R.W. Clarke

Interactions between cutaneous afferent inputs to a withdrawal reflex in the decerebrated rabbit and their control by descending and segmental systems

Previous studies have suggested that activation of nociceptive afferents from the heel recruits a supraspinal mechanism, which is modulated by adrenergic descending inhibition, that augments withdrawal reflexes in medial gastrocnemius (MG) motoneurones. To test this idea, we have studied the temporal evolution of reflexes evoked in MG by electrical stimulation of sural nerve A(beta)-, A(delta)- and C-fibre axons at 1 Hz, in decerebrated rabbits. Reflexes were analysed in three time bands, estimated to accord to afferent drive from A(beta)- (phase 1), A(delta)- (phase 2) and C-fibre (phase 3) inputs. Stimulation of A(delta)- and C-fibres gave significant temporal summation of all reflexes. The alpha(2)-adrenoceptor antagonist RX 821002 ((2-(2,3-dihydro-2-methoxy-1,4-benzodioxin-2-yl)-4,5-dihydro-1-H-imidazole)-HCl) (100 microg intrathecal (i.t.)) potentiated, and the alpha(2)-agonist dexmedetomidine (1-30 microg i.t.) depressed all reflexes per se, but the effects of these drugs on temporal summation were secondary to changes in baseline excitability. When C-fibres were stimulated, the N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine (1 mg i.t.) reduced temporal summation of phase 2 and 3 but not phase 1 reflexes. Spinalisation at L1 in the absence of drugs increased phase 2 and 3 reflexes but had no effect on phase 1, whereas spinalisation after RX 821002 resulted in decreased phase 1 responses with no significant change in later phases. Spinalisation in the presence of dizocilpine resulted in small reductions in phase 3 reflexes only. In all cases spinalisation virtually abolished temporal summation. In spinalised animals, dizocilpine selectively reduced late reflexes, and the opioid antagonist naloxone (100 microg i.t.) augmented all reflexes but gave rise to temporal subtraction of reflexes when C-fibres were stimulated.The present experiments have revealed a number of novel and important features of the sural-MG reflex pathway: (i) activity in fine afferent axons augments the reflexogenic potential of all subsequent afferent input, thereby allowing all afferent drive from the sural field to contribute to withdrawal of the heel; (ii) endogenous adrenergic control of this reflex pathway is completely non-selective; (iii) there is a non-adrenergic element of descending inhibition that is selective for the late components of MG reflex responses, and this element is directed particularly against transmission through NMDA receptors; (iv) temporal summation in this reflex is dependent on NMDA receptor-dependent and -independent mechanisms; and (v) this temporal summation is in some way dependent on the integrity of descending pathways.

Spinal 5-HT-receptors and tonic modulation of transmission through a withdrawal reflex pathway in the decerebrated rabbit

1 In decerebrated, non‐spinalized rabbits, intrathecal administration of either of the selective 5‐HT1A‐receptor antagonists (S)WAY‐100135 or WAY‐100635 resulted in dose‐dependent enhancement of the reflex responses of gastrocnemius motoneurones evoked by electrical stimulation of all myelinated afferents of the sural nerve. The approximate ED50 for WAY‐100635 was 0.9 nmol and that for (S)WAY‐100135 13 nmol. Intrathecal doses of the antagonists which caused maximal facilitation of reflexes in non‐spinalized rabbits had no effect in spinalized preparations. 2 In non‐spinalized animals, intravenous administration of (S)WAY‐100135 was significantly less effective in enhancing reflexes than when it was given by the intrathecal route. 3 When given intrathecally, the selective 5‐HT2A/2c‐receptor antagonist, ICI 170,809, produced a bell‐shaped dose‐effect curve, augmenting reflexes at low doses (≥ 44 nmol), but reducing them at higher doses (982 nmol). Idazoxan, the selective a2‐adrenoceptor antagonist, was less effective in enhancing reflex responses when given intrathecally after ICI 170,809 compared to when it was given alone. Intravenous ICI 170,809 resulted only in enhancement of reflexes and the facilitatory effects of subsequent intrathecal administration of idazoxan were not compromised. 4 The selective 5‐HT3‐receptor blocker ondansetron faciliated gastrocnemius medialis reflex responses in a dose‐related manner when given by either intrathecal or intravenous routes. This drug was slightly more potent when given i.v. and it did not alter the efficacy of subsequent intrathecal administration of idazoxan. 5 None of the antagonists had any consistent effects on arterial blood pressure or heart rate. 6 These data are consistent with the idea that, in the decerebrated rabbit, 5‐HT released from descending axons has multiple roles in controlling transmission through the sural‐gastrocnemius medialis reflex pathway. Thus, it appears 5‐HT tonically inhibits transmission between sural nerve afferents and gastrocnemius motoneurones by an action at spinal 5‐HT1A‐receptors. Spinal 5‐HT2A/2C‐receptors may mediate a weak inhibition of transmission in the spinal cord, but more convincing evidence was obtained for their involvement in descending facilitatory tone. Further, some of the facilitatory consequences of spinal α2‐adrenoceptor blockade may be mediated through 5‐HT2 type receptors. Spinal 5‐HT3 receptors do not appear to have a major role in tonic modulation of the sural‐gastrocnemius medialis reflex.

