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Hypertension is a major adverse effect of calcineurin inhibitors, such as tacrolimus (FK506) and cyclosporine, used clinically as immunosuppressants. Calcineurin inhibitor-induced hypertension (CIH) is linked to augmented sympathetic output from the hypothalamic paraventricular nucleus (PVN). GluA2-lacking, Ca 2+ -permeable AMPA receptors (CP-AMPARs) are a key feature of glutamatergic synaptic plasticity, yet their role in CIH remains elusive. Here, we found that systemic administration of FK506 in rats significantly increased serine phosphorylation of GluA1 and GluA2 in PVN synaptosomes. Strikingly, FK506 treatment reduced GluA1/GluA2 heteromers in both synaptosomes and endoplasmic reticulum-enriched fractions from the PVN. Blocking CP-AMPARs with IEM-1460 induced a larger reduction of AMPAR-mediated excitatory postsynaptic current (AMPAR-EPSC) amplitudes in retrogradely labelled, spinally projecting PVN neurons in FK506-treated rats than in vehicle-treated rats. Furthermore, FK506 treatment shifted the current-voltage relationship of AMPAR-EPSCs from linear to inward rectification in labelled PVN neurons. FK506 treatment profoundly enhanced physical interactions of α2δ-1 with GluA1 and GluA2 in the PVN. Inhibiting α2δ-1 with gabapentin, α2δ-1 genetic knockout, or disrupting α2δ-1-AMPAR interactions with an α2δ-1 C terminus peptide restored GluA1/GluA2 heteromers in the PVN and diminished inward rectification of AMPAR-EPSCs in labelled PVN neurons induced by FK506 treatment. Additionally, microinjection of IEM-1460 or α2δ-1 C terminus peptide into the PVN reduced renal sympathetic nerve discharges and arterial blood pressure elevated in FK506-treated rats but not in vehicle-treated rats. Thus, calcineurin in the hypothalamus constitutively regulates AMPAR subunit composition and phenotypes by controlling GluA1/GluA2 interactions with α2δ-1. Synaptic CP-AMPARs in PVN presympathetic neurons contribute to augmented sympathetic outflow in CIH. KEY POINTS: Systemic treatment with the calcineurin inhibitor increases serine phosphorylation of synaptic GluA1 and GluA2 in the PVN. Calcineurin inhibition enhances the prevalence of postsynaptic Ca 2+ -permeable AMPARs in PVN presympathetic neurons. Calcineurin inhibition potentiates α2δ-1 interactions with GluA1 and GluA2, disrupting intracellular assembly of GluA1/GluA2 heterotetramers in the PVN. Blocking Ca 2+ -permeable AMPARs or α2δ-1-AMPAR interactions in the PVN attenuates sympathetic outflow augmented by the calcineurin inhibitor., (© 2024 The Authors. The Journal of Physiology © 2024 The Physiological Society.)

