Rat Spinal Cord Slice Preparation Research Articles

Potentiation of NMDA-Mediated Responses by Amyloid-β Peptide 1-40 in Rat Sympathetic Preganglionic Neurons.

The abnormal accumulation of amyloid-β peptides (Aβ) is one of the main characteristics of Alzheimer's disease (AD). Cerebro- and cardiovascular diseases may be the risk factors for developing AD. The effect of Aβ on central sympathetic control of cardiovascular function remains unclear. The present study examines the acute effects of Aβ oligomers on the function of NMDA receptors, a subtype of ionotropic glutamate receptors, in rat sympathetic preganglionic neurons (SPNs). In the in vitro electrophysiological study, Aβ1-40 but not Aβ1-42 applied by superfusion for 5 min significantly potentiated NMDA-induced depolarizations in SPNs of neonatal rat spinal cord slice preparation. Application of Aβ1-40 had little effects on AMPA-induced depolarizations or GABA-induced hyperpolarizations. Treatment with a selective protein kinase C (PKC) inhibitor applied together with Aβ1-40 blocked the augmentation by Aβ1-40 of NMDA-induced depolarizations. Western blot analysis showed an increase in the levels of phosphoserine 896, selectively regulated by PKC, without significant changes in phosphoserine 897 on GluN1 subunits in lateral horn areas of spinal cord slices following treatment with Aβ1-40. In the in vivo study, intrathecal injection of Aβ1-40 (0.2 nmol) potentiated the pressor effects induced by NMDA (2 nmol) injected intrathecally in urethane-anesthetized rats. These results suggest that different fragments of Aβ may have differential effects on the NMDA receptor function and the selective augmentation of NMDA receptor function by Aβ1-40 may involve PKC-dependent mechanisms in sympathetic preganglionic neurons.

Effect of rat spinal cord injury (hemisection) on the ex vivo uptake and release of [3H]noradrenaline from a slice preparation.

We measured the ex vivo uptake and release of [3H]noradrenaline ([3H]NA) from perfused rat spinal cord slice preparations at 1, 3 and 14days after unilateral hemisection-induced spinal cord injury (SCI) compared with control slice preparations. After surgical hemisection under anaesthesia, the rats showed characteristic signs of hemiplegia, with no movement of the ipsilateral hindlimb. After 3days, the electron microscopy images showed overall degeneration of neuronal organelles and the myelin sheath, but the synapses seemed to be intact. In ex vivo experiments, the spinal cord injury did not influence uptake but increased [3H]NA release at rest and in response to axonal stimulation. The effect of a selective noradrenaline reuptake inhibitor, nisoxetine, was studied to identify the mechanisms underlying the increase in NA release. Nisoxetine potentiated stimulation-evoked [3H]NA release from the non-injured tissue, but it gradually lost its effectiveness after injury, depending on the time (1 and 3days) elapsed after hemisection, indicating that the noradrenaline transporter binding sites of the terminals become impaired after decentralisation.

Biochemical characterization and in vitro activity of AZ513, a noncovalent, reversible, and noncompetitive inhibitor of fatty acid amide hydrolase

Fatty acid amide hydrolase (FAAH) hydrolyzes several bioactive lipids including the endocannabinoid anandamide. Synthetic FAAH inhibitors are being generated to help define the biological role(s) of this enzyme, the lipids it degrades in vivo, and the disease states that might benefit from its pharmacological modulation. AZ513 inhibits human FAAH (IC 50 = 551 nM), is 20-fold more potent against rat FAAH (IC 50 = 27 nM), and is inactive at 10 μM against the serine hydrolases acetylcholinesterase, thrombin, and trypsin. In contrast to most other potent FAAH inhibitors, AZ513 showed no evidence of covalently modifying the enzyme and displayed reversible inhibition. In an enzyme cross-competition assay, AZ513 did not compete with OL-135, an inhibitor that binds to the catalytic site in FAAH, which indicates that AZ513 does not bind to the catalytic site and is therefore noncompetitive with respect to substrate. AZ513 has good cell penetration as demonstrated by inhibition of anandamide hydrolysis in human FAAH-transfected HEK293 cells (IC 50 = 360 nM). AZ513 was tested in a rat spinal cord slice preparation where CB 1 activation reduces excitatory post-synaptic currents (EPSCs). In this native tissue assay of synaptic activity, AZ513 reduced EPSCs, which is consistent with inhibiting endogenous FAAH and augmenting endocannabinoid tone. AZ513 has a unique biochemical profile compared with other published FAAH inhibitors and will be a useful tool compound to further explore the role of FAAH in various biological processes.

