Prevalence of psychological outcomes and its associated factors in healthcare personnel working during COVID-19 outbreak in India.

Background: Care of COVID-19 patients has been shown to affect the mental health of healthcare personnel (HCP), however, there is little data reflecting psychological health of HCP in India. Aims: The present study was undertaken to assess the prevalence of psychological outcomes and its association with various sociodemographic and occupational factors among the HCP in India. Methodology: A cross-sectional, online survey, using snowball sampling method was conducted between June 1, 2020, and June 22, 2020. The HCP working in COVID-19 designated hospitals across India were invited to participate. Patient Health Questionnaire-4 and 19-item stress-related questionnaire were used to evaluate symptoms of overall anxiety, depression, COVID-19 infection specific anxiety, exhaustion, and workload. Results: In this cross-sectional study with 2334 HCP from 27 states and 7 union territories of India; 17.9% of participants had depression, 18.7% had overall anxiety, 26.5% had exhaustion, 30.3% reported heavy workload, and 25.4% had COVID-19 infection-specific anxiety, respectively. The HCP working in states with higher caseload was a common risk factor for overall anxiety (odds ratio [OR], 1.7; P < 0.001), depression (OR, 1.6; P < 0.001), COVID-19 infection-specific anxiety (OR, 2.5; P < 0.001), exhaustion (OR, 3.1; P < 0.001), and heavy workload (OR, 2.6; P < 0.001). Nurses were more at risk for depression (OR, 2.2; P < 0.001), anxiety specific to COVID-19 infection (OR, 1.3; P = 0.034), and heavy workload (OR, 2.9; P < 0.001); while doctors were more at risk for overall anxiety (OR, 2.0; P = 0.001) and exhaustion (OR, 3.1; P < 0.001). Conclusions: Frontline workers, specifically nurses and doctors, and those working in states with high COVID-19 caseload are more at risk for adverse psychological outcomes. The relatively less prevalence compared with other countries, is perhaps a reflection of measures undertaken, including early lockdown, ensuring better all-round preparedness and social norms.

Reduced cerebral blood flow in an α-synuclein transgenic mouse model of Parkinson's disease.

There is increasing evidence that widespread cortical cerebral blood flow deficits occur early in the course of Parkinson's disease. Although cerebral blood flow measurement has been suggested as a potential biomarker for early diagnosis of Parkinson's disease, as well as a means for tracking response to treatment, the relationship of cerebral blood flow to α-synucleinopathy, a major pathological hallmark of Parkinson's disease, remains unclear. Therefore, we performed arterial spin-labeling magnetic resonance imaging and diffusion tensor imaging on transgenic mice overexpressing human wild-type α-synuclein and age-matched controls to measure cerebral blood flow and degenerative changes. As reported for early-stage Parkinson's disease, α-synuclein mice exhibited a significant reduction in cortical cerebral blood flow, which was accompanied by motor coordination deficits and olfactory dysfunction. Although no overt degenerative changes were apparent in diffusion tensor imaging images, magnetic resonance imaging volumetric analysis revealed a significant reduction in olfactory bulb volume, similar to that seen in Parkinson's disease patients. Our data, representing the first report of cerebral blood flow deficit in an animal model of Parkinson's disease, suggest a causative role for α-synucleinopathy in cerebral blood flow deficits in Parkinson's disease. Thus, α-synuclein transgenic mice comprise a promising model to study Parkinson's disease-related mechanisms of cerebral blood flow deficits and to investigate further its utility as a potential biomarker for Parkinson's disease.

Functional MRI of the mouse olfactory system.

Although olfactory dysfunction is an early warning sign of Alzheimer's and Parkinson's diseases, and is commonly present in a range of other neurodegenerative disorders, the mechanisms for its pathogenesis are not yet clear. Since fMRI allows the mapping of spatial and temporal patterns of activity in multiple brain regions simultaneously, it serves as a powerful tool to study olfactory dysfunction in animal models of neurodegenerative diseases. Nonetheless, there have been no reports to date of mapping odor-induced activation patterns beyond the olfactory bulb to the extended networks of olfactory and limbic archicortex, likely due to the small size of the mouse brain. Therefore, using an 11.7 T magnet and a blood volume-weighted fMRI technique, we mapped the functional neuroanatomy of the mouse olfactory system. Consistent with reports on imaging of the much larger human brain, we mapped activity in regions of the olfactory bulb, as well as olfactory and limbic archicortex. By using two distinct odorants, we further demonstrated odorant-specific activation patterns. Our work thus provides a methodological framework for fMRI studies of olfactory dysfunction in mouse models of neurodegeneration.

