Membrane potential oscillations are not essential for spontaneous firing generation in L4 Aß-afferent neurons after L5 spinal nerve axotomy and are not mediated by HCN channels.

NEW FINDINGS: What is the central question of this study? Is spontaneous activity (SA) in L4 dorsal root ganglion (DRG) neurons induced by L5 spinal nerve axotomy associated with membrane potential oscillations in these neurons, and if so, are these membrane oscillations mediated by HCN channels? What is the main finding and its importance? Unlike injured L5 DRG neurons, which have been shown to be incapable of firing spontaneously without membrane potential oscillations, membrane potential oscillations are not essential for SA generation in conducting 'uninjured' L4 neurons, and they are not mediated by HCN channels. These findings suggest that the underlying cellular mechanisms of SA in injured and 'uninjured' DRG neurons induced by spinal nerve injury are distinct. ABSTRACT: The underlying cellular and molecular mechanisms of peripheral neuropathic pain are not fully understood. However, preclinical studies using animal models suggest that this debilitating condition is driven partly by aberrant spontaneous activity (SA) in injured and uninjured dorsal root ganglion (DRG) neurons, and that SA in injured DRG neurons is triggered by subthreshold membrane potential oscillations (SMPOs). Here, using in vivo intracellular recording from control L4-DRG neurons, and ipsilateral L4-DRG neurons in female Wistar rats that had previously undergone L5 spinal nerve axotomy (SNA), we examined whether conducting 'uninjured' L4-DRG neurons in SNA rats exhibit SMPOs, and if so, whether such SMPOs are associated with SA in those L4 neurons, and whether they are mediated by hyperpolarization-activated cyclic nucleotide gated (HCN) channels. We found that 7 days after SNA: (a) none of the control A- or C-fibre DRG neurons showed SMPOs or SA, but 50%, 43% and 0% of spontaneously active cutaneous L4 Aß-low threshold mechanoreceptors, Aß-nociceptors and C-nociceptors exhibited SMPOs, respectively, in SNA rats with established neuropathic pain behaviors; (b) neither SMPOs nor SA in L4 Aß-neurons was suppressed by blocking HCN channels with ZD7288 (10 mg kg-1 , i.v.); and (c) there is a tendency for female rats to show greater pain hypersensitivity than male rats. These results suggest that SMPOs are linked to SA only in some of the conducting L4 Aß-neurons, that such oscillations are not a prerequisite for SA generation in those L4 A- or C-fibre neurons, and that HCN channels are not involved in their electrogenesis.

Inducible nitric oxide synthase inhibition by 1400W limits pain hypersensitivity in a neuropathic pain rat model.

NEW FINDINGS: What is the central question of this study? Can modulation of inducible NO synthase reduce pain behaviour and pro-inflammatory cytokine signalling in a rat model of neuropathic pain? What is the main finding and its importance? Nitric oxide synthase-based therapies could be effective for the treatment of peripheral neuropathic pain. ABSTRACT: Peripheral neuropathic pain (PNP), resulting from injury to or dysfunction of a peripheral nerve, is a major health problem that affects 7-8% of the population. It is inadequately controlled by current drugs and is characterized by pain hypersensitivity, which is believed to be attributable to sensitization of peripheral and CNS neurons by various inflammatory mediators. Here we examined, in a rat model of PNP: (i) whether reducing levels of nitric oxide (NO) with 1400W, a highly selective inhibitor of inducible NO synthase (iNOS), would prevent or attenuate pain hypersensitivity; and (ii) the effects of 1400W on plasma concentrations of several cytokines that are secreted after iNOS upregulation during chronic pain states. The L5 spinal nerve axotomy (SNA) model of PNP was used, and 1400W (20 mg kg-1 ) was administered i.p. at 8 h intervals for 3 days starting at 18 h post-SNA. Changes in plasma concentrations of 12 cytokines in SNA rats treated with 1400W were examined using multiplex enzyme-linked immunosorbent assay. The SNA rats developed behavioural signs of mechanical and heat hypersensitivity. Compared with the vehicle/control, 1400W significantly: (i) limited development of mechanical hypersensitivity at 66 h post-SNA and of heat hypersensitivity at 42 h and at several time points tested thereafter; and (ii) increased the plasma concentrations of interleukin (IL)-1α, IL-1ß and IL-10 in the SNA rats. The findings suggest that 1400W might exert its analgesic effects by reducing iNOS and altering the balance between the pro-inflammatory (IL-1ß and IL-1α) and anti-inflammatory (IL-10) cytokines and that therapies targeting NO or its enzymes might be effective for the treatment of PNP.

