Colocalization

Given a pair of adjoint functors between two arbitrary categories it induces mutually inverse equivalences between the full subcategories of the initial ones, consisting of objects for which the arrows of adjunction are isomorphisms. We investigate some cases in which these subcategories may be better characterized. One application is the construction of cellular approximations. Other is the definition and the characterization of (weak) *-objects in the non additive case.

The arcuate nucleus gives rise to approximately 80% of the neuropeptide Y (NPY)-immunoreactive (IR) innervation to thyrotropin-releasing hormone (TRH) neurons in the hypothalamic paraventricular nucleus (PVN). However, the source of the remaining 20% is unknown. Since medullary adrenergic neurons synthesize NPY and also innervate the hypophysiotropic TRH neurons, we raised the possibility that adrenergic neurons contribute to the NPY-ergic innervation of TRH neurons in the PVN. Triple-labeling immunofluorescence was performed to study the colocalization of NPY and phenylethanolamine N-methyltransferase (PNMT)-the key enzyme of adrenaline synthesis-in axons in association with hypophysiotropic TRH neurons. NPY-immunoreactivity was observed in 74% of PNMT-IR axon varicosities apposed to proTRH-IR neurons, comprising 26% of all NPY-IR axons in contact with proTRH perikarya and dendrites in the PVN. We conclude that at least two distinct populations of NPY neurons innervate hypophysiotropic TRH neurons, the NPY neurons of the hypothalamic arcuate nucleus and the medullary adrenergic neurons that co-contain NPY.

Abstract: One of the most routine uses of fluorescence microscopy is colocalization, ie, the demonstration of a relationship between pairs of biological molecules. Frequently this is presented simplistically by the use of overlays of red and green images, with areas of ...

The Pearson correlation coefficient (PCC) and the Mander's overlap coefficient (MOC) are used to quantify the degree of colocalization between fluorophores. The MOC was introduced to overcome perceived problems with the PCC. The two coefficients are mathematically similar, differing in the use of either the absolute intensities (MOC) or of the deviation from the mean (PCC). A range of correlated datasets, which extend to the limits of the PCC, only evoked a limited response from the MOC. The PCC is unaffected by changes to the offset while the MOC increases when the offset is positive. Both coefficients are independent of gain. The MOC is a confusing hybrid measurement, that combines correlation with a heavily weighted form of co-occurrence, favors high intensity combinations, downplays combinations in which either or both intensities are low and ignores blank pixels. The PCC only measures correlation. A surprising finding was that the addition of a second uncorrelated population can substantially increase the measured correlation, demonstrating the importance of excluding background pixels. Overall, since the MOC is unresponsive to substantial changes in the data and is hard to interpret, it is neither an alternative to nor a useful substitute for the PCC. The MOC is not suitable for making measurements of colocalization either by correlation or co-occurrence. '

The purpose of this study was to determine the presence of estrogen receptor alpha (ERa) and the relationship between neurons that express ERa and produce vasopressin (AVP) in the paraventricular nucleus of the hypothalamus (PVN) in new world mice of the genus Peromyscus. Brains were collected from male and female Peromyscus californicus, Peromyscus leucopus, Peromyscus maniculatus, and Peromyscus polionotus, and double labeled for the expression of ERa and AVP immunoreactivity (IR). The number of cells expressing ERa-IR and AVP-IR was determined in the medial and posterior region of the PVN. The results indicate that Peromyscus is the first taxonomic group reported to have ERa widely distributed in the PVN, occurring in both medial and posterior regions of the PVN. While estrogen can regulate the production of AVP, AVP and ERa were rarely colocalized. There was, however, a significant inverse relationship between the number of cells that expressed ERa-IR and the number expressing AVP-IR. There were no sex differences in the expression of ERa-IR or AVP-IR. D 2004 Elsevier B.V. All rights reserved.