Methylene Blue Ameliorates Olfactory Dysfunction and Motor Deficits in a Chronic MPTP/Probenecid Mouse Model of Parkinson's Disease.

Mitochondrial dysfunction and oxidative stress are very prominent and early features in Parkinson's disease (PD) and in animal models of PD. Thus, antioxidant therapy for PD has been proposed, but in clinical trials such strategies have met with very limited success. Methylene blue (MB), a small-molecule synthetic heterocyclic organic compound that acts as a renewable electron cycler in the mitochondrial electron transport chain, manifesting robust antioxidant and cell energetics-enhancing properties, has recently been shown to have significant beneficial effects in reducing nigrostriatal dopaminergic loss and motor impairment in acute toxin models of PD. However, no studies have investigated the impact of this promising agent in chronic models or for olfactory dysfunction, an early non-motor feature of PD. To test the efficacy of low-dose MB for olfactory dysfunction, motor symptoms, and dopaminergic neurodegeneration, mice were injected with ten subcutaneous doses of 25 mg/kg MPTP, plus 250 mg/kg intraperitoneal probenecid or saline/probenecid at 3.5-day intervals. Following the onset of olfactory dysfunction, MPTP/probenecid (MPTP/p) and saline/probenecid mice were provided drinking water with or without 1 mg/kg/day MB. Oral delivery of low-dose MB significantly ameliorated MPTP/p-induced deficits in motor coordination, as well as degeneration of tyrosine hydroxylase (TH)-positive neurons of the substantia nigra and TH-positive terminals in the striatum. Importantly, olfactory dysfunction was ameliorated by MB treatment, whereas this benefit is not observed with currently available anti-Parkinsonian medications. These results indicate that low-dose MB is a promising neuroprotective intervention for both motor and non-motor features of PD.

GDNF-expressing macrophages mitigate loss of dopamine neurons and improve Parkinsonian symptoms in MitoPark mice.

Glial cell line-derived neurotrophic factor (GDNF) is the most potent neuroprotective agent tested in cellular and animal models of Parkinson's disease (PD). However, CNS delivery of GDNF is restricted by the blood-brain barrier (BBB). Using total body irradiation as transplant preconditioning, we previously reported that hematopoietic stem cell (HSC) transplantation (HSCT)-based macrophage-mediated gene therapy could deliver GDNF to the brain to prevent degeneration of nigrostriatal dopamine (DA) neurons in an acute murine neurotoxicity model. Here, we validate this therapeutic approach in a chronic progressive PD model - the MitoPark mouse, with head shielding to avoid inducing neuroinflammation and compromising BBB integrity. Bone marrow HSCs were transduced ex vivo with a lentiviral vector expressing macrophage promoter-driven GDNF and transplanted into MitoPark mice exhibiting well developed PD-like impairments. Transgene-expressing macrophages infiltrated the midbrains of MitoPark mice, but not normal littermates, and delivered GDNF locally. Macrophage GDNF delivery markedly improved both motor and non-motor symptoms, and dramatically mitigated the loss of both DA neurons in the substantia nigra and tyrosine hydroxylase-positive axonal terminals in the striatum. Our data support further development of this HSCT-based macrophage-mediated GDNF delivery approach in order to address the unmet need for a disease-modifying therapy for PD.

Behavioral phenotypes associated with MPTP induction of partial lesions in common marmosets (Callithrix jacchus).

Parkinson's disease is a chronic neurodegenerative disorder with the core motor features of resting tremor, bradykinesia, rigidity, and postural instability. Non-motor symptoms also occur, and include cognitive dysfunction, mood disorders, anosmia (loss of smell), and REM sleep disturbances. As the development of medications and other therapies for treatment of non-motor symptoms is ongoing, it is essential to have animal models that aid in understanding the neural changes underlying non-motor PD symptoms and serve as a testing ground for potential therapeutics. We investigated several non-motor symptoms in 10 adult male marmosets using the MPTP model, with both the full (n=5) and partial (n=5) MPTP dosing regimens. Baseline data in numerous domains were collected prior to dosing; assessments in these same domains occurred post-dosing for 12 weeks. Marmosets given the partial MPTP dose (designed to mimic the early stages of the disease) differed significantly from marmosets given the full MPTP dose in several ways, including behavior, olfactory discrimination, cognitive performance, and social responses. Importantly, while spontaneous recovery of PD motor symptoms has been previously reported in studies of MPTP monkeys and cats, we did not observe recovery of any non-motor symptoms. This suggests that the neurochemical mechanisms behind the non-motor symptoms of PD, which appear years before the onset of symptoms, are independent of the striatal dopaminergic transmission. We demonstrate the value of assessing a broad range of behavioral change to detect non-motor impairment, anosmia, and differences in socially appropriate responses, in the marmoset MPTP model of early PD.