Blocking of cytokines signalling attenuates evoked and spontaneous neuropathic pain behaviours in the paclitaxel rat model of chemotherapy-induced neuropathy.

BACKGROUND: Chemotherapy-induced peripheral neuropathic pain (CIPNP) is a serious dose-limiting neurotoxic effect of cancer drug treatment. The underlying mechanism(s) of this debilitating condition, which lacks effective drug treatment, is incompletely understood. However, neural-immune interactions, involving increased expression and release of cytokines, are believed to be involved. Here, we examined, in the paclitaxel rat model of CIPNP, whether plasma levels of 24 cytokines/chemokines change after paclitaxel treatment, and whether blocking of signalling of some of those cytokines would reverse/attenuate behavioural signs of CIPNP. METHODS: To achieve these objectives luminex, pharmacological and behavioural experiments were performed on male Wistar rats (250-300 g) 31 days after the last injection of paclitaxel (1 mg/kg, i.p. on four alternate days) as well as on control (vehicle-treated) rats. RESULTS: Compared with control rats, plasma levels of IL-1α, IL-1ß, IL-6, TNF-α, INF-γ and MCP-1 were significantly upregulated in paclitaxel-treated rats. Blocking of TNF-α signalling with etanercept (2 mg/kg, i.p.) or IL-1ß with IL-1 receptor antagonist (IL-1ra; 3 mg/kg, i.p.), significantly attenuated established mechanical and cold hypersensitivity as well as spontaneous pain behaviour (spontaneous foot lifting) 24 and 48 h postdrug treatment. Pharmacological blockade of MCP-1/CCL2 signalling with a highly selective CCR2 receptor antagonist (S504393, 5 mg/kg, i.p.) also significantly reduced evoked, but not spontaneous, pain behaviours of CIPNP in paclitaxel-treated rats at the same time points. CONCLUSIONS: The findings support the notion that cytokines/chemokines, particularly TNF-α, IL-1 and MCP-1, are involved in the pathophysiology of CIPNP and suggest that strategies that target their inhibition may be effective in treating CIPNP. SIGNIFICANCE: This study demonstrates that paclitaxel-treated rats exhibit, in addition to indices of mechanical and cold hypersensitivity, a behavioural sign of spontaneous pain, the principal compliant of patients with neuropathic pain. This was accompanied by upregulation in plasma levels of key cytokines/chemokines (IL-1α, IL-1ß, IL-6, TNF-α, INF-γ and MCP-1) 31 days post-treatment. However, it is noteworthy that cytokine release, rather than nerve injury per se, may be causative of NP in this model of CIPNP. Nevertheless, our findings that pharmacological blockade of TNF-α, IL-1ß and MCP-1 attenuated both evoked and spontaneous pain suggest that strategies that target inhibition of these cytokines may be effective in treating CIPNP.

Persistent hindlimb inflammation induces changes in activation properties of hyperpolarization-activated current (Ih) in rat C-fiber nociceptors in vivo.

A hallmark of chronic inflammation is hypersensitivity to noxious and innocuous stimuli. This inflammatory pain hypersensitivity results partly from hyperexcitability of nociceptive dorsal root ganglion (DRG) neurons innervating inflamed tissue, although the underlying ionic mechanisms are not fully understood. However, we have previously shown that the nociceptor hyperexcitability is associated with increased expression of hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) protein and hyperpolarization-activated current (Ih) in C-nociceptors. Here we used in vivo voltage-clamp and current-clamp recordings, in deeply anesthetized rats, to determine whether activation properties of Ih in these C-nociceptors also change following persistent (not acute) hindlimb inflammation induced by complete Freund's adjuvant (CFA). Recordings were made from lumbar (L4/L5) C-nociceptive DRG neurons. Behavioral sensory testing was performed 5-7days after CFA treatment, and all the CFA-treated group showed significant behavioral signs of mechanical and heat hypersensitivity, but not spontaneous pain. Compared with control, C-nociceptors recorded 5-7days after CFA showed: (a) a significant increase in the incidence of spontaneous activity (from ∼5% to 26%) albeit at low rate (0.14±0.08Hz (Mean±SEM); range, 0.01-0.29Hz), (b) a significant increase in the percentage of neurons expressing Ih (from 35%, n=43-84%, n=50) based on the presence of voltage "sag" of >10%, and (c) a significant increase in the conductance (Gh) of the somatic channels conducting Ih along with the corresponding Ih,Ih, activation rate, but not voltage dependence, in C-nociceptors. Given that activation of Ih depolarizes the neuronal membrane toward the threshold of action potential generation, these changes in Ih kinetics in CFA C-nociceptors may contribute to their hyperexcitability and thus to pain hypersensitivity associated with persistent inflammation.

