Depletion of endogenous spinal 5-HT attenuates the... : PAIN (original) (raw)
1. Introduction
Peripheral nerve damage can result in severe chronic pain, characterised by symptoms such as allodynia and hyperalgesia as well as areas of sensory deficit. This distressing condition has stimulated significant efforts to understand the pathological mechanisms underlying neuropathic pain and there are numerous descriptions of changes in the peripheral and central nervous system that could account for the pain.
Central sensitization, initiated as a result of sustained barrage of afferent activity arriving in the spinal cord following nerve injury, is maintained long after the arrival of the initial peripheral barrage. One example of this hyperexcitability is an increased responsiveness of spinal neurones to peripheral stimuli, thought to be a key factor underlying chronic pain states. In addition to peripheral and central spinal mechanisms, it is well documented that descending influences, from brainstem areas, onto the spinal cord play a major role in the modulation of nociception in ascending pathways (for reviews, see Dubner and Ren, 1999; Millan, 2002).
Serotonergic neurones located within midbrain and brainstem nuclei such as the rostroventral medulla (RVM) can exert facilitatory or inhibitory influences onto dorsal horn neurones depending on the spinal 5-HT receptor subtype activated (Fields et al., 1991; Zhuo and Gebhart, 1992; Calejesan et al., 1998; Urban and Gebhart, 1999; Millan, 2002). Earlier studies principally focused on the role of descending inhibitions on the spinal cord. Thus, reduced pain behaviours in animals follow high intensity electrical stimulation or morphine injection into the periaqueductal gray (PAG) and/or RVM, and hyperalgesic/pronociceptive responses are observed following transection of the dorsolateral funiculus containing axons of descending pathways arising from the brainstem (Basbaum et al., 1976; Ren and Dubner, 1996). More recently, attention has turned towards the contribution of descending excitatory influences which may have a role in the development and maintenance of persistent pain states (Calejesan et al., 1998; Urban and Gebhart, 1999; Burgess et al., 2002; Porreca et al., 2002). In particular, tonic discharge of descending facilitation from the RVM was shown to be critical for the maintenance of neuropathic pain (Ossipov et al., 2001). In support of the findings of Ossipov et al. (2001), we have previously shown neuropathic pain to be associated with an overdrive or inappropriate activation of descending facilitation onto mechanically evoked spinal neuronal responses mediated by serotonin acting on spinal 5-HT3 receptors (Suzuki et al., 2004a). Furthermore, a single i.v. injection of ondansetron (selective 5HT3 receptor antagonist) significantly reduced pain scores in neuropathic pain patients compared with placebo control (McCleane et al., 2003). Interestingly, depletion of endogenous spinal 5-HT reduced at-level mechanical allodynia (pain which occurs at spinal levels just rostral to the lesion), in a model of spinal cord injury (Oatway et al., 2004).
The mounting evidence advocating a role for descending facilitatory mechanisms as the primary drive mediated by serotonergic pathways in chronic pain states led us to investigate the role of endogenous spinal 5-HT on neuronal activity in normal animals and on neuropathic pain behaviour and associated neurochemical changes, using the selective serotonergic neurotoxin, 5,7di-hydroxytryptamine (5,7DHT).
2. Materials and methods
2.1. Animals
Twenty-two male Sprague–Dawley rats (Central Biological Services, University College London, UK) were used for this study. Animals were housed at a maximum six per cage on a 12h day–12h night cycle and had unrestricted access to food and water. All experimental procedures were approved by the UK Home Office and follow the guidelines under the International Association for the Study of Pain (Zimmermann, 1983).
