Susumu Koyama

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.

Presynaptic 5-HT3 receptor-mediated modulation of synaptic GABA release in the mechanically dissociated rat amygdala neurons

1 Nystatin‐perforated patch recordings were made from mechanically dissociated basolateral amygdala neurons with preserved intact native presynaptic nerve terminals to study the mechanism of 5‐HT3 receptor‐mediated serotonergic modulation of GABAergic inhibition. 2 The specific 5‐HT3 agonist mCPBG (1 μM) rapidly facilitated the frequency of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) and this facilitation desensitized within 1 min. Tropisetron (30 nM), a specific 5‐HT3 antagonist, blocked the mCPBG effect. 3 mCPBG augmented mIPSC amplitude. However, no direct postsynaptic serotonergic currents were evoked by mCPBG. Neither GABA‐evoked current amplitude nor the kinetics of individual GABAergic mIPSCs were affected by mCPBG. Therefore, the augmentation is unlikely to be due to postsynaptic effects evoked by mCPBG. At higher concentrations mCPBG produced shorter‐duration facilitation of miniature events. 4 While mCPBG increased the mIPSC frequency in calcium‐containing solution with Cd2+, this increase was absent in Ca2+‐free external solution. It appears that the Ca2+ influx through voltage‐dependent calcium channels was not as crucial as that through 5‐HT3 receptors for synaptic GABA release. 5 When two pulses of mCPBG (each 1 μM, 1 min) were given, the response to the second pulse elicited full recovery when the interval between pulses was at least 9 min. Protein kinase A (PKA) activation by 8‐Br‐cAMP (300 μM) shortened and PKA inhibition by Rp‐cAMP (100 μM) prolonged the recovery time. PKA activity did not affect the time course of fast desensitization. 6 Our results suggest that a 5‐HT3‐specific agonist acts on presynaptic nerve terminals facilitating synaptic GABA release without postsynaptic effects. The facilitation requires calcium influx through presynaptic 5‐HT3 receptors. PKA modulates the recovery process from desensitization of presynaptic 5‐HT3 receptor‐mediated regulation of synaptic GABA release.

Synergistic μ-opioid and 5-HT1A presynaptic inhibition of GABA release in rat periaqueductal gray neurons

The periaqueductal gray (PAG) plays a critical role in descending antinociception. In mechanically dissociated rat PAG neurons, pharmacologically separated spontaneous GABAergic miniature inhibitory postsynaptic currents (mIPSCs) were recorded using the nystatin-perforated patch technique. Both DAMGO, a specific mu-opioid receptor agonist, and serotonin inhibited mIPSC frequency in a dose-dependent manner without affecting mIPSC amplitude, respectively, in the same PAG neurons. The presynaptic opioid effect was blocked by a specific mu-opioid receptor antagonist, CTOP. The presynaptic serotonergic effect was mimicked by a specific 5-HT(1A) receptor agonist, 8-OH-DPAT, and blocked by the specific antagonist, NAN-190. These opioidergic and serotonergic inhibitions of GABA release employed the similar intracellular mechanism of opening 4-AP-sensitive K(+) channels via GTP-binding proteins (G-proteins). Subthreshold concentrations of DAMGO (3 nM) significantly decreased mIPSC frequency with subthreshold concentrations of serotonin (3 nM) and this effect was completely blocked by pretreatment with N-ethylmaleimide (NEM), a PTX-sensitive G-protein inhibitor. In contrast, maximum doses of DAMGO (10 microM) did not further inhibit mIPSC frequency with maximum doses of serotonin (10 microM). In conclusion, activation of presynaptic mu-opioid and 5-HT(1A) receptors synergistically inhibited GABA release. These results suggest a cellular mechanism within PAG for the analgesic effectiveness of combined therapies using opioids in conjunction with classes of anti-depressants which increase synaptic serotonin levels.

Role of presynaptic 5-HT1A and 5-HT3 receptors in modulation of synaptic GABA transmission in dissociated rat basolateral amygdala neurons.

