Voltage-dependent calcium channel ß subunit-derived peptides reduce excitatory neurotransmission and arterial blood pressure.

AIMS: Voltage-dependent calcium channels (VDCCs) play an important role in various physiological functions in the nervous system and the cardiovascular system. In L-, N-, P/Q-, and R-type VDCCs, ß subunit assists the channels for membrane targeting and modulates channel properties. In this study, we investigated whether an inhibition of the ß subunit binding to α subunit, the pore-forming main subunit of VDCCs, have any effect on channel activation and physiological functions. MAIN METHODS: Peptides derived from the specific regions of ß subunit that bind to the α-interaction domain in I-II linker of α subunit were manufactured, presuming that the peptides interrupt α-ß subunit interaction in the channel complex. Then, they were tested on voltage-activated Ca2+ currents recorded in acutely isolated trigeminal ganglion (TG) neurons, excitatory postsynaptic currents (EPSCs) in the spinal dorsal horn neurons, and arterial blood pressure (BP) recorded from the rat femoral artery. KEY FINDINGS: When applied internally through patch pipettes, the peptides decreased the peak amplitudes of the voltage-activated Ca2+ currents. After fusing with HIV transactivator of transcription (TAT) sequence to penetrate cell membrane, the peptides significantly decreased the peak amplitudes of Ca2+ currents and the peak amplitudes of EPSCs upon the external application through bath solution. Furthermore, the TAT-fused peptides dose dependently reduced the rat BP when administered intravenously. SIGNIFICANCE: These data suggest that an interruption of α-ß subunit association in VDCC complex inhibits channel activation, thereby reducing VDCC-mediated physiological functions such as excitatory neurotransmission and arterial BP.

Endogenous TRPC channels mediate Ca2+ signals and trigeminal synaptic plasticity induced by mGluR5.

AIMS: Metabotropic glutamate receptor 5 (mGluR5), a member of group I mGluR, exerts its effect via elevation of intracellular Ca2+ level. We here characterized Ca2+ signals in the tsA201 cells transfected with mGluR5 and investigated the role of passages for mGluR5-induced Ca2+ signals in synaptic plasticity. MAIN METHODS: Using a genetically encoded Ca2+ indicator, GCamp2, Ca2+ signals were reliably induced by bath application of (S)-3,5-dihydroxyphenylglycine, the group I mGluR agonist, in the tsA201 cells transfected with mGluR5. Using whole-cell recordings in the substantia gelatinosa (SG) neurons of the spinal trigeminal subnucleus caudalis (Vc), excitatory postsynaptic currents were recorded by stimulating the trigeminal tract. KEY FINDINGS: Ca2+ signals were mediated by "classical" or "canonical" transient receptor potential (TRPC) channels, particularly TRPC1/3/4/6, but not TRPC5, naturally existing in the tsA201 cells. Interestingly, the induction of Ca2+ signals was independent of the phospholipase C signaling pathway; instead, it critically involves the cyclic adenosine diphosphate ribose/ryanodine receptor-dependent signaling pathway and only partially protein kinase C. On the other hand, both TRPC3 and TRPC4 mediated mGluR1/5-induced long-lasting potentiation of excitatory synaptic transmission from the trigeminal primary afferents to the SG neurons of the Vc. SIGNIFICANCE: This study demonstrates that endogenous TRPC channels contribute to mGluR5-induced Ca2+ signals in tsA201 cells and synaptic plasticity at excitatory synapses.

Postsynaptic N-type or P/Q-type calcium channels mediate long-term potentiation by group I metabotropic glutamate receptors in the trigeminal oralis.

AIMS: Both N-type and P/Q-type voltage-gated Ca2+ channels (VGCCs) are involved in the induction of long-term potentiation (LTP), the long-lasting increase of synaptic strength, in the central nervous system. To provide further information on the roles of N-type and P/Q-type VGCCs in the induction of LTP at excitatory synapses of trigeminal primary afferents in the spinal trigeminal subnucleus oralis (Vo), we investigated whether they contribute to the induction of LTP by activation of group I metabotropic glutamate receptors (mGluRs). MAIN METHODS: (S)-3,5-Dihydroxyphenylglycine (DHPG; 10µM for 5min), the group I mGluR agonist, was used to induce LTP of excitatory postsynaptic currents that were evoked in the Vo neurons by stimulating the trigeminal track. KEY FINDINGS: Weak blockade of the N-type or P/Q-type VGCCs by ω-conotoxin GVIA or ω-agatoxin IVA, respectively, which inhibited only 20-40% of Ca2+ currents recorded in isolated trigeminal ganglion neurons but had no effect on the basal excitatory synaptic transmission, completely blocked the induction of LTP. In contrast, stronger blockade of the channels, which inhibited >50% of Ca2+ currents and about 30% of basal synaptic transmission, resulted in the development of long-term depression (LTD), the long-lasting decrease of synaptic strength. Interestingly, the postsynaptic mechanism of DHPG-induced LTP, which was determined by paired-pulse ratio, disappeared when LTP was blocked, or LTD occurred, while a presynaptic mechanism still remained. SIGNIFICANCE: Our data suggest that postsynaptic N-type and P/Q-type VGCCs mediate the DHPG-induced LTP at the trigeminal afferent synapses in the Vo.

