Cortical nitric oxide required for presynaptic long-term potentiation in the insular cortex.

Nitric oxide (NO) is a key diffusible messenger in the mammalian brain. It has been proposed that NO may diffuse retrogradely into presynaptic terminals, contributing to the induction of hippocampal long-term potentiation (LTP). Here, we present novel evidence that NO is required for kainate receptor (KAR)-dependent presynaptic form of LTP (pre-LTP) in the adult insular cortex (IC). In the IC, we found that inhibition of NO synthase erased the maintenance of pre-LTP, while the induction of pre-LTP required the activation of KAR. Furthermore, NO is essential for pre-LTP induced between two pyramidal cells in the IC using the double patch-clamp recording. These results suggest that NO is required for homosynaptic pre-LTP in the IC. Our results present strong evidence for the critical roles of NO in pre-LTP in the IC. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

P2X receptor- and postsynaptic NMDA receptor-mediated long-lasting facilitation of inhibitory synapses in the rat insular cortex.

Adenosine triphosphate (ATP) changes the efficacy of synaptic transmission. Despite recent progress in terms of the roles of purinergic receptors in cerebrocortical excitatory synaptic transmission, their contribution to inhibitory synaptic transmission is unknown. To elucidate the effects of α,ß-methylene ATP (αß-mATP), a selective agonist of P2X receptors (P2XRs), on inhibitory synaptic transmission in the insular cortex (IC), we performed whole-cell patch-clamp recording from IC pyramidal neurons (PNs) and fast-spiking neurons (FSNs) in either sex of VGAT-Venus transgenic rats. αß-mATP increased the amplitude of miniature IPSCs (mIPSCs) under conditions in which NMDA receptors (NMDARs) are recruitable. αß-mATP-induced facilitation of mIPSCs was sustained even after the washout of αß-mATP, which was blocked by preincubation with fluorocitrate. The preapplication of NF023 (a P2X1 receptor antagonist) or AF-353 (a P2X3 receptor antagonist) blocked αß-mATP-induced mIPSC facilitation. Intracellular application of the NMDAR antagonist MK801 blocked the facilitation. d-serine, which is an intrinsic agonist of NMDARs, mimicked αß-mATP-induced mIPSC facilitation. The intracellular application of BAPTA a Ca2+ chelator, or the bath application of KN-62, a CaMKII inhibitor, blocked αß-mATP-induced mIPSC facilitation, thus indicating that mIPSC facilitation by αß-mATP required postsynaptic [Ca2+]i elevation through NMDAR activation. Paired whole-cell patch-clamp recordings from FSNs and PNs demonstrated that αß-mATP increased the amplitude of unitary IPSCs without changing the paired-pulse ratio. These results suggest that αß-mATP-induced IPSC facilitation is mediated by postsynaptic NMDAR activations through d-serine released from astrocytes. Subsequent [Ca2+]i increase and postsynaptic CaMKII activation may release retrograde messengers that upregulate GABA release from presynaptic inhibitory neurons, including FSNs. (250/250 words).

Descending projections from the insular cortex to the trigeminal spinal subnucleus caudalis facilitate excitatory outputs to the parabrachial nucleus in rats.

