We report a comparative immunofluorescence and immunoblotting study of GFA protein, the subunit of glial filaments, in nonmammalian vertebrates. The study was conducted with polyclonal antibodies raised to human and shark antigen and with monoclonal antibodies isolated from mice immunized with chicken and bovine antigen. With the exception of cyclostomes, glial filaments appeared remarkably conserved in vertebrate phylogeny, both with respect to the molecular weight and immunoreactivity of their protein subunit. In most species, the antibodies decorated a single band in brain, spinal cord, and optic nerve extracts by the immunoblotting procedure. This band had the same molecular weight in the different CNS regions. With the exception of the turtle, species differences in the molecular weight of the band were not greater than those observed among mammalian vertebrates (human, bovine, and rat). However, there were some exceptional findings in fish. In goldfish and trout brain and spinal cord extracts, the antibodies decorated with the same intensity two bands. In accordance with previous immunofluorescence findings, goldfish optic nerve extracts were negative by the immunoblotting procedure. In four fishes (sea bass, tautog, trout, and scup), optic nerves reacted with the antibodies. However, the band decorated by the antibodies was higher in molecular weight than that obtained from brain and spinal cord extracts. Glial fibers were demonstrated by immunofluorescence in the brain, spinal cord, optic nerve, and retina of most species studied. In amphibia immunofluorescent structures were comparatively few, probably accounting for the negative results by immunoblotting. A comparative immunohistological study of the cerebellum showed the presence of perpendicular glial fibers in the molecular layer of most species examined. Birds and amphibia were different in this respect. Bergmann glia in chicken were GFA negative. In the frog and the toad, immunofluorescent fibers in the molecular layer of the cerebellum were haphazardly oriented. Ependymal radial glia was GFA-negative in the cerebellum of subavian vertebrates. Antisera raised in rabbit to shark GFA protein reacted with the same bovine GFA fragments recognized by polyclonal and monoclonal antibodies raised to human and bovine antigens, respectively, i.e., 30-kDa N-bromosuccinimide fragment (tryptophan cleavage); 35-kDa 2-nitro-5-thiocyanobenzoic acid fragment (cysteine cleavage); 18-kDa cyanogen bromide fragment (methionine cleavage). Conversely, the chicken GFA monoclonal antibodies selected for this study only reacted with noncleaved protein.

Gap junctional proteins are important components of signaling pathways required for the development and ongoing functions of all animal tissues, particularly the nervous system, where they function in the intracellular and extracellular exchange of small signaling factors and ions. In animals whose genomes have been sufficiently sequenced, large families of these proteins, connexins, pannexins, and innexins, have been found, with 25 innexins in the nematode Caenorhabditis elegans Starich et al. (Cell Commun Adhes 8: 311-314, 2001) and at least 37 connexins in the zebrafish Danio rerio Cruciani and Mikalsen (Biol Chem 388:253-264, 2009). Having recently sequenced the medicinal leech Hirudo verbana genome, we now report the presence of 21 innexin genes in this species, nine more than we had previously reported from the analysis of an EST-derived transcriptomic database Dykes and Macagno (Dev Genes Evol 216: 185-97, 2006); Macagno et al. (BMC Genomics 25:407, 2010). Gene structure analyses show that, depending on the leech innexin gene, they can contain from 0 to 6 introns, with closely related paralogs showing the same number of introns. Phylogenetic trees comparing Hirudo to another distantly related leech species, Helobdella robusta, shows a high degree of orthology, whereas comparison to other annelids shows a relatively low level. Comparisons with other Lophotrochozoans, Ecdyzozoans and with vertebrate pannexins suggest a low number (one to two) of ancestral innexin/pannexins at the protostome/deuterostome split. Whole-mount in situ hybridization for individual genes in early embryos shows that ∼50% of the expressed innexins are detectable in multiple tissues. Expression analyses using quantitative PCR show that ∼70% of the Hirudo innexins are expressed in the nervous system, with most of these detected in early development. Finally, quantitative PCR analysis of several identified adult neurons detects the presence of different combinations of innexin genes, a property that may underlie the participation of these neurons in different adult coupling circuits.

