Genetic epidemiology of autoinflammatory disease variants in Indian population from 1029 whole genomes.

BACKGROUND: Autoinflammatory disorders are the group of inherited inflammatory disorders caused due to the genetic defect in the genes that regulates innate immune systems. These have been clinically characterized based on the duration and occurrence of unprovoked fever, skin rash, and patient's ancestry. There are several autoinflammatory disorders that are found to be prevalent in a specific population and whose disease genetic epidemiology within the population has been well understood. However, India has a limited number of genetic studies reported for autoinflammatory disorders till date. The whole genome sequencing and analysis of 1029 Indian individuals performed under the IndiGen project persuaded us to perform the genetic epidemiology of the autoinflammatory disorders in India. RESULTS: We have systematically annotated the genetic variants of 56 genes implicated in autoinflammatory disorder. These genetic variants were reclassified into five categories (i.e., pathogenic, likely pathogenic, benign, likely benign, and variant of uncertain significance (VUS)) according to the American College of Medical Genetics and Association of Molecular pathology (ACMG-AMP) guidelines. Our analysis revealed 20 pathogenic and likely pathogenic variants with significant differences in the allele frequency compared with the global population. We also found six causal founder variants in the IndiGen dataset belonging to different ancestry. We have performed haplotype prediction analysis for founder mutations haplotype that reveals the admixture of the South Asian population with other populations. The cumulative carrier frequency of the autoinflammatory disorder in India was found to be 3.5% which is much higher than reported. CONCLUSION: With such frequency in the Indian population, there is a great need for awareness among clinicians as well as the general public regarding the autoinflammatory disorder. To the best of our knowledge, this is the first and most comprehensive population scale genetic epidemiological study being reported from India.

Pharmacogenomic landscape of COVID-19 therapies from Indian population genomes.

Aim: Numerous drugs are being widely prescribed for COVID-19 treatment without any direct evidence for the drug safety/efficacy in patients across diverse ethnic populations. Materials & methods: We analyzed whole genomes of 1029 Indian individuals (IndiGen) to understand the extent of drug-gene (pharmacogenetic), drug-drug and drug-drug-gene interactions associated with COVID-19 therapy in the Indian population. Results: We identified 30 clinically significant pharmacogenetic variants and 73 predicted deleterious pharmacogenetic variants. COVID-19-associated pharmacogenes were substantially overlapped with those of metabolic disorder therapeutics. CYP3A4, ABCB1 and ALB are the most shared pharmacogenes. Fifteen COVID-19 therapeutics were predicted as likely drug-drug interaction candidates when used with four CYP inhibitor drugs. Conclusion: Our findings provide actionable insights for future validation studies and improved clinical decisions for COVID-19 therapy in Indians.

IndiGenomes: a comprehensive resource of genetic variants from over 1000 Indian genomes.

With the advent of next-generation sequencing, large-scale initiatives for mining whole genomes and exomes have been employed to better understand global or population-level genetic architecture. India encompasses more than 17% of the world population with extensive genetic diversity, but is under-represented in the global sequencing datasets. This gave us the impetus to perform and analyze the whole genome sequencing of 1029 healthy Indian individuals under the pilot phase of the 'IndiGen' program. We generated a compendium of 55,898,122 single allelic genetic variants from geographically distinct Indian genomes and calculated the allele frequency, allele count, allele number, along with the number of heterozygous or homozygous individuals. In the present study, these variants were systematically annotated using publicly available population databases and can be accessed through a browsable online database named as 'IndiGenomes' http://clingen.igib.res.in/indigen/. The IndiGenomes database will help clinicians and researchers in exploring the genetic component underlying medical conditions. Till date, this is the most comprehensive genetic variant resource for the Indian population and is made freely available for academic utility. The resource has also been accessed extensively by the worldwide community since it's launch.

Distribution of gonadotropin-inhibitory hormone (GnIH)-like immunoreactivity in the brain and pituitary of the frog (Pelophylax esculentus) during development.

