Motoneurone synchronization for intercostal and abdominal muscles: interneurone influences in two different species.

The contribution of branched-axon monosynaptic inputs in the generation of short-term synchronization of motoneurones remains uncertain. Here, synchronization was measured for intercostal and abdominal motoneurones supplying the lower thorax and upper abdomen, mostly showing expiratory discharges. Synchronization in the anaesthetized cat, where the motoneurones receive a strong direct descending drive, is compared with that in anaesthetized or decerebrate rats, where the direct descending drive is much weaker. In the cat, some examples could be explained by branched-axon monosynaptic inputs, but many others could not, by virtue of peaks in cross-correlation histograms whose widths (relatively wide) and timing indicated common inputs with more complex linkages, e.g., disynaptic excitatory. In contrast, in the rat, correlations for pairs of internal intercostal nerves were dominated by very narrow peaks, indicative of branched-axon monosynaptic inputs. However, the presence of activity in both inspiration and expiration in many of the nerves allowed additional synchronization measurements between internal and external intercostal nerves. Time courses of synchronization for these often consisted of combinations of peaks and troughs, which have never been previously described for motoneurone synchronization and which we interpret as indicating combinations of inputs, excitation of one group of motoneurones being common with either excitation or inhibition of the other. Significant species differences in the circuits controlling the motoneurones are indicated, but in both cases, the roles of spinal interneurones are emphasised. The results demonstrate the potential of motoneurone synchronization for investigating inhibition and have important general implications for the interpretation of neural connectivity measurements by cross-correlation.

Cardiac modulation of alpha motoneuron discharges.

Recordings of alpha motoneuron discharges from branches of the intercostal and abdominal nerves in anesthetized cats were analyzed for modulation during the cardiac cycle. Cardiac modulation was assessed by the construction of cross-correlation histograms between the R-wave of the ECG and the largest amplitude efferent spikes. In all but two recordings (which were believed to have either no or few alpha spikes), the histograms showed relatively short duration peaks and/or troughs (widths at half amplitude 4-50 ms) at lags of 10-150 ms. These observations were deduced to result from activity in oligosynaptic pathways, probably from muscle spindle afferents, whose discharges are known to be synchronized to the cardiac pulse. The results suggest that onward transmission of the cardiac signal from thoracic muscle afferents (and possibly from other dynamically sensitive afferents) to other parts of the central nervous system is highly likely and that therefore these afferents could contribute to cardiac interoception. NEW & NOTEWORTHY It has been recognized since 1933 that muscle spindles respond to the cardiac pulse, but it is unknown whether this cardiac signal is transmitted to other levels in the nervous system. Here we show that a cardiac signal, likely arising from muscle spindles, is present in the efferent activities of thoracic and abdominal muscle nerves, suggesting probable onward transmission of this signal to higher levels and therefore that muscle spindles could contribute to cardiac interoception.

Functional plasticity in the respiratory drive to thoracic motoneurons in the segment above a chronic lateral spinal cord lesion.

A previous neurophysiological investigation demonstrated an increase in functional projections of expiratory bulbospinal neurons (EBSNs) in the segment above a chronic lateral thoracic spinal cord lesion that severed their axons. We have now investigated how this plasticity might be manifested in thoracic motoneurons by measuring their respiratory drive and the connections to them from individual EBSNs. In anesthetized cats, simultaneous recordings were made intracellularly from motoneurons in the segment above a left-side chronic (16 wk) lesion of the spinal cord in the rostral part of T8, T9, or T10 and extracellularly from EBSNs in the right caudal medulla, antidromically excited from just above the lesion but not from below. Spike-triggered averaging was used to measure the connections between pairs of EBSNs and motoneurons. Connections were found to have a very similar distribution to normal and were, if anything (nonsignificantly), weaker than normal, being present for 42/158 pairs, vs. 55/154 pairs in controls. The expiratory drive in expiratory motoneurons appeared stronger than in controls but again not significantly so. Thus we conclude that new connections made by the EBSNs following these lesions were made to neurons other than α-motoneurons. However, a previously unidentified form of functional plasticity was seen in that there was a significant increase in the excitation of motoneurons during postinspiration, being manifest either in increased incidence of expiratory decrementing respiratory drive potentials or in an increased amplitude of the postinspiratory depolarizing phase in inspiratory motoneurons. We suggest that this component arose from spinal cord interneurons.

