可视膜片钳法记录初级传入纤维介导的脊髓背角神经元突触后电流

本文描述了用明胶半包埋法制备带背根脊髓薄片的实验步骤,和在脊髓背角记录由初级传入纤维介导的突触后电流的可视膜片钳法。手术制备一段带背根的脊髓标本,并用20％的明胶包埋在琼脂块上,再用振动切片机切片获得带背根的脊髓薄片。通过红外线可视的引导,在脊髓背角神经元上建立全细胞封接模式。在钳制电压为-70mV条件下,记录自发的和背根刺激引起的兴奋性突触后电流。以传入纤维的传导速度与刺激阈值为指标,可以区分A样纤维与C样纤维兴奋性突触后电流。在钳制电压为0mV条件下,记录自发的和背根刺激引起的抑制性突触后电流。用5μmol／L的士宁或20μmol／L的荷包牡丹碱分离出γ-氨基丁酸能或甘氨酸能的抑制性突触后电流。用可视膜片钳方法可以准确测量脊髓背角神经元的突触后电流,从而研究初级传入突触的传递过程。更重要的是,在红外线可视观察的帮助下,建立膜片钳封接的成功率显著提高,同时也使记录研究脊髓背角深层神经元变得更加容易。本研究为探索初级传入突触传递过程提供了一个有效的方法。     

Up-regulation of Cavβ3 subunit in primary sensory neurons increases voltage-activated Ca2+ channel activity and nociceptive input in neuropathic pain

High voltage-activated calcium channels (HVACCs) are essential for synaptic and nociceptive transmission. Although blocking HVACCs can effectively reduce pain, this treatment strategy is associated with intolerable adverse effects. Neuronal HVACCs are typically composed of α(1), β (Cavβ), and α(2)δ subunits. The Cavβ subunit plays a crucial role in the membrane expression and gating properties of the pore-forming α(1) subunit. However, little is known about how nerve injury affects the expression and function of Cavβ subunits in primary sensory neurons. In this study, we found that Cavβ(3) and Cavβ(4) are the most prominent subtypes expressed in the rat dorsal root ganglion (DRG) and dorsal spinal cord. Spinal nerve ligation (SNL) in rats significantly increased mRNA and protein levels of the Cavβ(3), but not Cavβ(4), subunit in the DRG. SNL also significantly increased HVACC currents in small DRG neurons and monosynaptic excitatory postsynaptic currents of spinal dorsal horn neurons evoked from the dorsal root. Intrathecal injection of Cavβ(3)-specific siRNA significantly reduced HVACC currents in small DRG neurons and the amplitude of monosynaptic excitatory postsynaptic currents of dorsal horn neurons in SNL rats. Furthermore, intrathecal treatment with Cavβ(3)-specific siRNA normalized mechanical hyperalgesia and tactile allodynia caused by SNL but had no significant effect on the normal nociceptive threshold. Our findings provide novel evidence that increased expression of the Cavβ(3) subunit augments HVACC activity in primary sensory neurons and nociceptive input to dorsal horn neurons in neuropathic pain. Targeting the Cavβ(3) subunit at the spinal level represents an effective strategy for treating neuropathic pain.     

Chronic opioid potentiates presynaptic but impairs postsynaptic N-methyl-D-aspartic acid receptor activity in spinal cords: implications for opioid hyperalgesia and tolerance

