Dong-ho Youn

Bone Marrow‐Derived Mesenchymal Stem Cells Promote Neuronal Networks with Functional Synaptic Transmission After Transplantation into Mice with Neurodegeneration

Recent studies have shown that bone marrow‐derived MSCs (BM‐MSCs) improve neurological deficits when transplanted into animal models of neurological disorders. However, the precise mechanism by which this occurs remains unknown. Herein we demonstrate that BM‐MSCs are able to promote neuronal networks with functional synaptic transmission after transplantation into Niemann‐Pick disease type C (NP‐C) mouse cerebellum. To address the mechanism by which this occurs, we used gene microarray, whole‐cell patch‐clamp recordings, and immunohistochemistry to evaluate expression of neurotransmitter receptors on Purkinje neurons in the NP‐C cerebellum. Gene microarray analysis revealed upregulation of genes involved in both excitatory and inhibitory neurotransmission encoding subunits of the ionotropic glutamate receptors (α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid, AMPA) GluR4 and GABAA receptor β2. We also demonstrated that BM‐MSCs, when originated by fusion‐like events with existing Purkinje neurons, develop into electrically active Purkinje neurons with functional synaptic formation. This study provides the first in vivo evidence that upregulation of neurotransmitter receptors may contribute to synapse formation via cell fusion‐like processes after BM‐MSC transplantation into mice with neurodegenerative disease.

Localized Calcineurin Confers Ca2+-Dependent Inactivation on Neuronal L-Type Ca2+ Channels

Excitation-driven entry of Ca2+ through L-type voltage-gated Ca2+ channels controls gene expression in neurons and a variety of fundamental activities in other kinds of excitable cells. The probability of opening of CaV1.2 L-type channels is subject to pronounced enhancement by cAMP-dependent protein kinase (PKA), which is scaffolded to CaV1.2 channels by A-kinase anchoring proteins (AKAPs). CaV1.2 channels also undergo negative autoregulation via Ca2+-dependent inactivation (CDI), which strongly limits Ca2+ entry. An abundance of evidence indicates that CDI relies upon binding of Ca2+/calmodulin (CaM) to an isoleucine–glutamine motif in the carboxy tail of CaV1.2 L-type channels, a molecular mechanism seemingly unrelated to phosphorylation-mediated channel enhancement. But our work reveals, in cultured hippocampal neurons and a heterologous expression system, that the Ca2+/CaM-activated phosphatase calcineurin (CaN) is scaffolded to CaV1.2 channels by the neuronal anchoring protein AKAP79/150, and that overexpression of an AKAP79/150 mutant incapable of binding CaN (ΔPIX; CaN-binding PXIXIT motif deleted) impedes CDI. Interventions that suppress CaN activity—mutation in its catalytic site, antagonism with cyclosporine A or FK506, or intracellular perfusion with a peptide mimicking the sequence of the phosphatases autoinhibitory domain—interfere with normal CDI. In cultured hippocampal neurons from a ΔPIX knock-in mouse, CDI is absent. Results of experiments with the adenylyl cyclase stimulator forskolin and with the PKA inhibitor PKI suggest that Ca2+/CaM-activated CaN promotes CDI by reversing channel enhancement effectuated by kinases such as PKA. Hence, our investigation of AKAP79/150-anchored CaN reconciles the CaM-based model of CDI with an earlier, seemingly contradictory model based on dephosphorylation signaling.

Pharmacological analysis of excitatory and inhibitory synaptic transmission in horizontal brainstem slices preserving three subnuclei of spinal trigeminal nucleus.

Spinal trigeminal nucleus (Vsp) consists of three subnuclei: oralis (Vo), interpolaris (Vi) and caudalis (Vc). Previous anatomical studies using antero-/retro-grade tracers have suggested that intersubnuclear ascending/descending synaptic transmissions exist between subnuclei. However, pharmacological properties of the intersubnuclear synaptic transmission have not been studied yet. Since three subnuclei are located in Vsp along rostro-caudal axis, it will be necessary to prepare horizontal brainstem slices to perform pharmacological analysis of the intersubnuclear synaptic transmission. We here show horizontal brainstem slices retaining three subnuclei, and that, using blind whole-cell recordings in the slices, synaptic transmission may be abundantly retained between subnuclei in the horizontal slices, except for the transmission from Vo to Vc. Finally, pharmacological analysis shows that excitatory and inhibitory synaptic responses, respectively, are mediated by AMPA and NMDA receptors and by GABA(A) and glycine receptors, with a differential contribution to the synaptic responses between subnuclei. We therefore conclude that horizontal brainstem slices will be a useful preparation for studies on intersubnuclear synaptic transmission, modulation and plasticity between subnuclei, as well as, further, other brainstem nuclei.