Recent advances in pain medicine: from bench to bed; August 17, 2009, Kobe, Japan

In late 2000, the US Congress passed into law a provision, which the President signed, that declared a 10-year period as the Decade of Pain Control and Research which began from January 1, 2001. The American Pain Society has actively supported the Decade of Pain Control and Research, and it has been a focal point for the development of numerous programs to heighten awareness and treatment of pain and research funding. Cutaneous sensation produced by noxious and innocuous stimuli is transmitted to the dorsal horn of the spinal cord via primary afferent fibers and recognized as pain and touch in the somatosensory cortex. While acute pain has a fundamental role as a protective system against the occurrence or threat of injuries and diseases, chronic pain associated with diseases such as postherpetic neuralgia, and rheumatoid arthritis often outlasts its biological usefulness and in itself causes functional disturbance. Neuropathic pain is a significant and largely unresolved medical problem characterized by spontaneous pain, hyperalgesia, and tactile pain (allodynia). Over several decades, non-steroidal antiinflammatory drugs (NSAIDs) and opioids have been used for clinical management of pain. Neuropathic pain is persistent and can be particularly problematic because they are often poorly managed by NSAIDs and conventional opioid analgesics. Kohno demonstrated that although excitatory transmission remained intact, presynaptic GABA release was reduced in lamina II neurons of an isolated adult rat spinal cord slice preparation in neuropathic pain models. He also reviewed the role of neuron–microglia interactions in the spinal cord in neuropathic pain [1]. Pain associated with cancer, particularly when tumors metastasize to bone, is often severe and debilitating. Better understanding of the neurobiological mechanisms underlying cancer pain are likely to lead to the development of more effective treatments. Kawamata et al. demonstrated that transient receptor potential vanilloid subfamily 1 (TRPV1) was up-regulated, and the l opioid receptor was down-regulated in peripheral neurons in bone cancer. Activation of spinal cannabinoid receptor type 1 (CB1) has been shown to inhibit glutamatergic excitatory postsynaptic currents in the spinal cord slices of naive rats and C-fiber-evoked neuronal response of the dorsal horn. He found that CB1 was located in the axon terminals of excitatory spinal interneuron, and its expression is preserved in bone cancer. Furthermore, he reported that spinal CB1 activation by an exogenously administered CB1 agonist reduced bone cancer-related pain. Therefore, spinal T. Minami (&) Department of Anesthesiology, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki, Osaka 569-8686, Japan e-mail: ane022@poh.osaka-med.ac.jp

Involvement of Reactive Oxygen Species in Long-Term Potentiation in the Spinal Cord Dorsal Horn

Recent studies suggest that reactive oxygen species (ROS) are functional messenger molecules in central sensitization, an underlying mechanism of persistent pain. Because spinal cord long-term potentiation (LTP) is the electrophysiological basis of central sensitization, this study investigates the effects of the increased or decreased spinal ROS levels on spinal cord LTP. Spinal cord LTP is induced by either brief, high-frequency stimulation (HFS) of a dorsal root at C-fiber intensity or superfusion of a ROS donor, tert-butyl hydroperoxide (t-BOOH), onto rat spinal cord slice preparations. Field excitatory postsynaptic potentials (fEPSPs) evoked by dorsal root stimulations with either Abeta- or C-fiber intensity are recorded from the superficial dorsal horn. HFS significantly increases the slope of both Abeta- and C-fiber evoked fEPSPs, thus suggesting LTP development. The induction, not the maintenance, of HFS-induced LTP is blocked by a N-methyl-D-aspartate (NMDA) receptor antagonist, D-2-amino-5-phosphonopentanoic acid (D-AP5). Both the induction and maintenance of LTP of Abeta-fiber-evoked fEPSPs are inhibited by a ROS scavenger, either N-tert-butyl-alpha-phenylnitrone or 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl. A ROS donor, t-BOOH-induced LTP is inhibited by N-tert-butyl-alpha-phenylnitrone but not by D-AP5. Furthermore, HFS-induced LTP and t-BOOH-induced LTP occlude each other. The data suggest that elevated ROS is a downstream event of NMDA receptor activation and an essential step for potentiation of synaptic excitability in the spinal dorsal horn.