Volume-regulated anion channels (VRACs) are critically involved in regulating cell volume, and leucine-rich repeat-containing protein 8A (LRRC8A, SWELL1) is an obligatory subunit of VRACs. Cell swelling occurs early after brain ischemia, but it is unclear whether neuronal LRRC8a contributes to ischemia-induced glutamate release and brain injury. We found that Lrrc8a conditional knockout (Lrrc8a-cKO) mice produced by crossing Nestin Cre+/- with Lrrc8a flox+/+ mice died 7-8 weeks of age, indicating an essential role of neuronal LRRC8A for survival. Middle cerebral artery occlusion (MCAO) caused an early increase in LRRC8A protein levels in the hippocampus in wild-type (WT) mice. Whole-cell patch-clamp recording in brain slices revealed that oxygen-glucose deprivation significantly increased the amplitude of VRAC currents in hippocampal CA1 neurons in WT but not in Lrrc8a-cKO mice. Hypotonicity increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in hippocampal CA1 neurons in WT mice, and this was abolished by DCPIB, a VRAC blocker. But in Lrrc8a-cKO mice, hypotonic solution had no effect on the frequency of sEPSCs in these neurons. Furthermore, the brain infarct volume and neurological severity score induced by MCAO were significantly lower in Lrrc8a-cKO mice than in WT mice. In addition, MCAO-induced increases in cleaved caspase-3 and calpain activity, two biochemical markers of neuronal apoptosis and death, in brain tissues were significantly attenuated in Lrrc8a-cKO mice compared with WT mice. These new findings indicate that cerebral ischemia increases neuronal LRRC8A-dependent VRAC activity and that VRACs contribute to increased glutamatergic input to hippocampal neurons and brain injury caused by ischemic stroke., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest with the contents of this article., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The hypothalamic paraventricular nucleus (PVN) plays a major role in generating increased sympathetic output in hypertension. Although group III metabotropic glutamate receptors (mGluRs) are expressed in the hypothalamus, little is known about their contribution to regulating PVN presympathetic neurons in hypertension. Here we show that activating group III mGluRs with L-2-amino-4-phosphonobutyric acid (L-AP4) consistently inhibited the firing activity of spinally projecting PVN neurons in normotensive rats. However, in spontaneously hypertensive rats (SHRs), L-AP4 inhibited 45% of PVN neurons but excited 37%. L-AP4 significantly reduced glutamatergic and GABAergic input to PVN neurons in both groups. Blocking postsynaptic G protein signaling eliminated the excitatory but not the inhibitory effect of L-AP4 on PVN neurons in SHRs. Remarkably, prior activation of group I mGluRs converted the L-AP4 effect from inhibitory to excitatory in PVN neurons, and L-AP4 consistently inhibited PVN neurons when mGluR5 was blocked in SHRs. Furthermore, the expression level of mGluR4 and mGluR6 in the PVN was significantly higher in SHRs than in normotensive rats. Microinjection of L-AP4 into the PVN decreased blood pressure and lumbar sympathetic nerve discharges in normotensive rats and SHRs. Additionally, blocking group I mGluRs in the PVN potentiated L-AP4's sympathoinhibitory effect in SHRs. Therefore, activation of presynaptic group III mGluRs inhibits the excitability of PVN presympathetic neurons to attenuate sympathetic vasomotor activity. Through crosstalk with mGluR5, postsynaptic group III mGluR stimulation paradoxically excites PVN presympathetic neurons in SHRs. Concurrently blocking mGluR5 and activating group III mGluRs in the PVN can effectively reduce sympathetic outflow in hypertension., Competing Interests: Declaration of competing interest The authors declare no conflict of interest with the contents of this article., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Ghrelin increases food intake and body weight by stimulating orexigenic agouti-related protein (AgRP)/neuropeptide Y (NPY) neurons and inhibiting anorexic pro-opiomelanocortin (POMC) neurons in the hypothalamus. Growth hormone secretagogue receptor (Ghsr) mediates the effect of ghrelin on feeding behavior and energy homeostasis. However, the role of Ghsr in the ghrelin effect on these two populations of neurons is unclear. We hypothesized that Ghsr mediates the effect of ghrelin on AgRP and POMC neurons. In this study, we determined whether Ghsr similarly mediates the effects of ghrelin on AgRP/NPY and POMC neurons using cell type-specific Ghsr-knockout mice. Perforated whole-cell recordings were performed on green fluorescent protein-tagged AgRP/NPY and POMC neurons in the arcuate nucleus in hypothalamic slices. In Ghsr +/+ mice, ghrelin (100 nM) significantly increased the firing activity of AgRP/NPY neurons but inhibited the firing activity of POMC neurons. In Ghsr -/- mice, the excitatory effect of ghrelin on AgRP/NPY neurons was abolished. Ablation of Ghsr also eliminated ghrelin-induced increases in the frequency of GABAergic inhibitory postsynaptic currents of POMC neurons. Strikingly, ablation of Ghsr converted the ghrelin effect on POMC neurons from inhibition to excitation. Des-acylated ghrelin had no such effect on POMC neurons in Ghsr -/- mice. In both Ghsr +/+ and Ghsr -/- mice, blocking GABA A receptors with gabazine increased the basal firing activity of POMC neurons, and ghrelin further increased the firing activity of POMC neurons in the presence of gabazine. Our findings provide unequivocal evidence that Ghsr is essential for ghrelin-induced excitation of AgRP/NPY neurons. However, ghrelin excites POMC neurons through an unidentified mechanism that is distinct from conventional Ghsr., (© 2017 International Society for Neurochemistry.)

Neurons in the vestibular nuclei have a vital function in balance maintenance, gaze stabilization, and posture. Although muscarinic acetylcholine receptors (mAChRs) are expressed and involved in regulating vestibular function, it remains unclear how individual mAChR subtypes regulate vestibular neuronal activity. In this study, we determined which specific subtypes of mAChRs control synaptic input and excitability of medial vestibular nucleus (MVN) neurons that project to the cerebellum. Cerebellum-projecting MVN neurons were labeled by a fluorescent retrograde tracer and then identified in rat brainstem slices. Quantitative PCR analysis suggested that M2 and M3 were the possible major mAChR subtypes expressed in the MVN. The mAChR agonist oxotremorine-M significantly reduced the amplitude of glutamatergic excitatory post-synaptic currents evoked by stimulation of vestibular primary afferents, and this effect was abolished by the M2-preferring antagonist AF-DX 116. However, oxotremorine-M had no effect on GABA-mediated spontaneous inhibitory post-synaptic currents of labeled MVN neurons. Furthermore, oxotremorine-M significantly increased the firing activity of labeled MVN neurons, and this effect was blocked by the M3-preferring antagonist J104129 in most neurons tested. In addition, AF-DX 116 reduced the onset latency and prolonged the excitatory effect of oxotremorine-M on the firing activity of labeled MVN neurons. Our findings suggest that M3 is the predominant post-synaptic mAChR involved in muscarinic excitation of cerebellum-projecting MVN neurons. Pre-synaptic M2 mAChR regulates excitatory glutamatergic input from vestibular primary afferents, which in turn influences the excitability of cerebellum-projecting MVN neurons. This new information has important therapeutic implications for treating vestibular disorders with mAChR subtype-selective agents. Medial vestibular nucleus (MVN) neurons projecting to the cerebellum are involved in balance control. We found that activation of pre-synaptic M2 muscarinic receptors inhibit glutamatergic input from vestibular primary afferents, whereas stimulation of post-synaptic M3 muscarinic receptors increases the firing activity of cerebellum-projecting MVN neurons. This new information advances our understanding of the cholinergic mechanism regulating the vestibular system., (© 2016 International Society for Neurochemistry.)

