Jeong-Seop Rhee

Mutations in a Cyclic Nucleotide-Gated Channel Lead to Abnormal Thermosensation and Chemosensation in C. elegans

The C. elegans tax-4 mutants are abnormal in multiple sensory behaviors: they fail to respond to temperature or to water-soluble or volatile chemical attractants. We show that the predicted tax-4 gene product is highly homologous to vertebrate cyclic nucleotide-gated channels. Tax-4 protein expressed in cultured cells functions as a cyclic nucleotide-gated channel. The green fluorescent protein (GFP)-tagged functional Tax-4 protein is expressed in thermosensory, gustatory, and olfactory neurons mediating all the sensory behaviors affected by the tax-4 mutations. The Tax-4::GFP fusion is partly localized at the sensory endings of these neurons. The results suggest that a cyclic nucleotide-gated channel is required for thermosensation and chemosensation and that cGMP is an important intracellular messenger in C. elegans sensory transduction.

Calcium channels in the GABAergic presynaptic nerve terminals projecting to meynert neurons of the rat.

Abstract : Effects of selective Ca2+ channel blockers on GABAergic inhibitory postsynaptic currents (IPSCs) were studied in the acutely dissociated rat nucleus basalis of Meynert (nBM) neurons attached with nerve endings, namely, the “synaptic bouton” preparation, and in the thin slices of nBM, using nystatin perforated and conventional whole‐cell patch recording modes, respectively. In the synaptic bouton preparation, nicardipine (3 × 10‐6M) and ω‐conotoxin‐MVIIC (3 × 10‐6M) reduced the frequency of spontaneous postsynaptic currents by 37 and 22%, respectively, whereas ω‐conotoxin‐GVIA had no effect. After blockade of L‐ and P/Q‐type Ca2+ channels, successive removal of Ca2+ from external solution had no significant effect on the residual spontaneous activities, indicating that N‐, R‐, and T‐type Ca2+ channels are not involved in the spontaneous GABA release. Thapsigargin, but not ryanodine, increased the frequency of spontaneous IPSCs in both the synaptic bouton and slice preparations, suggesting the partial contribution of the intracellular Ca2+ storage site to the spontaneous GABA release. In contrast, ω‐conotoxin‐GVIA (3 × 10‐6M) and ω‐conotoxin‐MVIIC (3 × 10‐6M) suppressed the evoked IPSCs by 31 and 37%, respectively, but nicardipine produced no significant effect. The residual evoked currents were abolished in Ca2+‐free external solution but not in the external solution containing 10‐5M Ni2+, suggesting the involvement of N‐, P/Q‐, and R‐type Ca2+ channels but not L‐ and T‐type ones in the evoked IPSCs. Neither thapsigargin nor ryanodine had any significant effects on the evoked IPSCs. It was concluded that Ca2+ channel subtypes responsible for spontaneous transmitter release are different from those mediating the transmitter release evoked by nerve stimulation.

Presynaptic serotonergic inhibition of GABAergic synaptic transmission in mechanically dissociated rat basolateral amygdala neurons

1 The basolateral amygdala (ABL) nuclei contribute to the process of anxiety. GABAergic transmission is critical in these nuclei and serotonergic inputs from dorsal raphe nuclei also significantly regulate GABA release. In mechanically dissociated rat ABL neurons, spontaneous miniature inhibitory postsynaptic currents (mIPSCs) arising from attached GABAergic presynaptic nerve terminals were recorded with the nystatin‐perforated patch method and pharmacological isolation. 2 5‐HT reversibly reduced the GABAergic mIPSC frequency without affecting the mean amplitude. The serotonergic effect was mimicked by the 5‐HT1A specific agonist 8‐OH DPAT (8‐hydroxy‐2‐(di‐n‐propylamino)tetralin) and blocked by the 5‐HT1A antagonist spiperone. 3 The GTP‐binding protein inhibitor N‐ethylmaleimide removed the serotonergic inhibition of mIPSC frequency. In either K+‐free or Ca2+‐free external solution, 5‐HT could inhibit mIPSC frequency. 4 High K+ stimulation increased mIPSC frequency and 8‐OH DPAT inhibited this increase even in the presence of Cd2+. 5 Forskolin, an activator of adenylyl cyclase (AC), significantly increased synaptic GABA release frequency. Pretreatment with forskolin prevented the serotonergic inhibition of mIPSC frequency in both the standard and high K+ external solution. 6 Ruthenium Red (RR), an agent facilitating the secretory process in a Ca2+‐independent manner, increased synaptic GABA release. 5‐HT also suppressed RR‐facilitated mIPSC frequency. 7 We conclude that 5‐HT inhibits GABAergic mIPSCs by inactivating the AC‐cAMP signal transduction pathway via a G‐protein‐coupled 5‐HT1A receptor and this intracellular pathway directly acts on the GABA‐releasing process independent of K+ and Ca2+ channels in the presynaptic nerve terminals.

