C fragment of tetanus toxin hybrid proteins evaluated for muscle-specific transsynaptic mapping of spinal motor circuitry in the newborn mouse.

We investigated whether the non-toxic C fragment of tetanus toxin (TTC) fused to either beta-galactosidase or green fluorescent protein could be utilized to transsynaptically trace muscle-specific spinal circuitry in the neonatal mouse after i.m. injection into a single hindlimb muscle. We found that even with careful low volume injection (0.2-1.0 microl) into a single muscle (medial gastrocnemius), the TTC hybrid proteins spread rapidly to many other hindlimb muscles and to trunk musculature such that retrograde labeling of motoneurons could not be constrained to a single motoneuron pool. Retrogradely labeled motoneurons in the lower lumbar segments harboring the medial gastrocnemius motoneuron pool were first observed two hours after the medial gastrocnemius injection. Within the next 10 h, additional lumbar and lower thoracic motoneurons became labeled, and punctate labeling in the neuropil surrounding the motoneurons appeared. Many of the TTC hybrid protein-labeled puncta in the neuropil co-localized synaptotagmin, indicating that they represent presynaptic axon terminals onto motoneurons. Although this is consistent with retrograde transsynaptic passage, we found no evidence that the TTC hybrid proteins were transported further along premotor axons to label interneuron somata. The i.m. TTC injection procedure described here therefore provides an important tool for the study of presynaptic terminals onto motoneurons. However, additional technical modifications will be required to utilize TTC tracers for transsynaptic mapping of muscle-specific spinal motor circuitry in the neonatal mouse. We provide here a set of criteria for assessing the i.m. delivery of TTC tracers as a basis for future improvements in this technique.

Organization of common synaptic drive to motoneurones during fictive locomotion in the spinal cat.

The basic locomotor rhythm in the cat is generated by a neuronal network in the spinal cord. The exact organization of this network and its drive to the spinal motoneurones is unknown. The purpose of the present study was to use time (cumulant density) and frequency domain (coherence) analysis to examine the organization of the last order drive to motoneurones during fictive locomotion (evoked by application of nialamide and dihydroxyphenylalanine (DOPA)) in the spinal cat. In all cats, narrow central synchronization peaks (half-width < 3 ms) were observed in cumulants estimated between electroneurograms (ENGs) of close synergists, but not between nerves belonging to muscles acting on different joints or to antagonistic muscles. Coherence was not observed at frequencies above 100 Hz and was mainly observed between synergists. Intracellular recording was obtained from a population of 70 lumbar motoneurones. Significant short-term synchronization was observed between the individual intracellular recordings and the ENGs recorded from nerves of the same pool and of close synergists. Recordings from 34 pairs of motoneurones (10 pairs belonged to the same motor pool, 11 pairs to close synergists and 13 pairs to antagonistic pools) failed to reveal any short-lasting synchronization. These data demonstrate that short-term synchronization during fictive locomotion is relatively weak and is restricted to close synergists. In addition, coherence analysis failed to identify any specific rhythmic component in the locomotor drive that could be associated with this synchronization. These results resemble findings obtained during human treadmill walking and imply that the spinal interneurones participating in the generation of the locomotor rhythm are themselves weakly synchronized. The restricted synchronization within the locomotor drive to motoneuronal pools may be a feature of the locomotor generating networks that is related to the ability of these networks to produce highly adaptive patterns of muscle activity during locomotion.

Presynaptic control of transmission along the pathway mediating disynaptic reciprocal inhibition in the cat.