Opioids are the most effective analgesics for the treatment of moderate to severe pain. However, chronic opioid treatment can cause both hyperalgesia and analgesic tolerance, which limit their clinical efficacy. In this study, we determined the role of pre- and postsynaptic NMDA receptors (NMDARs) in controlling increased glutamatergic input in the spinal cord induced by chronic systemic morphine administration. Whole-cell voltage clamp recordings of excitatory postsynaptic currents (EPSCs) were performed on dorsal horn neurons in rat spinal cord slices. Chronic morphine significantly increased the amplitude of monosynaptic EPSCs evoked from the dorsal root and the frequency of spontaneous EPSCs, and these changes were largely attenuated by blocking NMDARs and by inhibiting PKC, but not PKA. Also, blocking NR2A- or NR2B-containing NMDARs significantly reduced the frequency of spontaneous EPSCs and the amplitude of evoked EPSCs in morphine-treated rats. Strikingly, morphine treatment largely decreased the amplitude of evoked NMDAR-EPSCs and NMDAR currents of dorsal horn neurons elicited by puff NMDA application. The reduction in postsynaptic NMDAR currents caused by morphine was prevented by resiniferatoxin pretreatment to ablate TRPV1-expressing primary afferents. Furthermore, intrathecal injection of the NMDAR antagonist significantly attenuated the development of analgesic tolerance and the reduction in nociceptive thresholds induced by chronic morphine. Collectively, our findings indicate that chronic opioid treatment potentiates presynaptic, but impairs postsynaptic, NMDAR activity in the spinal cord. PKC-mediated increases in NMDAR activity at nociceptive primary afferent terminals in the spinal cord contribute critically to the development of opioid hyperalgesia and analgesic tolerance.     

Differential Regulation of Primary Afferent Input to Spinal Cord by Muscarinic Receptor Subtypes Delineated Using Knockout Mice

Stimulation of muscarinic acetylcholine receptors (mAChRs) inhibits nociceptive transmission at the spinal level. However, it is unclear how each mAChR subtype regulates excitatory synaptic input from primary afferents. Here we examined excitatory postsynaptic currents (EPSCs) of dorsal horn neurons evoked by dorsal root stimulation in spinal cord slices from wild-type and mAChR subtype knock-out (KO) mice. In wild-type mice, mAChR activation with oxotremorine-M decreased the amplitude of monosynaptic EPSCs in ∼67% of neurons but increased it in ∼10% of neurons. The inhibitory effect of oxotremorine-M was attenuated by the M₂/M₄ antagonist himbacine in the majority of neurons, and the remaining inhibition was abolished by group II/III metabotropic glutamate receptor (mGluR) antagonists in wild-type mice. In M₂/M₄ double-KO mice, oxotremorine-M inhibited monosynaptic EPSCs in significantly fewer neurons (∼26%) and increased EPSCs in significantly more neurons (33%) compared with wild-type mice. Blocking group II/III mGluRs eliminated the inhibitory effect of oxotremorine-M in M₂/M₄ double-KO mice. In M₂ single-KO and M₄ single-KO mice, himbacine still significantly reduced the inhibitory effect of oxotremorine-M. However, the inhibitory and potentiating effects of oxotremorine-M on EPSCs in M₃ single-KO and M₁/M₃ double-KO mice were similar to those in wild-type mice. In M₅ single-KO mice, oxotremorine-M failed to potentiate evoked EPSCs, and its inhibitory effect was abolished by himbacine. These findings indicate that activation of presynaptic M₂ and M₄ subtypes reduces glutamate release from primary afferents. Activation of the M₅ subtype either directly increases primary afferent input or inhibits it through indirectly stimulating group II/III mGluRs.     

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

Diabetic neuropathic pain is associated with increased glutamatergic input in the spinal dorsal horn. Group I metabotropic glutamate receptors (mGluRs) are involved in the control of neuronal excitability, but their role in the regulation of synaptic transmission in diabetic neuropathy remains poorly understood. Here we studied the role of spinal mGluR5 and mGluR1 in controlling glutamatergic input in a rat model of painful diabetic neuropathy induced by streptozotocin. Whole-cell patch-clamp recordings of lamina II neurons were performed in spinal cord slices. The amplitude of excitatory post-synaptic currents (EPSCs) evoked from the dorsal root and the frequency of spontaneous EPSCs (sEPSCs) were significantly higher in diabetic than in control rats. The mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) inhibited evoked EPSCs and sEPSCs more in diabetic than in control rats. Also, the percentage of neurons in which sEPSCs and evoked EPSCs were affected by MPEP or the group I mGluR agonist was significantly higher in diabetic than in control rats. However, blocking mGluR1 had no significant effect on evoked EPSCs and sEPSCs in either groups. The mGluR5 protein level in the dorsal root ganglion, but not in the dorsal spinal cord, was significantly increased in diabetic rats compared with that in control rats. Furthermore, intrathecal administration of MPEP significantly increased the nociceptive pressure threshold only in diabetic rats. These findings suggest that increased mGluR5 expression on primary afferent neurons contributes to increased glutamatergic input to spinal dorsal horn neurons and nociceptive transmission in diabetic neuropathic pain.     

