Identification of interneurons with contralateral, caudal axons in the lamprey spinal cord: synaptic interactions and morphology

1. As part of a continuing investigation of the organization of the spinal cord of the lamprey, propriospinal interneurons with axons projecting contralaterally and caudally (CC interneurons) were surveyed with intracellular recordings. 2. CC interneurons were identified by recording their axon spikes extracellularly in the spinal cord during intracellular stimulation of the cell body. The axon projections of Cc interneurons were confirmed after intracellular injection and development of horseradish peroxidase. 3. Intracellular stimulation of CC interneurons produced synaptic potentials in myotomal motoneurons, lateral interneurons and other CC interneurons that lay caudally on the opposite side of the spinal cord. Most CC interneurons were inhibitory, but some were excitatory. 4. CC interneurons were divided into three classes on the basis of reticulospinal Müller cell inputs. CC1 interneurons were excited by the ipsilateral Müller cell B1 and the contralateral Mauthner cell. CC1 interneurons were inhibitory. They were excited polysynaptically by ipsilateral sensory dorsal cells and were inhibited by contralateral dorsal cells. They were distinguished morphologically by having no rostral axon branch and no contralateral dendrites. CC1 interneurons were phasically active during fictive swimming with their peak depolarizations preceding those of myotomal motoneurons by about 0.15 cycle. 5. CC2 interneurons were also inhibitory, but they were distinguished from CC1 interneurons by their excitation from the ipsilateral Müller cells B2-4 nd by their thin rostral and thicker caudal axonal branches on the contralateral side of the spinal cord. 6. CC3 interneurons were excitatory, and they were inhibited by the ipsilateral Müller cell I1. CC3 interneurons could have contralateral dendrites and bifurcating axons, and they had lower average axonal conduction velocities than CC1 and CC2 interneurons. 7. Inhibitory CC interneurons may be important for motor coordination in the lamprey. Movements of the lamprey body during reflexes and swimming consist of contraction and relaxation of myotomal muscles on opposite sides of the body. By being coactive with ipsilateral myotomal motoneurons, inhibitory CC interneurons could contribute to the inhibition of contralateral motoneurons during these movements.     

Effects of groups of propriospinal interneurons on fictive swimming in the isolated spinal cord of the lamprey

Fictive swimming activity was induced in isolated spinal cords of adult lampreys Ichthyomyzon unicuspis and Petromyzon marinus by addition of D-glutamate or N-methyl-D,L-aspartate (NMA) to the bathing fluid. Propriospinal interneurons are defined as nerve cells within the spinal cord with projections longer than 1 segment. The hypothesis that propriospinal interneurons contribute to intersegmental coordination during fictive swimming was tested using electrical stimulation, extracellular recording, and separated compartments. Stimulation of the split caudal end of the spinal cord indirectly excited ascending propriospinal interneurons, which enhanced and entrained bursts in rostral contralateral ventral roots. Indirect electrical stimulation of descending propriospinal interneurons could delay and diminish bursts in caudal contralateral ventral roots. Extracellular recordings from the rostral and caudal split ends of the spinal cord sometimes showed spike activities in phase with contralateral or ipsilateral ventral roots. Inhibition of 1-3 segments by spot applications of glycine or gamma-aminobutyric acid (GABA) did not interrupt normal coordination or rostrocaudal phase lag. When a middle region of spinal cord was inhibited in a compartment with GABA or glycine, the caudal spinal cord could entrain the bursts in rostral ventral roots. In a few preparations the caudal region induced antiphasic bursts in previously silent rostral roots through the inhibited region. The maximum separation for caudal-upon-rostral antiphasic entrainment was approximately 20 segments in Ichthyomyzon and 36 segments in Petromyzon. Increased concentrations of an excitatory amino acid in a rostral compartment could produce descending entrainment of bursts in an adjacent caudal compartment at a higher frequency with rostrocaudal phase lag. The rostral-upon-caudal entrainment could still occur through spot applications of GABA or glycine but not through long inhibited regions. Two hypothetical groups of propriospinal interneurons are proposed for the coordination of swimming activities in the isolated spinal cords of adult lampreys. 1) Crossed, ascending interneurons may be excited in phase with nearby motoneurons and may excite and entrain rostral pattern generators on the opposite side. 2) Short, commissural interneurons may be excited in phase with nearby motoneurons and may inhibit contralateral generators.     