In cat lumbar motoneurones, disynaptic inhibitory postsynaptic potentials (IPSPs) evoked by stimulation of antagonist motor nerves were depressed for at least 150 ms following conditioning stimulation of flexor (1.7-2 times threshold (T)) and ankle extensor (5T) nerves. The aim of the present study was to investigate the possibility that this depression is caused by presynaptic inhibitory mechanisms acting at the terminals of group I afferent fibres projecting to the Ia inhibitory interneurones and/or the terminals of these interneurones to the target motoneurones. Conditioning stimulation of flexor, but not ankle extensor, nerves evoked a depression of the monosynaptic Ia excitatory postsynaptic potentials (EPSPs) recorded intracellularly in Ia inhibitory interneurones. This depression lasted between 200 and 700 ms and was not accompanied by a depression of the monosynaptic EPSPs evoked by stimulation of descending pathways. These results suggest that flexor, but not ankle extensor, group I afferent fibres can modulate sensory transmission at the synapse between Ia afferent fibres and Ia inhibitory interneurones. Conditioning stimulation of flexor muscle nerves, extensor muscle nerves and cutaneous nerves produced a long-lasting increase in excitability of the terminals of the Ia inhibitory interneurones. The increase in the excitability of the terminals was not secondary to an electrotonic spread of synaptic excitation at the soma. Indeed, concomitant with the excitability increase of the terminals there were signs of synaptic inhibition in the soma. The unitary IPSPs induced in target motoneurones following the spike activity of single Ia inhibitory interneurones were depressed by conditioning stimulation of muscle and cutaneous nerves. Since the conditioning stimulation also evoked compound IPSPs in those motoneurones, a firm conclusion as to whether unitary IPSP depression involved presynaptic inhibitory mechanism of the terminals of the interneurones could not be reached. The possibility that the changes in excitability of the Ia interneuronal terminals reflect the presence of a presynaptic inhibitory mechanism similar to that operating at the terminals of the afferent fibres (presynaptic inhibition) is discussed.1. In cat lumbar motoneurones, disynaptic inhibitory postsynaptic potentials (IPSPs) evoked by stimulation of antagonist motor nerves were depressed for at least 150 ms following conditioning stimulation of flexor (1.7-2 times threshold (T)) and ankle extensor (5T) nerves. The aim of the present study was to investigate the possibility that this depression is caused by presynaptic inhibitory mechanisms acting at the terminals of group I afferent fibres projecting to the Ia inhibitory interneurones and/or the terminals of these interneurones to the target motoneurones. Conditioning stimulation of flexor, but not ankle extensor, nerves evoked a depression of the monosynaptic Ia excitatory postsynaptic potentials (EPSPs) recorded intracellularly in Ia inhibitory interneurones. This depression lasted between 200 and 700 ms and was not accompanied by a depression of the monosynaptic EPSPs evoked by stimulation of descending pathways. These results suggest that flexor, but not ankle extensor, group I afferent fibres can modulate sensory transmission at the synapse between Ia afferent fibres and Ia inhibitory interneurones. Conditioning stimulation of flexor muscle nerves, extensor muscle nerves and cutaneous nerves produced a long-lasting increase in excitability of the terminals of the Ia inhibitory interneurones. The increase in the excitability of the terminals was not secondary to an electrotonic spread of synaptic excitation at the soma. Indeed, concomitant with the excitability increase of the terminals there were signs of synaptic inhibition in the soma. The unitary IPSPs induced in target motoneurones following the spike activity of single Ia inhibitory interneurones were depressed by conditioning stimulation

Proprioceptive control of extensor activity during fictive scratching and weight support compared to fictive locomotion.

At rest, extensor group I afferents produce oligosynaptic inhibition of extensor motoneurons. During locomotor activity, however, such inhibition is replaced by oligosynaptic excitation. Oligosynaptic excitation from extensor group I afferents plays a crucial role in the regulation of extensor activity during walking. In this study we investigate the possibility that this mechanism also regulates extensor muscle activity during other motor tasks. We show that the reflex pathways responsible for extensor group I oligosynaptic excitation during fictive locomotion can be activated during both fictive scratching and fictive weight support (tonic motor activity induced by contralateral scratching). These observations suggest that the excitatory group I oligosynaptic reflex pathways are open for transmission during several forms of motor activities. We also show that extensor group I input during fictive scratching can affect the amplitude and the timing of extensor activity in a pattern similar to that observed during locomotion. Most likely these effects involve the activation of the excitatory group I oligosynaptic reflex pathways. Accordingly, it is suggested that extensor group I oligosynaptic excitation during motor activities other than locomotion is also used to regulate extensor muscle activity. Furthermore, the similarity of effects from extensor group I input on the rhythmicity during scratching and locomotion supports the hypothesis that both rhythms are generated by a common network.

