The atypical 'hippocampal' glutamate receptor coupled to phospholipase D that controls stretch-sensitivity in primary mechanosensory nerve endings is homomeric purely metabotropic GluK2.

A metabotropic glutamate receptor coupled to phospholipase D (PLD-mGluR) was discovered in the hippocampus over three decades ago. Its pharmacology and direct linkage to PLD activation are well established and indicate it is a highly atypical glutamate receptor. A receptor with the same pharmacology is present in spindle primary sensory terminals where its blockade can totally abolish, and its activation can double, the normal stretch-evoked firing. We report here the first identification of this PLD-mGluR protein, by capitalizing on its expression in primary mechanosensory terminals, developing an enriched source, pharmacological profiling to identify an optimal ligand, and then functionalizing it as a molecular tool. Evidence from immunofluorescence, western and far-western blotting indicates PLD-mGluR is homomeric GluK2, since GluK2 is the only glutamate receptor protein/receptor subunit present in spindle mechanosensory terminals. Its expression was also found in the lanceolate palisade ending of hair follicle, also known to contain the PLD-mGluR. Finally, in a mouse model with ionotropic function ablated in the GluK2 subunit, spindle glutamatergic responses were still present, confirming it acts purely metabotropically. We conclude the PLD-mGluR is a homomeric GluK2 kainate receptor signalling purely metabotropically and it is common to other, perhaps all, primary mechanosensory endings.

Preventing yellow fever epidemics in Asian megacities: how can cities control mosquito-transmitted diseases?

The COVID-19 pandemic has reminded us of the ever present threat from infectious diseases, this includes the ones we know about already and future unknowns. The mosquito-transmitted disease yellow fever has claimed thousands of lives over the centuries and it hasn't gone away. It is still endemic in tropical areas of Africa and Latin America, where it is kept at bay through constant surveillance, mass vaccination campaigns and some natural immunity within local populations. Despite this there are serious outbreaks from time to time. The Aedes mosquitoes capable of transmitting the virus from person to person, are now widespread in warmer countries worldwide, moreover they thrive in urban areas. With increased international movement, the fear is that infected travellers could unwittingly introduce the virus into countries where people have little or no immunity. Densely populated Asian megacities are a major concern. There are simple measures citizens can take to protect themselves and their homes from the bite of infected mosquitoes, but city leaders must be at the forefront of a coordinated response bringing together diverse stakeholders to ensure a robust and sustainable defence.

The durability, functionality and acceptability of novel screened doors and windows after 4 years of use in a Gambian village: a cross-sectional survey.

BACKGROUND: The World Health Organization recommends house screening as a tool for malaria control, yet evidence of the long-term durability, functionality and acceptability of this intervention is lacking. In this study, the sustainability and use of novel types of screened doors and windows was examined 4 years after installation in a Gambian village. METHODS: A survey of 31 houses, each with two screened doors and two screened windows, was conducted in the rainy season. There were four types of screened door and two types of screened window. Trained staff carried out the survey and interviews of room owners were conducted in the local language before translation into English. RESULTS: Structurally, the manufactured doors and windows were highly durable and in excellent condition. Most doors shut smoothly 50/61 (82%), although only 25/61 (41%) shut fully automatically with the latch slotting into the hole on the frame and holding fast. Door locks were less robust, with only (24/61) 39% present and working. Blinds proved especially flimsy, with only 4/109 (4%) of door blinds and 10/56 (18%) of window blinds present and in working order. Householders hung curtains inside most doors 50/61 (82%) and in 26/61 (43%) of the windows. Front doors were commonly found propped open 21/31 (68%) and 23/27 (85%) of those with a front door curtain, put their curtains down at night. Doors and windows were well liked, 19/31 (61%) of respondents were happy with them because they kept mosquitoes out 14/31 (45%) and provided security 12/31 (39%). The main reason given for the use of curtains was to provide privacy 26/28 (93% of those with curtains), especially while the door was open or had 'see-through' panels. CONCLUSIONS: Overall, the screened doors and windows were in full-working order and undamaged after 4 years of use. The doors and windows were well liked, especially for their ability to reduce the entry of mosquitoes and for the security they afforded. Improvements to the lock design are needed before scale-up. Most householders hung curtains behind their doors for privacy. Installation of screening in buildings should be accompanied with recommendations that at night, when doors and windows are closed, curtains be lifted or drawn to one side-to improve ventilation and keep the house cool.