GABAB receptors decrease inhibitory synaptic transmission onto sympathetic preganglionic neurones (SPNs) in the rat spinal cord slice preparation.

Activation of GABAB receptors has a profound effect on sympathetic outflow from the spinal cord (Cheng et al 2005 J. Appl. Physiol. 99 –1667) but little is known about the role of these receptors on synaptic transmission in autonomic regions. We determined how baclofen affects inhibitory synaptic transmission onto SPNs. Wistar rats (11 days old) were anaesthetized with urethane and whole-cell patch clamp recordings made from SPNs in 300μm transverse slices of thoracic spinal cord. Bath application of baclofen (0.5–5μM) hyperpolarised 21/41 SPNs associated with a decrease in input resistance. These low concentrations hyperpolarised 4/4 interneurones but glial cells were not affected (n = 4). Inhibitory postsynaptic potentials (IPSPs) elicited by stimulating the lateral funiculus (Lf) or the central autonomic area (CAA) were significantly (p<0.05) reduced in amplitude by 1 μM baclofen (Lf 12.4± 1.7 mV (mean ± S.E.M.) to 9.4 ± 1.2 mV, n= 10; CAA 9.6 ± 1.6 mV to 7.0 ± 1.3 mV, n= 13). These decreases were accompanied by an increase in paired pulse ratio of amplitude of IPSPs elicited from both regions. Similar effects on IPSP amplitudes were observed regardless of the postsynaptic response and whether or not potassium channel blockers were included in the patch solution. Thus, these results indicate that presynaptic GABAB receptors can influence sympathetic outflow. Supported by The British Heart Foundation.

Involvement of an increased spinal TRPV1 sensitization through its up-regulation in mechanical allodynia of CCI rats

Vanilloid receptor 1 (TRPV1) antagonists are known to attenuate the neuropathic pain symptoms in peripheral nerve injury models, but the mechanism(s) of their effect remains unclear. At the same time, the role of spinal TRPV1 in pain transduction system has not been fully understood. In this study, the role of spinal TRPV1 in mechanical allodynia in rat chronic constriction injury (CCI) model was investigated. Intrathecal administration of a selective TRPV1 antagonist, N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl)tetrahydropryazine-1(2H)-carbox-amide (BCTC) significantly attenuated mechanical allodynia in CCI rats at 100 and 300 nmol. In vitro, BCTC inhibited capsaicin (300 nM)-induced releases of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) and substance P-like immunoreactivity (SP-LI) from the rat spinal cord slice preparations with IC 50s of 37.0 and 36.0 nM, respectively, confirming that BCTC potently inhibits TRPV1 function in the rat spinal cord. TRPV1 expression levels in the spinal cord following CCI were quantified in by Western blot analysis. TRPV1 protein levels were significantly increased in the ipsilateral side of the lumbar spinal cord at 7 and 14 days following CCI surgery, but not in the contralateral side. Furthermore, capsaicin (300 nM)-evoked release of CGRP-LI was significantly higher in the ipsilateral spinal cord of CCI rats (14 days after surgery) than that of sham-operated rats. These findings suggest that an increased sensitization of the spinal TRPV1 through its up-regulation is involved in the development and/or maintenance of mechanical allodynia in rat CCI model.