Increased expression of HCN2 channel protein in L4 dorsal root ganglion neurons following axotomy of L5- and inflammation of L4-spinal nerves in rats.

A hallmark of peripheral neuropathic pain (PNP) is chronic spontaneous pain and/or hypersensitivity to normally painful stimuli (hyperalgesia) or normally nonpainful stimuli (allodynia).This pain results partly from abnormal hyperexcitability of dorsal root ganglion (DRG) neurons. We have previously shown, using a modified version of the lumbar 5 (L5)-spinal nerve ligation model of PNP (mSNA model involving L5-spinal nerve axotomy plus loose ligation of the lumbar 4 (L4)-spinal nerve with neuroinflammation-inducing chromic-gut), that L4 DRG neurons exhibit increased spontaneous activity, the key characteristic of neuronal hyperexcitability. The underlying ionic and molecular mechanisms of the hyperexcitability of L4 DRG neurons are incompletely understood, but could result from changes in expression and/or function of ion channels including hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which are active near the neuron's resting membrane potential, and which produce an excitatory inward current that depolarizes the membrane potential toward the threshold of action potential generation. Therefore, in the present study we used the mSNA model to investigate whether: (a) expression of HCN1-HCN3 channels is altered in L4 DRG neurons which, in the mSNA model, are essential for transmission of the evoked pain, and which contribute to chronic spontaneous pain, and (b) local (intraplantar) blockade of these HCN channels, with a specific blocker, ZD7288, attenuates chronic spontaneous pain and/or evoked pain in mSNA rats. We found 7days after mSNA: (1) a significant increase in HCN2-immunoreactivity in small (<30µm) DRG neurons (predominantly IB4-negative neurons), and in the proportion of small neurons expressing HCN2 (putative nociceptors); (2) no significant change in HCN1- or HCN3-immunoreactivity in all cell types; and (3) attenuation, with ZD7288 (100µM intraplantar), of chronic spontaneous pain behavior (spontaneous foot lifting) and mechanical, but not, heat hypersensitivity. The results suggest that peripheral HCN channels contribute to mechanisms of spinal nerve injury-induced PNP, and that HCN channels, possibly HCN2, represent a novel target for PNP treatment.

Expression and properties of hyperpolarization-activated current in rat dorsal root ganglion neurons with known sensory function.

The hyperpolarization-activated current (I(h)) has been implicated in nociception/pain, but its expression levels in nociceptors remained unknown. We recorded I(h) magnitude and properties by voltage clamp from dorsal root ganglion (DRG) neurons in vivo, after classifying them as nociceptive or low-threshold-mechanoreceptors (LTMs) and as having C-, Aδ- or Aα/ß-conduction velocities (CVs). For both nociceptors andLTMs, I(h) amplitude and I(h) density (at -100 mV) were significantly positively correlated with CV.Median I(h) magnitudes and I(h) density in neuronal subgroupswere respectively:muscle spindle afferents(MSAs):-4.6 nA,-33 pA pF(-1); cutaneous Aα/ß LTMs: -2.2 nA, -20 pA pF(-1); Aß-nociceptors: -2.6 nA, -21 pA pF(-1); both Aδ-LTMs and nociceptors: -1.3 nA, ∼-14 pA pF(-1); C-LTMs: -0.4 nA, -7.6 pA pF(-1); and C-nociceptors: -0.26 nA, -5 pApF(-1). I(h) activation slow time constants (slow τ values) were strongly correlated with fast τ values; both were shortest in MSAs. Most neurons had τ values consistent with HCN1-related I(h); others had τ values closer to HCN1+HCN2 channels, or HCN2 in the presence of cAMP. In contrast, median half-activation voltages (V(0.5)) of -80 to -86 mV for neuronal subgroups suggest contributions of HCN2 to I(h). τ values were unrelated to CV but were inversely correlated with I(h) and I(h) density for all non-MSA LTMs, and for Aδ-nociceptors. From activation curves ∼2-7% of I(h)would be activated at normal membrane potentials. The high I(h) may be important for excitability of A-nociceptors (responsible for sharp/pricking-type pain) and Aα/ß-LTMs (tactile sensations and proprioception). Underlying HCN expression in these subgroups therefore needs to be determined. Altered high I(h) may be important for excitability of A-nociceptors (responsible for sharp/pricking-type pain) and Aα/ß-LTMs (tactile sensations and proprioception). Underlying HCN expression in these subgroups therefore needs to be determined. Altered Ih expression and/or properties (e.g. in chronic/pathological pain states) may influence both nociceptor and LTM excitability.expression and/or properties (e.g. in chronic/pathological pain states) may influence both nociceptor and LTM excitability.