Adrenergic and opioidergic modulation of a spinal reflex in the decerebrated rabbit.

1. In the decerebrated and spinalized rabbit, electrical stimulation of the sural nerve evokes a short‐latency reflex in the ipsilateral ankle extensor gastrocnemius medialis (GM) which is tonically suppressed by endogenous opioids. In the present study we have investigated the inhibitory influences affecting this reflex in non‐spinalized, decerebrated rabbits. 2. In non‐spinalized rabbits, the thresholds and latencies of the sural‐GM reflex were significantly higher than in spinalized preparations. The opioid antagonist naloxone and the alpha‐adrenoceptor antagonist idazoxan potentiated the reflex in both preparations. Naloxone was significantly more effective in spinalized rabbits whereas idazoxan had a much larger effect in non‐spinalized animals. 3. When the spinal cord was sectioned in the presence of naloxone alone, the GM reflex always increased in size. An ipsilateral hemisection of the cord was as effective as total section in this respect. When the section was performed in the presence of idazoxan and naloxone, the response usually decreased in size. 4. The alpha 2‐adrenoceptor agonist clonidine depressed the reflex in spinalized rabbits, an action that was reversed by idazoxan but not by naloxone. 5. These data show that in the decerebrated, non‐spinalized rabbit, the sural‐GM reflex is tonically suppressed by endogenous opioids, presumably acting at the segmental level, and by an ipsilateral descending pathway which involves an alpha‐adrenoceptor‐mediated synapse. Activity in this descending pathway masks the facilitatory effects of opioid antagonists on spinal reflexes in this preparation.

The contributions of μ‐, δ‐and κ‐opioid receptors to the actions of endogenous opioids on spinal reflexes in the rabbit

1 Spinal reflexes in the rabbit are suppressed tonically by endogenous opioids. The contributions made to this suppression by μ‐, δ‐and κ‐opioid receptors have been investigated by studying the actions of a range of opioid antagonists and agonists on reflexes evoked by sural nerve stimulation in the ankle extensor gastrocnemius medialis (g.m.), and in the knee flexor semitendinosus (s.t.). 2 When given at a total dose of 88.5 μg kg−1 i.v., either of the universal opioid receptor antagonists (–)‐naloxone and (–)‐quadazocine enhanced the g.m. response to more than 7 times the pre‐drug control values, and the s.t. reflex to 1.5 times controls. The effects of quadazocine were stereospecific. The selective δ antagonist ICI 174864 (3.5 mg kg−1 i.v. total) also augmented the g.m. reflex but only to twice pre‐drug controls. 3 The μ‐agonists fentanyl (100 μg kg−1) and morphine (50 mg kg−1) suppressed both g.m. and s.t. reflex responses to less than half control levels by a naloxone‐reversible mechanism. 4 The κ‐agonists bremazocine (50 μg kg−1 total), tifluadom (100 μg kg−1), ethylketocyclazocine (200 μg kg−1) and U50488H (1 mg kg−1) potentiated the g.m. reflex and had variable effects on the s.t. response. Naloxone usually added to the facilitatory actions of these drugs. κ‐Opioid receptor agonists also caused a profound, naloxone‐reversible depression of arterial blood pressure. 5 It may be concluded that the endogenous opioid‐mediated suppression of spinal reflexes in the rabbit is mediated mainly, if not exclusively, through μ‐receptors. There are no known endogenous ligands which are specific for the μ‐receptor, so in the present case it seems that selectivity is determined by the receptor population rather than by the ligand.