Extent and role of multisegmental coupling in the Lamprey spinal locomotor pattern generator

Timing of oscillatory activity along the longitudinal body axis is critical for locomotion in the lamprey and other elongated animals. In the lamprey spinal locomotor central pattern generator (CPG), intersegmental coordination is thought to arise from the pattern of extensive connections made by propriospinal interneurons. However, the mechanisms responsible for intersegmental coordination remain unknown, in large part because of the difficulty in obtaining quantitative information on these multisegmental fibers. System-level experiments were performed on isolated 50-segment preparations of spinal cord of adult silver lampreys, Ichthyomyzon unicuspis, to determine the dependence of CPG performance on multisegmental coupling. Coupling was manipulated through use of an experiment chamber with movable partitions, which allowed separate application of solution to rostral, middle, and caudal regions of the spinal cord preparation. During control trials, fictive locomotion, induced by bath application of D-glutamate in all three regions, was recorded extracellularly from ventral roots. Local synaptic activity in a variable number of middle segments was subsequently blocked with a low-Ca(2+), high-Mn(2+) saline solution in the middle compartment, whereas conduction in axons spanning the middle segments was unaffected. Spectral analysis was used to assess the effects of blocking propriospinal coupling on intersegmental phase lag, rhythm frequency, correlation, and variability. Significant correlation and a stable phase lag between the rostral and caudal regions of the spinal cord preparation were maintained during block of as many as 16 and sometimes 20 intervening segments. However, the mean value of this rostrocaudal phase decreased with increasing number of blocked segments from the control value of approximately 1% per segment. By contrast, phase lags within the rostral and caudal end regions remained unaffected. The cycle frequency in the rostral and caudal regions decreased with the number of blocked middle segments and tended to diverge when a large number of middle segments was blocked. The variability in cycle frequency and intersegmental phase both increased with increasing number of blocked segments. In addition, a number of differences were noted in the properties of the motor output of the rostral and caudal regions of the spinal cord. The results indicate that the maximal functional length of propriospinal coupling fibers is at least 16-20 segments in I. unicuspis, whereas intersegmental phase lags are controlled at a local level and are not dependent on extended multisegmental coupling. Other possible roles for multisegmental coupling are discussed.     

Disruption of left-right reciprocal coupling in the spinal cord of larval lamprey abolishes brain-initiated locomotor activity

In this study, contributions of left-right reciprocal coupling mediated by commissural interneurons in spinal locomotor networks to rhythmogenesis were examined in larval lamprey that had longitudinal midline lesions in the rostral spinal cord [8 --> 30% body length (BL), relative distance from the head] or caudal spinal cord (30 --> 50% BL). Motor activity was initiated from brain locomotor command systems in whole animals or in vitro brain/spinal cord preparations. After midline lesions in the caudal spinal cord in whole animals and in vitro preparations, left-right alternating burst activity could be initiated in rostral and usually caudal regions of spinal motor networks. In in vitro preparations, blocking synaptic transmission in the rostral cord abolished burst activity in caudal hemi-spinal cords. After midline lesions in the rostral spinal cord in whole animals, left-right alternating muscle burst activity was present in the caudal and sometimes the rostral body. After spinal cord transections at 30% BL, rhythmic burst activity usually was no longer generated by rostral hemi-spinal cords. For in vitro preparations, very slow burst activity was sometimes present in isolated right and left rostral hemi-spinal cords, but the rhythmicity for this activity appeared to originate from the brain, and the parameters of the activity were significantly different from those for normal locomotor activity. In summary, in larval lamprey under these experimental conditions, left and right hemi-spinal cords did not generate rhythmic locomotor activity in response to descending inputs from the brain, suggesting that left-right reciprocal coupling contributes to both phase control and rhythmogenesis.     

Segmental distribution of common synaptic inputs to spinal motoneurons during fictive swimming in the lamprey.