Depression of muscle and cutaneous afferent-evoked monosynaptic field potentials during fictive locomotion in the cat.

1. Monosynaptic extracellular field potentials evoked by electrical stimulation of ipsilateral hindlimb nerves carrying muscle group I, II and cutaneous afferents were examined during fictive locomotion. Fifty-eight field potentials were recorded in the dorsal and intermediate laminae throughout the mid-lumbar to first sacral segments and fictive locomotion was evoked by mesencephalic locomotor region (MLR) stimulation in paralysed decerebrate cats. 2. The majority (96 %) of group I, II and cutaneous-evoked field potentials were decreased during fictive locomotion. Group I, cutaneous and dorsal group II potentials were reduced on average to about 80 % of control values. Group II field potentials recorded in the intermediate laminae were reduced to a mean of 49 % of control values. Cyclic variations in field potential amplitude between the flexion and extension phases were observed in 24 of 45 cases analysed. Of those 24 field potentials, the two group I and four cutaneous field potentials were smaller during the flexion phase. All eleven group II and the remaining seven cutaneous fields were smaller during extension. In all but two cases, these cyclic variations were smaller than the tonic depression upon which they were superimposed. 3. In 7/9 group II field potentials examined, reductions (on average to 85 % of control) began with the onset of MLR stimulation that produced tonic activity in the motor nerves before the onset of rhythmic alternating, locomotor discharges. In six of the seven cases the field potential depression increased with the establishment of fictive locomotion. This observation and the cyclic modulation of field potentials during fictive locomotion suggests that the depression was strongly linked to the operation of the spinal locomotor circuitry. 4. Depression of the monosynaptic components of the field potentials suggests a reduction in synaptic transmission from primary afferents to first-order spinal interneurones during fictive locomotion. Accordingly, the larger depression of intermediate group II field potentials may indicate a preferential reduction in transmission from group II afferents to interneurones located in intermediate spinal laminae. 5. Flexion reflexes evoked by group II and cutaneous afferents were also depressed during MLR-evoked fictive locomotion. The possibility that this depression results from a reduction in transmission from primary afferents, and in particular from group II afferents, ending on interneurones in the intermediate laminae is discussed.

How do we approach the locomotor network in the mammalian spinal cord?

For a large number of vertebrate species it is now indisputable that spinal networks have the capability of generating the basic locomotor rhythm. However, because of technical difficulties, the rate of progress in defining the intrinsic properties of mammalian locomotor rhythm generators has been slow in comparison to that made in the study of such networks in lower vertebrates. Investigations on afferent and descending control of locomotor activity in mammals have demonstrated that many of these pathways interact with the rhythm generator. In this review we discuss how these interactions (resetting) can be used for outlining relevant spinal circuits as a basis for a future identification of individual neurons of the spinal locomotor networks. In this overview we have given particular emphasis to selected afferent systems to illustrate the possibilities and problems with this approach.

Ankle extensor group I afferents excite extensors throughout the hindlimb during fictive locomotion in the cat.