Sequence-specific single-stranded DNA-binding protein 2 (SSBP2) is a candidate tumor suppressor for human acute myelogenous leukemia (AML). Inducible expression of SSBP2 causes growth arrest and partial differentiation in AML cells. Here, we report that the adenoviral oncoprotein E1B55K directly binds to endogenous SSBP2 protein and sequesters it into juxtanuclear bodies in adenovirally transformed human embryonic kidney (HEK) 293 cells. Similarly, transient expression of E1B55K in IMR90 fibroblasts and HeLa cells result in the formation of juxtanuclear bodies containing SSBP2. When nuclear export of E1B55K is prevented, SSBP2 remains associated with E1B55K in nuclear foci. A requirement for intact microtubules to retain the integrity of the juxtanuclear bodies suggests them to be E1B55K containing aggresomes. The adenoviral E1B55K protein has been shown to localize to the Mre11 complex and p53 to aggresome structures; together with the viral E4orf6 protein, E1B55K recruits a cellular E3 ubiquitin ligase that induces degradation of Mre11 and p53. However, our present studies reveal that E1B55K does not degrade SSBP2. These data demonstrate that E1B55K targets the candidate leukemia suppressor SSBP2 and suggest that subverting its function may contribute to cell transformation by viral oncoproteins.

Hypothalamic dopaminergic periventricular and arcuate nuclei are known to project to the pituitary gland and contain serotonin in their terminals. In order to elucidate the potential of these neurons to synthesize serotonin, we studied immunohistochemically the possible tryptophan hydroxylase content of periventriculo-hypophyseal neurons, identified by retrograde tracing from the pituitary gland. These neurons were found to contain tryptophan hydroxylase-immunoreactivity (TpOH-IR), which was enhanced after colchicine treatment. All of the TpOH-IR neurons contained tyrosine hydroxylase-immunoreactivity as well. However, none of them were immunoreactive for serotonin in either intact animals or in animals pretreated with serotonin precursor L-tryptophan and MAO inhibitor pargyline. Thus, neurons of the dopaminergic periventriculo-hypophyseal pathway express tryptophan hydroxylase, but are unable to synthesize serotonin. These findings (i) raise the possibility that, in these nerves, serotonin might serve a function other than regular synaptic transmission, and (ii) suggest that expression of an enzyme synthesizing certain transmitter does not necessarily confirm the corresponding transmitter phenotype of that neuron.

It is generally accepted that the functional compartmentalization of eukaryotic cells is reflected by the differential occurrence of proteins in their compartments. The location and physiological function of a protein are closely related; local information of a protein is thus crucial to understanding its role in biological processes. The visualization of proteins residing on intracellular structures by fluorescence microscopy has become a routine approach in cell biology and is increasingly used to assess their colocalization with well-characterized markers. However, imageanalysis methods for colocalization studies are a field of contention and enigma. We have therefore undertaken to review the most currently used colocalization analysis methods, introducing the basic optical concepts important for image acquisition and subsequent analysis. We provide a summary of practical tips for image acquisition and treatment that should precede proper colocalization analysis. Furthermore, we discuss the application and feasibility of colocalization tools for various biological colocalization situations and discuss their respective strengths and weaknesses. We have created a novel toolbox for subcellular colocalization analysis under ImageJ, named JACoP, that integrates current global statistic methods and a novel object-based approach.

It is now well recognized that there are several groups of cholinergic neurons in the basal forebrain with direct projections to various cortical regions. Immunohistochemical investigations of the distribution of the neuropeptide galanin (GAL) have shown that two of these brain areas, the medial septum and diagonal band, contained large numbers of GAL-immunoreactive neurons. In the present study, double staining techniques using antibodies raised against choline acetyltransferase (CHAT) revealed that GAL-and CHATlike immunoreactivities are colocalized within a subpopulation of the cholinergic neurons within the medial septum and diagonal band. This colocalization of GAL-and ChAT-immunoreactivities was not seen to occur within other groups of forebrain cholinergic neurons. Immunohistochemistry carried out subsequent to injections of fluorescent retrograde tracers into the hippocampal formation revealed that both ChAT/GAL-and ChAT-containing neurons project to the hippocampat formation. The question of GAL as a modulator of cholinergic transmission in this projection is discussed.

