Amul J. Sakharkar

Astrocyte Leptin Receptor (ObR) and Leptin Transport in Adult-Onset Obese Mice

The agouti viable yellow (A vy) spontaneous mutation generates an unusual mouse phenotype of agouti-colored coat and adult-onset obesity with metabolic syndrome. Persistent production of agouti signaling protein in A vy mice antagonizes melanocortin receptors in the hypothalamus. To determine how this disruption of neuroendocrine circuits affects leptin transport across the blood-brain barrier (BBB), we measured leptin influx in A vy and B6 control mice after the development of obesity, hyperleptinemia, and increased adiposity. After iv bolus injection, (125)I-leptin crossed the BBB significantly faster in young (2 month old) B6 mice than in young A vy mice or in older (8 month old) mice of either strain. This difference was not observed by in situ brain perfusion studies, indicating the cause being circulating factors, such as elevated leptin levels or soluble receptors. Thus, A vy mice showed peripheral leptin resistance. ObRa, the main transporting receptor for leptin at the BBB, showed no change in mRNA expression in the cerebral microvessels between the age-matched (2 month old) A vy and B6 mice. Higher ObRb mRNA was seen in the A vy microvasculature with unknown significance. Immunofluorescent staining unexpectedly revealed that many of the ObR(+) cells were astrocytes and that the A vy mice showed significantly more ObR(+) astrocytes in the hypothalamus than the B6 mice. Although leptin permeation from the circulation was slower in the A vy mice, the increased ObR expression in astrocytes and increased ObRb mRNA in microvessels suggest the possibility of heightened central nervous system sensitivity to circulating leptin.

Neuropeptide Y in the forebrain of the adult male cichlid fish Oreochromis mossambicus: distribution, effects of castration and testosterone replacement.

We studied the organization of the neuropeptide Y (NPY)‐immunoreactive system in the forebrain of adult male cichlid fish Oreochromis mossambicus and its response to castration and testosterone replacement by using morphometric methods. Immunoreactivity for NPY was widely distributed in the forebrain, and the pattern generally resembled that in other teleosts. Whereas immunoreactivity was conspicuous in the ganglia of nervus terminalis (NT; or nucleus olfactoretinalis), a weak reaction was detected in some granule cells in the olfactory bulb and in the cells of area ventralis telencephali pars lateralis (Vl). Moderately to intensely immunoreactive cells were distinctly seen in the nucleus entopeduncularis (NE), nucleus preopticus (NPO), nucleus lateralis tuberis (NLT), paraventricular organ (PVO), and midbrain tegmentum (MT). NPY fibers were widely distributed in the forebrain. Castration for 10/15 days resulted in a drastic loss of immunoreactivity in the cells of NE (P < 0.001) and a significant decrease (P < 0.01) in their cell nuclear size. However, cell nuclei of the NT neurons showed a significant increase in size. A highly significant reduction in the NPY‐immunoreactive fiber density (P < 0.001) was observed in several areas of the forebrain. Although testosterone replacement reversed these changes, fibers in some areas showed supranormal responses. Immunoreactive cells in Vl, NPO, NLT, PVO, and MT and fiber density in some other areas did not respond to castration. We suggest that the NPY‐immunoreactive elements that respond to castration and testosterone replacement may serve as the substrate for processing the positive feedback action of the steroid hormone. J. Comp. Neurol. 489:148–165, 2005.

Immunohistochemical localization of cocaine- and amphetamine-regulated transcript peptide in the brain of the catfish, Clarias batrachus (Linn.)

