A dark quencher genetically encodable voltage indicator (dqGEVI) exhibits high fidelity and speed.

Voltage sensing with genetically expressed optical probes is highly desirable for large-scale recordings of neuronal activity and detection of localized voltage signals in single neurons. Most genetically encodable voltage indicators (GEVI) have drawbacks including slow response, low fluorescence, or excessive bleaching. Here we present a dark quencher GEVI approach (dqGEVI) using a Förster resonance energy transfer pair between a fluorophore glycosylphosphatidylinositol-enhanced green fluorescent protein (GPI-eGFP) on the outer surface of the neuronal membrane and an azo-benzene dye quencher (D3) that rapidly moves in the membrane driven by voltage. In contrast to previous probes, the sensor has a single photon bleaching time constant of ∼40 min, has a high temporal resolution and fidelity for detecting action potential firing at 100 Hz, resolves membrane de- and hyperpolarizations of a few millivolts, and has negligible effects on passive membrane properties or synaptic events. The dqGEVI approach should be a valuable tool for optical recordings of subcellular or population membrane potential changes in nerve cells.

Sensitive fluorescent hybridisation protocol development for simultaneous detection of microRNA and cellular marker proteins (in the retina).

Nowadays, increasing number of microRNAs are found to have crucial roles in various physiological processes through gene expression regulation via RNA silencing as a result of base pairing with complementary mRNA sequences. To reveal the spatial distribution of microRNA expression in tissues, in situ hybridisation is the only method developed to date. This work aims to provide a novel approach to obtain information on the possible involvement of microRNA-s in regulatory processes under experimental conditions by enhancing fluorescent detection of microRNA labelling. Developing Wistar rats were used as a model system to analyse retinal microRNA expression in the first 3 postnatal weeks. Using cryosections, the crucial elements of optimal labels were (1) the concentration and duration of proteinase K treatment, (2) hybridisation temperature of microRNA probes and (3) temperature of stringency washes. Further improvements made possible to combine our in situ hybridisation protocol with double-label immunofluorescence allowing for the simultaneous detection of microRNA-s with high sensitivity and a neuronal cell marker and/or a synaptic marker protein. Thus, the regulatory microRNA-s can be localised in an identified cell type along with its potential target protein. We believe that our protocol can be easily adapted for a variety of tissues of different origins, developmental stages and experimental conditions.

Local upregulation of transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1 ion channels in rectosigmoid deep infiltrating endometriosis.

Transient Receptor Potential Vanilloid 1 (TRPV1) and Transient Receptor Potential Ankyrin 1 (TRPA1) expressed mainly by primary sensory neurons function as major nociceptive integrators. They are also present on the rat endometrium in an oestrogen-regulated manner. TRPV1 is upregulated in peritoneal and ovarian endometriosis patients, but there is no information about TRPA1 and their pathophysiological significances. In this study, patients undergoing laparoscopic surgery were investigated: severe dysmenorrhoea due to rectosigmoid deep infiltrating endometriosis ( n = 15), uterine fibroid-induced moderate dysmenorrhoea ( n = 7) and tubal infertility with no pain ( n = 6). TRPA1 and TRPV1 mRNA and protein expressions were determined by quantitative polymerase chain reaction and semi-quantitative immunohistochemistry from the endometrium samples taken by curettage. Results were correlated with the clinical characteristics including pain intensity. TRPA1 and TRPV1 receptors were expressed in the healthy human endometrium at mRNA and protein levels. Sparse, scattered cytoplasmic TRPA1 and TRPV1 immunopositivities were found in the stroma and epithelial layers. We detected upregulated mRNA levels in deep infiltrating endometriosis lesions, and TRPV1 gene expression was also elevated in autocontrol endometrium of deep infiltrating endometriosis patients. Histological scoring revealed significant TRPA1 and TRPV1 difference between deep infiltrating endometriosis stroma and epithelium, and in deep infiltrating endometriosis epithelium compared to control samples. Besides, we measured elevated stromal TRPV1 immunopositivity in deep infiltrating endometriosis. Stromal TRPA1 and TRPV1 immunoreactivities strongly correlated with dysmenorrhoea severity, as well TRPV1 expression on ectopic epithelial cells and macrophages with dyspareunia. Epithelial TRPA1 and stromal TRPV1 immunopositivity also positively correlated with dyschezia severity. We provide the first evidence for the presence of non-neuronal TRPA1 receptor in the healthy human endometrium and confirm the expression of TRPV1 channels. Their upregulations in rectosigmoid deep infiltrating endometriosis lesions and correlations with pain intensity suggest potential roles in pathophysiological mechanisms of the disease.