ABSTRACT: Nociceptive information from the orofacial area projects to the trigeminal spinal subnucleus caudalis (Sp5C) and is then conveyed to several nuclei, including the parabrachial nucleus (PBN). The insular cortex (IC) receives orofacial nociceptive information and sends corticofugal projections to the Sp5C. The Sp5C consists of glutamatergic and GABAergic/glycinergic interneurons that induce excitatory postsynaptic currents and inhibitory postsynaptic currents, respectively, in projection neurons. Therefore, quantification of glutamatergic IC inputs in combination with identifying postsynaptic neuronal subtypes is critical to elucidate IC roles in the regulation of Sp5C activities. We investigated features of synaptic transmission from the IC to glutamatergic and GABAergic/glycinergic Sp5C neurons of laminae I/II using vesicular GABA transporter-Venus transgenic rats that received an injection of adeno-associated virus-channelrhodopsin-2-mCherry into the IC. Selective stimulation of IC axon terminals in Sp5C slice preparations induced monosynaptic excitatory postsynaptic currents in both excitatory glutamatergic and inhibitory GABAergic/glycinergic Sp5C neurons with a comparable amplitude. Paired whole-cell patch-clamp recordings showed that unitary inhibitory postsynaptic currents from inhibitory neurons influencing excitatory neurons, including neurons projecting to the PBN, exhibited a high failure rate and were suppressed by both bicuculline and strychnine, suggesting that excitatory neurons in the Sp5C receive both GABAergic and glycinergic inhibition with low impact. Moreover, selective stimulation of IC axons increased the firing rate at the threshold responses. Finally, we demonstrated that selective stimulation of IC axons in the Sp5C by a chemogenetic approach decreased the thresholds of both mechanical and thermal nociception. Thus, IC projection to the Sp5C is likely to facilitate rather than suppress excitatory outputs from the Sp5C.

Differential regulation of medium spiny and cholinergic neurons in the nucleus accumbens core by the insular and medial prefrontal cortices in the rat.

The nucleus accumbens (NAc) receives cortical projections principally from the insular cortex (IC) and medial prefrontal cortex (mPFC). Among NAc neurons, cholinergic interneurons (ChNs) regulate the activities of medium spiny neurons (MSNs), which make up ~ 95% of NAc neurons, by modulating their firing and synaptic properties. However, little is known about the synaptic mechanisms, including their cell-type-dependent corticoaccumbal projection properties and cholinergic effects on the NAc core. Here, we performed whole-cell patch-clamp recordings from NAc MSNs and ChNs in acute brain slice preparations obtained from rats that received an AAV5-hSyn-ChR2(H134R)-mCherry injection into the IC or mPFC. Light stimulation of IC or mPFC axons induced comparable phase-locked excitatory postsynaptic currents (EPSCs) in MSNs. On the other hand, ChNs showed consistent EPSCs evoked by light stimulation of mPFC axons, whereas light stimulation of IC axons evoked much smaller EPSCs, which often showed failure in ChNs. Light-evoked EPSCs were abolished by tetrodotoxin and were recovered by 4-aminopyridine, suggesting that corticoaccumbal projections monosynaptically induce EPSCs in MSNs and ChNs. Carbachol effectively suppressed the amplitude of EPSCs in MSNs and ChNs evoked by light stimulation of IC or mPFC axons and in ChNs evoked by stimulating mPFC axons. The carbachol-induced suppression was recovered by atropine or pirenzepine, while preapplication of gallamine, J104129, PD102807, or AF-DX384 did not block the carbachol-induced EPSC suppression. These results suggest that NAc MSNs and ChNs are differentially regulated by excitatory projections from the IC and mPFC and that these corticoaccumbal excitatory inputs are modulated by M1 receptor activation.

Synchronous inhibitory synaptic inputs to layer II/III pyramidal neurons in the murine barrel cortex.

The barrel cortex exhibits obvious columnar organization. Although GABAergic inhibition plays a critical role in regulating neural excitation in response to mechanical stimuli applied to whiskers, the profiles of synchronous events for inhibitory synaptic transmission in intracolumnar and transcolumnar pyramidal neurons remain unknown. To explore a functional mechanism of synchronous inhibition of pyramidal neurons, we performed paired whole-cell patch-clamp recordings and recorded spontaneous inhibitory postsynaptic currents (sIPSCs) from layer II/III pyramidal neurons. A cross-correlogram of sIPSCs (1 ms bin) was used to detect synchronous sIPSCs. Synchronous neuron pairs were defined as those whose peak number of sIPSCs between -3 and 3 ms exceeded the mean + 2 SD of the number of sIPSCs in the period of -50 to 50 ms minus the number in that of -3 to 3 ms period. In the recording of pyramidal neurons located in the same column (intracolumn), 61.5% of neuron pairs were classified as synchronous neuron pairs, while 52.6% of pyramidal neuron pairs in adjacent columns (transcolumn) were defined as synchronous neuron pairs. The amplitude of synchronous sIPSCs was comparable to that of asynchronous sIPSCs in asynchronous neuron pairs, whereas that of synchronous sIPSCs was larger than that of asynchronous sIPSCs in synchronous neuron pairs. Synchronicity of sIPSCs did not depend on the distance of neuron pairs. These results suggest that layer II/III pyramidal neurons receive synchronous inhibitory synaptic inputs generated by a certain type of GABAergic interneuron that induces large IPSCs in pyramidal neurons, likely to be fast-spiking cells.