Organisation of sensitisation of hind limb withdrawal reflexes from acute noxious stimuli in the rabbit

Spatial aspects of central sensitisation were investigated by studying the effects on three hind limb withdrawal reflexes of an acute noxious stimulus (20 % mustard oil) applied to a number of locations around the body in decerebrate and in anaesthetised rabbits. Reflex responses to electrical stimulation of the toes were recorded from the ankle flexor tibialis anterior (TA) and the knee flexor semitendinosus (ST), whereas responses to stimulation of the heel were recorded from the ankle extensor medial gastrocnemius (MG). In non‐spinalised, decerebrated, pentobarbitone‐sedated preparations, flexor reflexes were facilitated significantly from sites on the plantar surface of the ipsilateral foot but were either inhibited or unaffected by stimulation of sites away from this location. The heel–MG reflex was facilitated from the ipsilateral heel and was inhibited from a number of ipsilateral, contralateral and off‐limb sites. In decerebrated, spinalised, pentobarbitone‐sedated animals, mustard oil applied to any site on the ipsilateral hind limb enhanced both flexor reflexes, whereas the MG reflex was enhanced only after stimulation at the ipsilateral heel and was inhibited after stimulation of the toe tips or TA muscle. Mustard oil on the contralateral limb had no effect on any reflex. In rabbits anaesthetised with pentobarbitone and prepared with minimal surgical interference, the sensitisation fields for the heel–MG and toes–TA reflexes were very similar to those in non‐spinal decerebrates whereas that for toes–ST was more like the pattern observed in spinalised animals. In no preparation was sensitisation or inhibition of reflexes related to the degree of motoneurone activity generated in direct response to the sensitising stimulus. This study provides for the first time a complete description of the sensitisation fields for reflexes to individual muscles. Descending controls had a marked effect on the area from which sensitisation of flexor reflexes could be obtained, as the sensitisation fields for the flexor reflexes evoked from the toes were larger in spinalised compared to decerebrated, non‐spinalised animals. The intermediate sizes of sensitisation fields in anaesthetised animals suggests that the area of these fields can be dynamically controlled from the brain. On the other hand, the sensitisation field for the heel–MG reflex varied little between preparations and appears to be a function of spinal neurones.

Enhancement and depression of spinal reflexes by 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin in the decerebrated and spinalized rabbit: involvement of 5‐HT1A‐ and non‐5‐HT1A‐receptors

