 |
||||
|
||||
|
||||
|
||||
Previous Article | Next Article
The Journal of Neuroscience, November 15, 2002, 22(22):9961-9971
Firing Properties of Identified Interneuron Populations in the Mammalian Hindlimb Central Pattern Generator
Simon J. B. Butt, Ronald M. Harris-Warrick, and Ole Kiehn Mammalian Locomotor Laboratory, Department of Neuroscience, Karolinska Institutet, Stockholm S-171 77, Sweden
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
Little is known about the network structure of the central pattern generator (CPG) controlling locomotor movements in mammals. The present experiments aim at providing such knowledge by focusing on commissural interneurons (CINs) involved in left-right coordination. During NMDA and 5-HT-initiated locomotor-like activity, we recorded intracellularly from caudally or descending projecting L2 and L3 CINs (dCINs) located in the ventromedial area of the lumbar spinal cord in newborn rats. This region is crucial for rhythmic motor output and left-right coordination. The overall sample of dCINs represented a heterogenous population with neurons that fired in all phases of the locomotor cycle and exhibited varying degrees of rhythmicity, from strongly rhythmic to nonrhythmic. Among the rhythmic, putative CPG dCINs were populations that fired in-phase with the ipsilateral or with the contralateral L2 locomotor-like activity. There was a high degree of organization in the dorsoventral location of rhythmic dCINs, with neurons in-phase with the ipsilateral L2 activity located more ventrally. Spikes of rhythmically active dCINs were superimposed on membrane oscillations that were generated predominantly by synaptic input, with little direct contribution from the intrinsic pacemaker hyperpolarization-activated inward current. For both ipsilaterally and contralaterally firing dCINs the dominant synaptic drive was in-phase with the ipsilateral L2 motor activity. This study provides the first characterization of putative CPG interneurons in the mammalian spinal cord. Our results suggest an anatomical and physiological separation of CPG commissural interneurons in the ventral horn and demonstrate that it is possible to target specific interneuron subpopulations in the mammalian locomotor network.
Key words: commissural interneuron; locomotion; rhythmic; CPG; Ih current; 5-HT
INTRODUCTION
Vertebrate locomotor movements are organized by local neuronal networks, known as central pattern generators (CPGs), situated within the spinal cord. In swimming vertebrates such as the Xenopus tadpole and the lamprey, these CPG circuits have been characterized in some detail (Grillner et al., 1995
; Roberts, 2000
Although the basic principles of CPG structure might be phylogenetically preserved, it is evident that a layer of complexity must be added in limbed animals where the CPG is required to coordinate not only alternating movements between the left and right sides but also flexors and extensors in each limb. Relatively little is known about the network structure of the mammalian CPG, which to a large extent is regarded as a "black box." Our present experiments in the neonatal rat spinal cord aim to explore this black box by focusing on the CINs involved in coordination of the left and right hemicords. The fact that CINs have crossed projections makes them readily identifiable by electrical stimulation. Initial experiments with neonatal rats have shown that the ventromedial region of the lumbar spinal cord is crucial for rhythm generation and left-right coordination (Kjaerulff and Kiehn, 1996
In this study, whole-cell patch recordings were performed from CINs located in the L2-L3 segments with descending axons to the contralateral L4-L5 region. These CINs were monitored during NMDA- and 5-HT-induced locomotor-like activity to provide an insight into the functional role of interneurons in the mammalian hindlimb CPG.
Parts of this paper have been presented previously in abstract form (Butt et al., 2001
MATERIALS AND METHODS
Dissection. Neonatal (postnatal day 0-4) Wistar rats were used for all experiments, and the dissection was performed as described previously (Kjaerulff and Kiehn, 1996
Electrical stimulation of the contralateral hemicord. dCINs were identified by stimulating the contralateral L4-L5 hemicord at 1 Hz with 2 msec pulses up to 300 µA in amplitude (Isolator-11 stimulator; Axon Instruments). Strong stimulation of the L4-L5 hemicord resulted in sustained activity in the L2 ventral root (Fig. 1B). The threshold for such bursting (range, 30-267 µA) was termed the ventral root threshold (VRT) and provided a measure of the relative intensity of stimulation between preparations.
