Double-label Immunohistochemistry (double-label + immunohistochemistry)

Distribution by Scientific Domains


Selected Abstracts


Projections of RFamide-related Peptide-3 Neurones in the Ovine Hypothalamus, with Special Reference to Regions Regulating Energy Balance and Reproduction

JOURNAL OF NEUROENDOCRINOLOGY, Issue 8 2009
Y. Qi
RFamide-related peptide-3 (RFRP-3) is a neuropeptide produced in cells of the paraventricular nucleus and dorsomedial nucleus of the ovine hypothalamus. In the present study, we show that these cells project to cells in regions of the hypothalamus involved in energy balance and reproduction. A retrograde tracer (FluoroGold) was injected into either the arcuate nucleus, the lateral hypothalamic area or the ventromedial nucleus. The distribution and number of retrogradely-labelled RFRP-3 neurones was determined. RFRP-3 neurones projected to the lateral hypothalamic area and, to a lesser degree, to the ventromedial nucleus and the arcuate nucleus. Double-label immunohistochemistry was employed to identify cells receiving putative RFRP-3 input to cells in these target regions. RFRP-3 cells were seen to project to neuropeptide Y and pro-opiomelanocortin neurones in the arcuate nucleus, orexin and melanin-concentrating hormone neurones in the lateral hypothalamic area, as well as orexin cells in the dorsomedial nucleus and corticotrophin-releasing hormone and oxytocin cells in the paraventricular nucleus. Neurones expressing gonadotrophin-releasing hormone in the preoptic area were also seen to receive input from RFRP-3 projections. We conclude that RFRP-3 neurones project to hypothalamic regions and cells involved in regulation of energy balance and reproduction in the ovine brain. [source]


Evidence for a Stimulatory Action of Melanin-Concentrating Hormone on Luteinising Hormone Release Involving MCH1 and Melanocortin-5 Receptors

JOURNAL OF NEUROENDOCRINOLOGY, Issue 3 2006
J. F. Murray
Abstract The present series of studies aimed to further our understanding of the role of melanin-concentrating hormone (MCH) neurones in the central regulation of luteinising hormone (LH) release in the female rat. LH release was stimulated when MCH was injected bilaterally into the rostral preoptic area (rPOA) or medial preoptic area (mPOA), but not when injected into the zona incerta (ZI), of oestrogen-primed ovariectomised rats. In rats that were steroid-primed to generate a surge-like release of LH, MCH administration into the ZI blocked this rise in LH release: no such effect occurred when MCH was injected into the rPOA or mPOA. In vitro, MCH stimulated gonadotrophin-releasing hormone (GnRH) release from hypothalamic explants. Double-label immunohistochemistry showed GnRH-immunoreactive neurones in the vicinity of and intermingled with immunoreactive MCH processes. MCH is the endogenous ligand of the MCH type 1 receptor (MCH1-R). Previously, we have shown a role for melanocortin-5 receptors (MC5-R) in the stimulatory action of MCH, so we next investigated the involvement of both MCH1-R and/or MC5-R in mediating the actions of MCH on GnRH and hence LH release. The stimulatory action of MCH in the rPOA was inhibited by administration of antagonists for either MCH1-R or MC5-R. However, in the mPOA, the action of MCH was blocked only by the MC5-R antagonist. LH release was stimulated by an agonist for MC5-R injected into the rPOA or mPOA; this was blocked by the MC5-R antagonist but not the MCH1-R antagonist. These results indicate that both MCH1-R and MC5-R are involved in the central control of LH release by MCH. [source]


Oestrogen Receptor ,-Immunoreactivity in Gonadotropin Releasing Hormone-Expressing Neurones: Regulation by Oestrogen

