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Previous Article | Next Article
The Journal of Neuroscience, April 15, 1999, 19(8):3007-3022
Cholinergic and GABAergic Inputs Drive Patterned Spontaneous Motoneuron Activity before Target Contact
Louise D. Milner and Lynn T. Landmesser Department of Neurosciences, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
Patterned spontaneous electrical activity has been demonstrated in a number of developing neural circuits and has been proposed to play a role in refining connectivity once axons reach their targets. Using an isolated spinal cord preparation, we have found that chick lumbosacral motor axons exhibit highly regular bursts of activity from embryonic day 4 (E4) (stage 24-25), shortly after they exit the spinal cord and while still en route toward their target muscles. Similar bursts could be evoked by stimulating descending pathways at cervical or thoracic levels. Unlike older embryonic cord circuits, the major excitatory transmitter driving activity was not glutamate but acetylcholine, acting primarily though nicotinic non-
7 receptors. The circuit driving bursting was surprisingly robust and plastic, because bursting was only transiently blocked by cholinergic antagonists, and following recovery, was now driven by GABAergic inputs. Permanent blockade of spontaneous activity was only achieved by a combination of cholinergic antagonists and bicuculline, a GABAA antagonist. The early occurrence of patterned motor activity suggests that it could be playing a role in either peripheral pathfinding or spinal cord circuit formation and maturation. Finally, the characteristic differences in burst parameters already evident between different motoneuron pools at E4 would require that the combination of transcription factors responsible for specifying pool identity to have acted even earlier.
Key words: motoneurons; spontaneous neural activity; rhythmicity; nicotinic receptors; GABA; cholinergic inputs; spinal cord interneurons; embryonic networks
INTRODUCTION
The nervous system consists of complex neural circuits initially constructed during development by a combination of intrinsic molecular cues that guide neurons to their targets and activity-dependent cues that fine tune those connections (for review, see Goodman and Shatz, 1993
; Katz and Shatz, 1996
In the motor system, activity is important for modulating target innervation. Both the extent of initial synaptogenesis (Ding et al., 1983
In chick, the pattern and frequency of spontaneous motoneuron bursting episodes changes in a systematic way from initial target innervation [stages 29-30; embryonic day 6 (E6)] until after the motoneuron cell death period (stages 36-38; E10-E12) (O'Donovan and Landmesser, 1987
Because the nature of the circuit driving the earliest spontaneous activity occurring before target contact was unknown, we characterized early activity by recording from muscle nerves that were growing toward their targets. We show that motoneurons exhibit spontaneous recurrent episodes of bursting activity as early as E4 (stages 24-25). Unlike later stages, early activity appears to be driven primarily by cholinergic circuits, with minor roles from GABA and glutamate. These results indicate that, not only do motoneurons exhibit patterned activity earlier than previously shown, but that the circuit responsible undergoes a switch in transmitters during the second week of gestation. The major changes in activity and pharmacology coincided with target contact, suggesting that target influences may play a role in circuit maturation.
MATERIALS AND METHODS
Nerve recordings. All recordings of spontaneous motor nerve activity were made in White Leghorn chick embryos between stages 24 and 29.5 (Hamburger and Hamilton, 1951
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Figure 1. Motoneurons exhibit spontaneous episodes of bursting as they grow toward their targets. A, Schematic of the motor circuit in the chick lumbosacral spinal cord. Patterned motor activity is generated by local circuits in the cord (hypothetical CPG) and can be initiated by stimulating descending inputs from the rostral cord. Nerve recordings from specific muscle nerves were made using tight-fitting suction electrodes. In most figures, we have paired records from an extensor motor pool on the top trace with a flexor motor pool on the bottom trace. B, Example of spontaneous activity from the femorotibialis (top trace) and obturator (bottom trace) muscle nerves from a stage 25 embryo. Bursting activity occurred in episodes (bracket) containing multiple bursts (arrows). At stage 25, episodes occurred regularly, ~1-2 min apart (interepisode interval). C, The earliest activity was recorded from the sciatic nerve at early stage 24, when each episode contained a single burst and occurred every 3 min (top trace). The two bottom traces on an expanded time scale show that a burst evoked by stimulation of descending input from more rostral cord levels (bottom trace, arrow marks stimulus artifact) was similar to those occurring spontaneously (middle trace). CPG, Central pattern generator; SART, sartorius; FEMORO, femorotibialis.