Whole-cell tight-seal recording of ventromedial interneurons. A small slit was cut into the pial surface of segments L2-L3 (opposite to the side of L4-L5 stimulation), into which the patch electrode (resistance, 5-8 M
, borosilicate glass; Clark Electromedical Instruments, Pangbourne, UK) was lowered. Blind whole-cell tight-seal intracellular recordings of interneurons were performed in current-clamp mode (Axoclamp 2B; Axon Instruments). Locomotor-like activity was evoked by perfusion with Ringer's solution containing a combination of NMDA (6-8 µM) and 5-HT (6-10 µM). Rhythmic burst activity in the cL2 ventral root was recorded with a suction electrode and bandpass-filtered (100 Hz to 1 kHz). The following drugs were obtained from Sigma (St. Louis, MO): D(
)-2-amino-5-phosphonopentanoic acid (AP-5), 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt (CNQX), and tetrodotoxin (TTX). 4-Ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride (ZD 7288) was obtained from Tocris.
Calibration of recording locations. A counter on the microdrive was used to determine the ventral-dorsal depth of the recorded dCIN. The counter was zeroed on contact with the surface of the slit. Once recording was completed, the electrode was retracted and then lowered to the point of contact on either side of the slit. The average of these two values provided a measure of the depth to the initial point of contact within the slit. The distance to the midline was measured using the x-y scale on the micromanipulator stand. Both ventral depth and distance to midline were corrected for the angle of the micromanipulator with respect to the surface of the cord, which was kept constant throughout all the experiments.
To provide an indication of the error involved in such calculations, seven interneurons were labeled with 0.2% Lucifer yellow, and the spinal cords were sectioned transversely. Measurement of these filled neurons revealed that the initial measurement of the distance from the midline was incorrect laterally on average by 88 ± 13 µm (SEM), and the depth from the surface of the cord was incorrect in either direction by on average 61 ± 27 µm. Where recognizable in the transverse slices, the depth of the slit in the spinal cord was 98 ± 8 µm (n = 6). The interneuron positions in Figure 2C were not corrected for these values.
Protocols for identifying the hyperpolarization-activated inward current. Under control current-clamp conditions, 58 dCINs were stimulated at 0.25 Hz with 800 msec hyperpolarizing and depolarizing current pulses. The effect of the hyperpolarization-activated inward current (Ih) was detected in the slow depolarizing voltage sag during steady-state hyperpolarizing current injection at membrane potentials typically more negative than
Voltage-clamp analysis of Ih. For voltage-clamp analysis, neurons were typically maintained in normal Ringer's solution. Neurons were held at -50 mV and given a series of 1.5 sec steps from -60 to -130 mV in 5 mV increments. In general, the series access resistance was within 20-40 M
Data analysis. The locomotor pattern was analyzed off-line using DATAPAC 2000 version 2.41 (RUN technologies Co., Laguna Hills, CA) with the exception of the circular plots, which were calculated as described by Kjaerulff and Kiehn (1996)
Datapac analysis. For histograms of instantaneous spike frequency, the boundaries of each locomotor cycle were defined as being from the onset of a cL2 ventral root burst to the onset of the following burst. The threshold for spike events was normally set at 0 mV. To average intracellular dCIN membrane oscillations during locomotion, cL2 ventral root recordings were rectified, filtered (Butterworth filter, 100 Hz; rolloff, 5) and linear-smoothed with a time constant of 100-200 msec. dCIN intracellular recordings were filtered (Butterworth filter, 10 Hz; rolloff, 5) to remove fast transients such as spikes. The signals were averaged with respect to cL2 activity (for a minimum of 20 cycles) using the dual normalized function (1% increment, 50% break point).
Circular plots. Circular statistics were performed to ascertain the significance of the coupling between dCIN firing and the locomotor-like activity in the cL2. The latency (Kjaerulff and Kiehn, 1996
. The mean of 25
Analysis of Ih. Current amplitudes were measured from single exponential fits of the data performed in Clampfit version 8.1 (Axon Instruments), extrapolated back to the beginning of the hyperpolarizing step and forward to approximate the steady state at 2 sec. Currents were converted to conductance using a reversal potential for Ih (VRev) of -33 mV, which has been measured previously in neonatal rat spinal neurons (Takahashi, 1990
where g is the conductance, gmax is the maximal conductance, VAct is the voltage of half-activation, and s is the slope factor. The kinetics of activation were measured at
where IAct is the amplitude of the current,
Act is the time constant of activation, and C is an offset constant. Attempts were made to fit the data with a double-exponential relationship, but this did not improve the fit.
