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School of Biological Sciences, University of Bristol, Bristol BS8 1UG, United Kingdom
Submitted 8 January 2004; accepted in final form 10 March 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONSGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONSGRANTSACKNOWLEDGMENTSREFERENCES |
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; Helms and Johnson 2003). In particular, three groups of interneurons whose development depends on the presence of the roof-plate seem to provide sensory projection pathways. Two of these groups project to the opposite side of the cord and then on to the brain, like the adult spino-thalamic tract, while one projects on the same side, like the adult spino-cervical tract (Brown 1981; Willis and Coggeshall 1991). Interneurons with similar projection patterns are present as soon as frog tadpoles emerge from the egg (Li et al. 2001). As a final step in the structural and physiological definition of spinal sensory pathways for initiation of swimming in the developing frog, we examined the properties, connections, and responses of dorsal spinal cord interneurons with ipsilateral ascending projections.
The hatchling Xenopus tadpole (development stage 37/38) (Nieuwkoop and Faber 1956) can produce two main responses to trunk skin stimulation, even after spinalization. In the first, the tadpole bends its body away from the side where its tail skin is stroked and then swims forward (Boothby and Roberts 1995). In the second, the tadpole shows strong but slow rhythmic body flexions, called struggling, when it is held or the skin is stimulated repeatedly (Soffe 1991, 1993). The spinal cord has only eight anatomical classes of neuron (Fig. 1B), and the functions of six of these classes have been studied (Li et al. 2002; Roberts 2000). Functions for dorsolateral ascending interneurons (dlas) and Kolmer-Agdhur cells remain unknown. Two classes of possible sensory interneurons have been described (Fig. 1B; Clarke and Roberts 1984): dorsolateral commissural interneurons (dlcs) have dorsolateral somata and dorsal dendrites that receive inputs from skin sensory Rohon-Beard neurons (RB) axons and project axons to the contralateral side (Clarke et al. 1984; Roberts and Sillar 1990). They function as the relay neurons in the tadpole's disynaptic flexion reflex to contralateral skin stimulation (Li et al. 2003). Their activity can also increase swimming frequency when stimulation is applied during swimming (Sillar and Roberts 1988a). dlas are anatomically similar to dlcs, but their axons project rostrally on the same side, reaching the midbrain or forebrain (Clarke and Roberts 1984; Li et al. 2001). Are dlas also sensory projection interneurons?
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONSGRANTSACKNOWLEDGMENTSREFERENCES |
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). Xenopus tadpoles at stage 37/38 (see Fig. 1A) were anesthetized with 0.1% MS-222 (3-aminobenzoic acid ester) and pinned in a small bath of saline (concentrations in mM: 115 NaCl, 3 KCl, 2 CaCl2, 2.4 NaHCO3, 1 MgCl2, and 10 HEPES, adjusted with 5 M NaOH to pH 7.4). In many experiments, 1 mM MgCl2 was replaced by 1 mM CaCl2. The dorsal fin was cut, and the tadpole was transferred to 10 mM 
-bungarotoxin saline for immobilization (20–30 min). It was then re-pinned so skin and muscles over the right side of the spinal cord could be removed. A dorsal cut was made along the midline of the spinal cord to open the neurocoel and expose neuronal cell bodies. Some ependymal cells in the neurocoel were picked away to expose more ventral neurons. The animal was then re-pinned on a small rotatable Sylgard stage in a 700-µl recording chamber that allowed brightfield illumination from below on an upright Nikon E600FN microscope. The animal was tilted to an angle that allowed exposed neuronal cell bodies on the left and right sides of the cord to be seen using a 40x water immersion lens. Saline in the chamber was kept circulating at about 2 ml/min and was not oxygenated. Drops of antagonists were added to a 100-µl chamber upstream to the recording chamber (bath application) or applied close to the neuron being recorded by applying gentle pressure to solution in a pipette with tip diameter of 10–20 µm (microperfusion). NBQX, D-AP5, and bicuculline were from Tocris Cookson; strychnine and all other chemicals were from Sigma unless stated otherwise.