These experiments were designed to measure the degree of shared synaptic inputs coming to pairs of myotomal motoneurons during swimming activity in the isolated spinal cord of the lamprey. In addition, the experiments measured the decrease in the degree of shared synaptic inputs with the distance between the motoneurons to assess the segmental distribution of these shared inputs. Intracellular microelectrode recordings of membrane potential were made simultaneously on pairs of myotomal motoneurons during swimming activity induced with an excitatory amino acid. The swim cycle oscillations of motoneuron membrane potentials were removed with a digital notch filter, thus leaving the fast synaptic activities that underlie these slower oscillations. Cross-correlations of the fast synaptic activities in two simultaneously recorded motoneurons were made to measure the degree of shared inputs. The cross-correlation was done on time windows restricted to one swim cycle or to part of a swim cycle, and 50 consecutive swim cycle cross-correlograms then were averaged. The peak coefficients of the cross-correlations exhibited a wide range, even for pairs of motoneurons located near one another (range = 0.06-0.74, for pairs located within 2 segments). This observation suggests that there may be different functional classes of myotomal motoneurons with inputs originating from different sets of premotor interneurons. In spite of this variability, the mean peak correlation coefficients of motoneuron pairs clearly decreased with the distance between them. With separations of more than five segments, there was little or no clear correlation between the motoneurons (range = 0.04-0.10). These results suggest that common synaptic inputs to motoneurons during fictive swimming originate from local premotor interneurons and that beyond five spinal segments, common premotor inputs are rare or weak to motoneurons. Thus the premotor signals originating from the locomotor network have relatively short distribution lengths, on the order of 5 segments of 120 total spinal segments.     

Measurement and analysis of postsynaptic potentials using a novel voltage-deconvolution method

This study investigated cellular and synaptic mechanisms of cholinergic neuromodulation in the in vitro lamprey spinal cord. Most spinal neurons tested responded to local application of acetylcholine (ACh) with depolarization and decreased input resistance. The depolarization persisted in the presence of either tetrodotoxin or muscarinic antagonist scopolamine and was abolished with nicotinic antagonist mecamylamine, indicating a direct depolarization through nicotinic ACh receptors. Local application of muscarinic ACh agonists modulated synaptic strength in the spinal cord by decreasing the amplitude of unitary excitatory and inhibitory postsynaptic potentials. The postsynaptic response to direct application of glutamate was unchanged by muscarinic agonists, suggesting a presynaptic mechanism. Cholinergic feedback from motoneurons was assessed using stimulation of a ventral root in the quiescent spinal cord while recording intracellularly from spinal motoneurons or interneurons. Mainly depolarizing potentials were observed, a portion of which was insensitive to removal of extracellular Ca2+, indicating electrotonic coupling. Hyperpolarizing potentials were also observed and were attenuated by the glycinergic antagonist strychnine, whereas depolarizing responses were potentiated by strychnine. Mecamylamine also reduced hyperpolarizing responses. The pharmacology of these responses suggests a Renshaw-like feedback pathway in lamprey. Immunohistochemistry for choline acetyltransferase, performed in combination with retrograde filling of motoneurons, demonstrated a population of nonmotoneuron cholinergic cells in the lamprey spinal cord. Thus endogenous cholinergic modulation of the lamprey spinal locomotor network is likely produced by both motoneurons and cholinergic interneurons acting via combined postsynaptic and presynaptic actions.     

On the control of myotomal motoneurones during “fictive swimming” in the lamprey spinal cord in vitro

Intracellular recordings have been made from myotomal motoneurones during “fictive swimming” in the in vitro preparation of the lamprey spinal cord, while monitoring the efferent burst activity in the ventral roots. The pattern of rhythmic activity in the motoneurones is described, as well as how synaptic inputs from the premotoneuronal level exert their control of motoneurone activity. (1) All motoneurones investigated displayed rhythmic, symmetric oscillations of their membrane potential during “fictive swimming”. The period of depolarization occurred in phase with the burst discharge in the ventral root containing the motoneurone axon. (2) About one-third of the cells fired bursts of action potentials during the depolarized phase, while the remaining motoneurones exhibited subthreshold oscillations. (3) Intracellular injection of chloride ions reversed the sign of the hyperpolarized phase, demonstrating phasic active inhibition of the motoneurones during rhythmicity. (4) The depolarized phase was unaffected after chloride injection, showing that the motoneurones also received phasic active excitation. (5) “Pre-triggered” averaging of the motoneurone recording (using the ventral root spikes from other motoneurones for triggering), revealed that some degree of synchronous excitation of several motoneurones occurred, suggesting common excitation from the same premotor-interneurones. It is concluded that the rhythmic oscillations of membrane potential in lamprey myotomal motoneurones during “fictive locomotion” depend on phasic excitation alternating with phasic active inhibition. The premotoneuronal mechanism responsible for this control may consist of reciprocally organized groups of excitatory and inhibitory interneurones.     