RESULTS
Identification and initial characterization of dCINs
Ninety-nine dCINs were identified by stimulation of antidromic action potentials from the contralateral L4-L5 hemicord (Fig. 1A). All-or-nothing antidromic potentials were elicited in dCINs at constant latencies (range, 4.4-15.2 msec) by stimuli over the range of 21-312 µA (mean ± SEM, 103 ± 6.2 µA), corresponding to a mean VRT of 1.4 × VRT. Subthreshold stimulation (typically <1 × VRT) of the contralateral L4-L5 evoked short-latency PSPs in 72% of dCINs. A significant amount of these neurons received low-threshold, short-latency EPSPs or a mix of a short-latency EPSP followed by an IPSP (Fig. 2Ai). To confirm the antidromic nature of the spike and to prove that the spikes were not driven indirectly by EPSPs, supramaximal concentrations of the excitatory transmitter antagonists CNQX (30 µM) and AP-5 (20 µM) were coapplied. After 5-6 min, evoked EPSPs and polysynaptic IPSPs (see EPSP "hump"; Fig. 2Aii, arrowhead) were completely abolished (Fig. 2Aiii). In some instances, a voltage-independent, CNQX- and AP-5-resistant potential was observed at subspike thresholds, indicative of electrical coupling between dCINs (Kiehn and Tresch, 2002
View larger version (29K):[in this window]
[in a new window]
Figure 1. Experimental setup. A, Schematic of the lumbar region of the neonatal rat spinal cord and the setup used to investigate the locomotor properties of dCINs. Suction electrodes were placed on the contralateral L4-L5 hemicord for electrical stimulation of caudally projecting axons, and on the cL2 to record ventral root activity. The patch electrode (dCIN) was lowered into a slit made in the ventral surface of L2-L3. B, The intensity of L4-L5 stimulation was increased manually (top trace). At higher levels, the stimulation (stim.) caused sustained activity in the cL2 ventral root (lower trace). The threshold for such activity, as indicated by the dashed line, was termed the VRT.
View larger version (27K):[in this window]
[in a new window]
Figure 2. Identification of dCINs in the lumbar region by the presence of an antidromic action potential. Ai-Aiii, The top traces show the raw data superimposed, and the bottom traces show the averaged trace. Ai, EPSP-IPSP evoked at low stimulus levels (<1 × VRT) in a neuron subsequently identified as a dCIN by the presence of an antidromic spike (Aii, Aiii). Antidromic spikes were elicited at a short, constant latency (Aii). The hump in the spike, as indicated by the arrowheads in Aii, is the subthreshold EPSP that was abolished on incubation with 20 µM AP-5 and 30 µM CNQX (Aiii). B, Collision test to verify the antidromic nature of the spike. a, Orthodromic spike elicited by a 2 msec suprathreshold depolarizing current pulse; b, antidromic spike; c, initial segment spikelet; d, complete abolition of the antidromic action potential. C, Location of neurons recorded from in the ventromedial area (as indicated by the box, inset) of L2-L3. Black squares, Caudally projecting dCINs; white squares, non-dCINs.
Using these tests, we found that 34% (99 of 294) of the neurons recorded were dCINs with axons projecting at least to the contralateral L4-L5 segment. These dCINs were located throughout the ventromedial area sampled (Fig. 2C). Before addition of locomotor-inducing drugs, they had a mean resting potential of
Activity of dCINs during NMDA- and 5-HT-induced locomotion
To determine whether the dCINs were potential components of the hindlimb CPG, we measured their firing patterns during locomotor-like activity. Previous work has shown that locomotor-like activity can be induced by perfusion with a combination of 5-HT and NMDA (Cazalets et al., 1992
Application of locomotor drugs typically resulted in a significant depolarization of the dCIN membrane potential (by 6.5 ± 0.6 mV; n = 84) and an increase in spike firing or modulation of spontaneous firing (Fig. 3A). We found that dCINs exhibit a broad range of activities during locomotion. We have divided this continuous spectrum into three broad classes of dCINs on the basis of their firing pattern during NMDA and 5-HT: (1) highly rhythmic firing, often driven by pronounced oscillations in membrane potential, which were phase-related to cL2 ventral root bursting (Fig. 3Bi); (2) broadly tuned but rhythmically firing neurons with less distinct oscillations in membrane potential (Fig. 3Ci); and (3) dCINs that appeared to fire randomly with respect to cL2 bursting and that exhibited little or no underlying rhythmic oscillations in membrane potential (Fig. 3Di). Individual dCINs were monitored for a period of at least 10 min during locomotor-like activity. To determine the phase relationship of dCIN firing, we normalized the phasing of spike activity for 50 cycles of stable cL2 activity with respect to the onset of the cL2 burst. To provide a measure of instantaneous spike frequency, the normalized cycle periods were subdivided into 10 equal bins, the first five representing approximately the phase of cL2 bursting and the last five approximately equivalent to the iL2 phase (see Materials and Methods). Examples for the three categories of dCIN as represented in histogram form are shown in Figure 3Bii-Dii. The graphs give an indication of the spike tuning for a sustained period of ~2-3 min. The variability in the cL2 duty cycles within this time is represented by the error bar (±SD) below the x-axis; the end point of the cL2 burst was within one bin (10%) of the 50% duty cycle.
