S1 Dorsal Root Ganglia Research Articles

Arylacetamide kappa-opioid receptor agonists produce a tonic- and use-dependent block of tetrodotoxin-sensitive and -resistant sodium currents in colon sensory neurons.

We have previously reported that U50,488 [(trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide] enantiomers contribute to visceral antinociception by a nonopioid receptor-mediated blockade of sodium currents in colon sensory neurons. The present experiments were undertaken to examine the effect of arylacetamide kappa-opioid receptor agonists (kappa-ORAs) U50,488 and EMD 61,753 [(N-methyl-N-[1S)-1-phenyl)-2-(13S))-3-hydroxypyrrolidine-1-yl)-ethyl]-2,2-diphenylacetamide HCl] on tetrodotoxin-sensitive (TTX-S) and -resistant (TTX-R) sodium currents, and the mechanism of their sodium channel-blocking actions. Whole cell patch-clamp experiments were performed on colon sensory neurons from the S1 dorsal root ganglion identified by content of retrograde tracer 1.1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine metanesulfonate. The concentration-response curves of U50,488 and EMD 61,753, for tonic inhibition of total, TTX-S, and TTX-R sodium currents were similar (EC50 values for U50,488 and EMD 61,753 were 8.4 +/- 1.69 and 1.2 +/- 1.78 microM, respectively). In contrast, the peptide kappa-ORA dynorphin was without effect in these experiments. U50,488 (10 microM) shifted the voltage dependence of steady-state inactivation curves for total, TTX-S, and TTX-R currents to more negative potentials. Inhibition was present at holding potentials of -100 to -20 mV. After the tonic block elicited by 10 microM U50,488, repetitive stimulation with 5-ms depolarizing pulses at a frequency of 3 Hz further enhanced the inhibition of total, TTX-R, and TTX-S currents by 43.8 +/- 4.9, 46.2 +/- 4.9, and 40 +/- 3.2%, respectively. These results demonstrate that arylacetamide kappa-ORAs nonselectively inhibit voltage-evoked sodium currents in a manner similar to local anesthetics, by enhancing closed-state inactivation and induction of use-dependent block.

Bladder and cutaneous sensory neurons of the rat express different functional p2x receptors

The expression and functional responses of P2X receptors in bladder and cutaneous sensory neurons of adult rats and mice have been studied using immunohistochemistry and patch clamp techniques. Cell bodies of bladder pelvic afferents were identified in L6 and S1 dorsal root ganglia (DRG), following Fast Blue injection into the muscle wall of the urinary bladder. Similarly, cutaneous sensory neurons were identified in L3 and L4 DRG, following Fast Blue injection into the saphenous nerve innervating the skin. Bladder sensory neurons contained only weak to moderate P2X 3-immunoreactivity (IR), in contrast to strong P2X 3-IR observed in a sub-population of cutaneous afferents. Whole-cell patch-clamp recordings revealed that approximately 90% of bladder afferent neurons responded to αβ-methylene ATP (αβmeATP) and ATP (30 μM) with persistent currents, which were inhibited by 2′,3′- O-trinitrophenyl-ATP (TNP-ATP) (0.3 μM) to 6.4±1.9% and 8.0±2.6% of control, respectively ( n=8). The remaining bladder sensory neurons demonstrated biphasic, transient or no response to P2X agonists. In contrast, only 24% of cutaneous afferent neurons gave persistent currents to αβmeATP (30 μM), with 66% of cells giving transient or biphasic currents and the remaining 10% being non-responsive. Our results suggest that, in contrast to DRG neurons in general, bladder sensory neurons projecting via pelvic nerves express predominantly P2X 2/3 heteromeric receptors, which are likely to mediate the important roles of ATP as a signaling molecule of urinary bladder filling and nociception.