Immunostaining for the α3 isoform of the Na+/K+-ATPase is selective for functionally identified muscle spindle afferents in vivo.

Muscle spindle afferent (MSA) neurons can show rapid and sustained firing. Immunostaining for the α3 isoform of the Na(+)/K(+)-ATPase (α3) in some large dorsal root ganglion (DRG) neurons and large intrafusal fibres suggested α3 expression in MSAs (Dobretsov et al. 2003), but not whether α3-immunoreactive DRG neuronal somata were exclusively MSAs. We found that neuronal somata with high α3 immunointensity were neurofilament-rich, suggesting they have A-fibres; we therefore focussed on A-fibre neurons to determine the sensory properties of α3-immunoreactive neurons. We examined α3 immunointensity in 78 dye-injected DRG neurons whose conduction velocities and hindlimb sensory receptive fields were determined in vivo. A dense perimeter or ring of staining in a subpopulation of neurons was clearly overlying the soma membrane and not within satellite cells. Neurons with clear α3 rings (n = 23) were all MSAs (types I and II); all MSAs had darkly stained α3 rings, that tended to be darker in MSA1 than MSA2 units. Of 52 non-MSA A-fibre neurons including nociceptive and cutaneous low-threshold mechanoreceptive (LTM) neurons, 50 had no discernable ring, while 2 (Aα/ß cutaneous LTMs) had weakly stained rings. Three of three C-nociceptors had no rings. MSAs with strong ring immunostaining also showed the strongest cytoplasmic staining. These findings suggest that α3 ring staining is a selective marker for MSAs. The α3 isoform of the Na(+)/K(+)-ATPase has previously been shown to be activated by higher Na(+) levels and to have greater affinity for ATP than the α1 isoform (in all DRG neurons). The high α3 levels in MSAs may enable the greater dynamic firing range in MSAs.

Electrophysiological differences between nociceptive and non-nociceptive dorsal root ganglion neurones in the rat in vivo.

Intracellular recordings were made from 1022 somatic lumbar dorsal root ganglion (DRG) neurones in anaesthetized adult rats, classified from dorsal root conduction velocities (CVs) as C, Adelta or Aalpha/beta, and according to their responses to mechanical and thermal stimuli as nociceptive (including high-threshold mechanoreceptive (HTM) units), and non-nociceptive (including low-threshold mechanoreceptive (LTM) and cooling units). Of these, 463 met electrophysiological criteria for analysis of action potentials (APs) evoked by dorsal root stimulation. These included 47 C-, 71 Adelta- and 102 Aalpha/beta-nociceptive, 10 C-, 8 Adelta- and 178 Aalpha/beta-LTM, 18 C- and 19 Adelta- unresponsive, and 4 C-cooling units. Medians of AP and afterhyperpolarization (AHP) durations and AP overshoots were significantly greater for nociceptive than LTM units in all CV groups. AP overshoots and AHP durations were similar in nociceptors of all CV groups whereas AP durations were greater in slowly conducting, especially C-fibre, nociceptors. C-cooling units had faster CVs, smaller AP overshoots and shorter AP durations than C-HTM units. A subgroup of Aalpha/beta-HTM, moderate pressure units, had faster CVs and AP kinetics than other Aalpha/beta-HTM units. Of the Aalpha/beta-LTM units, muscle spindle afferents had the fastest CV and AP kinetics, while rapidly adapting cutaneous units had the slowest AP kinetics. AP variables in unresponsive and nociceptive units were similar in both C- and Adelta-fibre CV groups. The ability of fibres to follow rapid stimulus trains (fibre maximum following frequency) was correlated with CV but not sensory modality. These findings indicate both the usefulness and limitations of using electrophysiological criteria for identifying neurones acutely in vitro as nociceptive.

Spinal nerve injury increases the percentage of cold-responsive DRG neurons.