View larger version (53K):[in this window]
[in a new window]
Figure 3. Most dCINs are rhythmic during NMDA- and 5-HT-induced locomotor-like activity. A, Application of NMDA and 5-HT to the isolated spinal cord resulted in rhythmic locomotor-like activity in the cL2 (top trace) and a depolarization and rhythmic firing observed in the dCIN (bottom trace). Calibration: 20 mV, 24 sec. B-D, Analysis of the activity of the three types of dCIN during locomotor-like activity. Highly rhythmic hS-dCINs (B) were characterized first, on the basis that they exhibited pronounced oscillations in membrane potential (bottom trace, Bi; calibration, 40 mV) during locomotor activity (top trace, Bi). Second, their spikes were also locked to a specific phase as revealed by the spike frequency histogram of 50 locomotor cycles (Bii, top trace). Third, the circular plot vector for 25 spikes as shown by the bold arrow in the bottom trace of Bii was highly significant (p < 0.001). The duration of cL2 bursting is indicated in Bii-Dii by the gray bars (±SD; n = 50) under the histogram and the gray segment in the circular plot. C, Rhythmic S-dCIN that preferentially fires out-of-phase with cL2 activity. p values for S-dCINs were in the range of 0.001 < p < 0.05 (Cii, bottom trace). D, Nonrhythmic NS-dCIN with spikes not locked to any particular phase of the locomotor cycle and with p > 0.05 (Dii, bottom trace). In all subsequent plots, hS-dCIN data are shown in dark gray; S-dCIN data are light gray; and NS-dCIN data are white. E, Distribution of dCIN r values (n = 84). F, Analysis of the instantaneous firing frequency exhibited by the three classes of dCIN.
To provide a statistical measure of the spike tuning, spikes from every 10th cycle were analyzed using circular statistics (Kjaerulff and Kiehn, 1996
A plot of r values with respect to number of dCINs (Fig. 3E) revealed a broad distribution similar to that previously published for unidentified L2 neurons (Tresch and Kiehn, 1999
These results demonstrate that the set of CINs defined anatomically on the basis of axonal projections is heterogeneous with respect to firing properties during locomotor-like activity. The next section will show that rhythmically active cells can be further subclassified on the basis of their preferred phase of firing in the locomotor cycle.
Preferred phase of firing of rhythmically active dCINs
During locomotion, the iL2 alternates with the iL5 and with the cL2, and the iL2 and cL5 burst in-phase (Kiehn and Kjaerulff, 1996
The two rhythmic cells shown in Figure 3, B and C, have their preferred peak of firing in-phase with the iL2 (bins 6-10) and out-of-phase with the cL2 bursts (bins 1-5). This phasing was defined as ipsilateral firing. However, a number of dCINs fired in-phase with the cL2 activity, defined as contralateral firing. Within the total sample of dCINs recorded during locomotor-like activity, 61 significantly rhythmic (hS and S) neurons had their preferred phases of firing distributed throughout the locomotor cycle, but not equally so (Fig. 4A). Sixty-four percent of them fired with their peak of activity in-phase with the ipsilateral L2 bursts, whereas only 36% showed predominantly contralateral firing. A plot of the preferred phase of firing of dCINs by type revealed that >50% of the hS-dCINs fired in the final third of the ipsilateral L2 burst just at the peak of the iL2 burst (bin 8) and before the transition to the cL2 burst (bin 9) (Fig. 4Ai). This subset of hS neurons may be described as "transition neurons." The other hS interneurons fired at all other phases but predominantly just before or during the iL2 burst. In contrast, within the S-dCINs, most (62%) fire just before onset (bin 5) or at the midpoint (bins 7-8) of iL2 activity. For the contralaterally firing neurons, there was a less distinct pattern in preferred firing phase.