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.     

The excitability of dorsal horn neurons is affected by cerebrospinal fluid from humans with osteoarthritis

Changes in central neural processing are thought to contribute to the development of chronic osteoarthritis pain. This may be reflected as the presence of inflammatory mediators in the cerebral spinal fluid (CSF). We therefore exposed organotypically cultured slices of rat spinal cord to CSF from human subjects with osteoarthritis (OACSF) at a ratio of 1 part CSF in 9 parts culture medium for 5-6 days, and measured changes in neuronal electrophysiological properties by means of whole-cell recording. Although OACSF had no effect on the membrane properties and excitability of neurons in the substantia gelatinosa, synaptic transmission was clearly altered. The frequency of spontaneous excitatory postsynaptic currents (sEPSC) in delay-firing putative excitatory neurons was increased, as was sEPSC amplitude and frequency in tonic-firing inhibitory neurons. These changes could affect sensory processing in the dorsal horn, and may affect the transfer of nociceptive information. Although OACSF also affected inhibitory synaptic transmission (frequency of spontaneous inhibitory synaptic currents; sIPSC), this may have little bearing on sensory processing by substantia gelatinosa neurons, as sEPSC frequency is >3× greater than sIPSC frequency in this predominantly excitatory network. These results support the clinical notion that changes in nociceptive processing at the spinal level contribute to the generation of chronic osteoarthritis pain.     

Glucagon-like peptide-1 modulates synaptic transmission to identified pancreas-projecting vagal motoneurons

Using a brainstem slice preparation, we aimed to study the pre- and postsynaptic effects of glucagon-like peptide-1 (GLP-1) on synaptic transmission to identified pancreas-projecting vagal motoneurons. Following blockade of GABAergic mediated currents with bicuculline, perfusion with 100 nM GLP-1 increased both amplitude and frequency of excitatory postsynaptic currents (EPSCs) in 21 of 52 neurons. Perfusion with the GLP-1 selective agonist exendin-4 (100 nM), also increased the frequency of spontaneous EPSCs, while pretreatment with the GLP-1 selective antagonist, exendin 9-39, prevented the effects of GLP-1. In the presence of kynurenic acid to block ionotropic glutamatergic currents, perfusion with GLP-1 increased the frequency of inhibitory postsynaptic currents (IPSCs) in 28 of 74 neurons; in 14 of these responsive neurons, GLP-1 also increased IPSC amplitude, indicating actions at both pre- and postsynaptic sites. Perfusion with exendin-4 increased the frequency of spontaneous IPSCs, while pretreatment with exendin 9-39 prevented the effects of GLP-1. These results suggest that GLP-1 modulates both excitatory and inhibitory synaptic inputs to pancreas-projecting vagal motoneurons.     

Glial glutamate transporter and glutamine synthetase regulate GABAergic synaptic strength in the spinal dorsal horn

