Sang Jeong Kim

TRPV1 in GABAergic Interneurons Mediates Neuropathic Mechanical Allodynia and Disinhibition of the Nociceptive Circuitry in the Spinal Cord

Neuropathic pain and allodynia may arise from sensitization of central circuits. We report a mechanism of disinhibition-based central sensitization resulting from long-term depression (LTD) of GABAergic interneurons as a consequence of TRPV1 activation in the spinal cord. Intrathecal administration of TRPV1 agonists led to mechanical allodynia that was not dependent on peripheral TRPV1 neurons. TRPV1 was functionally expressed in GABAergic spinal interneurons and activation of spinal TRPV1 resulted in LTD of excitatory inputs and a reduction of inhibitory signaling to spinothalamic tract (STT) projection neurons. Mechanical hypersensitivity after peripheral nerve injury was attenuated in TRPV1(-/-) mice but not in mice lacking TRPV1-expressing peripheral neurons. Mechanical pain was reversed by a spinally applied TRPV1 antagonist while avoiding the hyperthermic side effect of systemic treatment. Our results demonstrate that spinal TRPV1 plays a critical role as a synaptic regulator and suggest the utility of central nervous system-specific TRPV1 antagonists for treating neuropathic pain.

Ca2+-channel-dependent and -independent inhibition of exocytosis by extracellular ATP in voltage-clamped rat adrenal chromaffin cells

Abstract Membrane currents and capacitance were measured to examine the effects of extracellular ATP on exocytosis in voltage-clamped rat adrenal chromaffin cells. ATP reversibly inhibited Ca2+ current (ICa) and exocytosis. The dependency of exocytosis on ICa evoked by 1-s depolarizations was determined. However, inhibition of exocytosis was 2.6 times larger than that estimated from the reduction of ICa, implying the existence of a Ca2+-channel-independent pathway. This inhibition did not rely on a further reduction of the intracellular Ca2+ concentration spike. ATP reduced the rate of exocytosis induced by clamping the intracellular Ca2+ concentration. Pertussis toxin blocked the inhibitory effects of ATP on ICa and exocytosis. Although RB-2, a P2Y antagonist, blocked the inhibitory effect of ATP on ICa, RB-2 itself produced large increase or decrease in membrane capacitance. Adenosine inhibited ICa via a pertussis-toxin-sensitive pathway but did not significantly inhibit exocytosis. Our data show that extracellular ATP inhibits exocytosis via inhibition of ICa by activation of a pertussis-toxin-sensitive G-protein linked to P2Y receptors. Furthermore, our data strongly suggest that ATP activates another pathway, which is also G-protein dependent and accounts for the majority of the inhibitory effect of ATP on exocytosis.

Effect of stretch on calcium channel currents recorded from the antral circular myocytes of guinea-pig stomach.

The effect of membrane stretch on voltage-activated Ba2+ current (IBa) was studied in antral circular myocytes of guinea-pig using the wholecell patch-clamp technique. The changes in cell volume were elicited by superfusing the myocytes with anisosmotic solutions. Hyposmotic superfusate (202 mosmol/1) induced cell swelling and increased peak values of IBa at 0 mV (from − 406.6 ± 45.5 pA to − 547.5 ± 65.6 pA, mean ± SEM, n = 8) and hypero-smotic superfusate (350 mosmol/1) induced cell shrinkage and decreased peak values of IBa at 0 mV (to − 269.5 ± 39.1 pA, n = 8). Such changes were reversible and the extent of change was dependent on the osmolarity of superfusate. The values of normalized IBa at OmV were 1.43 ±0.04, 1.30 ±0.06, 1.23 ±0.04, 1.19 ±0.04, 1 and 0.68 ±0.06 at 202, 220, 245, 267, 290 and 350 mosmol/1, respectively (n = 8). IBa was almost completely blocked by nicardipine (5 µM) under hyposmotic conditions. The values of steady-state half-inactivation voltage ( − 37.7 ± 3.3 and − 36.5 ± 2.6 mV, under control and hyposmotic conditions, respectively) or the half-activation voltage ( − 13.6 ± 2.3 and − 13.9 ±1.9 mV) of IBa were not significantly changed (P > 0.05, n = 6). Cell membrane capacitance was slightly increased from 50.00 ± 2.86 pF to 50.22 ± 2.82 pF by a hyposmotic superfusate (P < 0.05, n = 6). It is suggested that cell swelling increases voltage-operated L-type calcium channel current and that such a property is related to the response of gastric smooth muscle to mechanical stimuli.

