Adropin Stimulates Proliferation and Inhibits Adrenocortical Steroidogenesis in the Human Adrenal Carcinoma (HAC15) Cell Line.

Adropin is a multifunctional peptide hormone encoded by the ENHO (energy homeostasis associated) gene. It plays a role in mechanisms related to increased adiposity, insulin resistance, as well as glucose, and lipid metabolism. The low adropin levels are strongly associated with obesity independent insulin resistance. On the other hand, overexpression or exogenous administration of adropin improves glucose homeostasis. The multidirectional, adropin-related effects associated with the regulation of metabolism in humans also appear to be attributable to the effects of this peptide on the activity of various elements of the endocrine system including adrenal cortex. Therefore, the main purpose of the present study was to investigate the effect of adropin on proliferation and secretory activity in the human HAC15 adrenal carcinoma cell line. In this study, we obtained several highly interesting findings. First, GPR19, the main candidate sensitizer of adrenocortical cells to adropin, was expressed in HAC15 cells. Moreover, GPR19 expression was relatively stable and not regulated by ACTH, forskolin, or adropin itself. Our findings also suggest that adropin has the capacity to decrease expression levels of steroidogenic genes such as steroidogenic acute regulatory protein (StAR) and CYP11A1, which then led to a statistically significant inhibition in cortisol and aldosterone biosynthesis and secretion. Based on whole transcriptome study and research involving transforming growth factor (TGF)-ß type I receptor kinase inhibitor we demonstrated that attenuation of steroidogenesis caused by adropin is mediated by the TGF-ß signaling pathway likely to act through transactivation mechanism. We found that HAC15 cells treated with adropin presented significantly higher proliferation levels than untreated cells. Using specific intracellular inhibitors, we showed that adropin stimulate proliferation via ERK1/2 and AKT dependent signaling pathways. We have also demonstrated that expression of GPR19 is elevated in adrenocortical carcinoma in relation to normal adrenal glands. High level of GPR19 expression in adrenocortical carcinoma may constitute a negative prognostic factor of disease progression.

Characterization of Drosophila octopamine receptor neuronal expression using MiMIC-converted Gal4 lines.

Octopamine, the invertebrate analog of norepinephrine, is known to modulate a large variety of behaviors in Drosophila including feeding initiation, locomotion, aggression, and courtship, among many others. Significantly less is known about the identity of the neurons that receive octopamine input and how they mediate octopamine-regulated behaviors. Here, we characterize adult neuronal expression of MiMIC-converted Trojan-Gal4 lines for each of the five Drosophila octopamine receptors. Broad neuronal expression was observed for all five octopamine receptors, yet distinct differences among them were also apparent. Use of immunostaining for the octopamine neurotransmitter synthesis enzyme Tdc2, along with a novel genome-edited conditional Tdc2-LexA driver, revealed all five octopamine receptors express in Tdc2/octopamine neurons to varying degrees. This suggests autoreception may be an important circuit mechanism by which octopamine modulates behavior.