JOURNAL OF NEUROENDOCRINOLOGY, Issue 9 2001
I. Kalló
Abstract Double-label immunohistochemistry was employed to establish whether immunoreactivity for the , subtype of the oestrogen receptor (ER,-IR) is present in gonadotropin releasing hormone (GnRH)-containing cells. In the immortalized GnRH cell line, GT1-7, almost all nuclei were immunoreactive for ER,. In the preoptic area of ovariectomized rats, more than one-half of the GnRH neurones (52.0,63.5%) contained ER,-IR within the nucleus; a smaller proportion of these neurones (5,10%) displayed a particularly intense nuclear signal for ER,. The presence of ER,-IR in the nuclei of GT1-7 cells and GnRH neurones is consistent with recent reports of ER, mRNA in these cells. Oestrogen treatment reduced the percentage of GnRH neurones with detectable ER,-IR. The range of signal intensity for ER, and the incidence of the ER, signal in GnRH neurones were comparable following double-label immunohistochemistry involving either bright field or fluorescent techniques. These findings raise the possibility that ER, receptors mediate direct effects of oestrogen on GnRH neurones. [source]


Increased Fas ligand expression by T cells and tumour cells in the progression of actinic keratosis to squamous cell carcinoma

BRITISH JOURNAL OF DERMATOLOGY, Issue 1 2004
A.C. Satchell
Summary Background In the counterattack model of tumorigenesis, it has been proposed that tumours develop resistance to attack from Fas ligand (FasL)-expressing cytotoxic T cells by downregulating Fas (immune escape), while at the same time upregulating FasL expression to induce apoptosis in Fas-expressing T cells (counterattack). Objectives The aim of this study was to examine Fas and FasL expression on tumour cells and infiltrating T cells during the progression of actinic keratoses (AK), the benign precursor lesion, to squamous cell carcinoma (SCC). Patients and methods Samples of AK (n = 20) and SCC (n = 20) were collected from immunocompetent patients attending dermatology clinics. Double-label immunohistochemistry was performed on frozen sections using mouse monoclonal antibodies to Fas or FasL, simultaneously with a rabbit polyclonal antibody to either CD3 or cytokeratin, markers of T cells and keratinocytes, respectively. Cell densities and the optical density of tumour Fas expression were measured using image analysis. Results FasL-expressing T cells were observed in nine of 19 SCCs, compared with three of 20 AKs (P < 0·05). FasL-expressing tumour cells were found in nine of 18 SCCs, compared with only one of 20 AK specimens (P < 0·005). There was no difference in the number of Fas-expressing T cells infiltrating AK and SCC. Fas expression by keratinocytes, measured by optical density, was lower in SCC (range 0·1,40, median 17) compared with AK (range 4,62, median 25) (P < 0·05). Conclusions These results suggest that the greater numbers of FasL-expressing T cells infiltrating into SCC compared with AK are targeting Fas-expressing tumour cells. As AK cells progress to SCC, they subvert this T-cell-mediated killing of tumour cells by downregulating their Fas expression (immune escape). Furthermore, tumour cells upregulate their expression of FasL, possibly as a counterattack measure to induce apoptosis in the increased number of tumour-infiltrating T cells. Thus changes in Fas/FasL-mediated interactions between T cells and tumour cells occur during the progression of AK into SCC. [source]


Loss of Hypothalamic Response to Leptin During Pregnancy Associated with Development of Melanocortin Resistance

JOURNAL OF NEUROENDOCRINOLOGY, Issue 5 2009
S. R. Ladyman
Hypothalamic leptin resistance during pregnancy is an important adaptation that facilitates the state of positive energy balance required for fat deposition in preparation for lactation. Within the arcuate nucleus, pro-opiomelanocortin (POMC) neurones and neuropeptide Y (NPY)/agouti-related gene protein (AgRP) neurones are first-order leptin responsive neurones involved in the regulation of energy balance. The present study aimed to investigate whether the regulation of these neuropeptides is disrupted during pregnancy in association with the development of leptin resistance. As measured by quantitative in situ hybridisation, POMC and AgRP mRNA levels were not significantly different during pregnancy, whereas NPY mRNA levels increased such that, by day 21 of pregnancy, levels were significantly higher than in nonpregnant, animals. These data suggest that these neurones were not responding normally to the elevated leptin found during pregnancy. To further characterise the melanocortin system during pregnancy, double-label immunohistochemistry was used to quantify leptin-induced phosphorylation of signal transducer and activator of transcription 3 (pSTAT3) in POMC neurones, using ,-melanocyte-stimulating hormone (MSH) as a marker. The percentage of ,-MSH neurones containing leptin-induced pSTAT3 did not significantly differ from nonpregnant animals, indicating that there was no change in the number of POMC neurones that respond to leptin during pregnancy. Treatment with ,-MSH significantly reduced food intake in nonpregnant rats, but not in pregnant rats, indicating resistance to the satiety actions of ,-MSH during pregnancy. The data suggest that multiple mechanisms contribute to leptin resistance during pregnancy. As well as a loss of responses in first-order leptin-responsive neurones in the arcuate nucleus, there is also a downstream disruption in the melanocortin system. [source]


