Respiratory modulation of sympathetic activity is attenuated in adult rats conditioned with chronic hypobaric hypoxia.

Respiratory modulation of sympathetic nerve activity (SNA) depends on numerous factors including prior experience. In our studies, exposing naïve adult, male Sprague-Dawley rats to acute intermittent hypoxia (AIH) enhanced respiratory-modulation of splanchnic SNA (sSNA); whereas conditioning them to chronic hypobaric hypoxia (CHH) attenuated modulation. Further, AIH can evoke increased SNA in the absence phrenic long-term facilitation. We hypothesized that AIH would restore respiratory modulation of SNA in CHH rats. In anesthetized, CHH-conditioned (0.5 atm, 2 wks) rats (n=16), we recorded phrenic and sSNA before during and after AIH (8% O2 for 45s every 5min for 1h). At baseline, sSNA was not modulated with respiration. The sSNA was not recruited during a single brief exposure of hypoxia nor after 10 repetitive exposures. Further, the sSNA chemoresponse was not restored 1h after completing AIH. Thus, CHH-conditioning blocked the short-term plasticity expressed in sympatho-respiratory efferent activities and this was associated with reduced respiratory modulation of sympathetic activity and with attenuation of the sympatho-respiratory chemoresponse.

Respiratory and behavioral dysfunction following loss of the GABAA receptor α4 subunit.

γ-Aminobutyric acid type A (GABAA) receptor plasticity participates in mediating adaptation to environmental change. Previous studies in rats demonstrated that extrasynaptic GABAA receptor subunits and receptors in the pons, a brainstem region involved in respiratory control, are upregulated by exposure to sustained hypobaric hypoxia. In these animals, expression of the mRNA encoding the extrasynaptic α4 subunit rose after 3 days in sustained hypoxia, while those encoding the α6 and δ subunits increased dramatically by 2 weeks. However, the participation of extrasynaptic subunits in maintaining respiration in normoxic conditions remains unknown. To examine the importance of α4 in a normal environment, respiratory function, motor and anxiety-like behaviors, and expression of other GABAA receptor subunit mRNAs were compared in wild-type (WT) and α4 subunit-deficient mice. Loss of the α4 subunit did not impact frequency, but did lead to reduced ventilatory pattern variability. In addition, mice lacking the subunit exhibited increased anxiety-like behavior. Finally, α4 subunit loss resulted in reduced expression of other extrasynaptic (α6 and δ) subunit mRNAs in the pons without altering those encoding the most prominent synaptic subunits. These findings on subunit-deficient mice maintained in normoxia, in conjunction with earlier findings on animals maintained in chronic hypoxia, suggest that the expression and regulation of extrasynaptic GABAA receptor subunits in the pons is interdependent and that their levels influence respiratory control as well as adaptation to stress.

Hu proteins regulate alternative splicing by inducing localized histone hyperacetylation in an RNA-dependent manner.

Recent studies have provided strong evidence for a regulatory link among chromatin structure, histone modification, and splicing regulation. However, it is largely unknown how local histone modification patterns surrounding alternative exons are connected to differential alternative splicing outcomes. Here we show that splicing regulator Hu proteins can induce local histone hyperacetylation by association with their target sequences on the pre-mRNA surrounding alternative exons of two different genes. In both primary and mouse embryonic stem cell-derived neurons, histone hyperacetylation leads to an increased local transcriptional elongation rate and decreased inclusion of these exons. Furthermore, we demonstrate that Hu proteins interact with histone deacetylase 2 and inhibit its deacetylation activity. We propose that splicing regulators may actively modulate chromatin structure when recruited to their target RNA sequences cotranscriptionally. This "reaching back" interaction with chromatin provides a means to ensure accurate and efficient regulation of alternative splicing.

Sulfasalazine blocks the development of tactile allodynia in diabetic rats.