View larger version (28K):[in this window]
[in a new window]
Figure 4. Rhythmic dCIN populations with distinct ventral-dorsal locations exhibit differing phase relationships. A, Plot of dCIN circular statistic vectors over a locomotor cycle showing the distribution preferred phases of firing in rhythmic dCINs (hS-dCINs, Ai; S-dCINs, Aii). The small black arrowheads above the histograms indicate the bins containing most dCINs for each group. The dashed lines indicate the approximate transition points from contralateral bursts (cL2) to ipsilateral burst (iL2) (0.5) and from iL2 to cL2 (1.0). B, distribution of contralaterally firing (gray squares), ipsilaterally firing (black circles), and nonrhythmic (white circles) dCINs in the transverse plane of the lumbar spinal cord (the area magnified as shown in the inset). C, Histogram of the percentage of each cell type at a given depth (color coding as for B). Numbers shown above the bar graphs indicate the size of the sample at each depth. Memb., Membrane.
Because we did not use dual reference in our analysis (Berkowitz and Stein, 1994
dCIN subpopulations show a distinct ventral-dorsal distribution
It became apparent during our experiments, as more dCINs were identified, that we could preferentially target either ipsilaterally firing or contralaterally firing dCINs by selecting cells at either shallow or deep locations below the ventral surface, respectively. A plot of the locations of all dCINs based on their preferred phase of firing confirmed that cells firing rhythmically in-phase with their own ipsilateral side are preferentially located closer to the ventral surface than those that fire contralaterally (Fig. 4B). Nonrhythmic cells tended to occur in greater numbers at more dorsal locations. This overall distribution became even more apparent when plotted as a histogram of the percentage of the three types of dCINs at each depth: ipsilaterally firing dCINs were primarily found <200 µm below the surface, whereas contralaterally firing dCINs peaked at 200-300 µm. NS-dCINs increase in proportion the more dorsal the location (Fig. 4C). Thus, it appears that there is an anatomical separation of rhythmically active dCINs that may subserve different functional roles in the locomotor network.
Synaptic drive contributes to the rhythmicity of dCIN firing
As mentioned previously, hS-dCINs often displayed pronounced oscillations in membrane potential during the locomotor cycle, similar in nature to the locomotor drive potentials described previously in neonatal rat motor neurons (MNs) (Hochman and Schmidt, 1998
View larger version (31K):[in this window]
[in a new window]
Figure 5. Locomotor-related membrane oscillations of dCINs reflect synaptic input. Ai, Averaged membrane oscillation of an hS-dCIN (bold line, bottom traces) and NS-dCIN (thin line, bottom traces) with respect to the normalized and rectified cL2 cycle (top panel). Aii, Relationship between r value and average membrane oscillation (at 0 bias for a minimum of 20 cL2 cycles; n = 77). B, Membrane oscillations at different holding potentials for two different dCINs (Bi, Bii). Top traces show the normalized cL2 cycle, with the gray area corresponding to the ipsilateral L2 phase. Lower traces show the averaged, 10 Hz filtered intracellular recordings for 2 dCINs. Zero bias is indicated by the asterisk next to the membrane potential. The peak is indicated by the arrows. Bi, Neuron in which the phase of the peak reverses when hyperpolarized beyond the reversal potential for Cl . Bii, Cell with increased oscillation amplitudes at more hyperpolarized levels. Ci, Plot (black squares) of relative amplitude of the peak-trough oscillation at varying membrane potentials for the example shown in Bi. Note the sign reversal when the cell is hyperpolarized. Cii, Example of three dCINs driven predominantly by excitation: a, mixed AMPA and NMDA components (squares); b, mainly AMPA-driven (triangles); c, mainly NMDA-driven (circles). D, Distribution of average membrane oscillation for different membrane potential (in bins of 10 mV) ranges in neurons that are driven predominantly by inhibition and firing in-phase with the contralateral root (Di) and dCINs that receive phasic excitatory input and are firing in-phase with the ipsilateral root (Dii).
It has been shown previously that lumbar motor neurons and unidentified interneurons can be driven by phasic excitation alternating with inhibition during locomotion (Raastad et al., 1997
We then studied whether the sign of the predominant synaptic drive onto dCINs was related to either the ipsilateral or contralateral L2 ventral root phase. This analysis (Fig. 5D) revealed that the oscillations of five of the six cells that fired in the contralateral phase (i.e., with cL2) were shaped primarily by inhibitory rhythmic inputs (Fig. 5Di). These dCINs thus receive rhythmic inhibitory input during the ipsilateral motor neuron burst. In contrast, six of eight dCINs that fired ipsilaterally received predominantly rhythmic excitatory input (Fig. 5Dii), again during the ipsilateral motor neuron burst. Thus, in both cases, the predominant synaptic input onto the dCINs occurred in-phase with the ipsilateral L2 motor pool activity.
