is essential for coupled bone formation. Osteoclastic resorption increases interstitial fluid calcium locally from the normal 1.8 mM up to 5 mM. MC3T3-E1 osteoprogenitor cells, cultured in a 3.6 mM calcium medium, demonstrated that calcium signaling stimulated osteogenic cell proliferation, differentiation, and migration. Calcium channel knockdown studies implicated calcium channels, Cav1.2, store-operated calcium entry (SOCE), and calcium-sensing receptor (CaSR) in regulating bone cell anabolic activities. MC3T3-E1 cells cultured in a 3.6 mM calcium medium expressed increased gene expression of Wnt signaling and growth factors platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and bone morphogenic protein-2 (BMP 2). Our coupling model of bone formation, the receptor activator of nuclear factor-κΒ ligand (RANKL)-treated mouse calvaria, confirmed the role of calcium signaling in coupled bone formation by exhibiting increased gene expression for osterix and osteocalcin. Critically, dual immunocytochemistry showed that RANKL treatment increased osterix-positive cells and increased fluorescence intensity of Cav1.2 and CaSR protein expression per osterix-positive cell. The above data established that calcium released by osteoclasts contributed to the regulation of coupled bone formation. CRISPR/Cas-9 knockout of Cav1.2 in osteoprogenitor cells cultured in basal calcium medium caused a >80% decrease in the expression of downstream osteogenic genes, emphasizing the large magnitude of the effect of calcium signaling. Thus, calcium signaling is a major regulator of coupled bone formation.

P16INK4a/ki67 Immunocytochemistry in Improving the Predictive Value for High Grade Cervical Intraepithelial (≥CIN2) Neoplasia in Pap Smear.

Introduction:Cervical cytology has limited sensitivity to detect cervical pre-cancerous lesions. High-risk human papillomavirus (hr-HPV) DNA testing has high sensitivity but its specificity is limited. This study was done to assess the utility of p16INK4a/ki-67 dual stained cytology in improving the predictive value for high-grade cervical (CIN2+) lesions.Aim/Objective:To assess the significance of P16/Ki-67 immunocytochemistry in improving the predictive value for high-grade cervical intraepithelial (≥CIN 2+) lesions on Pap smear.Material and Methods:This was a prospective diagnostic study that included 93 patients with ASC-US/LSIL/ASC-H and HSIL on thin prep cervical smears and who also underwent hr-HPV DNA test and colposcopy-guided biopsy. Biopsy was the gold standard against which the performance of P16INK4a/Ki-67 and hr-HPV results were compared.Results:In women of all ages, sensitivity of (96.8%) hr-HPV test and p16/Ki-67 dual immunocytochemistry (≥1 positive cell) were similar and negative predictive value (NPV) was (97.1% vs. 97.9%) but the latter test showed better specificity (69.4% vs. 53.2%) and positive predictive value (PPV, 61.2% vs. 50.8%) for ≥CIN 2 lesions. A higher cut off of at least 10 positive cells gives a higher specificity and PPV, with slightly decreased sensitivity and NPV.Conclusion:Because high-risk HPV test has a high sensitivity and NPV, whereas P16/Ki-67 dual immunocytochemistry (≥10 positive cells) has a high specificity and PPV, the latter can be recommended as an ancillary test in hr-HPV-positive women to reduce the number of women going for colposcopy and biopsies.

Diagnostic algorithm for determining primary tumor sites using peritoneal fluid

This study was conducted to develop a novel algorithm for determining the origin of tumors by combining analysis of cluster patterns with immunocytochemistry (ICC) for markers in cells from fine-needle aspirates of ascites. We used LBC, based on SurePathTM (BD Diagnostics) technology, to screen 96 peritoneal fluid samples from patients with known malignancies and from 10 control patients with cirrhosis. Following dual ICC staining for cytokeratin 7 (CK7) and paired box gene 8 (PAX8), we developed an algorithm using immunoreactivity and three-dimensional (3D) cluster patterns to correlate staining and 3D cluster patterns with common primary origins that included stomach, ovarian, pancreatobiliary tract, colon, lung, and breast cancers. With the application of an automatic digitalized image analyzer, competence performance was analyzed using receiver operating characteristics (ROC) curve analysis. CK7 and PAX8 staining and 3D cluster patterns were used to differentiate primary origins. Samples from patients with stomach cancer were no 3D cluster /CK7+/PAX8- with area under the curve (AUC) of 0.8699 in ROC curve analysis. Samples from ovarian cancer patients were large 3D cluster/CK7+/PAX8+ with AUC of 0.9812. Samples from pancreatobiliary tract cancer patients were small 3D cluster/CK7+/PAX8- with AUC of 0.8772. The remaining cancer samples, including breast, lung and colon cancer samples, had similar patterns of large 3D clusters/CK7+/PAX8- with AUC of 0.882, especially for lung cancer. SurePathTM technology, using 3D cluster patterns and dual ICC for CK7 and PAX8 in peritoneal fluid samples, can provide important information for determining specific primary origins in cases of unknown primary carcinoma.