Modulation of action potential firing by iberiotoxin and NS1619 in rat dorsal root ganglion neurons

The present study investigated the effects of iberiotoxin (IbTx), a peptide toxin blocker of large-conductance Ca 2+-activated K + (BK Ca) channels and NS1619, a BK Ca channel opener, on action potential firing of small and medium size afferent neurons from L6 and S1 dorsal root ganglia of adult rats. Application of IbTx (100 nM) reduced whole-cell outward currents in 67% of small and medium size neurons. Analysis of action potential profile revealed that IbTx significantly prolonged the duration of action potential and increased firing frequency of afferent neurons. IbTx did not significantly alter the resting membrane potential, threshold for action potential activation and action potential amplitude. The benzimidazolone NS1619 (10 μM) increased opening activity of a Ca 2+-dependent channel as assessed by single channel measurements. In contrast to IbTx, NS1619 reversibly suppressed action potential firing, attributable to increases in threshold for evoking action potential, reduction in action potential amplitude and increases in amplitude of afterhyperpolarization. The effect of NS1619 on neuronal firing was sensitive to IbTx, indicating the attenuation of neuronal firing by NS1619 was mediated by opening BK Ca channels. NS1619 also reduced neuronal hyperexcitability evoked by 4-aminopyridine (4-AP), a transient-inactivated K + channel (A-current) blocker, in an IbTx-sensitive manner. These results indicate that IbTx-sensitive BK Ca channels exist in both small and medium diameter dorsal root ganglion (DRG) neurons and play important roles in the repolarization of action potential and firing frequency. NS1619 modulates action potential firing and suppresses 4-AP-evoked hyperexcitability in DRG neurons, in part, by opening BK Ca channels. These results suggest that opening BK Ca channels might be sufficient to suppress hyperexcitability of afferent neurons as those evoked by stimulants or by disease states.

Immunoneutralization of Nerve Growth Factor in Lumbosacral Spinal Cord Reduces Bladder Hyperreflexia in Spinal Cord Injured Rats.

We investigated the effects of intrathecal application of nerve growth factor (NGF) antibodies (Ab) on bladder hyperreflexia in chronic spinalized rats. In adult female rats an intrathecal catheter was implanted at the level of the L6 to S1 spinal cord, followed by complete transection of the Th8 to 9 spinal cord. At 10 days after spinalization the intrathecal catheter was connected to an osmotic pump for continuous delivery of vehicle or NGF Ab (10 microg daily) for 2 weeks. Awake cystometry was then performed. NGF levels in the L5 to S1 dorsal root ganglia, L6 spinal cord and bladder were also measured using enzyme-linked immunosorbent assay. The number of uninhibited bladder contractions per voiding cycle, maximal pressure of uninhibited bladder contraction and maximal voiding pressure were significantly decreased in NGF Ab treated versus vehicle treated spinalized rats. Intercontraction interval, baseline intravesical pressure, pressure threshold for voiding and voiding efficiency were not significantly changed by NGF Ab treatment. NGF levels in the bladder, L6 spinal cord and L5 to S1 dorsal root ganglia of vehicle treated spinalized rats was 1.6 to 4.8 times higher than in spinal cord intact rats. After intrathecal NGF Ab treatment NGF levels were significantly lower in the L6 to S1 dorsal root ganglia (30% to 35%) and L6 spinal cord (53%) but not in the bladder or L5 dorsal root ganglia compared with levels in vehicle treated spinalized rats. Increased levels of NGF in the bladder, spinal cord and dorsal root ganglia were associated with bladder hyperreflexia after spinal cord injury. Immuno-neutralization of NGF in the spinal cord suppressed NGF levels in the L6 to S1 dorsal root ganglia, which contain bladder afferent neurons, and also suppressed bladder hyperreflexia. Thus, suppression of NGF levels in afferent pathways could be useful for treating bladder hyperreflexia associated with spinal cord injury.

Cystitis-induced upregulation of tyrosine kinase (TrkA, TrkB) receptor expression and phosphorylation in rat micturition pathways.

