Insulin potentiates inhibitory synaptic currents between fast-spiking and pyramidal neurons in the rat insular cortex.

Insulin plays roles in brain functions such as neural development and plasticity and is reported to be involved in dementia and depression. However, little information is available on the insulin-mediated modulation of electrophysiological activities, especially in the cerebral cortex. This study examined how insulin modulates the neural activities of inhibitory neurons and inhibitory postsynaptic currents (IPSCs) in rat insular cortex (IC; either sex) by multiple whole-cell patch-clamp recordings. We demonstrated that insulin increased the repetitive spike firing rate with a decrease in the threshold potential without changing the resting membrane potentials and input resistance of fast-spiking GABAergic neurons (FSNs). Next, we found a dose-dependent enhancement of unitary IPSCs (uIPSCs) by insulin in the connections from FSNs to pyramidal neurons (PNs). The insulin-induced enhancement of uIPSCs accompanied decreases in the paired-pulse ratio, suggesting that insulin increases GABA release from presynaptic terminals. The finding of miniature IPSC recordings of the increased frequency without changing the amplitude supports this hypothesis. Insulin had little effect on uIPSCs under the coapplication of S961, an insulin receptor antagonist, or lavendustin A, an inhibitor of tyrosine kinase. The PI3-K inhibitor wortmannin or the PKB/Akt inhibitors, deguelin and Akt inhibitor VIII, blocked the insulin-induced enhancement of uIPSCs. Intracellular application of Akt inhibitor VIII to presynaptic FSNs also blocked insulin-induced enhancement of uIPSCs. In contrast, uIPSCs were enhanced by insulin in combination with the MAPK inhibitor PD98059. These results suggest that insulin facilitates the inhibition of PNs by increases in FSN firing frequency and IPSCs from FSNs to PNs. (250 words).

Presynaptic NK1 Receptor Activation by Substance P Suppresses EPSCs via Nitric Oxide Synthesis in the Rat Insular Cortex.

Substance P (SP) regulates inhibitory synaptic transmission mediated by GABAA receptors in the cerebral cortex; however, SP-mediated regulation of excitatory synaptic transmission remains poorly understood. We performed whole-cell patch-clamp recordings from pyramidal neurons to examine the effects of SP on excitatory postsynaptic currents (EPSCs) mediated via AMPA receptors in the insular cortex (IC), which is involved in nociceptive information processing. First, EPSCs evoked by minimal electrical stimulation (eEPSCs) including stepwise EPSCs and failure events, were examined. SP dose-dependently suppressed mean eEPSC amplitude, partially due to an increase in the failure rate of eEPSCs. The SP-induced suppression of eEPSCs was accompanied by an increase in the paired-pulse ratio and was inhibited by the preapplication of SR140333, an NK1 receptor antagonist. [Sar9,Met(O2)11]-substance P, an NK1 receptor-selective agonist, mimicked the effects of SP on eEPSCs and decreased the frequency of miniature EPSCs (mEPSCs) without changing the average mEPSC amplitude. Considering that most NK1 receptors in the cerebral cortex are expressed in nitric oxide synthase (NOS)-positive GABAergic neurons, the SP-induced suppressive effect on EPSCs may be mediated by nitric oxide (NO) in this subtype of GABAergic neurons. NO imaging using the fluorescent probe DAX-J2 Red supports this hypothesis: SP increased the fluorescence intensity of DAX-J2 Red in some GABAergic neurons. Furthermore, both L-NAME, an NOS inhibitor, and PTIO, an NO scavenger, diminished the SP-induced suppression of eEPSCs. These results suggest that the activation of presynaptic NK1 receptors contributes to SP-induced eEPSC suppression by activating the NO synthesis pathway in GABAergic neurons. (246 words).

Early Postnatal Treatment with Valproate Induces Gad1 Promoter Remodeling in the Brain and Reduces Apnea Episodes in Mecp2-Null Mice.

The deletion of Mecp2, the gene encoding methyl-CpG-binding protein 2, causes severe breathing defects and developmental anomalies in mammals. In Mecp2-null mice, impaired GABAergic neurotransmission is demonstrated at the early stage of life. GABAergic dysfunction in neurons in the rostral ventrolateral medulla (RVLM) is considered as a primary cause of breathing abnormality in Mecp2-null mice, but its molecular mechanism is unclear. Here, we report that mRNA expression levels of Gad1, which encodes glutamate decarboxylase 67 (GAD67), in the RVLM of Mecp2-null (Mecp2-/y, B6.129P2(C)-Mecp2tm1.1Bird/J) mice is closely related to the methylation status of its promoter, and valproate (VPA) can upregulate transcription from Gad1 through epigenetic mechanisms. The administration of VPA (300 mg/kg/day) together with L-carnitine (30 mg/kg/day) from day 8 to day 14 after birth increased Gad1 mRNA expression in the RVLM and reduced apnea counts in Mecp2-/y mice on postnatal day 15. Cytosine methylation levels in the Gad1 promoter were higher in the RVLM of Mecp2-/y mice compared to wild-type mice born to C57BL/6J females, while VPA treatment decreased the methylation levels in Mecp2-/y mice. Chromatin immunoprecipitation assay revealed that the VPA treatment reduced the binding of methyl-CpG binding domain protein 1 (MBD1) to the Gad1 promoter in Mecp2-/y mice. These results suggest that VPA improves breathing of Mecp2-/y mice by reducing the Gad1 promoter methylation, which potentially leads to the enhancement of GABAergic neurotransmission in the RVLM.