We tested the hypothesis that cold allodynia, observed following nerve injury reflects change(s) in the cold responsiveness of sensory neurons. To test this hypothesis we assessed the impact of the spinal nerve ligation (SNL) model of nerve injury on the responses of cutaneous sensory neurons to cooling in vitro. Nerve injury induced a significant increase in the incidence of cold responsive cutaneous neurons in uninjured but not injured ganglia. Because an increase in the percentage of cold responsive neurons in uninjured ganglia should increase the total neuronal response to cooling of peripheral tissue, these findings suggest that cold allodynia reflects, at least in part, a change in sensory neurons.

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, Adelta or Aalpha/beta 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, Adelta and Aalpha/beta 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 log(10) action potential duration, log(10) action potential rise time, log(10) afterhyperpolarisation duration, action potential amplitude and membrane potential and negatively (weakly) with log(10) 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.

Time course and nerve growth factor dependence of inflammation-induced alterations in electrophysiological membrane properties in nociceptive primary afferent neurons.

Novel findings of changes in nociceptive dorsal root ganglion (DRG) neurons during hindlimb inflammation induced by complete Freund's adjuvant (CFA) injections in the hindpaw and hindleg are reported. These include increased maximum fiber following frequency in nociceptive C- and Adelta-fiber units by 2.7 and 3 times, respectively, and increased incidence of ongoing (spontaneous) activity by 3.3 times (to 54%) and 2.4 times (to 27%), respectively. These changes and the CFA-induced changes in somatic action potential (AP) configuration in nociceptive neurons (Djouhri and Lawson, 1999) were incomplete 24 hr after CFA. The nerve growth factor (NGF) dependence of the inflammation-induced changes was examined by injecting a synthetic NGF sequestering protein [tyrosine receptor kinase A Ig2 (trkA Ig2)] with CFA and subsequently into the CFA injection sites. NGF sequestration prevented some CFA-induced changes in nociceptive neurons including: the increased fiber following frequency (C and Adelta), the increased proportions of units with ongoing activity (C and Adelta), the decreased AP duration (C and Adelta), but not the decreased afterhyperpolarization (AHP) durations (C, Adelta, and Aalpha/beta) (Djouhri and Lawson, 1999). AP variables of nociceptive units with spontaneous activity were examined. The time course of electrophysiological changes in nociceptive units is consistent with processes involving altered protein expression and/or retrograde transport of factors. These results (1) implicate NGF in regulating inflammation-induced decreases in AP duration and in increases in firing rate and spontaneous activity but not in decreases in AHP duration and (2) suggest clinical advantages of reducing NGF in some inflammatory pain states.

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 Adelta-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, Adelta or Aalpha/beta 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, Adelta and Aalpha/beta waves, with the upper border of the Adelta wave (i.e. the border between Adelta and Aalpha/beta 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 Adelta 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, Adelta or Aalpha/beta). 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 Adelta 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.

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.

Association of somatic action potential shape with sensory receptive properties in guinea-pig dorsal root ganglion neurones.