Integration of nonphaselocked exteroceptive information in the control of rhythmic flight in the locust

The integration of exteroceptive information in the flight control system of the locust was studied by determining the cellular basis of ocellar- (simple eye) mediated control of flight. Neural interactions that transform phase-independent sensory input into phase-specific motor output were characterized. Ocellar information about course deviations during flight was conveyed to the segmental thoracic ganglia by three pairs of large fast multimodal descending neurons. These made connections with thoracic motoneurons directly, via short-latency mono-or disynaptic pathways, and indirectly, via a population of intercalated thoracic interneurons. The synaptic potentials caused in the motoneurons by the direct pathway occurred at short latency and were adequate for summation with other types of sensory input. However, the strength of the synaptic effects of this pathway was weak compared with the central flight oscillator drive to the same motoneurons. In contrast, synaptic potentials evoked by the descending neurons in the thoracic interneurons were often large and brought these cells close to threshold. In turn, these interneurons always had stronger synaptic effects on postsynaptic flight motoneurons than did the descending neurons alone. We conclude that the indirect interneuronal pathway is more powerful in its effects on motoneurons than the direct pathway. Premotor thoracic interneurons, which received ocellar input appropriate for a role in correctional steering, were also rhythmically modulated during flight motor activity in phase with either depressor or elevator motoneurons. This phasic modulatory drive occurred in deafferented preparations, indicating that its source is the central oscillator for flight. Presentation of ocellar stimulation during flight motor activity showed that the central oscillatory modulation of the thoracic interneurons gated the transmission of sensory information through these interneurons. Ocellar-mediated postsynaptic potentials influenced the firing of thoracic interneurons only if they arrived during the proper phase of rhythmic drive. Thus the transmission of ocellar information from interneuron to motor neuron is possible only during appropriate phases of the flight cycle.     

Identification of excitatory interneurons contributing to generation of locomotion in lamprey: structure, pharmacology, and function

1. In the in vitro preparation of the lamprey spinal cord, paired intracellular recordings of membrane potential were used to identify interneurons producing excitatory postsynaptic potentials (EPSPs) on myotomal motoneurons. 2. Seventy-nine interneurons (8.4% of all neuron-motoneuron pairs tested) elicited unitary EPSPs that followed one-for-one at short, constant latencies and were therefore considered monosynaptic according to conventional criteria. Evidence was obtained for selectivity and divergence of excitatory interneuron (EIN) outputs and for convergence of EIN input to motoneurons. 3. The neurotransmitter released by EINs may be an excitatory amino acid such as glutamate, because the EPSPs were depressed by antagonists of excitatory amino acids. 4. Intracellular dye injection revealed that EINs have small cell bodies (average 11 x 27 microns), transversely oriented dendrites, and thin (less than 3 microns) slowly conducting axons (0.7 m/s) that project caudally and ipsilaterally. One EIN exhibited a system of thin multi-branching axon collaterals with periodic swellings. Ultrastructurally, these swellings contained clear spherical vesicles, and they apposed postsynaptic membrane specializations. 5. During fictive locomotion, the membrane-potential oscillations of EINs were greater in amplitude than, but similar in shape and timing to, those of their postsynaptic motoneurons. EINs fired action potentials during fictive locomotion and contributed to the depolarization of motoneurons. 6. These interneurons are proposed to be a source of excitation to motoneurons and interneurons in the lamprey spinal cord, participating in motor activity including locomotion.     

Coordination of locomotor activity in the lamprey: role of descending drive to oscillators along the spinal cord