Lobule-specific membrane excitability of cerebellar Purkinje cells

Non‐technical summary  Cerebellar vermis consists of 10 lobules, and each lobule receives different sensory information. Afferent inputs are integrated in cerebellar Purkinje cells (PCs) which are the sole output of the cerebellar cortex. We show that intrinsic membrane properties are widely different between PCs in the spinocerebellum (lobules III–V) and vestibulocerebellum (lobule X).

Cortical astrocytes rewire somatosensory cortical circuits for peripheral neuropathic pain

Long-term treatments to ameliorate peripheral neuropathic pain that includes mechanical allodynia are limited. While glial activation and altered nociceptive transmission within the spinal cord are associated with the pathogenesis of mechanical allodynia, changes in cortical circuits also accompany peripheral nerve injury and may represent additional therapeutic targets. Dendritic spine plasticity in the S1 cortex appears within days following nerve injury; however, the underlying cellular mechanisms of this plasticity and whether it has a causal relationship to allodynia remain unsolved. Furthermore, it is not known whether glial activation occurs within the S1 cortex following injury or whether it contributes to this S1 synaptic plasticity. Using in vivo 2-photon imaging with genetic and pharmacological manipulations of murine models, we have shown that sciatic nerve ligation induces a re-emergence of immature metabotropic glutamate receptor 5 (mGluR5) signaling in S1 astroglia, which elicits spontaneous somatic Ca2+ transients, synaptogenic thrombospondin 1 (TSP-1) release, and synapse formation. This S1 astrocyte reactivation was evident only during the first week after injury and correlated with the temporal changes in S1 extracellular glutamate levels and dendritic spine turnover. Blocking the astrocytic mGluR5-signaling pathway suppressed mechanical allodynia, while activating this pathway in the absence of any peripheral injury induced long-lasting (>1 month) allodynia. We conclude that reawakened astrocytes are a key trigger for S1 circuit rewiring and that this contributes to neuropathic mechanical allodynia.

A Role of Canonical Transient Receptor Potential 5 Channel in Neuronal Differentiation from A2B5 Neural Progenitor Cells

Store-operated Ca2+ entry (SOCE) channels are the main pathway of Ca2+ entry in non-excitable cells such as neural progenitor cells (NPCs). However, the role of SOCE channels has not been defined in the neuronal differentiation from NPCs. Here, we show that canonical transient receptor potential channel (TRPC) as SOCE channel influences the induction of the neuronal differentiation of A2B5+ NPCs isolated from postnatal-12-day rat cerebrums. The amplitudes of SOCE were significantly higher in neural cells differentiated from proliferating A2B5+ NPCs and applications of SOCE blockers, 2-aminoethoxy-diphenylborane (2-APB), and ruthenium red (RR), inhibited their rise of SOCE. Among TRPC subtypes (TRPC1-7), marked expression of TRPC5 and TRPC6 with turned-off TRPC1 expression was observed in neuronal cells differentiated from proliferating A2B5+ NPCs. TRPC5 small interfering RNA (siRNA) blocked the neuronal differentiation from A2B5+ NPCs and reduced the rise of SOCE. In contrast, TRPC6 siRNA had no significant effect on the neuronal differentiation from A2B5+ NPCs. These results indicate that calcium regulation by TRPC5 would play a key role as a switch between proliferation and neuronal differentiation from NPCs.

Mechanosensitive activation of K+ channel via phospholipase C-induced depletion of phosphatidylinositol 4,5-bisphosphate in B lymphocytes

