Ernest A. Jennings

Localization of P2X2 and P2X3 receptors in rat trigeminal ganglion neurons.

Purine receptors have been implicated in central neurotransmission from nociceptive primary afferent neurons, and ATP-mediated currents in sensory neurons have been shown to be mediated by both P2X3 and P2X2/3 receptors. The aim of the present study was to quantitatively examine the distribution of P2X2 and P2X3 receptors in primary afferent cell bodies in the rat trigeminal ganglion, including those innervating the dura. In order to determine the classes of neurons that express these receptor subtypes, purine receptor immunoreactivity was examined for colocalization with markers of myelinated (neurofilament 200; NF200) or mostly unmyelinated, non-peptidergic fibers (Bandeiraea simplicifolia isolectin B4; IB4). Forty percent of P2X2 and 64% of P2X3 receptor-expressing cells were IB4 positive, and 33% of P2X2 and 31% of P2X3 receptor-expressing cells were NF200 positive. Approximately 40% of cells expressing P2X2 receptors also expressed P2X3 receptors and vice versa. Trigeminal ganglion neurons innervating the dura mater were retrogradely labeled and 52% of these neurons expressed either P2X2 or P2X3 or both receptors. These results are consistent with electrophysiological findings that P2X receptors exist on the central terminals of trigeminal afferent neurons, and provide evidence that afferents supplying the dura express both receptors. In addition, the data suggest specific differences exist in P2X receptor expression between the spinal and trigeminal nociceptive systems.

Cannabinoid actions on rat superficial medullary dorsal horn neurons in vitro.

1 This study examined the cellular actions of cannabinoids on neurons in the substantia gelatinosa of the spinal trigeminal nucleus pars caudalis, using whole‐cell and perforated patch recording in brain slices. 2 The cannabinoid agonist WIN55,212‐2 (3 μm) decreased the amplitude of both GABAergic and glycinergic electrically evoked inhibitory postsynaptic currents (IPSCs) by 35 and 41%, respectively. This inhibition was completely reversed by the CB1 receptor‐selective antagonist N‐piperidino‐5‐(4‐chlorophenyl)‐l‐(2,4‐dichlorophenyl)‐4‐methyl‐3‐pyrazole‐carboxamide) (SR141716A, 3 μm). WIN55,212‐2 also produced relative facilitation of the second evoked IPSC to paired stimuli. 3 WIN55,212‐2 decreased the rate of both GABAergic and glycinergic miniature IPSCs by 44 and 34%, respectively, without changing their amplitude distributions or kinetics. 4 WIN55,212‐2 did not affect the amplitude of electrically evoked non‐NMDA glutamatergic excitatory postsynaptic currents (EPSCs). 5 WIN55,212‐2 produced no postsynaptic membrane current and had no significant effect on membrane conductance over a range of membrane potentials (–60 to –130 mV). 6 These results suggest that, within the superficial medullary dorsal horn, cannabinoids presynaptically inhibit GABAergic and glycinergic neurotransmission. At the cellular level, the analgesic action of cannabinoids on these medullary dorsal horn neurons therefore differs from that of μ‐opioids, which have both pre‐ and postsynaptic actions.

The actions of anandamide on rat superficial medullary dorsal horn neurons in vitro

Whole‐cell patch‐clamp recordings were made from neurons in the trigeminal nucleus caudalis and trigeminal ganglion, in vitro, to investigate the cellular actions of the endogenous cannabinoid, anandamide. Anandamide has been shown to act through both the cannabinoid receptor 1 (CB1) and the vanilloid receptor 1 (VR1). Anandamide (30 μm) caused a 54 % increase in the rate of miniature excitatory post‐synaptic currents (mEPSCs), without affecting their amplitude. The effect of anandamide was blocked by the VR1 antagonist capsazepine (20 μm), but not by the CB1‐specific antagonist AM251 (3 μm). Application of the VR1 receptor agonist capsaicin (300 nm) caused a 4200 % increase in the mEPSC rate. In dissociated trigeminal ganglion neurons, both anandamide and capsaicin caused an outward current in neurons that were voltage clamped at +40 mV. The maximal outward current produced by anandamide (EC50, 10 μm) was 45 % of that produced by capsaicin (10 μm). Co‐application of the VR1 antagonist capsazepine (30 μm) completely reversed the effects of both capsaicin and anandamide. The anandamide transport inhibitor, AM404 (30 μm) caused a 40 % increase in mEPSC rate in the slice preparation and an outward current in dissociated neurons. The latter current was reversed by the VR1 antagonist iodoresiniferatoxin (1 μm). The fatty acid amide hydrolase (FAAH) inhibitors phenylmethylsulfonyl fluoride (PMSF) (20 μm) and OL53 (1 μm) did not enhance the effect of anandamide in either the slice or dissociated neuron preparations. These results suggest that within the superficial medullary dorsal horn, anandamide (30 μm) acts presynaptically to enhance the release of glutamate via activation of the VR1 receptor.