0906 Natriuretic peptide mediated cgmp synthesis and phosphodiesterase activity in the rat spinal cord slice preparation

0906 Natriuretic peptide mediated cgmp synthesis and phosphodiesterase activity in the rat spinal cord slice preparation

A P2X Receptor-Mediated Nociceptive Afferent Pathway to Lamina I of the Spinal Cord

Of the six lamina regions in the dorsal horn of the spinal cord, lamina I is a major sensory region involved in nociceptive transmission under both physiological and pathological conditions. While P2X receptors have been shown to be involved in nociception, it remains unknown if P2X receptors are involved in nociceptive transmission to lamina I neurons. Using rat spinal cord slice preparations and patch-clamp recordings, we have demonstrated that the excitatory synaptic transmission between primary afferent fibers and lamina I neurons is significantly affected by ATP and α,β-methylene-ATP. The synaptic effects of them include the increases of the frequency of both miniature excitatory postsynaptic currents (mEPSCs) and spontaneous EPSCs (sEPSCs), and decreases of evoked EPSCs (eEPSCs). These effects were blocked by pyridoxalphosphate-6-azophenyl-2', 4'-disulfonic acid (PPADS, 10 μM) and suramin (30 μM). In the neurons for which ATP and α,β-methylene-ATP had effects on mEPSCs, sEPSCs and eEPSCs, capsaicin produced similar synaptic effects. Our results indicate that P2X receptors are expressed on many afferent fibers that directly synapse to lamina I neurons. Furthermore, these P2X receptor-expressing afferent fibers are capsaicin-sensitive nociceptive afferents. Thus, this study reveals a P2X receptor-mediated nociceptive afferent pathway to lamina I of the spinal cord and provides a new insight into the nociceptive functions of P2X receptors.

Activation of presynaptic group I metabotropic glutamate receptors enhances glutamate release in the rat spinal cord substantia gelatinosa*1

The activation of group I metabotropic glutamate receptors (mGluRs) produces a long-term potentiation of sensory transmission in the substantia gelatinosa (SG) region of the spinal cord (Prog. Brain Res. 129 (2000) 115). The mechanism(s) responsible for the induction of this potentiation is not known. Using rat spinal cord slice preparation and patch-clamp recordings, here we show, that the activation of the group I mGluRs by (S)-3,5-dihydroxyphenylglycine (DHPG, 1 μM), the mGluR1/5 agonist, increased the frequency of both activity-dependent spontaneous EPSCs, and activity-independent miniature EPSCs (mEPSCs). However, DHPG did not affect amplitude of mEPSCs. The effects of DHPG were not seen in the presence of the preferential mGluR1 antagonist CPCCOEt (10 μM). On the other hand, 2-methyl-6-(phenylethynyl)-pyridine (10 μM), a selective mGluR5 antagonist, blocked the DHPG facilitation present during the wash-out of the drug. This novel facilitating effect of the group I mGluR activation on glutamate release is the first report of a direct facilitatory action of both mGluR1 and mGluR5 subtypes on sensory transmission in the spinal cord SG region. These results indicate the potential contribution of synaptic activation of these facilitatory autoreceptors in plasticity of primary afferent neurotransmission.

Activation of presynaptic group I metabotropic glutamate receptors enhances glutamate release in the rat spinal cord substantia gelatinosa

The activation of group I metabotropic glutamate receptors (mGluRs) produces a long-term potentiation of sensory transmission in the substantia gelatinosa (SG) region of the spinal cord (Prog. Brain Res. 129 (2000) 115). The mechanism(s) responsible for the induction of this potentiation is not known. Using rat spinal cord slice preparation and patch-clamp recordings, here we show, that the activation of the group I mGluRs by ( S)-3,5-dihydroxyphenylglycine (DHPG, 1 μM), the mGluR1/5 agonist, increased the frequency of both activity-dependent spontaneous EPSCs, and activity-independent miniature EPSCs (mEPSCs). However, DHPG did not affect amplitude of mEPSCs. The effects of DHPG were not seen in the presence of the preferential mGluR1 antagonist CPCCOEt (10 μM). On the other hand, 2-methyl-6-(phenylethynyl)-pyridine (10 μM), a selective mGluR5 antagonist, blocked the DHPG facilitation present during the wash-out of the drug. This novel facilitating effect of the group I mGluR activation on glutamate release is the first report of a direct facilitatory action of both mGluR1 and mGluR5 subtypes on sensory transmission in the spinal cord SG region. These results indicate the potential contribution of synaptic activation of these facilitatory autoreceptors in plasticity of primary afferent neurotransmission.