This study examined tyrosine kinase receptor (Trk) expression and phosphorylation in lumbosacral dorsal root ganglia (DRG) after acute (8 or 48 hours) or chronic (10 days) cyclophosphamide (CYP)-induced cystitis. Increases in the number of TrkA-immunoreactive (IR) cell profiles were detected in the L1 and L6 DRG (four-fold; P < or = 0.01) and the S1 DRG (1.5-fold; P < or = 0.05) but not in the L2, L4, and L5 DRG with CYP-induced cystitis of acute and chronic duration compared with control rats. The number of TrkB-IR cell profiles increased in the L1 and L2 DRG (L1: 2.6-fold; L2: 1.4-fold; P < or = 0.05) and in the L6 and S1 DRG (L6: 2.2-fold; S1: 1.3-fold; P < or = 0.05) only after acute CYP treatment (8 hours). After CYP treatment, the percentage of bladder afferent cell profiles expressing TrkA-IR (approximately 50%; P < or = 0.05) increased in L1 and L6 DRG. The percentage of bladder afferent cell profiles expressing TrkB-IR (approximately 45%; P < or = 0.05) in L1, L2, L6, and S1 DRG also increased compared with control cell profiles. The increase in TrkA-IR in bladder afferent cells occurred 8 hours after CYP treatment and was maintained in L1 DRG with chronic (10 days) CYP-induced cystitis. However, the increase in bladder afferent cells expressing TrkB-IR only occurred at the most acute time point examined (8 hours). TrkA-IR and TrkB-IR cell profiles also demonstrated phosphorylated Trk-IR with acute and/or chronic CYP-induced cystitis. These results demonstrated that CYP-induced cystitis increases the expression and phosphorylation of Trk receptors in lumbosacral DRG. Expression of neurotrophic factors in the inflamed urinary bladder may contribute to this increased expression, and neurotrophic factor and Trk interactions may play unique roles in decreased urinary tract plasticity with CYP-induced cystitis.

Diabetic Cystopathy Correlates With a Long-Term Decrease in Nerve Growth Factor Levels in The Bladder and Lumbosacral Dorsal Root Ganglia

PURPOSE It has been proposed that a deficiency in the axonal transport of nerve growth factor (NGF) may have an important role in inducing diabetic neuropathy, which contributes to diabetic cystopathy. Therefore, in streptozotocin (Sigma Chemical Co., St. Louis, Missouri) induced diabetic rats we investigated the relationship of bladder function with NGF levels in the bladder and lumbosacral dorsal root ganglia, which contain afferent neurons innervating the bladder. MATERIALS AND METHODS At 6 and 12 weeks after the induction of diabetes with streptozotocin (65 mg./kg. intraperitoneally) the effects of diabetes on Adelta afferent fiber dependent, conscious voiding were evaluated by metabolic cage measurements and awake cystometry. The effects of diabetes on C-fiber mediated bladder nociceptive responses were also investigated by cystometry with intravesical instillation of 0.25% acetic acid in the rats under urethane anesthesia. NGF levels in the bladder and L6 to S1 dorsal root ganglia were measured by enzyme-linked immunosorbent assay 3, 6, 9 and 12 weeks after streptozotocin injection. RESULTS In diabetic rats NGF levels in the bladder and L6 to S1 dorsal root ganglia were significantly decreased 12 weeks after streptozotocin injection (p <0.01). In cystometry and metabolic cage studies bladder capacity and post-void residual volume were significantly increased 12 weeks after streptozotocin injection (p <0.01). Bladder nociceptive responses revealed by a reduction in inter-contraction intervals after acetic acid infusion were significantly decreased in a time dependent manner 12 weeks after streptozotocin injection.CONCLUSIONS Rats with streptozotocin induced diabetes mellitus showed a significant time dependent decrease in NGF levels in the bladder and L6 to S1 dorsal root ganglia that was associated with voiding dysfunction attributable to defects in Adelta and C-fiber bladder afferents. Therefore, reduced production of NGF in the bladder and/or impaired transport of NGF to L6 to S1 dorsal root ganglia, which contain bladder afferent neurons, may be an important mechanism inducing diabetic cystopathy.

Up-regulation of tyrosine kinase (Trka, Trkb) receptor expression and phosphorylation in lumbosacral dorsal root ganglia after chronic spinal cord (T8-T10) injury.

