The results were collected from different series of experiments in adult cats: 15 experiments with intracellular recording; five experiments with monosynaptic reflex testing and systematic analysis of local specificity of stimulation; some 40 experiments with monosynaptic reflex testing on the influence of opioids and l-DOPA (l-3′4-dihydroxyphenylalanine) on the transmission in the segmental reflex pathways; and four experiments with recording of neurograms during fictive locomotion. The experiments were carried out with official permission in accordance with national guidelines.
The basic procedure was identical in all experiments. Under general ether-halothane-nitrous oxide anaesthesia (O2:N2O, 1:2, halothane initially 2.5 %, then increasingly replaced by ether, to obtain full anaesthesia, as assessed by a complete loss of muscle tone and by a lack of blood pressure, heart rate or motor responses to any stimuli) the cats were anaemically decapitated by a permanent bilateral ligature of both common carotid arteries and their main branches, and of the ascending vertebral arteries. An irreversible interruption of the spontaneous respiration resulted from this procedure together with persistent large, non-reacting pupils. These effects were taken as a sign of the completion of anaemic decapitation.
To prove the reliability of the procedure, in another, former series of experiments 100 ml of an Evans Blue solution (0.5 g (100 ml)−1, 2000 i.u. heparin added) was infused over a period of 3 min. Then the cat was killed and dissected 5 min after the infusion. In contrast to spinal structures and the muscles of the body and limbs, no dye was found within the superficial (pial) or deep brain vessels (for further details see Kniffki et al. 1981).
After anaemic decapitation, the cat was artificially ventilated, spinalised at C1 and paralysed with pancuronium bromide (Pancuronium ‘Organon’ about 0.15 mg kg−1 every hour i.v. as required). An end-expiratory CO2 concentration of 3.5–4.5 % was regulated via the respiratory volume. The arterial blood pressure was maintained above 80 mmHg, if necessary by infusion of a dextran solution. Rectal temperature was maintained close to 37–38 °C. At the end of the experiments the cats were killed by injection of 5 ml of 3 m KCl solution, which induced immediate cardiac arrest.
Preparation and stimulation
One hindlimb was completely denervated except for the plantar division of the tibial nerve (Tib) which innervates the foot pad. For comparison, in six experiments the sural nerve (Sur, innervating the heel and the lateral side of the foot) or the cutaneous branch of the superficial peroneal nerve (SPC, innervating the dorsum of the foot) was also kept intact. The proximal branches of the transected nerves, posterior biceps semitendinosus (PBSt), anterior biceps semimembranosus (ABSm), quadriceps (Q), gastrocnemius-soleus (GS, partly separated as lateral gastrocnemius-soleus, LGS, and medial gastrocnemius, MG), plantaris (Pl), flexor digitorum et hallucis longus (FDL), deep peroneal (DP), superficial peroneal nerve muscular branch (SPm), saphenus (Saph), Sur and SPC (if not intact), posterior nerve to the knee joint (joint), as well as the Tib nerve (mobilised if left in continuity) were mounted on bipolar electrodes and electrically stimulated with single or double rectangular pulses of 0.1 ms duration, with a recurrence frequency of 1 Hz (intracellular recording) or 0.5 Hz (monosynaptic reflex testing) and a stimulation strength indicated in multiples of the threshold strength of the nerves (‘T‘) as indicated by the afferent volley recorded from the dorsal root L7 close to the root entry. Nociceptive cutaneous afferents were activated by noxious radiant heat (48–55 °C and up to 60 °C, in four specifically cited experiments). The radiated area was a circle of 1 cm2 and its temperature was measured at the skin surface (cf. Schomburg & Steffens, 1986). Heat stimulation was applied to areas innervated by the intact nerve or nerves, respectively, in the case of the intact Tib nerve to the central foot pad and for comparison to the different toe pads. Low threshold mechanoreceptors were activated by light stroking of the plantar side of the foot.
Except for the experiments concerning fictive locomotion, the ventral roots L5 or L6 to S2 were cut and the ventral roots L7/S1 were mounted for stimulation in experiments with intracellular recording and for recording in experiments with monosynaptic reflex testing.
