Il-Sung Jang

GABAA Receptor Phospho-Dependent Modulation Is Regulated by Phospholipase C-Related Inactive Protein Type 1, a Novel Protein Phosphatase 1 Anchoring Protein

GABAA receptors are critical in controlling neuronal activity. Here, we examined the role for phospholipase C-related inactive protein type 1 (PRIP-1), which binds and inactivates protein phosphatase 1α (PP1α) in facilitating GABAA receptor phospho-dependent regulation using PRIP-1-/- mice. In wild-type animals, robust phosphorylation and functional modulation of GABAA receptors containing β3 subunits by cAMP-dependent protein kinase was evident, which was diminished in PRIP-1-/- mice. PRIP-1-/- mice exhibited enhanced PP1α activity compared with controls. Furthermore, PRIP-1 was able to interact directly with GABAA receptor β subunits, and moreover, these proteins were found to be PP1α substrates. Finally, phosphorylation of PRIP-1 on threonine 94 facilitated the dissociation of PP1α-PRIP-1 complexes, providing a local mechanism for the activation of PP1α. Together, these results suggest an essential role for PRIP-1 in controlling GABAA receptor activity via regulating subunit phosphorylation and thereby the efficacy of neuronal inhibition mediated by these receptors.

Functional roles of presynaptic GABAA receptors on glycinergic nerve terminals in the rat spinal cord

GABAA receptor‐mediated presynaptic depolarization is believed to induce presynaptic inhibition of excitatory synaptic transmission. We report here the functional roles of presynaptic GABAA receptors in glycinergic transmission of the rat spinal cord. In mechanically dissociated rat sacral dorsal commissural nucleus (SDCN) neurons attached with native glycinergic and GABAergic nerve terminals, glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) were isolated from a mixture of both glycinergic and GABAergic sIPSCs by perfusing the SDCN nerve cell body with ATP‐free internal solution. Under such experimental conditions, exogenously applied muscimol (0.5 μM) depolarized glycinergic presynaptic nerve terminals and significantly increased glycinergic sIPSC frequency to 542.7 ± 47.3 % of the control without affecting the mean current amplitude. The facilitatory effect of muscimol on sIPSC frequency was completely blocked by bicuculline (10 μM) or SR95531 (10 μM), selective GABAA receptor antagonists. This muscimol‐induced presynaptic depolarization was due to a higher intraterminal Cl− concentration, which is maintained by a bumetanide‐sensitive Na‐K‐Cl cotransporter. On the contrary, when electrically evoked, this muscimol‐induced presynaptic depolarization was found to decrease the action potential‐dependent glycine release evoked by focal stimulation of a single terminal. The results suggest that GABAA receptor‐mediated presynaptic depolarization has two functional roles: (1) presynaptic inhibition of action potential‐driven glycinergic transmission, and (2) presynaptic facilitation of spontaneous glycinergic transmission.

Signaling pathways of bisphenol A–induced apoptosis in hippocampal neuronal cells: Role of calcium-induced reactive oxygen species, mitogen-activated protein kinases, and nuclear factor–κB

In the present study, we investigated the neurotoxicity of bisphenol A [BPA; 2,2‐bis‐(4 hydroxyphenyl) propane] and the underlying mechanisms of action in mouse hippocampal HT‐22 cells. BPA, known to be a xenoestrogen, is used in the production of water bottles, cans, and teeth suture materials. BPA‐treated HT‐22 cells showed lower cell viability than did controls at concentrations of BPA over 100 μM. BPA induced apoptotic cell death as indicated by staining with Hoechst 33258, costaining with Annexin V/propidium iodide, and activation of caspase 3. BPA regulated the generation of reactive oxygen species (ROS) by increasing intracellular calcium. BPA activated phosphorylation of extracellular signal–regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK), and nuclear translocation of nuclear factor (NF)‐κB. Pretreatment with specific inhibitors for calcium, ROS, ERK, and JNK decreased BPA‐induced cell death; however, inhibitor for NF‐κB increased BPA‐induced cell death. The results suggest that calcium, ROS, ERK, and JNK are involved in BPA‐induced apoptotic cell death in HT‐22 cells. In contrast, an NF‐κB cascade was activated for survival signaling after BPA treatment.

