Shao Rui Chen

Signalling pathway of nitric oxide in synaptic GABA release in the rat paraventricular nucleus

In the paraventricular nucleus (PVN) of the hypothalamus, nitric oxide (NO) inhibits sympathetic outflow through increased GABA release. However, the signal transduction pathways involved in its action remain unclear. In the present study, we determined the role of cGMP, soluble guanylyl cyclase, and protein kinase G in the potentiating effect of NO on synaptic GABA release to spinally projecting PVN neurones. The PVN neurones were retrogradely labelled by a fluorescent tracer injected into the thoracic spinal cord of rats. Whole‐cell voltage‐clamp recordings were performed on labelled PVN neurones in the hypothalamic slice. Bath application of the NO donor, S‐nitroso‐N‐acetyl‐penicillamine (SNAP), reproducibly increased the frequency of miniature GABAergic inhibitory postsynaptic currents (mIPSCs) without changing the amplitude and the decay time constant. Neither replacement of Ca2+ with Co2+ nor application of Cd2+ to block the Ca2+ channel altered the effect of SNAP on mIPSCs. Also, the effect of SNAP on mIPSCs was not significantly affected by thapsigargin, a Ca2+‐ATPase inhibitor that depletes intracellular Ca2+ stores. Application of a membrane‐permeant cGMP analogue, pCPT–cGMP, mimicked the effect of SNAP on mIPSCs in the presence of a phosphodiesterase inhibitor, IBMX. Furthermore, both the soluble guanylyl cyclase inhibitor, ODQ, and the specific protein kinase G inhibitor, Rp pCPT cGMP, abolished the effect of SNAP on mIPSCs. Thus, these data provide substantial new information that NO potentiates GABAergic synaptic inputs to spinally projecting PVN neurones through a cGMP–protein kinase G pathway.

Opioid-Induced Long-Term Potentiation in the Spinal Cord Is a Presynaptic Event

Opioids remain the mainstay of treatment for severe pain, but the associated hyperalgesia and tolerance are significant impediments to achieving adequate pain relief with opioids. Here we show that in the spinal cord, brief application of the μ-opioid receptor agonist (D-Ala2,N-Me-Phe4,Gly-ol5)-enkephalin (DAMGO) at 1 μm, but not at 1–10 nm, caused an initial decrease followed by a large and long-lasting increase in the amplitude of monosynaptic EPSCs evoked from the dorsal root in ∼50% of lamina I and II neurons. However, postsynaptic dialysis of the G-protein inhibitor had no effect on DAMGO-induced initial inhibition and long-term potentiation (LTP) in either lamina I or II neurons. DAMGO-induced LTP was associated with an increase in the paired-pulse depression ratio. Furthermore, DAMGO application and washout induced an initial decrease followed by a persistent increase in the frequency of miniature EPSCs. Bath application, but not postsynaptic dialysis, of an NMDA receptor antagonist or a calcium chelator abolished DAMGO-induced LTP. Strikingly, ablation of TRPV1-expressing primary afferents not only eliminated DAMGO-induced LTP but also prolonged DAMGO-induced inhibition of the miniature and evoked EPSCs (i.e., long-term depression). Thus, our study strongly suggests that TRPV1-expressing primary afferents play a prominent role in opioid-induced presynaptic LTP, which challenges a previous report suggesting the postsynaptic nature of this opioid-induced LTP. This excitatory effect of opioids on primary afferents can counteract the inhibitory effect of opioids on synaptic transmission at the spinal level and is likely involved in opioid-induced hyperalgesia and tolerance.

Effect of the μ Opioid on Excitatory and Inhibitory Synaptic Inputs to Periaqueductal Gray-Projecting Neurons in the Amygdala

Opioids are potent analgesics, but the sites of their action and cellular mechanisms are not fully understood. The central nucleus of the amygdala (CeA) is important for opioid analgesia through the projection to the periaquaductal gray (PAG). In this study, we examined the effects of μ opioid receptor stimulation on inhibitory and excitatory synaptic inputs to PAG-projecting CeA neurons retrogradely labeled with a fluorescent tracer injected into the ventrolateral PAG of rats. Whole-cell voltage-clamp recordings were performed on labeled CeA neurons in brain slices. The specific μ opioid receptor agonist, [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO, 1 μM), significantly reduced the frequency of miniature inhibitory postsynaptic currents (mIPSCs) without altering the amplitude and decay constant of mIPSCs in 47.6% (10 of 21) of cells tested. DAMGO also significantly decreased the peak amplitude of evoked IPSCs in 69% (9 of 13) of cells examined. However, DAMGO did not significantly alter the frequency of miniature excitatory postsynaptic currents (EPSCs) and the amplitude of evoked EPSCs in 69% (9 of 13) and 83% (10 of 12) of labeled cells, respectively. The IPSCs were blocked by the GABAA receptor antagonist bicuculline, whereas the EPSCs were largely abolished by the non-N-methyl-d-aspartate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. The immunoreactivity of μ opioid receptors was colocalized with synaptophysin, a presynaptic marker, in close appositions to labeled CeA neurons. These results suggest that activation of μ opioid receptors on presynaptic terminals primarily attenuates GABAergic synaptic inputs to PAG-projecting neurons in the CeA.