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. Small conductance calcium-activated K(+) (SK) channels, calmodulin, and protein kinase CK2 form a molecular complex and regulate neuronal excitability. Our study suggests that augmented CK2 activity in hypertension can increase calmodulin (CaM) phosphorylation, which leads to diminished SK channel function in pre-sympathetic neurons. Diminished SK channel activity plays a role in hyperactivity of pre-sympathetic neurons in the hypothalamus in hypertension., (© 2014 International Society for Neurochemistry.)

Nitric oxide (NO) increases glutamate release to the second-order neurons in the nucleus tractus solitarius (NTS). N-type Ca(2+) channel is essential for triggering glutamate release at synaptic terminals. In this study, we determined the role of Cav2.2 subunit in NO-induced increase in glutamate synaptic inputs to NTS neurons. The second-order NTS neurons and nodose ganglionic (NG) neurons were identified by applying DiA, a fluorescent lipophilic tracer, on aortic depressor nerve in rats. NO donor DEA/NO significantly increased tractus solitarius (TS)-evoked excitatory postsynaptic currents (EPSCs) in second-order NTS neurons, an effect was abolished by pretreatment of slice with ODQ, an inhibitor for soluble isoform of guanylyl cyclase. DEA/NO decreased the paired-pulse ratio of TS-evoked EPSCs, while increased the frequency, but not the amplitude, of miniature EPSCs in second-order NTS neurons. Furthermore, DEA/NO significantly increased Ba(2+) currents in identified baroreceptor NG neurons. However, DEA/NO had little effect on the Ba(2+) currents in the presence of specific N-type Ca(2+) blocker ω-conotoxin GVIA. In addition, immunocytochemistry staining revealed that Cav2.2 subunit immunoreactivates were colocalized with DiA-labeled baroreceptor nerve terminals in the NTS. Collectively, these findings suggest that NO stimulates glutamatergic synaptic inputs to second-order NTS neurons through augmentation of Cav2.2-mediated N-type Ca(2+) currents., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Barrington's nucleus (BN), commonly known as the pontine micturition center, controls micturition and other visceral functions through projections to the spinal cord. In this study, we developed a rat brain slice preparation to determine the intrinsic and synaptic mechanisms regulating pre-sympathetic output (PSO) and pre-parasympathetic output (PPO) neurons in the BN using patch-clamp recordings. The PSO and PPO neurons were retrogradely labeled by injecting fluorescent tracers into the intermediolateral region of the spinal cord at T13-L1 and S1-S2 levels, respectively. There were significantly more PPO than PSO neurons within the BN. The basal activity and membrane potential were significantly lower in PPO than in PSO neurons, and A-type K(+) currents were significantly larger in PPO than in PSO neurons. Blocking A-type K(+) channels increased the excitability more in PPO than in PSO neurons. Stimulting μ-opioid receptors inhibited firing in both PPO and PSO neurons. The glutamatergic EPSC frequency was much lower, whereas the glycinergic IPSC frequency was much higher, in PPO than in PSO neurons. Although blocking GABAA receptors increased the excitability of both PSO and PPO neurons, blocking glycine receptors increased the firing activity of PPO neurons only. Furthermore, blocking ionotropic glutamate receptors decreased the excitability of PSO neurons but paradoxically increased the firing activity of PPO neurons by reducing glycinergic input. Our findings indicate that the membrane and synaptic properties of PSO and PPO neurons in the BN are distinctly different. This information improves our understanding of the neural circuitry and central mechanisms regulating the bladder and other visceral organs., (© 2013 International Society for Neurochemistry.)