Drug treatments. Neurochemicals and receptor blockers were bath applied in the in vitro spinal cord-hindlimb preparation using a pump that superfused circulated oxygenated Tyrode's solution over the preparation. Each drug was evaluated either alone or in combination with other drugs for its effect on spontaneous bursting activity. The drugs were applied for a minimum of 20 min, and their effect on spontaneous activity was evaluated by quantifying the change in burst frequency, number of bursts per episode, and burst structure. As shown in Figure 1B, episodes of bursting activity consisting of one or more bursts (depending on the stage) occurred every several minutes. The interepisode interval was used to quantify the frequency of episodes, and the lengths of bursts and of interburst intervals were also measured in some cases. When comparing different drug treatments, 10 or more episodes were measured, and the data were displayed as mean ± SE. Except where indicated, a given drug treatment was performed on two or more embryos with similar results, and, in most cases, drugs were washed until burst parameters returned to control values (several minutes to 1-2 hr depending on the drug).
A list of the drugs used includes the following: cholinergic receptor blockers: d-tubocurarine (dTC), atropine, dihydro-B-erythroidine (DHBE),
RESULTS
Development of spontaneous bursting activity
Previous studies had inferred that chick limb motoneurons were spontaneously active in ovo by E6 (stages 28.5-29) because this activity resulted in recurrent episodes of hindlimb movements (Hamburger and Balaban, 1963
As early as stage 24 (E4), when motor axons have just begun to bundle together (Fig. 1A) after a period of defasciculation in the plexus region (Tang et al., 1994
At this stage, motoneurons produced a single burst every 1-2 min (Fig. 2A,G). Motor axons to flexor and extensor muscles burst simultaneously rather than in the alternating manner characteristic of more mature patterns of activity (O'Donovan and Landmesser, 1987
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Figure 2. Developmental changes in bursting activity recorded from muscle nerves from stages 25-28.5. A, At stage 25, each episode consists of a single burst, and characteristic differences between motoneuron pools are already apparent; the ischioflexorius/caudilioflexorius nerve (top trace) has longer duration bursts than the sartorius nerve (bottom trace). Extensors (top trace) and flexors (bottom trace) are activated synchronously. B, At stage 25.5, a second burst routinely occurs in each episode of activity, and differences in burst characteristics between muscle nerves are more distinct. Top trace, FEMORO nerve. Bottom trace, SART nerve. C, Stage 28 bursting patterns are similar to those at stage 25.5. Top trace, FEMORO nerve; bottom trace, SART nerve. D, At stage 28.5, bursting characteristics changed more dramatically, with a third or fourth burst occurring in each episode and with significant increases in burst duration of both extensors (top trace, FEMORO nerve) and flexors (bottom trace, SART nerve). E, Bursting can be elicited at stage 25 by stimulation of the rostral spinal cord (arrow marks stimulus). F, In the presence of low-calcium/high-magnesium Tyrode's solution, spontaneous activity is eliminated, and stimulation of the rostral cord (top trace) does not elicit a burst (arrows mark stimulus artifacts). However, direct stimulation of the motoneurons causes a compound action potential with a short latency (bottom trace, arrow marks stimulus artifact) but does not elicit a burst. G, Graph showing that the frequency of spontaneous bursting episodes, plotted as the interval between episodes in minutes, at different stages declines with increasing developmental age. Frequency measurements shown are from the embryos in A-D. Each bar represents the data from a single embryo, representative of that stage (mean ± SE). Traces from A, E, and F are from the same embryo. SART, Sartorius; FEMORO, femorotibialis. Calibration: A-E, 2 sec; F, top, 2 sec; F, bottom, 5 msec.
To determine whether this early activity was generated by a network of synaptically connected neurons or alternatively represented pacemaker-like activity of electrically coupled neurons, we bathed the embryo in Tyrode's solution with altered calcium (Ca2+) and magnesium (Mg2+) levels (0.2 mM Ca2+/7 mM Mg2+ or 2 mM Ca2+/12 mM Mg2+) to selectively block chemical transmission. Under these conditions, spontaneous bursts of activity ceased, although spontaneous unit activity was still present (data not shown). Low-calcium solution also inhibited our ability to elicit bursting by stimulation of the rostral cord (Fig. 2F, top trace, arrows mark stimulus artifacts), indicating that descending input drives the spinal circuit via chemical synapses. However, by positioning the stimulating electrode over the lateral motor column at the appropriate level, it was possible to directly activate the motoneurons, producing a large compound action potential in the muscle nerve (Fig. 2F, bottom trace, arrow marks stimulus). Such stimulation, however, did not elicit bursts. Blockade of bursting was reversed by returning to normal Tyrode's solution (3 mM Ca2+/1 mM Mg2+). These results suggest that early bursting activity is generated by a synaptically connected network of neurons in the spinal cord and can be driven by descending input from more rostral cord levels.