In the lamprey and most fish, locomotion is characterized by caudally propagating body undulations that result from a rostrocaudal phase lag for ipsilateral burst activity. One of the mechanisms that might contribute to rostrocaudal phase lags is a gradient of oscillator burst frequencies along the spinal cord that presumably are produced, in part, by descending drive from the brain. The purpose of the present study was to test whether a gradient of oscillator frequencies does exist along the lamprey spinal cord. First, during brain-initiated locomotor activity in in vitro brain/spinal cord preparations, the cycle times (=1/frequency) of locomotor activity generated by the functionally isolated rostral spinal cord (activity blocked in middle and caudal cord) were significantly shorter than control cycle times when the entire spinal cord was generating locomotor activity. Second, the cycle times of locomotor activity generated by the functionally isolated caudal cord (activity blocked in rostral and middle cord) were significantly longer than control cycle times for activity generated by the entire spinal cord. Thus, no one region of the spinal cord appears to dictate the overall cycle times of locomotor activity generated by the entire spinal cord, although overall cycle times tended to be closest to those of the isolated rostral spinal cord. Finally, although short- and long-distance coupling as well as oscillator frequency gradients probably contribute to rostrocaudal phase lags of spinal locomotor activity, the asymmetrical nature of short-distance coupling, in which the descending component dominates, appears to be the main factor. Received: 24 August 1998 / Accepted: 1 March 1999     

Commissural interneurons in rhythm generation and intersegmental coupling in the lamprey spinal cord.

Commissural interneurons in rhythm generation and intersegmental coupling in the lamprey spinal cord. To test the necessity of spinal commissural interneurons in the generation of the swim rhythm in lamprey, longitudinal midline cuts of the isolated spinal cord preparation were made. Fictive swimming was then induced by bath perfusion with an excitatory amino acid while recording ventral root activity. When the spinal cord preparation was cut completely along the midline into two lateral hemicords, the rhythmic activity of fictive swimming was lost, usually replaced with continuous ventral root spiking. The loss of the fictive swim rhythm was not due to nonspecific damage produced by the cut because rhythmic activity was present in split regions of spinal cord when the split region was still attached to intact cord. The quality of this persistent rhythmic activity, quantified with an autocorrelation method, declined with the distance of the split spinal segment from the remaining intact spinal cord. The deterioration of the rhythm was characterized by a lengthening of burst durations and a shortening of the interburst silent phases. This pattern of deterioration suggests a loss of rhythmic inhibitory inputs. The same pattern of rhythm deterioration was seen in preparations with the rostral end of the spinal cord cut compared with those with the caudal end cut. The results of this study indicate that commissural interneurons are necessary for the generation of the swimming rhythm in the lamprey spinal cord, and the characteristic loss of the silent interburst phases of the swimming rhythm is consistent with a loss of inhibitory commissural interneurons. The results also suggest that both descending and ascending commissural interneurons are important in the generation of the swimming rhythm. The swim rhythm that persists in the split cord while still attached to an intact portion of spinal cord is thus imposed by interneurons projecting from the intact region of cord into the split region. These projections are functionally short because rhythmic activity was lost within approximately five spinal segments from the intact region of spinal cord.     

Sacrocaudal afferents induce rhythmic efferent bursting in isolated spinal cords of neonatal rats

The ability of mammalian spinal cords to generate rhythmic motor behavior in nonlimb moving segments was examined in isolated spinal cords of neonatal rats. Stimulation of sacrocaudal afferents (SCA) induced alternating left-right bursts in lumbosacral efferents and in tail muscles. On each side of the tail, flexors, extensors, and abductors were coactive during each cycle of activity. This rhythm originated mainly in the sacrocaudal region because it persisted in sacrocaudal segments after surgical removal of the thoracolumbar cord. Sacrocaudal commissural pathways were sufficient to maintain the left-right alternation of lumbar efferent bursts, because their timing was unaltered after a complete thoracolumbar hemisection. The lumbar rhythm originated in part from sacrocaudal activity ascending in lateral and ventrolateral funiculi, because efferent bursts in rostral lumbar segments were nearly abolished on a particular side by lesions of the lateral quadrant of the cord at the L(4)-L(5) junction. Intracellular recordings from S(2)-S(3) motoneurons, obtained during the rhythm, revealed the presence of phasic oscillations of membrane potential superimposed on a tonic depolarization. Bursts of spikes occurred on the depolarizing phases of the oscillation. Between these bursts the membrane input conductance increased, and hyperpolarizing drive potentials were revealed. The inhibitory drive and the decreased input resistance coincided with contralateral efferent bursts, suggesting that crossed pathways controlled it. Our studies indicate that pattern generators are not restricted to limb-moving spinal segments and suggest that regional specializations of pattern-generating circuitry and their associated interneurons are responsible for the different motor patterns produced by the mammalian spinal cord.     

