Teruyuki Fukushima

Facilitatory Actions of Serotonin Type 3 Receptors on GABAergic Inhibitory Synaptic Transmission in the Spinal Superficial Dorsal Horn

Analgesic effects of serotonin (5-hydroxytryptamine [5-HT]) type 3 (5-HT3) receptors may involve the release of gamma-aminobutyric acid (GABA) in the spinal dorsal horn. However, the precise synaptic mechanisms for 5-HT3 receptor-mediated spinal analgesia are not clear. In this study, we investigated whether GABAergic neurons in the superficial dorsal horn (SDH) express functional 5-HT3 receptors and how these 5-HT3 receptors affect GABAergic inhibitory synaptic transmission in the SDH, by using slice preparations from adult glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mice. Tight-seal whole cell recordings from GFP-positive and -negative neurons showed that 5-HT3 receptor-specific agonist 2-methyl-serotonin (2-Me-5-HT) induced inward currents in a substantial population of both GFP-positive and -negative neurons. Additionally, we confirmed expression of 5-HT3 receptors in both types of neurons by single-cell reverse transcription-polymerase chain reaction (RT-PCR) analysis. Further, GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs)-both those evoked by electrical stimulation and those occurring spontaneously in tetrodotoxin (i.e., miniature IPSCs [mIPSCs])-were recorded from GFP-negative neurons. 2-Me-5-HT increased the amplitude of the evoked IPSCs and the frequency of mIPSCs. The amplitude of mIPSCs was not affected by 2-Me-5-HT, suggesting that 5-HT augments GABAergic synaptic transmission via presynaptic mechanisms. The present observations indicate that 5-HT3 receptors are expressed on both somadendritic regions and presynaptic terminals of GABAergic neurons and regulate GABAA receptor-mediated inhibitory synaptic transmission in the SDH. Taken together, these results provide clues for the underlying mechanisms of the antinociceptive actions of 5-HT3 receptors in the spinal dorsal horn.

Presynaptic large-conductance calcium-activated potassium channels control synaptic transmission in the superficial dorsal horn of the mouse.

Large-conductance calcium-activated potassium channels (BK channels) have been suggested to play a substantial role in synaptic transmission in the spinal cord dorsal horn. In the present experiments, we attempted to clarify the physiological significance of BK channels in the modulation of synaptic transmission in the superficial dorsal horn where nociceptive information is processed. Spontaneously occurring excitatory postsynaptic currents (sEPSCs) were recorded from the neurons located in the superficial dorsal horn of a mouse spinal cord slice, and the effects of iberiotoxin, a BK channel blocker, on sEPSCs were analyzed. The frequency of sEPSCs was significantly higher in the peripheral nerve-ligated neuropathic mice than in the sham-operated control mice, but the amplitude of sEPSCs was equivalent between the two groups. Iberiotoxin increased the frequency of sEPSCs in the control mice to the same level as that in the neuropathic mice without affecting the amplitude of sEPSCs. In contrast, iberiotoxin did not show any significant effects on the sEPSCs in the neuropathic mice. These findings suggest that the BK channels that are located in presynaptic terminals control synaptic transmission in the superficial dorsal horn, and that functional downregulation of BK channels accompanies the neuropathic pain induced by peripheral nerve injury. This downregulation was confirmed by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of the BK channel alpha subunit. Taken together, our present results indicate that BK channels play crucial roles in the synaptic transmission of nociceptive information in the superficial dorsal horn.

Enhancement of synaptic transmission and nociceptive behaviour in HPC-1/syntaxin 1A knockout mice following peripheral nerve injury

