Experiments were performed on 12 adult cats of either sex weighing 3.5–6.4 kg. All surgery and experimental protocols were conducted in accordance with EU regulations (Council Directive 86/609/EEC) and with National Institutes of Health guidelines for the care and use of laboratory animals (National Institutes of Health publication no. 86-23 revised 1985). All procedures were approved by the Danish Animal Experimentation Inspectorate. Our experimental procedures also comply with the polices set out by The Journal of Physiology (Drummond, 2009).
After the induction of anaesthesia with halothane/isoflurane–nitrous oxide (2–3% halothane, 70% N2O and 30% O2), the animals were intubated, and cannulas were inserted into the right carotid artery and the right jugular vein (near the trachea) for the monitoring of blood pressure and the administration of fluid and drugs. Atropine (0.1 mg kg−1, s.c.) and solumedrol (2.5 mg kg−1, i.v.) were administered at the beginning of the experiment whilst a buffer solution (10% dextrose and 1.7% NaHCO3) was infused continuously (∼4.5 ml h−1) after cannulation. The cats were decerebrated, either by an anaemic decerebration (n= 10) or by a precollicular transection and removal of the rostral parts of the brain (n= 2). The experiments using precollicular transections were mainly used for data collection for other studies.
Anaemic decerebrations were performed under anaesthesia by ligating the basilar and both common carotid arteries, a procedure that has been shown to produce a decerebration that includes all cortical tissue above the pons and the anterior part of the cerebellum (Pollock & Davis, 1930; Crone et al. 1988). Following the transfer of the cat to the recording frame, the last carotid artery was ligated and the anaesthetic removed. The decerebration was verified to be clinically complete by the development of tonic extensor muscle tone, lack of spontaneous movements, and large non-reactive pupils (Crone et al. 1988). In 1/10 cats, stereotyped stepping movements developed after the removal of anaesthetics and in this case the anaesthesia was immediately reinstated and the brain was removed rostral to a section at the level of the superior colliculi. In this case, the brain was found to be necrotic, and it was concluded that the stepping movements must have originated from the caudal brainstem centres. Following these procedures and tests, pancuronium bromide (0.6 mg h−1) was administered to block neuromuscular transmission, and artificial respiration was initiated.
A precollicular decerebration (2 cats) was made, during anaesthesia, at the end of the surgical procedure after the animal had been transferred to the recording frame with a stereotactic head-holder (Gossard et al. 1994). Following this decerebration, pancuronium bromide (0.6 mg h−1) was administered to block neuromuscular transmission, and artificial respiration was initiated.
The following nerves from the left hindlimb were cut and dissected for stimulation and recording: quadriceps (Q), sartorius (Sart), semimembranosus and anterior biceps (SmAB), posterior biceps and semitendinosus (PBSt), medial gastrocnemius (MG), lateral gastrocnemius plus soleus (LGS), tibialis anterior and extensor digitorum longus (TAEDL), flexor digitorum and hallucis longus (FDHL) and plantaris (Pl). On the right side, typically the following nerves were dissected: Sart, rectus femoris (RF), vastus lateralis, medialis, and intermedialis (Vasti), lateral and medial gastrocnemius and soleus (GS), and posterior tibial (Tib). The Q, RF, Vasti and Sart nerves were placed in cuff electrodes, and the other nerves were mounted on bipolar silver–silver chloride hook electrodes. The nerve branches of the right tibial and common peroneal nerves were sectioned but were usually not mounted. Other hindlimb nerves including the obturator nerves were all denervated bilaterally. The tendons around the hip were cut bilaterally to avoid sensory information of limb position reaching the spinal cord as it may interfere with the expression of fictive locomotion (Kriellaars et al. 1994). To expose the lumbar spinal cord a laminectomy over the L4–L7 vertebral columns was performed. As the animals were transferred to the recording frame (including a stereotactic head-holder), both the spinal column and head were mechanically secured. The skin flaps around the exposed areas of the spinal cord and the hindlimb were sewn and retracted to form pools that were filled with warm paraffin oil. A pneumothorax was performed bilaterally prior to recording to reduce movement artefacts. A decrease of blood pressure to <80 mmHg was compensated for by i.v. administration of additional amounts of buffer solution. The expired CO2 was maintained between 3 and 6% and the animals were kept at around 37°C core temperature by feedback-controlled heating lamps.
In 9/10 cats in which anaemic decerebration was performed, a complete spinalisation was performed at the first cervical (C1) level following a local injection of lidocaine (SAD, 20 mg ml−1) into the spinal cord.