Neuropeptide-mediated facilitation and inhibition of sensory inputs and spinal cord reflexes in the lamprey

The effects of neuromodulators present in the dorsal horn [tachykinins, neuropeptide Y (NPY), bombesin, and GABAB agonists] were studied on reflex responses evoked by cutaneous stimulation in the lamprey. Reflex responses were elicited in an isolated spinal cord preparation by electrical stimulation of the attached tail fin. To be able to separate modulator-induced effects at the sensory level from that at the motor or premotor level, the spinal cord was separated into three pools with Vaseline barriers. The caudal pool contained the tail fin. Neuromodulators were added to this pool to modulate sensory inputs evoked by tail fin stimulation. The middle pool contained high divalent cation or low calcium Ringer to block polysynaptic transmission and thus limit the input to the rostral pool to that from ascending axons that project through the middle pool. Ascending inputs and reflex responses were monitored by making intracellular recordings from motor neurons and extracellular recordings from ventral roots in the rostral pool. The tachykinin neuropeptide substance P, which has previously been shown to potentiate sensory input at the cellular and synaptic levels, facilitated tail fin-evoked synaptic inputs to neurons in the rostral pool and concentration dependently facilitated rostral ventral root activity. Substance P also facilitated the modulatory effects of tail fin stimulation on ongoing locomotor activity in the rostral pool. In contrast, NPY and the GABAB receptor agonist baclofen, both of which have presynaptic inhibitory effects on sensory afferents, reduced the strength of ascending inputs and rostral ventral root responses. We also examined the effects of the neuropeptide bombesin, which is present in sensory axons, at the cellular, synaptic, and reflex levels. As with substance P, bombesin increased tail fin stimulation-evoked inputs and ventral root responses in the rostral pool. These effects were associated with the increased excitability of slowly adapting mechanosensory neurons and the potentiation of glutamatergic synaptic inputs to spinobulbar neurons. These results show the possible behavioral relevance of neuropeptide-mediated modulation of sensory inputs at the cellular and synaptic levels. Given that the types and locations of neuropeptides in the dorsal spinal cord of the lamprey show strong homologies to that of higher vertebrates, these results are presumably relevant to other vertebrate systems.     

5-HT Modulation of identified segmental premotor interneurons in the lamprey spinal cord

Ipsilaterally projecting spinal excitatory interneurons (EINs) generate the hemisegmental rhythmic locomotor activity in lamprey, while the commissural interneurons ensure proper left-right alternation. 5-HT is a potent modulator of the locomotor rhythm and is endogenously released from the spinal cord during fictive locomotion. The effect of 5-HT was investigated for three segmental premotor interneuron types: EINs, commissural excitatory and commissural inhibitory interneurons. All three types of interneurons produced chemical postsynaptic potentials in motoneurons, but only those from EINs had an electrical component. The effect of 5-HT was studied on the slow afterhyperpolarization, involved in spike frequency regulation, and on the segmental synaptic transmission to motoneurons. 5-HT induced a reduction in the slow afterhyperpolarization and a depression of synaptic transmission in all three types of segmental interneurons. Thus 5-HT is a very potent modulator of membrane properties and synaptic transmission of last-order segmental premotor interneurons. Such modulation of locomotor network interneurons can partially account for the observed effects of 5-HT on the swimming pattern in lamprey.     

Inputs to intercostal motoneurons from ventrolateral medullary respiratory neurons in the cat