Patch pipettes were filled with 0.1% neurobiotin in intracellular solution (concentrations in mM: 100 K-gluconate, 2 MgCl2, 10 EGTA, 10 HEPES, 3 Na2ATP, and 0.5 NaGTP, adjusted to pH 7.3 with KOH) and had resistances around 10 M
. In some experiments, 0.1% Alexa Fluor 488 (Molecular Probes) was also used in the patch pipette solution to identify dlas in live tadpoles. Stimulating suction electrodes were placed on head and tail skin to apply 1-ms duration current pulses to start fictive swimming or struggling activity. Another suction electrode was usually placed on the 11th intermyotome cleft to record ventral root activity. Patch pipettes were manipulated under visual control to contact exposed neuron somata (Fig. 1A). Light positive pressure was always applied to the pipette solution before trying to get a seal. Signals were recorded with an Axoclamp 2B in conventional bridge or continuous single electrode voltage-clamp mode. Data were acquired with Signal software through a CED 1401 Plus (CED, Cambridge, UK) with sampling rate of 10 kHz. Stimuli to the skin were controlled using the CED 1401 Plus configured by Signal and given via an optically coupled isolator.
After the recording, tadpoles were fixed overnight in 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2 at 
4°C) and washed with 0.1 M PBS (120 mM NaCl in 0.1 M phosphate buffer pH 7.2). The animals were 1) washed twice in 1% triton X-100 in PBS for 15 min; 2) incubated in a 1:300 dilution of extravidin peroxidase conjugate in PBS containing 0.5% Triton X-100 for 2–3 h; 3) washed again in PBS; 4) presoaked in 0.08% diaminobenzidine in PBS (DAB solution) for 5 min; 5) moved to 0.075% hydrogen peroxide in DAB solution for 5 min; and 6) washed in running tap water. The brain and spinal cord were dissected free with the notochord and some ventral muscles, dehydrated, cleared in methyl benzoate and xylene, and mounted whole, between two coverslips using Depex (BDH Laboratory Supplies). Neurons filled with neurobiotin staining were observed using a 100x oil immersion lens and traced using a drawing tube. To compensate for shrinkage during dehydration, all neurobiotin staining measurements in this paper have been corrected by multiplying by 1.28 (Li et al. 2001). Fluorescent images were acquired using a Penguin 150 CLM camera (Pixera) with B-2A filter. All means are given with their SD unless otherwise stated. Off-line analyses were made with Minitab and Excel. Photos were processed with Photoshop.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONSGRANTSACKNOWLEDGMENTSREFERENCES |
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Recordings from 320 interneurons showed the short latency responses to skin stimulation that would be expected in sensory pathway interneurons (typically 3–8 ms depending on the spacing between stimulating and recording electrodes). Of these, 48 had the anatomical features of dlas defined by horseradish peroxidase backfilling and intracellular neurobiotin staining (Clarke and Roberts 1984; Li et al. 2001): a dorsolaterally located, usually multipolar soma, dorsolateral dendrites, and an ipsilateral axon projecting toward the brain (Figs. 3–8). The anatomical features used to identify the neurons were seen either during the experiment using fluorescent imaging of the Alexa Fluor 488 or after the experiment following processing to visualize neurobiotin. The recorded dlas had somata in the mid-trunk region at longitudinal positions between the 4th and 11th postotic muscle segments (1.2–2.4 mm from the midbrain) with a peak in numbers at the 7th segment (1.7 mm from the midbrain).
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, respectively.
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dlas are excited by skin stimulation and inhibited during swimming and struggling
The Xenopus tadpole at stage 37/38 responds to skin stimulation by producing two types of locomotion. When the skin is stimulated by a brief touch or single electric stimulus, neurons on both sides of the spinal cord are excited (Zhao et al. 1998), and the animal normally bends its body away from the touch and starts swimming forward with alternating contractions at 15–25 Hz (Roberts 1990). During fictive swimming, ventral roots show brief, tightly synchronized bursts of spikes (Fig. 3Aa) that alternate on left and right sides. Sustained pressure on the skin or repetitive electrical stimuli usually led to struggling with slower but stronger rhythmic contractions at 5–10 Hz (Soffe 1991). During fictive struggling, ventral root bursts are much longer (Fig. 3Ba). We therefore stimulated the skin and looked at the responses of dlas and their input during these two locomotion patterns.