Decreased GABAergic synaptic strength ('disinhibition') in the spinal dorsal horn is a crucial mechanism contributing to the development and maintenance of pathological pain. However, mechanisms leading to disinhibition in the spinal dorsal horn remain elusive. We investigated the role of glial glutamate transporters (GLT-1 and GLAST) and glutamine synthetase in maintaining GABAergic synaptic activity in the spinal dorsal horn. Electrically evoked GABAergic inhibitory post-synaptic currents (eIPSCs), spontaneous IPSCs (sIPSCs) and miniature IPSCs were recorded in superficial spinal dorsal horn neurons of spinal slices from young adult rats. We used (2S,3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA), to block both GLT-1 and GLAST and dihydrokainic acid to block only GLT-1. We found that blockade of both GLAST and GLT-1 and blockade of only GLT-1 in the spinal dorsal horn decreased the amplitude of GABAergic eIPSCs, as well as both the amplitude and frequency of GABAergic sIPSCs or miniature IPSCs. Pharmacological inhibition of glial glutamine synthetase had similar effects on both GABAergic eIPSCs and sIPSCs. We provided evidence demonstrating that the reduction in GABAergic strength induced by the inhibition of glial glutamate transporters is due to insufficient GABA synthesis through the glutamate-glutamine cycle between astrocytes and neurons. Thus, our results indicate that deficient glial glutamate transporters and glutamine synthetase significantly attenuate GABAergic synaptic strength in the spinal dorsal horn, which may be a crucial synaptic mechanism underlying glial-neuronal interactions caused by dysfunctional astrocytes in pathological pain conditions.     

Timing and specificity of feed-forward inhibition within the LGN

Local interneurons provide feed-forward inhibition from retinal ganglion cells (RGCs) to thalamocortical (TC) neurons, but questions remain regarding the timing, magnitude, and functions of this inhibition. Here, we identify two types of inhibition that are suited to play distinctive roles. We recorded excitatory and inhibitory postsynaptic currents (EPSCs/IPSCs) in TC neurons in mouse brain slices and activated individual RGC inputs. In 34% of TC neurons, we identified EPSCs and IPSCs with identical thresholds that were tightly correlated, indicating activation by the same RGC. Such "locked" IPSCs occurred 1 ms after EPSC onset. The remaining neurons had only "nonlocked" inhibition, in which EPSCs and IPSCs had different thresholds, indicating activation by different RGCs. Nonlocked inhibition may refine receptive fields within the LGN by providing surround inhibition. In contrast, dynamic-clamp recordings suggest that locked inhibition improves the precision of synaptically evoked responses in individual TC neurons by eliminating secondary spikes.     

Impaired Excitatory Drive to Spinal Gabaergic Neurons of Neuropathic Mice

Adequate pain sensitivity requires a delicate balance between excitation and inhibition in the dorsal horn of the spinal cord. This balance is severely impaired in neuropathy leading to enhanced pain sensations (hyperalgesia). The underlying mechanisms remain elusive. Here we explored the hypothesis that the excitatory drive to spinal GABAergic neurons might be impaired in neuropathic animals. Transgenic adult mice expressing EGFP under the promoter for GAD67 underwent either chronic constriction injury of the sciatic nerve or sham surgery. In transverse slices from lumbar spinal cord we performed whole-cell patch-clamp recordings from identified GABAergic neurons in lamina II. In neuropathic animals rates of mEPSC were reduced indicating diminished global excitatory input. This downregulation of excitatory drive required a rise in postsynaptic Ca²⁺. Neither the density and morphology of dendritic spines on GABAergic neurons nor the number of excitatory synapses contacting GABAergic neurons were affected by neuropathy. In contrast, paired-pulse ratio of Aδ- or C-fiber-evoked monosynaptic EPSCs following dorsal root stimulation was increased in neuropathic animals suggesting reduced neurotransmitter release from primary afferents. Our data indicate that peripheral neuropathy triggers Ca²⁺-dependent signaling pathways in spinal GABAergic neurons. This leads to a global downregulation of the excitatory drive to GABAergic neurons. The downregulation involves a presynaptic mechanism and also applies to the excitation of GABAergic neurons by presumably nociceptive Aδ- and C-fibers. This then leads to an inadequately low recruitment of inhibitory interneurons during nociception. We suggest that this previously unrecognized mechanism of impaired spinal inhibition contributes to hyperalgesia in neuropathy.     