Evidence for a non-canonical JAK/STAT signaling pathway in the synthesis of the brain's major ion channels and neurotransmitter receptors.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) is a major signaling molecule that the brain uses to control a vast network of intracellular cascades fundamental to properties of learning and memory, and cognition. While much is known about BDNF signaling in the healthy nervous system where it controls the mitogen activated protein kinase (MAPK) and cyclic-AMP pathways, less is known about its role in multiple brain disorders where it contributes to the dysregulated neuroplasticity seen in epilepsy and traumatic brain injury (TBI). We previously found that neurons respond to prolonged BDNF exposure (both in vivo (in models of epilepsy and TBI) and in vitro (in BDNF treated primary neuronal cultures)) by activating the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathway. This pathway is best known for its association with inflammatory cytokines in non-neuronal cells. RESULTS: Here, using deep RNA-sequencing of neurons exposed to BDNF in the presence and absence of well characterized JAK/STAT inhibitors, and without non-neuronal cells, we determine the BDNF transcriptome that is specifically regulated by agents that inhibit JAK/STAT signaling. Surprisingly, the BDNF-induced JAK/STAT transcriptome contains ion channels and neurotransmitter receptors coming from all the major classes expressed in the brain, along with key modulators of synaptic plasticity, neurogenesis, and axonal remodeling. Analysis of this dataset has revealed a unique non-canonical mechanism of JAK/STATs in neurons as differential gene expression mediated by STAT3 is not solely dependent upon phosphorylation at residue 705 and may involve a BDNF-induced interaction of STAT3 with Heterochromatin Protein 1 alpha (HP1α). CONCLUSIONS: These findings suggest that the neuronal BDNF-induced JAK/STAT pathway involves more than STAT3 phosphorylation at 705, providing the first evidence for a non-canonical mechanism that may involve HP1α. Our analysis reveals that JAK/STAT signaling regulates many of the genes associated with epilepsy syndromes where BDNF levels are markedly elevated. Uncovering the mechanism of this novel form of BDNF signaling in the brain may provide a new direction for epilepsy therapeutics and open a window into the complex mechanisms of STAT3 transcriptional regulation in neurological disease.

Transthoracic Pulmonary Artery Denervation for Pulmonary Arterial Hypertension.

Objective- Pulmonary arterial hypertension is characterized by progressive pulmonary vascular remodeling and persistently elevated mean pulmonary artery pressures and pulmonary vascular resistance. We aimed to investigate whether transthoracic pulmonary artery denervation (TPADN) attenuated pulmonary artery (PA) remodeling, improved right ventricular (RV) function, and affected underlying mechanisms. We also explored the distributions of sympathetic nerves (SNs) around human PAs for clinical translation. Approach and Results- We identified numerous SNs in adipose and connective tissues around the main PA trunks and bifurcations in male Sprague Dawley rats, which were verified in samples from human heart transplant patients. Pulmonary arterial hypertensive rats were randomized into TPADN and sham groups. In the TPADN group, SNs around the PA trunk and bifurcation were completely and accurately removed under direct visualization. The sham group underwent thoracotomy. Hemodynamics, RV function, and pathological changes in PA and RV tissues were measured via right heart catheterization, cardiac magnetic resonance imaging, and pathological staining, respectively. Compared with the sham group, the TPADN group had lower mean pulmonary arterial pressures, less PA and RV remodeling, and improved RV function. Furthermore, TPADN inhibited neurohormonal overactivation of the sympathetic nervous system and renin-angiotensin-aldosterone system and regulated abnormal expressions and signaling of neurohormone receptors in local tissues. Conclusions- There are numerous SNs around the rat and human main PA trunks and bifurcations. TPADN completely and accurately removed the main SNs around PAs and attenuated pulmonary arterial hypertensive progression by inhibiting excessive activation of the sympathetic nervous system and renin-angiotensin-aldosterone system neurohormone-receptor axes.

Characterization of neuromedin U (NMU), neuromedin S (NMS) and their receptors (NMUR1, NMUR2) in chickens.

Neuromedin U (NMU) and its structurally-related peptide, neuromedin S (NMS), are reported to regulate many physiological processes and their actions are mediated by two NMU receptors (NMUR1, NMUR2) in mammals. However, the information regarding NMU, NMS, and their receptors is limited in birds. In this study, we examined the structure, functionality, and expression of NMS, NMU, NMUR1 and NMUR2 in chickens. The results showed that: 1) chicken (c-) NMU cDNA encodes a 181-amino acid precursor, which may generate two forms of NMU peptide with 9 (cNMU-9) and 25 amino acids (cNMU-25), respectively. 2) Interestingly, two cNMS transcripts encoding two cNMS precursors of different lengths were identified from chicken pituitary, and both cNMS precursors may produce a mature cNMS peptide of 9 amino acids (cNMS-9). 3) cNMU-9, cNMU-25 and cNMS-9 could activate cNMUR1 expressed in HEK293 cells potently, as monitored by three cell-based luciferase reporter systems, indicating that cNMUR1 can act as a receptor common for cNMU and cNMS peptides, whereas cNMUR2 could be potently activated by cNMS-9, but not by cNMU-9/cNMU-25. 4) cNMU and cNMUR1 are widely expressed in chicken tissues with abundant expression noted in the gastrointestinal tract, as detected by quantitative real-time PCR, whereas cNMUR2 expression is mainly restricted to the brain and anterior pituitary, and cNMS is widely expressed in chicken tissues. Collectively, our data helps to elucidate the physiological roles of NMU/NMS peptides in birds and reveal the functional conservation and changes of NMU/NMS-NMUR axis across vertebrates.