OBJECTIVE: Diabetic neuropathy is manifested either by loss of nociception (painless syndrome) or by mechanical hyperalgesia and tactile allodynia (pain in response to nonpainful stimuli). While therapies with vasodilators or neurotrophins reverse some functional and metabolic abnormalities in diabetic nerves, they only partially ameliorate neuropathic pain. The reported link between nociception and targets of the anti-inflammatory drug sulfasalazine prompted us to investigate its effect on neuropathic pain in diabetes. RESEARCH DESIGN AND METHODS: We examined the effects of sulfasalazine, salicylates, and the poly(ADP-ribose) polymerase-1 inhibitor PJ34 on altered nociception in streptozotocin-induced diabetic rats. We also evaluated the levels of sulfasalazine targets in sciatic nerves and dorsal root ganglia (DRG) of treated animals. Finally, we analyzed the development of tactile allodynia in diabetic mice lacking expression of the sulfasalazine target nuclear factor-kappaB (NF-kappaB) p50. RESULTS: Sulfasalazine completely blocked the development of tactile allodynia in diabetic rats, whereas relatively minor effects were observed with other salicylates and PJ34. Along with the behavioral findings, sciatic nerves and DRG from sulfasalazine-treated diabetic rats displayed a decrease in NF-kappaB p50 expression compared with untreated diabetic animals. Importantly, the absence of tactile allodynia in diabetic NF-kappaB p50(-/-) mice supported a role for NF-kappaB in diabetic neuropathy. Sulfasalazine treatment also increased inosine levels in sciatic nerves of diabetic rats. CONCLUSIONS: The complete inhibition of tactile allodynia in experimental diabetes by sulfasalazine may stem from its ability to regulate both NF-kappaB and inosine. Sulfasalazine might be useful in the treatment of nociceptive alterations in diabetic patients.

Adaptation to hypobaric hypoxia involves GABA A receptors in the pons.

Survival in low-oxygen environments requires adaptation of sympathorespiratory control networks located in the brain stem. The molecular mechanisms underlying adaptation are unclear. In naïve animals, acute hypoxia evokes increases in phrenic (respiratory) and splanchnic (sympathetic) nerve activities that persist after repeated challenges (long-term facilitation, LTF). In contrast, our studies show that conditioning rats to chronic hypobaric hypoxia (CHH), an environment characteristic of living at high altitude, diminishes the response to hypoxia and attenuates LTF in a time-dependent manner. Phrenic LTF decreases following 7 days of CHH, and both sympathetic and phrenic LTF disappear following 14 days of CHH. Previous studies demonstrated that GABA is released in the brain stem during hypoxia and depresses respiratory activity. Furthermore, the sensitivity of brain stem neurons to GABA is increased following prolonged hypoxia. In this study, we demonstrate that GABA(A) receptor expression changes along with the CHH-induced physiological changes. Expression of the GABA(A) receptor alpha4 subunit mRNA increases two-fold in animals conditioned to CHH for 7 days. In addition, de novo expression of delta and alpha6, a subunit normally found exclusively in the cerebellum, is observed after 14 days. Consistent with these changes, diazepam-insensitive binding sites, characteristic of GABA(A) receptors containing alpha4 and alpha6 subunits, increase in the pons. Immunohistochemistry revealed that CHH-induced GABA(A) receptor subunit expression is localized in regions of sympathorespiratory control within the pons. Our findings suggest that a GABA(A) receptor mediated-mechanism participates in adaptation of the sympathorespiratory system to hypobaric hypoxia.

Association of PSD-95 with ErbB4 facilitates neuregulin signaling in cerebellar granule neurons in culture.