1 In decerebrated, spinalized and paralyzed rabbits, intravenous administration of the 5‐HT1A‐receptor agonists (±)‐8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT, 3–300 nmol kg−1, cumulative) and flesinoxan (22–2200 nmol kg−1, cumulative) significantly increased the short latency reflex evoked in gastrocnemius medialis motoneurones by electrical stimulation of all myelinated afferents (Aβ and Aδ fibres) of the sural nerve. Reflexes increased to median values of 198% (inter‐quartile range (IQR) 148–473%) and 296% (IQR 254–522%) of pre‐drug values with the highest doses of 8‐OH‐DPAT and flesinoxan, respectively. The enhancement of reflexes induced by 5‐HT1A‐receptor agonists was not reversed by the selective 5‐HT1A‐receptor antagonist (S)WAY‐100135 (2.05 μmol kg−1). 2 The effects of 8‐OH‐DPAT were tested after pretreatment with (S)WAY‐100135 (2.05 μmol kg−1), its more potent analogue WAY‐100635 (185 nmol kg−1), and the 5‐HT2/5‐HT1D‐/5‐HT7‐receptor ligand ritanserin (1.67 μmol kg−1). 8‐OH‐DPAT (300 nmol kg−1 single dose) significantly increased gastrocnemius reflex responses in the presence of (S)WAY‐100135 and WAY‐100635, to median values of 260% (IQR 171–295%) and 165% (IQR 136–170%) of pre‐drug levels, respectively. These values were not significantly different from each other, or from the effects of 8‐OH‐DPAT given alone. When 8‐OH‐DPAT was given after ritanserin, reflexes were a median of 102% (IQR 76–148%) of pre‐drug values: i.e. there was no significant increase in responses. Neither WAY‐100635 nor ritanserin had any effects on reflexes per se. 3 WAY‐100635 (185 nmol kg−1) and ritanserin (1.67 μmol kg−1) were given after 8‐OH‐DPAT (300 nmol kg−1). The agonist increased reflexes to a median value of 184% (IQR 135–289%), after which WAY‐100635 significantly reduced responses to 165% (IQR 130–254%) and ritanserin further decreased reflexes to a median of 107% (IQR 100–154%) of pre‐drug levels, i.e. not significantly different from controls. 4 Previous studies have shown that reflexes evoked by large myelinated axons tend to be suppressed, rather than enhanced, by 5‐HT1A‐receptor agonists. When tested against reflexes evoked by stimulation of the sural nerve at strengths between 1.5 and 2.5 times threshold, 8‐OH‐DPAT (3–300 nmol kg−1, cumulative) and flesinoxan (22–2200 nmol kg−1, cumulative) significantly reduced gastrocnemius responses to median values of 36% (IQR 15–75%) and 17% (IQR 12–38%) of pre‐drug levels, respectively. This inhibition was fully reversed by (S)WAY‐100135 (2.05 μmol kg−1). 5 These data show that drugs that are agonists at 5‐HT1A‐receptors increase polysynaptic spinal reflexes evoked by moderate to high stimulus intensities and depress responses to very low intensity stimuli. The inhibitory effects of these drugs were mediated through 5‐HT1A‐receptors as they were abolished by a selective antagonist for these sites. However, the facilitatory effects of 8‐OH‐DPAT could be completely blocked only by a combination of ritanserin, which has no significant affinity for 5‐HT1A‐receptors, with WAY‐100635. It appears that the enhancement of reflexes by 8‐OH‐DPAT arises from a combined action at 5‐HT1A‐receptors and other, ritanserin‐sensitive, sites which could be 5‐HT1D‐ or 5‐HT7‐receptors.

Prolonged inhibition of a spinal reflex after intense stimulation of distant peripheral nerves in the decerebrated rabbit

1. In decerebrated rabbits, repetitive stimulation of the high‐threshold afferents of the left common peroneal (CP) nerve evokes prolonged depression of the sural‐gastrocnemius medialis (GM) reflex recorded in the same limb. This inhibition is antagonized by co‐administration of the opioid antagonist naloxone with the alpha 2‐adrenoceptor antagonist idazoxan. The present study was designed to investigate whether such inhibition could be elicited from the contralateral hindlimb or the forelimbs. 2. The sural‐GM reflex of decerebrated rabbits was depressed for more than 15 min after stimulation of either ipsilateral or contralateral common peroneal (CP) or median nerves with 500 pulses of 20 V, 1 ms given at 5 Hz. The order of efficacy for generating this inhibition was ipsilateral CP greater than contralateral CP greater than or equal to ipsilateral median = contralateral median. In three of thirty‐nine rabbits, stimulation of the median nerves caused facilitation of the sural‐GM reflex. 3. Idazoxan (1‐2 mg/kg I.V.) did not significantly alter the depressant effect of ipsilateral CP stimulation but reduced that evoked by either median nerve and almost abolished the inhibition evoked from the contralateral CP nerve. 4. Naloxone (0.25 mg/kg I.V.) reduced the effects of ipsilateral CP stimulation, did not alter the inhibition evoked from contralateral CP, and had equivocal actions on the responses to median nerve stimulation. 5. When given together, the two antagonists almost abolished the effects of stimulating the median nerves and the contralateral CP nerve, and markedly reduced the inhibition evoked from the ipsilateral CP nerve. 6. These data show that prolonged inhibition of the sural‐GM reflex can be evoked by stimulation of nerves in all four limbs and that in each case the inhibition can be blocked or reduced by co‐administration of antagonists to opioid and alpha 2‐adrenergic receptors. Such persistent inhibition of reflexes may serve to inhibit withdrawal reflexes in situations where interruptions to normal movement would be disadvantageous.

