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The cerebellum is critically involved in motor control; however there is evidence to indicate that it also has a role in nociceptive processing. Previous studies suggest a dual role, whereby stimulation of the anterior lobe of the cerebellum invokes antinociception (1-3), and posterior lobe stimulation invokes pronociception (4,5). However, these data are difficult to interpret with respect to (i) somatic nociceptive pathways as they investigated visceral nociception and (ii) precise localisation of the origin of descending control as previous studies utilised electrical stimulation of the cerebellum, where current spread to neighbouring structures, activation of fibres of passage and antidromic activation are important confounds. The aim therefore of the present study was to investigate modulation of somatic nociception in rats following activation of the anterior (lobule V) and posterior (lobule VIII) lobes of the cerebellum using DL Homocysteic acid (DLH), an excitatory amino acid that allows cerebellar activation to be restricted to target neurons thereby overcoming some of the limitations of electrical stimulation. Rats were initially anaesthetised with halothane (2-3% in 02) delivered by face mask and maintained with a continuous infusion of alfaxalone (25mg/kg/hour i.v). EMG was recorded from the biceps femoris while pinching the ipsilateral hindpaw. The change in mechanical withdrawal threshold (the force required to elicit an EMG response) and magnitude of EMG activity at threshold was assessed following injection of either DLH (0.1M) or an equivalent volume of saline into lobule VIII (n=8 and n=7 respectively), or lobule V (n=4 and n=5 respectively) of the cerebellar cortex. Values are mean ± SEM compared by paired t test. Pronociception was observed following lobule VIII stimulation, indicated by a statistically significant decrease on average of 23% in mechanical withdrawal threshold following DLH injection (from 56.5 ± 5.0 g/mm² to 43.2 ± 4.3 g/mm², P=0.0038). There was also a statistically significant facilitation on average of 63% in EMG activity (from 0.007 ± 0.002 v/s to 0.012 ± 0.002 v/s, P=0.0077). In contrast, there was no statistically significant change in mechanical withdrawal threshold or EMG activity following DLH administration into lobule V or after injection of saline in either lobule VIII or V. These data indicate that stimulation of the anterior and posterior lobe of the cerebellum has a differential effect on nociceptive processing, which may be explained by activation of different cerebellar modules. However, recording EMG responses does not discriminate between effects due to modulation of sensory and/or motor components of the spinal pathways activated by the noxious stimulus. Further experiments will therefore investigate cerebellar modulation of sensory input by making recordings from dorsal horn neurons. [ABSTRACT FROM AUTHOR]

Delta and mu receptors (DOR & MOR) are differentially expressed by A- and C-nociceptors, and are hypothesised to differentially modulate mechanical and thermal nociceptive behaviours respectively¹. A- and C-nociceptors can be preferentially activated by controlled contact heating², allowing identification of the nociceptor population driving nociceptive changes, with no confound of different stimulus modalities. Neurons in the superficial dorsal horn (dh) process C-nociceptor inputs from the area of primary hyperalgesia. This is hypothesised to activate lamina I projection neurons, higher centres, and descending control pathways, which contribute to central sensitisation leading to enhanced responses of dh neurons to A-nociceptor input in secondary hyperalgesia. We hypothesised that sensitised responses to specific A- and/or C-nociceptor inputs, rather than to stimulus modalities (mechanical/thermal) are differentially controlled by MOR/DOR in inflammatory hyperalgesia. 