Orexin receptors mediate long-term depression of excitatory synaptic transmission in the spinal cord dorsal horn.

Neuropeptides orexin-A and -B are related to the regulation of sleep/wakefulness and feeding behaviors. Recently, the peptides have also been shown to yield antinociceptive effects in various pain models. However, it is not clear whether orexins are involved in forms of synaptic plasticity, such as long-term potentiation (LTP) and long-term depression (LTD), the increase and the decrease of synaptic efficacy, respectively. In the present study, we examined whether orexin receptor type 1 (OX1) and 2 (OX2) are involved in the induction or maintenance of LTD of excitatory synaptic transmission using transverse spinal cord slices of young rats. Repetitive electrical stimulation of Lissauer's tract zone at 2Hz for 5min (600 pulses), combined with a holding potential of -30mV, induced LTD of the amplitude of excitatory postsynaptic currents (EPSCs) which are evoked by the activation of primary afferent fibers. The maintenance of LTD was significantly prevented by bath application of SB674042 (1µM), an OX1 antagonist, or EMPA (1µM), an OX2 antagonist. In addition, LTD was dependent on the NMDA receptor, as the NMDA receptor antagonist D-AP5 blocked the maintenance of LTD. Our study suggests that orexins, via activation of both OX1 and OX2, play a significant role in the expression of NMDA-dependent LTD, thereby contributing to the spinal modulation of pain transmission.

Activated leukocyte cell adhesion molecule is involved in excitatory synaptic transmission and plasticity in the rat spinal dorsal horn.

Activated leukocyte cell adhesion molecule (ALCAM), a member of type I transmembrane immunoglobulin superfamily of cell adhesion molecule, is expressed in the surface membrane of various cell types including neurons. In the spinal cord dorsal horn (DH), the first gate for the sensory and pain transmission to the brain, the expression and function of ALCAM have not been known yet. Therefore, we here investigate the synaptic function of ALCAM in the substantia gelatinosa (lamina II) of the spinal DH, as well as its expression in the DH. Bath-application of ALCAM/Fc or CD6/Fc, the recombinant human IgG1-Fc chimeric proteins, specifically potentiated C-fiber-mediated excitatory synaptic transmission and predominantly increased spontaneous release of glutamate. In addition, the development of long-term potentiation, a form of synaptic plasticity, at excitatory synapses was significantly inhibited in the presence of the recombinant proteins. The functional roles of ALCAM in the spinal DH were further supported by immunohistochemical analysis; it showed that ALCAM intensely expressed through laminae I/II with the exception of lateral portion of the dorsal part of inner lamina II and distinctly co-localized with molecular markers of C-fibers, such as peptidergic calcitonin gene-related protein and transient receptor potential vanilloid type 1 and non-peptidergic isolectin B4. This study, for the first time, suggests the modulatory roles of ALCAM in the excitatory synaptic transmission and plasticity in the rat spinal DH.

Essential roles of mGluR1 and inhibitory synaptic transmission in NMDA-independent long-term potentiation in the spinal trigeminal interpolaris.

AIMS: Patterns of synaptic activity determine synaptic strengthening or weakening that is typically represented as long-term potentiation (LTP) and long-term depression (LTD), respectively. In the present study, we aim to test whether a conditioning stimulation of the spinal trigeminal subnucleus caudalis (Vc) induces LTP at excitatory synapses in the subnucleus interpolaris (Vi) and to characterize the LTP. MAIN METHODS: Generally, a presynaptic high-frequency stimulation (HFS) protocol can induce LTP at excitatory synapses in the brain, including the spinal cord. Therefore, LTP in the Vi was induced by the HFS (3 tetani at 100 Hz) of Vc in the horizontal brainstem slices. By pretreating slices with antagonists for NMDA receptors, metabotropic glutamate receptor subtype 1 or 5 (mGluR1 or 5), GABAA receptors, glycine receptors and Ca(2+) chelator, the LTP was characterized. KEY FINDINGS: The HFS reliably but slowly induced LTP of excitatory synaptic transmission in the Vi. This LTP was not dependent on NMDA receptor activation; however, it did require the activation of mGluR1, but not mGluR5, and an intracellular Ca(2+) rise. Interestingly, this LTP induction required inhibitory synaptic transmission mediated by GABAA and glycine receptors, and coincided with the slow development of LTD at GABAergic synapses. The GABAergic LTD was mediated by mGluR1 and the intracellular Ca(2+) rise. SIGNIFICANCE: These data suggest that the modulation of GABAergic synaptic transmission by conditioning synaptic activity contributes to the induction and expression of LTP at excitatory synapses in the Vi.