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).

Gap junction with MLO-A5 osteoblast-like cell line induces ALP and BSP transcription of 3T3-L1 pre-adipocyte like cell line via Hspb1 while retaining adipogenic differentiation ability.

In bone tissues, gap junctions form direct links between the cytoplasm of an osteocyte and another adjacent osteocyte or osteoblast, which underlie both bone formation and bone resorption. We have previously demonstrated that alkaline phosphatase (ALP) and bone sialoprotein (BSP), which are osteoblast markers, were induced in mesenchymal stem cells (MSCs) co-cultured with osteoblast-like cell line. However, the molecular mechanism of this process has not been fully addressed. Furthermore, few advances have been made toward elucidating the communication networks that link the status of committed cells such as (pre-) adipocytes that differentiated from MSCs as well as osteoblasts. Therefore, the objective of the present study was to investigate the mechanism underlying the communication network between pre-adipocytes and osteoblasts. We evaluated the effect of co-culture with osteoblast on the cell status of pre-adipocytes using murine osteoblast-like cell line, MLO-A5, and pre-adipocyte-like cell line, 3T3-L1, respectively. The results presented here demonstrated that osteoblasts and pre-adipocytes communicate via gap junctions, and the ensuing drastic increase in ALP and BSP transcription in co-cultured pre-adipocytes was induced, at least partly, via heat shock protein family B member 1 (Hspb1). In addition, terminal differentiation into adipocytes was suppressed in pre-adipocytes during co-culture with osteoblast without loss of adipogenic differentiation ability. Interestingly, after co-culture with osteoblasts, isolated co-cultured pre-adipocytes were able to differentiate to adipocytes as well as original pre-adipocytes. These results suggest that gap junctional communication with osteoblasts suppressed adipogenic differentiation of pre-adipocytes without loss of adipogenic differentiation ability.

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.

Trigeminal Nerve Transection-Induced Neuroplastic Changes in the Somatosensory and Insular Cortices in a Rat Ectopic Pain Model.

The primary sensory cortex processes competitive sensory inputs. Ablation of these competitive inputs induces neuroplastic changes in local cortical circuits. However, information concerning cortical plasticity induced by a disturbance of competitive nociceptive inputs is limited. Nociceptive information from the maxillary and mandibular molar pulps converges at the border between the ventral secondary somatosensory cortex (S2) and insular oral region (IOR); therefore, S2/IOR is a suitable target for examining the cortical changes induced by a disturbance of noxious inputs, which often causes neuropathic pain and allodynia. We focused on the plastic changes in S2/IOR excitation in a model of rats subjected to inferior alveolar nerve transection (IANX). Our optical imaging using a voltage-sensitive dye (VSD) revealed that the maxillary molar pulp stimulation-induced excitatory propagation was expanded one to two weeks after IANX at the macroscopic level. At the cellular level, based on Ca2+ imaging using two-photon microscopy, the amplitude of the Ca2+ responses and the number of responding neurons in S2/IOR increased in both excitatory and inhibitory neurons. The in vitro laser scanning photostimulation (LSPS) revealed that Layer II/III pyramidal and GABAergic fast-spiking neurons in S2/IOR received larger excitatory inputs from Layer IV in the IANX models, which supports the findings obtained by the macroscopic and microscopic optical imaging. Furthermore, the inhibitory postsynaptic inputs to the pyramidal neurons were decreased in the IANX models, suggesting suppression of inhibitory synaptic transmission onto excitatory neurons. These results suggest that IANX induces plastic changes in S2/IOR by changing the local excitatory and inhibitory circuits.