Serotonin (5-HT) is considered to play a significant role in anxiety-related behaviors in animals through actions on the amygdaloid complex. To evaluate this role from the point of neurotransmitter release regulation, nystatin-perforated patch recording was employed on mechanically dissociated basolateral amygdala neurons containing functional synaptic boutons. GABAAergic miniature inhibitory postsynaptic currents (mIPSCs) were pharmacologically separated. In subsets of neurons, 8-OH-DPAT (1 microM), a specific 5-HT1A agonist, continuously inhibited mIPSC frequency without effects on mIPSC amplitude. By comparison, mCPBG (1 microM), a specific 5-HT3 agonist, transiently facilitated mIPSC frequency without effects on mIPSC amplitude. Together these results suggest the presynaptic existence of both 5-HT receptor subtypes. In these neurons, application of 8-OH-DPAT and its subsequent removal still suppressed mCPBG-induced responses on mIPSCs. This suppression was not caused by a reduction of presynaptic 5-HT3 receptor affinities to mCPBG and was completely eliminated by pretreatment with N-ethylmaleimide, a pertussis toxin sensitive GTP-binding protein inhibitor. In the neurons exhibiting presynaptic modulation with mCPBG but not 8-OH-DPAT, such suppression by exposure to 8-OH-DPAT was not observed. In conclusion, activation of presynaptic 5-HT1A receptors inhibited mIPSC frequency and at the same time suppressed, via a G-protein-mediated mechanism, the transient facilitation of mIPSC frequency produced by activation of presynaptic 5-HT3 receptors.

Presynaptic modulation of synaptic γ-aminobutyric acid transmission by tandospirone in rat basolateral amygdala

Nystatin-perforated patch recordings were made from mechanically dissociated neurons (in which functional native presynaptic nerve terminals are preserved), isolated from the basolateral amygdala regions to investigate the effects of tandospirone on gamma-aminobutyric acidergic (GABAergic) inhibition. Two types of neurons, ovoid-shaped and pyramidal-shaped neurons, were obtained from the basolateral amygdala nuclei and the electrophysiological characteristics of these two types of neurons supported the morphological classification of these isolated neurons. From the ovoid-shaped neurons, bicuculline-sensitive GABA(A)ergic miniature inhibitory postsynaptic currents (miniature IPSC) were recorded in the presence of tetrodotoxin, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and DL-2-amino-5-phosphovaleric acid (DL-AP5). Tandospirone (10 microM) reversibly and continuously inhibited the GABAergic miniature synaptic events to 66.3+/-2.1% of control (P<0.01, n=17) without affecting the miniature IPSC amplitude (104.0+/-3.1% of control, n=17). The similar inhibition of miniature IPSC frequency was mimicked by a specific 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT, 1 microM), and the effects of tandospirone were prevented in the presence of a specific 5-HT1A receptor antagonist 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl] piperazine hydrobromide (NAN-190, 1 microM). Activation of 5-HT1A receptors by 8-OH-DPAT (1 microM) evoked no direct postsynaptic effects in enzyme-treated isolated basolateral amygdala neurons, suggesting that tandospirone acts at presynaptic 5-HT1A receptors. Furthermore, this presynaptic inhibition by tandospirone was prevented after treatment with a pertussis toxin-sensitive GTP-binding protein (G-protein) inhibitor, N-ethylmaleimide (at 3 microM for 5 min). In conclusion, in the basolateral amygdala nuclei, tandospirone activated presynaptic 5-HT1A receptors on the GABAergic nerve terminals projecting to ovoid-shaped neurons and inhibited synaptic GABA transmission via G-proteins.

ATP-sensitive and Ca2+-activated K+ channel activities in the rat locus coeruleus neurons during metabolic inhibition.

Locus coeruleus (LC) is the significant nucleus for consciousness and it is sensitive to metabolic inhibition. We investigated the effects of a metabolic inhibitor sodium cyanide (NaCN) on the rat dissociated LC neurons using nystatin-perforated patch recordings. Under voltage-clamp (VH=-40 mV), application of NaCN evoked outward currents composed of ATP-sensitive and Ca2+-dependent K+ channel currents (IKATP and IKCa2+). Onset of IKATP was faster than that of IKCa2+. Prolonged application of NaCN brought IKATP rundown but not IKCa2+ rundown. Okadaic acid prevented IKATP rundown, indicating that KATP channels are deactivated by dephosphorylation with protein phosphatase.