Dual p16 and Ki-67 Expression in Liquid-Based Cervical Cytological Samples Compared to Pap Cytology Findings, Biopsies, and HPV Testing in Cervical Cancer Screening: A Diagnostic Accuracy Study

Objective: Our objective was to verify the sensitivity and specificity of dual immunocytochemistry staining for p16 and Ki-67 in liquid-based samples (the “dual” assay) for cervical lesion screening, compared to biopsy findings and human papillomavirus (HPV) DNA molecular detection. Study Design: Sensitivity, specificity, and positive (PPV) and negative (NPV) predictive values for the “dual immunocytochemistry assay” were calculated and compared to histopathological results and to high-risk HPV DNA detection in adult women or teenagers submitted to cervical cancer screening. Results: A total of 151 women were included. The majority (96.2%) of those with negative dual assay results had lower biopsy grades (p < 0.001). Women with cytology results suggestive of cervical cancer had positive dual immunocytochemistry assay results more frequently (p < 0.001), and these positive results were also significantly associated with biopsy findings (p < 0.001) and with high-risk genotype HPV infection (p = 0.007). Specificity and PPV for the dual assay were 0.972 (0.855–0.999) and 0.800 (0.284–0.995), respectively, and 1.000 (0.590–1.000) and 1.000 (0.631–1.000) for HPV detection. Conclusions: The dual immunocytochemistry assay had high specificity and PPV. It reveals a persistent HPV infection, avoiding the need for new tissue collections for biopsies or hybrid capture.

Cytokeratin 20/p53 dual immunocytochemistry for improving the diagnostic accuracy of urine liquid-based cytology in the detection of urothelial neoplasm: A retrospective study.

Backgrounds:Dual immunocytochemistry (DIC) with cytokeratin (CK) 20 and p53 in liquid-based cytology is a tool for improving the accuracy of urine cytology (UC). This study was conducted to compare the diagnostic accuracy of UC alone with that of UC combined with CK20/p53 DIC.Methods:We retrieved urine samples collected between January 2015 and March 2016 stored in PreservCyt®solution that were from cases categorized as malignant, highly suspicious, suspicious, and atypical and that were matched with a subsequent biopsy. We re-prepared 63 samples of 28 patients for DIC and blindly evaluated 63 pairs of original Papanicolaou smears and DIC.Results:Of the 63 samples, 11 could not be analyzed because of the low number of atypical urothelial cells, and the results of the remaining 52 samples were as follows: 34 positive and 18 negative. The positive predictive value of DIC was 100%, and the negative predictive value was 78%. Fifteen DIC-positive cases, histologically proven as malignant were originally diagnosed as highly suspicious (4), suspicious (8), and atypical (3), which were strongly suggestive of “urothelial carcinoma”. Four negative cases, histologically confirmed as non-neoplastic cases, were filtered from false positivity.Conclusions:Despite the small sample size, this study demonstrated the diagnostic utility, high sensitivity, and positive predictive value of CK20/p53 DIC, especially in cases with a small number of single malignant cells or cellular clusters of reactive atypical urothelial cells. Thus, CK20/p53 DIC can be used for improving diagnostic accuracy of UC, either as an ancillary method to cytology or as a part of a potential future diagnostic panel to improve patient diagnosis and management.

Cellular Targets and Receptor of Sexual Transmission of Zika Virus.

