Voltage-gated sodium channel expression in rat spiral ganglion neurons.

The spiral ganglion neurons (SGN) provide the afferent innervation of the hair cells in the organ of Corti and relay auditory information from the inner ear to the brain. Voltage-gated sodium channels (Na(V)) initiate and propagate action potentials that encode this sensory information but little is known regarding the subtypes expressed in these cells. We have used RT-PCR and immunohistochemistry to study the compliment and anatomical distribution of Na(V) channels in rodent SGN. Na(V)1.1, Na(V)1.6 and Na(V)1.7 were all detected at the mRNA level. Fluorescence or streptavidin-horseradish peroxidase immunohistochemistry extended these findings, demonstrating predominant localisation of Na(V)1.6 and Na(V)1.7 on SGN cell bodies and Na(V)1.1 on axonal processes. Dual labelling with peripherin demonstrated higher Na(V)1.6 and Na(V)1.7 expression on Type I rather than Type II neurons. These results provide evidence for selective expression and variations in the distribution of VGSC in the rodent SGN, which may guide further studies into afferent function in the auditory pathway and therapeutic approaches for diseases such as hearing loss and tinnitus.

The distribution of small and intermediate conductance calcium-activated potassium channels in the rat sensory nervous system.

Small (SK) and intermediate (IK) conductance calcium-activated potassium channels are candidate ion channels for the regulation of excitability in nociceptive neurones. We have used unique peptide-directed antisera to describe the immunocytochemical distribution of the known isoforms of these ion channels in dorsal root ganglia (DRG) and spinal cord of the rat. These investigations sought to characterize further the phenotype and hence possible functions of nociceptive neurone subpopulations in the rat. In addition, using Western blotting, we sought to determine the level of protein expression of SK and IK channels in sensory nervous tissues following induction of inflammation (Freund's Complete Adjuvant (FCA) arthritis model) or nerve injury (chronic constriction injury model). We show that SK1, SK2, SK3 and IK1 are all expressed in DRG and spinal cord. Morphometric analysis revealed that SK1, SK2 and IK1 were preferentially localized to neurones having cell bodies <1000 microm2 (putative nociceptors) in DRG. Dual labeling immunocytochemistry showed that these ion channels co-localize with both CGRP and IB4, known markers of nociceptor sub-populations. SK2 was localized almost exclusively in the superficial laminae of the spinal cord dorsal horn, the region in which many sensory afferents terminate; the distribution of SK1 and IK1 was more widespread in spinal cord, although some preferential labeling within the dorsal horn was observed in the case of IK1. Here we show evidence for a distinctive pattern of expression for certain members of the calcium-activated potassium channel family in the rat DRG.

Identification of a prostaglandin E2 receptor splice variant and its expression in rat tissues.

The intercellular signalling actions of the lipid mediators, the eicosanoids, are transduced by a family of seven transmembrane domain receptors. Members of this receptor family with high affinity for PGE2 are termed EP receptors. There are four known EP receptor genes that are transcribed to generate EP1, EP2, EP3 and EP4 receptors. Two of these receptor transcripts, EP1 and EP3, are further modified by RNA splicing to give multiple receptor isoforms. The EP3 receptor is known to have multiple splice variants in human (9 variants), cow (4 variants), mouse (3 variants) and rat (3 variants). In the rat the three EP3 splice variants differ in the sequence of the intracellular C-terminus. We have identified a fourth splice variant of the rat prostaglandin EP3 receptor that has a greatly truncated intracellular C-terminus when compared to the other EP3 receptor isoforms. Using nested RT-PCR we have shown that this novel splice variant is strongly expressed in rat brain and is also found in spinal cord, kidney and spleen.

Expression and regulation of prostaglandin E receptor subtype mRNAs in rat sensory ganglia and spinal cord in response to peripheral inflammation.