Do MicroRNAs Modulate Visceral Pain?

Visceral pain, a common characteristic of multiple diseases relative to viscera, impacts millions of people worldwide. Although hundreds of studies have explored mechanisms underlying visceral pain, it is still poorly managed. Over the past decade, strong evidence emerged suggesting that microRNAs (miRNAs) play a significant role in visceral nociception through altering neurotransmitters, receptors and other genes at the posttranscriptional level. Under pathological conditions, one kind of miRNA may have several target mRNAs and several kinds of miRNAs may act on one target, suggesting complex interactions and mechanisms between miRNAs and target genes lead to pathological states. In this review we report on recent progress in examining miRNAs responsible for visceral sensitization and provide miRNA-based therapeutic targets for the management of visceral pain.

The neuropeptide NMU amplifies ILC2-driven allergic lung inflammation.

Type 2 innate lymphoid cells (ILC2s) both contribute to mucosal homeostasis and initiate pathologic inflammation in allergic asthma. However, the signals that direct ILC2s to promote homeostasis versus inflammation are unclear. To identify such molecular cues, we profiled mouse lung-resident ILCs using single-cell RNA sequencing at steady state and after in vivo stimulation with the alarmin cytokines IL-25 and IL-33. ILC2s were transcriptionally heterogeneous after activation, with subpopulations distinguished by expression of proliferative, homeostatic and effector genes. The neuropeptide receptor Nmur1 was preferentially expressed by ILC2s at steady state and after IL-25 stimulation. Neuromedin U (NMU), the ligand of NMUR1, activated ILC2s in vitro, and in vivo co-administration of NMU with IL-25 strongly amplified allergic inflammation. Loss of NMU-NMUR1 signalling reduced ILC2 frequency and effector function, and altered transcriptional programs following allergen challenge in vivo. Thus, NMUR1 signalling promotes inflammatory ILC2 responses, highlighting the importance of neuro-immune crosstalk in allergic inflammation at mucosal surfaces.

Neuronal Fibers and Neurotransmitter Receptor Expression in the Human Endolymphatic Sac.

INTRODUCTION: Recent studies suggest that the human endolymphatic sac (ES) may have multiple functions, including an ion-transport capacity comparable to the kidney, an immunological capacity and a possible natriuretic capacity. Further, there have been speculations of a yet undefined role in intracranial pressure homeostasis. The anatomical location towards the sigmoid sinus would suggest a possible endo- and/or paracrine signaling. However, neuronal connections may also apply, but it remains very scarcely explored in the human ES. STUDY DESIGN: DNA micro-arrays and immunohistochemistry were used for analyses of fresh human ES tissue samples. METHODS: A total of 30 tissue samples from the human ES were obtained during translabyrinthine surgery for vestibular schwannoma. Microarray technology was used to investigate tissue sample gene expression, using adjacent dura mater as control. The expression of genes specific for neuronal signaling was determined and results for selected key molecules verified by immunohistochemistry. Transmission electron microscopy was used for ultrastructural analysis. RESULTS: For the transmission electron microscopy analysis, a direct innervation of the ES was observed with unmyelinated fibers imbedded in the ES epithelial lining. The microarrays confirmed, that several molecules involved in neuronal signaling were found expressed significantly in the ES DNA profile, such as the Cholecystokinin peptide and related receptors, Dopamine receptors 2 and 5, vesicular monoamine transporter 2 (VMAT2), plasma monoamine transporter (PMAT), and Serotonin 1D. All peptides were verified by immunohistochemistry. CONCLUSIONS: Based on global gene expression profiling and immuno-histochemical labeling, we conclude that the human ES expresses neuropeptide receptors and monoamine transporters. Combined with the ultrastructural demonstration of unmyelinated axons imbedded within the epithelial lining, the findings suggest that neuro-signaling mechanisms are involved in functions exerted by the ES.