1. Intracellular voltage recordings were made from the somata of L6 and S1 dorsal root ganglion (DRG) neurones at 28.5-31 C in young guinea-pigs (150-300 g) anaesthetized with sodium pentobarbitone. Action potentials (APs) evoked by dorsal root stimulation were used to classify conduction velocities (CVs) as C, Adelta or Aalpha/beta. Units with overshooting APs and membrane potentials (Vm) more negative than -40 mV were analysed: 40 C-, 45 Adelta- and 94 Aalpha/beta-fibre units. 2. Sensory receptive properties were characterized as: (a) low-threshold mechanoreceptive (LTM) units (5 C-, 10 Adelta- and 57 Aalpha/beta-fibre units); (b) nociceptive units, responding to noxious mechanical stimuli, some also to noxious heat (40 C-, 27 Adelta- and 27 Aalpha/beta-fibre units); (c) unresponsive units that failed to respond to a variety of tests; and (d) C-fibre cooling-sensitive units (n = 4). LTM units made up about 8 % of identified C-fibre units, 36 % of identified Adelta-fibre units and > 73 % of identified Aalpha/beta-fibre units. Compared with LTM units, the nociceptive units had APs that were longer on average by 3 times (C-fibre units), 1.7 times (Adelta-fibre units) and 1.4 times (Aalpha/beta-fibre units). They also had significantly longer rise times (RTs) and fall times (FTs) in all CV ranges. Between Aalpha/beta-nociceptors and Aalpha/beta-LTMs there was a proportionately greater difference in RT than in FT. The duration of the afterhyperpolarization measured to 80 % recovery (AHP80) was also significantly longer in nociceptive than LTM neurones in all CV ranges: by 3 times (C-fibre units), 6.3 times (Adelta-fibre units) and 3.6 times (Aalpha/beta-fibre units). The mean values of these variables in unresponsive units were similar to those of nociceptive units in each CV range; in C- and Adelta-fibre groups their mean AHP duration was even longer than in nociceptive units. 3. A-fibre LTM neurones were divided into Adelta- (D hair units, n = 8), and Aalpha/beta- (G hair/field units, n = 22; T (tylotrich) hair units, n = 6; rapidly adapting (RA) glabrous units, n = 6; slowly adapting (SA) hairy and glabrous units, n = 2; and muscle spindle (MS) units n = 17). MS and SA units had the shortest duration APs, FTs and AHP80s of all these groups. The mean RT in D hair units was significantly longer than in all Aalpha/beta LTM units combined. T hair units had the longest mean FT and AHP of all the A-LTM groups. The mean AHP was about 10 times longer in T hair units than in all other A-LTM units combined (significant), and was similar to that of A-fibre nociceptive neurones. 4. These differences in somatic AP shape may aid in distinguishing between LTM and nociceptive or unresponsive C- and Adelta-fibre units but probably not between nociceptive and unresponsive units. The differences seen may reflect differences in expression or activation of different types of ion channel.

Indications for coupling between feline spinocervical tract neurones and midlumbar interneurones.

The possibility of collateral segmental actions of spinocervical tract (SCT) neurones upon interneurones with input from cutaneous and group II muscle afferents was investigated in deeply anaesthetized cats. To this end, intracellular and/or extracellular recordings were made from 35 dorsal horn and 15 intermediate zone interneurones in midlumbar segments of the spinal cord and effects of stimulation of the ipsilateral dorso-lateral funiculus (DLF) at C3 and C1 levels, i.e. below and above the lateral cervical nucleus where axons of SCT cells terminate, were compared. The stimuli applied at the C3 segment were within the range of stimuli (50-100 microA) required for antidromic activation of SCT neurones in the same experiment. Those applied at the C segment (200-500 microA) were at least 3 times stronger than C3 stimuli. Under the same experimental conditions, long ascending and descending tract neurones (dorsal spino-cerebellar and rubro-spinal tract neurones) with axons in the DLF were activated at similar thresholds from the C and C3 segments. Intracellular recordings were made from 29 interneurones of which 19 (65%) were dorsal horn and 10 (35%) were intermediate zone interneurones. Excitatory postsynaptic potentials (EPSPs) evoked by single stimuli applied at the C3 segment, but not the C segment, were found in 14 (48%) of those interneurones; their latencies (3.0-5.7 ms) and frequency following with only minimal temporal facilitation were as required for potentials being evoked monosynaptically by the fastest conducting SCT neurones. Extracellular recordings were made from 30 interneurones (24 dorsal horn and 6 intermediate zone interneurones), and in these neurones spike potentials induced from the C3, but not from the C segment, were evoked only by short trains of stimuli. However, their latencies from the first effective stimulus (4.3-5.4 ms) were compatible with mono- or oligosynaptically mediated collateral actions of SCT neurones. They were found in 10 (33%) of the 30 investigated interneurones. Similar effects of C3 stimuli were found in similar proportions of dorsal horn interneurones and intermediate zone interneurones. Indications were also found for synaptic actions evoked by C3 stimuli that could not be attributed to direct collateral actions of SCT neurones. In some intracellularly recorded dorsal horn interneurones, short-latency EPSPs were evoked from the C3 segment by the 2nd or 3rd stimulus in the train, but not by single stimuli. In other dorsal horn and intermediate zone interneurones, inhibitory postsynaptic potentials (IPSPs) were evoked from the C3 segment at minimal latencies (2.7-3.2 ms), which might be too short to allow their mediation via SCT neurones. We conclude that SCT neurones might be used to forward information from muscle group II and cutaneous afferents not only to neurones in the lateral cervical nucleus and via them to thalamus and cerebral cortex but also to interneurones in spinal reflex pathways. Thereby reflex actions evoked from group II and cutaneous afferents might be co-ordinated with responses mediated by supraspinal neurones. We conclude also that dorsal horn and intermediate zone mid-lumbar interneurones might contribute to the previously reported di-and poly-synaptic excitation or inhibition of postsynaptic dorsal column (PSDC), spinothalamic tract (STT) and spinomesencephalic tract (SMT) neurones by collateral actions of SCT cells. Thereby these interneurones might contribute to the co-ordination of responses mediated by various populations of supraspinal neurones.