When the tail skin was stimulated, all dlas tested received a strong compound EPSP (n = 40, Figs. 3, 5, 7C, and 10A). For stimuli less than twice threshold, these EPSPs could lead to a single action potential in most dlas (n = 36). The latencies measured for 10 EPSPs in each of 10 dlas were short and very consistent, (ranges, 3.45–6.65 and 0.065–0.164 ms). In a few cases (n = 5), this initial excitation was followed by an inhibitory postsynaptic potential (IPSP; Fig. 3Ab). The shortest latencies for these IPSPs were 11.2–14.0 ms after stimulation but before swimming started. During swimming, all 42 dlas tested were inhibited by IPSPs loosely in phase with the ventral root activity recorded on the same side (Figs. 3Aa, 6, C and D; we define this timing as "early-cycle"). When struggling was elicited by skin stimulation, all 22 dlas tested were also inhibited by IPSPs in phase with ventral root bursts (Fig. 3B). These results suggest that dlas are only active briefly prior to the initiation of swimming or struggling.
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Sensory RB neurons directly excite dlas
The short latency responses of dlas to skin stimulation implied that they were directly excited by sensory RB neurons. In 370 simultaneous recordings from pairs of neurons of a variety of types on the same side of the spinal cord, 16 were found to be between RB neurons and dla interneurons. The RB neurons were identified by their large dorsal soma without dendrites and ascending and descending axons in the dorsolat-eral tract. In six pairs, action potentials resulting from current injection into the RB neuron produced EPSPs in the dla at short latencies (1.6 ± 0.2 ms; Fig. 4A), suggesting that the interaction was direct and monosynaptic. These EPSPs were large (5.5 ± 1.8 mV; maximum amplitude, 12.3 ± 5.9 mV), had fast rise-times (10–90% in 1.8 ± 0.4 ms; time to peak, 3.4 ± 1.1 ms), and were of short duration (19.2 ± 16.3 ms at 50% amplitude; although most had a long, low-amplitude tail when recorded in 0 mM Mg2+). The neurons' anatomy, revealed by neurobiotin or fluorescent dye injection, confirmed the identity of the pre- and postsynaptic neurons and showed that RB neuron axons came into close contact with dla dendrites or somata (n = 4, Fig. 4B).
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The short latency excitation of dlas following skin stimulation and the paired recordings with RB neurons show that dla interneurons are directly excited through the activation of AMPARs and NMDARs following impulses in skin sensory RB neurons. dlas are therefore likely to be sensory pathway interneurons.
dlas are inhibited by premotor ascending interneurons
In six paired whole cell recordings of premotor ascending interneurons (aINs) and dlas, three of the aINs inhibited the postsynaptic dla (Figs. 6 and 7). In the 42 IPSPs from these recordings, the durations (52 ± 15.1 ms at 50% peak height) and the rise times (3.8 ± 0.3 ms from 10–90% of peak height) were similar to those in dlcs, another type of sensory interneuron, which are inhibited by aINs when the tadpole swims (Li et al. 2002, 2003). The latencies (2.5 ± 0.8 ms from the peak of the presynaptic spike), like the values for RB–dla interactions, all lay within the range found in previous cases where the connection was considered to be direct (see Li et al. 2002, 2003). In addition, the anatomy in all three pairs revealed possible contact sites between presynaptic aIN axons and dla dendrites or soma (Figs. 6B and 7B).
During swimming, some aIN spikes in paired recordings seemed to correlate closely with dla IPSPs (Fig. 6C; n = 3 pairs). We therefore compared the timing of aIN action potentials and dla IPSPs during swimming (Fig. 6D). For aIN spikes or dla IPSPs, phases were calculated by their timings relative to ventral root bursts in each normalized swimming cycle (668 spikes from 16 aINs and 691 IPSPs from 9 dlas). The expected longitudinal delays between the positions of recorded neurons and the ventral root electrode were compensated using a rostro-caudal delay of 3.5 ms/mm (Li et al. 2002). The correlation between the two distributions is significant (P < 0.001, Pearson). Since most of the other neurons active during swimming fire in a tightly synchronized burst and earlier in the swimming cycle than aINs (Li et al. 2002), this suggests that most of the IPSPs that dlas receive during swimming come from aINs.