1° & 2° inflammatory hyperalgesia were induced by injection of Freund's Complete Adjuvant into dorsal hindpaw and knee joint respectively under brief isofluorane anaesthesia (2-4% in O2). After 7 days, animals were re-anaesthetised (halothane 2-4% in O2, alfaxan maintenance (25.mg.kg-1 i.v.)) and DAMGO (MOR agonist 12 pmol/20µl) and SNC80 (DOR agonist 120 nmol/20µl) given intrathecally in inflamed or control animals. Withdrawal thresholds were determined in response to preferential activation of C- or A-nociceptors with contact heat stimulation, and to mechanical stimulation of the hindpaw. MOR and DOR agonists both inhibited A- and C-nociceptor evoked-responses in all groups (control, 1° & 2° hyperalgesia). SNC80 had a significantly greater effect on A-nociceptor-withdrawal thresholds in 2° hyperalgesia compared to controls, and a significantly smaller effect on A-nociceptor-evoked responses on 1º hyperalgesia (control, 8.6±0.4°C.hr; 1°, 5.9±0.6°C.hr; 2°, 13.2±1.4°C.hr; AUC mean±SEM, p<0.01). DAMGO had equivalent effects on preferential A- and C-nociceptor thermal stimulation under all conditions, but significantly increased withdrawal thresholds in response to mechanical stimuli in 2º but not 1º hyperalgesia compared to controls (control, 11.6±4.5°C.hr; 2°, 35.9±9.6°C.hr; p < 0.05). SNC80 had a greater effect on mechanical stimuli than DAMGO in all animals (p<0.05), but effects were equivalent in hyperalgesic and control animals. These results suggest that both MOR and DOR can modulate both unmyelinated and myelinated nociceptive inputs. DOR may have a greater contribution to the processing of A-nociceptor inputs in thermal 1° and 2° hyperalgesia than MOR, and MOR may be more important in modulation of responses to mechanical stimulation in 2° hyperalgesia. [ABSTRACT FROM AUTHOR]

Activation of neurons in the ventrolateral periaqueductal grey (vlPAG) matter has been shown to be associated with the expression of freezing responses to conditioned fear (1). These responses are reduced following lesions of the vlPAG, indicating that this structure plays an essential role in the freezing response to conditioned fear (2). Previous anatomical studies have demonstrated pathways from the vlPAG to the cerebellum (3,5). Data are presented here which suggest that such a functional connection, via the inferior olive, plays a role in the expression of conditioned freezing behaviour. Electrical stimulation of the vlPAG in anesthetised male Wistar rats (Sodium Pentobarbital; 60mg.kg-1 ip) was found to evoke climbing fibre field potentials in lateral lobule VIII of the cerebellar vermis. The functional significance of this climbing fibre mediated connection was investigated in relation to the finding that chemical stimulation of the vlPAG significantly increases hindlimb-evoked H-reflex amplitude (average increase = 45.2±11%, n=16, P<0.001, Repeated measures ANOVA with Newman-Keuls post-hoc test) in alphaxalone-anesthetised male Wistar rats (Alfaxan; 25mg.kg-1.hr-1iv). It was hypothesised that the cerebellum supports this descending effect upon spinal a-motoneurone excitability, which may be linked to the increased muscle tone associated with freezing behaviours. It was found that cerebellectomy (n=4) or neurotoxic lesion (n=4) of the caudal inferior olive abolished the effect of vlPAG stimulation upon H-reflex amplitude. In another set of rats localised lesions of connections to/from vermal lobule VIII, using the toxic tracer cholera toxin b-subunit- saporin (CTb-Sap) (4), were made under ketamine/medetomidine anaesthesia (Vetalar 50mg.kg-1ip and Domitor 300μg.kg-1ip). In these animals (n=12) fear-conditioned freezing was significantly disrupted (P<0.001, Mann-Whitney test) compared to sham controls (n=10), despite appearing otherwise normal in a battery of motor and affective behavioural tasks. The same CTb-Sap treated animals were subsequently alphaxalone-anesthetised (n=6) as previously and the effect of vlPAG stimulation upon H-reflex amplitude found to be abolished. Changes in freezing behaviour as a function of H-reflex facilitation were plotted for individual animals. Consistent with the concept of H-reflex excitability being a proxy measurement for muscle tone underlying freezing behaviour, a positive correlation between the two was found (r²= 0.75, P < 0.05, Linear regression). In summary, these data indicate that vermal lobule VIII of the cerebellum plays an important role in the expression of vlPAG mediated freezing behaviour. [ABSTRACT FROM AUTHOR]