Two proteolytic fragments of menin coordinate the nuclear transcription and postsynaptic clustering of neurotransmitter receptors during synaptogenesis between Lymnaea neurons.

Synapse formation and plasticity depend on nuclear transcription and site-specific protein targeting, but the molecular mechanisms that coordinate these steps have not been well defined. The MEN1 tumor suppressor gene, which encodes the protein menin, is known to induce synapse formation and plasticity in the CNS. This synaptogenic function has been conserved across evolution, however the underlying molecular mechanisms remain unidentified. Here, using central neurons from the invertebrate Lymnaea stagnalis, we demonstrate that menin coordinates subunit-specific transcriptional regulation and synaptic clustering of nicotinic acetylcholine receptors (nAChR) during neurotrophic factor (NTF)-dependent excitatory synaptogenesis, via two proteolytic fragments generated by calpain cleavage. Whereas menin is largely regarded as a nuclear protein, our data demonstrate a novel cytoplasmic function at central synapses. Furthermore, this study identifies a novel synaptogenic mechanism in which a single gene product coordinates the nuclear transcription and postsynaptic targeting of neurotransmitter receptors through distinct molecular functions of differentially localized proteolytic fragments.

Increased precipitation of spasms in an animal model of infantile spasms by prenatal stress exposure.

Infantile spasms (IS) represent a serious epileptic syndrome, called West syndrome (WS) that occurs in the early infantile age. Although several hypotheses and animal models have been proposed to explain the pathogenesis of IS, the pathophysiology of IS has not been elucidated. Recently, we proposed a hypothesis for IS under prenatal stress exposure (also called Zou's hypothesis) by correlating diverse etiologies and prenatal stresses with IS development. This research aims to determine the mechanism through which prenatal stress affects the offspring and establish the potential underlying mechanisms. Pregnant rats were subjected to forced swimming in cold water. Rat pups exposed to prenatal stress were administered with N-methyl-D-aspartate (NMDA). Exposure to prenatal stress sensitized the rats against development of NMDA-induced spasms. However, this phenomenon was altered by administering adrenocorticotropin. Prenatal stress exposure also altered the hormonal levels and neurotransmitter receptor expression of the developing rats as well as influenced the tissue structure of the brain. These findings suggest that maternal stress could alter the level of endogenous glucocorticoid, which is the basis of IS, and cerebral dysplasia, hypoxic-ischemic encephalopathy (HIE), inherited metabolic diseases, and other factors activated this disease in developmental brain.

Gene Expression Changes in Glutamate and GABA-A Receptors, Neuropeptides, Ion Channels, and Cholesterol Synthesis in the Periaqueductal Gray Following Binge-Like Alcohol Drinking by Adolescent Alcohol-Preferring (P) Rats.