Adenosine produces cardiovascular depressor effects in various brain regions. However, the cellular mechanisms underlying these effects remain unclear. The pre-sympathetic neurons in the hypothalamic paraventricular nucleus (PVN) play an important role in regulating arterial blood pressure and sympathetic outflow through projections to the spinal cord and brainstem. In this study, we performed whole-cell patch-clamp recordings on retrogradely labeled PVN neurons projecting to the intermediolateral cell column of the spinal cord in rats. Adenosine (10-100 microM) decreased the firing activity in a concentration-dependent manner, with a marked hyperpolarization in 12 of 26 neurons tested. Blockade of A(1) receptors with the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine or intracellular dialysis of guanosine 5'-O-(2-thodiphosphate) eliminated the inhibitory effect of adenosine on labeled PVN neurons. Immunocytochemical labeling revealed that A(1) receptors were expressed on spinally projecting PVN neurons. Also, blocking ATP-dependent K(+) (K(ATP)) channels with 100 microM glibenclamide or 200 microM tolbutamide, but not the G protein-coupled inwardly rectifying K(+) channels blocker tertiapin-Q, abolished the inhibitory effect of adenosine on the firing activity of PVN neurons. Furthermore, glibenclamide or tolbutamide significantly decreased the adenosine-induced outward currents in labeled neurons. The reversal potential of adenosine-induced currents was close to the K(+) equilibrium potential. In addition, adenosine decreased the frequency of both spontaneous and miniature glutamatergic excitatory post-synaptic currents and GABAergic inhibitory post-synaptic currents in labeled neurons, and these effects were also blocked by 8-cyclopentyl-1,3-dipropylxanthine. Collectively, our findings suggest that adenosine inhibits the excitability of PVN pre-sympathetic neurons through A(1) receptor-mediated opening of K(ATP) channels.

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 mu-opioid receptor agonist (D-Ala(2),N-Me-Phe(4),Gly-ol(5))-enkephalin (DAMGO) at 1 mum, 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 approximately 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.

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 mu 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 mu opioid receptor agonist, [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO, 1 microM), 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 GABA(A) 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 mu opioid receptors was colocalized with synaptophysin, a presynaptic marker, in close appositions to labeled CeA neurons. These results suggest that activation of mu opioid receptors on presynaptic terminals primarily attenuates GABAergic synaptic inputs to PAG-projecting neurons in the CeA.

Increased glutamatergic input to spinal dorsal horn neurons constitutes an important mechanism for neuropathic pain. However, the role of group III metabotropic glutamate receptors (mGluRs) in regulation of nociception and dorsal horn neurons in normal and neuropathic pain conditions is not fully known. In this study, we determined the effect of the group III mGluR specific agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4) on nociception and dorsal horn projection neurons in normal rats and a rat model of neuropathic pain. Tactile allodynia was induced by ligation of L5/L6 left spinal nerves in rats. Allodynia was determined by von Frey filaments in nerve-injured rats. The nociceptive threshold was tested using a radiant heat and a Randall-Selitto pressure device in normal rats. Single-unit activity of ascending dorsal horn neurons was recorded from the lumbar spinal cord in anesthetized rats. An intrathecal (5-30 microg) L-AP4 dose-dependently attenuated allodynia in nerve-injured rats but had no antinociceptive effect in normal rats. Topical spinal application of 5 to 50 microM L-AP4 also significantly inhibited the evoked responses of ascending dorsal horn neurons in nerve-ligated but not normal rats. Furthermore, blockade of spinal group III mGluRs significantly decreased the withdrawal threshold and increased the evoked responses of dorsal horn neurons in normal but not nerve-injured rats. These data suggest that group III mGluRs play distinct roles in regulation of nociception and dorsal horn neurons in normal and neuropathic pain states. Activation of spinal group III mGluRs suppresses allodynia and inhibits the hypersensitivity of dorsal horn projection neurons associated with neuropathic pain.

Neurons in the paraventricular nucleus (PVN) that project to the brain stem and spinal cord are important for autonomic regulation. The excitability of preautonomic PVN neurons is controlled by the noradrenergic input from the brain stem. In this study, we determined the role of alpha(2) adrenergic receptors in the regulation of excitatory and inhibitory synaptic inputs to spinally projecting PVN neurons. Excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) were recorded using whole cell voltage-clamp techniques on PVN neurons labeled by a retrograde fluorescence tracer injected into the thoracic spinal cord of rats. Bath application of 5-20 muM clonidine, an alpha(2) receptor agonist, significantly reduced the amplitude of evoked GABAergic IPSCs in a dose-dependent manner. Also, 10 microM clonidine significantly decreased the frequency (from 2.68 +/- 0.41 to 1.22 +/- 0.40 Hz) but not the amplitude of miniature IPSCs (mIPSCs), and this effect was blocked by the alpha(2) receptor antagonist yohimbine. Furthermore, clonidine increased the paired-pulse ratio of evoked IPSCs from 1.25 +/- 0.05 to 1.61 +/- 0.08 (P < 0.05). On the other hand, clonidine had little effect on evoked glutamatergic EPSCs, mEPSCs, and the paired-pulse ratio of evoked EPSCs in most labeled cells examined. Additionally, immunofluorescence labeling revealed that the alpha(2A) receptor and GABA immunoreactivities were co-localized in close apposition to labeled PVN neurons. Collectively, these data suggest that stimulation of alpha(2) adrenergic receptors primarily attenuates GABAergic inputs to PVN output neurons to the spinal cord. The presynaptic alpha(2) receptors function as heteroreceptors to modulate synaptic GABA release and contribute to the hypothalamic regulation of sympathetic outflow.