2.2. Surgical procedures
2.2.1. Intrathecal 5,7di-hydroxytryptamine administration
Endogenous spinal 5-HT was depleted in rats (130–150g) via intrathecal injection of 5,7di-hydroxytryptamine creatinine sulfate (5,7DHT) (Sigma–Aldrich) performed as previously described (Suzuki et al., 2002; Oatway et al., 2004). To prevent noradrenergic toxicity, rats were pre-treated with desipramine (Sigma–Aldrich) 45min prior to an intrathecal (i.t.) lumbar injection of 5,7DHT. After 45min, rats were deeply anaesthetised with ketamine (1.5mg/kg i.p.) and a small incision made in the atlanto-occipital membrane. A cannula was then inserted into the subarachnoid space, terminating in the L4-5 region. Animals received either an intrathecal injection of 0.9% saline or 5,7DHT (60μg/20μl) and flushed with 10μl saline. This procedure selectively and significantly depletes spinal 5-HT levels (Sawynok and Reid, 1994; Oatway et al., 2004). The cannula was withdrawn and the wound closed using 3-0 surgical sutures (Sherwood Davis & Geck, USA) and wound clips and the animals were allowed to recover.
2.2.2. Spinal nerve ligation
Two days post-injection of 5,7DHT, the injected animals underwent either ligation of L5 and L6 spinal nerves or sham operation. Two other groups that received spinal saline instead of 5,7DHT underwent either spinal nerve ligation or sham operation. This produced four experimental groups: SNL-5,7DHT, SNL-saline, sham-5,7DHT and sham-saline.
Neuropathic or sham surgery was carried out as previously described (Suzuki, 2004a). Briefly, the left L5 and L6 spinal nerves were isolated and tightly ligated with 6-0 silk thread under halothane anaesthesia (50% O2:50% N2O). Haemostasis was confirmed and the wound was sutured. The surgical procedure for the sham operated group was identical to that of the SNL group, except that the L5/L6 spinal nerves were not ligated.
After surgery, the foot posture and general behaviour of the operated rats were monitored throughout the post-operative period.
2.3. Immunohistochemistry
Immunohistochemical assessment of 5-HT depletion in lumbar cord sections was made on day 7 post-5,7DHT or saline injection only (these animals were used for in vivo electrophysiology only and did not undergo nerve injury). We also assessed 5-HT depletion in lumbar cord sections on days 7 and 14 post-SNL or sham surgery providing four groups: SNL-saline, SNL-5,7DHT and sham-5,7DHT, sham-saline. In addition, NK1 and IB4 like immunoreactivity was localised on lumbar cord sections of animals that underwent SNL or sham surgery as previously described (Suzuki et al., 2002) as well as Iba1 a marker of microglial activation.
Experimental animals were deeply anaesthetised with an intraperitoneal injection (600mg/kg) of pentobarbitone (Euthatal; Merial Animal Health), perfused transcardially with heparinised saline, followed by a solution of 4% paraformaldehyde in PBS (0.15M, pH 7.4). The spinal cord was removed and cryoprotected overnight in a 30% sucrose solution. Transverse sections (40μm thick) of cryoprotected lumbar spinal cord were cut and serially collected. Free-floating sections were pre-treated with 0.3% H2O2, rinsed three times in 0.1M phosphate-buffered saline containing 1% Triton X-100 followed by incubation in primary antibodies for 3 days at 4°C. Reactions were carried out on parallel sections with antibodies against NK1 (1:10,000, Polyclonal Rabbit, Chemicon), Iba1 (1:1000 polyclonal Rabbit mouse, Wako), 5-HT (1:100 monoclonal Rat, Chemicon) and Griffonia simplicifolia lectin (IB4) (10mg/ml; Sigma). Immunoreactive sites were revealed using biotinylated secondary antibodies (1:500; Vector Laboratories) followed by either avidin-conjugated CY3 (1:4000 Jackson Laboratories) or visualised using the Vectastain© (Vector Laboratories) avidin–biotin kit (ABC) with diaminobenzidine (DAB).