The growth factor neuregulin 1 (NRG) selectively induces an increase in the gamma-aminobutyric acid (GABA)(A) receptor beta2 subunit protein in rat cerebellar granule neurons in culture. We previously demonstrated that NRG acts by triggering ErbB4 receptor phosphorylation and subsequent signaling through the mitogen-activated kinase (MAPK), phosphatidyl inositol-3 kinase (PI-3K) and cyclin-dependent kinase 5 (cdk5) pathways. In this report we show that the scaffolding protein, PSD-95, plays a key role in mediating the effects of NRG and that reducing its level attenuates the NRG-induced increase in beta2 subunit expression. PSD-95 appears to facilitate the effects of NRG through its association with ErbB4, an interaction that is augmented by NRG-activated cdk signaling. Inhibition of cdk activity with roscovitine attenuates the association of PSD-95 with ErbB4. The effects of cdk5 are not blocked by U0126, an inhibitor of MAPK signaling, indicating that cdk5 functions independently of cross-talk with this pathway. These findings raise the possibility that NRG-induced activation of cdk5 works in part by recruiting PSD-95, a protein involved in regulating synaptic plasticity, to associate with ErbB4. This interaction may be a positive feedback loop that augments NRG signaling and its downstream effects on GABA(A) receptor beta2 subunit expression.

The serotonin type 3A receptor facilitates luteinizing hormone release and LHbeta promoter activity in immortalized pituitary gonadotropes.

The 5-hydroxytryptamine type 3A receptor (5-HT3AR) is a ligand-gated cation channel activated by serotonin. This receptor is expressed throughout the nervous system as well as in the pituitary gland. Although it has been documented that the 5-HT3AR modulates exocytosis in neurons, its role in the pituitary gland has not been determined. Previous research has shown that the 5-HT3AR modulates circulating gonadotropin levels in vivo. It is unclear, however, if its activation in the pituitary gland mediates these effects or if receptors elsewhere in the hypothalamus-pituitary-gondal axis are responsible. To investigate the potential for the 5-HT3AR to modulate gonadotropin release from pituitary gonadotropes, the gonadotrope-derived LbetaT2 cell line was used as a model system and radioimmunoassays were employed to investigate how 5-HT3AR activation influences luteinizing hormone (LH) release. Our studies demonstrate that gonadotropin releasing hormone (GnRH)-stimulated LH release was decreased by the 5-HT3AR-specific antagonist MDL 72222 in a concentration-dependent manner. In addition, it was found that overexpressing the 5-HT3AR in LbetaT2 cells enhanced both basal and GnRH-stimulated LH release and also increased LHbeta gene promoter activity. These results suggest that the 5-HT3AR may participate in the hypothalamus-pituitary-gonadal axis at the level of the pituitary gonadotrope to mediate pituitary hormone release.

Pontine GABAergic pathways: role and plasticity in the hypoxic ventilatory response.

The hypoxic ventilatory response (HVR) was compared before and after uni- and bi-lateral injections of bicuculline, a GABA(A) receptor antagonist, into the ventrolateral (vl) pons and before and after conditioning animals to chronic sustained hypoxia (CSH). The HVR was assessed by recording phrenic nerve activity (PNA) during and after brief exposures to hypoxia (8% O(2) and 92% N(2) for 45s). Inspiratory (T(I)) and expiratory (T(E)) durations were averaged before hypoxia, at the peak breathing frequency during hypoxia, before the end of hypoxia, immediately after hypoxia, and 60s after hypoxia. Blocking GABA(A) receptors in the vl pons prolonged T(E) during, but not after hypoxia. After CSH induced by 14 days in a hypobaric chamber (0.5atm), the HVR was attenuated compared to that in the naive animals. This plasticity of HVR was associated with selective induction of alpha6 and delta GABA(A) receptor subunit mRNAs specifically in the pons compared to the medulla. These physiological and molecular results illustrate the importance of pontine GABAergic pathways in shaping the response to hypoxia.

Neuregulin induces GABAA receptor beta2 subunit expression in cultured rat cerebellar granule neurons by activating multiple signaling pathways.