Menthol-induced facilitation of cerebrocortical excitatory propagation induced by air puff stimulation of the nasal cavity in the rat: An optical imaging study.

Temperature plays a critical role in the sensation of airflow in the nasal mucosa. Neural activities of the ethmoidal nerve, a trigeminal afferent, responding to airflow are suppressed by warm airflow, whereas cold airflow enhances the ethmoidal nerve activities, which is mimicked by application of menthol, a cold-sensitive TRPM8 receptor agonist. However, it has been an open issue how menthol modulates the spatiotemporal profiles of neural activities of somatosensory cortical neurons. In this study, we assessed neural responses to an air puff stimulation (100 ms) to the nasal cavity in the absence or presence of l-menthol using an optical imaging technique with a voltage-sensitive dye in the primary cortex (S1) of urethane-anesthetized rats. A weak air puff application (15 psi) without l-menthol induced neural excitation in a part of the contralateral S1. The air puff stimulation with l-menthol significantly increased the optical signal intensity, expanded the activated area, and shortened the latency, compared to those in the absence of l-menthol. These results suggest that activation of cold-sensitive TRPM8 receptors sharpens airflow sensation in the nasal cavity and expands the receptive field, especially toward the pharynx, which may contribute to enhanced flavor perception.

Opposite Roles in Short-Term Plasticity for N-Type and P/Q-Type Voltage-Dependent Calcium Channels in GABAergic Neuronal Connections in the Rat Cerebral Cortex.

Neurotransmitter release is triggered by Ca2+ influx through voltage-dependent Ca2+ channels (VDCCs). Distinct expression patterns of VDCC subtypes localized on the synaptic terminal affect intracellular Ca2+ dynamics induced by action potential-triggered Ca2+ influx. However, it has been unknown whether the expression pattern of VDCC subtypes depends on each axon terminal or neuronal subtype. Furthermore, little information is available on how these VDCC subtypes regulate the release probability of neurotransmitters. To address these questions, we performed multiple whole-cell patch-clamp recordings from GABAergic neurons in the insular cortex of either the male or the female rat. The paired-pulse ratio (PPR; 50 ms interstimulus interval) varied widely among inhibitory connections between GABAergic neurons. The PPR of unitary IPSCs was enhanced by ω-conotoxin GVIA (CgTx; 3 µm), an N-type VDCC blocker, whereas blockade of P/Q-type VDCCs by ω-agatoxin IVA (AgTx, 200 nm) decreased the PPR. In the presence of CgTx, application of 4 mm [Ca2+]o or of roscovitine, a P/Q-type activator, increased the PPR. These results suggest that the recruitment of P/Q-type VDCCs increases the PPR, whereas N-type VDCCs suppress the PPR. Furthermore, we found that charybdotoxin or apamin, blockers of Ca2+-dependent K+ channels, with AgTx increased the PPR, suggesting that Ca2+-dependent K+ channels are coupled to N-type VDCCs and suppress the PPR in GABAergic neuronal terminals. Variance-mean analysis with changing [Ca2+]o showed a negative correlation between the PPR and release probability in GABAergic synapses. These results suggest that GABAergic neurons differentially express N-type and/or P/Q-type VDCCs and that these VDCCs regulate the GABA release probability in distinct manners.SIGNIFICANCE STATEMENT GABAergic neuronal axons target multiple neurons and release GABA triggered by Ca2+ influx via voltage-dependent Ca2+ channels (VDCCs), including N-type and P/Q-type channels. Little is known about VDCC expression patterns in GABAergic synaptic terminals and their role in short-term plasticity. We focused on inhibitory synaptic connections between GABAergic neurons in the cerebral cortex using multiple whole-cell patch-clamp recordings and found different expression patterns of VDCCs in the synaptic terminals branched from a single presynaptic neuron. Furthermore, we observed facilitative and depressive short-term plasticity of IPSCs mediated by P/Q-type and N-type VDCCs, respectively. These results suggest that VDCC expression patterns regulate distinctive types of synaptic transmission in each GABAergic axon terminal even though they are branched from a common presynaptic neuron.