Presynaptic GABAA receptors facilitate spontaneous glutamate release from presynaptic terminals on mechanically dissociated rat CA3 pyramidal neurons

Mossy fiber-derived giant spontaneous miniature excitatory postsynaptic currents have been suggested to be large enough to generate action potentials in postsynaptic CA3 pyramidal neurons. Here we report on the functional roles of presynaptic GABA(A) receptors on excitatory terminals in contributing to spontaneous glutamatergic transmission to CA3 neurons. In mechanically dissociated rat hippocampal CA3 neurons with adherent presynaptic nerve terminals, spontaneous excitatory postsynaptic currents were recorded using conventional whole-cell patch clamp recordings. In most recordings, unusually large spontaneous excitatory postsynaptic currents up to 500 pA were observed. These large spontaneous excitatory postsynaptic currents were highly sensitive to group II metabotropic glutamate receptor activation, and were still observed even after the blockade of voltage-dependent Na(+) or Ca(2+) channels. Exogenously applied muscimol (0.1-3 microM) significantly increased the frequency of spontaneous excitatory postsynaptic currents including the large ones. This facilitatory effect of muscimol was completely inhibited in the presence of 10 microM 6-imino-3-(4-methoxyphenyl)-1(6H)-pyridazinebutanoic acid HBr, a specific GABA(A) receptor antagonist. Pharmacological data suggest that activation of presynaptic GABA(A) receptors directly depolarizes glutamatergic terminals resulting in the facilitation of spontaneous glutamate release. In the current-clamp condition, a subset of large spontaneous excitatory postsynaptic potentials triggered action potentials, and muscimol greatly increased the frequency of spontaneous excitatory postsynaptic potential-triggered action potentials in postsynaptic CA3 pyramidal neurons. The results suggest that presynaptic GABA(A) receptors on glutamatergic terminals play an important role in the excitability of CA3 neurons as well as in the presynaptic modulation of glutamatergic transmission onto hippocampal CA3 neurons.

Phenotypic Polarization of Activated Astrocytes: the Critical Role of Lipocalin-2 in the Classical Inflammatory Activation of Astrocytes

Astrocytes provide structural and functional support for neurons, as well as display neurotoxic or neuroprotective phenotypes depending upon the presence of an immune or inflammatory microenvironment. This study was undertaken to characterize multiple phenotypes of activated astrocytes and to investigate the regulatory mechanisms involved. We report that activated astrocytes in culture exhibit two functional phenotypes with respect to pro- or anti-inflammatory gene expression, glial fibrillary acidic protein expression, and neurotoxic or neuroprotective activities. The two distinct functional phenotypes of astrocytes were also demonstrated in a mouse neuroinflammation model, which showed pro- or anti-inflammatory gene expression in astrocytes following challenge with classical or alternative activation stimuli; similar results were obtained in the absence of microglia. Subsequent studies involving recombinant lipocalin-2 (LCN2) protein treatment or Lcn2-deficient mice indicated that the pro- or anti-inflammatory functionally polarized phenotypes of astrocytes and their intracellular signaling pathway were critically regulated by LCN2 under in vitro and in vivo conditions. Astrocyte-derived LCN2 promoted classical proinflammatory activation of astrocytes but inhibited IL-4–STAT6 signaling, a canonical pathway involved in alternative anti-inflammatory activation. Our results suggest that the secreted protein LCN2 is an autocrine modulator of the functional polarization of astrocytes in the presence of immune or inflammatory stimuli and that LCN2 could be targeted therapeutically to dampen proinflammatory astrocytic activation and related pathologies in the CNS.

Histaminergic modulation of GABAergic transmission in rat ventromedial hypothalamic neurones