Noxious stimulation of the toes evokes long-lasting, naloxone-reversible suppression of the sural-gastrocnemius reflex in the rabbit

Repetitive stimulation of the small myelinated and non-myelinated afferents of the common peroneal (c.p.) nerve evokes a long-lasting (20-25 min), naloxone-reversible inhibition of the sural-gastrocnemius reflex in the decerebrated and spinalized rabbit. Altering the number and frequency of stimuli applied to the c.p. nerve showed that this inhibition was dependent on temporal summation of afferent input from that nerve, and that the optimum frequency for producing the effect was between 2 and 10 Hz. Application of natural conditioning stimuli in and around the receptive field of the c.p. nerve showed that noxious, but not innocuous, mechanical and thermal stimuli could evoke long-lasting inhibition of the sural-gastrocnemius reflex. Thermal stimuli produced a biphasic change in the excitability of the reflex with facilitation followed by inhibition. The opioid antagonist naloxone (250 micrograms.kg-1) blocked all suppression resulting from these natural noxious stimuli. Chemical stimulation of the skin with mustard oil did not evoke naloxone-reversible inhibition of the reflex. These results indicate that intensely noxious stimuli can promote the release of opioid peptides in the spinal cord, and that one of the functions of these peptides may be to regulate the level of excitability in withdrawal reflex pathways.

The role of 5-HT1A-receptors in fentanyl-induced bulbospinal inhibition of a spinal withdrawal reflex in the rabbit

Abstract The sural to gastrocnemius withdrawal reflex is inhibited after injection of the OP3 (&mgr;)‐receptor‐selective opioid fentanyl into the fourth ventricle of decerebrated rabbits. This effect is abolished by complete section of the spinal cord but not by the selective &agr;2‐adrenoceptor antagonist RX 821002 (Clarke RW, Parry‐Baggott C, Houghton AK, Ogilvie J. The involvement of bulbo‐spinal pathways in fentanyl‐induced inhibition of spinal withdrawal reflexes in the decerebrated rabbit. Pain 1998;78:197–207). We have now investigated the role of 5‐HT1A receptors in mediating the descending inhibition activated by intraventricular fentanyl. In the control state, intraventricular fentanyl (3–30 &mgr;g/kg) inhibited gastrocnemius reflex responses to a median of 34% of pre‐drug levels. After intrathecal administration of the selective 5‐HT1A receptor antagonist WAY‐100635 (100 &mgr;g), fentanyl reduced reflex responses to 83% of pre‐fentanyl values, significantly less inhibition than in the control state. In a separate group of experiments, intravenous fentanyl (0.3–30 &mgr;g/kg) depressed the sural‐gastrocnemius reflex to 17% of pre‐drug controls. This inhibition was not affected by intrathecal WAY‐100635 (100 &mgr;g), but combined administration of the 5‐HT1A antagonist with RX 821002 (100 &mgr;g) significantly reduced the effectiveness of i.v. fentanyl. After the highest dose reflexes were 37% of pre‐fentanyl levels. These data show that the bulbospinal inhibition activated by fentanyl is mediated, at least in part, by activation of spinal 5‐HT1A receptors. That blockade of these receptors failed to influence the inhibition induced by i.v. fentanyl might be taken to mean that the brain‐stem action of fentanyl does not contribute significantly to the systemic actions of this opioid. A more probable explanation is that, in the preparation used in the present study, the bulbospinal and direct spinal actions of fentanyl occlude each other to produce an overall inhibition that is less than the sum of the two effects.

Prolonged potentiation of transmission through a withdrawal reflex pathway after noxious stimulation of the heel in the rabbit

&NA; The sural‐gastrocnemius reflex of the spinalized rabbit was potentiated to an average of 3–6 times control levels after the application of noxious mechanical, thermal or chemical stimuli to the skin of the heel. Facilitation of the reflex was maximal within 1 min of the noxious stimulus, and in many cases persisted for more than 1 h. Prolonged increases in the excitability of the sural‐gastrocnemius reflex were not seen after innocuous mechanical or thermal stimulation of the heel. Repetitive electrical stimulation of the sural nerve (100 shocks given at 0.5 Hz) caused persistent facilitation of the reflex when small myelinated A&dgr; fibres or non‐myelinated C‐fibres were recruited by the conditioning stimulus. Such protracted increases in the excitability of the sural‐gastrocnemius pathway would enhance the protective functions of this reflex. The mechanisms described here have probably evolved to provide a high level of reflex protection to the heel after tissue damage has occurred at that site.