Actions of nociceptin/orphanin FQ and other prepronociceptin products on rat rostral ventromedial medulla neurons in vitro.

1 Whole‐cell patch clamp recordings were made from rat rostral ventromedial medulla (RVM) neurons in vitro to investigate the cellular actions of the opioid‐like receptor ORL1 (NOP), ligand nociceptin/orphanin FQ and other putative prepronociceptin products. 2 Primary and secondary RVM neurons were identified as responding to the κ‐opioid receptor agonist U‐69593 (300 nm to 1 μm) and the μ‐ and δ‐opioid receptor agonist met‐enkephalin (10 μm), respectively. Both primary and secondary RVM neurons responded to nociceptin (3 nm to 1 μm) with an outward current that reversed polarity at –115 mV in brain slices and with inhibition of Ca2+ channel currents in acutely isolated cells. 3 The putative ORL1 antagonist J‐113397 (1 μm) produced no change in membrane current and abolished the outward current produced by nociceptin (100 nm). In contrast, Phe1ψ(CH2‐NH)Gly2]‐nociceptin‐(1‐13)NH2 (300 nm to 1 μm) alone produced an outward current and partially reduced the outward current produced by nociceptin (300 nm) when co‐applied. 4 In brain slices nociceptin (300 nm) reduced the amplitude of evoked GABAA receptor‐mediated inhibitory postsynaptic currents (IPSCs) but not non‐NMDA receptor‐mediated excitatory postsynaptic currents (EPSCs). 5 Met‐enkephalin (10 μm), but not nociceptin (300 nm), reduced the rate of spontaneous miniature IPSCs in normal external potassium solution (K+ 2.5 mm). In high external potassium (K+ 17.5 mm), nociceptin reduced the rate of miniature IPSCs in the presence (Ca2+ 2.4 mm, Mg2+ 1.2 mm) but not in the absence of external calcium (Ca2+ 0 mm, Mg2+ 10 mm, Cd2+ 10 μm). Nociceptin and met‐enkephalin had no effect on the amplitude of miniature IPSCs. 6 The putative nociceptin precursor products nocistatin (rat prepronociceptin125–132) and rat prepronociceptin154–181 had no effect on membrane currents, evoked IPSCs and evoked EPSCs. 7 These results indicate that nociceptin acts via the ORL1 receptor to directly inhibit both primary and secondary RVM neurons by activating a potassium conductance and by inhibiting calcium conductances. In addition, nociceptin inhibits GABA release within the RVM via a presynaptic Ca2+‐dependent mechanism. Thus, nociceptin has the potential to exert both disinhibitory and inhibitory effects on neuronal action potential firing within the RVM.

Effects of sumatriptan on rat medullary dorsal horn neurons

&NA; This study examined the cellular actions of the anti‐migraine drug sumatriptan, on neurons in the substantia gelatinosa of the spinal trigeminal nucleus pars caudalis. Sumatriptan inhibited the miniature EPSC (mEPSC) rate in a dose dependent fashion, with an EC50 of 250 nM. Sumatriptan (3 &mgr;M) inhibited the mEPSC rate by 36%, without altering the mEPSC amplitude. This effect was partially reversed by the 5HT1D specific antagonist BRL15572 (10 &mgr;M). In contrast, the 5HT1B agonist CP93129 (10 &mgr;m) did not alter the mEPSC rate. Furthermore, sumatriptan (3 &mgr;M) decreased the amplitude of electrically evoked EPSCs (eEPSC) by 40%. After incubating the slices in ketanserin (an antagonist which shows selectivity for 5HT1D over 5HT1B receptors) sumatriptan had little effect on eEPSC amplitude. In control conditions paired stimuli resulted in paired pulse depression (PPD; the ratio eEPSC2/eEPSC1=0.7±0.01), whilst in the presence of sumatriptan the PPD was blocked (ratio eEPSC2/eEPSC1=0.9±0.1). Sumatriptan produced no post‐synaptic membrane current and had no significant effect on membrane conductance over a range of membrane potentials (−60 to −130 mV). RT‐PCR experiments revealed the presence of mRNA for both 5HT1D and 5HT1B receptor subtypes in the trigeminal ganglia and subnucleus caudalis. These data suggest that sumatriptan acts pre‐synaptically on trigeminal primary afferent central terminals to reduce the probability of release of glutamate, and that this action is mediated through 5HT1D receptors.