1 The ventromedial nucleus of the hypothalamus (VMH) is a key nucleus in the homeostatic regulation of neuroendocrine and behavioural functions. In mechanically dissociated rat VMH neurones with attached native presynaptic nerve endings, GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded using the nystatin perforated patch recording mode under voltage‐clamp conditions. 2 Histamine reversibly inhibited the sIPSC frequency in a concentration‐dependent manner without affecting the mean current amplitude. The selective histamine receptor type 3 (H3) agonist imetit (100 nm) mimicked this effect and it was completely abolished by the selective H3 receptor antagonists clobenpropit (3 μm) and thioperamide (10 μm). 3 The GTP‐binding protein inhibitor N‐ethylmaleimide (10 μm) removed the histaminergic inhibition of GABAergic sIPSCs. 4 Elimination of external Ca2+ reduced the GABAergic sIPSC frequency without affecting the distribution of current amplitudes. In this condition, the inhibitory effect of imetit on the sIPSC frequency completely disappeared, suggesting that the histaminergic inhibition requires extracellular Ca2+. 5 The P/Q‐type Ca2+ channel blocker ω‐agatoxin IVA (300 nm) attenuated the histaminergic inhibition of the GABAergic sIPSC frequency, but neither the N‐type Ca2+ channel blocker ω‐conotoxin GVIA (3 μm) nor the L‐type Ca2+ channel blocker nicardipine (3 μm) was effective. 6 Activation of adenylyl cyclase with forskolin (10 μm) had no effect on histaminergic inhibition of the sIPSCs. 7 In conclusion, histamine inhibits spontaneous GABA release from presynaptic nerve terminals projecting to VMH neurones by inhibiting presynaptic P/Q‐type Ca2+ channels via a G‐protein coupled to H3 receptors and this may modulate the excitability of VMH neurones.

Activation of presynaptic glycine receptors facilitates glycine release from presynaptic terminals synapsing onto rat spinal sacral dorsal commissural nucleus neurons.

Glycine is a major inhibitory neurotransmitter in the spinal cord and brainstem. Here we report the novel finding that presynaptic glycine autoreceptors modulate release from terminals synapsing onto rat spinal sacral dorsal commissural nucleus (SDCN) neurons. In mechanically dissociated SDCN neurons, in which functional presynaptic nerve terminals remain adherent to the isolated neurons, exogenously applied glycine (3 μM) increased the frequency of glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) without affecting their amplitudes or decay times. This suggests that glycine acts presynaptically to increase glycine release probability. Picrotoxin, at a concentration that had little direct effect on sIPSC frequency and amplitude (30 μM), significantly attenuated glycine‐induced presynaptic sIPSC facilitation. The glycine‐induced sIPSC frequency facilitation was completely abolished either in a Ca2+‐free external solution or in the presence of 100 μM Cd2+, suggesting the involvement of extracellular Ca2+ influx into the nerve terminals. The glycine action was also completely occluded in the presence of 300 nM tetrodotoxin. In recordings from SDCN neurons in spinal cord slices, glycine (10 μM) increased evoked IPSC (eIPSC) amplitude and decreased the extent of paired‐pulse facilitation. In response to brief high frequency stimulus trains the eIPSCs displayed a profound frequency‐dependent facilitation that was greatly reduced by picrotoxin (30 μM). These results indicate that glycine acts at presynaptic autoreceptors, causing depolarization of the glycinergic nerve terminals, the subsequent activation of voltage‐dependent Na+ and Ca2+ channels, and facilitation of glycine release. Furthermore, this presynaptic facilitation was observed under more physiological conditions, suggesting that these glycinergic autoreceptors may contribute to the integration of local inhibitory inputs to SDCN neurons.

Developmental changes in P2X purinoceptors on glycinergic presynaptic nerve terminals projecting to rat substantia gelatinosa neurones

1 In mechanically dissociated rat spinal cord substantia gelatinosa (SG) neurones attached with native presynaptic nerve endings, glycinergic miniature inhibitory postsynaptic currents (mIPSCs) were recorded using nystatin perforated patch recording mode under voltage‐clamp conditions. Under these conditions, it was tested whether the changes in P2X receptor subtype on the glycinergic presynaptic nerve terminals occur during postnatal development. 2 ATP facilitated glycinergic mIPSC frequency in a concentration‐dependent manner through all developmental stages tested, whereas αβ‐methylene‐ATP (αβ‐me‐ATP) was only effective at later developmental stages. 3 αβ‐me‐ATP‐elicited mIPSC frequency facilitation was completely occluded in the Ca2+‐free external solution, but it was not affected by adding 10−4m Cd2+. 4 αβ‐me‐ATP still facilitated mIPSC frequency even in the presence of 10−6m thapsigargin, a Ca2+ pump blocker. 5 In later developmental stages, ATP‐elicited presynaptic or postsynaptic responses were reversibly blocked by 10−5m pyridoxal‐5‐phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS), but only partially blocked by 10−7m 2′,3′‐O‐(2,4,6‐trinitrophenyl)‐ATP (TNP‐ATP). However, αβ‐me‐ATP‐elicited presynaptic or postsynaptic responses were completely and reversibly blocked by either 10−5m PPADS or 10−7m TNP‐ATP. 6 αβ‐me‐ATP significantly reduced the evoked glycinergic IPSC amplitude in postnatal 28–30 day neurones, whereas it had no effect in 10–12 day neurones. 7 It was concluded that αβ‐me‐ATP‐sensitive P2X receptors were functionally expressed on the glycinergic presynaptic nerve terminals projecting to SG neurones in later developmental stages. Such developmental changes of presynaptic P2X receptor subtypes might contribute to synaptic plasticity such as the regulation of neuronal excitability and the fine controlling of the pain signal in spinal dorsal horn neurones.