2.4. In vivo electrophysiology
To study the effects of spinal 5-HT depletion on spinal neuronal response characteristics, in vivo electrophysiology was conducted on days 6–8 after intrathecal injection of 5,7DHT or saline. Animals were anaesthetised with 1.0–1.2% halothane (delivered in a gaseous mixture of 66% N2O and 33% O2) and a laminectomy was performed to expose the L4-5 segments of the spinal cord. Extracellular recordings were made from ipsilateral deep dorsal horn neurones (laminae V–VI) using parylene-coated tungsten electrodes (A-M Systems, USA). The evoked neuronal responses to peripheral electrical and a range of mechanical and heat stimuli were characterised as previously described (Rahman et al., 2004). Briefly, a train of 16 transcutaneous electrical stimuli (2ms wide pulses, 0.5Hz) was applied to the centre of the receptive field at 3 times the current C-fibre threshold. Responses evoked by Aβ- (0–20ms), Aδ- (20–90ms) and C-fibres (90–350ms) were separated and quantified on the basis of latency. Responses occurring after the C-fibre latency band were taken to be the post-discharge of the cell (350–800ms). The evoked responses to a range of natural stimuli (von Frey filaments, 2–60g and heat, 35–50°C) applied over a period of 10s were also quantified. Heat was applied with a constant water jet onto the centre of the receptive field. Data were captured and analysed by a CED 1401 interface coupled to a Pentium computer with Spike 2 software (Cambridge Electronic Design; PSTH and rate functions).
2.5. Mechanical and cold sensitivity
Behavioural testing was carried out over a 2-week period in the SNL-5,7DHT, SNL-saline, sham-5,7DHT and sham-saline groups. Mechanical sensitivity was assessed through measurement of foot withdrawal frequencies to a trial of 10 applications of calibrated von Frey filaments 2, 6 and 8g per paw. Cold allodynia was similarly assessed as the number of withdrawals out of a trial of 5 applications of acetone. Withdrawal frequency was quantified as=(number of foot withdrawals/10 or 5 trials as appropriate).
2.6. Data analysis
Data are presented as means±standard error of mean (sem) unless otherwise stated. Statistical analysis to compare the neuronal response characteristics of 5,7DHT-treated animals with saline controls was made using an unpaired Student’s _t_-test. Comparison of the behavioural responses between SNL-5,7DHT, SNL-saline, sham-5,7DHT and sham-saline groups was conducted using a Mann–Whitney _U_-test using Statview 4.5 software. Level of significance was set at P<0.05.
3. Results
3.1. Immunohistochemistry – depletion of spinal 5-HT
To assess the level of depletion of 5-HT in the lumbar spinal cord following 5,7DHT treatment, immunohistochemistry for 5-HT was carried out at day 7 post-intrathecal injection in normal (un-injured) animals. A marked reduction in 5-HT immunoreactivity was seen throughout the dorsal horn of the spinal cord 7 days following 5,7DHT treatment compared with the control group which received an intrathecal injection of saline (Fig. 1).
5-HT immunoreactivity in the dorsal horn transverse sections of the lumbar spinal cord 7 days after intrathecal injection of either (a) saline or (b) 5,7DHT injections. A marked reduction in 5-HT immunoreactive fibres (black arrows) was seen within the dorsal horn after 5,7DHT treatment compared with saline injected controls. Scale bar 50 μm.
5HT immunohistochemistry was also assessed in the lumbar cord of every animal used in the neuropathic study, i.e., SNL-saline, SNL-5,7DHT, sham-5,7DHT and sham-saline groups on days 7 and 14 post-nerve injury, which equates to 9 and 16 days following 5,7DHT treatment. Similar to the findings at day 7 post-5,7DHT treatment in non-injured animals, 5-HT immunoreactivity was almost completely lost in the dorsal horn of the SNL-5,7DHT and sham-5,7DHT groups compared with saline control groups (data not shown).