The investigation examined the synaptic input from medullary respiratory neurons in the nucleus retroambigualis (NRA) to external (EIM) and internal (IIM) intercostal motoneurons. Antidromic mapping revealed that 112/117 (96%) tested NRA units had axons descending into thoracic spinal cord with extensive arborizations at many thoracic segments, mainly contralaterally. The conduction velocities ranged from 10 to 105 m X s-1. The descending projections did not appear to be somatotopically arranged. Cross-correlation of the spike trains of NRA inspiratory units with the discharge of external intercostal nerves (performed usually with 4 contralateral nerves) showed significant narrow peaks only in 5 out of 40 averages. Of the 25 trigger units tested for the thoracic projection in this series of experiments, 24 were antidromically activated. Intracellular recordings were made from 52 IIMs [mean membrane potential 65.3 mV, central respiratory drive potentials (CRDPs) greater than 1 mV present in 23/52] and 53 EIM (mean membrane potential 54.3 mV, CRDPs in 31/53). During the depolarizing phase of the CRDPs, synaptic noise with frequent and apparently unitary EPSPs with amplitudes in excess of 1 mV was observed. Spike-triggered averages of synaptic noise were computed for 153 pairings between 137 NRA neurons and 105 contralateral intercostal motoneurons. Only four PSPs were revealed: two monosynaptic EPSPs between expiratory NRA units and IIMs and two probably disynaptic EPSPs between inspiratory NRA units and EIMs. When advancing the microelectrode down to the motoneuron pools, frequent recordings were made from interneurons with spontaneous respiratory discharge (inspiratory or expiratory) located dorsal and medial to the motor nuclei. The interneurons could be excited following stimulation of segmental afferents. It is concluded that monosynaptic connections between respiratory NRA neurons and intercostal motoneurons are rare (connectivity no more than approximately 4%). Segmental interneurons, interposed between the majority of descending respiratory axons and intercostal motoneurons, are likely to produce large unitary EPSPs and, thus, short-term synchronization in the discharge of intercostal motoneurons as observed by others.     

The activity of spinal commissural interneurons during fictive locomotion in the lamprey

Commissural interneurons in the lamprey coordinate activity of the hemisegmental oscillators to ensure proper left-right alternation during swimming. The activity of interneuronal axons at the ventral commissure was studied together with potential target motoneurons during fictive locomotion in the isolated lamprey spinal cord. To estimate the unperturbed activity of the interneurons, axonal recordings were chosen because soma recordings inevitably will affect the level of membrane depolarization and thereby spike initiation. Of 227 commissural axons recorded during locomotor activity, 14 produced inhibitory and 3 produced excitatory postsynaptic potentials (PSPs) in target motoneurons. The axons typically fired multiple spikes per locomotor cycle, with approximately 10 Hz sustained frequency. The average shortest spike interval in a burst corresponded to an instantaneous frequency of approximately 50 Hz for both the excitatory and inhibitory axons. The maximum number of spikes per locomotor cycle was inversely related to the locomotor frequency, in accordance with previous observations in the spinal hemicord preparation. In axons that fired multiple spikes per cycle, the mean interspike intervals were in the range in which the amplitude of the slow afterhyperpolarization (sAHP) is large, providing further support for the role of the sAHP in spike timing. One hundred ninety-five axons (86%) fired rhythmically during fictive locomotion, with preferred phase of firing distributed over either the segmental locomotor burst phase (40% of axons) or the transitional phase (between bursts; 60%). Thus in lamprey commissural interneurons, we found a broad distribution of firing rates and phases during fictive locomotion.     

Differential spinal projections from the forelimb areas of the rostral and caudal subregions of primary motor cortex in the cat

We used anterograde transport of WGA-HRP to examine the topography of corticospinal projections from the forelimb areas within the rostral and caudal motor cortex subregions in the cat. We compared the pattern of these projections with those from the somatic sensory cortex. The principal finding of this study was that the laminar distribution of projections to the contralateral gray matter from the two motor cortex subregions was different. The rostral motor cortex projected preferentially to laminae VI–VIII, whereas caudal motor cortex projected primarily to laminae IV–VI. Confirming earlier findings, somatic sensory cortex projected predominantly to laminae I–VI inclusive. We found that only rostral motor cortex projected to territories in the rostral cervical cord containing propriospinal neurons of cervical spinal segments C3-4 and, in the cervical enlargement, to portions presumed to contain Ia inhibitory interneurons. We generated contour maps of labeling probability on averaged segmental distributions of anterograde labeling for all analyzed sections using the same algorithm. For rostral motor cortex, heaviest label in the dorsal part of lamina VII in the contralateral cord was consistently located in separate medial and lateral zones. In contrast, no consistent differences in the mediolateral location of label was noted for caudal motor cortex. To summarize, laminae I–III received input only from the somatic sensory cortex, while laminae IV–V received input from both somatic sensory and caudal motor cortex. Lamina VI received input from all cortical fields examined. Laminae VII–IX received input selectively from the rostral motor cortex. For motor cortex, our findings suggest that projections from the two subregions comprise separate descending pathways that could play distinct functional roles in movement control and sensorimotor integration.     