The rostral ventromedial medulla (RVM) is a major locus for the descending control of nociception and opioid analgesia. However, it is not clear how opioids affect synaptic inputs to RVM neurons. In this study, we determined the effect of mu-opioid receptor activation on excitatory and inhibitory synaptic transmission in spinally projecting RVM neurons. RVM neurons were retrogradely labeled with a fluorescent tracer injected into the dorsal horn of the spinal cord in rats. Whole-cell voltage-clamp recordings were performed on labeled RVM neurons in brain slices in vitro. The mu-receptor agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO, 1 microM) significantly decreased the amplitude of evoked excitatory postsynaptic currents (EPSCs) in 52% (9 of 17) of labeled cells. DAMGO also significantly reduced the amplitude of evoked inhibitory postsynaptic currents (IPSCs) in 69% (11 of 16) of cells examined. Furthermore, DAMGO significantly decreased the frequency of miniature EPSCs in 55% (15 of 27) of cells and significantly decreased the frequency of miniature IPSCs in all 12 cells studied. Although most EPSCs and IPSCs were mediated by glutamate and GABA, the nicotinic and glycine receptor antagonists attenuated EPSCs and IPSCs, respectively, in some labeled RVM neurons. Immunocytochemical labeling revealed that only 35% of recorded RVM neurons were tryptophan hydroxylase-positive, and 15% cells had GABA immunoreactivity. Thus, this study provides important functional evidence that activation of mu-opioid receptors decreases the release of both excitatory and inhibitory neurotransmitters onto most spinally projecting RVM neurons.

The nucleus locus coeruleus (LC) plays an important role in analgesia produced by opioids and by modulation of the descending noradrenergic pathway. The functional role of micro-opioid receptors (muOR) in regulation of the excitability of spinally projecting LC neurons has not been investigated. In the present study, we tested the hypothesis that activation of presynaptic mu-opioid receptors excites a population of spinally projecting LC neurons through attenuation of gamma-aminobutyric acid (GABA)-ergic synaptic inputs. Spinally projecting LC neurons were retrogradely labeled by a fluorescent dye injected into the spinal dorsal horn of rats. Whole-cell current- and voltage-clamp recordings were performed on labeled LC neurons in brain slices. All labeled LC noradrenergic neurons were demonstrated by dopamine-beta-hydroxylase (DbetaH) immunofluorescence. In 37 labeled LC neurons, (D-Ala(2),N-Me-Phe(4),Gly-ol(5))-enkephalin (DAMGO) significantly increased the discharge activity of 17 (45.9%) neurons, but significantly inhibited the firing activity of another 15 (40.5%) cells. The excitatory effect of DAMGO on seven labeled LC neurons was diminished in the presence of bicuculline. DAMGO significantly decreased the frequency of GABA-mediated miniature inhibitory postsynaptic currents (mIPSCs) in all nine labeled LC neurons. However, DAMGO had no effect on glutamate-mediated miniature excitatory postsynaptic currents (mEPSCs) in 12 of 15 neurons. Furthermore, DAMGO significantly inhibited the peak amplitude of evoked inhibitory postsynaptic currents (eIPSCs) in all 11 labeled neurons, but had no significant effect on the evoked excitatory postsynaptic currents (eEPSCs) in 10 of these 11 neurons. Thus, data from this study suggest that activation of micro-opioid receptors excites a population of spinally projecting LC neurons by preferential inhibition of GABAergic synaptic inputs. These findings provide important new information about the descending noradrenergic modulation and analgesic mechanisms of opioids.

Paraventricular nucleus (PVN) neurons that project to the spinal cord are important in the control of sympathetic outflow. Angiotensin II (Ang II) can stimulate PVN neurons, but its cellular mechanisms are not clear. In this study, we determined the effect of Ang II on the excitatory and inhibitory synaptic inputs to spinally projecting PVN neurons. Whole-cell patch-clamp recordings were performed on PVN neurons labeled by a retrograde fluorescence tracer injected into the thoracic spinal cord of rats. Immunocytochemistry labeling revealed that the immunoreactivity of angiotensin type 1 (AT1) receptors was colocalized with a presynaptic marker, synaptophysin, in the PVN. Application of 0.1-5 microm Ang II significantly decreased the amplitude of evoked GABAergic IPSCs in a concentration-dependent manner. Also, Ang II decreased the frequency of miniature IPSCs from 2.56 +/- 0.45 to 1.05 +/- 0.20 Hz (p < 0.05; n = 12), without affecting the amplitude and the decay time constant. The effect of Ang II on miniature IPSCs was blocked by losartan but not PD123319. However, Ang II had no effect on the evoked glutamatergic EPSCs and did not alter the frequency and amplitude of miniature EPSCs at concentrations that attenuated IPSCs. Furthermore, Ang II increased the firing rate of PVN neurons from 3.75 +/- 0.36 to 7.89 +/- 0.85 Hz (p < 0.05; n = 9), and such an effect was abolished by losartan. In addition, Ang II failed to excite PVN neurons in the presence of bicuculline. Thus, this study provides substantial new evidence that Ang II excites spinally projecting PVN neurons by attenuation of GABAergic synaptic inputs through activation of presynaptic AT1 receptors.