Distribution and morphology of sensory and autonomic fibres in the subendocardial plexus of the rat heart.

Cardiac reflexes originating from sensory receptors in the heart ensure blood supply to vital tissues and organs in the face of constantly changing demands. Atrial volume receptors are mechanically sensitive vagal afferents which relay to the medulla and hypothalamus, affecting vasopressin release and renal sympathetic activity. To date, two anatomically distinct sensory endings have been identified which may subserve cardiac mechanosensation: end-nets and flower-spray endings. To map the distribution of atrial receptors in the subendocardial space, we have double-labelled rat right atrial whole mounts for neurofilament heavy chain (NFH) and synaptic vesicle protein 2 (SV2) and generated high-resolution maps of the rat subendocardial neural plexus at the cavo-atrial region. In order to elucidate the nature of these fibres, double labelling with synaptophysin (SYN) and either NFH, calcitonin gene-related peptide (CGRP), choline acetyltransferase (ChAT) or tyrosine hydroxylase (TH) was performed. The findings show that subendocardial nerve nets are denser at the superior cavo-atrial junction than the mid-atrial region. Adluminal plexuses had the finest diameters and stained positively for synaptic vesicles (SV2 and SYN), CGRP and TH. These plexuses may represent sympathetic post-ganglionic fibres and/or sensory afferents. The latter are candidate substrates for type B volume receptors which are excited by stretch during atrial filling. Deeper nerve fibres appeared coarser and may be cholinergic (positive staining for ChAT). Flower-spray endings were never observed using immunohistochemistry but were delineated clearly with the intravital stain methylene blue. We suggest that differing nerve fibre structures form the basis by which atrial deformation and hence atrial filling is reflected to the brain.

Recommendations for building out mosquito-transmitted diseases in sub-Saharan Africa: the DELIVER mnemonic.

In sub-Saharan Africa, most transmission of mosquito-transmitted diseases, such as malaria or dengue, occurs within or around houses. Preventing mosquito house entry and reducing mosquito production around the home would help reduce the transmission of these diseases. Based on recent research, we make key recommendations for reducing the threat of mosquito-transmitted diseases through changes to the built environment. The mnemonic, DELIVER, recommends the following best practices: (i) Doors should be screened, self-closing and without surrounding gaps; (ii) Eaves, the space between the wall and roof, should be closed or screened; (iii) houses should be Lifted above the ground; (iv) Insecticide-treated nets should be used when sleeping in houses at night; (v) houses should be Ventilated, with at least two large-screened windows to facilitate airflow; (vi) Environmental management should be conducted regularly inside and around the home; and (vii) Roofs should be solid, rather than thatch. DELIVER is a package of interventions to be used in combination for maximum impact. Simple changes to the built environment will reduce exposure to mosquito-transmitted diseases and help keep regions free from these diseases after elimination. This article is part of the theme issue 'Novel control strategies for mosquito-borne diseases'.

Environmental temperature and growth faltering in African children: a cross-sectional study.

BACKGROUND: Child growth faltering persists in sub-Saharan Africa despite the scale-up of nutrition, water, and sanitation interventions over the past 2 decades. High temperatures have been hypothesised to contribute to child growth faltering via an adaptive response to heat, reduced appetite, and the energetic cost of thermoregulation. We did a cross-sectional study to assess whether child growth faltering is related to environmental temperature in sub-Saharan Africa. METHODS: Data were extracted from 52 Demographic and Heath Surveys, dating from 2003 to 2016, that recorded anthropometric data in children aged 0-5 years, and were linked with remotely sensed monthly mean daytime land surface temperature for 2000-16. The odds of stunting (low height-for-age), wasting (low weight-for-height), and underweight (low weight-for-age) relative to monthly mean daytime land surface temperature were determined using multivariable logistic regression. FINDINGS: The study population comprised 656 107 children resident in 373 012 households. Monthly mean daytime land surface temperature above 35°C was associated with increases in the odds of wasting (odds ratio 1·27, 95% CI 1·16-1·38; p<0·0001), underweight (1·09, 1·02-1·16; p=0·0073), and concurrent stunting with wasting (1·23, 1·07-1·41; p=0·0037), but a reduction in stunting (0·90, 0·85-0·96; p=0·00047) compared with a monthly mean daytime land surface temperature of less than 30°C. INTERPRETATION: Children living in hotter parts of sub-Saharan Africa are more likely to be wasted, underweight, and concurrently stunted and wasted, but less likely to be stunted, than in cooler areas. Studies are needed to further investigate the relationship between temperature and child growth, including whether there is a direct effect not mediated by food security, regional wealth, and other environmental variables. Rising temperature, linked to anthropogenic climate change, might increase child growth faltering in sub-Saharan Africa. FUNDING: UK Medical Research Council and UK Global Challenges Research Fund.