Protective effects of the neuropeptide PACAP in diabetic retinopathy.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with highly potent neurotrophic and neuroprotective effects. PACAP and its receptors occur in the retina and PACAP has been applied in animal models of metabolic retinal disorders to reduce structural and functional damage. Furthermore, PACAP has been implicated as a potential anti-diabetic peptide. Our aim has been to investigate, by using a complex morphological, immunochemical and molecular biological approach, whether PACAP attenuates diabetic retinopathy. Diabetes was induced in rats with a single streptozotocin injection. PACAP was injected intravitreally into one eye (100 pmol) three times during the last week of a 3-week survival period. Retinas were processed for the following procedures: routine histology, immunohistochemistry (single and double labeling, whole-mount), quantitative reverse transcription with the polymerase chain reaction and Western blotting. Cone photoreceptors and dopaminergic amacrine and ganglion cells degenerated in diabetic retinas and glial fibrillary acidic protein were upregulated in Müller glial cells. The number of cones, the length of their outer segments and the cell number in the ganglion cell layer were decreased. PACAP ameliorated these structural changes. Moreover, PACAP increased the levels of PAC1-receptor and tyrosine-hydroxylase as detected by molecular biological methods. Thus, PACAP has significant protective effects in the diabetic retina. PACAP treatment attenuates neuronal cell loss in diabetic retinopathy, the protective effects of PACAP probably being mediated through the activation of PAC1-receptor. These results suggest that PACAP has a therapeutic potential in diabetic retinopathy.

Bringing to light the physiological and pathological firing patterns of human induced pluripotent stem cell-derived neurons using optical recordings.

Human induced pluripotent stem cells (hiPSCs) are a promising approach to study neurological and neuropsychiatric diseases. Most methods to record the activity of these cells have major drawbacks as they are invasive or they do not allow single cell resolution. Genetically encoded voltage indicators (GEVIs) open the path to high throughput visualization of undisturbed neuronal activity. However, conventional GEVIs perturb membrane integrity through inserting multiple copies of transmembrane domains into the plasma membrane. To circumvent large add-ons to the plasma membrane, we used a minimally invasive novel hybrid dark quencher GEVI to record the physiological and pathological firing patterns of hiPSCs-derived sensory neurons from patients with inherited erythromelalgia, a chronic pain condition associated with recurrent attacks of redness and swelling in the distal extremities. We observed considerable differences in action potential firing patterns between patient and control neurons that were previously overlooked with other recording methods. Our system also performed well in hiPSC-derived forebrain neurons where it detected spontaneous synchronous bursting behavior, thus opening the path to future applications in other cell types and disease models including Parkinson's disease, Alzheimer's disease, epilepsy, and schizophrenia, conditions associated with disturbances of neuronal activity and synchrony.

Presence of pituitary adenylate cyclase-activating polypeptide (PACAP) in the plasma and milk of ruminant animals.

Milk contains a variety of proteins and peptides that possess biological activity. Growth factors, such as growth hormone, insulin-like, epidermal and nerve growth factors are important milk components which may regulate growth and differentiation in various neonatal tissues and also those of the mammary gland itself. We have recently shown that pituitary adenylate cyclase-activating polypeptide (PACAP), an important neuropeptide with neurotrophic actions, is present in the human milk in much higher concentration than in the plasma of lactating women. Investigation of growth factors in the milk of domestic animals is of utmost importance for their nutritional values and agricultural significance. Therefore, the aim of the present study was to determine the presence and concentration of PACAP in the plasma and milk of three ruminant animal species. Furthermore, the presence of PACAP and its specific PAC1 receptor were investigated in the mammary glands. Radioimmunoassay measurements revealed that PACAP was present in the plasma and the milk of the sheep, goat and the cow in a similar concentration to that measured previously in humans. PACAP38-like immunoreactivity (PACAP38-LI) was 5-20-fold higher in the milk than in the plasma samples of the respective animals, a similar serum/milk ratio was found in all the three species. The levels did not show significant changes within the examined 3-month-period of lactation after delivery. Similar PACAP38-LI was measured in the homogenates of the sheep mammary gland samples taken 7 and 30 days after delivery. PAC1 receptor expression was detected in these udder biopsies by fluorescent immunohistochemistry suggesting that this peptide might have an effect on the mammary glands themselves. These data show that PACAP is present in the milk of various ruminant domestic animal species at high concentrations, the physiological implications of which awaits further investigation.