Characterization of Ih in dCINs
Intrinsic pacemaker currents can regulate rhythmic firing in both vertebrate and invertebrate preparations (Marder and Calabrese, 1996
View larger version (29K):[in this window]
[in a new window]
Figure 6. Characteristics of the dCIN Ih. A, Intracellular recording from S-dCINs showing a voltage sag (Ai) in response to current injection and a depolarizing rebound after termination of the current pulse. Data of the average Ih-induced sag amplitude ± SE from three control I-V trials are shown in Aii. B, Effect of 20 min incubation with 50 µM ZD 7288. Note the decrease in size of the injected current steps. C, Effect of ZD 7288 shown by comparison of averaged data for three runs at the most hyperpolarized step (control, bold line; ZD 7288, thin line). The control sag is indicated by the arrow, and the rebound depolarization is indicated by the arrowhead. Di, Activation of Ih measured under voltage-clamp conditions. Five millivolt steps were applied between
These current-clamp data were confirmed by voltage-clamp measurements of Ih in 24 dCINs. As was seen in the current-clamp data, all 24 dCINs had a detectable Ih. Figure 6Di shows typical currents activated from a holding potential of -50 mV with 1.5 sec steps between -60 and -130 mV. In parallel with the depolarizing sag voltage seen in current clamp, a very slowing activating inward current is detected at voltages below
The kinetics of activation were measured at -125 mV, at which Ih was well developed. The curves were well fit by a single-exponential relationship and were not improved by a double-exponential fit. Analysis of the time constant,
Boltzmann analysis of the voltage dependence of Ih activation supported the hypothesis of heterogeneity of Ih in different dCINs (Fig. 6Eii). The 20 neurons with short
In conclusion, all dCINs displayed an Ih. Furthermore, voltage-clamp experiments suggested that at least two classes of Ih are found in different dCINs. In the next analysis, we tested whether Ih plays a role in shaping the rhythmic spike output in individual dCINs.
Ih does not contribute significantly to dCIN rhythmicity
Comparison of the Ih-evoked voltage sag recorded under current-clamp conditions between hS-dCINs (n = 18), S-dCINs (n = 25), and NS-dCINs (n = 15) revealed that there was no significant difference in the amplitude of sag at a holding potential of
View larger version (28K):[in this window]
[in a new window]
Figure 7. Amplitude of Ih is not related to rhythmicity but to firing frequency. Ai, Plot of the average Ih-induced sag amplitude for the three different classes of dCIN as measured under current-clamp conditions (hyperpolarizing step to
To test this idea, seven neurons whose Ih had been measured under voltage-clamp conditions were analyzed for rhythmicity in NMDA and 5HT, as described previously. Three of these neurons were significantly rhythmic in-phase with the contralateral L2 root activity (p < 0.05; S-dCINs), whereas four were not (NS-dCINs). None of these cells had a slow
Ih amplitude does not relate to rhythmicity as determined by circular statistics; however, it was of interest to determine whether this current shows any relationship to firing frequency. As demonstrated for motor neurons in this preparation (Kiehn et al., 2000
1.0 Hz; n = 21). There was a significant increase in the size of the Ih-evoked sag between low- and high-frequency-firing and also between medium- and high-frequency-firing dCINs (p < 0.05) (Fig. 7B). This suggests that Ih, as previously suggested, contributes a tonic depolarizing conductance at membrane potentials at which dCINs are rhythmically modulated during locomotion.
DISCUSSION
Our investigation is the first comprehensive intracellular study of putative CPG interneurons in the developing mammalian spinal cord. The caudally projecting dCINs recorded in this study are located in the rhythm-generating areas of the neonatal rat spinal cord. Within this group, most of the cells exhibited significant rhythmicity, whereas fewer were nonrhythmic. The significantly rhythmic dCINs could be additionally divided into subpopulations on the basis of whether their preferred phase of firing was with the ipsilateral or contralateral motor pools. An important finding from this classification was that the division of rhythmic dCINs based on their phase relationship is reflected in their location in the transverse ventral-dorsal plane; cells in-phase with the cL2 occupy a more dorsal location than those in-phase with iL2. An additional finding is that rhythmicity of dCINs is generated to a significant extent by synaptic input, with little or no contribution to the rhythmicity per se from the endogenous pacemaker current, Ih. Furthermore, the rhythmic activity of the two distinct phase-related subpopulations is driven by different sources of synaptic input that appear to be temporally correlated to the synaptic input driving the ipsilateral motor neurons.