Absence of synergy for monosynaptic Group I inputs between abdominal and internal intercostal motoneurons.

Internal intercostal and abdominal motoneurons are strongly coactivated during expiration. We investigated whether that synergy was paralleled by synergistic Group I reflex excitation. Intracellular recordings were made from motoneurons of the internal intercostal nerve of T8 in anesthetized cats, and the specificity of the monosynaptic connections from afferents in each of the two main branches of this nerve was investigated. Motoneurons were shown by antidromic excitation to innervate three muscle groups: external abdominal oblique [EO; innervated by the lateral branch (Lat)], the region of the internal intercostal muscle proximal to the branch point (IIm), and muscles innervated from the distal remainder (Dist). Strong specificity was observed, only 2 of 54 motoneurons showing excitatory postsynaptic potentials (EPSPs) from both Lat and Dist. No EO motoneurons showed an EPSP from Dist, and no IIm motoneurons showed one from Lat. Expiratory Dist motoneurons fell into two groups. Those with Dist EPSPs and none from Lat (group A) were assumed to innervate distal internal intercostal muscle. Those with Lat EPSPs (group B) were assumed to innervate abdominal muscle (transversus abdominis or rectus abdominis). Inspiratory Dist motoneurons (assumed to innervate interchondral muscle) showed Dist EPSPs. Stimulation of dorsal ramus nerves gave EPSPs in 12 instances, 9 being in group B Dist motoneurons. The complete absence of heteronymous monosynaptic Group I reflex excitation between muscles that are synergistically activated in expiration leads us to conclude that such connections from muscle spindle afferents of the thoracic nerves have little role in controlling expiratory movements but, where present, support other motor acts.

Axon diameters and conduction velocities in the macaque pyramidal tract.

Small axons far outnumber larger fibers in the corticospinal tract, but the function of these small axons remains poorly understood. This is because they are difficult to identify, and therefore their physiology remains obscure. To assess the extent of the mismatch between anatomic and physiological measures, we compared conduction time and velocity in a large number of macaque corticospinal neurons with the distribution of axon diameters at the level of the medullary pyramid, using both light and electron microscopy. At the electron microscopic level, a total of 4,172 axons were sampled from 2 adult male macaque monkeys. We confirmed that there were virtually no unmyelinated fibers in the pyramidal tract. About 14% of pyramidal tract axons had a diameter smaller than 0.50 µm (including myelin sheath), most of these remaining undetected using light microscopy, and 52% were smaller than 1 µm. In the electrophysiological study, we determined the distribution of antidromic latencies of pyramidal tract neurons, recorded in primary motor cortex, ventral premotor cortex, and supplementary motor area and identified by pyramidal tract stimulation (799 pyramidal tract neurons, 7 adult awake macaques) or orthodromically from corticospinal axons recorded at the mid-cervical spinal level (192 axons, 5 adult anesthetized macaques). The distribution of antidromic and orthodromic latencies of corticospinal neurons was strongly biased toward those with large, fast-conducting axons. Axons smaller than 3 µm and with a conduction velocity below 18 m/s were grossly underrepresented in our electrophysiological recordings, and those below 1 µm (6 m/s) were probably not represented at all. The identity, location, and function of the majority of corticospinal neurons with small, slowly conducting axons remains unknown.

Connections between expiratory bulbospinal neurons and expiratory motoneurons in thoracic and upper lumbar segments of the spinal cord.