Stimulation of the noradrenergic nucleus locus coeruleus (LC) releases norepinephrine in the spinal cord, which inhibits dorsal horn neurons and produces analgesia. Activation of this descending noradrenergic pathway also contributes to the analgesic action produced by systemic opioids. The delta-opioid receptors are present presynaptically in the LC. However, their functional role in the control of the activity of spinally projecting LC neurons remains uncertain. In this study, we tested the hypothesis that activation of presynaptic delta-opioid receptors excites spinally projecting LC neurons through inhibition of GABA release. Spinally projecting LC neurons were retrogradely labeled by a fluorescent dye, DiI, injected into the spinal dorsal horn of rats. Whole cell voltage- and current-clamp recordings were performed on DiI-labeled LC neurons in brain slices in vitro. Retrogradely labeled LC noradrenergic neurons were demonstrated by dopamine-beta-hydroxylase immunofluorescence. [D-Pen(2), D-Pen(5)]-enkephalin (DPDPE, 1 microM) significantly decreased the frequency of GABA-mediated miniature inhibitory postsynaptic currents (IPSCs) of nine DiI-labeled LC neurons from 2.1 +/- 0.5 to 0.7 +/- 0.2 Hz without altering their amplitude and the kinetics. On the other hand, the miniature excitatory postsynaptic currents (EPSC) of nine DiI-labeled LC neurons were not significantly altered by DPDPE. Furthermore, DPDPE significantly inhibited the amplitude of evoked IPSC but not EPSC in eight DiI-labeled LC neurons. Under the current-clamp condition, the firing activity in 9 of 11 DiI-labeled LC neurons was significantly increased by 1 microM DPDPE from 4.6 +/- 0.7 to 6.2 +/- 1.0 Hz. Bicuculline (20 microM) also significantly increased the firing frequency in 13 of 20 neurons from 1.8 +/- 0.5 to 2.8 +/- 0.6 Hz. Additionally, the excitatory effect of DPDPE on LC neurons was diminished in the presence of bicuculline. Collectively, these data strongly suggest that activation of presynaptic delta-opioid receptors by DPDPE excites a population of spinally projecting LC neurons by preferential inhibition of GABA release. Thus presynaptic delta-opioid receptors likely play an important role in the regulation of the excitability of spinally projecting LC neurons and the descending noradrenergic inhibitory system.

Nitric oxide (NO) in the paraventricular nucleus (PVN) is involved in the regulation of the excitability of PVN neurons. However, the effect of NO on the inhibitory GABAergic and excitatory glutamatergic inputs to spinally projecting PVN neurons has not been studied specifically. In the present study, we determined the role of the inhibitory GABAergic and excitatory glutamatergic inputs in the inhibitory action of NO on spinally projecting PVN neurons. Spinally projecting PVN neurons were retrogradely labeled by a fluorescent dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocasbocyane (DiI), injected into the spinal cord of rats. Whole cell voltage- and current-clamp recordings were performed on DiI-labeled PVN neurons in the hypothalamic slice. The spontaneous miniature inhibitory postsynaptic currents (mIPSCs) recorded in DiI-labeled neurons were abolished by 20 microM bicuculline, whereas the miniature excitatory postsynaptic currents (mEPSCs) were eliminated by 20 microM 6-cyano-7-nitroquinoxaline-2,3-dione. Bath application of an NO donor, 100 microM S-nitroso-N-acetyl-penicillamine (SNAP), or the NO precursor, 100 microM L-arginine, both significantly increased the frequency of mIPSCs of DiI-labeled PVN neurons, without altering the amplitude and the decay time constant of mIPSCs. The effect of SNAP and L-arginine on the frequency of mIPSCs was eliminated by an NO scavenger, 2-(4-carboxypheny)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and an NO synthase inhibitor, 1-(2-trifluoromethylphenyl) imidazole, respectively. Neither SNAP nor L-arginine significantly altered the frequency and the amplitude of mEPSCs. Under current-clamp conditions, 100 microM SNAP or 100 microM L-arginine significantly decreased the discharge rate of the DiI-labeled PVN neurons, without significantly affecting the resting membrane potential. On the other hand, 20 microM bicuculline significantly increased the impulse activity of PVN neurons. In the presence of bicuculline, SNAP or L-arginine both failed to inhibit the firing activity of PVN neurons. This electrophysiological study provides substantial new evidence that NO suppresses the activity of spinally projecting PVN neurons through potentiation of the GABAergic synaptic input.