1. The effects of stimulating hindlimb extensor nerves (100-200 ms trains, 100 Hz, < or = 2 times threshold) during the flexor and extensor phases of the locomotor step cycle were analysed in the decerebrate, paralysed cat during fictive locomotion evoked by stimulation of the mesencephalic locomotor region. 2. Stimulation during extension of either the medial gastrocnemius (MG), lateral gastrocnemius-soleus (LGS) or plantaris (Pl) nerves was equally effective in increasing the duration and amplitude of electroneurogram (ENG) activity recorded in ipsilateral ankle, knee and hip extensor nerves. Enhancement of extensor ENG activity could be evoked with near threshold stimulation intensity and appeared within 10-40 ms of the onset of ankle extensor nerve stimulation. Stimulation of anterior biceps during extension occasionally evoked a modest increase in the duration of activity of hip, knee and ankle extensors. Stimulation of quadriceps during extension enhanced the activity of proximal extensors and soleus, but inhibited other ankle extensors. 3. Selective activation of ankle extensor Ia spindle afferents by muscle stretch also enhanced ipsilateral extension. It is argued that both muscle spindle and tendon organ afferents can contribute to the increase in extensor nerve activity evoked by group I stimulation intensity during fictive locomotion. 4. During flexion, stimulation of either the MG, Pl or LGS nerves at group I strength terminated on-going activity in ipsilateral flexors and initiated a burst of activity in ipsilateral hip, knee and ankle extensors, i.e. reset the step cycle to extension. 5. Low strength stimulation of the mixed muscle and cutaneous nerve innervating the plantar aspect of the foot produced extension enhancement and resetting similar to that evoked by group I muscle afferent stimulation. Stimulation of the cutaneous nerve supplying the dorsal aspect of the foot during extension enhanced extensor activity, and during flexion, enhanced the activity of flexors. 6. The effects reported here during fictive locomotion may also occur during overground locomotion with natural activation of group I muscle spindle and tendon organ afferents. Extensor spindle and tendon organ afferents may thus serve as an excitatory reflex system helping to shape the amplitude, duration and timing of ipsilateral extensor activity. Increased or unexpected activation of group I ankle extensor afferents or plantar foot afferents during locomotion could also compensate for increased loading of the limb.

Effects of stimulation of hindlimb flexor group II afferents during fictive locomotion in the cat.

1. This study examines the effects of electrical stimulation of hindlimb flexor nerves on the fictive locomotion pattern. Locomotion was initiated by stimulation of the mesencephalic locomotor region in the decerebrate paralysed cat and monitored by recording the electroneurogram from selected hindlimb flexor and extensor muscle nerves. Flexor nerves were stimulated using short trains (20-50 stimuli at 100 Hz) during either the flexor or the extensor phase of the fictive locomotor cycle. 2. Stimulation of tibialis anterior (TA), posterior biceps and semitendinosus (PBSt) or sartorius (Sart) nerves at 5 times threshold (T) during the flexor phase of the fictive locomotor cycle terminated on-going activity in flexor nerves and initiated activity in extensors. Thus, flexor nerve stimulation during flexion shortened the locomotor cycle by resetting to extension. The failure of lower intensity (2T) stimulation of PBSt or Sart nerves to reset the step cycle to extension suggests that group II afferents are responsible for these actions. Resetting evoked by 2T stimulation of the TA nerve may be due to a high proportion of group II afferents with low electrical threshold. 3. During extension, stimulation of TA and PBSt nerves at 5T did not perturb the locomotor rhythm whereas Sart stimulation prolonged the locomotor cycle. 4. Stimulation of cutaneous or knee joint afferents failed to produce effects similar to those evoked by stimulation of flexor muscle nerves at group II strength. These findings are at odds with those obtained elsewhere in the acute spinal, DOPA fictive locomotion preparation. The possibility that group II resetting during fictive locomotion is not mediated by flexion reflex pathways but by previously unknown pathways released in the present preparation is discussed. 5. Since many of the flexor afferents recruited by 5T electrical stimulation are the length-sensitive group II fibres, spindle secondaries may act to regulate the duration and onset of flexor and extensor activity during real locomotion. The resetting from flexion to extension also suggests that unexpected or enhanced activity of flexor secondaries during swing would promote a switch of the step cycle to stance.

Microstimulation of the medullary reticular formation during fictive locomotion.