BACKGROUND: Binge drinking of alcohol during adolescence is a serious public health concern with long-term consequences, including increased pain, fear, and anxiety. The periaqueductal gray (PAG) is involved in processing pain, fear, and anxiety. The effects of adolescent binge drinking on gene expression in this region have yet to be studied. METHODS: Male adolescent alcohol-preferring (P) rats were exposed to repeated binge drinking (three 1-hour sessions/d during the dark/cycle, 5 days/wk for 3 weeks starting at 28 days of age; ethanol intakes of 2.5 to 3 g/kg/session). We used RNA sequencing to assess the effects of ethanol intake on gene expression. RESULTS: Ethanol significantly altered the expression of 1,670 of the 12,123 detected genes: 877 (53%) decreased. In the glutamate system, 23 genes were found to be altered, including reduction in 7 of 10 genes for metabotropic and NMDA receptors. Subunit changes in the NMDA receptor may make it less sensitive to ethanol. Changes in GABAA genes would most likely increase the ability of the PAG to produce tonic inhibition. Five serotonin receptor genes, 6 acetylcholine receptor genes, and 4 glycine receptor genes showed decreased expression in the alcohol-drinking rats. Opioid genes (e.g., Oprk1, Oprm1) and genes for neuropeptides linked to anxiety and panic behaviors (e.g., Npy1r) had mostly decreased expression. Genes for 27 potassium, 10 sodium, and 5 calcium ion channels were found to be differentially expressed. Nine genes in the cholesterol synthesis pathway had decreased expression, including Hmgcr, encoding the rate-limiting enzyme. Genes involved in the production of myelin also had decreased expression. CONCLUSIONS: The results demonstrate that binge alcohol drinking during adolescence produces developmental changes in the expression of key genes within the PAG; many of these changes point to increased susceptibility to pain, fear, and anxiety, which could contribute to excessive drinking to relieve these negative effects.

A Follicle Rupture Assay Reveals an Essential Role for Follicular Adrenergic Signaling in Drosophila Ovulation.

Ovulation is essential for the propagation of the species and involves a proteolytic degradation of the follicle wall for the release of the fertilizable oocyte. However, the precise mechanisms for regulating these proteolytic events are largely unknown. Work from our lab and others have shown that there are several parallels between Drosophila and mammalian ovulation at both the cellular and molecular levels. During ovulation in Drosophila, posterior follicle cells surrounding a mature oocyte are selectively degraded and the residual follicle cells remain in the ovary to form a corpus luteum after follicle rupture. Like in mammals, this rupturing process also depends on matrix metalloproteinase 2 (Mmp2) activity localized at the posterior end of mature follicles, where oocytes exit. In the present study, we show that Mmp2 activity is regulated by the octopaminergic signaling in mature follicle cells. Exogenous octopamine (OA; equivalent to norepinephrine, NE) is sufficient to induce follicle rupture when isolated mature follicles are cultured ex vivo, in the absence of the oviduct or ovarian muscle sheath. Knocking down the alpha-like adrenergic receptor Oamb (Octoampine receptor in mushroom bodies) in mature follicle cells prevents OA-induced follicle rupture ex vivo and ovulation in vivo. We also show that follicular OA-Oamb signaling induces Mmp2 enzymatic activation but not Mmp2 protein expression, likely via intracellular Ca2+ as the second messenger. Our work develops a novel ex vivo follicle rupture assay and demonstrates the role for follicular adrenergic signaling in Mmp2 activation and ovulation in Drosophila, which is likely conserved in other species.

Expression Profiles of Neuropeptides, Neurotransmitters, and Their Receptors in Human Keratocytes In Vitro and In Situ.