Previous work from our laboratory has studied the functional roles of distinct types of nociceptors in acute pain processing [1, 2]. We have demonstrated that descending control can differentially modulate A- vs C-fibre evoked responses and we have shown that thermal nociception is under tonic regulation by descending noradrenergic control [3]. However, the different roles these nociceptors have in the development of heat hyperalgesia during chronic pain states and the changes descending control are poorly understood. Here we have investigated the descending noradrenergic control of A- and C-heat nociceptor evoked responses during the development of neuropathic hyperalgesia. Wistar rats (300-350g) had tibial nerve transection (TNT) under ketamine (50mg/kg) and medetomidine (300µg/kg) recovery anaesthesia [4]. Subsequently heat ramp withdrawal experiments were performed at day 3-5 or day 19-21 post-TNT under alphaxalone (9 - 15mg.kg-1.h-1 i.v.) anaesthesia. Fast (7.5°C/s) or slow (2.5°C/s) rates of skin heating were applied to the dorsal hindpaw to preferentially activate A- or C-heat nociceptors, respectively. EMG withdrawal response thresholds were recorded from the biceps femoris. A 32 gauge intrathecal catheter was inserted at the L5-L6 interspace for administration of yohimbine (α2-AR antagonist, 30µg) or clonidine (α2-AR agonist, 15µg). Changes in dopamine-beta-hydroxyalse (DBH) immunoreactivity were analysed at days 19-21. Data are expressed as mean withdrawal thresholds ± SEM or %DBH immunoreactivity analysed using either t-test or one-way ANOVA with Bonferroni's post tests. Following TNT animals showed substantial A- but not C-fibre evoked ipsilateral hyperalgesia by day 21 (TNT: A-fibre: day 3 55.2±0.7°C vs day 21 47.8±0.9°C P<0.001 n=5) when compared to sham. Intrathecal yohimbine revealed sensitivity of A- but not C-fibre evoked responses at day 3 (control 55.2±0.7°C vs 51.6±0.6°C post yohimbine P<0.001 n=4). Once hyperalgesia had developed (by day 19) yohimbine had no further sensitising effect on A-fibre responses however intrathecal clonidine increased A-fibre thresholds to beyond cut off (>58°C, P<0.01, n=3). Yohimbine was without effect in sham animals (n=3). Interestingly, yohimbine revealed contralateral heat hypersensitivity mediated by A- but not C-fibres at days 19-21 (A-fibre: 54.6±0.8°C to 49.0±0.8°C P<0.01, n=5). By days 19- 21 there was a loss of DBH-ir in the ipsilateral L4-L6 spinal cord when compared to sham (n=3).These data demonstrate that descending pontospinal noradrenergic tone preferentially modulates, both temporally and spatially, the development of fast, prickling, heat hypersensitivity mediated by A-fibres following a loss of descending noradrenergic innervation to the lumbar region. [ABSTRACT FROM AUTHOR]

Descending controls on spinal nociception, particularly descending facilitatory pathways from the midbrain periaqueductal grey (PAG), play major roles in the initiation and maintenance of chronic pain, and are important determinants of the pain experience. A better understanding of these systems may inform the development of more effective chronic pain treatments. Prostaglandin receptors EP1-4 in the ventrolateral periaqueductal grey (vlPAG)1 contribute to acute inflammatory pain and in nerve injury vlPAG EP1 receptors contribute to tonic descending facilitation in naïve and injured rats2. Inflammatory arthritis was induced in male rats under brief isoflurane anaesthesia (2% in O2) by left knee intra-articular injection of Freund's complete adjuvant (100µl/1mg.ml-1). Naïve animals were used as controls. After 7 days, rats were re-anesthetised with halothane (3% in O2) and maintained on alfaxan infusion (i.v. 25mg.kg.h 1). Bipolar steel wire electrodes were placed in the bicep femoris of the left hind limb to record electromyographic activity, and a micropipette positioned stereotaxically in the