Phasic variations of extracellular potassium during fictive swimming in the lamprey spinal cord in vitro

The lamprey spinal cord in vitro can generate the motor pattern underlying locomotion, which can be recorded with suction electrodes in the ventral roots. To test if the extracellular level of potassium changed during rhythmic activity, potassium-sensitive microelectrodes were used to systematically (every 25 μm) explore the level of extracellular potassium [K⁺]_e in different loci in the transverse plane of the spinal cord. During fictive locomotion the baseline level of [K⁺]_e increased with 0.08–0.4 mM in the grey matter. As a rule phasic variations of up to 0.2 mM, correlated to each ventral root burst, were superimposed on the tonic deviation of [K⁺]_e. Changes in this range may cause a moderate depolarization of the spinal neurones and might also affect other neuronal functions including the rhythm-generating circuits.     

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.     

Changes in electrophysiological properties of lamprey spinal motoneurons during fictive swimming

Electrophysiological properties of lamprey spinal motoneurons were measured to determine whether their cellular properties change as the spinal cord goes from a quiescent state to the active state of fictive swimming. Intracellular microelectrode recordings of membrane potential were made from motoneurons in the isolated spinal cord preparation. Electrophysiological properties were first characterized in the quiescent spinal cord, and then fictive swimming was induced by perfusion with D-glutamate and the measurements were repeated. During the depolarizing excitatory phase of fictive swimming, the motoneurons had significantly reduced rheobase and significantly increased input resistance compared with the quiescent state, with no significant changes in these parameters during the repolarizing inhibitory phase of swimming. Spike threshold did not change significantly during fictive swimming compared with the quiescent state. During fictive swimming, the slope of the spike frequency versus injected current (F-I) relationship decreased significantly as did spike-frequency adaptation and the amplitude of the slow after-spike hyperpolarization (sAHP). Serotonin is known to be released endogenously from the spinal cord during fictive swimming and is known to reduce the amplitude of the sAHP. Therefore the effects of serotonin on cellular properties were tested in the quiescent spinal cord. It was found that, in addition to reducing the sAHP amplitude, serotonin also reduced the slope of the F-I relationship and reduced spike-frequency adaptation, reproducing the changes observed in these parameters during fictive swimming. Application of spiperone, a serotonin antagonist, significantly increased the sAHP amplitude during fictive swimming but had no significant effect on F-I slope or adaptation. Because serotonin may act in part through reduction of calcium currents, the effect of calcium-free solution (cobalt substituted for calcium) was tested in the quiescent spinal cord. Similar to fictive swimming and serotonin application, the calcium-free solution significantly reduced the sAHP amplitude, the slope of the F-I relationship, and spike-frequency adaptation. These results suggest that there are significant changes in the firing properties of motoneurons during fictive swimming compared with the quiescent state, and it is possible that these changes may be attributed in part to the endogenous release of serotonin acting via reduction of calcium currents.     

Transmitter phenotypes of commissural interneurons in the lamprey spinal cord

The fundamental network for locomotion in all vertebrates contains a central pattern generator or CPG that produces the required motor output in the spinal cord. In the lamprey spinal cord different classes of interneuron's forming the core CPG circuitry have been characterized based on their morphological and electrophysiological features. The commissural interneuron's (C-INs) represent one essential component of CPG that have been implicated in controlling left–right alternation of the motor activity during swimming. However, it is still unclear if the C-INs displays a homogenous neurotransmitter phenotype and how they are distributed. In this paper we investigated the segmental distribution of glycine, glutamate and GABA-immunoreactive (ir) C-INs by combining retrograde Neurobiotin tracing with specific antibodies for these transmitters. The C-INs were more abundant in caudal and rostral segments adjacent to the injection site and their number gradually decreased in more distal segments, suggesting that these interneurons project over a short distance. The glycine-ir neurons represented around 50% of the total C-INs, while glutamate-ir neurons represented only 29%. Both types of C-INs were homogenously distributed over different segments along the spinal cord. Finally, no Neurobiotin labeled C-INs displayed GABA-ir, although many interneurons were ir to GABA, suggesting that GABAergic interneurons are not directly responsible for controlling left–right alternation of activity during locomotion in lamprey. Overall, these results show that the C-INs display a gradual rostrocaudal distribution and consist of both glycine- and glutamate-ir neurons. The difference in the proportion of inhibitory and excitatory C-INs represents an anatomical substrate that can ensure the predominance of alternating activity during locomotion.     

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.     

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.     

Effects of iontophoretically applied drugs on spinal interneurons of the lamprey

1. Intracellular records were obtained from giant interneurones in the isolated spinal cord of the sea lamprey. The cells had a mean resting potential of about 75 mV and action potentials with overshoots of about 35 mV. Their input resistances, measured by passing polarizing currents through the recording pipette, were in the range 3-7 MOmega.2. Iontophoretic ejection of gamma-aminobutyric acid (GABA) from a micropipette placed near the surface of a cell resulted in a slight hyperpolarization, accompanied by a marked reduction in input resistance. The reversal point for the potential change was about 5 mV greater than the resting membrane potential.3. Iontophoretic application of L-glutamate to the cells produced a depolarization with a decrease in input resistance much smaller than that accompanying a GABA potential of similar amplitude. The action potential amplitude was reduced by L-glutamate application. The reversal potential could not be determined accurately but appeared to be near zero membrane potential.4. Glutamate application produced, in addition, a burst of inhibitory synaptic potentials in the cell, presumably by depolarizing either inhibitory presynaptic nerve terminals or nearby inhibitory cell bodies.5. Acetylcholine (ACh) produced no detectable change in membrane resistance or potential.6. Application of the three drugs to first-order sensory cells in the spinal cord had no effect on their membrane properties.     