Although a number of pharmacological studies have shown the involvement of tachykinin type 2 receptors (NK2r) in the regulation of human colonic motility, few data are available so far on their pattern of expression. In this study this pattern was investigated in the myenteric plexuses, the longitudinal and circular muscle layers (external muscular layers), and the interstitial cells of Cajal (ICCs) using confocal microscopy immunofluorescence methods. NK2r immunoreactivity (NK2r-IR) was detected in the soma of myenteric neurons and in nerve varicosities located in myenteric plexuses as well as in external muscular layers. Colocalization analysis of NK2r-IR and synaptophysin-IR, showed significant regional differences in the distribution of NK2r-expressing nerve varicosities, the rate of occurrence was found to be 56.08% Ϯ 3% (mean Ϯ SE) in the external muscular layers and 30.22% Ϯ 1% (mean Ϯ SE) in the myenteric plexuses. NK2r-IR was found in membranes of most muscle cells previously incubated with a selective NK2r agonist, [␤-Ala 8 ] neurokinin A fragment 4-10, at 4°C, and then mainly relocated in the cytoplasm when heated to 37°C. A number of NK2r-IR nerve varicosities were close to NK2r-expressing neurons and muscle cells. Some of NK2r-expressing neurons and nerves were tachykinin-IR. No NK2r-IR was detected in ICCs. The present data indicate that presynaptic and postsynaptic neuroneuronal and neuromuscular regulatory processes mediated by tachykinins via NK2r may occur for modulating human colonic motility.

In this work we discuss how to use photophysical information for improved quantitative measurements using Photo Activated Localization Microscopy (PALM) imaging. We introduce a method that reliably estimates the number of photoblinking molecules present in a biological sample and gives a robust way to quantify proteins at the single-cell level from PALM images. We apply this method to determine the amount of b2 adrenergic receptor, a prototypical G Protein Coupled Receptor, expressed on the plasma membrane of HeLa cells.

Maltais, Stephane, Stephane C&e, Guy Drolet and Pierre Falardeau: Cellular colocalization of dopamine Dl mRNA and D2 receptor in rat brain using a D2 dopamine receptor specific polyclonal antibody. Pvog. Neuro-Psychoph~mt~co~. Viol. P~ychiuZ., 2OOO,a, pp. 1127-I 149.02000 Elsevier Science Inc. The main objective of this work was to investigate the extent of cellular colocalization of dopamine Dl and D2 receptors in the rat brain. A double labeling technique, that combined immunocytochemical labeling of the D2 receptor using polyclonal antibodies raised against the third intracellular loop of the short isoform of the human D2 receptor in combination with in situ hybridization detecting Dl mRNA expression, was designed to accomplish this goal. The specificity of the antisera obtained was confirmed by immunoprecipitation assay, Western blot analysis, and immunocytochemistry on D2R transfected cells and murine brain tissue. Western blot using the D2 receptor antibody revealed a specific broad band centered at 67 kDa in transfected cells and a major protein of 88 kDa corresponding to D2R expressed in the caudate-putamen, to a lesser extent in the cortex, and not at all detected in the hypothalamic region. The content of neurons double-labeled for Dln>2 receptors was observed at in differing intensities in the dorsal endopiroform nucleus, the intercalated nucleus of amygdala, the anterior part of the cortical nucleus amygdala, the nucleus of the lateral olfactory tract, the piriform cortex, the parabrachial nucleus, the supraoptic nucleus and the parabigeminal nucleus. All other regions of the brain revealed neurons expressing either Dl or D2 dopamine receptors but not both at that same time. These results clearly demonstrated that specific neurons expressed both receptors Dl and D2, and that this colocalization was restricted to particular regions of the rat brain.

The central control of growth hormone (GH) secretion from the pituitary gland is ultimately achieved by the interaction between two hypothalamic neurohormones, somatostatin which inhibits and growth hormone-releasing hormone (GHRH) which stimulates GH release. The regulation of the somatostatin and GHRH release from the hypothalamus is regulated by a range of other neuropeptides, neurotransmitters, neurohormones. In this mini review we attempt to provide a short summary covering the anatomy and chemical characteristics of the various cell populations regulating GH secretion as a tribute to Miklós Palkovits who pioneered the field of functional neuroanatomy of hypothalamic networks.