Previous studies have demonstrated changes in urinary bladder neurotrophic factors after bladder dysfunction. We have hypothesized that retrograde transport of neurotrophin(s) from the bladder to lumbosacral dorsal root ganglia (DRG) may play a role in bladder reflex reorganization after spinal cord injury (SCI). In this study, we determined whether the expression of tyrosine kinase receptors (TrkA, TrkB) is altered in lumbosacral DRG after SCI through immunofluorescence techniques. Complete transection of the spinal cord (T8-T10) was performed in female Wistar rats (120-150 g), and animals were studied 5-6 weeks after SCI. One week before killing, Fast Blue (FB) was injected into the bladder to label bladder afferent cells in the L1, L2, L6, and S1 DRG. After SCI, a significant increase in the number of TrkA-immunoreactive (IR) positive cells was detected in the L6-S1 DRG (L6: 1.9-fold, P < or = 0.01; S1: 1.7-fold, P < or = 0.05) and in the L1 DRG (3.0-fold; P < or = 0.01) but not in the L4-L5 DRG compared with spinal-intact (control) rats. After SCI, a significant increase in the number of TrkB-IR cells was also detected in the L6-S1 DRG (L6: 2.2-fold, P < or = 0.01; S1: 1.5-fold, P < or = 0.05) and in the L1-L2 DRG (L1: 1.5-fold, P < or = 0.01; L2: 1.3-fold, P < or = 0.05) but not in the L4-L5 DRG compared with control rats. After SCI, the percentage of FB-labeled cells expressing TrkA immunoreactivity (approximately 68%) or TrkB immunoreactivity (approximately 65%) in L1 and L6 DRG significantly (P < or = 0.01) increased compared with control (20-30%) DRG. After SCI, the percentage of TrkA-IR cells expressing phosphorylated (p)-Trk immunoreactivity significantly increased (1.5- to 2.3-fold increase) in the L1, L6, and S1 DRG. The percentage of TrkB-IR cells expressing p-Trk immunoreactivity after SCI also increased (1.3-fold increase) in the L1 and L6 DRG. These results demonstrate that (1) TrkA and TrkB immunoreactivity is increased in bladder afferent cells after SCI and (2) TrkA and TrkB receptors are phosphorylated in DRG after SCI. Neuroplasticity of lower urinary tract reflexes after SCI may be mediated by both nerve growth factor and brain-derived neurotrophic factor.

Estrogen receptor-α and -β coexist in a subpopulation of sensory neurons of female rat dorsal root ganglia

Immunoreactivities for estrogen receptor-α (ER-α) and ER-β are expressed in sensory neurons of the dorsal root ganglia (DRG). It has not been established, however, if the two receptor subtypes coexist in the same neuron. Double-staining immunohistochemical techniques were used to determine if subpopulations of neurons in the lumbosacral DRG exist based on their content of ERs. Results indicate that some neurons (approximately 17%) of the L6–S1 DRG contain ER-α –, some (approximately 23%) contain ER-β – immunoreactivity and some (approximately 5%) express immunoreactivity for both subtypes of the ER. These results suggest that many sensory neurons can respond to estrogens, but estrogens may produce different morphofunctional effects in different neurons based on their expression of ER subtypes.

Differences in the size of the somatic action potential overshoot between nociceptive and non-nociceptive dorsal root ganglion neurones in the guinea-pig

Intracellular action potentials evoked by dorsal root stimulation were intracellularly recorded from L6 and S1 dorsal root ganglion neurones in deeply anaesthetised guinea-pigs in vivo. Units were classed as C, Aδ or Aα/β units and as nociceptive, low-threshold mechanoreceptive or unresponsive. Units with membrane potentials of at least −40 mV and action potentials with an amplitude of >20 mV were included.Nociceptive neurones had significantly larger somatic action potential overshoots than low-threshold mechanoreceptors in C, Aδ and Aα/β units. A higher proportion of low-threshold mechanoreceptors than of nociceptors had action potentials that failed to overshoot in all conduction velocity groups. 60% of muscle spindle afferents failed to overshoot. The size of the overshoot was correlated positively with log10 action potential duration, log10 action potential rise time, log10 afterhyperpolarisation duration, action potential amplitude and membrane potential and negatively (weakly) with log10 conduction velocity.We conclude that nociceptive neurones are more likely to have somatic action potential overshoots than low-threshold mechanoreceptors in any conduction velocity group. This effect was not due to electrode properties or conduction failure at site(s) of failure of action potential regeneration. Differences in overshoot may affect the influence of neuronal firing on cellular processes. If an overshooting action potential is used as a selection criterion, a bias towards nociceptive neurones is likely to occur. An overshooting action potential coupled with a long afterhyperpolarisation or broad action potential may help in identifying sensory neurones as nociceptive.

Anatomical background of low back pain: variability and degeneration of the lumbar spinal canal and intervertebral disc

Fragestellung: Welche anatomischen Strukturen der lumbalen Wirbelsaule kommen als Auslosefaktoren fur Ruckenschmerz in Frage?

Nitric oxide modulates Ca(2+) channels in dorsal root ganglion neurons innervating rat urinary bladder.