Extracellular glucose-dependent IPSC enhancement by leptin in fast-spiking to pyramidal neuron connections via JAK2-PI3K pathway in the rat insular cortex.

Leptin is produced in the adipocytes and plays a pivotal role in regulation of energy balance by controlling appetite and metabolism. Leptin receptors are widely distributed in the brain, especially in the hypothalamus, hippocampus, and neocortex. The insular cortex (IC) processes gustatory and visceral information, which functionally correlate to feeding behavior. However, it is still an open issue whether and how leptin modulates IC neural activities. Our paired whole-cell patch-clamp recordings using IC slice preparations demonstrated that unitary inhibitory postsynaptic currents (uIPSCs) but not uEPSCs were potentiated by leptin in the connections between pyramidal (PNs) and fast-spiking neurons (FSNs). The leptin-induced increase in uIPSC amplitude was accompanied by a decrease in paired-pulse ratio. Under application of inhibitors of JAK2-PI3K but not MAPK pathway, leptin did not change uIPSC amplitude. Variance-mean analysis revealed that leptin increased the release probability but not the quantal size and the number of release site. These electrophysiological findings suggest that the leptin-induced uIPSC increase is mediated by activation of JAK2-PI3K pathway in presynaptic FSNs. An in vivo optical imaging revealed that leptin application decreased excitatory propagation in IC induced by electrical stimulation of IC. These leptin-induced effects were not observed under the low energy states: low glucose concentration (2.5 mM) in vitro and one-day-fasting condition in vivo. However, leptin enhanced uIPSCs under application of low glucose with an AMPK inhibitor. These results suggest that leptin suppresses IC excitation by facilitating GABA release in FSN→PN connections, which may not occur under a hunger state.

Hypoxia-induced upregulation of angiogenic factors in immortalized human periodontal ligament fibroblasts.

Hypoxia induces complex cellular responses that are mediated by a key transcription factor, hypoxia-inducible factor-1 (HIF-1). HIF-1 promotes production of cytokines and angiogenic factors and contributes to recovery of injured tissues. In the present study, expressions of angiogenin (ANG) and vascular endothelial growth factor (VEGF), which are potent angiogenic factors in mammalian tissues, were examined in immortalized fibroblasts exposed to hypoxia. After 24 h of exposure to hypoxia, ANG and VEGF mRNAs expressions were significantly elevated in periodontal ligament (PDL) fibroblasts but not in embryonic fibroblasts. Hypoxia also increased productions of ANG and VEGF proteins in PDL fibroblasts. HIF-1α mRNA expression was not affected by hypoxia in either fibroblast, although HIF-1α protein expression was enhanced after exposure to hypoxia. Treatment of PDL fibroblasts with dimethyloxaloylglycine, a prolyl hydroxylase inhibitor that stabilizes the HIF-1α protein, significantly increased expressions of ANG and VEGF mRNAs under normoxia. This suggests that stabilization of HIF-1α is crucial for upregulation of ANG and VEGF in PDL fibroblasts. These results indicate that, under hypoxic conditions, HIF-1α upregulates synthesis of ANG and VEGF in PDL fibroblasts and promotes angiogenesis.

Effect of hypoxia on the expression of CCAAT/enhancer-binding protein ß and receptor activator of nuclear factor kappa-B ligand in periodontal ligament cells.