Gonadotropin-inhibitory hormone (GnIH) is a newly discovered hypothalamic RFamide peptide that influences reproduction by regulating brain and pituitary neuroendocrine functions in vertebrates. We report here for the first time, the ontogenetic description of GnIH-like immunoreactivity in the brain, olfactory system, and pituitary of the frog, Pelophylax esculentus. GnIH-like immunoreactive (GnIH-ir) elements were first observed in larvae at stage 24 in the olfactory mucosa, ventral telencephalon, and diencephalon. GnIH-ir-positive staining progressively increased in frequency and intensity during larval growth and other ir perikarya appeared in the medial septum, anterior commissure, dorsal hypothalamus, and posterior tuberculum. A decline in GnIH-ir neurons was seen along the olfactory/vomeronasal/terminal nerve complex in the stages following the pre- and prometamorphosis, while other GnIH-ir neurons showed positivity in the ventromedial surface of the olfactory bulbs and into the habenular nuclei, but the latter are no longer observed in the following stages of development. The anterior-posterior axis in several brain areas, along with the median eminence and pars intermedia of the hypophysis had the appearance of GnIH-ir fibers from early stages, with a progressive increase in the number till metamorphosis in all major subdivisions of the brain. After premetamorphosis, GnIH-ir fibers arising from labeled neurons in the suprachiasmatic nucleus could be seen contacting the ventricular lumen. The transient appearance of GnIH-ir elements in the olfactory system may hint at an olfactory placode origin in the extracranial region. The distribution of GnIH in several brain regions throughout development suggests important involvement of GnIH in multiple brain functions during development.

Knockdown of calcium-binding calb2a and calb2b genes indicates the key regulator of the early development of the zebrafish, Danio rerio.

The present study initiates our investigation regarding the role of calb2a and calb2b genes that are expressed in the central nervous system, including the multiple tissues during early embryonic development of zebrafish. In this study, we have adopted individual and combined morpholino-mediated inactivation approach to investigate the functions of calb2a and calb2b in early development of the zebrafish. We have found that calb2a and calb2b morpholino alone failed to generate an obvious phenotype; however, morphological inspection in early developmental stages of calb2a and calb2b combined knockdown morphants show abnormal neural plate folding in midbrain-hindbrain region. In addition to this, combinatorial loss of these mRNA leads to severe hydrocephalus, axial curvature defect, and yolk sac edema in later developmental stages. Also, the combined knockdown of calb2a and calb2b are found to be associated with an impaired touchdown and swimming performance in the zebrafish. Co-injection of the calb2a and calb2b morpholino oligonucleotide cocktail with human CALB2 mRNA leads to the rescue of the strong phenotype. This study provided the first comprehensive analyses of the zebrafish Calb2a and Calb2b proteins; we have found that Calb2a and Calb2b are highly conserved across vertebrate species and originated from the same ancestral gene long back in the evolution. Homology modeling and docking with the similar structure and Ca2+ binding sites for both proteins provide the evidence that both the proteins may have similar function and one can compensate for the loss of other. Collectively, these findings confirm the unique and essential functions of calb2a and calb2b genes in the early development of the zebrafish.

Distribution of NADPH-diaphorase reactivity in the central nervous system of the common toad (Bufo bufo).

We examined the distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d)-reactive elements in the central nervous system (CNS) of the common toad, Bufo bufo. The investigation involved adult male and female toads collected during the breeding season. Labeled neurons of different morphological appearances (weakly or darkly stained, unipolar, bipolar, and multipolar) and fibers were observed across all subdivisions of the amphibian brain. Overall, a similar distribution of NADPH-d-labeled neurons was observed in the brain of male and female toads. In the secondary prosencephalon NADPH-d-labeled neurons were observed in the olfactory bulbs, pallial regions, nucleus accumbens, diagonal band of Broca, septum, striatum, amygdala, suprachiasmatic and magnocellular preoptic nuclei, dorsal and ventral hypothalamus. In the diencephalon, NADPH-d-positive neurons were seen in the anterior thalamic nuclei, ventromedial and ventrolateral nuclei, central and lateral thalamic nuclei, posterior tubercle, posterodorsal division of the lateral thalamic nucleus, and in the pretectal and pretoral gray. In the mesencephalon, heavily stained neurons were present in the anterodorsal and anteroventral tegmental nuclei, magnocellular, principal and laminar nuclei of the torus semicircularis, and nucleus profundus mesencephali. In the isthmus, stained cells were observed medially and ventrally in the posterodorsal and posteroventral tegmental nuclei. In the rhombencephalon, numerous NADPH-d-stained neurons were distributed in the cerebellar nucleus, sensory and descending trigeminal nuclei, motor nuclei of the glossopharyngeal and vagus nerves, the nucleus of the solitary tract, nuclei of the hypoglossal and octaval nerves, dorsal column nucleus, central gray region, and in reticular formation. However, the complete absence of NADPH-d-stained neurons in the cerebellar cortex was an unusual feature observed in this study. The widespread distribution of NADPH-d staining in diverse cell types, belonging to a variety of neuronal systems suggests a widespread role for NADPH-d in modulating diverse functions, including sensory coding in the amphibian nervous system.