What is the mechanism of sexual transmission of Zika virus (ZIKV)? By utilizing exquisite reverse transcriptase-initiated in situ polymerase chain reaction (RT-in situ PCR), which enables an improved visualization of spermatozoa's subcellular compartment, we precisely localized the mid-piece of sperm that carry receptors for ZIKV. ZIKV is transmitted sexually and recent studies have verified ZIKV presence in semen of previously Zika-infected patients for >6-month postinfection when ZIKV had disappeared from blood, saliva, and urine. Strong serial analyses of various body fluids suggest that ZIKV can be transmitted between sexual partners. Currently, there is limited information on the association of the virus with human semen cell types that may carry the virus. Analyses were carried out to localize ZIKV for subcellular localization of ZIKV on cell types. The Tyro3 receptor for ZIKV was colocalized by dual immunocytochemistry with specific monoclonal antibodies. Three semen specimens were purchased from a commercial sperm bank. Motile sperm was separated from nonmotile cells by the "swim-up" technique. Each of the semen fractions was infected with ZIKV at the multiplicity of infection of 0.1.0 and 1.0 and evaluated for the primary targets of ZIKV in the semen cells by RT-in situ PCR and confirmed by real-time RT-PCR. ZIKV was present primarily at the mid-piece of mature spermatozoa in about 30% of the sperm. In addition, we determined that Tyro3 receptors, primarily expressed on mid-piece of human spermatozoa, play a role in ZIKV-binding and entry into spermatozoa. Our data strongly suggest a potential sexual/horizontal route of transmission for ZIKV primarily via infected sperms; most likely ZIKV enters the sperm via the Tyro3 receptor found at the mid-piece of the mature spermatozoa. We are uncertain as to what phase of spermatogenesis, that in human takes about 120 days, sperms are permissive to ZIKV. If permissiveness was very early during spermatogenesis males could be infectious for ∼120 days after the disappearance of viremia in an infected man. Our findings bring a new focus on the current affords to develop ZIKV vaccine. Why in the presence of anti-ZIKV antibodies infected men are still able to transmit the virus sexually? We suggest that only certain subclass of immunoglobulin (Ig)G (ie, IgG4) can cross the blood-Sertoli barrier therefore, a successful vaccine must provoke a subclass of IgG can quell ZIKV inside the seminiferous tubules.

Cardiogenic differentiation of mesenchymal stem cells on elastomeric poly (glycerol sebacate)/collagen core/shell fibers

To facilitate engineering of suitable biomaterials to meet the challenges associated with myocardial infarction. Poly (glycerol sebacate)/collagen (PGS/collagen) core/shell fibers were fabricated by core/shell electrospinning technique, with core as PGS and shell as collagen polymer; and the scaffolds were characterized by scanning electron microscope (SEM), fourier transform infrared spectroscopy (FTIR), contact angle and tensile testing for cardiac tissue engineering. Collagen nanofibers were also fabricated by electrospinning for comparison with core/shell fibers. Studies on cell-scaffold interaction were carried out using cardiac cells and mesenchymal stem cells (MSCs) co-culture system with cardiac cells and MSCs separately serving as positive and negative controls respectively. The co-culture system was characterized for cell proliferation and differentiation of MSCs into cardiomyogenic lineage in the co-culture environment using dual immunocytochemistry. The co-culture cells were stained with cardiac specific marker proteins like actinin and troponin and MSC specific marker protein CD 105 for proving the cardiogenic differentiation of MSCs. Further the morphology of cells was analyzed using SEM. PGS/collagen core/shell fibers, core is PGS polymer having an elastic modulus related to that of cardiac fibers and shell as collagen, providing natural environment for cellular activities like cell adhesion, proliferation and differentiation. SEM micrographs of electrospun fibrous scaffolds revealed porous, beadless, uniform fibers with a fiber diameter in the range of 380 ± 77 nm and 1192 ± 277 nm for collagen fibers and PGS/collagen core/shell fibers respectively. The obtained PGS/collagen core/shell fibrous scaffolds were hydrophilic having a water contact angle of 17.9 ± 4.6° compared to collagen nanofibers which had a contact angle value of 30 ± 3.2°. The PGS/collagen core/shell fibers had mechanical properties comparable to that of native heart muscle with a young's modulus of 4.24 ± 0.7 MPa, while that of collagen nanofibers was comparatively higher around 30.11 ± 1.68 MPa. FTIR spectrum was performed to confirm the functional groups present in the electrospun scaffolds. Amide I and amide II of collagen were detected at 1638.95 cm(-1) and 1551.64 cm(-1) in the electrospun collagen fibers and at 1646.22 cm(-1) and 1540.73 cm(-1) for PGS/collagen core/shell fibers respectively. Cell culture studies performed using MSCs and cardiac cells co-culture environment, indicated that the cell proliferation significantly increased on PGS/collagen core/shell scaffolds compared to collagen fibers and the cardiac marker proteins actinin and troponin were expressed more on PGS/collagen core/shell scaffolds compared to collagen fibers alone. Dual immunofluorescent staining was performed to further confirm the cardiogenic differentiation of MSCs by employing MSC specific marker protein, CD 105 and cardiac specific marker protein, actinin. SEM observations of cardiac cells showed normal morphology on PGS/collagen fibers and providing adequate tensile strength for the regeneration of myocardial infarction. Combination of PGS/collagen fibers and cardiac cells/MSCs co-culture system providing natural microenvironments to improve cell survival and differentiation, could bring cardiac tissue engineering to clinical application.