3.2. Effect of 5,7DHT on deep dorsal horn neuronal characteristics
To assess the effects of spinal 5-HT depletion on deep dorsal horn neuronal responses, we compared the evoked neuronal responses to electrical and a range of natural stimuli, and also receptive field area to von Frey filaments 8 and 60g in 5,7DHT-treated animals to saline controls. Intrathecal administration of 5,7DHT did not produce any significant effects on any of the evoked responses of deep dorsal horn neurones to electrical stimulation of the peripheral receptive field compared with saline controls (Table 1). Remarkably, the depletion of 5-HT resulted in significant reductions in the evoked responses to punctate mechanical stimuli that were very marked and disrupted the stimulus coding. In addition, a rightward shift in the stimulus–response curve for heat stimuli of lamina V–VI neurones was seen in the 5-HT depleted group compared with controls (P<0.05 for all von Frey hairs and heat stimuli tested, unpaired _t_-test; Figs. 2a and b). Similarly a concomitant reduction in receptive field area for evoking a response to von Frey filaments 8 and 60g (56% and 43% reduction, respectively) was seen in the 5,7DHT group compared with saline controls (Fig. 2c).
A comparison of the mean cell depth and the evoked neuronal responses to peripheral electrical stimuli in normal animals given intrathecal 5,7DHT or saline
Depletion of spinal 5-HT in normal (un-injured) animals results in marked reduction in the electrophysiological responses of deep dorsal horn neurones compared with the saline injected control group. Significant reductions were seen in (a) the mechanically evoked neuronal responses (von Frey 6–60 g) and (b) the neuronal responses evoked by all heat stimuli tested here in the 5,7DHT group (black squares; n = 32) compared with saline injected controls (open squares; n = 21). Data are presented as mean number of action potentials ± sem. (c) A comparison of the mean receptive field areas from which stimulation with von Frey 8 and 60 g evokes neuronal responses in the saline (white bars; n = 21) and 5,7DHT (black bars; n = 32) treated animals. Data are presented as mean (%) of the projected area of the hindpaw. *Significant difference when compared with saline injected control rats (P < 0.05 Student’s _t_-test unpaired).
This overall reduction in deep dorsal horn neuronal responsiveness to peripheral natural stimulation lead us to investigate the consequences of 5,7DHT treatment on neuropathic pain behaviour after spinal nerve ligation.
3.3. Mechanical and cooling hypersensitivity following nerve injury is reduced after 5,7DHT treatment
Probing with all three von Frey hairs used in this study on the ipsilateral hindpaw resulted in a high frequency of paw withdrawals and also of paw licking behaviour in the SNL-saline-treated animals compared with the sham-saline group. This was seen most clearly with von Frey hairs 6 and 8g (Fig. 3). Similarly SNL-saline animals responded vigorously to the trials of acetone applied to the ipsilateral hindpaw compared with the sham-saline group from day 2 onwards post-nerve injury. These hyper-responsive behaviours are indicative of mechanical and cooling allodynia (Fig. 4). In comparison, very little sensitivity to either stimulus type was seen on the contralateral hindpaw of these animals. The degree of behavioural hypersensitivity seen ipsilaterally in the SNL-saline group and the lack of abnormal responses in the sham-saline group are in line with our previous reports using this model (Chapman et al., 1998; Matthews and Dickenson, 2001; Suzuki et al., 2004a; Suzuki et al., 2005). A comparison of the evoked responses to mechanical or cooling stimulation of the ipsilateral hindpaw of the sham-5,7DHT animals compared with the sham-saline group control group revealed a non-significant tendency towards greater paw withdrawal frequencies in the former group (_P_>0.05, Mann–Whitney _U_-test).