Our previous analysis of HPC‐1/syntaxin 1A knockout (KO) mice indicated that HPC‐1/syntaxin 1A plays an important role in the synaptic plasticity of the hippocampus in vitro and learning behaviour in vivo. In order to gain further insights into the physiological functions of HPC‐1/syntaxin 1A, we studied the changes in the plasticity of synaptic transmission in the superficial dorsal horn of the spinal cord following a peripheral nerve injury in HPC‐1/syntaxin 1A KO and wild‐type (WT) mice. The von Frey filament test revealed that partial ligation of the sciatic nerve caused neuropathic pain in both WT and KO mice. However, KO mice showed significant enhancement of mechanical allodynia as compared with WT mice. Tight‐seal whole‐cell recordings were obtained from neurons in the superficial dorsal horn of the spinal cord slices. Electrical stimulus‐evoked excitatory postsynaptic currents (EPSCs), asynchronous EPSCs (aEPSCs) in the presence of strontium, and spontaneously occurring miniature EPSCs (mEPSCs) were analysed. Prior to peripheral nerve ligation, no significant differences were observed in the properties of evoked EPSCs, aEPSCs and mEPSCs in KO and WT mice. Seven−14 days after partial ligation, the amplitude of evoked EPSCs and the frequency of aEPSCs and mEPSCs in KO mice were significantly greater than those in WT mice; however, the amplitude of aEPSCs and mEPSCs remained unchanged in both groups. Enhanced allodynia behaviour and significant enhancement of excitatory synaptic transmission following peripheral nerve ligation in KO mice suggest that HPC‐1/syntaxin 1A might play a role in synaptic plasticity in the nociceptive pathway.

Physiological properties of enkephalin-containing neurons in the spinal dorsal horn visualized by expression of green fluorescent protein in BAC transgenic mice

BackgroundEnkephalins are endogenous opiates that are assumed to modulate nociceptive information by mediating synaptic transmission in the central nervous system, including the spinal dorsal horn.ResultsTo develop a new tool for the identification of in vitro enkephalinergic neurons and to analyze enkephalin promoter activity, we generated transgenic mice for a bacterial artificial chromosome (BAC). Enkephalinergic neurons from these mice expressed enhanced green fluorescent protein (eGFP) under the control of the preproenkephalin (PPE) gene (penk1) promoter. eGFP-positive neurons were distributed throughout the gray matter of the spinal cord, and were primarily observed in laminae I-II and V-VII, in a pattern similar to the distribution pattern of enkephalin-containing neurons. Double immunostaining analysis using anti-enkephalin and anti-eGFP antibodies showed that all eGFP-expressing neurons contained enkephalin. Incubation in the presence of forskolin, an activator of adenylate cyclase, increased the number of eGFP-positive neurons. These results indicate that eGFP expression is controlled by the penk1 promoter, which contains cyclic AMP-responsive elements. Sections obtained from sciatic nerve-ligated mice exhibited increased eGFP-positive neurons on the ipsilateral (nerve-ligated side) compared with the contralateral (non-ligated side). These data indicate that PPE expression is affected by peripheral nerve injury. Additionally, single-neuron RT-PCR analysis showed that several eGFP positive-neurons in laminae I-II expressed glutamate decarboxylase 67 mRNA and that some expressed serotonin type 3 receptors.ConclusionsThese results suggest that eGFP-positive neurons in laminae I-II coexpress enkephalin and γ-aminobutyric acid (GABA), and are activated by forskolin and in conditions of nerve injury. The penk1-eGFP BAC transgenic mouse contributes to the further characterization of enkephalinergic neurons in the transmission and modulation of nociceptive information.

Syntaxin 1A occludes GABAB receptor-induced inhibition of exocytosis downstream of Ca2+ entry in mouse hippocampal neurons

The mechanisms underlying gamma-amino butyric acid (GABA(B)) receptor-mediated inhibition of exocytosis have been characterized in a variety of synapses. Using patch-clamp recording methods, we attempted to clarify the intracellular mechanisms underlying presynaptic inhibition in autaptic synapses of isolated mouse hippocampal neurons. Baclofen, a selective GABA(B) receptor agonist, decreased the frequency of glutamatergic miniature excitatory postsynaptic currents (mEPSCs) without changing their amplitude in Ca(2+)-free extracellular solution, suggesting that baclofen inhibits exocytosis downstream of Ca(2+) entry. Syntaxin 1A is known to modulate exocytosis and suppress neuronal sprouting. Antisense oligonucleotide-mediated knockdown of syntaxin 1A increased the frequency of mEPSCs under Ca(2+)-free condition. Estimation of the number of functional release sites by staining with FM1-43 indicated that the increased frequency of mEPSCs was induced by facilitation of exocytosis at each site, rather than by an increased number of release sites due to neuronal sprouting. Baclofen reduced mEPSC frequency in syntaxin 1A-knockdown neurons to the same level as that in nonsense oligonucleotide transfected neurons under Ca(2+)-free condition. These results suggest that the GABA(B) receptor- and syntaxin 1A-induced inhibitions of exocytosis occlude one another and that the GABA(B) receptor shares a common intracellular pathway with syntaxin 1A in inhibiting transmitter release downstream of Ca(2+) entry.