During struggling, aINs are rhythmically active in phase with ventral root activity, again at the time when dlas receive inhibition. They may be the source of this inhibition too, but detailed correlation between aIN spikes and dla IPSPs was not attempted during struggling because it is not possible to compare the phases of these events with sufficient precision.
Bath application of the glycine antagonist strychnine blocked the inhibition from aINs onto dlas in two paired recordings, and when one of these was tested further, the GABAA antagonist bicuculline did not have any obvious effect (Fig. 7A). During swimming, the rhythmic IPSPs in dlas were also blocked by strychnine (Fig. 7C, n = 6), and again bicuculline had no effect (n = 1). We conclude that the transmission from aIN to dla is probably glycinergic and that aINs produce glycinergic inhibition of dlas during swimming.
dlas excite rostral central pattern generator neurons
Paired recordings.
Having found the main sources of synaptic inputs to dlas, excitation from sensory RBs and inhibition from aINs, we investigated their output connections. dla cell bodies are mainly located in the mid-trunk region of the spinal cord between 1.25 and 2 mm (segments 5–9) from the midbrain (Li et al. 2001). Their axons project rostrally in the dorsal half of the spinal cord, but are often quite wavy, so they could contact dendrites in a wide range of dorso-ventral positions. In paired recordings from dlas and 17 more rostral ipsilateral CPG neurons (9 cINs, 6 aINs, 2 dINs), current-evoked dla spikes excited two aINs and one cIN. The two aINs also made reciprocal inhibitory synapses back onto the recorded dlas. An example of a paired recording with reliable interaction between a dla and an aIN is shown in Fig. 8. The mean latencies from the peak of the dla spike to the start of the CPG neuron EPSPs were between 1.4 and 1.8 ms (3 neurons, 12 EPSPs). This indicates direct, monosynaptic connections from dlas to these CPG neurons (Li et al. 2003).
Because the total number of dlas is small (Li et al. 2001), it has been difficult to get paired recordings with interaction between dlas and ipsilateral CPG neurons. To identify the neuronal types that may receive dla excitation and investigate the pharmacology of this excitation, we analyzed CPG neuron responses to ipsilateral tail skin stimulation below swimming threshold. We looked for evidence that CPG neurons are excited di-synaptically via dlas (sensory RB neurons 
dlas CPG neurons).
Indirect evidence for dla output connections.
Following skin stimulation, sensory pathway interneurons receive reliable, large, short-latency, monosynaptic EPSPs from RB neurons that usually lead to an action potential (Clarke and Roberts 1984; Roberts and Sillar 1990; Sillar and Roberts 1988a; Figs. 3– 5). These EPSPs are glutamatergic (Sillar and Roberts 1988a), with a strong AMPAR-mediated component (Li et al. 2003) (Fig. 5). As a result, when 5 µM NBQX was applied in the bathing saline onto 12 dlcs and 5 dlas, EPSPs were strongly attenuated so that firing in response to skin stimulation was abolished in 14 of them (88%, Fig. 5A). The spikes produced by NMDAR-mediated EPSPs in the other two dlcs and one dla were unreliable and had long and variable latencies. In NBQX, therefore, neither dlcs nor dlas would be able to produce reliable, short latency synaptic excitation of CPG neurons. By applying NBQX globally in the saline, we therefore predicted that disynaptic excitation of CPG neurons in response to ipsilateral skin stimulation would disappear as a result of the failure in spiking of dla sensory interneurons (Fig. 9B). We tested the effect of bath-applied 5 µM NBQX on EPSPs produced in 25 CPG neurons by ipsilateral tail skin stimulation below swimming threshold.