Actions of endomorphins on synaptic transmission of aδ-fibers in spinal cord dorsal horn neurons

The effects of endogenous mu-opioid ligands, endomorphins, on Adelta-afferent-evoked excitatory postsynaptic currents (EPSCs) were studied in substantia gelatinosa neurons in spinal cord slices. Under voltage-clamp conditions, endomorphins blocked the evoked EPSCs in a dose-dependent manner. To determine if the block resulted from changes in transmitter release from glutamatergic synaptic terminals, the opioid actions on miniature excitatory postsynaptic currents (mEPSCs) were examined. Endomorphins (1 microM) reduced the frequency but not the amplitude of mEPSCs, suggesting that endomorphins directly act on presynaptic terminals. The effects of endomorphins on the unitary (quantal) properties of the evoked EPSCs were also studied. Endomorphins reduced unitary content without significantly changing unitary amplitude. These results suggest that in addition to presynaptic actions on interneurons, endomorphins also inhibit evoked EPSCs by reducing transmitter release from Adelta-afferent terminals.     

Molecular Basis of Regulating High Voltage-Activated Calcium Channels by S-Nitrosylation

Nitric oxide (NO) is involved in a variety of physiological processes, such as vasoregulation and neurotransmission, and has a complex role in the regulation of pain transduction and synaptic transmission. We have shown previously that NO inhibits high voltage-activated Ca²⁺ channels in primary sensory neurons and excitatory synaptic transmission in the spinal dorsal horn. However, the molecular mechanism involved in this inhibitory action remains unclear. In this study, we investigated the role of S-nitrosylation in the NO regulation of high voltage-activated Ca²⁺ channels. The NO donor S-nitroso-N-acetyl-dl-penicillamine (SNAP) rapidly reduced N-type currents when Cav2.2 was coexpressed with the Cavβ1 or Cavβ3 subunits in HEK293 cells. In contrast, SNAP only slightly inhibited P/Q-type and L-type currents reconstituted with various Cavβ subunits. SNAP caused a depolarizing shift in voltage-dependent N-type channel activation, but it had no effect on Cav2.2 protein levels on the membrane surface. The inhibitory effect of SNAP on N-type currents was blocked by the sulfhydryl-specific modifying reagent methanethiosulfonate ethylammonium. Furthermore, the consensus motifs of S-nitrosylation were much more abundant in Cav2.2 than in Cav1.2 and Cav2.1. Site-directed mutagenesis studies showed that Cys-805, Cys-930, and Cys-1045 in the II-III intracellular loop, Cys-1835 and Cys-2145 in the C terminus of Cav2.2, and Cys-346 in the Cavβ3 subunit were nitrosylation sites mediating NO sensitivity of N-type channels. Our findings demonstrate that the consensus motifs of S-nitrosylation in cytoplasmically accessible sites are critically involved in post-translational regulation of N-type Ca²⁺ channels by NO. S-Nitrosylation mediates the feedback regulation of N-type channels by NO.     

Presynaptic inhibition of miniature excitatory synaptic currents by baclofen and adenosine in the hippocampus.

Presynaptic inhibition of neurotransmitter release is thought to be mediated by a reduction of axon terminal Ca2+ current. We have compared the actions of several known inhibitors of evoked glutamate release with the actions of the Ca2+ channel antagonist Cd2+ on action potential-independent synaptic currents recorded from CA3 neurons in hippocampal slice cultures. Baclofen and adenosine decreased the frequency of miniature excitatory postsynaptic currents (mEPSCs) without affecting the distribution of their amplitudes. Cd2+ blocked evoked synaptic transmission, but had no effect on the frequency or amplitude of either mEPSCs or inhibitory postsynaptic currents (IPSCs). Inhibition of presynaptic Ca2+ current therefore appears not to be required for the inhibition of glutamate release by adenosine and baclofen. Baclofen had no effect on the frequency of miniature IPSCs, indicating that gamma-aminobutyric acid B-type receptors exert distinct presynaptic actions at excitatory and inhibitory synapses.     

