Mapping the distributions and quantifying the labelling intensities of cell compartments by immunoelectron microscopy: progress towards a coherent set of methods

An important tool in cell biology is the combination of immunogold labelling and transmission electron microscopy (TEM) by which target molecules (e.g. antigens) are bound specifically to affinity markers (primary antibodies) and then detected and localised with visualisation probes (e.g. colloidal gold particles bound to protein A). Gold particles are electron-dense, punctate and available in different sizes whilst TEM provides high-resolution images of particles and cell compartments. By virtue of these properties, the combination can be used also to quantify one or more defined targets in cell compartments. During the past decade, new ways of quantifying gold labelling within cells have been devised. Their efficiency and validity rely on sound principles of specimen sampling, event counting and inferential statistics. These include random selection of items at each sampling stage (e.g. specimen blocks, thin sections, microscopical fields), stereological analysis of cell ultrastructure, unbiased particle counting and statistical evaluation of a suitable null hypothesis (no difference in the intensity or pattern of labelling between compartments or groups of cells). The following approaches are possible: (i) A target molecule can be tested for preferential labelling by mapping the localisation of gold particles across a set of compartments. (ii) Data from wild-type and knockdown/knockout control cells can be used to correct raw gold particle counts, estimate specific labelling densities and then test for preferential labeling. (iii) The same antigen can be mapped in two or more groups of cells to test whether there are experimental shifts in compartment labelling patterns. (iv) A variant of this approach uses more than one size of gold particle to test whether or not different antigens colocalise in one or more compartments. (v) In studies involving antigen translocation, absolute numbers of gold particles can be mapped over compartments at specific positions within polarised, oriented or dividing cells. Here, the current state of the art is reviewed and approaches are illustrated with virtual datasets.     

The impact of more visible standard drink labelling on youth alcohol consumption: Helping young people drink (ir)responsibly?

Introduction and Aims . In response to increasing concerns about excessive drinking among young people the Australian alcohol industry announced that it will introduce more visible standard drink labels. This study sought to examine whether young people use this information in a way that decreases, or increases, alcohol‐related harms. Design and Methods. Six focus groups with students enrolled in an undergraduate university course in a large regional city in New South Wales, recruited by direct approach on the university grounds and via an online message posted on the university bulletin board. Results: The majority of the participants reported that they are aware of the existence of standard drink labelling; notice standard drink labels; and take these into account when choosing what to purchase. However, this was predominantly to help them choose the strongest drinks for the lowest cost. Discussion and Conclusions. This study provides initial evidence to support the view that standard drink labelling, in isolation of other modifications to product packaging and marketing, is likely to serve to further increase heavy drinking among young people.[Jones SC, Gregory P. The impact of more visible standard drink labelling on youth alcohol consumption: Helping young people drink (ir)responsibly? Drug Alcohol Rev 2009]     

Dye Labelling of Nucleosides

Dye‐labelled nucleosides were obtained in 30–79% (average 45%) yields by treating N‐(4‐arylazobenzoyl)‐1H‐benzotriazoles 3a–b with appropriate nucleosides. Similarly, 3a–b afforded dye‐labelled threoninol conjugates in 55–89% (average 67%) yields. All novel products were characterized by NMR and elemental analysis.     

Histological changes and involvement of apoptosis after photodynamic therapy for actinic keratoses

BACKGROUND: Photodynamic therapy (PDT), which employs a combination of a tumour-localizing photosensitizer and visible light, has been used to treat superficial malignancies in the epidermis. OBJECTIVES: To examine histological changes and the role of apoptosis in lesions of actinic keratosis (AK) after PDT using 5-aminolaevulinic acid (ALA) and excimer dye laser. METHODS: After topical ALA-PDT, biopsy specimens were collected from 18 skin lesions in 15 patients with AK. Paraffin-embedded sections of the skin specimens were stained with haematoxylin and eosin. The detection of apoptosis was performed using a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling (TUNEL) method, antiactivated caspase-3 antibody and anti-Fas antibody. RESULTS: One hour after PDT, cells with eosinophilic cytoplasm and markedly stained nuclei were found, and vacuolation of some tumour cells was noted in the lower layer of the epidermis. An infiltrate of lymphocytes and neutrophils was observed in the upper layer of the dermis. One day after PDT, all layers of the epidermis exhibited slightly degenerative necrosis, with shadow cell formation and chromatin condensation around the nuclear membrane in the lower layer of the epidermis. Necrosis in all layers of the epidermis and lymphocyte infiltration in the dermis were found 3 days after PDT. Tumour cells had disappeared and regenerative thickening of the epidermis was observed 7 days after PDT. TUNEL staining revealed apoptosis-positive cells in the epidermis in 8 of 11 specimens obtained 1 day after PDT. Activated caspase-3 expression was noted in the lower layer of the epidermis in four of these eight TUNEL-positive specimens. CONCLUSIONS: Results suggested that apoptosis is involved in tumour cell death after PDT in patients with AK, and that it occurs within 1 day after PDT.     