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To confirm that the disynaptic EPSPs following skin stimulation in the 11 CPG neurons resulted from dla spikes, we checked that the latencies of these two events occurred in a strict sequence. We compared the distribution of latencies of EPSPs (n = 140 EPSPs, 10 stimuli in each of the 11 neurons) and dla spikes (n = 126 spikes, 10 stimuli in each of 11 dlas tested, Fig. 10A) produced by ipsilateral tail skin stimulation. The combined distribution histograms for both dla spikes and CPG EPSPs (Fig. 10B) had very similar skewed shapes, and as expected, CPG EPSPs were slightly delayed relative to dla spikes (median value 9.5 vs. 6.8 ms). After correcting for this delay by subtracting the difference in median values between the two distributions from the EPSP latency values, the two latency distributions were highly significantly correlated (coefficient = 0.796, P < 0.001). The difference in median latencies between the distributions (2.7 ms) was also very similar to the latencies measured between dla spikes and CPG neuron EPSPs in paired recordings: a combination of synaptic delay and a conduction delay resulting from the more caudal recording positions of dlas compared with CPG neurons.
We produced EPSP latency distribution histograms for 14 individual CPG neurons where a sufficient number of skin stimuli were delivered. If the EPSPs were from dlas, their latency distribution should be very similar to the combined distribution described above, produced by using EPSPs from many neurons (hatched bars in Fig. 10B). This was the case for 1/2 mns, 2/2 dINs, 1/3 aINs, and 4/7 cINs (3cINs were from the above 11 CPG neurons; Fig. 10C). Together, these results give us confidence that the EPSPs in CPG neurons having a latency of around 10 ms in response to skin stimulation come from ipsilateral dlas and that dlas can excite all types of CPG neurons found in the tadpole spinal cord swim circuitry.
We studied the pharmacology of the 11 cases of disynaptic EPSPs, presumed to be from dlas, using very local application of glutamatergic antagonists. Microperfusion was used to minimize the risk of a direct effect of the drugs on RB to dla transmission and therefore dla firing (Fig. 9Aa). In each case, microperfusion of either NBQX or D-AP5 could only partially block the EPSPs. When they were applied together, however, the EPSPs were totally blocked (Fig. 9Ab).
We conclude that after being excited to fire by RB sensory neurons, dla interneurons excite ipsilateral CPG neurons located more rostrally. This excitation is mediated by both NMDARs and AMPARs.
Role for ipsilateral sensory ascending excitation in swimming
When the tail skin is stimulated with a brief current pulse (<1 ms), a few RB sensory neuron peripheral neurites are excited (Clarke et al. 1984; Soffe 1997). The action potentials propagate via the soma to the central, longitudinal RB axons that excite dlc and dla interneurons to fire action potentials. Despite their ipsilateral excitatory connections, dlas do not appear to mediate ipsilateral reflex responses, and previous work suggests that this is partly the result of early ipsilateral inhibition following skin stimulation (Roberts et al. 1984; Zhao et al. 1998). After some period of time, depending on the stimulation intensity (30–100 ms, 12 tadpoles, data not shown), swimming starts (Figs. 3A and 7C). Direct evidence suggests that dlas play a role in the initiation of swimming activity. In one case, current induced multiple firing of a single dla was capable of starting fictive swimming (4 observations, Fig. 11Aa). This phenomenon was seen more often in artificial 0 Mg2+ saline (7 observations in 4 dlas, data not shown) where magnesium's blocking effect on NMDARs was absent.
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, 1993). Recruitment of silent contralateral CPG neurons by dlcs was proposed to be the mechanism for this increase in frequency (Sillar and Roberts 1992b, 1993). Like dlcs, dlas receive early-cycle IPSPs from aINs during swimming (Figs. 3A and 6, C and D), and can also be excited to fire spikes by skin stimulation during swimming (Fig. 11B). Excitation from dlas could be important in recruiting silent ipsilateral CPG neurons when skin stimulation is delivered during swimming. For example, in one dla recorded in 1 mM Mg2+ saline, current-induced multiple firing could result in faster swimming (Fig. 11Ab). We therefore looked at the responses of eight cINs and two aINs that fired unreliably during swimming to see if they could be recruited by ipsilateral skin stimulation during swimming. Nine of these neurons were reliably recruited by a skin stimulus given during swimming. Analysis of the latencies of EPSPs in these neurons following skin stimulation suggested that EPSPs with shorter and consistent latencies (5–7 ms) were produced directly by RB neurons, while those with longer and more variable latencies (7–13 ms) were most likely produced by dlas (Figs. 10C and 11, C and D). We conclude that dlas are a component of the swim initiation pathway and can also contribute to speeding up swimming when stimuli are given during swimming. |
DISCUSSION |
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We provide the first evidence on the responses and physiology of Xenopus tadpole dorsolateral ascending interneurons (dlas). Both the anatomical features of dye filled neurons and their location, in a longitudinal region of the spinal cord 1.2–2.2 mm from the midbrain (muscle segments 4–10; cf. Fig. 9B in Li et al. 2001), matched those of dlas defined previously (Li et al. 2001; Roberts and Clarke 1982). This evidence gives us confidence that our recordings were from a single class of spinal interneuron.