Photosensitizing properties of 5,10,15,20-tetrakis(4-hydroxyphenyl)porphyrin (p-THPP) functionalized by covalent attachment of one chain of poly(ethylene glycol) (PEG) with a molecular weight of 350, 2000, or 5000 Da (p-THPP-PEG 350 , p-THPP-PEG 2000 , p-THPP-PEG 5000) were studied in vitro. Dark and photo cytotoxicity of these photosensitizers delivered in solution or embedded in liposomes were evaluated on two cell lines: a human colorectal carcinoma cell line (HCT 116) and a prostate cancer cell line (DU 145), and compared with these treated with free p-THPP. The attachment of PEG chains results in the pronounced reduction of the dark cytotoxicity of the parent porphyrin. Cell viability tests have demonstrated that the phototoxicity of pegylated porphyrins is dependent on the length of PEG chain and p-THPP-PEG 2000 exhibited the highest photodynamic efficacy for both cell lines. The encapsulation into liposomes did not improve the PDT effect. However, the liposomal formulation of p-THPP-PEG 2000 showed a greater tendency to induce apoptosis in both cell lines than the parent or pegylated porphyrin delivered in solution. The colocalization of p-THPP, p-THPP-PEG 2000 and p-THPP-PEG 2000 enclosed in liposomes with fluorescent markers for lysosomes, mitochondria, endoplasmatic reticulum (ER) and Golgi apparatus (GA) was determined in the HCT 116 line. The p-THPP exhibited ubiquitous intracellular distribution with a preference for membranes: mitochondria, ER, GA, lysosomes and plasma membrane. Fluorescence of p-THPP-PEG 2000 was observed within the cytoplasm, with a stronger signal detected in membranous organelle: mitochondria, ER, GA and lysosomes. In contrast, p-THPP-PEG 2000 delivered in liposomes gave a distinct lysosomal pattern of localization.

h i g h l i g h t s A new immunohistochemical method was developed to detect B[a]P and TCDD in worms. Cryostat sections of E. andrei were reacted with antibodies for PAHs and dioxins. Immunofluorescence was observed even at minimal concentrations of the test chemicals (ppb). The gut has proved to be the major route of toxicant absorption in exposed organisms. Double immunolabelling demonstrated the lysosomal accumulation of the test chemicals.

The mammalian lung may have an important endocrine function besides being involved in gas exchange mechanisms. A number of peptide hormones have been localized to neurons and endocrine cells in the lung where they may contribute to the regulation of local pulmonary functions. We have investigated the presence of calcitonin gene-related peptide (CGRP), in the hamster lung by radioimmunoassay and by immunocytochemistry. Measurable quantities of CGRP were detected in lung tissue. Females had higher lung tissue levels of CGRP-like immunoreactivity (IR) than males. This was not reflected in an observable increase in the intensity or distribution of CGRP-like reactivity with immunocytochemistry. Distinct CGRP-like IR was recorded in clustered (NEB) and solitary (NEC) neuroendocrine cells in neonates, weanlings and adults, including all airways from trachea (NEC only) to bronchi, bronchioles, and alveolar ducts to the level of alveoli (NEC and NEB). In adult hamsters, there seemed to be fe...

The coexistence of 7-aminobutyric acid (GABA) and glycine in axon terminals impinging on rat abducens motoneurones was investigated using a double staining procedure combining retrograde labelling of the motoneurones with HRP and post-embedding immunocytochemical staining of axon terminals. Adjacent ultrathin sections of cell bodies of identified motoneurones were individually treated with GABA or glycine antibodies. The terminals single labelled for GABA represented 11.4% of the terminals analyzed, while 8% of them were glycine immunoreactive and 9% were both GABA and glycine immunoreactive. All the labelled terminals contained pleomorphic vesicles. The mean length of apposition of the double labelled terminals was statistically larger (2.20 _+ 0.97/~m) than the GABA (1.65-+ 0.57/~m) or glycine immunoreactive ones (1.37 + 0.35/~m).

öIn brain, signaling pathways initiated by atrial natriuretic peptide, or transmitters which stimulate nitric oxide synthesis, increase cGMP as their second messenger. One important class of target molecules for cGMP is cGMPdependent protein kinases, and in the present study, biochemical and immunocytochemical analyses demonstrate the widespread distribution of type II cGMP-dependent protein kinase in rat brain, from the cerebral cortex to the brainstem and cerebellum. Also, colocalization of cGMP-dependent protein kinase type II with its activator, cGMP, was found in several brain regions examined after in vitro stimulation of brain slices with sodium nitroprusside. In western blots, cGMP-dependent protein kinase type II was observed in all brain regions examined, although cerebellar cortex and pituitary contained comparatively less of the kinase. Immunocytochemistry revealed cGMP-dependent protein kinase type II in certain neurons, and occasionally in putative oligodendrocytes and astrocytes, however, its most striking and predominant localization was in neuropil. Electron microscopy examination of neuropil in the medial habenula showed localization of the kinase in both axon terminals and dendrites. As a membrane-associated protein, cGMP-dependent protein kinase type II often appeared to be transported to cell processes to a greater extent than being retained in the cell body. Thus, immunocytochemical labeling of cGMP-dependent protein kinase type II often did not coincide with the localization of kinase mRNA previously observed by others using in situ hybridization. We conclude that in contrast to cGMP-dependent protein kinase type I, which has a very restricted localization to cerebellar Purkinje cells and a few other sites, cGMP-dependent protein kinase type II is a very ubiquitous brain protein kinase and thus a more likely candidate for relaying myriad cGMP e¡ects in brain requiring protein phosphorylation.