The ventromedial area: a kernel for left-right coordination
The neurons presented in this study are located in the ventromedial area of lumbar segments 2 and 3, an area known from both activity-dependent labeling studies and microlesion experiments to be important for rhythmogenesis (for review, see Kiehn and Kjaerulff, 1998
Comparison with rhythmic CIN populations in other vertebrates
Although there is a wealth of information on the overall structure, our understanding of the mammalian spinal CPGs at the cellular and network levels is still superficial (Kiehn et al., 1997
The limited understanding of locomotor CPG networks in the mammalian spinal cord stands in contrast to the considerable amount already known about the locomotor CPGs of a number of aquatic vertebrates, notably those of the Xenopus tadpole and lamprey. In particular, in these preparations, the CINs play an important functional role, using reciprocal inhibition to maintain the alternating phase relationship between bilateral motor pools (Soffe et al., 1984
Putative functional role of dCINs
We can begin to speculate on the roles of the rhythmically active dCINs on the basis of their preferred phase of firing. During locomotion, ipsilateral iL2 flexor bursts are in-phase with contralateral cL4-cL5 extensor bursts to promote alternating movement of the limbs (Kiehn and Kjaerulff, 1996
View larger version (43K):[in this window]
[in a new window]
Figure 8. Hypothetical model for the role of rhythmic dCINs during locomotor-like activity. A, The phase relationship of dCINs can be related to not only the recorded cL2 activity (black trace) but also to the likely activity of the ipsilateral motor pool (iL2; top gray trace) and target MNs (cL5; bottom gray trace). The expected role of the L2-L5 dCINs is shown in the bottom trace and expanded in B. Exc, Phase of predominantly crossed iL2 to cL5 excitatory information; Inhib, phase of mainly inhibitory information. The gradient of the line, positive (+ve), negative (
In this context, the ventral-dorsal distribution of ipsilaterally and contralaterally firing dCINs becomes important, showing that the dCINs can be anatomically separated along the dorsal-ventral plane on the basis of their function. Our arbitrary binning of neurons into depths of 100 µm (Fig. 4B) is unlikely to reflect the true patterns. However, it allows us to specifically record from either ipsilaterally or contralaterally firing dCINs with a reasonable degree of accuracy depending on the depth of the electrode. It remains to be seen whether this organization is true for other commissural interneuron subtypes located in the ventral horn.
The preferred phase of firing of most dCINs was in-phase with the iL2 in bins 7 and 8 (Fig. 4B). One can envisage a role for the broadly tuned but rhythmic firing of these "midphase" cells to stabilize the cL4-L5 extensor MN bursts and to define their burst duration. dCINs firing in the midphase of cL2 could have a similar effect on the bursts in cL4-L5 flexor MNs. Such a stabilizing role is in contrast to that of the "transition" cells firing at the cL2-iL2 and iL2-cL2 transitions (Fig. 3B). These cells might be specialized to determine the exact timing of switching between extensor and flexor phases, as suggested previously (Edgerton et al., 1976
Factors influencing rhythmic discharge of CPG interneurons
Evidence from the experiments in which the cells were held at various holding potentials indicates that the rhythmicity is shaped to a significant extent by synaptic input, either predominantly excitation in the case of ipsilaterally firing dCINs or predominantly inhibition in contralaterally firing cells. It is unlikely that these excitatory and inhibitory inputs operate in complete isolation; rather, they are likely to exist in varying proportions (Raastad et al., 1997
Because the pacemaker current (Ih) is present in nearly all dCINs, we sought to determine whether this current was involved in generating rhythmic activity. There was heterogeneity in the voltage and kinetic parameters of Ih in different dCINs. This may represent differential expression, or coexpression, of the four genes (HCN1-4) that encode Ih with somewhat different parameters (Santoro et al., 2000
Conclusions
Our results reveal a high degree of organization in the mammalian hindlimb CPG. The ability to target specific interneuronal subpopulations, even at the general level of phasing with ipsilateral or contralateral L2 flexor-related activity, is a considerable step forward in determining the overall function of the locomotor network. Further analysis of synaptic drive and endogenous properties is likely to reveal subtle differences between subpopulations, reflecting their differing inputs and roles in the CPG. Developing such knowledge is fundamental to our understanding of locomotion and neural circuits in general.
FOOTNOTES Received June 28, 2002; revised Aug. 28, 2002; accepted Sept. 3, 2002.
This work was supported by National Institutes of Health (NIH) Grant 1R01NS40795-01 and a grant from Karolinska Institutet (O.K.). R.M.H.-W. was supported by NIH Senior Postdoctoral Fellowship NS42405-01. We thank Ole Kjaerulff for participating in initial experiments.