ATP facilitates spontaneous glycinergic IPSC frequency at dissociated rat dorsal horn interneuron synapses.

1 The ATP action on spontaneous miniature glycinergic inhibitory postsynaptic currents (mIPSCs) was investigated in rat substantia gelatinosa (SG) neurons mechanically dissociated from the 2nd layer of the dorsal horn in which their presynaptic glycinergic nerve terminals remained intact. 2 ATP reversibly facilitated the frequency of the mIPSCs in a concentration‐dependent manner without affecting their amplitude distribution. The ATP agonist, 2‐methylthioATP (2MeSATP), mimicked the ATP action, while another ATP receptor agonist, αβ‐methylene‐ATP (α,β‐meATP), had no effect on mIPSCs. 3 The ATP receptor antagonists, suramin (1 × 10−6 M) and pyridoxal‐5‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) (1 × 10−5 M), completely blocked the facilitatory effect of ATP on glycine release (102·0 ± 11·2 % and 99·3 ± 16·2 %, n= 6, respectively) without altering the current amplitude distributions. 4 N‐Ethylmaleimide (NEM), a sulphydryl alkylating agent, suppressed the inhibitory effect of adenosine on mIPSC frequency (111·2 ± 13·3 %, n= 4) without altering the current amplitude distribution. However, ATP still facilitated the mIPSC frequency (693·3 ± 245·2 %, n= 4) even in the presence of NEM. 5 The facilitatory effect of ATP (1 × 10−5 M) on mIPSC frequency was not affected by adding 1 × 10−4 M Cd2+ to normal external solution but was eliminated in a Ca2+‐free external solution. 6 These results suggest that ATP enhances glycine release from nerve terminals, presumably resulting in the inhibition of SG neurons which conduct nociceptive signals to the CNS. This presynaptic P2X‐type ATP receptor may function to prevent excess excitability in SG neurons, thus preventing an excessive pain signal and/or SG cell death.

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.

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.

Regional difference of high voltage-activated Ca2+ channels in rat CNS neurones

The diversity of high voltage-activated (HVA) Ca2+ channels in rat CNS neurones was investigated with the nystatin perforated patch recording configuration. The neurones were freshly dissociated from rat substantia nigra, ventromedial hypothalamus, tuberomammillary nucleus, nucleus tractus solitarius, hippocampal CA1 region and cerebellum. Five different types of HVA Ca2+ channels were distinguished pharmacologically; dihydro-pyridine sensitive L-type, ω-conotoxin-GVIA sensitive N-type, ω-agatoxin-IVA sensitive P-type, ω-conotoxin-MVIIC sensitive Q-type, and R-type which is insensitive to these organic Ca2+ antagonists. The results showed clearly that the five subtypes of HVA Ca2+ channels differ considerably in their distribution among various CNS regions.