The distribution of gonadotropin-releasing hormone (GnRH)-immunoreactive (ir) cells in the preoptic area (POA) of the dwarf gourami, Colisa lalia, was immunohistochemically studied. These neurons form cell columns on both sides of the common ventricle, and their axons project to the pituitary gland by forming distinct bundles. Also examined was the distribution of isotocin (IST) cells in the POA by using an anti-oxytocin (OXT) serum which has been proven to crossreact with IST. These two kinds of immunoreactive cells were distributed quite similarly in the POA. However, by using an immunofluorescence double-labeling method on thinner sections we found that a population of small IST cells in the ventral POA were also immunoreactive to GnRH, but that large IST cells in the dorsal POA were not immunoreactive to GnRH, and small GnRH-ir cells in the most ventral POA were not immunoreactive to the OXT antiserum. In the pituitary gland, GnRH-ir fibers were found in both the neurohypophysis and proximal pars distalis, but IST fibers were found only in the neurohypophysis.

The interaction between S100 and annexin proteins was initially identified in porcine intestinal brush border-derived membranes, as a complex formed between S100A10 and annexin A2. Annexin A2 (previously named p36 or calpactin I, etc.) is a substrate of srcrelated viral tyrosine kinase [1,2], which raises the possibility that this complex may be involved in cancer-related pathology. The complex of S100A10 and annexin A2 (S100A10-annexin A2 complex) has been found to bind to cytoskeletal components and to colocalize in submembranous compartments , suggesting that this complex may play a role in subcellular vesicle organization to exert its biological function.

Melanin-concentrating hormone (MCH)-containing neurons have recently been localized in the dorsolateral region of the rat hypothalamus, an area where the second a-MSH system is found which contains only a-MSH and none of the pro-opiomelanocortin (POMC)-related peptides. In order to study the morphological relationships between the MCH and a-MSH neuronal systems, we have studied the immunocytochemical localization of both MCH and a-MSH in the rat hypothalamus. The same study was also performed in the human hypothalamus where there is only one a-MSH system which contains a-MSH as well as the other POMC-related peptides (first a-MSH system), In the rat dorsolateral hypothalamus, we could demonstrate that most neuronal cell bodies stained for MCH also contained immunoreactive a-MSH. In the human hypothalamus, neuronal cell bodies stained for MCH were observed only in the periventricular area whereas cell bodies containing a-MSH were exclusively located in the infundibular (arcuate) nucleus. In the rat, immunoelectron microscopy showed labelling for MCH in the dense core vesicles of positive neurons and double-staining techniques clearly demonstrated that both immunoreactive MCH and a-MSH could be consistently detected in the same dense core vesicles. These ultrastructurai studies then suggest that these two peptides should be released simultaneously from neurons located in the rat dorsolateral hypothalamus.

It is widely recognized that the accuracy of colocalization measurements is dependent upon the quality of the source images. We demonstrate that, as the image quality increases, the measured colocalization, using the Pearson and Spearman rank correlation coefficients, approaches the true colocalization asymptotically. This means that in practice it is difficult to obtain images of sufficient quality for accurate measurements. We introduce the replicate-based noise corrected correlation (RBNCC) which aligns the measured colocalization with the true colocalization: a noise measurement is made for each fluorophore from a pair of replicate images, the two noise measurements are combined to generate a correction factor which is applied to the measured colocalization between the two fluorophores. In consequence, accurate measurements can be obtained even with noisy images, making RBNCC especially attractive for live imaging. Even with images of apparently good quality we found an average discrepancy of about 20% between the measured and corrected colocalization. A case is made for using the Spearman rank coefficient instead of the Pearson coefficient to measure colocalization.