Cross-correlation of neural discharges was used to investigate the connections between expiratory bulbospinal neurons (EBSNs) in the caudal medulla and expiratory motoneurons innervating thoracic and abdominal muscles in anesthetized cats. Peaks were seen in the cross-correlation histograms for around half of the EBSN-nerve pairs for the following: at T8, the nerve branches innervating internal intercostal muscle and external abdominal oblique muscle and a more distal branch of the internal intercostal nerve; and at L1, a nerve branch innervating internal abdominal oblique muscle and a more distal branch of the ventral ramus. Fewer peaks were seen for the L1 nerve innervating external abdominal oblique, but a paucity of presumed α-motoneuron discharges could explain the rarity of the peaks in this instance. Taking into account individual EBSN conduction times to T8 and to L1, as well as peripheral conduction times, nearly all of the peaks were interpreted as representing monosynaptic connections. Individual EBSNs showed connections at both T8 and L1, but without any discernible pattern. The overall strength of the monosynaptic connection from EBSNs at L1 was found to be very similar to that at T8, which was previously argued to be substantial and responsible for the temporal patterns of expiratory motoneuron discharges. However, we argue that other inputs are required to create the stereotyped spatial patterns of discharges in the thoracic and abdominal musculature.

Voltage-dependent amplification of synaptic inputs in respiratory motoneurones.

The role of persistent inward currents (PICs) in cat respiratory motoneurones (phrenic inspiratory and thoracic expiratory) was investigated by studying the voltage-dependent amplification of central respiratory drive potentials (CRDPs), recorded intracellularly, with action potentials blocked with the local anaesthetic derivative, QX-314. Decerebrate unanaesthetized or barbiturate-anaesthetized preparations were used. In expiratory motoneurones, plateau potentials were observed in the decerebrates, but not under anaesthesia. For phrenic motoneurones, no plateau potentials were observed in either state (except in one motoneurone after the abolition of the respiratory drive by means of a medullary lesion), but all motoneurones showed voltage-dependent amplification of the CRDPs, over a wide range of membrane potentials, too wide to result mainly from PIC activation. The measurements of the amplification were restricted to the phase of excitation, thus excluding the inhibitory phase. Amplification was found to be greatest for the smallest CRDPs in the lowest resistance motoneurones and was reduced or abolished following intracellular injection of the NMDA channel blocker, MK-801. Plateau potentials were readily evoked in non-phrenic cervical motoneurones in the same (decerebrate) preparations. We conclude that the voltage-dependent amplification of synaptic excitation in phrenic motoneurones is mainly the result of NMDA channel modulation rather than the activation of Ca2+ channel mediated PICs, despite phrenic motoneurones being strongly immunohistochemically labelled for CaV1.3 channels. The differential PIC activation in different motoneurones, all of which are CaV1.3 positive, leads us to postulate that the descending modulation of PICs is more selective than has hitherto been believed.

Electrophysiological and morphological characterization of propriospinal interneurons in the thoracic spinal cord.

Propriospinal interneurons in the thoracic spinal cord have vital roles not only in controlling respiratory and trunk muscles, but also in providing possible substrates for recovery from spinal cord injury. Intracellular recordings were made from such interneurons in anesthetized cats under neuromuscular blockade and with the respiratory drive stimulated by inhaled CO(2). The majority of the interneurons were shown by antidromic activation to have axons descending for at least two to four segments, mostly contralateral to the soma. In all, 81% of the neurons showed postsynaptic potentials (PSPs) to stimulation of intercostal or dorsal ramus nerves of the same segment for low-threshold (≤ 5T) afferents. A monosynaptic component was present for the majority of the peripherally evoked excitatory PSPs. A central respiratory drive potential was present in most of the recordings, usually of small amplitude. Neurons depolarized in either inspiration or expiration, sometimes variably. The morphology of 17 of the interneurons and/or of their axons was studied following intracellular injection of Neurobiotin; 14 axons were descending, 6 with an additional ascending branch, and 3 were ascending (perhaps actually representing ascending tract cells); 15 axons were crossed, 2 ipsilateral, none bilateral. Collaterals were identified for 13 axons, showing exclusively unilateral projections. The collaterals were widely spaced and their terminations showed a variety of restricted locations in the ventral horn or intermediate area. Despite heterogeneity in detail, both physiological and morphological, which suggests heterogeneity of function, the projections mostly fitted a consistent general pattern: crossed axons, with locally weak, but widely distributed terminations.

Further observations on cardiac modulation of thoracic motoneuron discharges.