The GABAA receptor beta subunit is required to confer sensitivity to gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the CNS. In previous studies we demonstrated that the growth and differentiation factor neuregulin 1 (NRG1) selectively induced expression of the beta2 subunit mRNA and encoded protein in rat cerebellar granule neurons in culture. In the present report we examine the signaling pathways that mediate this effect. These studies demonstrate that the effects of NRG1 on beta2 subunit polypeptide expression require activation of the ErbB4 receptor tyrosine kinase; its effects are inhibited by pharmacological blockade of ErbB4 phosphorylation or reduction of receptor level with an antisense oligodeoxynucleotide. The NRG1-induced activation of ErbB4 stimulates the mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K) and cyclin-dependent kinase-5 (cdk5) pathways. Pharmacological blockade of any of these pathways inhibits increased beta2 subunit expression, demonstrating that all three pathways are required to mediate the effects of NRG1 on GABAA receptor subunit expression in cerebellar granule neurons. These studies provide novel information concerning the actions of NRG1 on GABAA receptor expression in the CNS.

Three putative N-glycosylation sites within the murine 5-HT3A receptor sequence affect plasma membrane targeting, ligand binding, and calcium influx in heterologous mammalian cells.

The serotonin type 3(A) receptor (5-HT3(A)R) is a ligand-gated ion channel (LGIC) that modulates a diverse set of cognitive and physiological functions. The 5-HT3(A)R, as with other LGICs, is a pentameric ion channel comprising five glycoprotein subunits. Although the N-terminal of the 5-HT3(A)R contains three putative N-linked glycosylation sites, the importance of each glycosylation site has not yet been established. To address this question, we used tunicamycin treatment and site-directed mutagenesis to inhibit selectively N-linked glycosylation at each site and then examined the effects of these treatments on receptor expression and function in transiently transfected heterologous cells. We show that the murine 5-HT3(A)R is glycosylated and that each N-linked glycosylation site plays a role in receptor regulation. Our findings suggest that N109 is necessary for receptor assembly, whereas N174 and N190 are important for plasma membrane targeting and ligand binding. Furthermore, we demonstrate that each site is necessary for 5-HT3(A)R-mediated Ca(2+) influx. We conclude that N-glycosylation is a critical step in the maturation, trafficking, and function of the murine 5-HT3(A)R.

Signaling by bone morphogenetic proteins and Smad1 modulates the postnatal differentiation of cerebellar cells.

Previous studies have demonstrated that bone morphogenetic proteins (BMPs) activate the Smad1 signaling pathway to regulate cell determination and differentiation in the embryonic nervous system. Studies examining gene and protein expression in the rat cerebellum suggest that this pathway also regulates postnatal differentiation. Using microarrays, we found that Smad1 mRNA expression in the cerebellum increases transiently at postnatal day 6 (P6). Immunohistochemistry and Western blots showed that Smad1 and BMP4 proteins are present in the cerebellum, and that their expression also changes postnatally. The proteins are detectable at P4-P6, a stage at which most cerebellar cells reside in the external germinal layer (EGL), where they extensively differentiate. The levels become maximal at P8-P10, when neurons begin to migrate from the EGL into their mature positions in the internal granule layer. In cerebellar cultures prepared at P6 or P10, BMP4 activates Smad1 signaling to modulate cell differentiation. Brief BMP4 application caused Smad1 translocation from the neuronal cytoplasm into the nucleus, where it is known to regulate transcription in association with Smad4. Longer BMP4 treatment promoted the differentiation of both neuronal and non-neuronal cells. By 3 d, neuronal processes appeared more fasciculated, and the level of synaptotagmin, a protein found in synaptic vesicles, increased. In addition, many astroglial cells became more branched and stellate in morphology. The BMP-induced changes were reduced by treatment with antisense oligonucleotides to Smad1 or Smad4. These findings in vivo and in culture suggest that BMP4 and Smad1 signaling participate in regulating postnatal cerebellar differentiation.