Histamine regulates arousal, circadian rhythms, and thermoregulation. Activation of H3 histamine receptors expressed by preoptic GABAergic neurons results in a decrease of their firing rate and hyperthermia. Here we report that an increase in the A-type K+ current in preoptic GABAergic neurons in response to activation of H3 histamine receptors results in decreased firing rate and hyperthermia in mice. The Kv4.2 subunit is required for these actions in spite of the fact that Kv4.2−/− preoptic GABAergic neurons display A-type currents and firing characteristics similar to those of wild-type neurons. This electrical remodeling is achieved by robust upregulation of the expression of the Kv4.1 subunit and of a delayed rectifier current. Dynamic clamp experiments indicate that enhancement of the A-type current by a similar amount to that induced by histamine is sufficient to mimic its robust effect on firing rates. These data indicate a central role played by the Kv4.2 subunit in histamine regulation of body temperature and its interaction with pERK1/2 downstream of the H3 receptor. We also reveal that this pathway provides a mechanism for selective modulation of body temperature at the beginning of the active phase of the circadian cycle.

Histamine acts centrally to increase energy expenditure and reduce body weight by mechanisms not fully understood. It has been suggested that in the obese state hypothalamic histamine signaling is altered. Previous studies have also shown that histamine acting in the preoptic area controls thermoregulation. We aimed to study the influence of preoptic histamine on body temperature and energy homeostasis in control and obese mice. Activating histamine receptors in the preoptic area by increasing the concentration of endogenous histamine or by local injection of specific agonists induced an elevation of core body temperature and decreased respiratory exchange ratio (RER). In addition, the food intake was significantly decreased. The hyperthermic effect was associated with a rapid increase in mRNA expression of uncoupling proteins in thermogenic tissues, the most pronounced being that of uncoupling protein (UCP) 1 in brown adipose tissue and of UCP2 in white adipose tissue. In diet induced obese mice histamine had much diminished hyperthermic effects as well as reduced effect on RER. Similarly, the ability of preoptic histamine signaling to increase the expression of uncoupling proteins was abolished. We also found that the expression of mRNA encoding the H1 receptor subtype in the preoptic area was significantly lower in obese animals. These results indicate that histamine signaling in the preoptic area modulates energy homeostasis by regulating body temperature, metabolic parameters and food intake and that the obese state is associated with a decrease in neurotransmitter’s influence.

Histamine is involved in the central control of arousal, circadian rhythms and metabolism. The preoptic area, a region that contains thermoregulatory neurons is the main locus of histamine modulation of body temperature. Here we report that in mice histamine activates H2 subtype receptors in the medial preoptic nucleus (MPON) and induces hyperthermia. We also found that a population of glutamatergic MPON neurons express H2 receptors and are excited by histamine or H2 specific agonists. The agonists decreased the input resistance of the neuron and increased the depolarizing “sag” observed during hyperpolarizing current injections. Furthermore, at −60 mV holding potential activation of H2 receptors induced an inward current that was blocked by ZD7288, a specific blocker of the hyperpolarization activated cationic current (Ih). Indeed, activation of H2 receptors resulted in increased Ih amplitude in response to hyperpolarizing voltage steps and a depolarizing shift in its voltage-dependent activation. The neurons excited by H2 specific agonism expressed the HCN1 and HCN2 channel subunits. Our data indicate that at the level of the MPON histamine influences thermoregulation by increasing the firing rate of glutamatergic neurons that express H2 receptors.

Background Chronic kidney disease (CKD) is an important cause of morbidity and mortality worldwide. There is lack of information on epidemiology and progression of CKD in low-middle income countries. The Indian Chronic Kidney Disease (ICKD) study aims to identify factors that associate with CKD progression, and development of kidney failure and cardiovascular disease (CVD) in Indian CKD patients . Methods ICKD study is prospective, multicentric, cohort study enrolling patients with estimated glomerular filtration rate (eGFR) 15-60 ml/min/1.73m2 or > 60 ml/min/1.73m2 with proteinuria. Clinical details and biological samples are collected at annual visit. We analysed the baseline characteristics including socio-demographic details, risk factors, disease characteristics and laboratory measurements. In addition, we compared characteristics between urban and rural participants. Results A total of 4056 patients have been enrolled till March 31, 2020. The age was 50.3 ±11.8 years, 67.2% were males, 2/3 lived in rural areas and median eGFR was 40 ml/min/1.73m2. About 87% were hypertensive, 37% had diabetes, 22% had CVD, 6.7% had past history of acute kidney injury and 23% reported prior use of alternative drugs. Diabetic kidney disease, chronic interstitial nephritis and CKD-cause unknown were the leading causes. Rural participants had more occupational exposure and tobacco use but lower educational status and income. CIN and unknown categories were leading causes in rural participants. Conclusions The ICKD study is the only large cohort study of patients with mild-to-moderate CKD in a lower-middle income country. Baseline characteristics of study population reveal differences as compared to other cohorts.

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