The Na+/H+ exchanger is a major pH regulator in GABAergic presynaptic nerve terminals synapsing onto rat CA3 pyramidal neurons

The effects of pHi on GABAergic miniature inhibitory postsynaptic currents (mIPSCs) were studied in mechanically dissociated CA3 pyramidal neurons, by use of ammonium prepulse and whole‐cell patch‐clamp techniques, under the voltage‐clamp condition. NH4Cl itself, which is expected to alkalinize pHi, increased GABAergic mIPSC frequency in a concentration‐dependent manner. In contrast, NH4Cl decreased mIPSC frequency, either in the presence of 200 µm Cd2+ or in Ca2+‐free external solution, suggesting that intraterminal alkalosis decreased GABAergic mIPSC frequency while [NH4+] itself may activate Ca2+ channels by depolarizing the terminal. On the other hand, GABAergic mIPSC frequency was greatly increased immediately after NH4Cl removal, a condition expected to acidify pHi, and recovered to the control level within 2 min after NH4Cl removal. This explosive increase in mIPSC frequency observed after NH4Cl removal was completely eliminated after depletion of Ca2+ stores with 1 µm thapsigargin in the Ca2+‐free external solution, suggesting that acidification increases in intraterminal Ca2+ concentration via both extracellular Ca2+ influx and Ca2+ release from the stores. However, the acidification‐induced increase in mIPSC frequency had not recovered by 10 min after NH4Cl removal either in the Na+‐free external solution or in the presence of 10 µm 5‐(N‐ethyl‐N‐isopropyl)‐amiloride (EIPA), a specific Na+/H+ exchanger (NHE) blocker. The present results suggest that NHEs are major intraterminal pH regulators on GABAergic presynaptic nerve terminals, and that the NHE‐mediated regulation of pHi under normal physiological or pathological conditions might play an important role in the neuronal excitability by increasing inhibitory tones.

Feed-forward facilitation of glutamate release by presynaptic GABAA receptors

Disynaptic GABAergic inputs from Schaffer collateral (SC) afferents on to the soma of glutamatergic CA1 pyramidal neurons are involved in feed-forward inhibition in the hippocampal neural circuits. Here we report the functional roles of presynaptic GABA(A) receptors on SC afferents projecting to CA1 pyramidal neurons. Muscimol (0.5 microM), a selective GABA(A) receptor agonist, increased SC-evoked EPSC amplitude and decreased paired-pulse ratio in the slice preparation, in addition, it facilitated spontaneous glutamate release on to mechanically dissociated CA1 pyramidal neurons in an external Ca2+-dependent manner. In field recordings, muscimol at low concentrations (< or = 0.5 microM) increased not only the excitability of SC afferents but glutamate release, however, it at high concentrations (> or = 1 microM) changed bidirectionally. These results suggest that the moderate activation of presynaptic GABA(A) receptors depolarizes SC afferents and enhances SC-mediated glutamatergic transmission. When endogenous GABA was disynaptically released by brief trains of stimulation of SC afferents, the axonal excitability in addition to glutamate release was increased. The effects of endogenous GABA on the excitability of SC afferents were blocked by either SR95531 or AMPA receptor blockers, which would be expected to block disynaptic feed-forward neural circuits. The present results provide a novel form of presynaptic modulation (feed-forward facilitation) of glutamatergic transmission by presynaptic GABA(A) receptors within the intrinsic hippocampal neural circuits.