Hypoxia after traumatic injuries to a tooth is one of the causes of subsequent root resorption. Inflammatory cytokines produced under hypoxic conditions are associated with root resorption, but the mechanism has not been fully understood. In this study, the role of hypoxia-inducible factor-1 (HIF-1) signaling in the regulation of CCAAT (cytosine-cytosine-adenosine-adenosine-thymidine)/enhancer-binding protein-ß (C/EBPß) and the receptor activator of nuclear factor kappa-B ligand (RANKL) expressions in immortalized human periodontal ligament (PDL) cells was investigated. PDL cells cultured under a hypoxic condition showed an increase in the expression of C/EBPß and RANKL messenger RNAs (mRNAs), whereas the expression of osteoprotegerin and HIF-1α mRNAs was unaffected. Hypoxia had no effects on the secretion of interleukin (IL)-1ß, IL-6, IL-8, IL-17A, tumor necrosis factor-alpha, macrophage migration inhibitory factor, monocyte chemoattractant protein-1, and macrophage colony-stimulating factor in the culture media. Treatment of the cells with dimethyloxaloylglycine, a competitive HIF prolyl hydroxylase inhibitor, significantly increased the expression of C/EBPß and RANKL mRNAs. This suggested that the hypoxia-induced elevation of C/EBPß and RANKL mRNAs was dependent on the HIF-1 activity. PDL cells transfected with a specific small interfering RNA designed to target the C/EBPß gene showed a significant suppression of the RANKL mRNA. These findings indicated that C/EBPß may play an important role in tooth root resorption via RANKL activation in hypoxia-exposed PDL cells.

Histamine H3 Heteroreceptors Suppress Glutamatergic and GABAergic Synaptic Transmission in the Rat Insular Cortex.

Histamine H3 receptors are autoreceptors that regulate histamine release from histaminergic neuronal terminals. The cerebral cortex, including the insular cortex (IC), expresses abundant H3 receptors; however, the functions and mechanisms of H3 receptors remain unknown. The aim of this study was to elucidate the functional roles of H3 in synaptic transmission in layer V of the rat IC. Unitary excitatory and inhibitory postsynaptic currents (uEPSCs and uIPSCs) were obtained through paired whole-cell patch-clamp recording in cerebrocortical slice preparations. The H3 receptor agonist, R-α-methylhistamine (RAMH), reduced the uEPSC amplitude obtained from pyramidal cell to pyramidal cell or GABAergic interneuron connections. Similarly, RAMH reduced the uIPSC amplitude in GABAergic interneuron to pyramidal cell connections. RAMH-induced decreases in both the uEPSC and uIPSC amplitudes were accompanied by increases in the failure rate and paired-pulse ratio. JNJ 5207852 dihydrochloride or thioperamide, H3 receptor antagonists, inhibited RAMH-induced suppression of uEPSCs and uIPSCs. Unexpectedly, thioperamide alone increased the uIPSC amplitude, suggesting that thioperamide was likely to act as an inverse agonist. Miniature EPSC or IPSC recordings support the hypothesis that the activation of H3 receptors suppresses the release of glutamate and GABA from presynaptic terminals. The colocalization of H3 receptors and glutamate decarboxylase or vesicular glutamate transport protein 1 in presynaptic axon terminals was confirmed through double pre-embedding microscopy, using a combination of pre-embedding immunogold and immunoperoxidase techniques. The suppressive regulation of H3 heteroreceptors on synaptic transmission might mediate the regulation of sensory information processes, such as gustation and visceral sensation, in the IC.

An acyl-CoA-binding protein from grape that is induced through ER stress confers morphological changes and disease resistance in Arabidopsis.

We here report characterization of a grape (Vitis vinifera) acyl-CoA-binding protein (VvACBP). Expression of VvACBP was detected in grape leaves exposed to tunicamycin-induced endoplasmic reticulum (ER) stress as well as cold and heat shock treatments. In tendrils and peduncles, however, high-temperature treatment induced BiP (luminal binding protein) expression, a marker of ER stress in berry skin, but not VvACBP expression. We hypothesize that VvACBP may be sorted to the periphery of plant cells. Transgenic Arabidopsis plants, expressing VvACBP, exhibited slowed-down floral transition. The gene expression of proteins related to the photoperiodic pathway, CONSTANS, FLOWERING LOCUS T (FT), and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), was down-regulated in transgenic seedlings. These results underscore the possibility that VvACBP may affect the regulation of floral transition in Arabidopsis by suppressing the photoperiodic pathway. The transgenic Arabidopsis plants also exhibited morphological changes such as thicker inflorescences and rosette leaves. In addition, the rosette leaves of the transgenic plants had higher anthocyanin, total phenol, and chlorophyll contents than those of the control plants. Finally, the transgenic plants showed disease resistance to Pseudomonas syringae and Colletotrichum higginsianum, suggesting that VvACBP may also enhance disease resistance in grapevine.

Histaminergic effects on the frequency of repetitive spike firing in rat insular cortex.