Neuroanatomical demonstration of calbindin 2a- and calbindin 2b-like calcium binding proteins in the early embryonic development of zebrafish: mRNA study.

Certain calcium binding proteins (CaBPs) are essential for metabolic processes but the role of these proteins in the development is not well known. We have investigated the mRNA expression of CaBPs, calbindin 2a (Calb2a) and calbindin 2b (Calb2b) in the zebrafish embryos 24, 36, 48 and 72h post fertilization (hpf). We have seen very high Calb2a mRNA expression in the tegmentum (Tg), midbrain-hindbrain boundary (Mhb), hindbrain (Hb), spinal cord (Sc), retina and cranial ganglion (Crg). Also very high Calb2b mRNA expression was noted in olfactory cells, cerebellum, Tg, Mhb, Hb, optic tectum, retina, retinal ganglion cell layer, retinal inner nuclear layer, Sc, Neural crest, infraorbital neuromasts, pharyngeal arch 3-7 skeleton and mandibular neuromasts. It is known that many factors are involved in the differentiation of Mhb. Here we are reporting for the first time the mRNA expression of CaBPs (Calb2a and Calb2b) in the Mhb indicating their role in the differentiation of Mhb and development of the brain, eyes and other tissues in the zebrafish. We suggest that Calb2a and Calb2b play an important role in the regulation of zebrafish early embryonic development.

The distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) in the medulla oblongata, spinal cord, cranial and spinal nerves of frog, Microhyla ornata.

Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) enzymatic activity has been reported in few amphibian species. In this study, we report its unusual localization in the medulla oblongata, spinal cord, cranial nerves, spinal nerves, and ganglions of the frog, Microhyla ornata. In the rhombencephalon, at the level of facial and vagus nerves, the NADPH-d labeling was noted in the nucleus of the abducent and facial nerves, dorsal nucleus of the vestibulocochlear nerve, the nucleus of hypoglossus nerve, dorsal and lateral column nucleus, the nucleus of the solitary tract, the dorsal field of spinal grey, the lateral and medial motor fields of spinal grey and radix ventralis and dorsalis (2-10). Many ependymal cells around the lining of the fourth ventricle, both facial and vagus nerves and dorsal root ganglion, were intensely labeled with NADPH-d. Most strikingly the NADPH-d activity was seen in small and large sized motoneurons in both medial and lateral motor neuron columns on the right and left sides of the brain. This is the largest stained group observed from the caudal rhombencephalon up to the level of radix dorsalis 10 in the spinal cord. The neurons were either oval or elongated in shape with long processes and showed significant variation in the nuclear and cellular diameter. A massive NADPH-d activity in the medulla oblongata, spinal cord, and spinal nerves implied an important role of this enzyme in the neuronal signaling as well as in the modulation of motor functions in the peripheral nervous systems of the amphibians.

Gonadotropin-inhibitory hormone (GnIH) in the amphibian brain and its relationship with the gonadotropin releasing hormone (GnRH) system: An overview.