Regulation of increased glutamatergic input to spinal dorsal horn neurons by mGluR5 in diabetic neuropathic pain.

J. Neurochem. (2010) 112, 162–172.

The glutamatergic nature of TRPV1‐expressing neurons in the spinal dorsal horn

The transient receptor potential vanilloid receptor 1 (TRPV1) is expressed on primary afferent terminals and spinal dorsal horn neurons. However, the neurochemical phenotypes and functions of TRPV1‐expressing post‐synaptic neurons in the spinal cord are not clear. In this study, we tested the hypothesis that TRPV1‐expressing dorsal horn neurons are glutamatergic. Immunocytochemical labeling revealed that TRPV1 and vesicular glutamate transporter‐2 were colocalized in dorsal horn neurons and their terminals in the rat spinal cord. Resiniferatoxin (RTX) treatment or dorsal rhizotomy ablated TRPV1‐expressing primary afferents but did not affect TRPV1‐ and vesicular glutamate transporter‐2‐expressing dorsal horn neurons. Capsaicin significantly increased the frequency of glutamatergic spontaneous excitatory post‐synaptic currents and miniature excitatory post‐synaptic currents in almost all the lamina II neurons tested in control rats. In RTX‐treated or dorsal rhizotomized rats, capsaicin still increased the frequency of spontaneous excitatory post‐synaptic currents and miniature excitatory post‐synaptic currents in the majority of neurons examined, and this effect was abolished by a TRPV1 blocker or by non‐NMDA receptor antagonist. In RTX‐treated or in dorsal rhizotomized rats, capsaicin also produced an inward current in a subpopulation of lamina II neurons. However, capsaicin had no effect on GABAergic and glycinergic spontaneous inhibitory post‐synaptic currents of lamina II neurons in RTX‐treated or dorsal rhizotomized rats. Collectively, our study provides new histological and functional evidence that TRPV1‐expressing dorsal horn neurons in the spinal cord are glutamatergic and that they mediate excitatory synaptic transmission. This finding is important to our understanding of the circuitry and phenotypes of intrinsic dorsal horn neurons in the spinal cord.

Adenosine inhibits paraventricular pre-sympathetic neurons through ATP-dependent potassium channels

J. Neurochem. (2010) 113, 530–542.

Increased C-Fiber Nociceptive Input Potentiates Inhibitory Glycinergic Transmission in the Spinal Dorsal Horn

Glycine is an important inhibitory neurotransmitter in the spinal cord, but it also acts as a coagonist at the glycine site of N-methyl-d-aspartate (NMDA) receptors to potentiate nociceptive transmission. However, little is known about how increased nociceptive inflow alters synaptic glycine release in the spinal dorsal horn and its functional significance. In this study, we performed whole-cell recordings in rat lamina II neurons to record glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs). The transient receptor potential vanilloid receptor 1 agonist capsaicin caused a prolonged increase in the frequency of sIPSCs in 17 of 25 (68%) neurons tested. The potentiating effect of capsaicin on sIPSCs was blocked by ionotropic glutamate receptor antagonists or tetrodotoxin in most lamina II neurons examined. In contrast, the P2X agonist αβ-methylene-ATP increased sIPSCs in only two of 16 (12.5%) neurons. The glutamate transporter inhibitor l-trans-pyrrolidine-2,4-dicarboxylic acid either increased or reduced the basal frequency of sIPSCs but did not significantly alter the potentiating effect of capsaicin on sIPSCs. Furthermore, the groups II and III metabotropic glutamate receptor antagonists had no significant effect on the capsaicin-induced increase in the sIPSC frequency. Although capsaicin reduced the amplitude of evoked excitatory postsynaptic currents at high stimulation currents, it did not change the ratio of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/NMDA currents. This study provides the important new information that increased nociceptive inflow augments synaptic glycine release to spinal dorsal horn neurons through endogenous glutamate release. Potentiation of inhibitory glycinergic tone by stimulation of nociceptive primary afferents may function as a negative feedback mechanism to attenuate nociceptive transmission at the spinal level.