Mechanical allodynia following peripheral nerve injury is attenuated with pre-treatment with intrathecal 5,7DHT. (a, c and e) A comparison of the paw withdrawal frequencies to innocuous mechanical stimuli (von Frey 2, 6 and 8 g) applied to the ipsilateral hindpaw of SNL-saline (n = 8), SNL-5,7DHT (n = 10), sham-5,7DHT (n = 7) and sham-saline (n = 6). Paw withdrawal frequencies were significantly greater in the SNL-saline group compared with the sham-saline to (a) stimulation with von Frey 2 g from day 7 post-injury and (c, d) to von Frey 6 and 8 g from day 2 onwards. In the nerve injured groups a significant reduction in paw withdrawal frequencies to von Frey hairs 2, 6 and 8 g was seen from day 7 post-injury in the 5-HT depleted group compared with the SNL-saline controls. No difference in paw withdrawal frequency was seen between SNL-5,7DHT compared with sham-5,7DHT except with von Frey 8 g at day 2 post-injury. (b, d and f) A comparison of the paw withdrawal frequencies to von Frey 2, 6 and 8 g applied to the contralateral hindpaw of all groups. The paw withdrawal frequencies were similar in all three groups. Data are presented as means ± sem number of paw withdrawals out of a trial of 10 stimulations. *Significant difference between SNL-5,7DHT compared to the SNL-saline group, +significant difference between SNL-saline compared with sham-saline. §Significant difference between SNL-5,7DHT compared with the sham-5,7DHT group. P < 0.05 Mann–Whitney _U_-test.
Allodynia to cooling stimulation is reduced following depletion of spinal 5-HT in nerve injured animals. Paw withdrawal response frequency to cooling stimulation (acetone) applied to the (a) ipsilateral and (b) contralateral hindpaw of SNL-saline (n = 8), SNL-5,7DHT (n = 10), sham-5,7DHT (n = 7) and sham-saline (n = 6). Similar to the effects seen on behavioural responses to mechanical stimulation, a reduction in the paw withdrawal responses was seen in the SNL-5,7DHT compared with SNL-saline group. Paw withdrawal frequencies to acetone application were significantly greater in the SNL-saline group compared with the sham-saline group from day 2–14 post-injury. Data are presented as means ± sem number of paw withdrawals out of a trial of 5 stimulations. *Significant difference between SNL-5,7DHT compared to the SNL-saline group, +significant difference between SNL-saline compared with sham-saline. P < 0.05 Mann–Whitney _U_-test.
In contrast, the response frequencies to von Frey and acetone stimulation of the ipsilateral hindpaw were attenuated in the SNL-5,7DHT group compared with the SNL-saline group. Significant reductions in paw withdrawal frequencies were observed from days 7 to 12 post-nerve injury to von Frey 2g, and from days 7 to 14 post-SNL to von Frey hairs 6 and 8g (P<0.05, Mann–Whitney _U_-test) (Fig. 3). With regard to paw withdrawals to acetone (cooling) stimulation of ipsilateral hindpaw, significant reductions in response frequency were observed on days 5, 7 and 9 only post-nerve injury in the SNL-5,7DHT group compared with SNL-saline (P<0.05, Mann–Whitney _U_-test) (Fig. 6).
NK1 receptor immunoreactivity in the ipsilateral dorsal horn of the lumbar spinal cord at day 14 post-nerve injury in SNL-5,7DHT, SNL-saline and sham-5,7DHT groups. Nerve injury results in an increase in NK1 receptor immunoreactivity within the superficial lamina of the dorsal horn ipsilateral to the side of nerve injury (a) SNL-5,7DHT and (b) SNL-saline as compared to the level of immunoreactivity in the ipsilateral dorsal horn of the non-injured (c) sham-5,7DHT group and (d) the contralateral dorsal horn of SNL-5,7DHT group. The upregulation of NK1 immunoreactivity in the dorsal horn ipsilateral to nerve injury was similar in both SNL-saline and SNL-5,7DHT groups. Scale bar 500 μm.
Paw withdrawal responses to von Frey hairs or acetone stimuli of either the ipsilateral or contralateral hindpaw were either not present or very low in the sham-5,7DHT and sham-saline animals (Figs. 3 and 4). Overall, no significant differences were observed between response frequencies to mechanical or cooling stimulation of the ipsilateral hindpaw in SNL-5,7DHT and sham-5,7DHT groups, except for response frequency to von Frey 8g at day 2 post-nerve injury which was significantly greater in the SNL-5,7DHT group compared with the sham-5,7DHTgroup (P<0.05, Mann–Whitney _U_-test). This finding may suggest a maintenance role for 5-HT in the development of pain behaviour to low intensity mechanical stimulation.