Prostaglandins are known to act via seven transmembrane domain receptors to exert actions on both peripheral and central neurons resulting in changes in neuronal excitability. Prostaglandin E2, the prostaglandin most often associated with inflammation, itself acts on a family of closely related receptors, the EP receptors. Using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), we have shown that rat primary afferent neurons express the mRNA for all EP receptor subtypes, and that some, but not all EP receptor subtype mRNAs are down-regulated in sensory neurons in response to an acute peripheral inflammation. We also show for the first time that all EP receptor subtype mRNAs are expressed in rat lumbar spinal cord. Spinal cord EP receptor subtype mRNAs are also regulated in acute inflammation in a pattern distinct from the changes seen in sensory ganglia in response to the same inflammatory stimulus.

Cyclooxygenase-1 is a marker for a subpopulation of putative nociceptive neurons in rat dorsal root ganglia.

Immunocytochemical and morphometric techniques were used to quantify the distribution of cyclooxygenase (cox)-containing neurons in rat L5 dorsal root ganglia (DRG). Cox-1 immunolabelling was almost exclusively restricted to small diameter DRG neurons (< 1000 microm2), and was extensively colocalized with calcitonin gene-related peptide (CGRP) and isolectin B4 (IB4). Cox-1 was present in 65% and 70% of CGRP- and IB4-labelled neurons, respectively. Cox-1 labelling was also found in neurons expressing the sensory neuron-specific (SNS) Na+ channel. Cox-2 labelling was absent in DRG from normal rats. In the Freund's adjuvant model of monoarthritis, the proportion of cox-1-positive DRG neurons was unchanged and no neurons were found to be labelled for cox-2. In primary tissue culture, cox-1 immunolabelling persisted in vitro for up to 9 days and was present in morphologically identical neurons. The selective expression of cox-1 in peripheral ganglia was confirmed by the small number of nodose ganglion neurons and superior cervical ganglion (SCG) neurons labelled for cox-1. These data suggest that cox-1 is a marker for a subpopulation of putative nociceptive neurons in vitro and in vivo, and suggests that the prostaglandins synthesized by these neurons may be important for nociceptor function. These data may have important implications for the mode and mechanism of action of non-steroidal anti-inflammatory drugs (NSAIDs).

The neurotoxicity of the venom phospholipases A(2), notexin and taipoxin.

The presynaptically active, toxic phospholipases known as notexin and taipoxin are principal components of the venom of the Australian tiger snake and the Australian taipan respectively. The inoculation of the toxins into one hind limb of rats caused, within 1 h, the depletion of transmitter from the motor nerve terminals of the soleus muscle. This was followed by the degeneration of the motor nerve terminals and of the axonal cytoskeleton. By 24 h 70% of muscle fibers were completely denervated. Regeneration and functional reinnervation were almost fully restored by 5 days, but collateral innervation was common in the regenerated muscles, and this abnormality persisted for at least 9 months. The data provide an explanation for both the severity of neuromuscular paralysis that can accompany envenoming bites by tiger snakes and taipans and the difficulty experienced by physicians in managing the envenomed subjects.

The intraspinal release of prostaglandin E2 in a model of acute arthritis is accompanied by an up-regulation of cyclo-oxygenase-2 in the spinal cord.

In anaesthetized rats, the intraspinal release of immunoreactive prostaglandin E2 was measured using antibody microprobes. We addressed the question of whether the release of immunoreactive prostaglandin E2 is altered during development of acute inflammation in the knee evoked by intra-articular injections of kaolin and carrageenan. We also examined cyclo-oxygenase-1 and cyclo-oxygenase-2 protein levels in the spinal cord during the development of inflammation using the same model of arthritis. Densitometric analysis of microprobes showed that basal release of immunoreactive prostaglandin E2 in the period 175-310 min after kaolin was slightly higher than in the absence of inflammation. A pronounced enhancement of basal release of immunoreactive prostaglandin E2 was observed 430-530 min after kaolin. Enhanced levels of immunoreactive prostaglandin E2 were observed throughout the dorsal and ventral horns. Release of immunoreactive prostaglandin E2 was not altered further by the application of innocuous and noxious pressure onto the inflamed knee. Western blot analysis revealed that cyclo-oxygenase-2 but not cyclo-oxygenase-1 protein levels were elevated in the spinal cords of animals with inflammation compared to normal animals. This effect was evident as early as 3 h after the induction of arthritis. The maximum elevation of cyclo-oxygenase-2 protein levels (six-fold) was observed 12 h after the induction of arthritis. The results show that there is a tonic release of immunoreactive prostaglandin E2 from the spinal cord following the induction of arthritis, which is accompanied by enhanced expression of cyclo-oxygenase-2 protein in the spinal cord. We suggest that intraspinal prostaglandins may play a role in inflammation-evoked central sensitization of spinal cord neurons.