Direct Effect of Remifentanil and Glycine Contained in Ultiva? on Nociceptive Transmission in the Spinal Cord: In Vivo and Slice Patch Clamp Analyses

Background

Ultiva® is commonly administered intravenously for analgesia during general anaesthesia and its main constituent remifentanil is an ultra-short-acting μ-opioid receptor agonist. Ultiva® is not approved for epidural or intrathecal use in clinical practice. Previous studies have reported that Ultiva® provokes opioid-induced hyperalgesia by interacting with spinal dorsal horn neurons. Ultiva® contains glycine, an inhibitory neurotransmitter but also an N-methyl-D-aspartate receptor co-activator. The presence of glycine in the formulation of Ultiva® potentially complicates its effects. We examined how Ultiva® directly affects nociceptive transmission in the spinal cord.

Methods

We made patch-clamp recordings from substantia gelatinosa (SG) neurons in the adult rat spinal dorsal horn in vivo and in spinal cord slices. We perfused Ultiva® onto the SG neurons and analysed its effects on the membrane potentials and synaptic responses activated by noxious mechanical stimuli.

Results

Bath application of Ultiva® hyperpolarized membrane potentials under current-clamp conditions and produced an outward current under voltage-clamp conditions. A barrage of excitatory postsynaptic currents (EPSCs) evoked by the stimuli was suppressed by Ultiva®. Miniature EPSCs (mEPSCs) were depressed in frequency but not amplitude. Ultiva®-induced outward currents and suppression of mEPSCs were not inhibited by the μ-opioid receptor antagonist naloxone, but were inhibited by the glycine receptor antagonist strychnine. The Ultiva®-induced currents demonstrated a specific equilibrium potential similar to glycine.

Conclusions

We found that intrathecal administration of Ultiva® to SG neurons hyperpolarized membrane potentials and depressed presynaptic glutamate release predominantly through the activation of glycine receptors. No Ultiva®-induced excitatory effects were observed in SG neurons. Our results suggest different analgesic mechanisms of Ultiva® between intrathecal and intravenous administrations.     

Cyclic AMP-mediated long-term facilitation of glycinergic transmission in developing spinal dorsal horn neurons

cAMP is known to regulate neurotransmitter release via protein kinase A (PKA)-dependent and/or PKA-independent signal transduction pathways at a variety of central synapses. Here we report the cAMP-mediated long-lasting enhancement of glycinergic transmission in developing rat spinal substantia gelatinosa neurons. Forskolin, an adenylyl cyclase activator, elicited a long-lasting increase in the amplitude of nerve-evoked glycinergic inhibitory postsynaptic currents (IPSCs), accompanied by a long-lasting decrease in the paired-pulse ratio in immature substantia gelatinosa neurons, and this forskolin-induced increase in glycinergic IPSCs decreased with postnatal development. Forskolin also decreased the failure rate of glycinergic IPSCs evoked by minimal stimulation, and increased the frequency of glycinergic miniature IPSCs. All of these data suggest that forskolin induces the long-lasting enhancement of glycinergic transmission by increasing in the presynaptic release probability. This pre-synaptic action of forskolin was mediated by hyperpolarization and cyclic nucleotide-activated cation channels and an increase in intraterminal Ca²⁺ concentration but independent of PKA. The present results suggest that cAMP-dependent signal transduction pathways represent a dynamic mechanism by which glycinergic IPSCs could potentially be modulated during postnatal development.     

Selective Targeting of TRPV1 Expressing Sensory Nerve Terminals in the Spinal Cord for Long Lasting Analgesia

Chronic pain is a major clinical problem and opiates are often the only treatment, but they cause significant problems ranging from sedation to deadly respiratory depression. Resiniferatoxin (RTX), a potent agonist of Transient Receptor Potential Vanilloid 1 (TRPV1), causes a slow, sustained and irreversible activation of TRPV1 and increases the frequency of spontaneous excitatory postsynaptic currents, but causes significant depression of evoked EPSCs due to nerve terminal depolarization block. Intrathecal administration of RTX to rats in the short-term inhibits nociceptive synaptic transmission, and in the long-term causes a localized, selective ablation of TRPV1-expressing central sensory nerve terminals leading to long lasting analgesia in behavioral models. Since RTX actions are selective for central sensory nerve terminals, other efferent functions of dorsal root ganglion neurons can be preserved. Preventing nociceptive transmission at the level of the spinal cord can be a useful strategy to treat chronic, debilitating and intractable pain.     