Piezo Is Essential for Amiloride-Sensitive Stretch-Activated Mechanotransduction in Larval Drosophila Dorsal Bipolar Dendritic Sensory Neurons.

Stretch-activated afferent neurons, such as those of mammalian muscle spindles, are essential for proprioception and motor co-ordination, but the underlying mechanisms of mechanotransduction are poorly understood. The dorsal bipolar dendritic (dbd) sensory neurons are putative stretch receptors in the Drosophila larval body wall. We have developed an in vivo protocol to obtain receptor potential recordings from intact dbd neurons in response to stretch. Receptor potential changes in dbd neurons in response to stretch showed a complex, dynamic profile with similar characteristics to those previously observed for mammalian muscle spindles. These profiles were reproduced by a general in silico model of stretch-activated neurons. This in silico model predicts an essential role for a mechanosensory cation channel (MSC) in all aspects of receptor potential generation. Using pharmacological and genetic techniques, we identified the mechanosensory channel, DmPiezo, in this functional role in dbd neurons, with TRPA1 playing a subsidiary role. We also show that rat muscle spindles exhibit a ruthenium red-sensitive current, but found no expression evidence to suggest that this corresponds to Piezo activity. In summary, we show that the dbd neuron is a stretch receptor and demonstrate that this neuron is a tractable model for investigating mechanisms of mechanotransduction.

Heterogeneity of histaminergic neurons in the tuberomammillary nucleus of the rat.

Histaminergic neurons of the hypothalamic tuberomammillary nuclei (TMN) send projections to the whole brain. Early anatomical studies described histaminergic neurons as a homogeneous cell group, but recent evidence indicates that histaminergic neurons are heterogeneous and organized into distinct circuits. We addressed this issue using the double-probe microdialysis in freely moving rats to investigate if two compounds acting directly onto histaminergic neurons to augment cell firing [thioperamide and bicuculline, histamine H(3)- and gamma-aminobutyric acid (GABA)(A)-receptor (R) antagonists, respectively] may discriminate groups of histaminergic neurons impinging on different brain regions. Intra-hypothalamic perfusion of either drug increased histamine release from the TMN and cortex, but not from the striatum. Thioperamide, but not bicuculline, increased histamine release from the nucleus basalis magnocellularis (NBM), bicuculline but not thioperamide increased histamine release from the nucleus accumbens (NAcc). Intra-hypothalamic perfusion with thioperamide increased the time spent in wakefulness. To explore the local effects of H(3)-R blockade in the histaminergic projection areas, each rat was implanted with a single probe to simultaneously administer thioperamide and monitor local changes in histamine release. Thioperamide increased histamine release from the NBM and cortex significantly, but not from the NAcc or striatum. The presence of H(3)-Rs on histaminergic neurons was assessed using double-immunofluorescence with anti-histidine decarboxylase antibodies to identify histaminergic cells and anti-H(3)-R antibodies. Confocal analysis revealed that all histaminergic somata were immunopositive for the H(3)-R. This is the first evidence that histaminergic neurons are organized into functionally distinct circuits that influence different brain regions, and display selective control mechanisms.

Molecular and biochemical pharmacology of the histamine H4 receptor.

The elucidation of the human genome has had a major impact on histamine receptor research. The identification of the human H4 receptor by several groups has been instrumental for a new appreciation of the role of histamine in the modulation of immune function. In this review, we summarize the historical developments and the molecular and biochemical pharmacology of the H4 receptor.

Cloning and characterization of dominant negative splice variants of the human histamine H4 receptor.