Sustained Inhibition of Neurotransmitter Release from Nontransient Receptor Potential Vanilloid Type 1-Expressing Primary Afferents by μ-Opioid Receptor Activation-Enkephalin in the Spinal Cord

Removing transient receptor potential vanilloid type 1 (TRPV1)-expressing primary afferent neurons reduces presynaptic μ-opioid receptors but potentiates opioid analgesia. However, the sites and underlying cellular mechanisms for this paradoxical effect remain uncertain. In this study, we determined the presynaptic and postsynaptic effects of the μ-opioid receptor agonist [d-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO) using whole-cell patch-clamp recordings of lamina II neurons in rat spinal cord slices. Treatment with the ultrapotent TRPV1 agonist resiniferotoxin (RTX) eliminated TRPV1-expressing dorsal root ganglion neurons and their central terminals in the spinal dorsal horn and significantly reduced the basal amplitude of glutamatergic excitatory postsynaptic currents (EPSCs) evoked from primary afferents. Although RTX treatment did not significantly alter the concentration-response effect of DAMGO on evoked monosynaptic and polysynaptic EPSCs, it causes a profound long-lasting inhibitory effect of DAMGO on evoked EPSCs. Subsequent naloxone treatment did not reverse the prolonged inhibitory effect of DAMGO on evoked EPSCs. Furthermore, brief application of DAMGO produced a sustained inhibition of miniature EPSCs in RTX-treated rats. However, the concentration response and the duration of the effects of DAMGO on G protein-coupled inwardly rectifying K+ currents in lamina II neurons were not significantly different between vehicle- and RTX-treated groups. These data suggest that stimulation of μ-opioid receptors on non-TRPV1 afferent terminals causes extended inhibition of neurotransmitter release to spinal dorsal horn neurons. The differential effect of μ-opioid receptor agonists on different phenotypes of primary afferents provides a cellular basis to explain why the analgesic action of opioids on mechanonociception is prolonged when TRPV1-expressing primary afferents are removed.

Control of Glycinergic Input to Spinal Dorsal Horn Neurons by Distinct Muscarinic Receptor Subtypes Revealed Using Knockout Mice

Muscarinic acetylcholine receptors (mAChRs) play an important role in the tonic regulation of nociceptive transmission in the spinal cord. However, how mAChR subtypes contribute to the regulation of synaptic glycine release is unknown. To determine their role, glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in lamina II neurons by using whole-cell recordings in spinal cord slices of wild-type (WT) and mAChR subtype knockout (KO) mice. In WT mice, the mAChR agonist oxotremorine-M dose-dependently decreased the frequency of sIPSCs in most neurons, but it had variable effects in other neurons. In contrast, in M3-KO mice, oxotremorine-M consistently decreased the glycinergic sIPSC frequency in all neurons tested, and in M2/M4 double-KO mice, it always increased the sIPSC frequency. In M2/M4 double-KO mice, the potentiating effect of oxotremorine-M was attenuated by higher concentrations in some neurons through activation of GABAB receptors. In pertussis toxin-treated WT mice, oxotremorine-M also consistently increased the sIPSC frequency. In M2-KO and M4-KO mice, the effect of oxotremorine-M on sIPSCs was divergent because of the opposing functions of the M3 subtype and the M2 and M4 subtypes. This study demonstrates that stimulation of the M2 and M4 subtypes inhibits glycinergic inputs to spinal dorsal horn neurons of mice, whereas stimulation of the M3 subtype potentiates synaptic glycine release. Furthermore, GABAB receptors are involved in the feedback regulation of glycinergic synaptic transmission in the spinal cord. This study revealed distinct functions of mAChR subtypes in controlling glycinergic input to spinal dorsal horn neurons.

Protein kinase CK2 contributes to diminished small conductance Ca2+-activated K+ channel activity of hypothalamic pre-sympathetic neurons in hypertension.

Small conductance calcium‐activated K+ (SK) channels regulate neuronal excitability. However, little is known about changes in SK channel activity of pre‐sympathetic neurons in the hypothalamic paraventricular nucleus (PVN) in essential hypertension. SK channels, calmodulin, and casein kinase II (CK2) form a molecular complex. Because CK2 is up‐regulated in the PVN in spontaneously hypertensive rats (SHRs), we hypothesized that CK2 increases calmodulin phosphorylation and contributes to diminished SK channel activity in PVN pre‐sympathetic neurons in SHRs. Perforated whole‐cell recordings were performed on retrogradely labeled spinally projecting PVN neurons in Wistar‐Kyoto (WKY) rats and SHRs. Blocking SK channels with apamin significantly increased the firing rate of PVN neurons in WKY rats but not in SHRs. CK2 inhibition restored the stimulatory effect of apamin on the firing activity of PVN neurons in SHRs. Furthermore, apamin‐sensitive SK currents and depolarization‐induced medium after‐hyperpolarization potentials of PVN neurons were significantly larger in WKY rats than in SHRs. CK2 inhibition significantly increased the SK channel current and medium after‐depolarization potential of PVN neurons in SHRs. In addition, CK2‐mediated calmodulin phosphorylation level in the PVN was significantly higher in SHRs than in WKY rats. Although SK3 was detected in the PVN, its expression level did not differ significantly between SHRs and WKY rats. Our findings suggest that CK2‐mediated calmodulin phosphorylation is increased and contributes to diminished SK channel function of PVN pre‐sympathetic neurons in SHRs. This information advances our understanding of the mechanisms underlying hyperactivity of PVN pre‐sympathetic neurons and increased sympathetic vasomotor tone in hypertension.