Keratocytes, the quiescent cells of the corneal stroma, play a crucial role in corneal wound healing. Neuropeptides and neurotransmitters are usually associated with neuronal signaling, but have recently been shown to be produced also by non-neuronal cells and to be involved in many cellular processes. The aim of this study was to assess the endogenous intracellular and secreted levels of the neuropeptides substance P (SP) and neurokinin A (NKA), and of the neurotransmitters acetylcholine (ACh), catecholamines (adrenaline, noradrenaline and dopamine), and glutamate, as well as the expression profiles of their receptors, in human primary keratocytes in vitro and in keratocytes of human corneal tissue sections in situ. Cultured keratocytes expressed genes encoding for SP and NKA, and for catecholamine and glutamate synthesizing enzymes, as well as genes for neuropeptide, adrenergic and ACh (muscarinic) receptors. Keratocytes in culture produced SP, NKA, catecholamines, ACh, and glutamate, and expressed neurokinin-1 and -2 receptors (NK-1R and NK-2R), dopamine receptor D2, muscarinic ACh receptors, and NDMAR1 glutamate receptor. Human corneal sections expressed SP, NKA, NK-1R, NK-2R, receptor D2, choline acetyl transferase (ChAT), M3, M4 and M5 muscarinic ACh receptors, glutamate, and NMDAR1, but not catecholamine synthesizing enzyme or the α1 and ß2 adrenoreceptors, nor M1 receptor. In addition, expression profiles assumed significant differences between keratocytes from the peripheral cornea as compared to those from the central cornea, as well as differences between keratocytes cultured under various serum concentrations. In conclusion, human keratocytes express an array of neuropeptides and neurotransmitters. The cells furthermore express receptors for neuropeptides/neurotransmitters, which suggests that they are susceptible to stimulation by these substances in the cornea, whether of neuronal or non-neuronal origin. As it has been shown that neuropeptides/neurotransmitters are involved in cell proliferation, migration, and angiogenesis, it is possible that they play a role in corneal wound healing.

Cingulate area 32 homologies in mouse, rat, macaque and human: cytoarchitecture and receptor architecture.

Homologizing between human and nonhuman area 32 has been impaired since Brodmann said he could not homologize with certainty human area 32 to a specific cortical domain in other species. Human area 32 has four divisions, however, and two can be structurally homologized to nonhuman species with cytoarchitecture and receptor architecture: pregenual (p32) and subgenual (s32) in human and macaque monkey and areas d32 and v32 in rat and mouse. Cytoarchitecture showed that areas d32/p32 have a dysgranular layer IV in all species and that areas v32/s32 have large and dense neurons in layer V, whereas a layer IV is not present in area v32. Areas v32/s32 have the largest neurons in layer Va. Features unique to humans include large layer IIIc pyramids in both divisions, sparse layer Vb in area p32, and elongated neurons in layer VI, with area s32 having the largest layer Va neurons. Receptor fingerprints of both subdivisions of area 32 differed between species in size and shape, although AMPA/GABAA and NMDA/GABAA ratios were comparable among humans, monkeys, and rats and were significantly lower than in mice. Layers I-III of primate and rodent area 32 subdivisions share more similarities in their receptor densities than layers IV-VI. Monkey and human subdivisions of area 32 are more similar to each other than to rat and mouse subdivisions. In combination with intracingulate connections, the location, cytoarchitecture, and ligand binding studies demonstrate critical homologies among the four species.

Alcohol administration blocks stress-induced impairments in memory and anxiety, and alters hippocampal neurotransmitter receptor expression in male rats.

Chronic exposure to stress has many deleterious effects on behavior, which can often lead to self-medication with anxiolytics, antidepressants, or alcohol. We determined the effects of alcohol administration following a stressor on established behavioral, physiological, and neural responses to stress. Male Sprague-Dawley rats received: No alcohol/No stress (CON), Alcohol alone (ALC), Stress alone (STR), or Stress plus Alcohol (STR+ALC). For seven consecutive days, two cohorts received an oral dose of 2.0 g/kg of either 20% ethanol or saline. In Cohort 1, behavioral testing began after the final treatment (day-8). Memory was tested using the object recognition (OR) and Y-maze, anxiety on the plus maze, and depression on the forced swim task. Memory on OR and Y-maze tasks was impaired in the ALC and STR groups. This deficit was reversed in the STR+ALC group, which performed not differently from the CON group. Stress alone was associated with increased anxiety, which was alleviated with alcohol treatment. No treatment effects were found in the forced swim task. In Cohort 2, hippocampal GABAα4 was upregulated in the STR+ALC group and GluN2B was upregulated in the ALC and STR+ALC groups. The STR+ALC group in Cohort 1 showed enhanced corticosterone levels after forced swim. The STR+ALC group in Cohort 2 showed increased corticosterone levels on day-1 of treatment and a habituation by day-7. In conclusion, this study found a reversal of stress-induced deficits in cognition and anxiety when alcohol was given post-stress, and changes in neurotransmitter receptor expression may contribute to these behavioral effects.