Within a few hours of development (stage 25.5), two bursts of activity began to occur in each spontaneous episode (Fig. 2B), indicating that the circuit was now capable of generating more than one cycle of activity per episode, although the interburst interval varied considerably between episodes. In addition, the differences between the patterns of activity in different nerves became more pronounced (Fig. 2, compare A, B). However, the frequency of episodes remained similar to that in early stage 25 embryos (Fig. 2G).
By stage 28 (E6), when motor axons first contact their targets (Landmesser, 1978
As summarized in Figure 2G, at all stages studied, the frequency of spontaneous bursting episodes occurred with great regularity, although the interepisode interval increased with increasing age. Interestingly, the greatest change in frequency and bursting characteristics occurred shortly after initial target contact, but whether target contact is necessary for these changes will require additional experiments.
Pharmacology of the developing circuit driving spontaneous bursting
The mechanisms underlying spontaneous rhythm generation in the spinal cord are not fully understood. At later stages of development (E10-E12), spontaneous activity of chick lumbosacral motoneurons appears to be driven primarily by excitatory connections, especially glutamate (Barry and O'Donovan, 1987
Glutamatergic transmission is not required for normal bursting activity
The excitatory amino acid glutamate has been shown to stimulate patterned bursts of activity in a number of locomotor circuits, including chick. In many systems, the effect of glutamate can be mimicked by NMDA, an agonist of NMDA-type glutamate receptors, suggesting that glutamate drives activity in large part through activating NMDA receptors (Barry and O'Donovan, 1987
To determine whether NMDA receptors are also important in the early chick cord, we bath applied NMDA (30 µM) to stages 25 and 28 isolated cord preparations while recording from specific muscle nerves (Fig. 3). Similar to observations in older (stage 36) chick cords (Barry and O'Donovan, 1987
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Figure 3. Activation of NMDA receptors elicits rhythmic bursting but is unnecessary for the generation of spontaneous bursting episodes. Traces on the left are shown at a slow time scale to demonstrate the effect of drug treatments on the frequency of bursting. Corresponding traces on the right show a subset of bursts on an expanded time scale to show details of the burst structure. Unlike control bursting (A), application of 30 µM NMDA at stage 25 (B, arrow) elicits multiple bursts of activity that continue for the duration of drug treatment. Initially, NMDA-induced bursts are well formed (B, right, left two bursts) and resemble controls (A, right). After prolonged NMDA exposure, unit activity increased (B, left, asterisk), and bursts increased in duration and looked more ragged (B, right, far right burst). C, Pretreatment with 100 µM APV does not alter frequency or structure of spontaneous bursts (left and right) but does block the ability of NMDA to induce bursting (arrows mark application of NMDA). A-C are from the same embryo. Top traces in each pair, FEMORO nerve. Bottom traces in each pair, obturator nerve. D, At stage 28, NMDA application induced more frequent bursting than at stage 25 (B). NMDA bursts were also well formed (D, right). In D, top traces are FEMORO nerves, and bottom traces are SART nerves. FEMORO, Femorotibialis; SART, sartorius. Calibration: A-D, left, 20 sec; right, 4 sec.
Despite the presence of activatable NMDA receptors in these early circuits, application of APV alone did not affect the frequency (Fig. 4F) or the pattern (Fig. 3, compare A, C) of spontaneous activity at either stages 25 or 28. Thus, although NMDA receptors are present and their activation evokes rhythmic stereotyped bursts of activity, they are not necessary for the normal generation of bursting at these early stages.