Multimodal MRI Evaluation of the MitoPark Mouse Model of Parkinson's Disease.

The MitoPark mouse, a relatively new genetic model of Parkinson's disease (PD), has a dopaminergic neuron-specific knock-out that inactivates the mitochondrial transcription factor A (Tfam), a protein essential for mitochondrial DNA expression and maintenance. This study used multimodal MRI to characterize the neuroanatomical correlates of PD-related deficits in MitoPark mice, along with functional behavioral tests. Compared with age-matched wild-type animals, MitoPark mice at 30 weeks showed: i) reduced whole-brain volume and increased ventricular volume, indicative of brain atrophy, ii) reduced transverse relaxation time (T2*) of the substantia nigra and striatum, suggestive of abnormal iron accumulation, iii) reduced apparent diffusion coefficient in the substantia nigra, suggestive of neuronal loss, iv) reduced fractional anisotropy in the corpus callosum and substantia nigra, indicative of white-matter damages, v) cerebral blood flow was not significantly affected, and vi) reduced motor activity in open-field tests, reduced memory in novel object recognition tests, as well as decreased mobility in tail suspension tests, an indication of depression. In sum, MitoPark mice recapitulate changes in many MRI parameters reported in PD patients. Multimodal MRI may prove useful for evaluating neuroanatomical correlates of PD pathophysiology in MitoPark mice, and for longitudinally monitoring disease progression and therapeutic interventions for PD.

Hematopoietic knockdown of PPARδ reduces atherosclerosis in LDLR-/- mice.

PPARδ (peroxisome proliferator-activated receptor δ) mediates inflammation in response to lipid accumulation. Systemic administration of a PPARδ agonist can ameliorate atherosclerosis. Paradoxically, genetic deletion of PPARδ in hematopoietic cells led to a reduction of atherosclerosis in murine models, suggesting that downregulation of PPARδ expression in these cells may mitigate atherogenesis. To advance this finding forward to potential clinical translation through hematopoietic stem cell transplantation-based gene therapy, we employed a microRNA (miRNA) approach to knock down PPARδ expression in bone marrow cells followed by transplantation of the cells into LDLR-/- mice. We found that knockdown of PPARδ expression in the hematopoietic system caused a dramatic reduction in aortic atherosclerotic lesions. In macrophages, a key component in atherogenesis, knockdown of PPARδ led to decreased expression of multiple pro-inflammatory factors, including monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-1ß and IL-6. Expression of CCR2, a receptor for MCP-1, was also decreased. The downregulation of pro-inflammatory factors is consistent with significant reduction of macrophage presence in the lesions, which may also be attributable to elevation of ABCA1 (ATP-binding cassette, subfamily A, member 1) and depression of adipocyte differentiate-related protein. Furthermore, the abundance of both MCP-1 and matrix metalloproteinase-9 proteins was reduced in plaque areas. Our results demonstrate that miRNA-mediated PPARδ knockdown in hematopoietic cells is able to ameliorate atherosclerosis.

Odor enrichment sculpts the abundance of olfactory bulb mitral cells.

Mitral cells are the primary output cell from the olfactory bulb conveying olfactory sensory information to higher cortical areas. Gene-targeted deletion of the Shaker potassium channel Kv1.3 alters voltage-dependence and inactivation kinetics of mitral cell current properties, which contribute to the "Super-smeller" phenotype observed in Kv1.3-null mice. The goal of the current study was to determine if morphology and density are influenced by mitral cell excitability, olfactory environment, and stage of development. Wildtype (WT) and Kv1.3-null (KO) mice were exposed to a single odorant (peppermint or citralva) for 30 days. Under unstimulated conditions, postnatal day 20 KO mice had more mitral cells than their WT counterparts, but no difference in cell size. Odor-enrichment with peppermint, an olfactory and trigeminal stimulus, decreased the number of mitral cells in three month and one year old mice of both genotypes. Mitral cell density was most sensitive to odor-stimulation in three month WT mice. Enrichment at the same age with citralva, a purely olfactory stimulus, decreased cell density regardless of genotype. There were no significant changes in cell body shape in response to citralva exposure, but the cell area was greater in WT mice and selectively greater in the ventral region of the OB in KO mice. This suggests that trigeminal or olfactory stimulation may modify mitral cell area and density while not impacting cell body shape. Mitral cell density can therefore be modulated by the voltage and sensory environment to alter information processing or olfactory perception.