The effect of a nitric oxide (NO) donor on high-voltage-activated Ca(2+) channel currents (I(Ca)) was examined using the whole cell patch-clamp technique in L(6)-S(1) dorsal root ganglion (DRG) neurons innervating the urinary bladder. The neurons were labeled by axonal transport of a fluorescent dye, Fast Blue, injected into the bladder wall. Approximately 70% of bladder afferent neurons exhibited tetrodotoxin (TTX)-resistant action potentials (APs), and 93% of these neurons were sensitive to capsaicin, while the remaining neurons had TTX-sensitive spikes and were insensitive to capsaicin. The peak current density of nimodipine-sensitive L-type Ca(2+) channels activated by depolarizing pulses (0 mV) from a holding potential of -60 mV was greater in bladder afferent neurons with TTX-resistant APs (39.2 pA/pF) than in bladder afferent neurons with TTX-sensitive APs (28.9 pA/pF), while the current density of omega-conotoxin GVIA-sensitive N-type Ca(2+) channels was similar (43-45 pA/pF) in both types of neurons. In both types of neurons, the NO donor, S-nitroso-N-acetylpenicillamine (SNAP) (500 microM), reversibly reduced (23.4-26.6%) the amplitude of I(Ca) elicited by depolarizing pulses to 0 mV from a holding potential of -60 mV. SNAP-induced inhibition of I(Ca) was reduced by 90% in the presence of omega-conotoxin GVIA but was unaffected in the presence of nimodipine, indicating that NO-induced inhibition of I(Ca) is mainly confined to N-type Ca(2+) channels. Exposure of the neurons for 30 min to 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM), an inhibitor of NO-stimulated guanylyl cyclase, prevented the SNAP-induced reduction in I(Ca). Extracellular application of 8-bromo-cGMP (1 mM) mimicked the effects of NO donors by reducing the peak amplitude of I(Ca) (28.6% of reduction). Action potential configuration and firing frequency during depolarizing current pulses were not altered by the application of SNAP (500 microM) in bladder afferent neurons with TTX-resistant and -sensitive APs. These results indicate that NO acting via a cGMP signaling pathway can modulate N-type Ca(2+) channels in DRG neurons innervating the urinary bladder.

Gene therapy of bladder pain with herpes simplex virus (HSV) vectors expressing preproenkephalin (PPE)

Interstitial cystitis (IC) and bladder pain have been a major challenge to understand and treat. We propose a new concept in the treatment of IC and bladder pain. We hypothesize that targeted and localized expression of enkephalin in afferent nerves that innervate the urinary bladder by preproenkephalin (PPE) gene transfer using herpes simplex virus (HSV) vectors can treat bladder pain. SHZ vectors (HSV-1 with lacZ insert, 5 ×108 plaque forming units [pfu]) or SHPE vectors (HSV-1 with PPE cDNA insert, 5 × 108 pfu) were injected into the bladder wall of adult female rats. β-Galactosidase staining, which detects lacZ expression, was observed in the bladder and L6 dorsal root ganglia (DRG) 1 week after bladder injection with SHZ. At 7, 14, and 30 days after bladder injection with SHPE, PPE transgene levels in the bladder and L4, L6, S1 DRGs, which were quantified by PCR techniques using primers specific for human PPE, revealed that PPE transgene expression elevated in the bladder and L6>S1>>L4 DRGs at all time points, consistent with afferent innervation of the rat urinary bladder. Cystometrograms under urethane anesthesia exhibited that intrathecal met-enkephalin (10 μg) blocks bladder hyperactivity induced by intravesical capsaicin (15 μmol/L) in untreated rats. This effect was antagonized by intrathecal naloxone, an opioid antagonist (4 μg). SHPE-injected rats demonstrated a significantly increased intercontraction interval (ICI), compared with SHZ-injected rats (15.1 ± 3.0 vs 6.1 ± 0.97 minutes; P = 0.009). Moreover, reduction in the ICI induced by intravesical capsaicin was significantly smaller in SHPE-injected rats than SHZ-injected rats (24% vs 35% change; P = 0.04). This reduced response to capsaicin in the ICI of SHPE-injected rats was antagonized by intramuscular naloxone (0.5 mg/kg), indicating that enkephalinergic mechanisms were upregulated to suppress capsaicin-induced bladder hyperactivity in SHPE-injected animals. These results suggest that the PPE gene can be transferred and maintained in the bladder and bladder afferent nerves using HSV vectors and that increased expression of enkephalin in bladder afferent pathways can suppress nociceptive responses induced by bladder irritation. This technique of gene transfer may be useful for treating IC and other types of visceral pain.