Neurokinin receptor 1-expressing spinal cord neurons in lamina I and III/IV of postnatal rats receive inputs from capsaicin sensitive fibers

Dorsal horn neurons expressing receptor for substance P (SP), the neurokinin 1 (NK1) receptor, play an important role in transmission and processing of nociceptive stimuli. To identify and study these neurons in the rat spinal cord slice preparation, we used fluorescence-conjugated SP to label NK1 receptor-expressing neurons. Labeled neurons in lamina I and III/IV were patch clamped and the vanilloid receptor 1 (TRPV1) agonist, capsaicin, was applied to evoke glutamate release from central terminals of peripheral nociceptors. Capsaicin induced an increase in the frequency of miniature excitatory postsynaptic currents in 73% of lamina I and 43% of lamina III/IV neurons expressing NK1-receptor indicating that these neurons receive direct input from capsaicin and heat sensitive nociceptors.

Neurokinin receptor 1-expressing spinal cord neurons in lamina I and III/IV of postnatal rats receive inputs from capsaicin sensitive fibers

Dorsal horn neurons expressing receptor for substance P (SP), the neurokinin 1 (NK1) receptor, play an important role in transmission and processing of nociceptive stimuli. To identify and study these neurons in the rat spinal cord slice preparation, we used fluorescence-conjugated SP to label NK1 receptor-expressing neurons. Labeled neurons in lamina I and III/IV were patch clamped and the vanilloid receptor 1 (TRPV1) agonist, capsaicin, was applied to evoke glutamate release from central terminals of peripheral nociceptors. Capsaicin induced an increase in the frequency of miniature excitatory postsynaptic currents in 73% of lamina I and 43% of lamina III/IV neurons expressing NK1-receptor indicating that these neurons receive direct input from capsaicin and heat sensitive nociceptors.

Partial Peripheral Nerve Injury Promotes a Selective Loss of GABAergic Inhibition in the Superficial Dorsal Horn of the Spinal Cord

To clarify whether inhibitory transmission in the superficial dorsal horn of the spinal cord is reduced after peripheral nerve injury, we have studied synaptic transmission in lamina II neurons of an isolated adult rat spinal cord slice preparation after complete sciatic nerve transection (SNT), chronic constriction injury (CCI), or spared nerve injury (SNI). Fast excitatory transmission remains intact after all three types of nerve injury. In contrast, primary afferent-evoked IPSCs are substantially reduced in incidence, magnitude, and duration after the two partial nerve injuries, CCI and SNI, but not SNT. Pharmacologically isolated GABA(A) receptor-mediated IPSCs are decreased in the two partial nerve injury models compared with naive animals. An analysis of unitary IPSCs suggests that presynaptic GABA release is reduced after CCI and SNI. Partial nerve injury also decreases dorsal horn levels of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD) 65 kDa ipsilateral to the injury and induces neuronal apoptosis, detected by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling staining in identified neurons. Both of these mechanisms could reduce presynaptic GABA levels and promote a functional loss of GABAergic transmission in the superficial dorsal horn.

Partial peripheral nerve injury promotes a selective loss of GABAergic inhibition in the superficial dorsal horn of the spinal cord.

To clarify whether inhibitory transmission in the superficial dorsal horn of the spinal cord is reduced after peripheral nerve injury, we have studied synaptic transmission in lamina II neurons of an isolated adult rat spinal cord slice preparation after complete sciatic nerve transection (SNT), chronic constriction injury (CCI), or spared nerve injury (SNI). Fast excitatory transmission remains intact after all three types of nerve injury. In contrast, primary afferent-evoked IPSCs are substantially reduced in incidence, magnitude, and duration after the two partial nerve injuries, CCI and SNI, but not SNT. Pharmacologically isolated GABA(A) receptor-mediated IPSCs are decreased in the two partial nerve injury models compared with naive animals. An analysis of unitary IPSCs suggests that presynaptic GABA release is reduced after CCI and SNI. Partial nerve injury also decreases dorsal horn levels of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD) 65 kDa ipsilateral to the injury and induces neuronal apoptosis, detected by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling staining in identified neurons. Both of these mechanisms could reduce presynaptic GABA levels and promote a functional loss of GABAergic transmission in the superficial dorsal horn.