Morphological Substrate of the Catecholaminergic Input of the Vasopressin Neuronal System in Humans

JOURNAL OF NEUROENDOCRINOLOGY, Issue 12 2006
B. Dudás
It has been postulated that the stress response is associated with water balance via regulating vasopressin release. Nausea, surgical stress and insulin-induced hypoglycaemia were shown to stimulate vasopressin secretion in humans. Increased vasopressin release in turn induces water resorption through the kidneys. Although the mechanism of the stress-mediated vasopressin release is not entirely understood, it is generally accepted that catecholamines play a crucial role in influencing water balance by modulating the secretion of vasopressin. However, the morphological substrate of this modulation has not yet been established. The present study utilised double-label immunohistochemistry to reveal putative juxtapositions between tyrosine hydroxylase (TH)-immunoreactive (IR) catecholaminergic system and the vasopressin systems in the human hypothalamus. In the paraventricular and supraoptic nuclei, numerous vasopressin-IR neurones received TH-IR axon varicosities. Analysis of these juxtapositions with high magnification combined with oil immersion did not reveal any gaps between the contacted elements. In conclusion, the intimate associations between the TH-IR and vasopressin-IR elements may be functional synapses and may represent the morphological basis of vasopressin release modulated by stressors. Because certain vasopressin-IR perikarya receive no detectable TH innervations, it is possible that additional mechanisms may participate in the stress-influenced vasopressin release. [source]


The Percentage of Pituitary Gonadotropes with Immunoreactive Oestradiol Receptors Increases in the Follicular Phase of the Ovine Oestrous Cycle

JOURNAL OF NEUROENDOCRINOLOGY, Issue 10 2001
V. A. Tobin
Abstract During the oestrous cycle, there is an alteration in gonadotrope responsiveness to gonadotropin releasing hormone (GnRH). One cellular mechanism that may be involved in these changes at the pituitary level is the hormonal regulation of oestrogen receptor (ER) expression. Using double-label immunohistochemistry, we examined the proportion of gonadotropes, lactotropes and somatotropes with immunoreactive (ir) oestrogen receptor alpha (ER,) in pituitary sections from ewes at three stages of the ovine oestrous cycle (n = 8 per group). The percentage of ER, positive cells that also stained positive for luteinizing hormone (LH) increased in the transition from the luteal phase to the follicular phase (n = 8), with no further increase at the time of oestrus (n = 8). In the pituitaries from the luteal phase sheep, only a small number (15%) of lactotropes and 4% of somatotropes were found to contain ir-ER, and there were no alterations across the oestrous cycle. When we examined pituitaries from ovariectomized (OVX) ewes treated (i.m.) with either oestradiol benzoate (50 µg) or oil vehicle for 2, 4, 6 or 16 h (n = 4 per group), there was no effect of treatment. In fact, the percentage of gonadotropes that were ER,-positive in OVX ewes was similar to that observed in the pituitaries from the follicular phase ewes, both of which display a high frequency of pulsatile GnRH secretion. We conclude that the number of gonadotropes that contain ir-ER, increases in the follicular phase of the oestrous cycle and this may enhance the responsiveness of these cells to oestrogen and GnRH. We suggest that this may be due to increased pulsatile GnRH input rather than rising oestrogen levels. [source]