The H(4)R (histamine H(4) receptor) is the latest identified member of the histamine receptor subfamily of GPCRs (G-protein-coupled receptors) with potential functional implications in inflammatory diseases and cancer. The H(4)R is primarily expressed in eosinophils and mast cells and has the highest homology with the H(3)R. The occurrence of at least twenty different hH(3)R (human H(3)R) isoforms led us to investigate the possible existence of H(4)R splice variants. In the present paper, we report on the cloning of the first two alternatively spliced H(4)R isoforms from CD34+ cord blood-cell-derived eosinophils and mast cells. These H(4)R splice variants are localized predominantly intracellularly when expressed recombinantly in mammalian cells. We failed to detect any ligand binding, H(4)R-ligand induced signalling or constitutive activity for these H(4)R splice variants. However, when co-expressed with full-length H(4)R [H(4)R((390)) (H(4)R isoform of 390 amino acids)], the H(4)R splice variants have a dominant negative effect on the surface expression of H(4)R((390)). We detected H(4)R((390))-H(4)R splice variant hetero-oligomers by employing both biochemical (immunoprecipitation and cell-surface labelling) and biophysical [time-resolved FRET (fluorescence resonance energy transfer)] techniques. mRNAs encoding the H(4)R splice variants were detected in various cell types and expressed at similar levels to the full-length H(4)R((390)) mRNA in, for example, pre-monocytes. We conclude that the H(4)R splice variants described here have a dominant negative effect on H(4)R((390)) functionality, as they are able to retain H(4)R((390)) intracellularly and inactivate a population of H(4)R((390)), presumably via hetero-oligomerization.

Immunolocalization of histamine H3 receptors on endocrine cells in the rat gastrointestinal tract.

The histamine H3 receptor (H3R) has been identified in the gastrointestinal tract of the rat by immunohistochemistry, using the first validated anti-H3 receptor antibody. Immunoreactivity to H3R was exclusively localized to the endocrine cells scattered in the gastrointestinal mucosa, with positive cells being prominently abundant in the gastric fundus, while they were rarely found in the other regions. In the fundus, positive cells were distributed in the lower half of the mucosa and their number significantly decreased after a 24 h-fasting period. Double-labeling studies were undertaken to identify the H3R-immunoreactive cell types in the fundic and antral mucosa. The H3R-immunoreactive cells were positive for chromogranin A. In the fundus, approximately 90% of cells positive to H3R were also positive to the histamine-forming enzyme, histidine decarboxylase. None of the cells expressing H3R displayed immunoreactivity for gastrin, somatostatin or ghrelin. Location, the influence of food deprivation and colocalization with histidine decarboxylase indicate that H3R positive cells correspond to the enterochromaffin-like cells (ECL).

Human inflammatory dendritic epidermal cells express a functional histamine H4 receptor.

Expression of histamine H(4) receptor (H(4)R) on leukocytes suggests an immunomodulatory role of this receptor. Here we investigated the expression and function of H(4)R on human inflammatory dendritic epidermal cells (IDECs). H(4)R is expressed by IDEC of the skin. On monocyte-derived IDECs (Mo-IDECs), H(4)R is also expressed and upregulated by IFN-gamma. Functionally, histamine and H(4)R agonists clobenpropit and 4-methylhistamine downregulated the production of the Th2-linked chemokine CCL2 and the Th1 cytokine IL-12 on Mo-IDEC, whereas agonists for the other histamine receptors did not. An H(4)R-selective antagonist (JNJ7777120) blocked the effect of H(4)R agonists. Downregulation of CCL2 also led to a decreased migration of monocytes. Thus, IDEC express a functionally active H(4)R, which upon stimulation leads to downregulation of CCL2 and IL-12. This might have implications for the treatment of atopic dermatitis, since H(4)R agonists may have beneficial effects in downregulating inflammation.

Histamine downregulates monocyte CCL2 production through the histamine H4 receptor.