Signal Transduction Mechanisms Underlying Group I mGluR-mediated Increase in Frequency and Amplitude of Spontaneous EPSCs in the Spinal Trigeminal Subnucleus Oralis of the Rat

Group I mGluRs (mGluR1 and 5) pre- and/or postsynaptically regulate synaptic transmission at glutamatergic synapses. By recording spontaneous EPSCs (sEPSCs) in the spinal trigeminal subnucleus oralis (Vo), we here investigated the regulation of glutamatergic transmission through the activation of group I mGluRs. Bath-applied DHPG (10 μM/5 min), activating the group I mGluRs, increased sEPSCs both in frequency and amplitude; particularly, the increased amplitude was long-lasting. The DHPG-induced increases of sEPSC frequency and amplitude were not NMDA receptor-dependent. The DHPG-induced increase in the frequency of sEPSCs, the presynaptic effect being further confirmed by the DHPG effect on paired-pulse ratio of trigeminal tract-evoked EPSCs, an index of presynaptic modulation, was significantly but partially reduced by blockades of voltage-dependent sodium channel, mGluR1 or mGluR5. Interestingly, PKC inhibition markedly enhanced the DHPG-induced increase of sEPSC frequency, which was mainly accomplished through mGluR1, indicating an inhibitory role of PKC. In contrast, the DHPG-induced increase of sEPSC amplitude was not affected by mGluR1 or mGluR5 antagonists although the long-lasting property of the increase was disappeared; however, the increase was completely inhibited by blocking both mGluR1 and mGluR5. Further study of signal transduction mechanisms revealed that PLC and CaMKII mediated the increases of sEPSC in both frequency and amplitude by DHPG, while IP3 receptor, NO and ERK only that of amplitude during DHPG application. Altogether, these results indicate that the activation of group I mGluRs and their signal transduction pathways differentially regulate glutamate release and synaptic responses in Vo, thereby contributing to the processing of somatosensory signals from orofacial region.