Diabetic neuropathic pain is often considered to be caused by peripheral neuropathy. The involvement of the CNS in this pathological condition has not been well documented. Development of hypersensitivity of spinal dorsal horn neurons is involved in neuropathic pain induced by traumatic nerve injury. In the present study, we determined the functional changes of identified spinothalamic tract (STT) neurons and their correlation to diabetic neuropathic pain. Diabetes was induced in rats by intraperitoneal injection of streptozotocin. Hyperalgesia and allodynia were assessed by the withdrawal responses to pressure, radiant heat, and von Frey filaments applied to the hindpaw. Single-unit activity of STT neurons was recorded from the lumbar spinal cord in anesthetized rats. The responses of STT neurons to mechanical and thermal stimuli and the sensitivity to intravenous morphine were determined in diabetic and normal rats. In 12 diabetic rats, mechanical allodynia and hyperalgesia, but not thermal hyperalgesia, developed within 2 wk after streptozotocin injection and lasted for >/=7 wk. Compared to the 32 STT neurons recorded in normal animals, the 37 STT neurons in diabetic rats displayed a higher spontaneous discharge activity and enlarged receptive fields. Also, the STT neurons in diabetic rats exhibited lower thresholds and augmented responses to mechanical stimulation. Intravenous injection of 2.5 mg/kg of morphine suppressed significantly the responses of STT neurons to noxious stimuli in 12 nondiabetic rats. However, such an inhibitory effect of morphine on the evoked response of STT neurons was diminished in 14 diabetic animals. This electrophysiological study provides new information that development of hypersensitivity of spinal dorsal horn projection neurons may be closely related to neuropathic pain symptoms caused by diabetes. Furthermore, the attenuated inhibitory effects of morphine on evoked responses of STT neurons in diabetes likely accounts for its reduced analgesic efficacy in this clinical form of neuropathic pain.

Inflammatory pain is associated with peripheral and central sensitization, but the underlying synaptic plasticity at the spinal cord level is poorly understood. Transient receptor potential (TRP) channels expressed at peripheral nerve endings, including TRP subtypes ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1), can detect nociceptive stimuli. In this study, we determined the contribution of presynaptic TRPA1 and TRPV1 at the spinal cord level to regulating nociceptive drive in chronic inflammatory pain induced by complete Freund's adjuvant (CFA) in rats. CFA treatment caused a large increase in the frequency of spontaneous excitatory postsynaptic currents (EPSCs) in lamina I, but not lamina II outer zone, dorsal horn neurons. However, blocking NMDA receptors had no effect on spontaneous EPSCs in lamina I neurons of CFA‐treated rats. Application of a specific TRPA1 antagonist, AM‐0902, or of a specific TRPV1 antagonist, 5′‐iodoresiniferatoxin, significantly attenuated the elevated frequency of spontaneous EPSCs and miniature EPSCs, the amplitude of monosynaptic EPSCs evoked from the dorsal root in lamina I neurons of CFA‐treated rats. AM‐0902 and 5′‐iodoresiniferatoxin had no effect on evoked or miniature EPSCs in lamina I neurons of vehicle‐treated rats. In addition, intrathecal injection of AM‐0902 or 5′‐iodoresiniferatoxin significantly reduced pain hypersensitivity in CFA‐treated rats but had no effect on acute nociception in vehicle‐treated rats. Therefore, unlike neuropathic pain, chronic inflammatory pain is associated with NMDA receptor–independent potentiation in glutamatergic drive to spinal lamina I neurons. Endogenous presynaptic TRPA1 and TRPV1 activity at the spinal level contributes to increased nociceptive input from primary sensory nerves to dorsal horn neurons in inflammatory pain. Open science badges: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. [ABSTRACT FROM AUTHOR]

Vesicular glutamate transporter‐2 (VGluT2) mediates the uptake of glutamate into synaptic vesicles in neurons. Spinal cord dorsal horn interneurons are highly heterogeneous and molecularly diverse. The functional significance of VGluT2‐expressing dorsal horn neurons in physiological and pathological pain conditions has not been explicitly demonstrated. Designer receptors exclusively activated by designer drugs (DREADDs) are a powerful chemogenetic tool to reversibly control neuronal excitability and behavior. Here, we used transgenic mice with Cre recombinase expression driven by the VGluT2 promoter, combined with the chemogenetic approach, to determine the contribution of VGluT2‐expressing dorsal horn neurons to nociceptive regulation. Adeno‐associated viral vectors expressing double‐floxed Cre‐dependent Gαq‐coupled human M3 muscarinic receptor DREADD (hM3D)‐mCherry or Gαi‐coupled κ‐opioid receptor DREADD (KORD)‐IRES‐mCitrine were microinjected into the superficial spinal dorsal horn of VGluT2‐Cre mice. Immunofluorescence labeling showed that VGluT2 was predominantly expressed in lamina II excitatory interneurons. Activation of excitatory hM3D in VGluT2‐expressing neurons with clozapine N‐oxide caused a profound increase in neuronal firing and synaptic glutamate release. Conversely, activation of inhibitory KORD in VGluT2‐expressing neurons with salvinorin B markedly inhibited neuronal activity and synaptic glutamate release. In addition, chemogenetic stimulation of VGluT2‐expressing neurons increased mechanical and thermal sensitivities in naive mice, whereas chemogenetic silencing of VGluT2‐expressing neurons reversed pain hypersensitivity induced by tissue inflammation and peripheral nerve injury. These findings indicate that VGluT2‐expressing excitatory neurons play a crucial role in mediating nociceptive transmission in the spinal dorsal horn. Targeting glutamatergic dorsal horn neurons with inhibitory DREADDs may be a new strategy for treating inflammatory pain and neuropathic pain. Many excitatory interneurons in the spinal cord dorsal horn express vesicular glutamate transporter‐2 (VGluT2), but their functional significance in physiological and pathological pain has not been shown specifically. Using chemogenetic approaches, we showed that stimulating VGluT2‐expressing dorsal horn neurons with hM3Dq‐CNO elicits pain hypersensitivity. Conversely, inhibiting VGluT2‐expressing dorsal horn neurons with KORD‐SALB attenuates persistent pain hypersensitivity caused by tissue inflammation and peripheral nerve injury. These findings indicate that VGluT2‐expressing neurons in the spinal dorsal horn play a pivotal role in transmitting painful information. [ABSTRACT FROM AUTHOR]