Total neuronal numbers of rat lumbosacral primary afferent neurons do not change with age

Total cell numbers and neuronal diameters of L6 and S1 dorsal root ganglia, which provide a sensory innervation to pelvic viscera were determined in young adult (3-months-old) and compared to those in old (24-months-old) male rats. Two methods of cell counting, serial (section) reconstructions and total profile counting, were used in this study. Our data showed that the total number of L6 and S1 dorsal root ganglia (DRG) cells and their diameters remain essentially constant from 3 to 24 months of age. These results have shown that rat DRG cell numbers do not change during adult life and that neurogenesis of DRG cells in adult rats or neuronal cell death in aged rats cannot be supported. These findings are also consistent with other data supporting the maintenance of pelvic sensory innervation in old age.

HERPES SIMPLEX VIRUS MEDIATED NERVE GROWTH FACTOR EXPRESSION IN BLADDER AND AFFERENT NEURONS: POTENTIAL TREATMENT FOR DIABETIC BLADDER DYSFUNCTION

Diabetic cystopathy resulting from sensory neuropathy may potentially be treated by direct gene therapy. It has been suggested that nerve growth factor (NGF) has an ameliorative effect in preventing the death in diabetes of afferent dorsal root ganglion neurons, which control bladder function. We investigated NGF gene transfer to the bladder and bladder afferent pathways for treating diabetic cystopathy. We used replication competent and replication defective herpes simplex virus type 1 (HSV-1) vectors that express a functionally active form of the beta-subunit of mouse NGF (beta-NGF) to examine the level and duration of therapeutic gene expression after administration of the vectors. NGF expression during acute (3 days) and latent (21 days) infections was assessed by enzyme-linked immunosorbent assay (ELISA) and immunohistochemical testing after the injection of 1 x 106 to 1 x 108 pfu HSV-NGF expression vectors into the bladder wall of adult rats. HSV vectors with the strong human cytomegalovirus immediate early promoter used to drive beta-NGF gene expression exhibited increased NGF 3 days after infection in the bladder and L6 to S1 dorsal root ganglia, where bladder afferent neurons are located. ELISA analysis revealed that NGF in the bladder tissue and dorsal root ganglia was increased 7 to 9 and 2 to 4-fold, respectively, over the control vector. Increased NGF expression in L6 to S1 dorsal root ganglia neurons was also detected by immunohistochemical staining with antiNGF antibodies. Extended NGF expression was detected by ELISA 21 days after injection. Replication defective vectors containing HSV-1 latency promoter (LAP-2) driving NGF expressed NGF in the bladder and dorsal root ganglia 21 days after bladder injection. ELISA analysis confirmed an approximate 2 to 3-fold increase of NGF expression in the bladder and L6 to S1 dorsal root ganglia. The NGF gene may be transferred and expressed in the bladder and bladder afferent pathways using HSV vectors. To our knowledge our study represents the first demonstration of the effectiveness of gene therapy for altering neurotrophic expression in visceral sensory neurons. This technique of gene transfer may be useful for treating certain types of neurogenic bladder dysfunction, such as diabetic cystopathy, in which decreased NGF transport may be a causative factor.

Increased conduction velocity of nociceptive primary afferent neurons during unilateral hindlimb inflammation in the anaesthetised guinea-pig