Ion Channel Modulation in Spinal/Trigeminal Synaptic Plasticity

The sensory experience of pain provides an early warning sign to protect the body from tissue injury. Although pain is a straightforward symptom found in virtually every field of medicine, the cellular and molecular bases underlying the sensation of pain are complex and involve diverse mechanisms in a variety of areas of the brain and spinal cord. In 1965, Melzack and Wall proposed the gate control theory of pain [1], which posited the role of the dorsal horn (DH) of the spinal cord as a site for gating and brain control of pain. In the decades since then, research on pain mechanisms in the spinal DH has exploded, as has research in the spinal trigeminal nucleus (Vsp) for orofacial and head pain. The spinal DH has a laminated structure, consisting of superficial (laminae I and II) and deep (laminae III–VI) layers. The Vsp is, in a rostrocaudal sense, divided into oralis, interpolaris, and caudalis subnuclei. The caudalis of Vsp has particularly captured the attention of orofacial pain researchers, as this area is analogous to the DH of the spinal cord. Neurons in the spinal DH and Vsp form synapses with primary afferent fibers from peripheral and trigeminal ganglia, descending fibers from higher brain areas, and axonal fibers arising from other local neurons. The neurons of the spinal DH and Vsp employ a wide variety of ligand- and voltage-gated ion channels to support synaptic transmission, neuronal excitability, and proper relay of sensory and nociceptive information. Short-term and long-term modulation of the properties of these ion channels provide mechanisms for neural plasticity and changes in gene expression that can in turn lead to structural modification of neurons in pain pathways of the spinal DH and Vsp. The modulation of ion channels includes changes in their phosphorylation/dephosphorylation state, the composition of subunits contributing to channel formation, and interactions with other signaling molecules and scaffolding proteins that target modulators to channels. In this special issue, we focus on the roles of ion channel modulation in spinal/trigeminal mechanisms of neural plasticity that contribute to chronic pain. Advances in understanding mechanisms of ion channel modulation are expected to lead to improved approaches for management of chronic pain. The first paper “Ionotropic glutamate receptors and voltage-gated Ca2+ channels in long-term potentiation of spinal dorsal horn synapses and pain hypersensitivity” in this issue introduces the anatomical and synaptic organization of the spinal DH. Although the anatomical location of the spinal DH is easily identified in transverse or parasagittal sections, identifying specific neuronal types and synaptic circuitry is challenging for two reasons. The first dilemma is that neurons in this area are not arranged in a precisely-organized structure; instead, neurons of any given functional subtype are scattered in a seemingly random way within the DH. Determining the pattern of synaptic connections between functional neuronal subtypes is as a consequence exceedingly difficult. The second challenge is that DH neurons are multimodal, meaning that any given subpopulation of DH neurons is involved in conveying more than one form of sensory information. Among the sensory modalities supported by DH neurons are mechanical touch, pinch, thermal heat, and cold. In this issues first paper, the authors synthesize findings in the recent literature to present an integrated model of the synaptic organization that supports pain processing in the spinal DH. The model also incorporates the synaptic circuitry suggested in the gate control theory of pain [1]. The paper proceeds to discuss recent published work regarding the contributions in spinal DH of ionotropic glutamate receptors (ion channels) and voltage-gated Ca2+ channels (VGCCs) to both long-term potentiation (LTP), an increase in the strength of synaptic transmission, and/or pain hypersensitivity. Consideration of the subtypes in both of these groups of ion channels distinguishes the differential contributions made by each type of ion channel to LTP and pain. Next, A-R. Park et al. “Dual effect of exogenous nitric oxide on neuronal excitability in rat substantia gelatinosa neurons” report a remarkable effect of nitric oxide on the excitability of substantia gelatinosa (SG) neurons located in lamina II of the spinal DH. The directionality of the effect of nitric oxide is concentration-dependent: 10 μM sodium nitroprusside, a nitric oxide donor, causes depolarization of neurons, but 1 mM causes hyperpolarization. Both effects are mediated by soluble guanylyl cyclases. However, the hyperpolarizing effect of nitric oxide involves activation of various types of K+ channels, while the depolarizing effect involves activation of certain types of Ca2+ channels. These findings may correlate with the complex effect of nitric oxide on pain behaviors, that is, a concentration-dependent switch between analgesic and hyperalgesic actions of nitric oxide. T. T. H. Nguyen et al. “Activation of glycine and extrasynaptic GABAA receptors by taurine on the substantia gelatinosa neurons of the trigeminal subnucleus caudalis” report an effect of the free amino acid taurine, present at a high concentration in the brain, on the excitability of SG neurons located in the caudalis of Vsp. These authors report that taurine action on SG neurons of Vsp is mediated by glycine and GABAA receptors, but owing to the high concentration of chloride ions they employed in their recording pipettes, taurine application caused neuronal depolarization. With a lower, physiological level of internal chloride, taurine will exert an inhibitory action on neuronal excitability, which predicts an antinociceptive action of taurine on orofacial pain behaviors. Kwi-H. Choi et al. “Presynaptic glycine receptors increase GABAergic neurotransmission in rat periaqueductal gray neurons” report that the periaqueductal gray (PAG) plays a role in the regulation of pain transmission via a descending inhibitory pathway to the spinal DH, thereby participating in the system postulated in the gate control theory. In this study, mechanically dissociated PAG neurons were voltage-clamped and spontaneous excitatory postsynaptic currents (EPSCs) were recorded. The axonal terminals, but not cell bodies, of presynaptic neurons remained attached to the neurons recorded, ruling out any contribution to the experimental results from presynaptic cell bodies. Using this method, the authors report facilitation by glycine of glutamate release, an effect that is mediated by glycine receptors and by voltage-gated Na+ and Ca2+ channels. Facilitation of glutamate release by glycine may be due to glycine receptor-mediated depolarization of presynaptic terminals, an action which relies upon the high concentration of chloride ions found within the presynaptic terminals. Because facilitation of excitatory, glutamatergic synaptic input to PAG neurons is expected to increase activity in the descending inhibitory pathway from PAG to the spinal DH, the results of this study suggest glycine-induced facilitation of PAG output could suppress the sensation of pain. In the final paper in this special issue, E. V. Khomula et al. “Nociceptive neurons differentially express fast and slow T-type Ca2+ currents in different types of diabetes neuropathy” report that two different kinds of T-type Ca2+ currents are present in isolectin B4 (IB4)-positive neurons of dorsal root ganglia (DRG). These neurons are small-diameter DRG neurons and respond to capsaicin application, suggesting that they are nonpeptidergic, C-type nociceptive neurons that are involved in the sensation of thermal pain. Based on measurements of the inactivation time constant (τ0.5) for T-type Ca2+ currents, IB4-positive neurons could be divided into two groups: ~70% of IB4-positive neurons exhibited fast-inactivating T-type current (τ0.5 50 ms). Among T-type Ca2+ channel isoforms, the CaV3.2 isoform is more sensitive to block by Ni2+, a property exploited by these authors to test the relative contribution of this isoform to fast or slow T-type currents. The authors found that fast-inactivating T-currents are more sensitive to Ni+ than are the slow-inactivating T-currents, suggesting that fast-inactivating T-currents are carried by CaV3.2 channels. The work was extended to investigate the role of fast- and slow-inactivating T-currents in hyperalgesia, using streptozotocin-induced diabetic rats that displayed neuropathic hyperalgesia. Whereas 30% of the IB4-positive DRG neurons obtained from control animals exhibited slow-inactivating T-current, none of the IB4-positive neurons from the hyperalgesic rats possessed slow-inactivating T-type current; instead, IB4-positive neurons from hyperalgesic rats possessed exclusively fast-inactivating T-current and at increased density. Moreover, a depolarizing shift of steady-state inactivation of fast T-type current was found in IB4-positive neurons from hyperalgesic rats. Thus, this study suggests that a change in the functional properties of ion channels may cause a pathological state such as neuropathic pain. This special issue highlights the importance of ion channel modulation in neuronal excitability, synaptic transmission, and synaptic plasticity of spinal DH and Vsp neurons, and it provides examples of ion channel modulation by chemicals and etiological factors. We hope that this issue will inspire further studies regarding various aspects of ion channel modulation in the spinal/trigeminal areas and other brain areas involved in pain transmission, thereby revealing new targets for pain treatment.