Accelerated retinal aging in PACAP knock-out mice.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurotrophic and neuroprotective peptide. PACAP and its receptors are widely distributed in the retina. A number of reports provided evidence that PACAP is neuroprotective in retinal degenerations. The current study compared retina cell type-specific differences in young (3-4months) and aged adults (14-16months), of wild-type (WT) mice and knock-out (KO) mice lacking endogenous PACAP production during the course of aging. Histological, immunocytochemical and Western blot examinations were performed. The staining for standard neurochemical markers (tyrosine hydroxylase for dopaminergic cells, calbindin 28 kDa for horizontal cells, protein kinase Cα for rod bipolar cells) of young adult PACAP KO retinas showed no substantial alterations compared to young adult WT retinas, except for the specific PACAP receptor (PAC1-R) staining. We could not detect PAC1-R immunoreactivity in bipolar and horizontal cells in young adult PACAP KO animals. Some other age-related changes were observed only in the PACAP KO mice only. These alterations included horizontal and rod bipolar cell dendritic sprouting into the photoreceptor layer and decreased ganglion cell number. Also, Müller glial cells showed elevated GFAP expression compared to the aging WT retinas. Furthermore, Western blot analyses revealed significant differences between the phosphorylation state of ERK1/2 and JNK in KO mice, indicating alterations in the MAPK signaling pathway. These results support the conclusion that endogenous PACAP contributes to protection against aging of the nervous system.

Estrogen-dependent up-regulation of TRPA1 and TRPV1 receptor proteins in the rat endometrium.

Transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) receptors expressed predominantly in sensory nerves are activated by inflammatory stimuli and mediate inflammation and pain. Although they have been shown in the human endometrium, their regulation and function are unknown. Therefore, we investigated their estrogen- and progesterone-dependent alterations in the rat endometrium in comparison with the estrogen-regulated inflammatory cytokine macrophage migration inhibitory factor (MIF). Four-week-old (sexually immature) and four-month-old (sexually mature) female rats were treated with the non-selective estrogen receptor (ER) agonist diethylstilboestrol (DES), progesterone and their combination, or ovariectomized. RT-PCR and immunohistochemistry were performed to determine mRNA and protein expression levels respectively. Channel function was investigated with ratiometric [Ca(2+)]i measurement in cultured primary rat endometrial cells. Both TRP receptors and MIF were detected in the endometrium at mRNA and protein levels, and their localizations were similar. Immunostaining was observed in the immature epithelium, while stromal, glandular and epithelial positivity were observed in adults. Functionally active TRP receptor proteins were shown in endometrial cells by activation-induced calcium influx. In adults, Trpa1 and Trpv1 mRNA levels were significantly up-regulated after DES treatment. TRPA1 increased after every treatment, but TRPV1 remained unchanged following the combined treatment and ovariectomy. In immature rats, DES treatment resulted in increased mRNA expression of both channels and elevated TRPV1 immunopositivity. MIF expression changed in parallel with TRPA1/TRPV1 in most cases. DES up-regulated Trpa1, Trpv1 and Mif mRNA levels in endometrial cell cultures, but 17ß-oestradiol having ERα-selective potency increased only the expression of Trpv1. We provide the first evidence for TRPA1/TRPV1 expression and their estrogen-induced up-regulation in the rat endometrium in correlation with the MIF.

The taxon-specific paralogs of grapevine PRLIP genes are highly induced upon powdery mildew infection.

PRLIP (pathogenesis-related lipase) is a gene family encoding class 3 lipase-like proteins originally described and first characterized in Arabidopsis thaliana. Nine paralog genes of Arabidopsis can be separated into two groups based on expression characteristics and pathogen responses. Genes of Group 1 are clustered on chromosome 5 and show either high inducibility to different stress hormones and in response to pathogen attack or are undetectable at the transcript level. Group 2 contains the remaining genes, spread over the genome and are expressed constitutively in all the tissues tested. The aim of the present study was to determine the distribution of these two groups among plants, and to verify their differential expression. Orthologs of constitutively active members (Group 2) were found in all angiosperms, with available genome sequences. They are referred to as "core PRLIPs". In contrast, the gene cluster containing the pathogen-inducible PRLIPs (Group 1) was unique for Arabidopsis. Among other angiosperms, grapevine also possesses such a unique genome-specific group of PRLIP genes. To investigate whether these genes are also counterparts in pathogen responses, their expression pattern was tested under stress conditions. Two of the specific Vitis PRLIPs were highly induced in response to both powdery mildew infection and benzothiadiazole (BTH) treatment. Core Vitis PRLIPs, however, were not responsive to either pathogen attack or the chemical inducer. Our data provide insights into the distribution of a pathogenesis-related gene family in different plant lineages, and might reveal common characteristics with other inducible defense-related gene families.