Experiments were terminated by an overdose of pentobarbital.
Fictive motor activity
Nialamide, a monoamine oxidase inhibitor (i.v. 50 mg kg−1, Sigma), was administered at a slow rate during the hindlimb surgery in experiments aimed at investigating DOPA-locomotion (anaemic decerebration was used in these instances). l-DOPA (50 mg kg−1) (Grillner & Zangger, 1979) was administered i.v. after the animals were mounted in the frame and spinalised at the C1 level. In some animals, a second dose of l-DOPA (given >6 h after the first dose) was also successful in evoking rhythmic activity lasting for another 4–6 h period.
In animals in which brainstem stimulation was used to evoke fictive locomotion, tungsten electrodes (0.1–0.3 MΩ, World Precision Instruments, Sarasota, FL, USA) were placed according to stereotaxic coordinates of the mesencephalic locomotor region (MLR) (Shik et al. 1969; Gossard et al. 1994) and the final electrode positioning was determined by monitoring rhythmic hindlimb ENG activity. Pulses of 0.1–0.2 ms duration were applied at frequencies of 10–20 Hz and at strengths of 50–150 μA.
The C1–C2 spinal segments were exposed in order to apply d-tubocurarine (0.1–0.3%) (Domer & Feldberg, 1960), and/or bicucculine (0.1–1%) (Baev et al. 1981) topically at the C1 and C2 dorsal root entry zones prior to mechanical stimulation of the skin covering the ears and the surrounding face in order to evoke fictive scratch activity.
Recording of interneurone activity
Extracellular recordings from identified Ia inhibitory INs were collected in three experiments. The criteria for identifying Ia inhibitory INs included the following: (1) action potentials could be evoked by peripheral nerve stimulation (low threshold group I fibres) at latencies <0.9 ms and could follow >200 Hz stimuli, (2) an inhibition of IN activity could be evoked by ventral root stimulation (i.e. recurrent inhibition; Hultborn et al. 1971; Hultborn, 1972). We preferentially aimed to record from INs receiving input from hip or ankle flexors and MNs innervating hip or ankle extensors. After identification, a bout of fictive motor activity was evoked and repeated typically 2–3 times. In a few cases, locomotion and scratch activity could be evoked consecutively while maintaining stable recording conditions for the same IN.
As one of the aims of this experiment was to clarify the relationship between the activity of Ia INs and MNs, we also recorded pairs of identified INs and MNs (Jankowska & Roberts, 1972) during bouts of fictive motor activity. This consisted of identifying a Ia IN (activated from Sart or Q) in the 5th lumbar segment at a depth of 2.9–3.8 mm, by the criteria stated above, and then intracellularly recording from an antidromically identified MN (SmAB or PBSt) in the 7th lumbar segment using a separate glass microelectrode.
Recording of disynaptic IPSPs/IPSCs during the locomotor cycle
Intracellular recordings were obtained (with a microelectrode amplifier; Axoclamp 2A, Molecular Devices, Sunnyvale, CA, USA) from antidromically identified MNs innervating the left hindlimb. Glass micropipettes (tip diameter, 1.2–2 μm; resistance, 2–4 MΩ) filled with the lidocaine derivative QX-314 (100–200 mm) in potassium acetate (2 m) were used. QX-314 blocks sodium spikes, allowing the recording of Ia IPSPs throughout the step/scratch cycle without interference of spike potentials. Only MNs with a stable membrane potential more hyperpolarised than −50 mV and spike potentials of more than 60 mV (before the action by QX-314) were accepted.
The recordings from MNs were used for recording (1) locomotor drive potentials, (2) unitary IPSPs as part of the most rigid identification of the Ia inhibitory INs (see preceding section), (3) Ia IPSPs to assess the transmission in the Ia inhibitory pathway, and (4) Ia IPSCs in voltage clamp mode.
The threshold (T) was defined as the intensity required for activating the largest diameter afferents in each individual nerve. The disynaptic reciprocal Ia IPSPs were evoked by stimulating group I afferents in the nerves from antagonist muscles. The electrical stimulus strength (single pulse) was adjusted to evoke IPSPs both at minimal amplitude (with stimulation about 1.1 ×T) and also as large as possible (with supramaximal stimulation for the Ia afferents; i.e. 2–5 ×T, see below).