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Figure 4. Activation of non-NMDA receptors by kainate induces bursting activity but is not necessary for the generation of spontaneous bursting episodes. Traces are from stage 25 embryos treated with kainate (B, E), with (D, E) or without (B) glutamate receptor blockers. Addition of 10 µM kainate (B) induces a series of bursts that resemble control bursts (A). Extended application causes an increase in unit activity, especially marked in extensor motoneurons (asterisk). Top traces in A, B, FEMORO nerve. Bottom traces in A, B, SART nerve. In another embryo, application of a cocktail of glutamate receptor blockers APV (100 µM), CNQX (50 µM), and kyurinate (1 mM) had no significant effect on bursting (compare with control in C) but did prevent kainate (E, arrow) from eliciting bursts. The burst in E that follows kainate application was the second in a spontaneous episode and was not induced by kainate. C-E are from one experiment. Top traces, FEMORO nerve; bottom traces, obturator nerve. Calibration: 2 sec. F, Bar graph showing the effect of glutamate drugs on frequency of spontaneous bursting episodes. Glutamate blockers APV, CNQX, and kyurinate have no significant effect on frequency of spontaneous bursting at stages 25 or 28. Similarly, DHK, a glutamate uptake inhibitor, had no effect on bursting at stage 25. Three separate experiments are shown, two at stage 25 and one at stage 28 (mean ± SE). C, Control. APV, 100 µM. CNQX, used at 50 µM at stage 25, 20 µM at stage 28. Kyurinate, 1 mM. DHK, 200 µM.
Endogenously released glutamate could also be acting via kainate-AMPA receptors, and bath application of 10 µm kainate was able to induce bursting activity (Fig. 4B). The pattern of activity induced by kainate differed from that induced by NMDA in two ways. First, kainate induced only a few bursts of activity rather than the continuous bursting elicited by NMDA. Second, these bursts were then followed by a significant increase in background unit activity, especially in extensor motoneurons (Fig. 4B, asterisk), and the cessation of further bursting activity. Background activity remained pronounced during drug application but returned to baseline within 1-2 min after the onset of a wash in normal Tyrode's solution (data not shown). Bursting activity also returned shortly after the beginning of the rinse. To determine whether kainate receptors were necessary for normal spontaneous bursting, we treated embryos with 20 µM CNQX, a selective kainate receptor blocker. CNQX did not affect the frequency of bursting at either stages 25 or 28 (Fig. 4F). However, in some cases, it did induce a slight inhibitory period in extensor bursts shortly after burst onset, which subsequently disappeared after washout of the drug (data not shown).
Because it remained possible that glutamate was acting through both NMDA and non-NMDA glutamate receptors and that blocking alone was insufficient to alter normal bursting, we blocked both with a combination of APV (100 µM), CNQX (20 µM), and in one case, kyurinate (1 mM). As seen in Figures 4 and 5, this treatment did not significantly alter either the frequency of bursting (Fig. 4F) or the burst shape (Fig. 4, compare C, D). However, it did block the ability of kainate to induce multiple bursts (Fig. 4E, arrow marks addition of kainate to embryo pretreated with APV, CNQX, and kyurinate).
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Figure 5. Effect of GABA agonists and antagonists on the pattern of spontaneous bursting. A, Bicuculline (50 µM) has little effect on the structure or pattern of bursting at stage 25 (top trace in each pair, SART nerve; bottom trace in each pair, obturator nerve). However, at stage 28 (B), bicuculline prevented the generation of multiple bursts in an episode and altered the burst structure, which became triangular in shape (top traces in each pair, FEMORO nerves; bottom traces in each pair, SART nerves). C, At stage 25, episodes in the presence of nipecotic acid (1 mM) contained only a single burst, which was, however, of similar duration to control bursts (top traces in each pair, FEMORO nerves; bottom traces in each pair, obturator nerves). D, At stage 28, although nipecotic acid prevented spontaneous bursting (see Fig. 6B, open circles), a burst could still be elicited by stimulation of the rostral spinal cord (Fig. 5D, arrow, bottom traces). However, in contrast to stage 25 (C), burst structure was markedly altered and considerably lengthened. In addition, flexor pools, such as the sartorius (bottom trace in each pair), exhibited a long period of quiescence after initial activation, after which they resumed bursting. Such a quiescent period did not occur in extensor pools (top trace in each pair, FEMORO). E, In the presence of 5 µM muscimol, a GABAA agonist, stimulation of rostral cord (top trace, arrow marks stimulus artifact) no longer elicits a burst in the FEMORO nerve. However, a compound action potential in this nerve was produced by stimulation directly over the motor pool in LS3 (second trace, asterisk). FEMORO motoneurons could also be activated synaptically by cord stimulation of LS1 (third trace, asterisk). This synaptic response could be blocked by a train of stimuli at 1 Hz (fourth trace, arrow marks stimulus) and in other experiments by low-Ca+2 Tyrode's solution (data not shown). FEMORO, Femorotibialis; SART, sartorius. Arrows mark stimulus artifacts in each trace where applicable. Calibration: A-D, 2 sec; E, 5 msec.