Bone marrow-derived microglia-based neurturin delivery protects against dopaminergic neurodegeneration in a mouse model of Parkinson's disease.

Although neurotrophic factors have long been recognized as potent agents for protecting against neuronal degeneration, clinical success in treating Parkinson's disease and other neurodegenerative disorders has been hindered by difficulties in delivery of trophic factors across the blood brain barrier (BBB). Bone marrow hematopoietic stem cell-based gene therapy is emerging as a promising tool for overcoming drug delivery problems, as myeloid cells can cross the BBB and are recruited in large numbers to sites of neurodegeneration, where they become activated microglia that can secrete trophic factors. We tested the efficacy of bone marrow-derived microglial delivery of neurturin (NTN) in protecting dopaminergic neurons against neurotoxin-induced death in mice. Bone marrow cells were transduced ex vivo with lentivirus expressing the NTN gene driven by a synthetic macrophage-specific promoter. Infected bone marrow cells were then collected and transplanted into recipient animals. Eight weeks after transplantation, the mice were injected with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropuridine (MPTP) for seven days to induce dopaminergic neurodegeneration. Microglia-mediated NTN delivery dramatically ameliorated MPTP-induced degeneration of tyrosine hydroxylase (TH)-positive neurons of the substantia nigra and their terminals in the striatum. Microglia-mediated NTN delivery also induced significant recovery of synaptic marker staining in the striatum of MPTP-treated animals. Functionally, NTN treatment restored MPTP-induced decline in general activity, rearing behavior, and food intake. Thus, bone marrow-derived microglia can serve as cellular vehicles for sustained delivery of neurotrophic factors capable of mitigating dopaminergic injury.

Macrophage LXRα gene therapy ameliorates atherosclerosis as well as hypertriglyceridemia in LDLR(-/-) mice.

Liver X receptors (LXRs) are implicated in the regulation of cholesterol homeostasis, inflammatory response and atherogenesis. Administration of LXR agonists inhibits the progress of atherosclerosis, and also increases plasma triglyceride levels, representing an obstacle to their use in treating this disease. The objective of this study was to develop an alternative approach that could overcome this obstacle. Eight-week-old low-density lipoprotein receptor-deficient (LDLR(-/-)) mice were transplanted with hematopoietic stem cell (HSC)-enriched bone marrow cells transduced with lentivectors expressing either green fluorescent protein (GFP) (Lenti-SP-GFP, control) or LXRα (Lenti-SP-LXRα) driven by a synthetic macrophage promoter. At 4 weeks post-transplant, the mice were fed with a Western diet for 8 weeks and then killed. Compared with Lenti-SP-GFP mice, the Lenti-SP-LXRα mice had a 30% reduction in atherosclerotic lesions, which was accompanied by increases in levels of macrophage expression of cholesterol efflux genes apolipoprotein E and ATP-binding cassette A1, as well as decreases in plasma inflammatory cytokines interleukin-6 and tumor necrosis factor-α. Intriguingly, a 50% reduction of plasma triglyceride level was also observed. We conclude that HSC-based macrophage LXRα gene therapy ameliorates the development of atherosclerosis along with an unexpected concomitant reduction of plasma triglyceride levels in LDLR(-/-) mice. These findings highlight the potential value of macrophage LXR expression as an avenue for therapeutic intervention against atherosclerosis.

The Olfactory Bulb: A Metabolic Sensor of Brain Insulin and Glucose Concentrations via a Voltage-Gated Potassium Channel.