Highlights: [•] Nitric oxide increases presynaptic glutamate release to baroreceptor NTS neurons. [•] Nitric oxide increases N-type Ca2+ currents in baroreceptor NTS neurons in nodose ganglion. [•] Cav2.2 subunit is present on the baroreceptor afferent nerve terminals. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2012) 121, 944-953. Abstract Abnormal hyperexcitability of primary sensory neurons contributes to neuropathic pain development after nerve injury. Nerve injury profoundly reduces the expression of big conductance Ca2+ -activated K+ (BK) channels in the dorsal root ganglion (DRG). However, little is known about how nerve injury affects BK channel activity in DRG neurons. In this study, we determined the changes in BK channel activity in DRG neurons in a rat model of neuropathic pain and the contribution of brain-derived neurotrophic factor (BDNF) to reduced BK channel activity. The BK channel activity was present predominantly in small and medium DRG neurons, and ligation of L5 and L6 spinal nerves profoundly decreased the BK current density in these neurons. Blocking BK channels significantly increased neuronal excitability in sham control, but not in nerve-injured, rats. The BDNF concentration in the DRG was significantly greater in nerve-injured rats than in control rats. BDNF treatment largely reduced BK currents in DRG neurons in control rats, which was blocked by either anti-BDNF antibody or K252a, a Trk receptor inhibitor. Furthermore, either anti-BDNF antibody or K252a reversed reduction in BK currents in injured DRG neurons. BDNF treatment reduced the mRNA levels of BKα1 subunit in DRG neurons, and anti-BDNF antibody attenuated the reduction in the BKα1 mRNA level in injured DRG neurons. These findings suggest that nerve injury primarily diminishes the BK channel activity in small and medium DRG neurons. Increased BDNF levels contribute to reduced BK channel activity in DRG neurons through epigenetic and transcriptional mechanisms in neuropathic pain. [ABSTRACT FROM AUTHOR]

Abstract: Most δ-opioid receptors are located on the presynaptic terminals of primary afferent neurons in the spinal cord. However, their presence in different phenotypes of primary afferent neurons and their contribution to the analgesic effect of δ-opioid agonists are not fully known. Resiniferatoxin (RTX) is an ultra-potent transient receptor potential vanilloid type 1 channel (TRPV1) agonist and can selectively remove TRPV1-expressing primary afferent neurons. In this study, we determined the role of δ-opioid receptors expressed on TRPV1 sensory neurons in the antinociceptive effect of the δ-opioid receptor agonists [d-Pen2,d-Pen5]-enkephalin and [d-Ala2,Glu4]-deltorphin. Nociception was measured by testing the mechanical withdrawal threshold in the hindpaw of rats. Changes in the δ-opioid receptors were assessed using immunocytochemistry and the [3H]-naltrindole radioligand binding. In RTX-treated rats, the δ-opioid receptor on TRPV1-immunoreactive dorsal root ganglion neurons and afferent terminals in the spinal cord was diminished. RTX treatment also significantly reduced the maximal specific binding sites (31%) of the δ-opioid receptors in the dorsal spinal cord. Interestingly, intrathecal injection of [d-Pen2,d-Pen5]-enkephalin or [d-Ala2,Glu4]-deltorphin produced a large and prolonged increase in the nociceptive threshold in RTX-treated rats. These findings indicate that loss of TRPV1-expressing afferent neurons leads to a substantial reduction in presynaptic δ-opioid receptors in the spinal dorsal horn. However, the effect of δ-opioid agonists on mechano-nociception is paradoxically potentiated in the absence of TRPV1-expressing sensory neurons. This information is important to our understanding of the cellular sites and mechanisms underlying the spinal analgesic effect of δ-opioid agonists. [Copyright &y& Elsevier]