Characterization of cultured dorsal root ganglion neuron P2X receptors.

P2X receptors for adenosine 5'-triphosphate (ATP) comprise a family of ligand-gated cation channels with distinct characteristics which are dependent on the receptor subunits (P2X1-7) expressed, and the homomeric or heteromeric assembly of protein subunits in individual cells. We describe the properties of P2X receptors expressed by cultured adult rat dorsal root ganglion cells on the basis of the time course of responses to ATP, alpha, beta-methylene adenosine 5'-triphosphate (alpha, beta-meATP) and 2-methyl-thioadenosine 5'-triphosphate (2-meSATP), and using the antagonists 2',3'-O-(2,4,6-trinitrophenyl) ATP (TNP-ATP), a novel and highly selective purinoceptor antagonist, suramin and iso-pyridocalphosphate-6-azophenyl-2',5' disulphonic acid (PPADS). ATP (10 microM) evoked inward currents in approximately 95% of neurons tested and > 80% responded with a fast transient inward current that rapidly inactivated during the continued presence of ATP. Of the remaining neurons, approximately 4% showed a sustained response and approximately 10% showed a combination of transient and sustained components. Rapid application of ATP, alpha,beta-meATP and 2meSATP demonstrated these to be full agonists of the rapidly inactivating P2X receptor (pA50 values = 5.83, 5.86 and 5.55, respectively), whilst uridine triphosphate (UTP) and 1-beta,gamma-methyleneadenosine 5'-triphosphate (1-beta,gamma-meATP) were ineffective as agonists. These rapidly inactivating responses could be inhibited by TNP-ATP, suramin and PPADS (pIC50 = 9.5, 6.5, 6.4, respectively). Using inactivation protocols, we demonstrate the presence of homomeric P2X3-like receptors and non-inactivating P2X receptors, which indicates that individual subsets of adult dorsal root ganglion neurons have distinct P2X receptor phenotypes, and that individual DRG neurons may express multiple P2X receptor subtypes.

Prostanoids synthesized by cyclo-oxygenase isoforms in rat spinal cord and their contribution to the development of neuronal hyperexcitability.

1. The responses of wide dynamic range spinal dorsal horn neurones to noxious mechanical stimulation of the ankle or knee joint were tested before and after spinal administration of the non-selective cyclooxygenase (COX) inhibitors, indomethacin and meclofenamic acid. Neither of these drugs altered the responses of these neurones to noxious mechanical stimulation. 2. Wind-up of a spinal nociceptive reflex evoked by electrical stimulation of the sural nerve at C-fibre strength was dose-dependently inhibited by intravenous administration of indomethacin, a non-selective COX inhibitor, and SC58125, a selective COX-2 inhibitor. Intrathecal administration of indomethacin also reduced the wind-up of this nociceptive reflex. 3. Western blot analysis of proteins extracted from normal rat spinal cord revealed the presence of both cyclo-oxygenase (COX)-1 and COX-2 proteins. 4. Immunocytochemistry of sections of normal rat spinal cord with specific COX-1 antiserum revealed little specific COX-1-like immunoreactivity in the grey matter. With the same antiserum, intense COX-1-like immunoreactivity was observed in the cytoplasm, nuclear membrane and axonal processes of small to medium sized (< 1000 microns2) dorsal root ganglion (DRG) cell bodies. 5. Immunocytochemistry of sections of normal rat spinal cord incubated with specific COX-2 antiserum showed intense COX-2-like immunoreactivity (COX-2-li) in the superficial dorsal horn of the spinal cord (laminae I and II) and around the central canal (lamina X). COX-2-li was also observed in some neurones in deep dorsal horn and in individual motor neurones in ventral horn. COX-2-li was not observed in the cell bodies of DRG. 6. Superfusion of the lumbar spinal cord of normal rats with artificial CSF and subsequent radioimmunoassay revealed the presence of prostaglandin D2 (PGD2) < PGE2, but not PGI2 (determined by measurement of the stable metabolite, 6-keto-PGF1 alpha) or PGF2 alpha. 7. These data suggest that eicosanoids synthesized by an active COX pathway in the spinal cord of normal animals may contribute to nociceptive processing, but only when the spinal cord neurones are rendered hyperexcitable following C-fibre stimulation. Selective inhibition of one or both of the COX isoforms in normal animals may represent a novel target for spinal analgesia.