The voltage-gated potassium channel, Kv1.3, contributes a large proportion of the current in mitral cell neurons of the olfactory bulb where it assists to time the firing patterns of action potentials as spike clusters that are important for odorant detection. Gene-targeted deletion of the Kv1.3 channel, produces a "super-smeller" phenotype, whereby mice are additionally resistant to diet- and genetically-induced obesity. As assessed via an electrophysiological slice preparation of the olfactory bulb, Kv1.3 is modulated via energetically important molecules - such as insulin and glucose - contributing to the body's metabolic response to fat intake. We discuss a biophysical characterization of modulated synaptic communication in the slice following acute glucose and insulin stimulation, chronic elevation of insulin in mice that are in a conscious state, and induction of diet-induced obesity. We have discovered that Kv1.3 contributes an unusual nonconducting role - the detection of metabolic state.

State-dependent sculpting of olfactory sensory neurons is attributed to sensory enrichment, odor deprivation, and aging.

Gene-targeted deletion of the predominant Shaker potassium channel, Kv1.3, in the mitral cells of the olfactory bulb, decreases the number of presynaptic, odorant receptor (OR)-identified olfactory sensory neurons (OSNs) in the main olfactory epithelium (MOE) and alters the nature of their postsynaptic connections to mitral cell targets. The current study examined whether OSN density was state-dependent by examining the impact of (1) odor enrichment, (2) sensory deprivation, and (3) aging upon the number of P2- or M72-expressing neurons. Histological approaches were used to quantify the number of OSNs across entire epithelia for wildtype (WT) vs. Kv1.3-null (KO) mice bred onto an ORtauLacZ reporter background. Following either odor enrichment or early unilateral naris-occlusion, the number of M72-expressing OSNs was significantly decreased in WT mice, but was unchanged in KO animals. Following naris-occlusion, the number of P2-expressing OSNs was decreased regardless of genotype. Animals that were reared to 2 years of age demonstrated loss of both P2- and M72-expressing OSNs in WT mice and a concomitant loss of only M72-expressing neurons in KO mice. These findings suggest that voltage-gated activity of the mitral cells is important for OSN plasticity, and can prevent neuronal loss via sensory- and OR-dependent mechanisms.

Neuronal nitric oxide synthase in the olfactory system, forebrain, pituitary and retina of the adult teleost Clarias batrachus.

Immunocytochemical application of antibodies against nNOS to the brain sections of Clarias batrachus revealed intense immunoreactivity in several olfactory receptor neurons (ORNs), in their axons over the olfactory nerve, and terminals in the olfactory glomeruli. Several basal cells in the olfactory epithelium showed NOS immunoreactivity. Application of post-embedding immunoelectron microscopy showed nNOS labeled gold particles in apical cilia, dendrites and soma of the ORNs and also in the axon terminals in the glomeruli of the olfactory bulb. nNOS containing fibers were also encountered in the medial olfactory tracts (MOTs). Bilateral ablation of the olfactory organ resulted in total loss of nNOS immunoreactivity in the fascicles of the olfactory nerve layer and also in the MOT. nNOS immunoreactivity was seen in several cells of the nucleus preopticus (NPO) and their axons that innervate the pituitary gland. Some cells in the floor of the tuberal area were stained positive with nNOS antibodies. nNOS immunolabeled cells were seen in all the three components of the pituitary gland with light as well as post-embedding immunoelectron microscopy. While several nNOS immunoreactive fibers were seen in rostral pars distalis, a much limited fiber population was seen in the proximal pars distalis. In addition, conspicuous immunoreactivity was noticed in some ganglion cells in the retina and in some fibers of the optic nerve traceable to the optic tectum. The NO containing system in this fish appears to be similar to that in other fishes.

Olfactory sensory deprivation increases the number of proBDNF-immunoreactive mitral cells in the olfactory bulb of mice.

In the olfactory bulb, apoptotic cell-death induced by sensory deprivation is restricted to interneurons in the glomerular and granule cell layers, and to a lesser extent in the external plexiform layer, whereas mitral cells do not typically undergo apoptosis. With the goal to understand whether brain-derived neurotrophic factor (BDNF) mediates mitral cell survival, we performed unilateral naris occlusion on mice at postnatal day one (P1) and examined the subsequent BDNF-immunoreactive (BDNF-ir) profile of the olfactory bulb at P20, P30, and P40. Ipsilateral to the naris occlusion, there was a significant increase in the number of BDNF-ir mitral cells per unit area that was independent of the duration of the sensory deprivation induced by occlusion. The number of BDNF-ir juxtaglomerular cells per unit area, however, was clearly diminished. Western blot analysis revealed the presence of primarily proBDNF in the olfactory bulb. These data provide evidence for a neurotrophic role of proBDNF in the olfactory system of mice and suggest that proBDNF may act to protect mitral cells from the effects of apoptotic changes induced by odor sensory deprivation.