Modulation of glycine-induced chloride current in acutely dissociated rat periaqueductal gray neurons by μ-opioid agonist, DAGO

Effect of a mu-opioid agonist (D-Ala2,N-MePhe4,Gly5-ol-enkephalin, DAGO), on glycine (Gly)-induced chloride current (IGly) was investigated in the periaqueductal gray (PAG) neurons acutely dissociated 1-2-week-old Wistar rats by the use of nystatin-perforated patch recording configuration under voltage-clamp condition. At a holding potential (VH) of -40 mV, DAGO caused a sustained potentiation of IGly at the low concentrations (10(-6)-10(-5) M) but reduced slightly the Gly response at the high concentration (10(-4) M). The reversal potential of IGly was equal to the Cl- equilibrium potential (ECl) and was not changed in the presence of 10(-6) M DAGO. The 10(-5) M Gly response was inhibited by the simultaneous treatment of forskolin and 3-isobutyl-1-methylxanthine (IBMX). H-89, a protein kinase A (PKA) inhibitor, increased the 10(-5) M Gly response but had little effect on the 10(-4) M Gly response. DAGO increased 10(-5) M Gly response in the presence of forskolin and IBMX but, not more than in the absence of forskolin and IBMX. The 10(-5) M Gly response augumented by DAGO was not affected by adding H-89. The present results suggest that the glycine-induced chloride current is cAMP dependent and is inhibited by PKA, and that the potentiation of the glycine response by DAGO is also cAMP dependent and is due to the inhibition of PKA as that of H-89. We conclude that the potentiation of glycine response by DAGO is mediated by an inhibition of cAMP-dependent PKA in the PAG neurons.

Serotonin modulates high-voltage-activated CA2+ channels in rat ventromedial hypothalamic neurons

The modulation of high-voltage-activated (HVA) Ca2+ channels by serotonin (5-HT) was studied in ventromedial hypothalamic (VMH) neurons acutely dissociated from 12-14-day-old Wistar rats using the whole-cell and nystatin perforated-patch recording configurations. 5-HT inhibited the HVA Ca2+ channels in a concentration-, time- and voltage-dependent manner. This inhibition was mimicked by the 5-HT1A agonist 8-hydroxy-dipropylaminotetralin and was prevented by pretreatment with pertussis toxin (PTX). omega-Conotoxin-GVIA, omega-agatoxin-IVA, nicardipine and omega-conotoxin-MVIIC blocked each fraction of HVA Ca2+ channel currents, suggesting the existence of N-, P-, L- and Q-types of HVA Ca2+ channels. A component of the current resistant to these Ca2+ channel antagonists also existed in the VMH neurons. Among these five components of HVA Ca2+ channel currents, the N- and Q-type currents were significantly inhibited by 5-HT. These findings suggest that the activation of 5-HT1A receptors produces the selective inhibition of N- and Q-type Ca2+ channels through a PTX-sensitive G-protein in rat VMH neurons.

α2-adrenoceptor-mediated enhancement of glycine response in rat sacral dorsal commissural neurons

The effect of noradrenaline on the glycine response was investigated in neurons acutely dissociated from the rat sacral dorsal commissural nucleus using nystatin perforated patch recording configuration under voltage-clamp conditions. Noradrenaline reversibly potentiated the 10(-5)M glycine-induced Cl- current in a concentration-dependent manner. Single channel recordings in a cell-attached mode revealed that noradrenaline decreased the closing time of the glycine-activated channel activity. Noradrenaline neither changed the reversal potential of the glycine response nor affected the affinity of glycine to its receptor. Clonidine mimicked and yohimbine blocked the noradrenaline action on glycine response. N-[2(methylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride, protein kinase A inhibitor, mimicked the effect of noradrenaline on glycine response. Noradrenaline failed to affect the glycine response in the presence of these intracellular cyclic AMP and protein kinase A modulators. However, noradrenaline further enhanced the glycine response even in the presence of phorbol-12-myristate-13-acetate and chelerythrine, a protein kinase C inhibitor. Pertussis toxin treatment for 6-8 h blocked the noradrenaline facilitatory effect on the glycine response. In addition, noradrenaline potentiated the strychnine-sensitive postsynaptic currents evoked in a slice preparation of sacral dorsal commissural nucleus. These results suggest that the activation of alpha2-adrenoceptor by noradrenaline coupled with pertussis toxin-sensitive G-proteins reduces intracellular cyclic AMP formation through the inhibition of adenyl cyclase. The reduction of cyclic AMP decreases the protein kinase A activity, thus resulting in the potentiation of the glycinergic inputs to the sacral dorsal commissural neurons. It is thus feasible that the noradrenergic input to the sacral dorsal commissural nucleus modulates such nociceptive signals as pain by intracellular enhancing the glycine response.