It is well known that the hypothalamic neuropeptide gonadotropin-releasing hormone (GnRH) plays an important role as a primary factor regulating gonadotropin secretion in reproductive processes in vertebrates. The discovery of the presence of a gonadotropin-inhibitory hormone (GnIH) in the brains of birds has further contributed to our understanding of the reproduction control by the brain. GnIH plays a key role in inhibition of reproduction and acts on the pituitary gland and GnRH neurons via a novel G protein-coupled receptor (GPR147). GnIH decreases gonadotropin synthesis and release, thus inhibiting gonadal development and maintenance. The GnRH and GnIH neuronal peptidergic systems are well reported in mammals and birds, but limited information is available regarding their presence and localization in the brains of other vertebrate species, such as reptiles, amphibians and fishes. The aim of this review is to compile and update information on the localization of GnRH and GnIH neuronal systems, with a particular focus on amphibians, summarizing the neuroanatomical distribution of GnIH and GnRH and emphasizing the discovery of GnIH based on RFamide peptides and GnIH orthologous peptides found in other vertebrates and their functional significance.

Neuroanatomical localization of nitric oxide synthase (nNOS) in the central nervous system of carp, Labeo rohita during post-embryonic development.

Nitric oxide (NO) is a chemically diffusible molecular messenger playing various roles in both vertebrates and invertebrates. Nitric oxide synthase (NOS) is the key enzyme in synthesis of NO. The neuroanatomical distribution pattern of neuronal nitric oxide synthase (nNOS) was studied and developing stages of Labeo rohita such as hatchlings (10-15mm), frys (15-35mm), semi-fingerlings (35-65mm), fingerlings (65-100mm) and adults (350-370mm) were used. In the telencephalon, nitrergic cells were observed in both pallial and subpallial regions along with entopeduncular nucleus suggesting the involvement of NO in the control of sensory functions throughout the development. In the diencephalon, nNOS positive neurons were localized in the nucleus preopticus periventricularis and preopticus parvocellularis throughout development while nucleus preopticus magnocellularis was found immunopositive only in adult specimens who suggest the involvement of NO in the hormonal regulation. nNOS immunoreaction was also noted in suprachaismatic nucleus, habenula, lateral tuberal nucleus, paraventricular organ and anterior division of preglomerular nucleus throughout development. In the mesencephalic region, nNOS immunoreactivity was seen in the optic tectum, torus longitudinalis, nucleus of median longitudinal fascicle and occulomotor nucleus indicate the role of NO in integration of visual inputs and modulates motor control of the eyes and movements. Caudally, in the rhombencephalon, the cerebellum, the nucleus reticularis, the octaval nucleus and the motor nucleus of vagal nerve were nNOS positive during development. nNOS reactive cells and fibers were noted in the spinal motor column, thus suggesting a role of NO in gestation and startle response from early development.

Short-term exposure to L-type calcium channel blocker, verapamil, alters the expression pattern of calcium-binding proteins in the brain of goldfish, Carassius auratus.

The influx of calcium ions (Ca(2+)) is responsible for various physiological events including neurotransmitter release and synaptic modulation. The L-type voltage dependent calcium channels (L-type VDCCs) transport Ca(2+) across the membrane. Calcium-binding proteins (CaBPs) bind free cytosolic Ca(2+) and prevent excitotoxicity caused by sudden increase in cytoplasmic Ca(2+). The present study was aimed to understand the regulation of expression of neuronal CaBPs, namely, calretinin (CR) and parvalbumin (PV) following blockade of L-type VDCCs in the CNS of Carassius auratus. Verapamil (VRP), a potent L-type VDCC blocker, selectively blocks Ca(2+) entry at the plasma membrane level. VRP present in the aquatic environment at a very low residual concentration has shown ecotoxicological effects on aquatic animals. Following acute exposure for 96h, median lethal concentration (LC50) for VRP was found to be 1.22mg/L for goldfish. At various doses of VRP, the behavioral alterations were observed in the form of respiratory difficulty and loss of body balance confirming the cardiovascular toxicity caused by VRP at higher doses. In addition to affecting the cardiovascular system, VRP also showed effects on the nervous system in the form of altered expression of PV. When compared with controls, the pattern of CR expression did not show any variations, while PV expression showed significant alterations in few neuronal populations such as the pretectal nucleus, inferior lobes, and the rostral corpus cerebellum. Our result suggests possible regulatory effect of calcium channel blockers on the expression of PV.