left PAG. A and C nociceptors were preferentially activated by application of fast (7.5°C.s-1) or slow (2.5°C.s-1) rates of contact skin heating applied to the dorsal surface of the left hind-paw3 respectively. Withdrawal thresholds to A- and C-nociceptor stimulation (interstimulus interval 8 min) were determined before and after delivery of drug/vehicle into the PAG by pressure ejection. There was a prolonged secondary (hindpaw) hyperalgesia 7 days after induction of inflammation, indicated by a reduction in A- but not C-nociceptor withdrawal thresholds (naïve vs. arthritic A-nociceptor thresholds: 57±1oC vs 54±1.2oC . p<0.0001 unpaired t test. n=16 naïve, 18 arthritic). In naïve animals EP3 receptor antagonism (GW671021B 250nM/200nl) in the vlPAG increased C- but not A-nociceptor evoked-reflexes. In contrast in secondary hyperalgesia, EP3 receptor antagonist increased A-nociceptor responses (AUC GW A-nociceptor effect vs AUC Vehicle A-nociceptor effect: 352.1±85.0oC.min vs 116.8±26.10oC.min. p<0.0357 Mann Whitney. n=5 for GW, n=3 for Vehicle.) with minimal effect on C-nociceptor reflexes. Inflammatory knee joint arthritis produces secondary hyperalgesia of the hind paw involving sensitisation of A- but not C-nociceptor-evoked spinal reflexes. Blockade of prostaglandin EP3 receptors in the vlPAG predominantly modulates A-nociceptor withdrawals in the hyperalgesic state but selectively alters C-nociceptor reflexes in the normal rat. In the transition to chronic inflammatory pain, descending EP3 receptor-mediated facilitation from vlPAG switches from C-nociceptor facilitation in naïve rats to A-nociceptor facilitation in inflammatory secondary hyperalgesia. [ABSTRACT FROM AUTHOR]

Specific patterns of survival (defence) behaviours that are coordinated by the different functional columns of the midbrain periaqueductal grey (PAG) may be triggered by A- and C-nociceptive inputs that have different behavioural significance, in that they mediate 'first' and 'slow' pain respectively, and have different roles in the initiation and maintenance of chronic pain. It is of considerable importance therefore to determine to the columnar organisation of A- and C-nociceptor inputs in normal animals and in animal models of chronic pain. Our previous studies have shown that, in uninjured animals, inputs arising from these different fibre types target different regions of the PAG: C-fibre inputs target the ventrolateral column of the PAG (VL-PAG) and A-nociceptor inputs, the dorsolateral/lateral column (DL/L-PAG). This current study was designed to determine the activation of different regions of the PAG following either A- or C-nociceptor stimulation in an area of secondary hyperalgesia in a rat model of inflammatory arthritis. Arthritis was induced by injection of Freund's Complete Adjuvant into the left knee joint under brief isoflurane/O2 anaesthesia (2% in O2). Animals (4-6/group) were re-anaesthetised 7 days later (halothane (2% in O2), alfaxan maintenance (i.v. 25.mg.kg.h-1) and either C- or A-nociceptors preferentially activated respectively, by slow (2.5°C.s-1, 30-55°C) or fast (7.5°C.s-1, 30-57°C) contact heating of the left hindpaw dorsum (area of secondary hyperalgesia). Some inflamed rats did not receive any stimulation (control group). Two hours after stimulation, animals were perfuse fixed and brains processed immunocytochemically for visualisation of Fos-like immunoreactivity (FLI). Fos-positive PAG neurones were counted (Image J, NIH) and their locations assigned to the different functional columns of the PAG throughout its rostrocaudal extent. FLI was greater in caudal than rostral PAG in stimulated and control animals. Hindpaw A-nociceptor stimulation increased FLI in the caudal right (contralateral) L-PAG but in no other area, in arthritic rats. These results demonstrate that caudal L-PAG activation is greater in response to A-nociceptor input from the area of secondary hyperalgesia than in inflamed unstimulated rats. These data may indicate that processing of 'first' pain in L-PAG is enhanced in secondary inflammatory hyperalgesia. [ABSTRACT FROM AUTHOR]