Apamin reduces the late afterhyperpolarization of lamprey spinal neurons, with little effect on fictive swimming.

The role of the late afterhyperpolarization (late AHP) in the firing properties of lamprey spinal neurons was tested by bath application of apamin, a selective blocker of the sk calcium-dependent potassium current. Intracellular recordings of identified motoneurons and interneurons were made with micropipette electrodes in the isolated lamprey spinal cord. Apamin reversibly reduced the amplitude of the late afterhyperpolarization without affecting other aspects of the action potential or the resting potential. The firing frequencies of the neurons were enhanced by apamin over a range of depolarizing current pulse injections. The effect of apamin was also tested on fictive swimming, which was induced in the isolated spinal cord by bath application of an excitatory amino acid (D-glutamate or N-methyl-D,L-aspartate). A concentration of apamin (10 microM) sufficient to substantially reduce the late AHP had no significant effect on the ventral root burst rate, intensity, or phase lag during fictive swimming.     

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.     

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.     

Interaction between disinhibited bursting and fictive locomotor patterns in the rat isolated spinal cord

Using a transverse barrier that allowed discrete application of neurochemicals to certain lumbar regions of the rat isolated spinal cord, we studied the intersegmental organization of rhythmic patterns recorded extracellularly from ventral roots and intracellularly from single motoneurons. Fictive locomotor patterns were elicited by serotonin (5-HT) and/or N-methyl-D-aspartate (NMDA) or high K(+) solution applied to the rostral or caudal lumbar region of the cord. Neither 4-aminopyridine nor Mg(2+)-free solution shared this property. Coapplication of strychnine and bicuculline (blockers of fast synaptic inhibition) to the caudal part elicited slow bursting in the whole cord. These bursts could trigger episodes of fictive locomotion patterns in the rostral roots. When the rostral region was exposed to 5-HT and/or NMDA (during continuous application of strychnine and bicuculline caudally) a standard locomotor-like pattern was generated during each interburst interval and was surprisingly expressed with its typical pattern alternation even in the caudal area despite the local presence of strychnine and bicuculline. Midsagittal splitting of the caudal region did not change this alternating pattern, indicating that it was driven by rostral regions above the surgical cut. Discrete changes in the concentrations of NMDA rostrally modulated the burst amplitude recorded in the same region after caudal application of strychnine and bicuculline. The period of fictive locomotor patterns changed bimodally depending on the temporal relation with disinhibited bursts, indicating a tight interaction between these two rhythmic activities. These results are interpreted on the basis of a model that assumes a modular arrangement for the locomotor central pattern generator, made up by a series of unit burst generators with serial and crossed connections.     

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.     

Synaptic potentials in motoneurons during fictive swimming in spinal Xenopus embryos

Embryos spinalized at the 3rd to 6th postotic myotome and immobilized in 10(-4) M tubocurarine can respond to a brief skin stimulus with motor root activity suitable for swimming. Embryos spinalized at the more caudal levels give shorter episodes of fictive swimming. We have previously described the synaptic inputs to motoneurons during fictive swimming in intact embryos (23). In the present paper we look to see if similar synaptic inputs are present in spinal embryos and are therefore spinal in origin. All motoneuron firing during fictive swimming is associated with a tonic depolarization that falls away slowly once firing stops, is increased by hyperpolarizing current, and is reduced by depolarizing current. A slow depolarizing potential evoked by lower levels of skin stimulation has similar properties and rate of fall. In 1-2 mM PDA, an excitatory amino acid antagonist, only a small remnant of the depolarization remains, and motoneuron firing stops. The NMDA antagonist 50 microM APV reduces the depolarization less but also blocks firing. Motoneurons fire one spike per swimming cycle, in phase with nearby motor root discharge. Spikes are preceded by a depolarizing prepotential. This increases with hyperpolarizing current, which can block the spike to reveal an underlying depolarizing potential. In phase with motor root discharge on the opposite side of the body, motoneurons receive a midcycle inhibitory postsynaptic potential, which increases with depolarizing current, decreases with hyperpolarizing current, and is blocked by 10(-6) M strychnine. Strychnine, 5 X 10(-7) M, leads first to broadening of motor root bursts then to loss of the alternating swimming pattern of activity, which is replaced by synchronous bursts on both sides of the body. We conclude that the synaptic inputs to motoneurons during fictive swimming in spinal embryos are very similar in properties and pharmacology to those in intact embryos. These inputs, including the tonic depolarization always associated with motoneuron firing during swimming, must be at least partly spinal in origin.