The response frequencies to all three von Frey hairs and acetone stimulation of the contralateral hindpaw were minimal and not significantly different between any of the groups (_P_>0.05, Mann–Whitney _U_-test), (Figs. 3 and 4).
3.4. Immunohistochemistry – NK1 receptor and Iba1 (marker for activated microglia) labelling after nerve injury in relation to behaviour
Immunoreactivity for NK1 receptors and Iba1 (a marker for activated microglia) within the dorsal horn of the lumbar spinal cord was carried out. An increase in immunofluorescence intensity was observed for both NK1 and Iba1 in the dorsal horn ipsilateral to the side of injury compared with the contralateral side within each subject for SNL-saline and SNL-5,7DHT groups, and also when comparing the ipsilateral dorsal horn of these groups to the ipsilateral side of the sham operated group (Figs. 5 and 6). A marked loss of IB4 staining was observed on the ipsilateral side in both nerve injured groups as is expected after ligation of L5/6 nerves (data not shown). No differences in NK1 or Iba1 immunofluorescence were seen in the dorsal horn of sham-operated animals when comparing the ipsilateral to the contralateral side, suggesting that changes in these systems were specific to the nerve injury state (Figs. 5 and 6; sham-saline data not shown). Similar increases in NK1 and Iba1 levels were seen in both SNL-saline and SNL-5,7DHT groups when comparing the ipsilateral side to injury to the contralateral dorsal horn although the depletion of 5-HT in the latter group led to significantly reduced mechanical and cooling allodynia. Thus, the ability of 5-HT to modulate the abnormal behaviour seen after nerve injury is independent of microglial activation and NK1 receptor upregulation.
Immunoreactivity for Iba1 (a marker for microglial activation) within the dorsal horn of the lumbar spinal cord at 14 days post-nerve injury. Under pathological conditions such as nerve injury microglia become activated, this is seen here as a significant increase in immunofluorescence for Iba1 in the spinal cord dorsal horn ipsilateral to the side of nerve injury (picture – right-hand side) in the SNL-5,7 DHT (a, b) and SNL-saline groups (c, d) as compared to the sham-5,7DHT group (e, f) where very little immunoreactivity for Iba1 was seen in the dorsal horn. The level of microglial activation appears to be similar in the nerve injured groups. (a, c and e) Scale bar 500 μm and (b, d and f) scale bar 20 μm.
4. Discussion
We provide electrophysiological and behavioural evidence supporting the hypothesis that descending brainstem serotonergic facilitation plays a critical role in modulating spinal nociceptive circuits. Depletion of spinal 5-HT in normal (un-injured) animals produced deficits in neuronal responsiveness to mechanical and thermal stimuli and reduced receptive field areas. Similarly, 5,7DHT pretreatment attenuated behavioural hypersensitivity (“allodynic response”) to mechanical and cooling stimuli in the SNL-5,7DHT compared with SNL-saline group. Our findings complement earlier studies which suggested that an inappropriate and sustained activation of descending facilitation from the brainstem plays a major role in maintaining chronic pain (Pertovaara et al., 1996; Ossipov et al., 2000; Porreca et al., 2001; Burgess et al., 2002; Suzuki et al., 2004a). The effect of 5-HT depletion on neuropathic behaviour was seen at later time points (day 5 onwards) in accordance with an earlier study on descending RVM facilitation where the impact appears to be related more to the maintenance stage than induction (Burgess et al., 2002). The failure of 5,7DHT treatment to alter the development of the neuropathic behaviour at earlier stages is unlikely to be due to an insufficient depletion of 5HT as a marked loss (≈82%) in 5HT levels 3–5 days post-5,7DHT injection has been previously demonstrated (Liu et al., 1988). Further studies will be necessary to dissect out the mechanisms underlying the initiation and maintenance of neuropathic pain states, but here and as seen previously (Suzuki et al., 2002; Rahman et al., 2004; Suzuki et al., 2004a), these 5-HT pronociceptive drives are present in normal animals suggesting that any increase in these controls could trigger the altered persistent pain state.