The burst-like firing of spinal neurons in rats with peripheral inflammation is reduced by an antagonist of N-methyl-D-aspartate.

Ankle inflammation was induced in rats by subcutaneous injection of complete Freund's adjuvant and the firing properties of spinal neurons receiving afferent input from the inflamed areas were studied four to six days later. Comparable neurons in normal rats were also studied. In normal animals the response of neurons to ankle compression consisted of a brief burst of action potentials followed by sustained firing during stimulus application. On cessation of the stimulus there was no prolonged afterdischarge. In rats with an inflamed ankle, compression of the ankle produced firing while the stimulus was applied, but with 17 of 22 neurons there was a prolonged (219 +/- 55 s) post-stimulus afterdischarge. All neurons studied in rats with peripheral inflammation fired with intermittent bursts of action potentials, particularly during the afterdischarge and spontaneous firing. The N-methyl-D-aspartate receptor antagonist DL-2-amino-5-phosphonopentanoate was ejected microiontophoretically near the cells studied. The major effect was a near abolition of bursts present in spontaneous firing and post-stimulus afterdischarges with a lesser reduction in firing during stimulus application. Effects on afterdischarge duration were variable. Since firing in bursts is known to increase transmitter release at some sites in the brain, it is proposed that when the relevant spinal neurons fire in bursts, additional intraspinal pathways are recruited and this contributes to the expanded receptive fields of neurons and possibly to the enhanced pain experienced by manipulation of inflamed peripheral tissues.

Lack of effect of microinjection of noradrenaline or medetomidine on stimulus-evoked release of substance P in the spinal cord of the cat: a study with antibody microprobes.

1. Experiments were performed on barbiturate anaesthetized, spinalized cats to investigate the effect of microinjected noradrenaline or medetomidine on the release of immunoreactive substance P in the dorsal spinal cord following peripheral nerve stimulation. The presence of immunoreactive substance P was assessed with microprobes bearing C-terminus-directed antibodies to substance P. 2. Noradrenaline or medetomidine were microinjected into the grey matter of the spinal cord, near microprobe insertion sites, at depths of 2.5, 2.0, 1.5 and 1.0 mm below the spinal cord surface with volumes of approximately 0.125 microliters and a concentration of 10(-3) M. 3. In the untreated spinal cord, electrical stimulation of the ipsilateral tibial nerve (suprathreshold for C-fibres) elicited release of immunoreactive substance P which was centred in and around lamina II. Neither noradrenaline nor medetomidine administration in the manner described produced significant alterations in this pattern of nerve stimulus-evoked release. 4. In agreement with recent ultrastructural studies these results do not support a control of substance P release by catecholamines released from sites near to the central terminals of small diameter primary afferent fibres.

Release of immunoreactive galanin in the spinal cord of rats with ankle inflammation: studies with antibody microprobes.

Antibody microprobes bearing antibodies to the carboxy-terminus of rat galanin were inserted into the spinal cords of anaesthetized normal rats and those in which ankle inflammation had been induced by the unilateral subcutaneous injection of Freund's adjuvant four to six days previously. In normal rats, a basal presence of immunoreactive galanin was detected in the dorsal horn. Similar levels of immunoreactive galanin were found in the dorsal horn of both sides of the spinal cord in animals with unilateral ankle inflammation. Flexing the ankle or compressing the foot in normal rats failed to alter levels of immunoreactive galanin detected by microprobes. In animals with ankle inflammation, prolonged periods of ankle flexion did release immunoreactive galanin in the ipsilateral dorsal horn. Subsequent noxious ankle compression in these animals did not increase but rather decreased immunoreactive galanin in the dorsal horn to below basal levels. The reason for this decrease is unknown but it may represent an inhibition of release or a depletion of spinal stores of galanin.