Organization of the human inferior parietal lobule based on receptor architectonics.

Human inferior parietal lobule (IPL) plays a key role in various cognitive functions. Its functional diversity, including attention, language, and action processing, is reflected by its structural segregation into 7 cytoarchitectonically distinct areas, each with characteristic connectivity patterns. We hypothesized that commonalities of the cytoarchitectonic, connectional, and functional diversity of the IPL should be reflected by a correlated transmitter receptor-based organization. Since the function of a cortical area requires a well-tuned receptor balance, the densities of 15 different receptors were measured in each IPL area. A hierarchical cluster analysis of the receptor balance revealed a tripartite segregation of the IPL into a rostral, middle, and caudal group. Comparison with other cortical areas showed strong similarities with Broca's region for all 3 groups, with the superior parietal cortex for the middle, and with extrastriate visual areas for the caudal group. Notably, caudal-most area PGp has a receptor fingerprint very similar to that of ventral extrastriate visual cortex. We therefore propose a new organizational model of the human IPL, consisting of 3 clusters, which corresponds to its known cytoarchitectonic, connectional, and functional diversity at the molecular level. This might reflect a general organizational principle of human IPL, beyond specific functional domains.

Effects of subchronic benzo(a)pyrene exposure on neurotransmitter receptor gene expression in the rat hippocampus related with spatial learning and memory change.

Exposure of laboratory rats to Benzo(a)pyrene (BaP), an environmental contaminant with its high lipophilicify which is widely dispersed in the environment and can easily cross the blood brain barrier presenting in the central nervous system, is associated with impaired learning and memory. The purpose of the research was to examine whether subchronic exposure to BaP affects spatial learning and memory, and how it alters normal gene expression in hippocampus, as well as selection of candidate genes involving neurotransmitter receptor attributed to learning and memory. Morris water maze (MWM) was used to evaluate behavioral differences between BaP-treated and vehicle-treated groups. To gain a better insight into the mechanism of BaP-induced neurotoxicity on learning and memory, we used whole genome oligo microarrays as well as Polymerase Chain Reaction (PCR) to assess the global impact of gene expression. Male Sprague-Dawley rats were intraperitoneally injected with 6.25mg/kg of BaP or vehicle for 14 weeks. The results from the Morris water maze (MWM) test showed that rats treated with BaP exhibited significantly higher mean latencies as compared to vehicle controls. BaP exposure significantly decreased the number of crossing the platform and the time spent in the target area. After the hippocampus was collected from each rat, total RNA was isolated. Microarray and PCR revealed that exposure to BaP affected mRNA expression of neurotransmitter receptors. The web tool DAVID was used to analyze the significantly enriched gene ontology (GO) and KEGG pathways in the differentially expressed genes. Analysis showed that the most significantly affected gene ontology category was behavior. Furthermore, the fourth highest significantly affected gene ontology category was learning and memory. KEGG molecular pathway analysis showed that "neuroactive ligand-receptor interaction" was affected by BaP with highest statistical significance, and 9 candidate neurotransmitter receptor genes involving learning and memory were selected out. Our results revealed a close link between behavioral changes and altered neurotransmitter receptor gene expression in BaP-treated rats.

Dual role for Drosophila lethal of scute in CNS midline precursor formation and dopaminergic neuron and motoneuron cell fate.