The organization of cocaine‐ and amphetamine‐regulated transcript peptide (CARTp, 54–102) immunoreactivity was investigated in the brain of the catfish, Clarias batrachus. CARTp‐immunoreactivity was observed in several granule cells of the olfactory bulbs, in dot‐like terminals around mitral cells, and in the fibers of the medial olfactory tracts. While several groups of discrete cells in the telencephalon showed CARTp‐immunoreactivity, the immunostained fibers were widely distributed in the area dorsalis and ventralis telencephali. Immunoreactivity was seen in several periventricular and a few magnocellular neurons, and in a dense fiber network throughout the preoptic area. Varying degrees of immunoreactive fibers were seen in the periventricular region in the thalamus, hypothalamus, and pituitary. Some neurons in the nucleus preglomerulosus medialis and lateralis, central nucleus of the inferior lobes, nucleus lobobulbaris of the posterior tuberculum, and nucleus recessus posterioris showed distinct CARTp‐immunoreactivity. Considerable immunoreactivity was seen in the optic tectum, rostral torus semicircularis, central pretectal area, and granule cells of the cerebellum. While only isolated immunoreactive cells were seen at three distinct sites in the metencephalon, a fiber network was seen in the facial and vagal lobes and periventricular and ventral regions of the medulla oblongata. The pattern of the CARTp distribution in the brain of C. batrachus suggests that it may play an important role in the processing of sensory information, the regulation of hormone secretion by hypophysial cell types, and motor and vegetative function. Finally, as in other animal species, CARTp seems to play a role in the processing of gustatory information. J. Comp. Neurol. 502:215–235, 2007.

Neuronal nitric oxide synthase in the olfactory system of an adult teleost fish Oreochromis mossambicus.

The aim of the present study is to explore the distribution of nitric oxide synthase in the olfactory system of an adult teleost, Oreochromis mossambicus using neuronal nitric oxide synthase (nNOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry methods. Intense nNOS immunoreactivity was noticed in several olfactory receptor neurons (ORNs), in their axonal extensions over the olfactory nerve and in some basal cells of the olfactory epithelium. nNOS containing fascicles of the ORNs enter the bulb from its rostral pole, spread in the olfactory nerve layer in the periphery of the bulb and display massive innervation of the olfactory glomeruli. Unilateral ablation of the olfactory organ resulted in dramatic loss of nNOS immunoreactivity in the olfactory nerve layer of the ipsilateral bulb. In the olfactory bulb of intact fish, some granule cells showed intense immunoreactivity; dendrites arising from the granule cells could be traced to the glomerular layer. Of particular interest is the occurrence of nNOS immunoreactivity in the ganglion cells of the nervus terminalis. nNOS containing fibers were also encountered in the medial olfactory tracts as they extend to the telencephalon. The NADPHd staining generally coincides with that of nNOS suggesting that it may serve as a marker for nNOS in the olfactory system of this fish. However, mismatch was encountered in the case of mitral cells, while all are nNOS-negative, few were NADPHd positive. The present study for the first time revealed the occurrence of nNOS immunoreactivity in the ORNs of an adult vertebrate and suggests a role for nitric oxide in the transduction of odor stimuli, regeneration of olfactory epithelium and processing of olfactory signals.

Reproduction Phase-Related Expression of β-Endorphin-Like Immunoreactivity in the Nucleus Lateralis Tuberis of the Female Indian Major Carp Cirrhinus mrigala: Correlation with the Luteinising Hormone Cells-Ovary Axis

The present study aimed to determine whether β‐endorphin immunoreactivity (bEP‐ir) in the neurones of the nucleus lateralis tuberis (NLT) is linked to the seasonal cycle and shows correlation with the number of luteinising hormone (LH) cells in the pituitary gland and ovaries in the teleost, Cirrhinus mrigala. Although LH cells were moderately immunostained during the resting phase (December to January), the morphological profile suggested increased synthetic and secretory activity during the preparatory (February to April) and prespawning (May to June) phases. However, LH immunoreactivity was greatly reduced (P < 0.001) in the spawning (July to August) phase, suggesting massive discharge of the hormone; this pool was partly replenished in the postspawning (September to November) phase. The ovaries grew rapidly in the preparatory and prespawning phases; maximal size was attained during spawning, when ovulation occurred. Thereafter, the ovaries regressed. The NLT of C. mrigala is divisible into the pars lateralis (NLTl) and medialis (NLTm). During the postspawning and resting phases, bEP‐ir was readily detectable in the NLTm as well as NLTl neurones. However, a steady reduction in the immunoreactivity was observed in the NLTm neurones during the preparatory through spawning phases (P < 0.001), suggesting a negative correlation with the LH cells‐ovary axis. Thus, the inhibitory influence of β‐endorphin on the gonadotrophin‐releasing hormone (GnRH)‐LH axis appears to be attenuated during the preparatory through spawning phases. This may be necessary for the rapid stimulation of the axis culminating in spawning. Neurones of the NLTl also showed a gradual reduction in bEP‐ir during the preparatory and prespawning phases (P < 0.01) and may therefore play a similar role. However, significant augmentation of the immunoreactivity was noticed in these neurones during the spawning phase (P < 0.001), the physiological significance of which is unknown. Although the present study demonstrated a temporal correlation between the β‐endorphin in the NLT, LH cells and the ovary, we suggest that the peptide in the NLTl and NLTm may show functional duality during the spawning phase.