Prothrombin kringle-2 induces death of mesencephalic dopaminergic neurons in vivo and in vitro via microglial activation

JOURNAL OF NEUROSCIENCE RESEARCH, Issue 7 2010
Sang Ryong Kim
Abstract We have shown that prothrombin kringle-2 (pKr-2), a domain of human prothrombin distinct from thrombin could activate cultured rat brain microglia in vitro. However, little is known whether pKr-2-induced microglial activation could cause neurotoxicity on dopaminergic (DA) neurons in vivo. To address this question, pKr-2 was injected into the rat substantia nigra (SN). Tyrosine hydroxylase (TH) immunohistochemistry experiments demonstrate significant loss of DA neurons seven days after injection of pKr-2. In parallel, pKr-2-activated microglia were detected in the SN with OX-42 and OX-6 immunohistochemistry. Reverse transcription PCR and double-label immunohistochemistry revealed that activated microglia in vivo exhibit early and transient expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and several proinflammatory cytokines. The pKr-2-induced loss of SN DA neurons was partially inhibited by the NOS inhibitor NG -nitro-L-arginine methyl ester hydrochloride, and the COX-2 inhibitor DuP-697. Extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase were activated in the SN as early as 1 hr after pKr-2 injection, and localized within microglia. Inhibition of these kinases led to attenuation of mRNA expression of iNOS, COX-2 and several proinflammatory cytokines, and rescue of DA neurons in the SN. Intriguingly, following treatment with pKr-2 in vitro, neurotoxicity was detected exclusively in co-cultures of mesencephalic neurons and microglia, but not microglia-free neuron-enriched mesencephalic cultures, indicating that microglia are required for pKr-2 neurotoxicity. Our results strongly suggest that microglia activated by endogenous compound(s), such as pKr-2, are implicated in the DA neuronal cell death in the SN. © 2009 Wiley-Liss, Inc. [source]


PGE2 receptor EP1 renders dopaminergic neurons selectively vulnerable to low-level oxidative stress and direct PGE2 neurotoxicity

JOURNAL OF NEUROSCIENCE RESEARCH, Issue 14 2007
Emilce Carrasco
Abstract Oxidative stress and increased cyclooxygenase-2 (COX-2) activity are both implicated in the loss of dopaminergic neurons from the substantia nigra (SN) in idiopathic Parkinson's disease (PD). Prostaglandin E2 (PGE2) is one of the key products of COX-2 activity and PGE2 production is increased in PD. However, little is known about its role in the selective death of dopaminergic neurons. Previously, we showed that oxidative stress evoked by low concentrations of 6-hydroxydopamine (6-OHDA) was selective for dopaminergic neurons in culture and fully dependent on COX-2 activity. We postulated that this loss was mediated by PGE2 acting through its receptors, EP1, EP2, EP3, and EP4. Using double-label immunohistochemistry for specific EP receptors and tyrosine hydroxylase (TH), we identified EP1 and EP2 receptors on dopaminergic neurons in rat SN. EP2 receptors were also found in non-dopaminergic neurons of this nucleus, as were EP3 receptors, whereas the EP4 receptor was absent. PGE2, 16-phenyl tetranor PGE2 (a stable synthetic analogue), and 17-phenyl trinor PGE2 (an EP1 receptor,selective agonist) were significantly toxic to dopaminergic cells at nanomolar concentrations; EP2- and EP3-selective agonists were not. We challenged dopaminergic neurons in embryonic rat mesencephalic primary neuronal cultures and tested whether these receptors mediate selective 6-OHDA toxicity. The nonselective EP1,3 receptor antagonist AH-6809 and two selective EP1 antagonists, SC-19220 and SC-51089, completely prevented the 40%,50% loss of dopaminergic neurons caused by exposure to 5 ,M 6-OHDA. Together, these results strongly implicate PGE2 activation of EP1 receptors as a mediator of selective toxicity in this model of dopaminergic cell loss. © 2007 Wiley-Liss, Inc. [source]