Coordination of spinal locomotor activity in the lamprey: long-distance coupling of spinal oscillators

 The extent and strength of long-distance coupling between locomotor networks in the rostral and caudal spinal cord of larval lamprey were examined with in vitro brain/spinal cord preparations, in which spinal locomotor activity was initiated by chemical microstimulation in the brain, as well as with computer modeling. When locomotor activity and short-distance coupling were blocked in the middle spinal cord for at least 40 segments, burst activity in the rostral and caudal spinal cord was still coupled 1:1, indicating that long-distance coupling is extensive. However, in the absence of short-distance coupling, intersegmental phase lags were not constant but decreased significantly with increasing cycle times, suggesting that long-distance coupling maintains a relatively constant delay rather than a constant phase lag between rostral and caudal bursts. In addition, under these conditions, intersegmental phase lags, measured between rostral and caudal burst activity, were significantly less than normal, and the decrease was greater for longer distances between rostral and caudal locomotor networks. The above result could be mimicked by a computer model consisting of pairs of oscillators in the rostral, middle, and caudal spinal cord that were connected by short- and long-distance coupling. With short-distance coupling blocked in the middle spinal cord, strychnine was applied to either the rostral or caudal spinal cord to convert the pattern locally from right-left alternation to synchronous burst activity. Synchronous burst activity in the rostral spinal cord resulted in a reduction in right-left phase values for burst activity in the caudal cord. These results also could be mimicked by the computer model. Strychnine-induced synchronous burst activity in the caudal spinal cord did not appear to alter the right-left phase values of rostral burst activity. Taken together, the experimental and modeling results suggest that the descending and ascending components of long-distance coupling, although producing qualitatively different effects, are comparatively weak. In particular, the descending component of long-distance coupling appears to become progressively weaker with increasing distance between two given regions of spinal cord. Therefore, short-distance coupling probably contributes substantially to normal rostrocaudal phase lags for locomotor activity along the spinal cord. However, short-distance coupling may be more extensive than ”nearest neighbor coupling.” Received: 4 June 1998 / Accepted: 6 November 1998     

Spinal locomotor inputs to individually identified reticulospinal neurons in the lamprey

Locomotor feedback signals from the spinal cord to descending brain stem neurons were examined in the lamprey using the uniquely identifiable reticulospinal neurons, the Müller and Mauthner cells. The same identified reticulospinal neurons were recorded in several preparations, under reduced conditions, to address whether an identified reticulospinal neuron shows similar locomotor-related oscillation timing from animal to animal and whether these timing signals can differ significantly from other identified reticulospinal neurons. Intracellular recordings of membrane potential in identified neurons were made in an isolated brain stem-spinal cord preparation with a high-divalent cation solution on the brain stem to suppress indirect neural pathways and with D-glutamate perfusion to the spinal cord to induce fictive swimming. Under these conditions, the identified reticulospinal neurons show significant clustering of the timings of the peaks and troughs of their locomotor-related oscillations. Whereas most identified neurons oscillated in phase with locomotor bursting in ipsilateral ventral roots of the rostral spinal cord, the B1 Müller cell, which has an ipsilateral descending axon, and the Mauthner cell, which has a contralateral descending axon, both had oscillation peaks that were out of phase with the ipsilateral ventral roots. The differences in oscillation timing appear to be due to differences in synaptic input sources as shown by cross-correlations of fast synaptic activity in pairs of Müller cells. Since the main source of the locomotor input under these experimental conditions is ascending neurons in the spinal cord, these experiments suggest that individual reticulospinal neurons can receive locomotor signals from different subsets of these ascending neurons. This result may indicate that the locomotor feedback signals from the spinal locomotor networks are matched in some way to the motor output functions of the individual reticulospinal neurons, which include command signals for turning and for compensatory movements.     

Whole-cell patch clamp recordings from rhythmically active motoneurons in the isolated spinal cord of the chick embryo

Whole-cell patch clamp recordings were obtained during motor activity from electrically identified motoneurons within the spinal cord of the chick embryo maintained in vitro. Most recordings were performed on E11-E13 motoneurons although it was also possible to record from younger cells (E7-E9). Voltage clamp recordings were used to characterize the synaptic currents expressed in femoro-tibialis (extensor) motoneurons during motor activity. These motoneurons exhibited rhythmic excitatory currents with reversal potentials near 0 mV. This powerful technique enables high resolution recordings from identified motoneurons in situ and allows investigation of the membrane and synaptic mechanisms involved in the development of embryonic motility.