Modulation of responses of four types of feline ascending tract neurons by serotonin and noradrenaline.

Modulation of responses of four types of ascending tract cells by noradrenaline and serotonin was compared in order to investigate how information forwarded by these cells may be gated by monoaminergic tract neurons. Spinocervical tract, postsynaptic dorsal column and dorsal spinocerebellar tract neurons located in Clarke's column and in the dorsal horn were identified by their axonal projections. Noradrenaline and serotonin were applied ionophoretically close to a selected neuron, and their effects were tested on extracellularly recorded responses of this neuron to electrical stimulation of low-threshold skin afferents and group II muscle spindle afferents. The modulatory actions of noradrenaline and serotonin were estimated from changes in the number of responses evoked by 30 successive stimuli, the minimal latencies of these responses, and their firing frequency. All four populations of ascending tract neurons investigated were modulated by serotonin and noradrenaline, but not in the same way. The responses were most often depressed by noradrenaline and facilitated by serotonin, but in some types of neuron they were affected in the same direction. Transmission from low-threshold skin and group II muscle afferents changed in the same direction in some types of neuron but in the opposite direction in other types. The results indicate that transfer of information from skin and group II muscle afferents to supraspinal centres may be gated by descending monoaminergic pathways in a highly differentiated manner, and is adjusted to the requirements of various behavioural situations.

Electrophysiological evidence that spinomesencephalic neurons in the cat may be excited via spinocervical tract collaterals.

Extracellular microelectrode recordings were made from spinomesencephalic tract (SMT) neurons in the lumbosacral spinal cord of cats anaesthetized with chloralose and paralysed with gallamine triethiodide. The SMT cells were antidromically fired from the posterolateral parts of the superior colliculus and the intercollicular region, were located in laminae IV to VIII, and had response properties and axonal conduction velocities similar to those described previously. The effects of stimulating the dorsolateral funiculus of the cervical cord at C3 and rostral C1, below and above the termination of spinocervical tract (SCT) axons in the lateral cervical nucleus, were examined on 33 SMT cells. The strength of stimulation was adjusted so that at C3 it was above threshold for antidromic activation of SCT cells and at C1 was below threshold for activation of the same cells. Seven (21%) SMT neurons were excited from C3 but not from C1. The remaining 26 (79%) were excited from both C3 and rostral C1 and 23 (70% of these) were excited significantly more from C3. That is, 91% of the total sample were either excited only from C3 or more strongly from C3 than from rostral C1. We discuss the possible neuronal systems involved and conclude that the greater excitatory effects from C3 are most likely due to antidromic activation of the SCT. The shortest latency effects from C3 indicate a monosynaptic linkage between SCT cells with the fastest axons and the SMT. The longer latency actions may be due to monosynaptic connexions from SCT cells with slower conducting axons, to di- or polysynaptic actions from SCT cells with fast axons, or a combination of both. SMT cells are another population of spinal neurons, in addition to postsynaptic dorsal column, spinothalamic and dorsal horn spinocerebellar neurons, which receive excitation via SCT collaterals.

Effects of upper cervical spinal cord stimulation on neurons in the lumbosacral enlargement of the cat: spinothalamic tract neurons.

Extracellular microelectrode recordings were made from deep spinothalamic tract neurons in the lumbosacral spinal cord of cats anaesthetized with chloralose and paralyzed with gallamine triethiodide. The effects of upper cervical spinal cord stimulation were tested on 43 spinothalamic tract neurons, by stimulation of the ipsilateral dorsolateral funiculus at C3 and rostral C1 using five or six shocks at 333 Hz. The strength of cervical stimulation was adjusted so that the C3 shock was above threshold for antidromic activation of spinocervical tract neurons but the same strength of shock applied at C1 was below threshold for the same neurons. Four of the 43 spinothalamic cells (9%) were not influenced by upper cervical stimulation. The remaining 39 spinothalamic tract cells (91%) were all excited from the upper cervical cord. Twenty-seven of these (63%) were excited more strongly from C3 than from C1, 4 (9%) were excited more strongly from C1 than from C3, and the remaining eight cells (19%) showed no significant differences between their responses to stimulation at C1 and C3. There were no obvious differences between those spinothalamic tract neurons showing differential effects from C1 and C3 and those showing no such effects. The neuronal systems possibly responsible for the differential effects from C3 and C1 on spinothalamic tract neurons are discussed. We conclude that the most likely candidate system for the greater excitation from C3 compared with C1 is the subset of spinocervical tract neurons with axon collaterals in the lumbosacral enlargement and that the spinothalamic tract is a further ascending path, in addition to the postsynaptic dorsal column path, that receives excitatory input from spinocervical axon collaterals. The greater excitation from C1 compared with C3 is interpreted as due to excitation from C1 and a mixture of excitation and inhibition from C3. The responsible neuronal systems seem likely to be either the spinocervical neurons with axon collaterals operating on the spinothalamic tract via inhibitory interneurons, or cells in the lateral cervical nucleus with axons descending to the lumbosacral cord.