Finally, as an additional test of whether endogenously released glutamate was involved in locomotor activity, we bath applied DHK, which blocks the reuptake of glutamate at the synapse. We found that DHK (200 µM) had no effect on the frequency (Fig. 4F) or pattern (data not shown) of spontaneous activity.
Together, these results demonstrate that although glutamate receptors are present in the spinal cord at early stages (E4-E5), they are not important for the initiation of rhythmic bursting. However, because CNQX induced a slight inhibitory period in extensor but not flexor bursts and treatment with the agonist kainate increased spontaneous background activity in extensors more than flexors, glutamate, acting through kainate-AMPA receptors, may play a minor role in the circuit responsible for generating burst shape, especially in extensor motor pools. Finally, because 1 mM glutamate was still able to induce several bursts, even in the presence of APV (100 µM), CNQX (20 µM), and kyurinate (1 mM) (data not shown), glutamate receptors not blocked by these antagonists may be present. Nevertheless, in striking contrast to stage 36 (Chub and O'Donovan, 1998
Role of GABA in spontaneous bursting episodes
At stage 36, blockade of inhibitory transmission with the GABAA receptor blocker bicuculline and the glycine receptor blocker strychnine results in a reduction in the frequency and regularity of spontaneous episodes, as well as a loss of flexor-extensor alternation (Sernagor et al., 1995
To test whether endogenous GABA circuits are also important for the frequency or pattern of early spontaneous activity, we first treated stages 25-29 embryos with the GABAA receptor antagonist bicuculline. Bath application of bicuculline (50 µM) tended to slow the frequency of bursting episodes at both stages 25 and 28, although some variability was observed, with frequency in some cases increasing and in other cases decreasing or remaining unchanged (Fig. 6A). In all cases, however, the frequency of bursting episodes in the presence of bicuculline occurred at ~5-7 min intervals, regardless of the starting rate of control activity. Together, these observations indicate that by stage 25, GABA circuits are present and can modulate patterned activity.
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Figure 6. The effect of GABA antagonists and uptake inhibitors on the frequency of bursting episodes. A, The GABAA antagonist bicuculline altered the frequency of bursting episodes at both stages 25 and 28, in some cases decreasing and in others increasing the interval between episodes. The bar graphs show the results from three separate embryos, and data are presented as mean ± SE interval. B, The effect of 1 mM nipecotic acid (a GABA uptake inhibitor) on the interval between bursting episodes at stages 25 (filled circles) and 28 (open circles). In both cases, activity was blocked, but it resumed after 25 min at stage 25, whereas at stage 28, only one spontaneous episode occurred (open circle) during prolonged nipecotic acid exposure. Such "single" spontaneous bursts that occasionally occurred during 1 or more hour of drug application may actually have been set off by extrinsic stimuli. Because bursts could still be evoked by stimulation of descending input in nipecotic acid, it is possible that this single burst might have resulted from some stimulated activation of the cord generator and may not reflect spontaneous activity. Arrow and dashed line show addition of nipecotic acid to the bath. The x symbol marks an episode that was induced by electrical stimulation of the rostral spinal cord of the stage 28 embryo.
In contrast to its effect on frequency, bicuculline at stage 25 did not appreciably affect burst structure (Fig. 5A). However, by stage 28, bicuculline altered both the number and structure of bursts in each episode (Fig. 5B). First, it prevented the generation of multiple bursts; regardless of the number of bursts generated in control Tyrode's solution, only one burst per episode occurred in the presence of bicuculline (Fig. 5B). Second, unlike controls, bursts appeared triangular in shape and were longer in duration.
To confirm that GABA was being released during early activity, we treated embryos with nipecotic acid (Fig. 5C,D), which blocks the reuptake of GABA and should increase the amount of transmitter at GABAergic synapses. We reasoned that if GABA is playing a role in spontaneous activity then blocking GABA reuptake should interfere with the normal pattern or frequency of bursting episodes. Results in Figures 5, C and D, and 6B confirm that endogenous GABA is being released during spontaneous activity. Specifically, at stage 25, bath application of nipecotic acid (1 mM) caused an initial cessation of spontaneous bursting episodes (Fig. 6B, filled circles), but bursting returned after 15 min at a slightly lower frequency. In addition, only one burst occurred during each episode, and it was slightly shorter in duration than control bursts (Fig. 5C).