Ionotropic Glutamate Receptors and Voltage-Gated Ca2+ Channels in Long-Term Potentiation of Spinal Dorsal Horn Synapses and Pain Hypersensitivity

Over the last twenty years of research on cellular mechanisms of pain hypersensitivity, long-term potentiation (LTP) of synaptic transmission in the spinal cord dorsal horn (DH) has emerged as an important contributor to pain pathology. Mechanisms that underlie LTP of spinal DH neurons include changes in the numbers, activity, and properties of ionotropic glutamate receptors (AMPA and NMDA receptors) and of voltage-gated Ca2+ channels. Here, we review the roles and mechanisms of these channels in the induction and expression of spinal DH LTP, and we present this within the framework of the anatomical organization and synaptic circuitry of the spinal DH. Moreover, we compare synaptic plasticity in the spinal DH with classical LTP described for hippocampal synapses.

GABAA receptor-mediated tonic currents in substantia gelatinosa neurons of rat spinal trigeminal nucleus pars caudalis

In the present study, we describe GABAA receptor-mediated tonic inhibitory currents in the substantia gelatinosa (SG) region of rat spinal trigeminal nucleus pars caudalis (Vc). The GABA(A) receptor-mediated tonic currents were identified by bath-application of the GABAA receptor antagonists, picrotoxin (1mM), SR95531 (100microM) and bicuculline (100microM). All three antagonists completely blocked outward spontaneous (phasic) inhibitory postsynaptic currents, but only picrotoxin and bicuculline induced a significant (>5pA) inward shift of holding currents at a holding potential (Vh) of 0mV in 60-70% of SG neurons, revealing the existence of tonic outward currents. The tonic currents were resistant to further the blockades of glycine receptors or those in addition to glutamate receptors and voltage-dependent sodium channels. An acute bath-application of THDOC (0.1microM), the stress-related neurosteroid, did enhance tonic currents, but only in a small population of SG neurons. In addition, slices incubated with THDOC for 30min increased the probability of neurons with significant tonic currents. The GABAergic tonic inhibition demonstrated in this study may play a significant role in the sensory processing system of the Vc.