Neuroanatomical organization of the brain gonadotropin-inhibitory hormone and gonadotropin-releasing hormone systems in the frog Pelophylax esculentus.

Growing evidence suggests that gonadotropin-inhibitory hormone (GnIH) may play a key role in mediating vertebrate reproduction. GnIH inhibits gonadotropin synthesis and release by decreasing the activity of gonadotropin-releasing hormone (GnRH) neurons as well as by directly regulating gonadotropin secretion from the pituitary. Whereas the presence of GnIH has been widely investigated in various classes of vertebrates, there are very few immunohistochemical reports focusing on GnIH in amphibians. The aim of this study was to assess the presence and neuroanatomical distribution of GnIH-like immunoreactivity in the brain of the anuran amphibian Pelophylax (Rana) esculentus (esculenta) and to explore any potential anatomical relationship with mammalian GnRH-immunoreactive (mGnRH-ir) elements. The GnIH-like immunoreactive (GnIH-ir) system constitutes two distinct subpopulations in the telencephalon and diencephalon, with the highest number of immunoreactive cells located in the preoptic and suprachiasmatic areas. GnIH-ir neurons were also observed in the medial septum, the anterior commissure, the dorsal hypothalamus, the periventricular nucleus of the hypothalamus, and the posterior tuberculum. Scattered GnIH-ir fibers were present in all major subdivisions of the brain but only occasionally in the median eminence. mGnRH-ir neurons were distributed in the mediobasal telencephalon, the medial septal area, and the anterior preoptic area. Double-label immunohistochemistry revealed that the GnRH and GnIH systems coexist and have overlapping distributions at the level of the anterior preoptic area. Some GnIH-ir fibers were in close proximity to mGnRH-ir cell bodies. Our results suggest that both the neuroanatomy and the functional regulation of GnRH release are conserved properties of the hypothalamic GnIH-ir system among vertebrate species.

GnIH and GnRH expressions in the central nervous system and pituitary of Indian major carp, Labeo rohita during ontogeny: An immunocytochemical study.

Gonadotropin-releasing hormone (GnRH) is the major hypothalamic neuropeptide stimulating gonadotropin secretion in vertebrates. In 2000, gonadotropin-inhibitory hormone (GnIH) was discovered as a hypothalamic neuropeptide that inhibits gonadotropin secretion in birds. Subsequent studies have shown that GnIH is present in the brain of other vertebrates. We show for the first time GnIH immunoreactivity in the central nervous system and pituitary during development of Indian major carp, Labeo rohita and compare it with the localization of GnRH. GnIH and GnRH immunoreactivities were observed from the olfactory system to spinal cord throughout development. In the brain, both neuropeptides were localized in the telencephalon, diencephalon including the preoptic area and rhombencephalon. The localization of GnIH and GnRH in the pituitary suggests that these neuropeptides are involved in the regulation of pituitary hormones by an autocrine manner during development. In addition, the presence of GnIH and GnRH in several other brain regions including the olfactory system suggests their involvement in the regulation of other physiological functions.

Calcium binding protein calretinin (29kD) localization in the forebrain of the cichlid fish: An immunohistochemical study.

Ionic regulation is essential for the metabolism and cellular function. For many physiological processes, ionic calcium (Ca(+2)) is important for example muscle contractions, nerve signaling, membrane permeability, cell division and hormone release. In nerve cells, the excess intracellular concentration of Ca(+2) causes cell death. It has been shown that certain calcium binding proteins (CaBPs) are essential for Ca(+2) homeostasis and protect neurons from excess Ca(+2) influx. We are for the first time showing an unusual calretinin (CR) expression and significant differences in its occurrence in the forebrain of the cichlid fish (Cynotilapia sp.) compared to other teleosts. CR labeled neurons were seen in the dorsal and lateral part of the dorsal telencephalic area, entopeduncular nucleus (EN), nucleus preopticus (NPO), diffuse nucleus of lateral torus (NDTL), ventral hypothalamic nucleus (VH), preglomerular nucleus (NPG) and optic tectum. Surprisingly, large numbers of CR immunoreactive perikarya were noted in the optic chiasma (Oc). These neurons were oval with elongated processes and forming a huge fiber network in the Oc. Enormously CR stained fibers were seen in the lateral and medial olfactory tract. Widespread distributions of strongly CR labeled fibers were observed around the EN projecting dorsally into the telencephalon, Oc and optic nerve. Presence of CR in the NPO suggests that it may be involved in the hormonal regulation by the pituitary. As in vertebrates EN plays an important role in sensory functions, massive localization CR in the EN may suggests role of CR in sensory functions of the cichlid fish.