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.

Ablation of C-fibers decreases quantal size of GABAergic synaptic transmission in the insular cortex.

The primary sensory cortex exhibits neuroplastic changes responding to sensory disturbances, and GABAergic synaptic transmission plays a critical role in the regulation of plasticity. The insular cortex (IC) integrates orofacial nociceptive signals conveyed via myelinated Aδ- and unmyelinated C-fibers. However, it has been unknown whether a disturbance of nociceptive inputs, such as a deletion of the peripheral nerves, alters GABAergic local circuit in IC. The present study elucidated GABAergic synaptic transmission in the model rat whose C-fibers were ablated by capsaicin injection 1-2 days after birth. In vivo optical imaging revealed that capsaicin-treated rats showed a facilitative excitatory propagation in IC responding to dental pulp stimulation. Whole-cell patch-clamp recording from pyramidal neurons (Pyr) demonstrated that capsaicin-treated rats showed the smaller amplitude of miniature inhibitory postsynaptic currents (IPSCs) than sham-treated rats without changing the frequency. Furthermore, replacement of extracellular Ca2+ to Sr2+, which induces an asynchronous release of neurotransmitters in the quantal size, induced a smaller amplitude of asynchronous unitary IPSCs recorded from fast-spiking GABAergic interneuron to Pyr connections in capsaicin-treated rats than sham-treated rats. These results suggest that capsaicin treatment depresses IPSCs via a postsynaptic mechanism. To confirm this possibility, the variance-mean analysis of unitary IPSCs was employed and we found that quantal size of GABAergic synaptic transmission was smaller in capsaicin-treated rats than in sham-treated rats. These results suggest that ablation of C-fibers induces plastic changes in GABAergic synaptic transmission by decreasing postsynaptic GABAA receptor-mediated conductance, which is a possible mechanism of the facilitative excitation in IC of capsaicin-treated rats.

Opioid subtype- and cell-type-dependent regulation of inhibitory synaptic transmission in the rat insular cortex.

The insular cortex (IC) plays a principal role in the regulation of pain processing. Although opioidergic agonists depress cortical excitatory synaptic transmission, little is known about opioidergic roles in inhibitory synaptic transmission. In the IC, the opioid receptors differentially regulate the excitatory propagation: agonists of the mu (MOR), delta (DOR), and kappa (KOR) exhibit suppressive, facilitative, and little effects, respectively. Thus, we aimed to examine the effects of opioid receptor agonists on unitary inhibitory postsynaptic currents (uIPSCs) in the IC. Pyramidal and GABAergic neurons in the rat IC were recorded by a multiple whole-cell patch-clamp technique. [D-Ala2,N-Me-Phe4,Gly5-ol]-Enkephalin acetate salt (DAMGO), an MOR agonist, reduced uIPSC amplitude by 74% in fast-spiking GABAergic interneuron (FS)→FS connections without a significant effect on FS→pyramidal cell (Pyr) connections. These effects of DAMGO were also observed in non-FS→FS and non-FS→Pyr connections: DAMGO reduced the uIPSC amplitude in non-FS→FS but not in non-FS→Pyr connections. DAMGO-induced depression of uIPSCs was blocked by the MOR antagonist, D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2. The DOR agonist, [D-Pen2,5]-Enkephalin hydrate (DPDPE), reduced uIPSC amplitude by 39% in FS→FS and by 49% in FS→Pyr connections, which was antagonized by the DOR antagonist, naltrindole. However, DPDPE had little effect on non-FS→FS/Pyr connections. (±)-trans-U-50488 methanesulfonate salt (U50488), a KOR agonist, had little effect on uIPSC in FS→FS/Pyr connections. These results suggest that MOR-induced uIPSC depression in FS→FS and non-FS→FS, but not FS→Pyr and non-FS→Pyr connections, results in the depression of excitatory propagation in the IC, which may be an underlying mechanism of the powerful analgesic effects of MOR agonists.