Capsaicin-sensitive extrinsic afferents are involved in acid-induced activation of distinct myenteric neurons in the rat stomach

Challenge of the rat gastric mucosa with 0.5 mol L(-1) HCl activates nitrergic neurons in the myenteric plexus as visualized by c-Fos immunohistochemistry. In the present study, we characterized the activated neurons more extensively by their chemical coding and investigated whether a neural pathway that involves capsaicin-sensitive extrinsic afferents and/or cholinergic neurons transmitting via nicotinic receptors contributes to the activation of myenteric neurons. In multiple labelling experiments, c-Fos was examined for co-localization with nitric oxide synthase (NOS), vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), enkephalin (ENK), gastrin-releasing peptide (GRP), substance P (SP), calbindin D-28k (CALB) and neurofilament 145 (NF 145). All c-Fos-positive neurons were immunoreactive for NOS, VIP, NPY and NF 145, but not for SP, ENK, GRP and CALB. Nerve fibres co-expressing NOS, VIP and NPY were predominantly found in the external muscle layer and in the muscularis mucosae but rarely in the mucosa. Pre-treatment with capsaicin or hexamethonium or a combination of both pre-treatments reduced HCl-induced c-Fos expression by 54, 66 and 63%, respectively. Acid challenge of the stomach, therefore, leads to activation of presumably inhibitory motor neurons responsible for muscle relaxation. Activation of these neurons is partly mediated by capsaicin-sensitive afferents and involves ganglionic transmission via nicotinic receptors.     

Rhenium-188 and technetium-99m nitridobis(N-ethoxy-N-ethyldithiocarbamate) leucocyte labelling radiopharmaceuticals: [ReN(NOET)2] and [TcN(NOET)2], NOET = Et(EtO)NCS2: Their in vitro localization and chemical behaviour

In this study, we have investigated the preparation of rhenium-188 nitridobis(N-ethoxy-N-ethyldithiocarbamate) [¹⁸⁸ReN(NOET)₂] (NOET = Et(EtO)NCS₂), analogous to the known technetium-99m radiopharmaceutical. The new ¹⁸⁸Re complex was synthesized in good yield with a satisfactory radiochemical purity, using a kit method. The subcellular localization of both radiopharmaceuticals in granulocytes was observed by microautoradiography. The uptake was independent of the radionuclide and predominantly nuclear. Furthermore, HPLC was used to characterize the ^99mTc complex before and after blood cell labelling and revealed that the intact radiopharmaceutical was involved.     

The lacteridian reticular thalamic nucleus projects topographically upon the dorsal thalamus: Experimental study in Gallotia galloti

The projection pattern of the ventral thalamic reticular nucleus onto the dorsal thalamus was studied in the lizard Gallotia gallotiusing in vitro horseradish peroxidase and fluorescent carbocyanine labelling techniques. Localized label deposits at three dorsoventrally spaced sites in the dorsal thalamus elicited retrograde transport into separate, though partly overlapping, medial, dorsolateral and ventrolateral sectors within an extended cytoarchitectonic complex which may be globally identifiable as the reticular nucleus. Neurons found in the dorsolateral and ventrolateral sectors mainly corresponded to the cell group named nucleus ventromedialis (or nucleus of the dorsal supraoptic decussation) in the literature, whereas neurons labelled in the medial sector corresponded to the so-called dorsal hypothalamic nucleus. Sparser cells appear labelled in the superficially placed nucleus suprapeduncularis. Thalamotelencephalic fibers arising from the injected dorsal thalamic nuclei also project on the corresponding retrogradely labeled sectors within the reticular nucleus. These findings reveal a rough topographic organization in the connections of the extended reticular nucleus complex with the whole dorsal thalamus. This supports the hypothesis of hodological homology between this ventral thalamic formation in Gallotiaand the mammalian thalamic reticular nucleus. © 1994 Wiley-Liss, Inc.     

Afferent and efferent connections of the habenula in the larval sea lamprey (Petromyzon marinus L.): An experimental study

The habenula is an integrative center between the striatum and the limbic and motor systems. With the aim of achieving further understanding of the evolution of this structure in vertebrates, we carried out an experimental study of the afferent and efferent connections of the habenula of larval sea lamprey. Experimental procedures included in vivo and in vitro transport after injections of horseradish peroxidase (HRP) into the habenula, telencephalon, pineal organ, dorsal thalamus, and posterior tubercle as well as carbocyanine dye tracing (DiI). The combined results of these experiments showed that the pattern of habenular connections is very simple. Most afferents appear to originate from the lobus subhippocampalis and neighboring area, whereas the only efferents found coursed in the fasciculus retroflexus to the neuropil of the nucleus interpeduncularis. This neuropil comprises a commissural region in the rostral mesencephalon, two long bilateral areas extending in the basal mesencephalon and medulla oblongata to the trigeminal level, and, finally, a caudal commissural zone. The conspicuous habenular commissure contains interhemispheric fibers that appear to form occasional contacts within the habenulae. The lamprey habenula also receives a few immunocytochemically identified fibers (somatostatinergic, catecholaminergic, and serotoninergic fibers) from other sources. © 1994 Wiley-Liss, Inc.     

Morphological and electrophysiological properties of pelvic ganglion cells in the rat

Intracellular recording and dye injection were used to study the morphological and electrophysiological properties of rat pelvic ganglion cells. The dye-injected cells measured on the average 37 micron by 22.5 micron and had a mean number of 1.5 primary processes (axon and dendrites). The cells received unmyelinated preganglionic inputs from either the pelvic (parasympathetic) or the hypogastric (sympathetic) nerves, but no cells received inputs from both nerves. The number of synaptic inputs to each cell varied between 1 and 5 with a mean of 2. Each cell had at least one large amplitude suprathreshold EPSP which always initiated an action potential. These properties, namely, morphological simplicity, small number of inputs, security of synaptic transmission and lack of convergence between sympathetic and parasympathetic inputs, suggest that the capacity for synaptic modulation and integration in this ganglion is minimal. Such a structure should therefore relay preganglionic information to target organs with little or no alteration.