Evidence That Dopamine Acts via Kisspeptin to Hold GnRH Pulse Frequency in Check in Anestrous Ewes

Recent work has implicated stimulatory kisspeptin neurons in the arcuate nucleus (ARC) as important for seasonal changes in reproductive function in sheep, but earlier studies support a role for inhibitory A15 dopaminergic (DA) neurons in the suppression of GnRH (and LH) pulse frequency in the nonbreeding (anestrous) season. Because A15 neurons project to the ARC, we performed three experiments to test the hypothesis that A15 neurons act via ARC kisspeptin neurons to inhibit LH in anestrus: 1) we used dual immunocytochemistry to determine whether these ARC neurons contain D2 dopamine receptor (D2-R), the receptor responsible for inhibition of LH in anestrus; 2) we tested the ability of local administration of sulpiride, a D2-R antagonist, into the ARC to increase LH secretion in anestrus; and 3) we determined whether an antagonist to the kisspeptin receptor could block the increase in LH secretion induced by sulpiride in anestrus. In experiment 1, 40% of this ARC neuronal subpopulation contained D2-R in breeding season ewes, but this increased to approximately 80% in anestrus. In experiment 2, local microinjection of the two highest doses (10 and 50 nmol) of sulpiride into the ARC significantly increased LH pulse frequency to levels 3 times that seen with vehicle injections. Finally, intracerebroventricular infusion of a kisspeptin receptor antagonist completely blocked the increase in LH pulse frequency induced by systemic administration of sulpiride to anestrous ewes. These results support the hypothesis that DA acts to inhibit GnRH (and LH) secretion in anestrus by suppressing the activity of ARC kisspeptin neurons.

Spatial organization of RYRs and BK channels underlying the activation of STOCs by Ca2+ sparks in airway myocytes

Short-lived, localized Ca2+ events mediate Ca2+ signaling with high efficiency and great fidelity largely as a result of the close proximity between Ca2+-permeable ion channels and their molecular targets. However, in most cases, direct evidence of the spatial relationship between these two types of molecules is lacking, and, thus, mechanistic understanding of local Ca2+ signaling is incomplete. In this study, we use an integrated approach to tackling this issue on a prototypical local Ca2+ signaling system composed of Ca2+ sparks resulting from the opening of ryanodine receptors (RYRs) and spontaneous transient outward currents (STOCs) caused by the opening of Ca2+-activated K+ (BK) channels in airway smooth muscle. Biophysical analyses of STOCs and Ca2+ sparks acquired at 333 Hz demonstrate that these two events are associated closely in time, and approximately eight RYRs open to give rise to a Ca2+ spark, which activates ∼15 BK channels to generate a STOC at 0 mV. Dual immunocytochemistry and 3-D deconvolution at high spatial resolution reveal that both RYRs and BK channels form clusters and RYR1 and RYR2 (but not RYR3) localize near the membrane. Using the spatial relationship between RYRs and BK channels, the spatial-temporal profile of [Ca2+] resulting from Ca2+ sparks, and the kinetic model of BK channels, we estimate that an average Ca2+ spark caused by the opening of a cluster of RYR1 or RYR2 acts on BK channels from two to three clusters that are randomly distributed within an ∼600-nm radius of RYRs. With this spatial organization of RYRs and BK channels, we are able to model BK channel currents with the same salient features as those observed in STOCs across a range of physiological membrane potentials. Thus, this study provides a mechanistic understanding of the activation of STOCs by Ca2+ sparks using explicit knowledge of the spatial relationship between RYRs (the Ca2+ source) and BK channels (the Ca2+ target).

Neuroglial and synaptic rearrangements associated with photic entrainment of the circadian clock in the suprachiasmatic nucleus

AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL In the published paper of Girardet et al. (2010), the graphs comparing the mean synaptic innervation of VIP dendrites by GABAergic terminals (GABA+) and non-GABAergic terminals (GABA−) have been inverted (Fig. 4E). The correct data were those that had been described in the text (no day-night variations vs 62% increase in the respective synaptic density of GABAergic terminals and non-GABAergic terminals. The authors apologize for this error. Figure 4Open in figure viewerPowerPoint Quantitative electron-microscopic evaluation of the effect of time on the density of GABAergic and non-GABAergic synapses onto SCN VIP dendrites. Data were obtained using the unbiaised dissector method after analysis of pairs of serial ultrathin sections. According to the method (Sterio, 1984), only the synapses in the first ‘reference’ section which disappeared in the second section (‘look-up’ section) were counted. The GABAergic endings were gold-labelled with a polyclonal anti-GABA antiserum (kindly provided by Dr P. Somogyi; see Hodgson et al., 1985), according to a post-embedding procedure (Andréet al., 1987). The gold labelling was carried out on SCN sections containing peroxidase-stained VIP dendrites (see Becquet et al., 2008). (A–D) Examples of pairs of GABA–VIP double-labelled ultrathin sections. The synapse shown in A (filled arrowheads) between a GABAergic (gold-labelled) terminal and a VIP (peroxidase-stained) dendrite is no longer visible in B. In C and D, the VIP-positive dendrite shares a synapse with a GABA-positive ending which is visible on the two paired sections (filled arrowheads). It receives another synapse from a nonimmunoreactive ending (empty arrowheads) which has disappeared on section D. (E) Day–night variations in the mean synaptic innervation of VIP dendrites by GABAergic terminals (GABA+) and non-GABAergic terminals (GABA−). Data are related to the volume of the postsynaptic partner. The quantitative analysis was carried out after sampling of 127 VIP-stained dendritic profiles in rats killed at ZT02 (daytime, n = 4 rats) and of 111 such profiles in rats killed at ZT18 (nighttime, n = 4 rats). It involved 139 serially sectioned terminals in contact with at least one VIP dendrite at ZT02, among which 98 were GABA-positive, and 124 such terminals including 82 GABA-positive ones at ZT18. The mean numbers of synapses (Nv) per μm3 dendrite was estimated according to the equation Nv = ΣQ−/Vdis, where ΣQ− represents the total number of synapses present in the ‘reference’ section which disappeared in the ‘look-up’ section, and Vdis the dissector volume, derived from the area of the dendrites (as measured using the NIH ImageJ image processing software) multiplied by the section thickness. *P < 0.05 with the two-way Kruskal–Wallis anova test (Statistica, Statsoft). Scale bars, 0.5 μm. References André, D., Vuillon-Cacciuttolo, G. & Bosler, O. (1987) GABA nerve endings in the rat red nucleus combined detection with serotonin terminals using dual immunocytochemistry. Neuroscience, 23, 1095– 1102. Becquet, D., Girardet, C., Guillaumond, F., François-Bellan, A. & Bosler, O. (2008) Ultrastructural plasticity in the rat suprachiasmatic nucleus. Possible involvement in clock entrainment. Glia, 56, 294– 305. Girardet, C., Becquet, D., Blanchard, M.-P., François-Bellan, A.-M. & Bosler, O. (2010) Neuroglial and synaptic rearrangements associated with photic entrainment of the circadian clock in the suprachiasmatic nucleus. Eur. J. Neurosci., 32, 2133– 2142. Hodgson, A.J., Penke, B., Erdei, A., Chubb, I.W. & Somogyi, P. (1985) Antisera to gamma-aminobutyric acid. I. Production and characterization using a new model system. J. Histochem. Cytochem., 33, 229– 239. Sterio, D.C. (1984) The unbiased estimation of number and sizes of arbitrary particles using the disector. J. Microsc., 134, 127– 136. Volume33, Issue8April 2011Pages 1561-1561 FiguresReferencesRelatedInformation

Estradiol Negative Feedback Regulation by Glutamatergic Afferents to A15 Dopaminergic Neurons: Variation with Season

It is now clear that seasonal breeding in ewes is due to an increase in response to estradiol (E(2)) negative feedback in the nonbreeding season (anestrus) that is mediated by the A15 group of dopaminergic (DA) neurons. Because A15 cells do not contain estrogen receptors, we have postulated the presence of estrogen-responsive afferents and recently reported evidence that input from neurons containing gamma-aminobutyric acid (GABA) contribute to the control of A15 activity by E(2). However, GABAergic afferents account for only a fraction of A15 synaptic input and do not appear to vary with season. We therefore investigated the possible role of stimulatory glutamatergic input to A15 neurons. In experiments 1 and 2, local administration into the A15 of either a N-methyl-D-aspartate (NMDA) receptor or a kainate/alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor antagonist stimulated episodic LH secretion in a dose-dependent manner in ovary-intact anestrous ewes. In experiment 3, we examined the number of glutamatergic close contacts onto A15 neurons using dual immunocytochemistry in tissue from E(2)-treated ovariectomized anestrous and breeding season ewes. All A15 DA neurons were contacted by glutamatergic vesicles, and the number of close contacts was significantly higher in anestrus than the breeding season. Finally, using a triple-label immunocytochemistry procedure, we did not observe any colocalization of markers for GABA and glutamate in vesicles contacting A15 neurons. These results support the hypothesis that glutamatergic afferents actively stimulate A15 DA neurons in ovary-intact anestrous ewes and raise the possibility that alterations in this input may contribute to increased A15 neural activity during anestrus.