Sexually dimorphic distribution of calcium-binding protein, calretinin in the preoptic area of the freshwater catfish, Clarias batrachus (Linn.).

Preoptic area (POA) plays an important role in the hormonal regulation of the pituitary gland in vertebrates. In this study we report the sexually dimorphic distribution of calcium-binding proteins calretinin (CR) in the POA in the freshwater catfish, Clarias batrachus. Nissl staining highlighted the presence of the nucleus praeopticus periventricularis (NPP) and other subdivisions of the nucleus praeopticus (NPO), including supraoptic (NPOs), paraventricular (NPOp) and magnocellular (NPOm) divisions. In NPO, CR immunoreactivity was noted only in females but not in males. In both sexes, CR stained perikarya were found in the NPP. Sexually dimorphic localization of CR in the POA supports the notion that CR may play a gender-specific role and may be involved in hormonal regulation in fishes.

A comparative cluster analysis of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry in the brains of amphibians.

Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) is a key enzyme in the synthesis of the gaseous neurotransmitter nitric oxide. We compare the distribution of NADPH-d in the brain of four species of hylid frogs. NADPH-d-positive fibers are present throughout much of the brain, whereas stained cell groups are distributed in well-defined regions. Whereas most brain areas consistently show positive neurons in all species, in some areas species-specific differences occur. We analyzed our data and those available for other amphibian species to build a matrix on NADPH-d brain distribution for a multivariate analysis. Brain dissimilarities were quantified by using the Jaccard index in a hierarchical clustering procedure. The whole brain dendrogram was compared with that of its main subdivisions by applying the Fowlkes-Mallows index for dendrogram similarity, followed by bootstrap replications and a permutation test. Despite the differences in the distribution map of the NADPH-d system among species, cluster analysis of data from the whole brain and hindbrain faithfully reflected the evolutionary history (framework) of amphibians. Dendrograms from the secondary prosencephalon, diencephalon, mesencephalon, and isthmus showed some deviation from the main scheme. Thus, the present analysis supports the major evolutionary stability of the hindbrain. We provide evidence that the NADPH-d system in main brain subdivisions should be cautiously approached for comparative purposes because specific adaptations of a single species could occur and may affect the NADPH-d distribution pattern in a brain subdivision. The minor differences in staining pattern of particular subdivisions apparently do not affect the general patterns of staining across species.

Role of catecholamines and nitric oxide on pigment displacement of the chromatophores of freshwater snakehead teleost fish, Channa punctatus.

We are reporting for the first time that the catecholamines (adrenaline and noradrenaline) inhibit the effect of nitric oxide (NO) on melanosome dispersion in freshly isolated scales of the freshwater snakehead fish, Channa punctatus. We studied the effect of NO and catecholamines on the pigment displacement by observing the changes in the melanophore index. The scales when treated with solution containing NO donor sodium nitroprusside (SNP) showed dispersion of melanosomes, whereas NO synthase blocker N-omega-Nitro-L-arginine suppresses this action of SNP. Treatment with adrenaline and noradrenaline on the isolated scales caused aggregation of melanosomes. Scales treated with solution containing catecholamines and SNP resulted in aggregation of melanosomes suggesting that catecholamines mask the effect of SNP. These results suggest that the catecholamines are inhibiting the effect of NO and causing the aggregation of the melanosomes may be via surface receptors.

Nitric oxide inhibited the melanophore aggregation induced by extracellular calcium concentration in snakehead fish, Channa punctatus.