Dopaminergic neurons play important behavioral roles in locomotion, reward and aggression. The Drosophila H-cell is a dopaminergic neuron that resides at the midline of the ventral nerve cord. Both the H-cell and the glutamatergic H-cell sib are the asymmetric progeny of the MP3 midline precursor cell. H-cell sib cell fate is dependent on Notch signaling, whereas H-cell fate is Notch independent. Genetic analysis of genes that could potentially regulate H-cell fate revealed that the lethal of scute [l(1)sc], tailup and SoxNeuro transcription factor genes act together to control H-cell gene expression. The l(1)sc bHLH gene is required for all H-cell-specific gene transcription, whereas tailup acts in parallel to l(1)sc and controls genes involved in dopamine metabolism. SoxNeuro functions downstream of l(1)sc and controls expression of a peptide neurotransmitter receptor gene. The role of l(1)sc may be more widespread, as a l(1)sc mutant shows reductions in gene expression in non-midline dopaminergic neurons. In addition, l(1)sc mutant embryos possess defects in the formation of MP4-6 midline precursor and the median neuroblast stem cell, revealing a proneural role for l(1)sc in midline cells. The Notch-dependent progeny of MP4-6 are the mVUM motoneurons, and these cells also require l(1)sc for mVUM-specific gene expression. Thus, l(1)sc plays an important regulatory role in both neurogenesis and specifying dopaminergic neuron and motoneuron identities.

Quantitative gene-expression analysis of the ligand-receptor system for classical neurotransmitters and neuropeptides in hippocampal CA1, CA3, and dentate gyrus.

We have shown quantitative expression levels of genes coding for the "ligand-receptor system" for classical neurotransmitters and neuropeptides in hippocampal subregions CA1, CA3, and dentate gyrus (DG). Using a combination of DNA microarray and quantitative PCR methods, we found that the three subregions have relatively similar expression patterns of ionotropic receptors for classical neurotransmitters. Expression of ionotropic receptors for glutamate and GABA represents more than 90% of all ionotropic receptors for classical neurotransmitters, and the expression ratio between ionotropic receptors for glutamate and GABA is constant (1.2:1-1.6:1) in each subregion. Meanwhile, the three subregions have different expression patterns of neuropeptide receptors. Furthermore, there are asymmetric expression patterns between neuropeptides and their receptors. Expression of Cck, Npy, Sst, and Penk1 represents 90% of neuropeptides derived locally in the hippocampus, whereas expression of these four neuropeptide receptors accounts for 50% of G protein-coupled receptors for neuropeptides. We propose that CA1, CA3, and DG have different modalities based on the ligand-receptor system, particularly the "neuropeptidergic system." Our quantitative gene-expression analysis provides fundamental data to support functional differences between the three hippocampal subregions regarding ligand-receptor interactions.

Brain derived neurotrophic factor and neurotrophic factor 3 modulate neurotransmitter receptor expressions on developing spiral ganglion neurons.

Cochlear spiral ganglion neurons (SGN) provide the only pathway for transmitting sound evoked activity from the hair cells to the central auditory system. Neurotrophic factor 3 (NT-3) and brain derived neurotrophic factor (BDNF) released from hair cells and supporting cells exert a profound effect on SGN survival and neural firing patterns; however, it is unclear what the effects NT-3 and BDNF have on the type of neurotransmitter receptors expressed on SGN. To address this question, the whole-cell patch clamp recording technique was used to determine what effect NT-3 and BDNF had on the function and expression of glutamate, GABA and glycine receptors (GlyR) on SGN of cochlea from postnatal C57 mouse. Receptor currents induced by the agonist of each receptor were recorded from SGN cultured with or without BDNF or NT-3. NT-3 and BDNF exerted different effects. NT-3, and to a lesser extent BDNF, enhanced the expression of GABA receptors and had comparatively little effect on glutamate receptors. Absence of BDNF and NT-3 resulted in the emergence of glycine-induced currents; however, GlyR currents were absent from the short term cultured SGN. In contrast, NT-3 and BDNF suppressed GlyR expression on SGN. These results indicate that NT-3 and BDNF exert a profound effect on the types of neurotransmitter receptors expressed on postnatal SGN, results that may have important implications for neural development and plasticity.