One confounding factor when judging the convergence onto the pool of Ia inhibitory INs using changes in the amplitude of the Ia IPSPs recorded from the MNs is introduced when the membrane potential of the recorded MN is changing significantly. This is indeed the case during fictive motor behaviour. When the membrane potential is depolarised (i.e. during the active phase of the locomotor and scratch drive potentials) the difference between the current membrane potential and the equilibrium potential for IPSPs is of course increased resulting in larger amplitudes even with activation of the same number of inhibitory INs (Coombs et al. 1955). In order to resolve this complication we also recorded the Ia inhibition at a fixed membrane potential by recording the Ia IPSCs in single electrode voltage-clamp mode of the microelectrode amplifier (Stuart & Redman, 1990). The switching frequency was typically 3 kHz, and thus the efficiency of the clamp was not optimal for the beginning of the IPSP.
Another confounding factor is related to changes in the subliminal fringe (the number of neurones that are excited, but have not yet reached firing threshold) versus the discharge zone (the number of neurones that have reached firing threshold) in the pool of Ia inhibitory INs during the step/scratch cycle. To allow a facilitation of Ia IPSPs as the pool of Ia INs are excited by the locomotor circuits, it is important to keep input from the Ia afferents as weak as possible. Indeed, if the Ia volley is large enough to discharge most of the INs, an additional excitation in a particular phase of locomotion or scratch may actually cause a decreased Ia IPSP due to occlusion. In order to assess this factor, we often used two kinds of test Ia IPSPs, the first by weak stimulation of the antagonist nerve to evoke a Ia IPSP of minimal amplitude, and the second by a supramaximal stimulation for Ia afferents (2–5 ×T) to discharge most (or all) Ia inhibitory INs.
Data capture and analysis
ENG recordings were digitized at a rate of 10 kHz and filtered (5 Hz to 1 kHz). Potentials recorded from the dorsum of the spinal cord (CDP), IN recordings (AC coupled) and MN intracellular (i.c.) recordings (DC coupled) were digitized at a rate of 10–20 kHz. All recordings were collected and stored on a PC for offline analysis (CED 1401+ with Spike 2.5 software, Cambridge Electronic Design, Cambridge, UK). The ENG recordings from each nerve were rectified and smoothed in order to determine the onset of flexor and extensor bursts by visual inspection.
The extracellular recordings of the activity of Ia INs were low-pass filtered. Built-in spike sorting algorithms within the Spike 2.5 software were used to follow the activity of the neuron identified by the criteria listed above as a Ia inhibitory IN. Extracellular (e.c.) IN recordings as well as i.c. recordings from MNs were selected for further analysis from a bout of rhythmic activity with a minimum of five consecutive cycles with alternating flexion and extension. Cycle-based analysis of IN firing and IPSP amplitude was based on flexor bursts as determined by the onset of a flexor (either Sart or TA) ENG when passing a threshold, which was selected by visual inspection of the bout. Comparisons across different experiments were possible by normalizing the step cycles. Cycle duration was determined as the time between consecutive onsets of flexor activity; flexor phase duration was determined as the time period when the flexor ENG activity was larger than the determined thresholds. The time points when the minimum and maximum MN membrane potentials were measured were determined for each cycle and expressed as a percentage of the cycle (i.e. normalised). The time points at which IN firing started and stopped as well as the time point of maximal instantaneous firing were determined for each cycle and they were also normalised. Spike counts are reported as per 50 ms long bins during non-normalised cycles and as per cycle for normalised cycles and as mean firing frequency for flexion and extension averaged separately from several cycles. We verified that IPSPs were evoked disynaptically by confirming that the onset latency was between 1.2 and 1.5 ms from the arrival of the incoming volley at the cord dorsum. The membrane potential just prior to stimulation and the peak amplitude of the IPSPs were measured using averaged records during the active (i.e. depolarised) and the inactive (i.e. hyperpolarised) phases of each MN. Detailed analysis of IPSP modulation was performed in some cases by dividing the step or scratch cycles into 10 bins. The IPSPs evoked by near threshold stimulation were expressed both in absolute values (mV) and as percentages of the maximal IPSPs evoked by supramaximal stimulation. In voltage clamp mode, the peak amplitude of the IPSCs was measured during both the active and the inactive phases. Statistical comparisons between the amplitudes measured in the two phases were performed using Student's t test for paired data during each type of rhythmic motor activity. The IPSCs during rest (i.e. no ENG activity in any of the recorded nerves) and during tonic flexor activity preceding rhythmic scratch were also compared to the two phases during rhythmic scratch when these periods were sufficiently long that at least 10 single records could be averaged.