Deletion of voltage-gated channel affects glomerular refinement and odorant receptor expression in the mouse olfactory system.

Olfactory sensory neurons (OSNs) expressing a specific odorant receptor (OR) gene send axonal projections to specific glomeruli, creating a stereotypic olfactory sensory map. Odorant receptor sequence, G-protein cAMP signaling, and axon guidance molecules have been shown to direct axons of OSNs toward central targets in the olfactory bulb (OB). Although the OR sequence may act as one determinant, our objective was to elucidate the extent by which voltage-dependent activity of postsynaptic projection neurons in the OB centrally influences peripheral development and target destination of OSNs. We bred OR-tagged transgenic mice to homozygosity with mice that had a gene-targeted deletion of the Shaker potassium ion channel (Kv1.3) to elucidate how activity modulates synaptic connections that formulate the sensory map. Here we report that the Kv1.3 ion channel, which is predominantly expressed in mitral cells and whose gene-targeted deletion causes a "super-smeller" phenotype, alters synaptic refinement of axonal projections from OSNs expressing P2, M72, and MOR28 ORs. Absence of Kv1.3 voltage-gated activity caused the formation of small, heterogeneous, and supernumerary glomeruli that failed to undergo neural pruning over development. These changes were accompanied by a significant decrease in the number of P2-, M72-, and MOR28-expressing OSNs, which contained an overexpression of OR protein and G-protein G(olf) in the cilia of the olfactory epithelium. These findings suggest that voltage-gated activity of projection neurons is essential to refine primary olfactory projections and that it regulates proper expression of the transduction machinery at the periphery.

Neurotrophin B receptor kinase increases Kv subfamily member 1.3 (Kv1.3) ion channel half-life and surface expression.

Kv subfamily member 1.3 (Kv1.3), a member of the Shaker family of potassium channels, has been found to play diverse roles in immunity, metabolism, insulin resistance, sensory discrimination, and axonal targeting in addition to its traditional role in the stabilization of the resting potential. We demonstrate that the neurotrophin B receptor (TrkB) causes an upregulation of Kv1.3 ion channel protein expression in the absence of the preferred ligand for the receptor (brain-derived neurotrophic factor; BDNF) and oppositely downregulates levels of Kv subfamily member 1.5. Although the effect occurs in the absence of the ligand, Kv1.3 upregulation by TrkB is dependent upon the catalytic domain of the TrkB kinase as well as tyrosine (Y) residues in the N and C terminus of the Kv1.3 channel. Using pulse-chase experiments we find that TrkB alters the half-life residence of the channel by approximately 2x and allows it to sustain activity as reflected in an increased current magnitude without alteration of kinetic properties. TrkB and Kv1.3 co-immunoprecipitate from tissue preparations of the mouse olfactory bulb and olfactory cortex, and by immunocytochemical approaches, are found to be co-localized in the glomerular, mitral cell, and internal plexiform layers of the olfactory bulb. These data suggest that Kv1.3 is not only modulated by direct phosphorylation in the presence of BDNF-activated TrkB kinase, but also may be fine tuned via regulation of surface expression while in the proximity of neurotrophic factor receptors. Given the variability of TrkB expression during development, regeneration, or neuronal activation, modulation of surface expression and turnover of Kv channels could significantly impact neuronal excitability, distinct from that of tyrosine kinase phosphorylation.

Role of neuropeptide Y in the regulation of gonadotropin releasing hormone system in the forebrain of Clarias batrachus (Linn.): immunocytochemistry and high performance liquid chromatography-electrospray ionization-mass spectrometric analysis.