1. The present study was designed to determine the effects of microstimulation of the medullary reticular formation (MRF) on the locomotor activity of the cat in the absence of phasic afferent feedback from the limbs. To this end, both short (33 ms) and long (200 ms) trains of stimuli (trains of 0.2-ms pulses at 330 Hz, 35 microA) were applied at 43 loci in the MRF (P:6-12 mm; L:0.5-1.5 mm), and in 3 loci in the medial longitudinal fasciculus (P7.5, L < 0.5 mm) during fictive locomotion in the decerebrate and paralyzed cat. The locomotor pattern was monitored by recording the activity of representative flexor and extensor muscle nerves from each of the four limbs. 2. Short trains of stimuli evoked transient excitatory and/or inhibitory responses in extensor and flexor nerves of each limb that were incorporated into the locomotor pattern. In the majority of sites, excitatory responses were obtained in the motor nerves to both flexor and extensor muscles of the fore- and hindlimbs. The exception to this rule was the ipsilateral triceps, in which the predominant response was inhibitory. The amplitude of these responses was dependent on the time of the locomotor cycle at which the stimulus was delivered, and it was always maximum during the period of activity of the respective nerve. 3. The shortest latency response in the nerves to different muscles of the forelimb averaged between 5.6 and 7.3 ms; for the hindlimbs the values were between 6.9 and 9.3 ms. 4. Changing the depth at which the stimulation was applied in any one trajectory usually produced changes only in the amplitude of the evoked responses but occasionally also caused a change in the sign of these responses, especially in the most ventral regions of the MRF. 5. At 72% of the loci (31/43), short trains of stimulation also changed the duration of the activity in the recorded nerves. These changes were often (20/31 loci) sufficiently strong to alter the duration of the overall locomotor cycle. If one considers only the largest changes produced at each locus, stimulation during the period of ipsilateral extensor activity produced an average reduction in the ipsilateral locomotor cycle duration of 12.8 +/- 8.8% (mean +/- SD), whereas stimulation when the ipsilateral flexor nerve was active produced an average increase in locomotor cycle duration of 27.1 +/- 20.8%. 6. Long trains of stimuli produced similar but larger effects than the shorter trains and always reset the locomotor rhythm.(ABSTRACT TRUNCATED AT 400 WORDS)

Activity of medullary reticulospinal neurons during fictive locomotion.

1. The pattern of discharge of medullary reticulospinal neurons, identified by antidromic stimulation applied at the L1-L2 segment of the spinal cord, was studied during fictive locomotion, occurring spontaneously, or evoked by stimulation of the mesencephalic locomotor region in high-decerebrate, paralyzed cats. Unitary recordings were made in the medial reticular formation (P5.0-14.0 mm; L0.5-2.0 mm), and the fictive locomotor pattern was monitored by recording the electroneurogram (ENG) of representative flexor and extensor muscle nerves from each of the four limbs. 2. In total, 117 reticulospinal neurons were recorded in 15 cats. Among these, 73.5% (86/117) modified their discharge at the onset of locomotion. These cells were divided into three subpopulations: 34/86 of the cells always maintained a fixed temporal relationship with the activity of one of the recorded nerves (ENG-related = 39.6%); the pattern of discharge of 42/86 cells was related to the locomotor rhythm [(LR-related-48%)] but was not temporally correlated with any of the recorded nerves; and the remaining 10 cells increased their firing frequency at the onset of locomotion but remained tonic (TONIC-11.6%). 3. Of the ENG-related neurons, 64.8% were temporally correlated to extensor nerve activity, whereas the remaining 35.2% were correlated to flexor nerves. These neurons were either related to forelimb (55.9%) or hindlimb (44.1%) nerves lying either ipsilateral (38.2%) or contralateral (61.8%) to the recording site. A few neurons (n = 3; 8.8%) were related to nerve activity of more than one limb. 4. The pattern of discharge of the LR-related neurons, although not correlated to the activity of any one recorded nerve, could be preferentially related to the locomotor rhythm in either the forelimbs (12/23) or hindlimbs (11/23). 5. ENG- and LR-related reticulospinal neurons were intermingled in the medial reticular formation. In both cases, cells related to the forelimbs were located more dorsally than those related to the hindlimbs. It is suggested that both the ENG- and LR-related neurons represent a single functional population of reticulospinal neurons that is part of an intrinsically organized reticulospinal system that functions to coordinate the activity of the skeletal musculature. 6. The present results show that the majority of reticular neurons projecting as far as the lumbar spinal cord are phasically modulated during locomotion, even in the absence of phasic peripheral afferent inputs. Moreover, the complexity of the discharge patterns in paralyzed animals was found to be similar to that observed in the intact cat.(ABSTRACT TRUNCATED AT 400 WORDS)