Reproduction phase‐related variations in neuropeptide Y immunoreactivity in the olfactory system, forebrain, and pituitary of the female catfish, Clarias batrachus (Linn.)

The aim of this study was to determine whether neuropeptide Y (NPY) immunoreactivity in the cells and fibers in the forebrain and pituitary of Clarias batrachus is linked to the annual reproductive cycle. A steady rise in luteinizing hormone (LH) immunoreactivity was seen in the pituitary through preparatory (February–April) and prespawning (May–June) phases; it was greatly reduced during spawning (July–August; P < 0.001) and partially replenished during postspawning (September–November; P < 0.01) through resting (December–January) phases. Although NPY immunoreactivity in olfactory receptor neurons and olfactory nerve layer in olfactory bulb was gradually augmented during resting through prespawning phases (P < 0.001), attaining a peak in spawning phase (P < 0.001), a dramatic decline was encountered during postspawning phase (P < 0.001). A similar pattern was also observed in NPY‐containing fibers of the medial olfactory tract (MOT) and pituitary. However, a different pattern of NPY immunoreactivity was observed in the neurons of nucleus entopeduncularis (NE) and nucleus preopticus periventricularis (NPP). Whereas these neurons and fibers in the forebrain showed significant augmentation during the resting through prespawning phases (P < 0.001), the immunoreactivity dramatically declined during spawning (P < 0.001) and was partially replenished in the postspawning phase. Testosterone injection of juveniles significantly augmented (P < 0.001) NPY immunoreactivity in NE neurons. We suggest that NPY cells of NE and NPP, and related fiber systems, might be involved in processing of sex steroid‐borne information and regulation of the gonadotropin‐releasing hormone‐LH axis. J. Comp. Neurol. 504:450–469, 2007.

Neuronal nitric oxide synthase immunoreactivity in forebrain, pineal, and pituitary of Oreochromis mossambicus (Tilapia)

Ultrastructural localization of neuronal nitric oxide synthase (nNOS) in olfactory receptor neurons (ORNs) and immunohistochemical detection of the enzyme in forebrain, pituitary, and pineal were undertaken in the teleost Oreochromis mossambicus. Application of post-embedding immunoelectron microscopy revealed nNOS-labeled gold particles on the cilia, microvilli, mitochondria, and Golgi complex of the ORNs. Gold particles were also seen adhered to microtubules in the axons that extend to the olfactory nerve layer in the olfactory bulb. With light microscopy, nNOS-immunoreactive neurons were seen in preoptic area, nucleus entopeduncularis, and parvocellular, and magnocellular subdivisions of nucleus preopticus (NPO). Numerous cerebrospinal fluid-contacting cells lining the wall of the third ventricle at the level of the NPO showed intense immunoreactivity. Intense to moderate immunoreactivity was observed in the neurons of suprachiasmatic nucleus, nucleus lateralis tuberis pars lateralis, and nucleus recessus lateralis. While several immunoreactive fibers were detected in medial olfactory tract, suprachiasmatic area, and hypothalamo-hypophyseal tract, a few were seen throughout the telencephalon, in the optic chiasma, tuberal area, and inferior lobes. In the pituitary, nNOS-containing fibers were seen in the neurohypophysis, rostral pars distalis, proximal pars distalis, and pars intermedia. While intense immunoreactivity was noticed in some cells in the pineal, immunoreactive fibers were detected in the pineal stalk as well as parenchyma. We suggest that nitric oxide may play a role in processing olfactory and photic information, circadian rhythms, and neuroendocrine regulation in tilapia.

Reproduction phase-related variations in the GnRH immunoreactive fibers in the pineal of the Indian major carp Cirrhinus mrigala (Ham.)