In various types of cells mechanical stimulation of the plasma membrane activates phospholipase C (PLC). However, the regulation of ion channels via mechanosensitive degradation of phosphatidylinositol 4,5‐bisphosphate (PIP2) is not known yet. The mouse B cells express large conductance background K+ channels (LKbg) that are inhibited by PIP2. In inside‐out patch clamp studies, the application of MgATP (1 mm) also inhibited LKbg due to the generation of PIP2 by phosphoinositide (PI)‐kinases. In the presence of MgATP, membrane stretch induced by negative pipette pressure activated LKbg, which was antagonized by PIP2 (> 1 μm) or higher concentration of MgATP (5 mm). The inhibition by PIP2 was partially reversible. However, the application of methyl‐β‐cyclodextrin, a cholesterol scavenger disrupting lipid rafts, induced the full recovery of LKbg activity and facilitated the activation by stretch. In cell‐attached patches, LKbg were activated by hypotonic swelling of B cells as well as by negative pressure. The mechano‐activation of LKbg was blocked by U73122, a PLC inhibitor. Neither actin depolymerization nor the inhibition of lipid phosphatase blocked the mechanical effects. Direct stimulation of PLC by m‐3M3FBS or by cross‐linking IgM‐type B cell receptors activated LKbg. Western blot analysis and confocal microscopy showed that the hypotonic swelling of WEHI‐231 induces tyrosine phosphorylation of PLCγ2 and PIP2 hydrolysis of plasma membrane. The time dependence of PIP2 hydrolysis and LKbg activation were similar. The presence of LKbg and their stretch sensitivity were also proven in fresh isolated mice splenic B cells. From the above results, we propose a novel mechanism of stretch‐dependent ion channel activation, namely, that the degradation of PIP2 caused by stretch‐activated PLC releases LKbg from the tonic inhibition by PIP2.

In vivo patch-clamp recording from locus coeruleus neurones in the rat brainstem

Abstract  Locus coeruleus (LC) neurones extend noradrenergic projections throughout the neuroaxis and are involved in homeostatic functions such as pain modulation, arousal and cardio‐respiratory control. To address the cellular mechanisms underlying pain modulation we have developed a patch‐clamp recording technique from LC neurones in anaesthetized rats. These recordings showed LC discharge in vivo to be driven by both spontaneous membrane potential oscillations and CNQX‐sensitive EPSCs opposed by bicuculine‐sensitive IPSCs. Hindlimb pinch evoked a biphasic action potential response underpinned by a slow monophasic excitatory current. This approach allows detailed characterisation of the synaptic and integrative mechanisms of LC responses to naturalistic stimulation.

Agonist‐induced internalization of mGluR1α is mediated by caveolin

Agonist‐induced internalization of metabotropic glutamate receptors (mGluRs) plays an important role in neuronal signaling. Although internalization of mGluRs has been reported to be mediated by clathrin‐dependent pathway, studies describing clathrin‐independent pathways are emerging. Here, we report that agonist‐induced internalization of mGluR1α is mediated by caveolin. We show that two caveolin‐binding motifs of mGluR1α interact with caveolin1/2. Using cell surface‐immunoprecipitation and total internal reflection fluorescence imaging, we found that agonist‐induced internalization of mGluR1α is regulated by caveolin‐binding motifs of the receptor in heterologous cells. Moreover, in the cerebellum, group I mGluR agonist dihydroxyphenylglycol increased the interaction of phosphorylated caveolin with mGluR1α. This interaction was blocked by methyl‐β‐cyclodextrin, known to disrupt caveolin/caveolae‐dependent signaling by cholesterol depletion. Methyl‐β‐cyclodextrin also blocked the agonist‐induced internalization of mGluR1α. Thus, these findings represent the evidence for agonist‐induced internalization of mGluR1α via caveolin and suggest that caveolin might play a role in synaptic metaplasticity by regulating internalization of mGluR1α in the cerebellum.

Impaired learning and memory in CD38 null mutant mice.

CD38 is an enzyme that catalyzes the formation of cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate, both of which are involved in the mobilization of Ca2+ from intracellular stores. Recently, CD38 has been shown to regulate oxytocin release from hypothalamic neurons. Importantly, CD38 mutations are associated with autism spectrum disorders (ASD) and CD38 knockout (CD38−/−) mice display ASD-like behavioral phenotypes including deficient parental behavior and poor social recognition memory. Although ASD and learning deficits commonly co-occur, the role of CD38 in learning and memory has not been investigated. We report that CD38−/− mice show deficits in various learning and memory tasks such as the Morris water maze, contextual fear conditioning, and the object recognition test. However, either long-term potentiation or long-term depression is not impaired in the hippocampus of CD38−/− mice. Our results provide convincing evidence that CD38−/− mice show deficits in various learning and memory tasks including spatial and non-spatial memory tasks. Our data demonstrate that CD38 is critical for regulating hippocampus-dependent learning and memory without modulating synaptic plasticity.