We studied the role of nitric oxide (NO) and extra-cellular Ca(2+) on the melanophores in Indian snakehead teleost, Channa punctatus. Increase of Ca(2+) level in the external medium causes pigment aggregation in melanophores. This pigment-aggregating effect was found to be inhibited when the external medium contained spontaneous NO donor, sodium nitro prusside (SNP) at all the levels of concentration tested. Furthermore, it has been observed that SNP keeps the pigment in dispersed state even after increasing the amount of Ca(2+). In order to test whether NO donor SNP causes dispersion of pigments or not is checked by adding the inhibitor of nitric oxide synthase, N-omega-Nitro-L-arginine (L-NNA) in the medium. It has been noted that the inhibitor L-NNA blocked the effect of NO donor SNP causing aggregation of pigments. In that way NO is inhibiting the effect of extracellular Ca(2+), keeping the pigment dispersed.

The ontogeny of Mauthner cells in the brain of Labeo rohita as revealed by NADPH-d and nNOS immunohistochemistry.

Nitric oxide (NO) is well demonstrated to act as a neuronal messenger in neurotransmission in vertebrate animals. We are for the first time reporting nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and neuronal nitric oxide synthase (nNOS) in the Mauthner cells (MC) of the carp, Labeo rohita brain during post-embryonic development. The NADPH-d and nNOS-positive MC were seen intensely labeled in all young stages on either side of the ventricle in the hindbrain. In adult, these cells were moderately stained with NADPH-d but not with nNOS. Further, the morphometeric analysis of the staining intensities showed the decrease in the optical densities of the MC as the development progresses. The gradual increase in cell diameter was evident from hatchling to adult (18-22 µm to 98-106 µm), while the nuclear diameter remained nearly unchanged (10-12 µm to 18-20 µm). In fishes, MC are known to be involved in the control of startle response. The NADPH-d enzymatic activity seen in the MC of early development may play an important role to activate the startle response to fast escape from the predators and unfavorable environmental conditions to survive during early phase of life. As nNOS is absent in the adult MC, we assume that the adult MC express a different splice variant of nNOS which is detected by NADPH-d, but not by the currently used antibody. Hence, the presence of nNOS (enzyme required for the synthesis of NO) in the MC suggests the involvement of NO in neurotransmission to generate the startle response rapidly. Furthermore, we also suggest that NO may be involved in other aspects such as pathway finding, neuronal cell migration, signaling and refinement during the development of the brain of L. rohita.

Localization of calcium-binding protein (calretinin, 29 kD) in the brain and pituitary gland of teleost fish: an immunohistochemical study.

Immunocytochemical techniques were used to investigate the distribution of calretinin in the brain and pituitary gland of the hardhead catfish Arius felis. Calretinin immunoreactive neurons were found in the telencephalon (lateral nucleus of ventral telencephalic area), diencephalon (around the medial forebrain bundle, lateral tuberal nucleus, central pretectal nucleus, posterior periventricular hypothalamic nucleus, medial preglomerular nucleus, diffuse nucleus of the inferior lobe), mesencephalon (nucleus of the medial longitudinal fascicle, ventral nucleus of the semicircular torus), cerebellum (valvula cerebelli, eurydendroid cells) and rhombencephalon (secondary gustatory nucleus, isthmic nucleus, trigeminal motor nucleus, medial auditory nucleus of the medulla, medial and inferior reticular formation, anterior, descending, posterior and tangential octaval nuclei). Calretinin-labeled fibers were observed in the optic nerve and at the levels of the central pretectal nucleus, the nucleus of the medial longitudinal fascicle, the ventral nucleus of the semicircular torus, the secondary gustatory nucleus, the trigeminal motor nucleus, the eurydendroid cells, the medial auditory nucleus of the medulla and the octaval nucleus. For the first time, we are reporting on calretinin-positive cells in the rostral and proximal pars distalis of the adenohypophysis. Although, it seems speculatory, calretinin-expressing cells in the pituitary gland may be involved in hormonal regulation and hence, calretinin might play a significant role in governing hypophysial functions in fishes. Our results suggest that calretinin shows species-specific variations also among the teleost fish, similar to mammals.