Neurophysiological evidence for increased kappa opioidergic control of spinal cord neurons in rats with unilateral inflammation at the ankle.

The role of the endogenous kappa opioid system in the control of neuronal activity has been studied in the spinal cord of normal rats and in rats with Freund's adjuvant induced unilateral inflammation of the ankle under barbiturate anaesthesia. During recordings from neurons with ankle input the kappa receptor agonist U50,488H and/or the kappa antagonist nor-binaltorphamine were administered ionophoretically using multibarrel electrodes. In most neurons tested U50,488H reduced the responses evoked by pressure applied across the ankle whereas smaller proportions of neurons showed increased activity or were not affected. The kappa opioid antagonist nor-binaltorphamine affected more neurons in rats with inflammation than in control rats. Ongoing activity was increased in 7 of 19 (37%) neurons in control rats, in 16 of 24 (67%) neurons in the acute phase of inflammation (2 days post inoculation) and in 15 of 23 (65%) neurons in the chronic phase of inflammation (16-20 days post inoculation). During application of nor-binaltorphamine in control rats, the responses to pressure were increased in 9 cells (36%), reduced in 7 cells (28%) and unaffected in 9 cells (36%). In the acute phase of inflammation significantly more neurons (11 of 15, 73%) showed enhanced responses to pressure during ionophoresis of nor-binaltorphamine but not in the chronic phase. These results show that spinal cord neurons with ankle input are influenced by the endogenous kappa opioid system particularly under inflammatory conditions. The upregulation of this system under inflammatory conditions may serve to counteract inflammation-induced hyperexcitability.

Prostanoid-induced potentiation of the excitatory and sensitizing effects of bradykinin on articular mechanonociceptors in the rat ankle joint.

Responses of articular mechanonociceptors to intra-arterial injections of either bradykinin alone or in combination with prostaglandin E2, prostaglandin I2 or the selective I-type prostaglandin receptor agonist cicaprost were investigated electrophysiologically in anaesthetized rats. Bradykinin excited 76% of the mechanonociceptors studied and increased their responsiveness to repeated mechanical stimuli in 70% of units. Tachyphylaxis of these responses was evident in all cases. Injections of minimally effective doses of prostaglandin I2 or cicaprost excited the mechanonociceptors and increased their responsiveness to mechanical stimuli. Injections of prostaglandin E2 evoked only small increases in spontaneous discharge. Potentiation of bradykinin-evoked excitation was seen for combined injections of bradykinin with minimally effective or subthreshold doses of cicaprost in 86% of units, prostaglandin I2 in 40% of units and prostaglandin E2 in 56% of units. Combined injections of bradykinin and minimally effective or subthreshold doses of prostanoid agonist caused an increase in the responsiveness of mechanonociceptors to mechanical stimuli greater than that caused by either drug alone in 80% of units for cicaprost, 80% for prostaglandin I2 and 100% for prostaglandin E2. The relative potencies of prostaglandin I2, cicaprost and prostaglandin E2, suggest that prostanoid-induced excitation and sensitization of articular mechanonociceptors is mediated primarily by receptors for the naturally occurring prostanoid prostaglandin I2 (I-type P-receptors). Prostaglandin E2 may be important in potentiation of the sensitizing effects of bradykinin on mechanonociceptor responsiveness.

Acute and chronic phases of unilateral inflammation in rat's ankle are associated with an increase in the proportion of calcitonin gene-related peptide-immunoreactive dorsal root ganglion cells.