Nociceptin-induced outward current in substantia gelatinosa neurones of the adult rat spinal cord

Nociceptin (NOC), also known as orphanin FQ, is a newly discovered endogenous ligand for the opioid receptor-like1 (ORL1) receptor. Although NOC has been shown to modulate nociceptive transmission, mechanisms for this action are still unknown. In the present study, actions of NOC on substantia gelatinosa (SG) neurones were examined in adult rat spinal cord slice preparations by using the whole-cell patch-clamp technique. NOC at a concentration of 1 μM induced an outward current having an amplitude of 26±5 pA ( n=68) at a holding potential of −70 mV; this action was dose-dependent with an EC 50 value of 0.23 μM (Hill coefficient: 1.5). The NOC current reversed its polarity at a potential which was close to the equilibrium potential for K +, as calculated by the Nernst equation ( n=4). The NOC current had slope conductances of 0.80±0.15 nS and 0.50±0.13 nS ( n=4) in voltage ranges of −120 to −140 mV and of −60 to −90 mV, respectively. The NOC current was inhibited by Ba 2+ (100 μM; by 56±8%, n=4) but not by 4-aminopyridine (4-AP; 1 mM; n=4) and tetraethylammonium (TEA; 5 mM; n=4). The NOC current was not affected by tetrodotoxin (TTX; 1 μM; n=4) and also by a non-specific opioid receptor antagonist, naloxone (1 μM; n=4). When examined using some inhibitors with respect to the ORL1 receptor, the NOC (1 μM) current was depressed in amplitude by a putative NOC precursor product, nocistatin (1 μM; by 18±4%, n=6) and also by a non-peptidyl ORL1 receptor antagonist, CompB (1 μM; by 64±10%, n=7) without a change in holding currents. On the other hand, a putative ORL1 receptor antagonist, [Phe 1ψ(CH 2-NH)Gly 2]nociceptin-(1-13)-NH 2 (1 μM; which is a derivative of NOC), by itself induced an outward current (7±3 pA, n=8), during which the NOC current was suppressed in amplitude by 56±8% ( n=8). We conclude that NOC activates in SG neurones a K + channel exhibiting a mild inwardly rectification through the activation of ORL1 receptor; this hyperpolarising action of NOC might contribute to at least a part of its antinociceptive effect.

Modulatory actions of serotonin, norepinephrine, dopamine, and acetylcholine in spinal cord deep dorsal horn neurons.

The deep dorsal horn represents a major site for the integration of spinal sensory information. The bulbospinal monoamine transmitters, released from serotonergic, noradrenergic, and dopaminergic systems, exert modulatory control over spinal sensory systems as does acetylcholine, an intrinsic spinal cord biogenic amine transmitter. Whole cell recordings of deep dorsal horn neurons in the rat spinal cord slice preparation were used to compare the cellular actions of serotonin, norepinephrine, dopamine, and acetylcholine on dorsal root stimulation-evoked afferent input and membrane cellular properties. In the majority of neurons, evoked excitatory postsynaptic potentials were depressed by the bulbospinal transmitters serotonin, norepinephrine, and dopamine. Although, the three descending transmitters could evoke common actions, in some neurons, individual transmitters evoked opposing actions. In comparison, acetylcholine generally facilitated the evoked responses, particularly the late, presumably N-methyl-D-aspartate receptor-mediated component. None of the transmitters modified neuronal passive membrane properties. In contrast, in response to depolarizing current steps, the biogenic amines significantly increased the number of spikes in 14/19 neurons that originally fired phasically (P < 0.01). Together, these results demonstrate that even though the deep dorsal horn contains many functionally distinct subpopulations of neurons, the bulbospinal monoamine transmitters can act at both synaptic and cellular sites to alter neuronal sensory integrative properties in a rather predictable manner, and clearly distinct from the actions of acetylcholine.