Possible involvement of syntaxin 1A downregulation in the late phase of allodynia induced by peripheral nerve injury

Syntaxin 1A is a membrane protein playing an integral role in exocytosis and membrane trafficking. The superficial dorsal horn (SDH) of the spinal cord, where nociceptive synaptic transmission is modulated, is rich in this protein. We recently reported that peripheral nerve ligation-induced nociceptive responses are considerably enhanced in syntaxin 1A-knockout mice [Takasusuki T, Fujiwara T, Yamaguchi S, Fukushima T, Akagawa K, Hori Y (2007) Eur J Neurosci 26:2179-2187]. On the basis of this earlier finding, we hypothesized that syntaxin 1A is involved in peripheral nerve injury-induced nociceptive plasticity. In this study, we examined this hypothesis by using nociceptive behavioral studies and tight-seal whole-cell recordings from neurons in the SDH of adult mouse spinal cord slices. Partial sciatic nerve ligation (PSNL) in adult male Institute of Cancer Research (ICR) mice increased the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs). The amplitude of the mEPSCs did not exhibit any changes, suggesting that peripheral nerve injury is associated with increased synaptic release of excitatory neurotransmitters. Western blot and real-time quantitative reverse transcription-polymerase chain reaction analyses revealed that PSNL gradually decreased the expression level of syntaxin 1A in the spinal SDH. This downregulation of syntaxin 1A took several days to develop, whereas behavioral allodynia developed within one day after PSNL. Syntaxin 1A knockdown by intrathecal injection of an antisense oligodeoxynucleotide against the syntaxin 1A gene led to the gradual development of allodynia. These results indicate a possible involvement of syntaxin 1A downregulation in the late maintenance phase of peripheral nerve injury-induced allodynia. In addition, syntaxin 1A knockdown by ribonucleic acid interference enhanced the axonal elongation and sprouting of spinal dorsal horn neurons in culture, suggesting that PSNL-induced syntaxin 1A downregulation may result in the rearrangement of the synaptic connections between neurons in the spinal dorsal horn. Taken together, it is possible to conclude that syntaxin 1A might be involved in spinal nociceptive plasticity induced by peripheral nerve injury.

Effects of pregabalin on spinal d-serine content and NMDA receptor-mediated synaptic transmission in mice with neuropathic pain

Pregabalin (PGB) is a chemical derivative of the inhibitory neurotransmitter γ-aminobutyric acid, and is successfully used for the treatment of neuropathic pain. Substantial evidence suggests that d-serine, an endogenous co-agonist at the strychnine-insensitive glycine site of the NMDA receptor, counteracts the antinociceptive actions of PGB at the level of the spinal cord. In the present study, we examined the impact of PGB treatment on spinal d-serine content and NMDA receptor-mediated synaptic transmission in the superficial dorsal horn of peripheral nerve-ligated neuropathic mice. Mechanical allodynia was assessed using von Frey filaments. On post-surgical day 9 (after 5days of treatment with PGB [50mg/kg] or saline vehicle), the lumbar spinal cord was removed, homogenized, and ultrafiltrated. Supernatant samples were treated with Marfeys reagent and analyzed with liquid chromatography-mass spectrometry to measure d-serine content. In the electrophysiological experiments, tight-seal whole-cell recording was performed on neurons in the superficial dorsal horn of spinal cord slices. Partial sciatic nerve ligation increased spinal d-serine content, increased the NMDA/non-NMDA ratio of EPSC amplitudes, and slowed the decay phase of the NMDA component of EPSCs (NMDA-EPSCs). PGB treatment attenuated mechanical allodynia and reduced spinal d-serine content, decreased the NMDA/non-NMDA ratio, and shortened the decay time of NMDA-EPSCs. Furthermore, bath-applied d-serine attenuated the effects of PGB treatment. Although the precise mechanism for the effect of PGB on d-serine metabolism and abundance is unknown, the antinociceptive action of PGB likely involves the reduction of spinal d-serine content and subsequent attenuation of NMDA receptor-mediated synaptic transmission in the superficial dorsal horn.