BACKGROUND: The expression of the recently cloned histamine H(4) receptor (H(4)R) by leukocytes suggests a role in immunomodulation. OBJECTIVE: The expression and function of the H(4)R on human monocytes obtained from peripheral blood was investigated. METHODS: H(4)R expression was studied by using flow cytometry. Effects of H(4)R stimulation on Ca(2+) mobilization was determined fluorometrically, CCL2 production was determined by means of ELISA, intracellular CCL2 staining was measured with flow cytometry, and CCL2 mRNA was measured by using real-time quantitative LightCycler PCR. The relevance of CCL2 production was determined in chemotaxis transmigration assays. RESULTS: H(4)R protein was expressed by monocytes and upregulated by IFN-gamma. H(4)R agonists (clobenpropit and 4-methylhistamine) induce a Ca(2+) mobilization in monocytes, which could be blocked with the selective H(4)R antagonist JNJ7,777,120. Furthermore, H(4)R agonists downregulated CCL2 protein production. This effect could also be blocked by JNJ7,777,120. Supernatants of H(4)R agonist-stimulated monocytes attracted less monocytes in transmigration assays. The downregulation of CCL2 production was regulated at different levels. First, the synthesis of CCL2 mRNA was significantly decreased. Second, intracellular staining suggested an inhibition of CCL2 secretion after stimulation with H(4)R agonists. CONCLUSION: Human monocytes express the H(4)R, and its stimulation leads to a Ca(2+) influx and an inhibition of CCL2 production, resulting in a reduction of monocyte recruitment. CLINICAL IMPLICATIONS: The H(4)R could represent an important anti-inflammatory receptor on monocytes and could be an interesting target for drug development.

Oligomerization of recombinant and endogenously expressed human histamine H(4) receptors.

In this study, we report the homo- and hetero-oligomerization of the human histamine H(4)R by both biochemical (Western blot and immobilized metal affinity chromatography) and biophysical [bioluminescence resonance energy transfer and time-resolved fluorescence resonance energy transfer (tr-FRET)] techniques. The H(4)R receptor is the most recently discovered member of the histamine family of G-protein-coupled receptors. Using specific polyclonal antibodies raised against the C-terminal tail of the H(4)R, we demonstrate the presence of H(4)R oligomers in human embryonic kidney 293 and COS-7 cells heterologously overexpressing H(4)Rs and putative native H(4)R oligomers in human phytohaemagglutinin blasts endogenously expressing H(4)Rs. Moreover, we show that H(4)R homo-oligomers are formed constitutively, are formed at low receptor densities (300 fmol/mg of protein), and are present at the cell surface, as detected by tr-FRET. The formation of these oligomers is independent of N-glycosylation and is not modulated by H(4)R ligands, covering the full spectrum of agonists, neutral antagonists, and inverse agonists. Although we show H(4)R homo-oligomer formation at physiological expression levels, the detection of H(1)R-H(4)R hetero-oligomers was achieved only at higher H(1)R expression levels and are most likely not physiologically relevant.

Discovery of naturally occurring splice variants of the rat histamine H3 receptor that act as dominant-negative isoforms.

We described previously the cDNA cloning of three functional rat histamine H3 receptor (rH3R) isoforms as well as the differential brain expression patterns of their corresponding mRNAs and signaling properties of the resulting rH3A, rH3B, and rH3C receptor isoforms (Mol Pharmacol 59:1-8). In the current report, we describe the cDNA cloning, mRNA localization in the rat central nervous system, and pharmacological characterization of three additional rH3R splice variants (rH3D, rH3E, and rH3F) that differ from the previously published isoforms in that they result from an additional alternative-splicing event. These new H3R isoforms lack the seventh transmembrane (TM) helix and contain an alternative, putatively extracellular, C terminus (6TM-rH3 isoforms). After heterologous expression in COS-7 cells, radioligand binding or functional responses upon the application of various H3R ligands could not be detected for the 6TM-rH3 isoforms. In contrast to the rH3A receptor (rH3AR), detection of the rH3D isoform using hemagglutinin antibodies revealed that the rH3D isoform remains mainly intracellular. The expression of the rH3D-F splice variants, however, modulates the cell surface expression-levels and subsequent functional responses of the 7TM H3R isoforms. Coexpression of the rH3AR and the rH3D isoforms resulted in the intracellular retention of the rH3AR and reduced rH3AR functionality. Finally, we show that in rat brain, the H3R mRNA expression levels are modulated upon treatment with the convulsant pentylenetetrazole, suggesting that the rH3R isoforms described herein thus represent a novel physiological mechanism for controlling the activity of the histaminergic system.