Although the importance of neuropeptide Y (NPY) in the regulation of gonadotropin releasing hormone (GnRH) and reproduction has been highlighted in recent years, the neuroanatomical substrate within which these substances might interact has not been fully elucidated. Present work was undertaken with a view to define the anatomical-physiological correlates underlying the role exercised by NPY in the regulation of GnRH in the forebrain of the teleost Clarias batrachus. Application of double immunocytochemistry revealed close associations as well as colocalizations of the two peptides in the olfactory receptor neurons (ORNs), olfactory nerve fibers and their terminals in the glomeruli, ganglion cells of nervus terminalis, medial olfactory tract, fibers in the area ventralis telencephali/pars supracommissuralis and cells as well as fibers in the pituitary. NPY containing axons were found to terminate in the vicinity of GnRH cells in the pituitary with light as well as electron microscopy. Double immunoelectron microscopy demonstrated gold particles for NPY and GnRH colocalized on the membrane and in dense core of the secretory granules in the cells distributed in all components of the pituitary gland. To assess the physiological implication of these observations, NPY was injected via the intracranial route and the response of GnRH immunoreactive system was evaluated by relative quantitative morphometry as well as high performance liquid chromatography (HPLC) analysis. Two hours following NPY (20 ng/g body weight) administration, a dramatic increase was observed in the GnRH immunoreactivity in the ORNs, in the fibers of the olfactory bulb (163%) and medial olfactory tract (351%). High performance liquid chromatography-electrospray ionization-mass spectrometric analysis confirmed the immunocytochemical data. Significant rise in the salmon GnRH (sGnRH)-like peptide content was observed in the olfactory organ (194.23%), olfactory bulb (146.64%), telencephalon+preoptic area (214.10%) and the pituitary (136.72%) of the NPY-treated fish. However, GnRH in the hypothalamus was below detection limit in the control as well as NPY-treated fish. Present results suggest the involvement of NPY in the up-regulation of sGnRH containing system at different level of neuraxis extending from the olfactory epithelium to the pituitary in the forebrain of C. batrachus.

Ontogeny of GnRH-like immunoreactive neuronal systems in the forebrain of the Indian major carp, Cirrhinus mrigala.

GnRH immunoreactivity appeared in the medial olfactory placode very early in the development of Cirrhinus mrigala. The immunoreactive elements were divisible into distinct migratory and non-migratory components. The migratory component appeared as a patch of intensely immunoreactive cells located close to the olfactory epithelium in day 6 post-fertilization larvae. Subsequently, these neurons migrate caudally along the ventromedial aspect of the developing forebrain and enroute give rise to GnRH immunoreactive neurons in the (1) nervus terminalis located in ventral and caudal part of the olfactory bulb (day 8), and (2) basal telencephalon (day 9). The non-migratory GnRH immunoreactive component appeared in the olfactory placode of day 1 post-fertilization larvae. It consisted of few olfactory receptor neuron (ORN)-like cells with distinct flask-shaped somata, dendrites that communicate with the periphery and a single axon on the basal side; GnRH immunoreactivity was seen throughout the neuron. Considerable increase in the number of immunoreactive ORNs was encountered in day 2 post-fertilization larvae. On day 3, the dendrites of ORNs sprout bunches of apical cilia, while on the basal side the axonal outgrowths can be traced to the olfactory bulb. GnRH immunoreactive fibers were distributed in the olfactory nerve layer in the periphery of the bulb and glomeruli-like innervation was clearly established in 5 days old larvae. The innervation to the olfactory bulb showed a considerable increase in GnRH immunoreactivity in 9 and 19 days old larvae. However, GnRH immunoreactivity in non-migratory as well as migratory components gradually diminished and disappeared altogether by the age of 68 days. Results of the present study suggest that GnRH may serve a neurotransmitter role in the ORNs during early stages of development in C. mrigala.

Role of neuropeptide Y (NPY) in the regulation of reproduction: study based on catfish model.

Significance of NPY in the regulation of GnRH-LH axis was evaluated. Considerable NPY immunoreactivity was seen in the components like olfactory system, basal telencephalon, preoptic and tuberal areas, and the pituitary gland that serve as neuroanatomical substrates for processing reproductive information. Close anatomical association as well as colocalizations of NPY and GnRH were seen in the olfactory receptor neurons, olfactory nerve fibers and their terminals in the glomeruli, ganglion cells of nervus terminalis, medial olfactory tracts, fibers in the ventral telencephalon and pituitary. In the pituitary, NPY fibers seem to innervate the GnRH as well as LH cells. Intracranial administration of NPY resulted in significant increase in the GnRH immunoreactivity in all the components of the olfactory system. In the pituitary, NPY augmented the population of GnRH fibers and LH cells. HPLC analysis showed that salmon GnRH content in the olfactory organ, bulb, preoptic area+telencephalon and pituitary was also significantly increased following NPY treatment. NPY may play a role in positive regulation of GnRH throughout the neuraxis and also up-regulate the LH cells in the pituitary.