Nociceptin, Phe1ψ‐nociceptin1–13, nocistatin and prepronociceptin154–181 effects on calcium channel currents and a potassium current in rat locus coeruleus in vitro

The actions of the neuropeptide nociceptin, the putative nociceptin receptor antagonist [Phe1ψ(CH2‐NH)Gly2]‐nociceptin‐(1–13)NH2 (Phe1ψ‐nociceptin1–13) and the putative nociceptin precursor products nocistatin (rat prepronociceptin125–132) and rat prepronociceptin154–181 were examined on membrane properties of rat locus coeruleus (LC) neurons using whole cell patch clamp techniques. Nociceptin inhibited IBa in all LC neurons, (pD2 of 8.9, maximum inhibition 50%). The inhibition of IBa by nociceptin was associated with slowing of the activation of IBa and could be significantly reversed by a strong depolarizing prepulse. Phe1ψ‐nociceptin1–13 also inhibited IBa in LC neurons (notional pD2 of 7.6, maximum inhibition 18%). Application of Phe1ψ‐nociceptin1–13 (1 μM) significantly occluded the subsequent effects of a co‐application of nociceptin (3 nM) on IBa. As previously reported for nociceptin, Phe1ψ‐nociceptin1–13 caused an outward current in LC neurons voltage clamped at −60 mV (pD2 of 7.1, maximum current 50% of that of methionine enkephalin, 10 μM). The Phe1ψ‐nociceptin1–13 induced current reversed polarity at −112 mV and exhibited pronounced inward rectification. Phe1ψ‐nociceptin1–13 (1 μM) reversibly inhibited the current caused by nociceptin (300 nM) by 30%. Neither nocistatin nor rat prepronociceptin154–181 inhibited IBa in LC neurons, or prevented the subsequent inhibition by nociceptin. Neither nocistatin or prepronociceptin154–181 affected the membrane properties of LC neurons. This study demonstrates that nociceptin modulates somatic IBa in rat LC neurons. The putative ORL1 antagonist Phe1ψ‐nociceptin1–13 exhibited partial agonist activity at inhibiting IBa and opening K+ channels in LC. Other putative nociceptin precursor products were without effect on LC cells.

N-glycosylation determines ionic permeability and desensitization of the TRPV1 capsaicin receptor

Background: We studied effects of N-linked sugar residues on the sensory ion channel, TRPV1. Results: Glycosylation of TRPV1 did not alter cell surface expression but was necessary for sustained cell calcium responses to allow uptake of YO-PRO-1 dye. Conclusion: N-Glycosylation regulated inactivation and ion selectivity but not expression of TRPV1. Significance: N-Glycosylation is a basic regulatory mechanism of TRPV1. The balance of glycosylation and deglycosylation of ion channels can markedly influence their function and regulation. However, the functional importance of glycosylation of the TRPV1 receptor, a key sensor of pain-sensing nerves, is not well understood, and whether TRPV1 is glycosylated in neurons is unclear. We report that TRPV1 is N-glycosylated and that N-glycosylation is a major determinant of capsaicin-evoked desensitization and ionic permeability. Both N-glycosylated and unglycosylated TRPV1 was detected in extracts of peripheral sensory nerves by Western blotting. TRPV1 expressed in HEK-293 cells exhibited various degrees of glycosylation. A mutant of asparagine 604 (N604T) was not glycosylated but did not alter plasma membrane expression of TRPV1. Capsaicin-evoked increases in intracellular calcium ([Ca2+]i) were sustained in wild-type TRPV1 HEK-293 cells but were rapidly desensitized in N604T TRPV1 cells. There was marked cell-to-cell variability in capsaicin responses and desensitization between individual cells expressing wild-type TRPV1 but highly uniform responses in cells expressing N604T TRPV1, consistent with variable levels of glycosylation of the wild-type channel. These differences were also apparent when wild-type or N604T TRPV1-GFP fusion proteins were expressed in neurons from trpv1−/− mice. Capsaicin evoked a marked, concentration-dependent increase in uptake of the large cationic dye YO-PRO-1 in cells expressing wild-type TRPV1, indicative of loss of ion selectivity, that was completely absent in cells expressing N604T TRPV1. Thus, TRPV1 is variably N-glycosylated and glycosylation is a key determinant of capsaicin regulation of TRPV1 desensitization and permeability. Our findings suggest that physiological or pathological alterations in TRPV1 glycosylation would affect TRPV1 function and pain transmission.