4.1. Alterations in spinal NK1 receptors and microglial activity are not essential for the maintenance of allodynia seven days following nerve injury
Allodynia resulting from nerve injury has also been attributed to activation of microglia (Tsuda et al., 2003). We observed marked increases in Iba1 labelling in the ipsilateral spinal cord compared to the contralateral side in both SNL-saline and SNL-5,7DHT groups. Furthermore, the level of microglial activity seen in the nerve injury side appeared similar in both these groups, yet, allodynic behaviour was significantly reduced in the latter group. Thus, our data may indicate that changes in descending serotonergic activity can modulate allodynia independently of enduring microglial activation.
Activation of lamina I projection neurones, the majority of which express the NK1 receptor, is one mechanism by which nociceptive primary afferents can activate descending influences back onto spinal neurones (Todd et al., 2000; Suzuki et al., 2002; Khasabov et al., 2005). Superficial NK1 receptor expressing neurones can enhance nociception by increasing spinal neuronal excitability via the activation of a descending serotonergic pathway (Suzuki et al., 2002). Since these neurones play a critical role in the initiation and maintenance of central sensitization (Mantyh and Hunt, 2004), an upregulation of NK1 receptors observed after nerve injury presents a possible mechanism for increasing spinal neuronal excitability. However, the behavioural data here suggest that increases in spinal NK1 receptors, from day 7 post-nerve injury, are unlikely to underlie allodynia (at least without co-operation from descending serotonergic fibres) since a marked decrease in sensitivity to innocuous stimuli was observed in the 5HT depleted group, despite the increase in NK1 immunofluorescence remaining similar to the SNL-saline group. It is possible that an upregulation of spinal NK1 receptors is important in the earlier stages of neuropathic pain development.
4.2. Descending serotonergic facilitation is an essential component in driving neuropathic pain
The RVM is a primary source of descending serotonergic modulation of ascending nociceptive transmission from the spinal cord (see Millan, 2002). Neurones within this area are classified into three types based upon their firing patterns in response to noxious thermal stimuli. ON-cells increase their firing immediately before a nocifensive response and are thought to facilitate nociception, whilst OFF-cells, considered to mediate inhibition, pause in their firing just prior to a nociceptive withdrawal reflex. Neutral cells do not appear to play a role in physiological pain, although a role in neuropathic pain has been reported (Fields et al., 1983; Fields and Heinricher, 1985; Pertovaara et al., 2001). The pharmacology of descending facilitatory pathways remains unclear, as recordings from RVM neurones suggest that 5-HT containing neurones are neither ON nor OFF cells (Gao and Mason, 2000), however recent immunohistochemical data suggest that a proportion of RVM cells activated by noxious stimuli are labelled as serotonergic (Suzuki et al., 2002).
Descending controls from the RVM (excitatory/inhibitory) can be modulated by different manipulations (e.g., electrical stimulation, pharmacological agents) suggesting that anatomically distinct pathways are involved (Urban and Gebhart, 1997) which could differentially activate ON or OFF cells. The varying levels of activity in supraspinal descending pathways in concert with peripheral input and intrinsic spinal mechanisms determine the level of neuronal excitability within the spinal cord. A shift towards a more dominant role for descending facilitation could thus manifest as hyperalgesia and/or allodynia. The results here suggest that after nerve injury facilitation dominates and a re-appraisal of the effects of electrical stimulation of the RVM shows facilitation with the lowest stimulus levels with inhibitions only being produced by increased currents (Zhuo and Gebhart, 1997).