Osteocytes up-regulate the terminal differentiation of pre-osteoblasts via gap junctions.

We examined cell-to-cell interaction between pre-osteoblasts and osteocytes using MC3T3-E1 and MLO-Y4, respectively. First, GFP expressing MC3T3-E1 (E1-GFP) cells were generated to isolate the cells from co-culture with MLO-Y4. No changes were observed in the expression of osteogenic transcription factors Runx2, Osterix, Dlx5 and Msx2, but expression of alkaline phosphatase (ALP) and bone sialoprotein (BSP) in E1-GFP co-cultured with MLO-Y4 was 300-400-fold greater than that in mono-cultured E1-GFP. In addition, mineralized nodule formation was drastically increased in co-cultured E1-GFP cells compared to mono-cultured cells. Patch clamp assay showed the presence of gap junctions between E1-GFP and MLO-Y4. Furthermore, when the gap junction inhibitor carbenoxolone (CBX) was added to the culture, increased expression of ALP and BSP in E1-GFP co-cultured with MLO-Y4 was suppressed. These results suggest that gap junction detected between pre-osteoblasts and osteocytes plays an important role on the terminal differentiation of pre-osteoblasts.

Osteogenic gene transcription is regulated via gap junction-mediated cell-cell communication.

An analytical study of cell-cell communications between murine osteoblast-like MLO-A5 cells and bone marrow mesenchymal stem cell (BMSC)-like C3H10T1/2 cells was performed. C3H10T1/2 cells expressing green fluorescent protein (10T-GFP cells) were generated to enable the isolation of the BMSC-like cells from co-cultures with MLO-A5 cells. The mRNA expression levels of several osteogenic transcription factors (Runx2, Osterix, Dlx5, and Msx2) did not differ between the co-cultured and mono-cultured 10T-GFP cells, but those of alkaline phosphatase (ALP) and bone sialoprotein (BSP) were 300- to 400-fold higher in the co-cultured cells. Patch clamp and biocytin transfer assays revealed gap junction-mediated communication between co-cultured 10T-GFP and MLO-A5 cells. The addition of a gap junction inhibitor suppressed the increases in the expression levels of the ALP and BSP mRNAs in co-cultured 10T-GFP cells. Furthermore, the histone acetylation levels were higher in co-cultured 10T-GFP cells than in mono-cultured 10T-GFP cells. These results suggest that osteoblasts and BMSCs associate via gap junctions, and that gap junction-mediated signaling induces histone acetylation that leads to elevated transcription of the genes encoding ALP and BSP in BMSCs.

Fast-spiking cell to pyramidal cell connections are the most sensitive to propofol-induced facilitation of GABAergic currents in rat insular cortex.

BACKGROUND: Propofol facilitates γ-aminobutyric acid-mediated inhibitory synaptic transmission. In the cerebral cortex, γ-aminobutyric acidergic interneurons target both excitatory pyramidal cells (Pyr) and fast-spiking (FS) and non-FS interneurons. Therefore, the propofol-induced facilitation of inhibitory transmission results in a change in the balance of excitatory and inhibitory inputs to Pyr. However, it is still unknown how propofol modulates γ-aminobutyric acidergic synaptic transmission in each combination of Pyr and interneurons. METHODS: The authors examined whether propofol differentially regulates inhibitory postsynaptic currents (IPSCs) depending on the presynaptic and postsynaptic cell subtypes using multiple whole cell patch clamp recording from γ-aminobutyric acidergic interneurons and Pyr in rat insular cortex. RESULTS: Propofol (10 µM) consistently prolonged decay kinetics of unitary IPSCs (uIPSCs) in all types of inhibitory connections without changing paired-pulse ratio of the second to first uIPSC amplitude or failure rate. The FS→Pyr connections exhibited greater enhancement of uIPSC charge transfer (2.2 ± 0.5 pC, n = 36) compared with that of FS→FS/non-FS connections (0.9 ± 0.2 pC, n = 37), whereas the enhancement of charge transfer in non-FS→Pyr (0.3 ± 0.1 pC, n = 15) and non-FS→FS/non-FS connections (0.2 ± 0.1 pC, n = 36) was smaller to those in FS→Pyr/FS/non-FS. Electrical synapses between FS pairs were not affected by propofol. CONCLUSIONS: The principal inhibitory connections (FS→Pyr) are the most sensitive to propofol-induced facilitation of uIPSCs, which is likely mediated by postsynaptic mechanisms. This preferential uIPSC enhancement in FS→Pyr connections may result in suppressed neural activities of projection neurons, which in turn reduces excitatory outputs from cortical local circuits.