Previous analyses of recordings of alpha motoneuron discharges from branches of the intercostal and abdominal nerves in anesthetized cats under neuromuscular blockade demonstrated modulation with the cardiac cycle. This modulation was interpreted as evidence that thoracic somatosensory afferents, most likely muscle spindles, provide a signal to the CNS that could contribute to cardiac interoception. Here, two aspects of these observations have been extended. First, new measurements of thoracic and abdominal EMG activity in spontaneously breathing dogs show that a very similar modulation exists in these rather different circumstances. Second, further analysis of the cat recordings shows that cardiac modulation of the discharges of bulbospinal neurons that transmit the expiratory drive to thoracic motoneurons is weak and of an inappropriate time-course to be a contributor to the effect seen in the motoneurons.

Respiratory drive in hindlimb motoneurones of the anaesthetized female cat.

Anatomical studies have shown a monosynaptic projection from nucleus retroambiguus (NRA) to semimembranosus (Sm) motor nucleus in female cats, which is stronger in oestrus. Expiratory bulbospinal neurones are the best documented functional cell type in the NRA. If these cells participate in this projection, an expiratory drive would be expected in Sm motoneurones and this drive would be expected to be stronger in oestrus. In anaesthetized, paralyzed, ovariohysterectomized female cats, artificially ventilated to produce a strong respiratory drive (as monitored by phrenic nerve discharges), intracellular recordings were made from Sm motoneurones and from motoneurones in the surrounding hindlimb motor nuclei that are outside the focus of the NRA projection. The animals comprised two groups: either treated for 7 days with oestradiol benzoate (oestrous) or untreated (non-oestrous). Central respiratory drive potentials (CRDPs) were observed in most motoneurones of both groups, with amplitudes larger for the oestrous than for the non-oestrous group (1.58+/-1.34 mV versus 0.89+/-0.79 mV, mean+/-S.D.). However, the CRDPs most often consisted of a maximum depolarization in early expiration, which declined in late expiration and into inspiration. This pattern is different from the incrementing firing pattern of most expiratory bulbospinal neurones. The CRDPs in Sm and semitendinosus motoneurones (located in the same motor column) were of similar size and frequency to CRDPs in motoneurones outside that column. The hypothesis that expiratory bulbospinal neurones are significantly involved in the projection was rejected. Alternative sources and possible functional roles for the CRDPs are discussed.

Ultrastructural evidence for direct excitatory retroambiguus projections to cutaneous trunci and abdominal external oblique muscle motoneurons in the cat.

The nucleus retroambiguus (NRA) is a group of neurons, located laterally in the caudal medulla oblongata. The NRA is thought to modulate abdominal pressure in the framework of respiration, vomiting, vocalization, probably parturition, and, in all likelihood mating behavior. The NRA exerts this control through its projections to motoneurons to the nucleus ambiguus in the lateral medulla (innervating pharynx, larynx), and spinal cord (innervating cutaneous trunci, intercostal, abdominal, pelvic floor, and lower limb muscles). The nature of these NRA-motoneuronal projections is unknown. In this study we have determined the ultrastructure of the NRA-motoneuronal projections, and especially those to the abdominal external oblique and cutaneous trunci muscles. In four cats 0.1% cholera toxin subunit b was injected in the external oblique and cutaneous trunci muscles to retrogradely label their motoneurons in the spinal cord. Wheat germ agglutinin-conjugated horseradish peroxidase was injected into the NRA to anterogradely label its contralaterally descending fibers to the motoneurons of both muscles. In order to prevent anterograde labeling of ipsilaterally descending systems not originating from the NRA, a hemisection was made at the level of C2 prior to the NRA injection. The ultrastructural results indicate that the majority (60-74%) of the anterogradely labeled NRA-terminals made monosynaptic contacts with retrogradely labeled dendrites of the external oblique and the cutaneous trunci muscle motoneurons. The majority (86-95%) of the NRA terminals made asymmetric synaptic contacts and 79-84% contained round vesicles. These results demonstrate the existence of direct, presumably excitatory, projections from NRA to external oblique and cutaneous trunci muscle motoneurons.

Macaque ventral premotor cortex exerts powerful facilitation of motor cortex outputs to upper limb motoneurons.