Mixed reports exist on the function of descending controls from the RVM and of 5-HT in nociceptive transmission (Oyama et al., 1996; Dubner and Ren, 1999; Ossipov et al., 2001; Millan, 2002; Suzuki et al., 2004b). Traditionally, inhibition of RVM activity or loss of 5-HT modulation has pointed to a loss of inhibitory control resulting in increased pain behaviours (for review, see Dubner and Ren, 1999). Yet, equally, there is evidence for a key role for facilitation from the RVM affecting spinal transmission, particularly after neuropathy since disruption of descending pathways prevents allodynia and/or hyperalgesia (Urban and Gebhart, 1999; Ossipov et al., 2001; Porreca et al., 2002; Suzuki et al., 2004b). Further, the pronociceptive drive from the RVM is likely to involve the activation of serotonergic pathways (Zhuo and Gebhart, 1991; Green et al., 2000; Pertovaara et al., 2001; Suzuki et al., 2002, 2004a,b). Indeed, it has been reported that antinociception produced by stimulation of the nucleus raphe magnus or PAG does not appear to require the activation of descending serotonergic pathways (Sawynok, 1989; Matos et al., 1992; Sorkin et al., 1993).
Early studies on the effects of neurotoxic destruction of spinal serotonergic pathways revealed a loss of inhibition or no change in nociceptive thresholds. However, these studies were in normal animals and only studied threshold responses rather than suprathreshold measures such as neuronal activity (Mohrland and Gebhart, 1980; Howe and Yaksh, 1982; Berge et al., 1983; Fasmer et al., 1983b; Kuraishi et al., 1983; Liu et al., 1988). However, there have been reports of a loss of facilitation after spinal 5-HT depletion; interestingly, acting on measures of pain such as allodynia and the formalin test rather than for brief noxious stimuli such as tail-flick (Fasmer et al., 1985; Oatway et al., 2004).
Consistent with our hypothesis for a facilitatory role for 5-HT (alongside its inhibitory effects), intrathecal injection of 5-HT or substance P produced similar behavioural responses, effects which were blocked by a substance P antagonist (Fasmer et al., 1983a; Fasmer and Post, 1983). Further, a role for RVM neutral cells in neuropathy has been suggested which, at least in part, is thought to be due to a contribution of serotonergic mechanisms of descending facilitation (Pertovaara et al., 2001). More recently, in a model of spinal cord injury, depletion of spinal 5-HT reduced the at-level mechanical allodynia thought to be due to reduced activity at spinal 5-HT3 receptors (Oatway et al., 2004). This supports the idea that nerve injury results in an enhanced descending facilitatory drive onto the mechanical evoked responses of dorsal horn neurones partly mediated by spinal 5-HT3 receptors (Suzuki et al., 2004a).
Our current finding of reduced nocifensive responses to innocuous stimuli following spinal 5-HT depletion in nerve injured animals supports the idea of an active participation of supraspinal sites in driving sustained facilitatory influences onto the spinal cord following peripheral nerve injury. This is in keeping with the evidence of earlier work where we demonstrated an increased efficacy of ondansetron (selective 5-HT3 receptor antagonist) on formalin responses, with similar findings in transgenic animals on mechanically evoked responses after nerve injury (Green et al., 2000; Zeitz et al., 2002). Thus targeting serotonergic systems, and specifically the 5-HT3 receptor system, with brain penetrant drugs, may have a potential clinical use for the treatment of neuropathic pain states, particularly for patients with tactile allodynia.
These serotonergic systems will also participate in brainstem and midbrain systems involved in mood, anxiety and the sleep–wake cycle. Disturbances in these circuits produced by ascending pathways may explain these common co-morbidities in chronic pain patients (Meyer-Rosberg et al., 2001). However, internally generated activity in these same areas could drive descending 5-HT and other facilitatory systems, diffuse and non-segmental, that in turn may contribute to pains such as fibromyalgia, IBS and migraine where widespread pains are observed (Goadsby, 1998; Haus et al., 2004; Mertz, 2005).
Acknowledgement
This work was supported by the Wellcome Trust.
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Keywords:
Spinal cord; 5-HT; Descending facilitation; Neuropathic; Behaviour; Electrophysiology
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