Evidence that γ-Aminobutyric Acid Is Part of the Neural Circuit Mediating Estradiol Negative Feedback in Anestrous Ewes

Seasonal anestrus in ewes is driven by an increase in response to estradiol (E2) negative feedback. Compelling evidence indicates that inhibitory A15 dopaminergic (DA) neurons mediate the increased inhibitory actions of E2 in anestrus, but these neurons do not contain estrogen receptors. Therefore, we have proposed that estrogen-responsive afferents to A15 neurons are part of the neural circuit mediating E2 negative feedback in anestrus. This study examined the possible role of afferents containing gamma-aminobutyric acid (GABA) and nitric oxide (NO) in modulating the activity of A15 neurons. Local administration of NO synthase inhibitors to the A15 had no effect on LH, but GABA receptor ligands produced dramatic changes. Administration of either a GABA A or GABA B receptor agonist to the A15 increased LH secretion in ovary-intact ewes, suggesting that GABA inhibits A15 neural activity. In ovariectomized anestrous ewes, the same doses of GABA receptor agonist had no effect, but combined administration of a GABA A and GABA B receptor antagonist to the A15 inhibited LH secretion. These data are consistent with the hypothesis that endogenous GABA release within the A15 is low in ovary-intact anestrous ewes and elevated after ovariectomy. Using dual immunocytochemistry, we observed that GABAergic varicosities make close contacts on to A15 neurons and that A15 neurons contain both the GABA A-alpha1 and the GABA B-R1 receptor subunits. Based on these data, we propose that in anestrous ewes, E2 inhibits release of GABA from afferents to A15 DA neurons, increasing the activity of these DA neurons and thus suppressing episodic secretion of GnRH and LH.

CVL‐projecting NTS neurons receive input from a distinct subset of vagal afferents

The vagus nerve contains primary visceral afferents that convey information from cardiovascular, pulmonary, and gastrointestinal tissues to the nucleus tractus solitarii (NTS). We previously showed that two tract tracers, cholera toxin B subunit (CTb) and isolectin B4 (IB4), label distinct populations of vagal afferents. In this study, we tested whether these fibers contact different populations of NTS neurons, including NTS neurons that project to the caudal ventrolateral medulla (CVL). To label CVL‐projecting NTS neurons, the retrograde tracer Fluorogold (2% in 0.9% saline; 50 nl, Fluorochrome Inc.) was injected into CVL; vagal afferents were labeled with injections of CTb and IB4 (2% solution, 1–4 ul) into the vagus nerve. Male Sprague‐Dawley rats (350–425 g) were used and surgeries were performed under isoflurane anesthesia. Seven days later, rats were perfused and vibratome sections through NTS were examined using dual immunocytochemistry for Fluorogold (rabbit anti‐FG, Chemicon) and either CTb (goat anti‐CTb, List Biological Laboratories) or IB4 (goat anti‐IB4, Vector Laboratories). Primary antibodies were visualized using fluorescent secondary antibodies. By confocal microscopy, we found that CVL‐projecting NTS neurons were more than twice as likely to receive somatic input from CTb‐labeled afferents (79%) than IB4‐labeled afferents (34%). In somatic nerves, CTb and IB4 label myelinated and unmyelinated afferents, respectively. Therefore, our findings suggest that CVL‐projecting NTS neurons receive input mainly from myelinated vagal afferents that contain a distinct population of transporters, neurotransmitters, and receptors.Support: NIH HL56301

Kisspeptin Neurons in the Arcuate Nucleus of the Ewe Express Both Dynorphin A and Neurokinin B