The relaxing effect of Poncirus fructus and its flavonoid content on porcine coronary artery

Coronary artery disease is a common occurrence in human, and causes enormous social cost. Poncirus fructus (PF), the dried immature fruits of Poncirus trifoliata Rafinesquem, is used in the treatment of womb contraction and dyspepsia, as a prokinetic, and in improving blood circulation. This study was performed to investigate the effects of PF and some of its flavonoids components on the coronary from the pig. The arterial ring was suspended by a pair of stainless steel stirrups in an organ bath. The end of the upper stirrup was connected to an isometric force transducer. A dose-dependent induction of relaxation was observed by both water and 70% ethanol extracts of PF in the porcine coronary artery precontracted with U46619 (100 nM), a stable analogue of the potent vasoconstrictor thromboxane A2. The 70% ethanol extract showed more efficacy than the water extract. Pretreatment of the artery with L-NAME (100 µM), a nitric oxide synthase inhibitor, resulted in a significant reduction in the relaxation induced by PF extract. In addition, ODQ (10 µM), a soluble guanylate cyclase inhibitor, also significantly reduced the effects of PF extracts. Hesperidin, a flavonoid present in PF, induced very weak relaxation of the porcine coronary artery at a high concentration (100 µM), while its aglycone, hesperetin, demonstrated a dose-dependent relaxation. In conclusion, PF extracts induced relaxation in the porcine coronary artery, partially through the nitric oxide-cGMP pathway, and the aglycones of flavonoids might be also involved in the relaxation of the same artery.

Long-term potentiation by activation of group I metabotropic glutamate receptors at excitatory synapses in the spinal trigeminal subnucleus oralis.

In this study, I examined if activation of group I metabotropic glutamate receptors (mGluRs; mGluR1 and 5) induces long-term potentiation (LTP) at excitatory synapses in the ascending pathway from the spinal trigeminal subnuclei caudalis (Vc) to oralis (Vo), in which group I mGluRs are strongly expressed. As a result, the activation of group I mGluRs produced an initial short-lasting depression and subsequently a delayed type of long-term potentiation (LTP) of excitatory synaptic transmission. Analyses of paired pulse ratio and coefficient of variation indicated that the initial short-lasting depression was induced presynaptically, whereas LTP was expressed postsynaptically. In addition, the short-lasting depression and LTP were mostly mediated by mGluR1, and only partially by mGluR5 and the N-methyl-d-aspartate receptor. Thus, this study suggests that group I mGluRs play an important role in the expression of LTP in the Vc-to-Vo pathway.

Orexin-A modulates excitatory synaptic transmission and neuronal excitability in the spinal cord substantia gelatinosa.

Although intrathecal orexin-A has been known to be antinociceptive in various pain models, the role of orexin-A in antinociception is not well characterized. In the present study, we examined whether orexin-A modulates primary afferent fiber-mediated or spontaneous excitatory synaptic transmission using transverse spinal cord slices with attached dorsal root. Bath-application of orexin-A (100nM) reduced the amplitude of excitatory postsynaptic currents (EPSCs) evoked by electrical stimulation of Aδ- or C-primary afferent fibers. The magnitude of reduction was much larger for EPSCs evoked by polysynaptic C-fibers than polysynaptic Aδ-fibers, whereas it was similar in EPSCs evoked by monosynaptic Aδ- or C-fibers. SB674042, an orexin-1 receptor antagonist, but not EMPA, an orexin-2 receptor antagonist, significantly inhibited the orexin-A-induced reduction in EPSC amplitude from mono- or polysynaptic Aδ-fibers, as well as from mono- or polysynaptic C-fibers. Furthermore, orexin-A significantly increased the frequency of spontaneous EPSCs but not the amplitude. This increase was almost completely blocked by both SB674042 and EMPA. On the other hand, orexin-A produced membrane oscillations and inward currents in the SG neurons that were partially or completely inhibited by SB674042 or EMPA, respectively. Thus, this study suggests that the spinal actions of orexin-A underlie orexin-A-induced antinociceptive effects via different subtypes of orexin receptors.