Using immunocytochemical methods, the proportion of calcitonin gene-related peptide immunoreactive perikarya was determined in dorsal root ganglia L4-L6 in four control rats and in ten rats with a unilateral inflammation in the ankle region of the left hindlimb. The inflammation was induced by subdermal injection of Freund's complete adjuvant at the ankle. Swelling and cellular infiltration of the ankle region developed within 2 days, and were stable and restricted to the injected ankle for the duration of the 3-week study. In control rats approximately 24% of 20,419 perikarya showed calcitonin gene-related peptide (CGRP)-like immunoreactivity. In rats with unilateral inflammation the proportion of CGRP-positive neurons was increased on the inflamed side to approximately 32% of 11,454 cells at day 2 (P < 0.001 with respect to ganglia in normal rats) and approximately 29% of 10,739 perikarya at day 20 post inoculation (P < 0.01). By contrast, no significant changes were found between ganglia in the non-injected side (approximately 25% at day 2 and approximately 24% at day 20). These results demonstrate that peripheral inflammation is associated with an increase in the proportion of neurons in the dorsal root ganglia that synthetize CGRP. This up-regulation is already present at an early stage of inflammation but also at later stages, suggesting that the increased synthesis of CGRP is an important neurobiological reaction associated with the acute and chronic phases of inflammation.

Dynamic changes in the receptive field properties of spinal cord neurons with ankle input in rats with chronic unilateral inflammation in the ankle region.

The aim of this study was to determine the discharge and receptive field properties of spinal cord neurons with ankle input in spinal segments L4-6 in the rat, both under control conditions and during the course of an adjuvant-induced unilateral inflammation in the ankle. The extent of receptive fields in the skin and deep tissue was assessed using brush, pinch and compression stimuli. Neurons were categorized as nociceptive-specific or wide-dynamic-range neurons on the basis of their response thresholds and responses to suprathreshold stimuli. At all stages of inflammation (2, 6, 13 and 20 days post inoculation) the population of neurons with ankle input showed differences from the population of neurons with ankle input in control rats. There was a reduction in the number of neurons that appeared as nociceptive specific and a concomitant increase in the number of neurons showing a wide-dynamic-range response profile. The receptive fields of the neurons with ankle input were markedly larger in rats with inflammation in the ankle region and mainly spread proximally on the ipsilateral hindlimb and also to the abdomen and tail in some cases. There was also an increase in the number of neurons with contralateral excitatory inputs. The mechanical thresholds at the ankle joint and proximal parts of the ipsilateral hindlimb were less in arthritic rats than in controls. The proportion of spontaneously active neurons was also increased in rats during the initial and later stages of inflammation, although there was no significant increase in the mean spontaneous discharge frequency. These data show that there are long-term changes in the receptive field and response properties of neurons in intact rats with chronic unilateral adjuvant-induced inflammation similar to those described previously in spinal cats with acute inflammation (Neugebauer and Schaible 1990). It is presumed that similar afferent and spinal mechanisms are at work under acute and chronic inflammatory conditions which produce hyperexcitability in spinal neurons with joint input.

Effects of paracetamol and aspirin on neural activity of joint mechanonociceptors in adjuvant arthritis.

1. The effects of paracetamol and lysine acetylsalicylate (L-AS) on high-threshold mechanonociceptors have been investigated by recording neural activity from the inflamed ankle joint in anaesthetized rats with mild adjuvant-induced monoarthritis. 2. Paracetamol (50 mg kg-1, i.v.) and L-AS (100 mg kg-1, i.v., equivalent to 50 mg kg-1 aspirin) both caused a maximal reduction of about 40% in mechanically-evoked discharge and of 30% in ongoing (spontaneous) activity by about 15 min after the injection: a second dose of either drug did not have any significant additional effect on discharge. 3. The prostanoid IP receptor agonist, cicaprost (0.1-0.5 micrograms), increased both mechanically-evoked and ongoing discharge to pre-paracetamol levels when injected close-arterially 30-50 min after paracetamol, whereas prostaglandin E2 (PGE2) was relatively ineffective at restoring activity. 4. The results suggest that prostacyclin (PGI2) contributes to the sensitization of high-threshold joint mechanonociceptors in adjuvant-induced monoarthritis, and that paracetamol and L-AS both act to reduce discharge by inhibiting the synthesis of prostacyclin in the joint capsule. 5. Paracetamol has a direct peripheral action affecting joint capsule mechanonociceptors in rat adjuvant-induced arthritis which is very similar to that of the soluble aspirin preparation, L-AS. These findings, together with the existing literature concerning the anti-arthritic effects of paracetamol, are relevant to the treatment of chronic inflammatory disorders such as rheumatoid arthritis.