Kisspeptin is a potent stimulator of GnRH secretion that has been implicated in the feedback actions of ovarian steroids. In ewes, the majority of hypothalamic kisspeptin neurons are found in the arcuate nucleus (ARC), with a smaller population located in the preoptic area. Most arcuate kisspeptin neurons express estrogen receptor-alpha, as do a set of arcuate neurons that contain both dynorphin and neurokinin B (NKB), suggesting that all three neuropeptides are colocalized in the same cells. In this study we tested this hypothesis using dual immunocytochemistry and also determined if kisspeptin neurons contain MSH or agouti-related peptide. To assess colocalization of kisspeptin and dynorphin, we used paraformaldehyde-fixed tissue from estrogen-treated ovariectomized ewes in the breeding season (n = 5). Almost all ARC, but no preoptic area, kisspeptin neurons contained dynorphin. Similarly, almost all ARC dynorphin neurons contained kisspeptin. In experiment 2 we examined colocalization of kisspeptin and NKB in picric-acid fixed tissue collected from ovary intact ewes (n = 9). Over three quarters of ARC kisspeptin neurons also expressed NKB, and a similar percentage of NKB neurons contained kisspeptin. In contrast, no kisspeptin neurons stained for MSH or agouti-related peptide. These data demonstrate that, in the ewe, a high percentage of ARC kisspeptin neurons also produce dynorphin and NKB, and we propose that a single subpopulation of ARC neurons contains all three neuropeptides. Because virtually all of these neurons express estrogen and progesterone re-ceptors, they are likely to relay the feedback effects of these steroids to GnRH neurons to regulate reproductive function.

Treatment with trimethyltin promotes the formation of cleaved tau in the rat brain

Trimethyltin (TMT) is a well-documented neurotoxin that affects primarily limbic system structures. Most previous studies have relied on histological approaches to examine TMT neurotoxicity, so the aim of this study was to employ the novel biomarker cleaved MAP-tau (C-tau) to assess TMT-induced CNS injury both quantitatively and qualitatively. Immunoblot studies indicated that cleaved MAP-tau proteins with molecular weights of 45-50 kD were present in the hippocampus of rats treated with TMT but not vehicle 21 days after treatment. Quantitative ELISA revealed that C-tau concentration in rats treated with TMT was greatest at 14 and 21 days in the piriform cortex and hippocampus, respectively; TMT did not significantly increase C-tau concentration in the mesencephalon. C-tau immunocytochemistry demonstrated the greatest TMT-induced damage in the hippocampus and piriform cortex. Additional studies utilizing dual immunocytochemistry revealed that C-tau-labeled cells were also glial fibrillary acidic protein-positive, leading to identification of these cells as astrocytes. Although the origin of C-tau in astrocytes of rats treated with TMT is currently unknown, increased C-tau concentration and the presence of C-tau positive cells in limbic system structures of TMT-treated rats further supports the view that C-tau is a reliable marker of CNS toxicity.

The KCNQ Channel Opener Retigabine Inhibits the Activity of Mesencephalic Dopaminergic Systems of the Rat

Homo- and heteromeric complexes of KCNQ channel subunits are the molecular correlate of the M-current, a neuron-specific voltage-dependent K(+) current with a well established role in control of neural excitability. We investigated the effect of KCNQ channel modulators on the activity of dopaminergic neurons in vitro and in vivo in the rat ventral mesencephalon. The firing of dopaminergic neurons recorded in mesencephalic slices was robustly inhibited in a concentration-dependent manner by the KCNQ channel opener N-(2-amino-4-(4-fluorobenzylamino)-phenyl) carbamic acid ethyl ester (retigabine). The effect of retigabine persisted in the presence of tetrodotoxin and simultaneous blockade of GABA(A) receptors, small-conductance calcium-activated K(+) (SK) channels, and hyperpolarization-activated (I(h)) channels, and it was potently reversed by the KCNQ channel blocker 4-pyridinylmethyl-9(10H)-anthracenone (XE991), indicating a direct effect on KCNQ channels. Likewise, in vivo single unit recordings from dopaminergic neurons revealed a prominent reduction in spike activity after systemic administration of retigabine. Furthermore, retigabine inhibited dopamine synthesis and c-Fos expression in the striatum under basal conditions. Retigabine completely blocked the excitatory effect of dopamine D(2) autoreceptor antagonists. Again, the in vitro and in vivo effects of retigabine were completely reversed by preadministration of XE991. Dual immunocytochemistry revealed that KCNQ4 is the major KCNQ channel subunit expressed in all dopaminergic neurons in the mesolimbic and nigrostriatal pathways. Collectively, these observations indicate that retigabine negatively modulates dopaminergic neurotransmission, likely originating from stimulation of mesencephalic KCNQ4 channels.