The ventral premotor area (F5) is part of the cortical circuit controlling visuomotor grasp. F5 could influence hand motor function through at least two pathways: corticospinal projections and corticocortical projections to primary motor cortex (M1). We found that stimulation of macaque F5, which by itself evoked little or no detectable corticospinal output, could produce a robust modulation of motor outputs from M1. Arrays of fine microwires were implanted in F5 and M1. During terminal experiments under chloralose anesthesia, single stimuli delivered to M1 electrodes evoked direct (D) and indirect (I1,I2, and I3) corticospinal volleys. In contrast, single F5 shocks were ineffective; double shocks (3 msec separation) evoked small I waves but no D wave. However, when the test (T) M1 shock was conditioned (C) by single or double F5 shocks, there was strong facilitation of I2 and I3 waves from M1, with C-T intervals of <1 msec. Intracellular recordings from 79 arm and hand motoneurons (MNs) revealed no postsynaptic effects from single F5 shocks. In contrast, these stimuli produced a robust facilitation of I2 and I3 EPSPs evoked from M1 (60% of MNs); this was particularly marked in hand muscle MNs (92%). Muscimol injection in M1 reduced I waves from F5 and abolished the F5-induced facilitation of late I waves from M1, and of EPSPs associated with them. Thus, some motor effects evoked from F5 may be mediated by corticocortical inputs to M1 impinging on interneurons generating late corticospinal I waves. Similar mechanisms may allow F5 to modulate grasp-related outputs from M1.

The importance of the cortico-motoneuronal system for control of grasp.

Our recent work has revealed new evidence of the importance of direct cortico-motoneuronal (CM) connections for voluntary control of the hand. Most of these connections are derived from corticospinal neurons located in the M1 hand area, although there are some much smaller contributions from other secondary motor areas, such as the supplementary motor area (SMA). Intracellular recordings show that 75% of upper limb motoneurons in the chloralose-anaesthetized macaque monkey receive a monosynaptic projection from the corticospinal tract; evidence for non-monosynaptic, propriospinal excitatory influences from the corticospinal tract was conspicuously lacking in these anaesthetized preparations. Moreover, in the conscious monkey, hand and arm muscle motor unit responses to corticospinal tract input are dominated by single, brief peaks compatible with monosynaptic excitation. CM excitatory post-synaptic potentials, recorded from a comparable sample of hand and arm motoneurons in anaesthetized macaque and squirrel monkeys, were found to be larger and faster rising in the macaque, which is by far the more dexterous of the two species. CM cells facilitating a given muscle in the conscious macaque are distributed over a wide region of M1 cortex, and each contributes a particular pattern of discharge during a skilled task. In addition to their direct effects on target muscles there may be weaker but potentially important effects that derive from the synchronous binding of assemblies of output neurons. Synchronous oscillations between these neurons are particularly prevalent during steady grip, but disappear during digit movement.

Focal blockade of single unit synaptic transmission by iontophoresis of antagonists.

We have assessed the use of iontophoresis for investigating the pharmacology of synaptic transmission from individual presynaptic neurones in-vivo, by modifying extracellular focal synaptic potentials (FSPs) recorded by spike-triggered averaging. FSPs from two types of excitatory neurone (muscle spindle primary afferents and expiratory bulbospinal neurones) and from one inhibitory interneurone were studied in the thoracic ventral horn of anaesthetized cats. The antagonist of excitatory amino acids at non-NMDA receptors, DNQX, blocked the FSPs from the first two, as did bicuculline for the third. Thus the FSPs were generated by excitatory amino acids acting via non-NMDA receptors and GABA, respectively. The postsynaptic neurones were probably motoneurones. Merits and limitations of the method are discussed.

Intercostal muscles and purring in the cat: the influence of afferent inputs.

Feline purring has previously been reported as originating in a central oscillator, independent of afferent inputs, and also as not involving expiratory muscles. Here we show, via electromyographic recordings from intercostal muscles, quantified by cross-correlation, that expiratory muscles can be involved and that even if the oscillator is central, reflex components nevertheless play a considerable part in the production of the periodic pattern of muscle activation seen during purring.

Respiratory recovery following high cervical hemisection.