Excessive release of [formula omitted]noradrenaline and glutamate in response to simulation of ischemic conditions in rat spinal cord slice preparation: effect of NMDA and AMPA receptor antagonists

In the present study we investigated the effects of NMDA and non-NMDA glutamate receptor antagonists on the ischemia-evoked release of [ 3 H] noradrenaline from rat spinal cord slices. An in vitro ischemia model (oxygen and glucose deprivation) was used to simulate the ischemic conditions known to cause neuronal injury. Spinal cord slices were loaded with [ 3 H] noradrenaline and superfused with Krebs solution in a micro-organ bath. Both axonal stimulation and ischemia increased the release of [ 3 H] noradrenaline, but the release in response to glucose and oxygen deprivation was [Ca 2+] o independent. Dizocilpine (MK-801), an NMDA receptor antagonist, suppressed the release of [ 3 H] noradrenaline produced by ischemia, while it enhanced the release of [ 3 H] noradrenaline evoked by electrical field stimulation. In contrast, LY300168 (GYKI-53655) [(±)-3- N-methylcarbamyde-1-(4-aminophenyl)-4-methyl-1.8-methylene-dioxy-5 H-2.3-benzodiazepine] and its (−)isomer LY303070 (GYKI-53784) [(−)-3- N-methylcarbamyde-1-(4-aminophenyl)-4-methyl-1.8-methylene-dioxy-5 H-2.3-benzodiazepine] AMPA receptor antagonists, had no effect on the release of [ 3 H] noradrenaline evoked by either electrical stimulation or ischemia. Desipramine, a noradrenaline uptake inhibitor, potentiated the release of [ 3 H] noradrenaline evoked by ischemia, while in the absence of [Ca 2+] o but under conditions when [ 3 H] noradrenaline release was further increased, it reduced the release. Dizocilpine also decreased glutamate and aspartate release, measured by high performance liquid chromatography, during ischemia. It is concluded that glutamate release and NMDA receptors, but not AMPA receptors, are involved in the acute effect of oxygen and glucose deprivation on the excessive release of noradrenaline and that this release is not related to physiological axonal conduction.

Effects of the P 2-purinoceptor antagonists suramin and pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid on glutamatergic synaptic transmission in rat dorsal horn neurons of the spinal cord

The effects of suramin and pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) on glutamatergic synaptic transmission were studied on dorsal horn lamina II neurons of rat spinal cord slice preparation and cultured dorsal horn neurons. Suramin at 100 μM significantly suppressed the amplitude of the evoked excitatory postsynaptic currents (EPSCs) by 33%, miniature EPSC (mEPSC) amplitude was decreased by 46% and the mEPSC frequency also decreased by 41%. PPADS at 50 μM had little effect on either the evoked EPSCs or mEPSCs. The lack of effect of PPADS on glutamatergic synaptic transmission suggests that the effect of suramin is less likely to be mediated by P 2x receptors. When whole-cell (±)- α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) currents evoked by glutamate were examined, both suramin and PPADS showed no inhibition of peak amplitude. However, the onset of glutamate-evoked whole-cell currents became significantly slowed by suramin but not by PPADS. The suppression of synaptic transmission by suramin may be due, in part, to the slowed onset of glutamate-evoked AMPA currents. These results suggest that the analgesic effects of suramin shown in cancer patients and animal pain models may not be solely due to its antagonism to purinoceptors. PPADS is probably a more suitable antagonist for the study of synaptic P2 x receptor function at excitatory synapses mediated by AMPA receptors.

Electrophysiological phenomena recorded from spinal cord slice preparation in the adult rat

The dorsal root potential (DRP-V) is believed to have the same origin as the slow positive potential of the segmental spinal cord potential. Furthermore it is also thought to be a reflection of primary afferent depolarization (PAD) which is an index of presynaptic inhibition1. It is believed that primary afferent terminals are depolarized by GABAergic neurons in the substantia gelatinosa (lamina-II). However, intracellular recording from substantia gelatinosa (SG) neurons has been impossible in vivo and very difficult in vitro, because of the high vulnerability of SG neurons to anoxic and mechanical damage in adult rat together with their small size. An in vitro spinal preparation has been developed in the newborn rat. However, it is known that the mode of synaptic transmissions in the adult rat is not the same as in the newborn rat2,3. We therefore developed an in vitro adult rat spinal cord slice preparation to achieve stable intracellular recordings from SG neurons simultaneously with electrotonic potentials from the dorsal root for studying the relationship between them.