dla interneurons are excited by skin stimulation and form an ipsilateral cutaneous sensory projection pathway. They show strong parallels with dorsolateral commissural interneurons (dlcs), which we previously identified as the major crossed sensory projection pathway in the developing Xenopus spinal cord (Clarke and Roberts 1984; Li et al. 2002; Roberts and Sillar 1990; Sillar and Roberts 1988b, 1992a). Both types of interneurons 1) receive strong, direct, mainly AMPAR-mediated glutamatergic excitation that can lead to action potentials, from cutaneous sensory RB neurons; 2) receive glycinergic inhibition early on each swimming cycle from premotor aINs and do not fire during swimming; 3) are inhibited and do not fire during struggling; and 4) make excitatory glutamatergic synapses onto spinal neurons that are components of the central pattern generator for swimming. Surprisingly, dlas have a significantly higher input resistance than dlcs (dla, 1224 ± 541 M; dlc, 569 ± 282 M; P = 0.04; W.-C. Li, unpublished whole cell recordings from dlcs). Despite this, both types of sensory interneurons share similar firing properties, giving adapting trains of spikes to depolarizing current injection (see also Aiken et al. 2003). The major difference between dlas and dlcs is that their postsynaptic target neurons are on opposite sides of the spinal cord. We propose that dlas and dlcs form two primitive ascending cutaneous sensory pathways in the tadpole central swimming circuitry. Since dlas do not produce a significant short latency ipsilateral reflex response, we now need to consider their role in the initiation of locomotion.
Role of dlas in initiation of swimming
Current injection that evokes spikes in individual sensory RB neurons can often start fictive swimming activity in immobilized tadpoles (Soffe 1997). Current evoked multiple firing of single dla or dlc sensory interneurons (Roberts and Sillar 1990) can also start fictive swimming. Following skin stimulation, dlcs and dlas are the only two groups of interneurons that fire reliably before swimming starts. Our paired recordings have confirmed the monosynaptic excitation of dlcs and dlas by skin sensory RB neurons (Li et al. 2003) (Fig. 4). All these data support the proposal that the dlas and dlcs are the first order sensory interneurons in the swimming-initiation pathway. When the skin is stimulated locally, a few sensory RB neurons will fire (Clarke et al. 1984). RB neurons have central axons about 2 mm long that all pass the mid-trunk region (segments 4–10; Hartenstein 1993) wherever their somata lie. RB neurons innervating all parts of the body surface can therefore make synapses to excite dlas located in the mid-trunk. The first role of dlas is therefore to distribute this excitation rostrally through the spinal cord, hindbrain, and to the midbrain, independent of the site of stimulation. Because the axons of more caudal sensory RB neurons only reach the mid-trunk region (Hartenstein 1993), dlas are necessary to ensure that excitation from these RB neurons reaches more rostral parts of the CNS. The second role of dlas, like dlcs (Clarke and Roberts 1984), is to amplify the skin stimulation signal. A few RB neuron spikes can lead to the excitation of many dlas, which would normally be active together to initiate swimming.
We have examined the output connections of dlas within the spinal cord, although we assume that their axons in the brain make further en passant synapses here too. In the spinal cord, we have direct evidence from paired recordings and less direct evidence from responses to skin stimulation that dlas activate AMPAR and NMDAR to excite all classes of central pattern generator neurons (motoneurons, reciprocal inhibitory cINs, recurrent inhibitory aINs, and excitatory dINs; see Fig. 1B). Again, these connections are similar to those made by dlcs (Li et al. 2003). When the tadpole is at rest and receives a skin stimulus, swimming normally starts after a 30- to 100-ms delay. The dla-derived EPSPs in CPG neurons appear after a 10-ms delay and are long-lasting (Figs. 8 and 9). It is highly likely that these EPSPs contribute to ipsilateral CPG neuron firing when swimming starts. Together with some RB-derived EPSPs (Fig. 11D), dla EPSPs may partly account for the more reliable firing of many CPG neurons at the beginning of swimming.