In this paper we review respiratory recovery following C2 spinal cord hemisection (C2HS) and introduce evidence for ipsilateral (IL) and contralateral (CL) phrenic motor neuron (PhrMN) synchrony post-C2HS. Rats have rapid, shallow breathing after C2HS but ventilation ( logical or (E)) is maintained. logical or (E) deficits occur during hypercapnic challenge reflecting reduced tidal volume (VT), but modest recovery occurs by 12 wks post-injury. IL PhrMN activity recovers in a time-dependent manner after C2HS, and neuroanatomical evidence suggests that this may involve both mono- and polysynaptic pathways. Accordingly, we used cross-correlation to examine IL and CL PhrMN synchrony after C2HS. Uninjured rats showed correlogram peaks consistent with synchronous activity and common synaptic input. Correlogram peaks were absent at 2 wks post-C2HS, but by 12 wks 50% of rats showed peaks occurring with a 1.1+/-0.19ms lag from zero on the abscissa. These data are consistent with prolonged conduction time to IL (vs. CL) PhrMNs and the possibility of polysynaptic inputs to IL PhrMNs after chronic C2HS.

The respiratory drive to thoracic motoneurones in the cat and its relation to the connections from expiratory bulbospinal neurones.

The descending control of respiratory-related motoneurones in the thoracic spinal cord remains the subject of some debate. In this study, direct connections from expiratory bulbospinal neurones to identified motoneurones were investigated using spike-triggered averaging and the strengths of connection revealed were related to the presence and size of central respiratory drive potentials in the same motoneurones. Intracellular recordings were made from motoneurones in segments T5-T9 of the spinal cord of anaesthetized cats. Spike-triggered averaging from expiratory bulbospinal neurones in the caudal medulla revealed monosynaptic EPSPs in all groups of motoneurones, with the strongest connections to expiratory motoneurones with axons in the internal intercostal nerve. In the latter, connection strength was similar irrespective of the target muscle (e.g. external abdominal oblique or internal intercostal) and the EPSP amplitude was positively correlated with the amplitude of the central respiratory drive potential of the motoneurone. For this group, EPSPs were found in 45/83 bulbospinal neurone/motoneurone pairs, with a mean amplitude of 40.5 microV. The overall strength of the connection supports previous measurements made by cross-correlation, but is about 10 times stronger than that reported in the only previous similar survey to use spike-triggered averaging. Calculations are presented to suggest that this input alone is sufficient to account for all the expiratory depolarization seen in the recorded motoneurones. However, extra sources of input, or amplification of this one, are likely to be necessary to produce a useful motoneurone output.

Functional heterogeneity among neurons in the nucleus retroambiguus with lumbosacral projections in female cats.

Nucleus retroambiguus (NRA), in the caudal medulla, projects to all spinal levels. One physiological role is abdominal pressure control, evidenced by projections to intercostal and abdominal motoneurons from expiratory bulbospinal neurons (EBSNs) within NRA. The roles of NRA projections to the lumbosacral cord are less certain, although those to limb motoneurons may relate to mating behavior and those to Onuf's nucleus (ON) to maintaining continence. To clarify this we physiologically characterized NRA projections to the lumbosacral cord. Extracellular recordings were made in NRA under anesthesia and paralysis in estrus cats. Administered CO(2) gave a strong respiratory drive. Antidromic unit responses were recorded to stimulation of the contralateral ventrolateral funiculus of L(6), L(7), or sacral segments and to microstimulation in the region of semimembranosus motor nucleus or ON. All units were found at sites showing expiratory discharges. Units that showed collisions between antidromic and spontaneous spikes (all in late expiration) were identified as EBSNs. These were common from the ventrolateral funiculus (VLF) of L(6) (42.5%) or L(7) (32.9%), but rare from the sacral VLF or the motor nuclei. Antidromic latencies revealed a subthreshold respiratory drive in some non-EBSNs. This group had lower conduction velocities than the EBSNs. The remainder, with a negligible respiratory drive, had even lower conduction velocities. A new population of NRA neurons has thus been defined. They are not active even with a strong respiratory drive, but may provide most of the synaptic input from NRA to lower lumbar and sacral segments and could subserve functions related to mating behavior.