Correspondence should be addressed to Ole Kiehn at the above address. E-mail: Ole.Kiehn{at}neuro.ki.se.
Dr. Harris-Warrick's present address: Department of Neurobiology and Behavior, Cornell University, Ithaca, NY 14852.
REFERENCES
- Andersen P, Moser EI (1995) Brain temperature and hippocampal function. Hippocampus 5:491-498[ISI][Medline].
- Beierholm U, Nielsen CD, Ryge J, Alstrom P, Kiehn O (2001) Characterization of reliability of spike timing in spinal interneurons during oscillating inputs. J Neurophysiol 86:1858-1868
[Abstract/Free Full Text] .- Berkowitz A, Stein PSG (1994) Activity of descending propriospinal axons in the turtle hindlimb enlargement during two forms of fictive scratching: phase analyses. J Neurosci 14:5105-5119[Abstract].
- Buchanan JT (1982) Identification of interneurons with contralateral, caudal axons in the lamprey spinal cord: synaptic interactions and morphology. J Neurophysiol 47:961-975
[Abstract/Free Full Text] .- Buchanan JT (1996) Lamprey spinal interneurons and their roles in swimming activity. Brain Behav Evol 48:287-296[ISI][Medline].
- Buchanan JT (1999) Commissural interneurons in rhythm generation and intersegmental coupling in the lamprey spinal cord. J Neurophysiol 81:2037-2045
[Abstract/Free Full Text] .- Buchanan JT, Kasicki S (1995) Activities of spinal neurons during brain stem-dependent fictive swimming in lamprey. J Neurophysiol 73:80-87
[Abstract/Free Full Text] .- Buchanan JT, McPherson DR (1995) The neuronal network for locomotion in the lamprey spinal cord: evidence for the involvement of commissural interneurons. J Physiol (Paris) 89:221-233[ISI][Medline].
- Butt SJB, Lebret JM, Kiehn O (2001) Identification of commissural interneurons in the lumbar region of the neonatal rat spinal cord. Soc Neurosci Abstr 27:306.9.
- Carr PA, Noga BR, Nance DM, Jordan LM (1994) Intracellular labeling of cat spinal neurons using a tetramethylrhodamine-dextran amine conjugate. Brain Res Bull 34:447-451[ISI][Medline].
- Cazalets JR, Sqalli-Houssaini Y, Clarac F (1992) Activation of the central pattern generators for locomotion by serotonin and excitatory amino acids in neonatal rat. J Physiol (Lond) 455:187-204
[Abstract/Free Full Text] .- Chen S, Wang J, Siegelbaum SA (2001) Properties of hyperpolarization-activated pacemaker current defined by coassembly of HCN1 and HCN2 subunits and basal modulation by cyclic nucleotide. J Gen Physiol 117:491-504
[Abstract/Free Full Text] .- Dale N (1985) Reciprocal inhibitory interneurones in the Xenopus embryo spinal cord. J Physiol (Lond) 363:61-70
[Abstract/Free Full Text] .- Edgerton VR, Grillner S, Sjöström A, Zangger P (1976) Central generation of locomotion in vertebrates. In: Neural control of locomotion (Herman RM, Grillner S, Stein PSG, Stuart DG, eds), pp 439-464. New York: Plenum.
- Eide AL, Glover J, Kjaerulff O, Kiehn O (1999) Characterization of commissural interneurons in the lumbar region of the neonatal rat. J Comp Neurol 403:332-345[ISI][Medline].
- Grillner S, Deliagina T, Ekeberg O, El Manira A, Hill RH, Lansner A, Orlowsky GN, Wallen P (1995) Neural networks that co-ordinate locomotion and body orientation in lamprey. Trends Neurosci 18:270-279[ISI][Medline].
- Hochman S, Schmidt BJ (1998) Whole cell recordings of lumbar motoneurons during locomotor-like activity in the in vitro neonatal rat spinal cord. J Neurophysiol 79:743-752
[Abstract/Free Full Text] .- Huang A, Noga BR, Carr PA, Fedirchuk B, Jordan LM (2000) Spinal cholinergic neurons activated during locomotion: localization and electrophysiological characterization. J Neurophysiol 83:3537-3547
[Abstract/Free Full Text] .- Hultborn H, Conway B, Gossard J-P, Brownstone R, Fedirchuk B, Schomburg ED, Enriquez-Denton M, Perreault MC (1998) How do we approach the locomotor network in the mammalian s