Differential ascending projections from neurons in the cat's lateral cervical nucleus.

Extracellular microelectrode recordings were made from single cells of the lateral cervical nucleus (LCN) in cats anaesthetized with chloralose and paralysed with gallamine triethiodide. The cells were tested for antidromic activation from the contralateral medial lemniscus and the contralateral tectum. Seventy-two LCN units were recorded which projected to one or both targets. Sixty (83%) projected through the medial lemniscus, and of these 36 (50% of the total) also projected to the tectum, whereas 24 (33%) projected through the medial lemniscus only; 12 (17%) projected only to the tectum. Twenty-nine units (40%) were excited by moving hairs of the coat but not by pinch of the skin, and 9 (31%) of these projected to the tectum, 11 (38%) through the medial lemniscus and 9 (31%) to both targets. Forty units (56%) were excited by hair movement and noxious pinch, and 3 (7%) of these projected to the tectum, 10 (25%) through the medial lemniscus and 27 (68%) to both targets. Three units (4%) had no discernible receptive fields and they all projected through the medial lemniscus, but not to the tectum. Of the 12 units projecting only to the tectum, 11 had receptive fields completely or partially on the trunk. Units projecting either through the medial lemniscus only, or through the medial lemniscus and also into the tectum, had receptive fields more widely distributed: these included small fields on the fore- and hind feet, on the limbs and also, a minority, on the trunk. Units with glove- or stocking-like receptive fields projected through the medial lemniscus. The results show that while most LCN cells project through the medial lemniscus, those excited by hair movement alone preferentially project either to the tectum or through the medial lemniscus, but not by both routes. The differences in receptive field properties of the differently projecting units are discussed in terms of the possible functions of the spinocervical system.

Lumbosacral spinal neurons in the cat that are candidates for being activated by collaterals from the spinocervical tract.

Lumbosacral spinal neurons activated via the spinocervical tract were stained by intracellular injection of horseradish peroxidase in cats anaesthetized with chloralose and paralysed with gallamine triethiodide. The neurons were activated orthodromically by single shock stimulation of the ipsilateral dorsolateral funiculus at the second to third cervical segment, but not from the rostral part of the first cervical segment. Twenty nine cells were recovered from the histological material and subsequently reconstructed from transverse sections. Sixteen cells (55%) had axons that projected ipsilaterally to the lateral funiculus and their somata were located in two regions of the spinal cord, one group in the dorsal horn (laminae IV-V) and the other in the intermediate gray matter (laminae VI-VII). The axons of 10 of these cells gave off collaterals, and in seven of them the collaterals ramified in the grey matter deep to the cell body. The axons of five cells (17%) projected medially towards the central canal, four crossing the mid line in the ventral white commissure and ascending in the contralateral ventral funiculus. Only one of these cells had an axon collateral that crossed into the contralateral dorsal horn. Of the remaining eight cells, three had no obvious long axons but had many local axon collaterals, the axons of three cells were not stained, one had an axon projecting towards the ipsilateral ventral funiculus and one was a motoneuron and its axon projected into a ventral root. A feature of the dendritic trees of many cells was their wide spread in the mediolateral and/or the dorsoventral directions, although no dendrites reached dorsally into lamina II. Twenty-two cells (76%) were excited by moving hairs and by noxious pinch, three (10%) by hair movement alone, two (7%) by noxious pinch and pressure, and for two cells (7%) no receptive field could be found. It is concluded that not only postsynaptic dorsal column neurons receive input from the spinocervical tract but also other cells in the dorsal and ventral horns and the intermediate gray matter. Possible identities for these cells are discussed.