Another similarity to the dlc interneurons in the crossed sensory pathway is that dlas receive gating inhibition during swimming (Sillar and Roberts 1988a, 1992a). We have shown that in both neuron classes, this inhibition comes from glycinergic ascending interneurons (aINs; Li et al. 2002; Figs. 6 and 7). The effect of the inhibition is strong when swimming starts and may effectively close down the sensory pathway but later, as the inhibition weakens, stimulation at certain phases can escape inhibition and lead to higher frequency swimming (Sillar and Roberts 1993). Because the timing of modulatory inhibition is similar in both types of sensory interneuron, sensory stimuli that escape inhibition will excite CPG neurons on both sides via dlcs and dlas. Such phasic inhibitory gating during locomotion is a general feature of sensory pathways in most animals (Pearson and Collins 1993).
Relation of dlas to neurons in other vertebrates
dla interneurons in Xenopus have very long ascending axons so seem suited to a role as sensory projection neurons. At early stages in their development, the spinal cords of the newt Triturus vulgaris and zebrafish have interneurons that may be homologues of Xenopus tadpole dlas, with ascending axons and similar morphology (Roberts 2000). If they are homologous, we would expect them all to have similar sensory relay functions. Dorsal horn interneurons excited by cutaneous stimulation and with long distance ipsilateral ascending projections are a basic feature of the vertebrate spinal cord (Brown 1981; Willis and Coggeshall 1991). In adult mammals, these interneurons fall into two groups, having axons in the dorso-lateral funiculus (spinocervical tract neurons, Morin 1955) or in the dorsal columns (postsynaptic dorsal column pathway, Brown and Fyffe 1981). Interneurons that are possibly homologous to those forming the mammal spinocervical tract have also been described in adult Xenopus (Munoz et al. 1996).
Anatomically, dlcs and dlas in Xenopus tadpole spinal cord may be the counterparts of classes of interneurons recently described in the developing mammal dorsal spinal cord (Goulding et al. 2002; Helms and Johnson 2003; Lee and Pfaff 2001; Sharma and Peng 2001). Classes of possible somatosensory relay interneurons are defined first by the dependence of their progenitors on the presence of the roof plate and second by a lack of expression of the transcription factor Lbx1. Individual populations are defined by the combinatorial expression of other transcription factors, the position of the soma, and their axonal projections (Gowan et al. 2001; Gross et al. 2002; Lee and Pfaff 2001; Muller et al. 2002). Particularly intriguing is the possible relationship between Xenopus dlas and the dl3 class. Neurons in this class lie deep in the dorsal horn and are thought to have ipsilateral ascending axon projections and to be excited by sensory input.
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CONCLUSIONS |
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). Since both classes are inhibited when pressure stimulation to the skin leads to violent struggling movements, they cannot be responsible for the initiation of this motor output (Soffe 1993).
We believe that the ability to define anatomical and physiological classes of neurons remains a key issue in understanding how the vertebrate spinal cord works in controlling behavior. Since we are close to understanding the function of each class of neuron in the Xenopus tadpole spinal cord, this primitive vertebrate system has great potential for establishing links between genetically marked neuronal types and function (Sharma and Peng 2001).
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONSGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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Address for reprint requests and other correspondence: W.-C. Li, School of Biological Sciences, Univ. of Bristol, Woodland Rd., Bristol BS8 1UG, UK (E-mail: wenchang.li{at}bristol.ac.uk).
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REFERENCES |
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) and 807 M
) current pulses. The 2 regression lines cross at –58.9 mV (resting membrane potential is –60.3 mV). C: the same dlas firing to 3 different levels of current injection. D: the same dlas firing frequency changes vs. time to 7 rectangular positive current pulses (105–350 pA; E, 