Early Emergence of Neural Activity in the Developing Mouse Enteric Nervous System

Neurons of the enteric nervous system (ENS) arise from neural crest cells that migrate into and along the developing gastrointestinal tract. A subpopulation of these neural-crest derived cells express pan-neuronal markers early in development, shortly after they first enter the gut. However, it is unknown whether these early enteric “neurons” are electrically active. In this study we used live Ca2+ imaging to examine the activity of enteric neurons from mice at embryonic day 11.5 (E11.5), E12.5, E15.5, and E18.5 that were dissociated and cultured overnight. PGP9.5-immunoreactive neurons from E11.5 gut cultures responded to electrical field stimulation with fast [Ca2+]i transients that were sensitive to TTX and ω-conotoxin GVIA, suggesting roles for voltage-gated Na+ channels and N-type voltage-gated Ca2+ channels. E11.5 neurons were also responsive to the nicotinic cholinergic agonist, dimethylphenylpiperazinium, and to ATP. In addition, spontaneous [Ca2+]i transients were present. Similar responses were observed in neurons from older embryonic gut. Whole-cell patch-clamp recordings performed on E12.5 enteric neurons after 2–10 h in culture revealed that these neurons fired both spontaneous and evoked action potentials. Together, our results show that enteric neurons exhibit mature forms of activity at early stages of ENS development. This is the first investigation to directly examine the presence of neural activity during enteric neuron development. Along with the spinal cord and hindbrain, the ENS appears to be one of the earliest parts of the nervous system to exhibit electrical activity.

Inflammation-induced increase in hyperpolarization-activated, cyclic nucleotide-gated channel protein in trigeminal ganglion neurons and the effect of buprenorphine

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are active at resting membrane potential and thus contribute to neuronal excitability. Their increased activity has recently been demonstrated in models of nerve injury-induced pain. The major aim of the current study was to investigate altered HCN channel protein expression in trigeminal sensory neurons following inflammation of the dura. HCN1 and HCN2 channel immunoreactivity was observed on the membranes of medium- to large-sized trigeminal ganglion neurons with 76% and 85% of HCN1 and HCN2 expressing neurons also containing the 200 kDa neurofilament protein (associated with myelinated fibers). Western immunoblots of lysates from rat trigeminal ganglia also showed bands with appropriate molecular weights for HCN1 and HCN2. Three days after application of complete Freunds adjuvant (CFA) to the dura mater, Western blot band densities were significantly increased; compared to control, to 166% for HCN1 and 284% for HCN2 channel protein. The band densities were normalized against alpha-actin. In addition, the number of retrogradely labeled neurons from the dura expressing HCN1 and HCN2 was significantly increased to 247% (HCN1) and 171% (HCN2), three days after inflammation. When the opioid receptor partial agonist, buprenorphine, was given systemically, immediately after CFA, the inflammation-induced increase in HCN protein expression in both Western blot and immunohistochemical experiments was not observed. These results suggest that HCN1 and HCN2 are involved in inflammation-induced sensory neuron hyperexcitability, and indicate that an opioid receptor agonist can reverse the protein upregulation.

5-HT1D Receptor Immunoreactivity in the Sphenopalatine Ganglion: Implications for the Efficacy of Triptans in the Treatment of Autonomic Signs Associated With Cluster Headache

Objective.— To determine if 5‐HT1D receptors are located in the sphenopalatine ganglion.