Ionotropic Glutamate Receptors in Spinal Nociceptive Processing

Glutamate is the predominant excitatory transmitter used by primary afferent synapses and intrinsic neurons in the spinal cord dorsal horn. Accordingly, ionotropic glutamate receptors mediate basal spinal transmission of sensory, including nociceptive, information that is relayed to supraspinal centers. However, it has become gradually more evident that these receptors are also crucially involved in short- and long-term plasticity of spinal nociceptive transmission, and that such plasticity have an important role in the pain hypersensitivity that may result from tissue or nerve injury. This review will cover recent findings on pre- and postsynaptic regulation of synaptic function by ionotropic glutamate receptors in the dorsal horn and how such mechanisms contribute to acute and chronic pain.     

Nonstationary fluctuation analysis and direct resolution of single channel currents at postsynaptic sites.

In order to measure unitary properties of receptor channels at the postsynaptic site, the noise within the decay phases of inhibitory postsynaptic currents (IPSCs) and of N-methyl-D-aspartate (NMDA)-dependent excitatory postsynaptic currents (EPSCs) in rat hippocampal neurons was studied by nonstationary fluctuation analysis. Least squares scaling of the mean current was used to circumvent the wide variation in amplitude of postsynaptic currents. The variance of fluctuations around the expected current was analyzed to calculate single channel conductance, and fluctuation kinetics were studied with power spectra. The single channel conductance underlying the IPSC was measured as 14 pS, whereas that underlying the EPSC was 42 pS. Openings of the EPSC channel could also be resolved directly in low-noise whole-cell recordings, allowing verification of the accuracy of the fluctuation analysis. The results are the first measurements of the properties of single postsynaptic channels activated during synaptic currents, and suggest that the technique can be widely applicable in investigations of synaptic mechanism and plasticity.     

Plasticity of pre- and postsynaptic GABAB receptor function in the paraventricular nucleus in spontaneously hypertensive rats

GABA(B) receptor function is upregulated in the paraventricular nucleus (PVN) of the hypothalamus in spontaneously hypertensive rats (SHR), but it is unclear whether this upregulation occurs pre- or postsynaptically. We therefore determined pre- and postsynaptic GABA(B) receptor function in retrogradely labeled spinally projecting PVN neurons using whole cell patch-clamp recording in brain slices in SHR and Wistar-Kyoto (WKY) rats. Bath application of the GABA(B) receptor agonist baclofen significantly decreased the spontaneous firing activity of labeled PVN neurons in both SHR and WKY rats. However, the magnitude of reduction in the firing rate was significantly greater in SHR than in WKY rats. Furthermore, baclofen produced larger membrane hyperpolarization and outward currents in labeled PVN neurons in SHR than in WKY rats. The baclofen-induced current was abolished by either including G protein inhibitor GDPbetaS in the pipette solution or bath application of the GABA(B) receptor antagonist in both SHR and WKY rats. Blocking N-methyl-d-aspartic acid receptors had no significant effect on baclofen-elicited outward currents in SHR. In addition, baclofen caused significantly greater inhibition of glutamatergic excitatory postsynaptic currents (EPSCs) in labeled PVN neurons in brain slices from SHR than WKY rats. By contrast, baclofen produced significantly less inhibition of GABAergic inhibitory postsynaptic currents (IPSCs) in labeled PVN neurons in SHR than in WKY rats. Although microinjection of the GABA(B) antagonist into the PVN increases sympathetic vasomotor tone in SHR, the GABA(B) antagonist did not affect EPSCs and IPSCs of the PVN neurons in vitro. These findings suggest that postsynaptic GABA(B) receptor function is upregulated in PVN presympathetic neurons in SHR. Whereas presynaptic GABA(B) receptor control of glutamatergic synaptic inputs is enhanced, presynaptic GABA(B) receptor control of GABAergic inputs in the PVN is attenuated in SHR. Changes in both pre- and postsynaptic GABA(B) receptors in the PVN may contribute to the control of sympathetic outflow in hypertension.