Intracellular recording of α-motoneurones (DC recording bandwidth 0–3 kHz, AC recording 0.1–10 kHz) was performed with microelectrodes filled with 2 m potassium citrate. Results were obtained from 126 motoneurones of PBSt (41), GS (38), ABSm (23), Tib (12), Pl (9) and SPm (3).
Monosynaptic reflex testing. In order to be able to test different reflex pathways to different motor nuclei in parallel for a systematic analysis of local specificity of noxious stimulation or before, during and after drug application, the technique of monosynaptic reflex testing was used. The monosynaptic reflexes were recorded from the ventral roots L7/S1. The reflex recordings were rectified and averaged over eight samples with a time resolution of 20 μs per point.
Different peripheral motor nerves (PBSt, GS, Pl, Tib) and occasionally FDL, SPm and DP were stimulated alternately. The strength of the test stimuli was routinely 5T i.e. well above group I maximum in order to avoid any influence of a changing group I excitability during the experiment. Single pulses were used. Only if single pulses were insufficient to evoke a reflex were double pulses applied, at an interval of 1.0–1.2 ms. Any polysynaptic responses which occurred at the stimulus strength used and identified by their longer delay, were excluded from the data analysis.
The conditioning stimuli were as follows: noxious radiant heat applied to the central foot pad (and also to the different toe pads for analysis of local specificity) or to the dorsum or the lateral side of the foot for activation of nociceptive cutaneous afferents; stimulation of cutaneous nerves (Saph, Sur, SPC) with low strength (generally less than 1.2T) for activation of low threshold cutaneous afferents; stimulation of the joint nerve (strength not higher than 2.2T) for activation of mainly non-nociceptive joint afferents; and stimulation of the Q and the GS nerve (5.5T) for activation of group Ib and group II muscle afferents (conditioning-test interval with electrical conditioning nerve stimulation 5–7 ms). A differentiation between group Ib and group II effects was performed by grading the strength (cf. Fu et al. 1974; Lundberg et al. 1987a). Antagonistic group Ia effects from Q to PBSt were largely excluded by the long conditioning-test interval, but a contribution of Ia fibres to the Ib effects from Q and GS via common interneurones (Jankowska, 1979; Harrison et al. 1983) cannot be excluded.
The influence of the following drugs was tested: (d-Ser2)-leuenkephalin (Thr6) (DSLET) an opioid with a δ-morphine receptor agonistic action; (d-Ala2,N-Me-Phe4,Gly5-ol)-enkephalin (DAMGO), an opioid with a μ-morphine receptor agonistic action; a benzomorphan derivate (MR 2034) with a prevailing κ-morphine receptor agonistic action, l-DOPA (40–100 mg kg−1, i.v.), and the α2-agonist clonidine (0.02–0.045 mg kg−1, i.v.). The application of the opioids was performed in two different ways. The lumbar spinal cord (segment L3 to cauda equina) was suffused with opioids (‘local application’, seven experiments with DSLET, five experiments with DAMGO) diluted in Ringer solution (concentration of 10−6-10−3m). After suffusion the exposed spinal cord remained covered with the solution. In other experiments the drugs were injected intravenously (vena cava superior, dosage: DSLET, 0.5–3.6 mg kg−1; DAMGO, 1.2–2 mg kg−1; MR2034, 1–3 mg kg−1).
In most experiments, after recovery from the first application (generally after about 3–10 h, average time 7.7 h) the drug was applied a second, sometimes even a third, time. Between local applications the opioid solution was washed out and the spinal cord was covered with Ringer's solution for at least 1 h. If not mentioned otherwise, in the figures, according to the technique used, the time course of the influence of the opioids (and naloxone) is shown for the different reflex pathways in parallel, i.e. before, during and after the same application of a drug.
Fictive locomotion. In experiments with investigations on Pl activity during fictive locomotion, neurograms were recorded from the nerves to PBSt, GS, Pl, FDL and DP (partly on both sides). Stimulation of nociceptive afferents and cutaneous nerves was performed in the same way as in the other experiments. Fictive locomotion was induced by i.v. injection of 100 mg kg−1 nialamide and 40–100 mg kg−1l-DOPA (cf. Koehler et al. 1984).