Decreases in durations of action potentials (C- and Aδ-fibre units) and afterhyperpolarisations (A-fibre units) occur in somata of nociceptive dorsal root ganglion neurons during hindlimb inflammation induced in young guinea-pigs by intradermal injections of Complete Freund’s Adjuvant into the ipsilateral leg and foot. Here we present evidence that the single-point conduction velocity (i.e. estimated over a single conduction distance) of these nociceptive neurons is increased during this type of inflammation. The single-point conduction velocities in anaesthetised untreated guinea-pigs (control) were compared with those two and four days after Complete Freund’s Adjuvant treatment in two types of experiment. The first involved intracellular voltage recordings from somata of ipsilateral L6 and S1 dorsal root ganglion neurons. Units were classified as C, Aδ or Aα/β on the basis of their dorsal root conduction velocities and characterised as nociceptive, low-threshold mechanoreceptive or unresponsive according to their responses to mechanical and thermal stimuli. Compared with untreated animals, significant increases of 54% for C-fibre nociceptive units and 46% for A-fibre nociceptive units in the medians of dorsal root single-point conduction velocities were found four days after Complete Freund’s Adjuvant treatment. These increases were greater at four days than at two days after Complete Freund’s Adjuvant. A slight tendency in the same direction (10%) that was not significant was also seen in low-threshold mechanoreceptors four days after treatment, but not after two days. The increased velocities were confirmed with compound action potential recordings from ipsilateral S2 dorsal roots and sural nerves, in treated and control animals. Recordings showed a tendency for increased single-point velocities in C, Aδ and Aα/β waves, with the upper border of the Aδ wave (i.e. the border between Aδ and Aα/β waves) falling at a significantly higher conduction velocity in treated than control animals. This was seen both in S2 dorsal roots and in sural nerves. There was also a significant decrease in the mean electrical threshold for eliciting the C and Aδ components of compound action potentials of both dorsal root and sural nerves during inflammation. No evidence was found for a reduction in utilisation time for any components of the sural nerve compound action potential (C, Aδ or Aα/β). The conduction velocity increases may be due to altered expression or activation/inactivation of certain ion channel types, such as Na + channels. The present experiments demonstrate that hindlimb inflammation caused a significant increase in conduction velocity of nociceptive but not of low-threshold mechanoreceptive primary afferent neurons during inflammation, as well as a significant decrease in the mean electrical threshold for eliciting the C and Aδ components of compound action potentials of both dorsal root and sural nerves. These changes, together with the previously described changes in the action potential shape of nociceptive neurons during inflammation, probably reflect alterations in membrane function that contribute to inflammatory hyperalgesia.

Glial cell line-derived neurotrophic factor is expressed in penis of adult rat and retrogradely transported in penile parasympathetic and sensory nerves.

Glial cell line-derived neurotrophic factor (GDNF), a member of the GDNF family of neurotrophic factors, promotes the survival and function of several neuronal populations in the peripheral and central nervous system. In the present study, expression of GDNF mRNA in the shaft of adult rat penis is demonstrated. In situ hybridization revealed GDNF mRNA expression in cells lying in the narrow zone between the tunica albuginea and the cavernous tissue. Most subtunical cells exhibited immunoreactivity for vimentin and S100 beta, but they did not stain for smooth muscle alpha actin or PGP9.5. This suggests that the GDNF mRNA-expressing cells may have a mesenchymal origin. Also retrograde axonal transport of intracavernously injected 125I-labeled GDNF in penile parasympathetic and sensory neurons is shown. The transport was inhibited by excess unlabeled GDNF, whereas excess cytochrome c had no effect. This is in agreement with the view that the transport was mediated by binding to specific receptors located on axon terminals. In addition, this study demonstrates expression of GDNF family receptor-alpha 3 (GFR alpha 3) mRNA in most adrenergic, but only in a minor part (5.3%) of the penis-projecting adult rat major pelvic ganglion neurons, as well as in almost half (45.6%) of the penile S1 dorsal root ganglion neurons. In conclusion, the present data suggest that GDNF may act as a neurotrophic factor for subpopulations of adult rat penile parasympathetic and sensory neurons.

Mechanosensitive potassium channels in rat colon sensory neurons.

Single-channel recording techniques were used to characterize mechanosensitive channels in identified (1.1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine methanesulfonate labeled) colon sensory neurons dissociated from adult S1 dorsal root ganglia. Channels were found in 30% (7/23) of patches in a cell-attached configuration and in 43% (48/111) of excised inside-out patches. Channels were highly selective for K(+), had a slope conductance of 54 pS in symmetrical solutions, and were blocked by tetraethylammonium, amiloride, and benzamil. Channels were also seen under Ca(2+)-free conditions. Gadolinium (Gd(3+)), a known blocker of mechanosensitive ion channels, did not block channel activity. Tetrodotoxin and 4-aminopyridine were also ineffective. The cytoskeletal disrupters colchicine and cytochalasin D reduced the percentage of patches containing mechanosensitive channels. These results indicate that rat colon sensory neurons contain K(+)-selective mechanosensitive channels that may modulate the membrane excitability induced by colonic distension.