The insular cortex (IC) processes multimodal sensory information including gustatory, visceral, nociceptive, and thermal sensation, and is considered to play a role in the regulation of homeostasis. The IC receives dense histaminergic projection from the tuberomamillary nucleus in the hypothalamus, and recent studies have demonstrated that the blockage of histaminergic receptors impairs physiological functions in the IC. However, little is known about the effects of histamine on the electrophysiological properties of the IC. To explore the effects of histamine on the subthreshold responses and action potential properties in the IC, intracellular recording with a sharp glass electrode was obtained from IC pyramidal cells in cortical slice preparations. Application of histamine (30 µM) increased the frequency of repetitive spike firing in response to a long depolarizing current pulse injection; accompanied by an increase in input resistance. The frequency of repetitive spike firing was estimated by the slope of the frequency-current (f/I) curve. Histamine caused an increase from 23.3±2.3 Hz/nA to 40.3±4.3 Hz/nA. The histamine-induced facilitation of repetitive spike firing was blocked by pre-application of 50 µM cimetidine, an H(2) receptor antagonist, but not 30 µM pyrilamine, an H(1) receptor antagonist. R-α-methylhistamine (10 µM), an H(3) autoreceptor agonist, had little effect on the slope of the f/I curve. These results suggest that the histamine-induced facilitation of firing frequency is mediated via H(2) and not H(1) receptors. In addition, H(3) receptors have a minor role in the intrinsic membrane and firing properties of IC pyramidal cells.

Kinetics of GABAB autoreceptor-mediated suppression of GABA release in rat insular cortex.

Release of GABA is controlled by presynaptic GABA receptor type B (GABA(B)) autoreceptors at GABAergic terminals. However, there is no direct evidence that GABA(B) autoreceptors are activated by GABA release from their own terminals, and precise profiles of GABA(B) autoreceptor-mediated suppression of GABA release remain unknown. To explore these issues, we performed multiple whole-cell, patch-clamp recordings from layer V rat insular cortex. Both unitary inhibitory and excitatory postsynaptic currents (uIPSCs and uEPSCs, respectively) were recorded by applying a five-train depolarizing pulse injection at 20 Hz. In connections from both fast-spiking (FS) and non-FS interneurons to pyramidal cells, the GABA(B) receptor antagonist CGP 52432 had little effect on the initial uIPSC amplitude. However, uIPSCs, responding to later pulses, were effectively facilitated. This CGP 52432-induced facilitation was prominent in the fourth uIPSCs, which were evoked 150 ms after the first uIPSC. The facilitation of uIPSCs was accompanied by an increase in the paired-pulse ratio. In addition, analysis of the coefficient of variation suggests the involvement of presynaptic mechanisms in CGP 52432-induced uIPSC facilitation. Paired-pulse stimulation (interstimulus interval = 150 ms) of presynaptic FS cells revealed that the second uIPSC was also facilitated by CGP 52432, which had little effect on the amplitude and interevent interval of miniature IPSCs. In contrast, uEPSCs, responding to all five stimulations of a presynaptic pyramidal cell, were less affected by CGP 52432. These results suggest that a single presynaptic action potential is sufficient to activate GABA(B) autoreceptors and to suppress GABA release in the cerebral cortex.

Functional mapping of gustatory neurons in the insular cortex revealed by pERK-immunohistochemistry and in vivo optical imaging.

Neural plasticity in the gustatory area of the insular cortex (IC) plays a critical role in detecting novel taste and taste memory formation, which require extracellular signal-regulated kinase 1-2 (ERK1-2) phosphorylation. However, the distribution patterns of phosphorylated ERK1-2 (pERK) responses to gustatory stimulation remain unknown. This study examined distribution patterns of gustatory stimulation-driven pERK expression in the IC of anesthetized and alert rats. In both pentobarbital-anesthetized and alert rats, gustatory stimulation (10% sucrose) induced pERK-like immunoreactivity in pyramidal cells of all IC subdivisions: agranular (AI), dysgranular (DI), and granular IC (GI). Alert naïve rats exhibited approximately 10-fold larger number of pERK-like immunopositive (pERK-LI) cells than anesthetized naïve rats in response to sucrose application. Most pERK-LI cells were located in layers II/III but not deeper layers and almost no parvalbumin/somatostatin-immunopositive cells expressed pERK. In the AI, rostral regions exhibited more pERK-LI cells than caudal regions, whereas most pERK-LI cells existed in the DI/GI around the intersection of the rhinal fissure and middle cerebral artery (MCA), where in vivo optical imaging revealed activation during sucrose application in addition to the ventral primary and secondary somatosensory cortices. Gustatory experience affected the number of pERK-LI cells in the IC: sucrose stimulation induced more pERK-LI cells in the DI/GI of alert naïve rats than sucrose-exposed rats, which had received sucrose solution for 1 week. These results suggest that pyramidal cells in the upper layers of the gustatory region are highly susceptible to ERK1-2 phosphorylation by gustatory stimulation, which may induce neuroplastic changes in the IC.