Cholera toxin B subunit and isolectin B4 label distinct populations of vagal afferents projecting to the nucleus tractus solitarii

The vagus nerve contains primary visceral afferents that convey information from cardiovascular, pulmonary, and gastrointestinal tissues to nucleus tractus solitarii (NTS). We tested the hypothesis that different anterograde tracers will label distinct populations of primary afferents. In somatic nerves, cholera toxin B subunit (CTb) and isolectin B4 (IB4) have been shown to label myelinated and unmyelinated fibers, respectively. We also examined the vesicular glutamate transporter 2 (VGlut2) content of these afferents. Male Sprague-Dawley rats (275–350 g) received injections of either CTb or IB4 (2% solution, 1–4 μl) into the vagus nerve under isoflurane anesthesia. Seven days later, rats were perfused and vibratome sections through NTS were examined with dual immunocytochemistry for either CTb (goat anti-CTb, List) or IB4 (goat anti-IB4, Vector) and VGlut2 (guinea pig anti-VGlut2, Chemicon), visualized using fluorescent secondary antibodies. CTb and IB4 produced distinct patterns of afferent labeling in NTS following vagal nerve injections. CTb produced both anterograde labeling in NTS and retrograde labeling of vagal motor neurons, while IB4 labeled only afferents. By confocal microscopy, we found VGlut2 in a subset of CTb-labeled vagal afferents, but VGlut2 was rarely found in IB4-labeled afferents. By electron microscopy we found that CTb afferents were both myelinated and unmyelinated, but IB4 afferents were only unmyelinated. These findings suggest that these tracers label largely distinct, but partially overlapping, populations of vagal afferents to NTS with distinct phenotypes. Support: NIH HL56301

Progesterone-Receptive Dopaminergic and Neuropeptide Y Neurons Project from the Arcuate Nucleus to Gonadotropin-Releasing Hormone-Rich Regions of the Ovine Preoptic Area

Progesterone inhibits gonadotropin-releasing hormone (GnRH) secretion in sheep through an interneuronal system located in the mediobasal hypothalamus. This study focused on known inhibitors of GnRH secretion in sheep, dopamine and neuropeptide Y (NPY). As the distributions of tyrosine hydroxylase (TH)- and NPY-immunoreactive neurons overlap with progesterone receptors (PR) in the arcuate nucleus, we hypothesized that, if these neurons mediate, at least partially, the inhibitory feedback signal of progesterone, then they should co-express PRs. Fluorogold (FG), a retrograde tracer, was injected into the preoptic area of ovariectomized ewes pretreated with estrogen and progesterone. When the FG injection site encompassed at least 80 GnRH neurons, sections from the arcuate nucleus were processed using dual immunocytochemistry for PR and either TH or NPY. We found that 30% of PR-immunoreactive, 12% of TH-containing and 21% of NPY-synthesizing neurons project toward this GnRH-rich region. Of the PR/TH dual-labeled cells, which represent 21% of PR and 31% of TH cells, respectively, 22% displayed FG labeling. Of the PR/NPY neurons, which account for 19% of PR and 67% of NPY neurons, respectively, 26% were FG fluorescent. This study suggests that subsets of arcuate nucleus dopaminergic and NPY neurons may transduce, at least in part, the progesterone-mediated inhibition of GnRH secretion.

Developmental regulation of gonadotropin-releasing hormone neurons by fibroblast growth factor signaling.

GnRH neurons are central to the initiation and maintenance of reproductive function in diverse vertebrates. The formation of a functional GnRH system during development is a highly complex event that likely requires extensive guidance by neurotrophic factors. In this study, we examined whether members of the fibroblast growth factor (FGF) family are critically involved in the development of endogenous GnRH neurons. Immunocytochemistry revealed the presence of FGF receptors (FGFRs) 1, 2, and 3, but not 4, in embryonic day (E) 10.5 medial nasal placode, an area and time consistent with the first appearance of GnRH neurons in mice. Dual immunocytochemistry confirmed the presence of FGFRs 1 and 3, but not 2 and 4, in a substantial fraction of E15.5 and postnatal day (P) 3 GnRH neurons. To examine whether FGF signaling was essential for the specification of GnRH neuronal fate, a nasal explant culture that supported the in vitro emergence of GnRH neurons from E10.5 noses was established. In this system, the addition of SU5402, a FGFR antagonist, suppressed the emergence of GnRH neurons. Lastly, we investigated whether FGF signaling altered the extension of neurites in cultures of dispersed GnRH neurons. The addition of FGF2 to E15.5 and P3 GnRH neurons expressing the green fluorescent protein significantly stimulated neurite outgrowth (E15.5 and P3) and branching (P3), suggesting a regulatory role of FGFs in GnRH axon targeting. Together these results demonstrated that FGF signaling critically regulates multiple phases of development in a neuroendocrine system essential for vertebrate reproduction.