Reciprocal regulation of inhibitory synaptic transmission by nicotinic and muscarinic receptors in rat nucleus accumbens shell.

Medium spiny neurones (MSNs) in the nucleus accumbens (NAc) are the principal neurones whose activities are regulated by GABAergic inputs from MSNs and fast-spiking interneurones (FSNs). Cholinergic interneurones play important roles in the regulation of activity in MSNs; however, how acetylcholine modulates inhibitory synaptic transmission from MSNs/FSNs to MSNs remains unknown. We performed paired whole-cell patch-clamp recordings from MSNs and FSNs in rat NAc shell slice preparations and examined cholinergic effects on unitary inhibitory postsynaptic currents (uIPSCs). Carbachol (1 µM) suppressed uIPSC amplitude by 58.3 ± 8.0% in MSN→MSN connections, accompanied by increases in paired-pulse ratio and failure rate, suggesting that acetylcholine reduces the probability of GABA release from the synaptic terminals of MSNs. Carbachol-induced uIPSC suppression was antagonised by 100 µM atropine, and was mimicked by pilocarpine (1 µM) and acetylcholine (1 µM) but not nicotine (1 µM). Application of AM251 slightly reduced carbachol-induced uIPSC suppression (30.8 ± 8.9%), suggesting an involvement of endocannabinoid signalling in muscarinic suppression of uIPSCs. In contrast, FSN→MSN connections showed that pilocarpine had little effect on the uIPSC amplitude, whereas both nicotine and acetylcholine facilitated uIPSC amplitude, with decreases in failure rate and paired-pulse ratio, suggesting that nicotine-induced uIPSC facilitation is mediated by presynaptic mechanisms. Miniature IPSC recordings support these hypotheses of presynaptic cholinergic mechanisms. These results suggest a differential role for muscarinic and nicotinic receptors in GABA release, which depends on presynaptic neuronal subtypes in the NAc shell.

Functional neuroimaging of aversive taste-related areas in the alert rat revealed by positron emission tomography.

Among noninvasive functional brain imaging techniques, (18) F-fluorodeoxyglucose (FDG)-positron emission tomography (PET) has a comparative advantage in detecting active brain regions in freely locomoting animals. We developed an [(18) F]FDG-PET protocol that visualizes active brain regions that respond preferentially to citrate-induced multiple behaviors in freely locomoting rats. In addition, c-Fos immunohistochemistry, an activity-dependent mapping, was performed to examine whether the areas detected by PET correspond to regions with c-Fos-immunopositive neurons. Citrate (0.1 M) was intraorally applied to detect activated brain regions responding to gustation and the rejection behaviors including gaping and tongue protrusion, which would potently activate the limbic system. PET images during citrate stimulation were subtracted from those obtained during free locomotion or during application of distilled water. Citrate increased FDG signals in multiple gustation-related regions: the nucleus accumbens (core and shell), the ventromedial nucleus of the thalamus, the basolateral and central nuclei of the amygdala, the hypothalamus, and the insular cortex. In addition, the ventrolateral striatum and the cingulate and entorhinal cortices, which have received less attention in the field of gustatory studies, also showed an increase in FDG signals. As expected, c-Fos-immunopositive cells were also found in these regions, suggesting that increased FDG signals induced by intraoral citrate injection are likely to reflect neural activity in these regions. Our [(18) F]FDG-PET protocol reveals the contributions of multiple brain regions responding to aversive taste in freely locomoting rats, and this approach may aid in the identification of unknown neural networks especially relating to the limbic information processing.

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.