We studied GnRH immunoreactivity in the pineal gland of the Indian major carp Cirrhinus mrigala during different phases of reproductive cycle. In the resting phase (December–January), GnRH immunoreactive (-ir) fibers were organized as paired fascicles above the posterior commissure that ascend in the stalk and distribute widely in the pineal gland. The GnRH-ir fiber density significantly declined (P<0.001) during the preparatory phase (February–April) and the fibers disappeared thereafter. While no GnRH fibers were seen during the prespawning (May–June) and spawning (July–August), isolated GnRH-ir fibers reappeared in the postspawning phase. Since no GnRH cell bodies were detected in the pineal, these GnRH-ir fibers seem to be of central origin. The results reveal a distinct reciprocal relationship between the GnRH immunoreactivity in the pineal and the status of the ovarian maturity; the fibers appeared in the pineal only during the period of ovarian quiescence. While the functional significance of these cyclic changes in GnRH is yet to be determined, we suggest that the decapeptide may serve as a transmitter of central origin that modulates the activity of the pineal gland.

Neurosecretory neurons of the nucleus preopticus (NPO) express salmon GnRH mRNA and show reproduction phase-related variation in the female Indian major carp, Cirrhinus cirrhosus

We studied the expression of sGnRH mRNA in the neurons of the nucleus preopticus (NPO) of the Indian major carp, Cirrhinus cirrhosus, and their correlation with the reproductive status of the fish. Non-radioisotopic in situ hybridization histochemistry protocol employing biotinylated-oligonucleotide probes complementary to salmon GnRH, cichlid GnRH I, catfish GnRH, chicken GnRH II (from cichlid and catfish), and mammalian GnRH, were applied to the sections through the POA of the female Indian major carp Cirrhinus cirrhosus. Incubation with the probe complimentary to salmon GnRH (sGnRH) mRNA from salmon, produced distinct hybridization signal in the cytosol of several neurosecretory neurons of the magnocellular and parvocellular subdivisions of the NPO of the fish collected during February-April (preparatory phase) and May-June (prespawning phase). However, no signal was detected in the NPO of fish collected during July-August (spawning phase). Application of other antisense probes, or sense probe for salmon GnRH mRNA, produced no signal. We suggest that NPO neurons in C. cirrhosus may express sGnRH mRNA, produce GnRH peptide, and play a role in regulation of pituitary-ovary axis.

Neuropeptide CART prevents memory loss attributed to withdrawal of nicotine following chronic treatment in mice: CART in nicotine withdrawal

Although chronic nicotine administration does not affect memory, its withdrawal causes massive cognitive deficits. The underlying mechanisms, however, have not been understood. We test the role of cocaine‐ and amphetamine‐regulated transcript peptide (CART), a neuropeptide known for its procognitive properties, in this process. The mice on chronic nicotine treatment/withdrawal were subjected to novel object recognition task. The capability of the animal to discriminate between the novel and familiar objects was tested and represented as discrimination index (DI); reduction in the index suggested amnesia. Nicotine for 49 days had no effect on DI, but 8‐hour withdrawal caused a significant reduction, followed by full recovery at 24‐hour withdrawal timepoint. Bilateral CART infusion in dorsal hippocampus rescued deficits in DI at 8‐hours, whereas CART‐antibody infusion into the dorsal hippocampus attenuated the recovery at 24‐hours. Commensurate changes were observed in the CART as well as CART mRNA profiles in the hippocampus. CART mRNA expression and the peptide immunoreactivity did not change significantly following chronic nicotine treatment. However, there was a significant reduction at 8‐hour withdrawal, followed by a drastic increase in CART immunoreactivity as well as CART mRNA at 24‐hour withdrawal, compared with 8‐hour withdrawal. Distinct α7‐nicotinic receptor immunoreactivity was detected on the hippocampal CART neurons, suggesting cholinergic inputs. An increase in the synaptophysin immunoreactive elements around CART cells in the dentate gyrus, cornu ammonis 3 and subiculum at 24‐hour post‐withdrawal timepoint suggested neuronal plasticity. CART circuit dynamics in the hippocampus seems to modulate short‐term memory associated with nicotine withdrawal.