Changes in somatic action potential shape in guinea-pig nociceptive primary afferent neurones during inflammation in vivo.

We have examined whether there are changes during inflammation in the membrane properties of nociceptive primary afferent neurones in the guinea-pig that might contribute to hyperalgesia. Inflammation was induced by intradermal injections of complete Freund's adjuvant (CFA) in the left leg. Intracellular voltage recordings were made from the somata of ipsilateral L6 and S1 dorsal root ganglion neurones in anaesthetised untreated guinea-pigs at 2 or 4 days after CFA treatment. 2. Units were classified as C, Adelta or Aalpha/beta on the basis of their dorsal root conduction velocities (CVs). Units with receptive fields on the left leg were characterized as nociceptive, low- threshold mechanoreceptive (LTM) or unresponsive according to their responses to mechanical and thermal stimuli. The shapes of their somatic action potentials (APs) evoked by dorsal root stimulation were recorded. 3. Comparisons of data from nociceptive neurones recorded in CFA treated animals after 2 and 4 days with data from CFA untreated (control) animals showed the following significant changes: in C-fibre nociceptors, decreased AP duration at base, AP rise time and AP fall time, and increased maximum rates of AP rise and fall with no change in afterhyperpolarization measured to 80 % recovery (AHP80); in Adelta-fibre nociceptors, decreased AP duration at base, AP fall time and a reduction in AHP80; and in Aalpha/beta-fibre nociceptors, a decreased AHP80 but no change in AP duration. Apart from a more negative membrane potential and AHP depth below 0 mV in Aalpha/beta nociceptors at 4 days compared with 2 days post-CFA, none of the above variables differed significantly between units recorded 2 or 4 days after CFA. Therefore the two groups were pooled and called CFA2 + 4d. 4. The reduction in AP duration in C-fibre nociceptors was apparent both in high threshold mechanoreceptor and polymodal nociceptors and also in units with either cutaneous or subcutaneous receptive fields. 5. No significant changes in AP duration at base or AHP80 were seen 2 or 4 days after CFA compared with control in either LTM or unresponsive neurones, although some of the latter may have become classified as nociceptors after CFA treatment. 6. The alterations in membrane properties of nociceptors should permit higher discharge frequencies, thus contributing to inflammatory hyperalgesia. They suggest active changes in the expression or activation of cation channels during peripheral inflammation.

Primary afferent fibers of the pelvic nerve terminate in the gracile nucleus of the rat

After injection of horseradish peroxidase (HRP) into the pelvic nerve of the rat, a small number of HRP-labeled axon terminals were found in the gracile nucleus. Double labeling experiments were also performed: Fluoro-Gold (FG) was injected into the pelvic nerve, while cholera toxin B subunit (CTb) was injected into the gracile nucleus or dorsal faciculus at the fifth and sixth cervical cord segments ipsilateral to the FG injection. About 5% of FG-labeled neurons were labeled with CTb in the L6-, S1- and S2-dorsal root ganglia ipsilateral to the tracer injection. The results suggest that some primary afferent information from pelvic visceral organs may be directly conveyed to gracile nucleus by the primary afferent neurons.

Is sympathetic sprouting in the dorsal root ganglia responsible for the production of neuropathic pain in a rat model?

Partial peripheral nerve injury often results in neuropathic pain that is aggravated by sympathetic excitation and induces sympathetic nerve sprouting in both the injured nerve and corresponding dorsal root ganglia (DRGs). Presently, the functional mechanisms of the interactions between the sprouting and injured somatic afferents remain uncertain. This study was performed to see whether the sprouting in the DRGs plays a key role in the development of neuropathic pain. To this aim, we compared two groups of rats, both of which were subjected to unilateral transection of the superior and inferior caudal trunks at the level between the S1 and S2 spinal nerves, with respect to sympathetic fiber sprouting; one group showed well-developed neuropathic pain behaviors (i.e. mechanical, cold and warm allodynia signs) and the other group showed poorly-developed ones. Immuno-histochemical staining with tyrosine hydroxylase (TH) antibody of the injured S1 DRG taken from both groups of rats after behavioral tests revealed that the magnitude of penetration of TH-positive fibers into the S1 DRG was not significantly different between the two groups. These results suggest that sympathetic nerve sprouting in the injured DRG is not a key factor in the development of neuropathic pain.