In contrast to stage 25, the loss of spontaneous activity induced by nipecotic acid (1 mM) at stage 28 remained for the duration of drug treatment (Fig. 6B, open circles). However, as shown in Figure 5D, stimulation of the rostral cord could still elicit a single burst, in contrast to the multiple bursts characteristic of stage 28 controls. Bursts in the presence of nipecotic acid were also much longer than control (30 vs 1 sec), and flexor and extensor motoneurons responded differently, with flexors exhibiting a long inhibitory period after the onset of the burst (Fig. 5D, bottom, bottom trace), whereas extensors did not (Fig. 5D, bottom, top trace). These results suggest that GABAergic connections influencing flexor and extensor bursts differ or that flexor and extensor motoneurons respond differently to exogenously released GABA. The blocking effect of nipecotic acid on spontaneous activity was reversed quickly (within 3 min) after return to normal Tyrode's solution, and bursting rebounded at an accelerated rate (interval between episodes, 40 sec vs 4-5 min in controls) for the first hour of wash before returning to control levels (data not shown). Together, these observations show that GABA can modulate motoneuron activity even at stage 25 and that its role in pattern generation increases and changes with increasing embryonic age.
To confirm that GABAA receptors were present in the cord at stages 25 and 28, we treated embryos with muscimol, a GABAA agonist. At both stages, muscimol blocked spontaneous bursting, and activity did not resume until washout of the drug (data not shown). Muscimol treatment differed from nipecotic acid treatment in that muscimol sometimes stimulated a series of bursts before shutting off activity (data not shown). This was followed by a transient increase in background unit activity lasting several minutes, after which spontaneous bursting ceased. At this point, bursts could no longer be evoked by electrical stimulation to the rostral cord (Fig. 5E, top trace). However, stimulation directly over the lumbar lateral cord, although not eliciting a burst, directly activated motoneurons, producing a large short latency compound action potential in the muscle nerve (Fig. 5E, second trace, asterisk). Motoneurons could also be activated synaptically by stimulating the cord more medially. Stimulation at this site evoked a compound action potential (Fig. 5E, third trace, asterisk) of 6 msec longer latency than that evoked by direct stimulation. Consistent with synaptic activation, this compound action potential recorded from the muscle nerve was blocked by 1 Hz stimulation (a frequency that blocks early synapses) (Fig. 5E, bottom trace) and by low Ca2+/high Mg2+ Tyrode's solution (data not shown). In contrast, the compound action potential elicited by direct stimulation of the motoneurons was not blocked at 20 Hz stimulation nor by low Ca2+ Tyrode's solution (data not shown). The effects of muscimol were similar throughout stages 25-28, indicating that GABAA receptors are present at these stages and that their indiscriminate activation can disrupt spontaneous activity more severely than GABAA receptor antagonists or GABA reuptake blockers.
Despite the similarity in the effect of muscimol at stages 25 and 28, the change in sensitivity to GABAA receptor blockers over this period indicates that the circuit responsible for generating rhythmic activity is changing and that GABA receptors are being used differently at these two stages in the developing locomotor circuit.
Cholinergic input strongly modulates early spontaneous activity
As shown above, neither blockade of glutamate nor GABA receptors with specific antagonists blocked recurrent spontaneous bursting activity in motoneurons, indicating that other transmitters must be involved in driving spontaneous activity. At later stages, both ACh and glycine have been shown to affect patterned activity (Sernagor et al., 1995
To test this hypothesis, we bath applied dTC, a nicotinic ACh receptor (nAChR) antagonist, at stages 25-29. At all stages tested, dTC (5 × 10
6 M) transiently stopped spontaneous bursting activity (Fig. 7A), although bursting episodes could still be elicited by electrical stimulation of the descending input (data not shown). Interestingly, spontaneous bursting resumed after 10-25 min in the presence of drug, but at a lower frequency than control. The number and structure of bursts were also altered with the amplitude and number of bursts being increased in both flexor and extensor motoneurons (Fig. 7B,C). Extensors and flexors were differentially affected by dTC, with the burst duration of extensors being increased (Fig. 7C, top trace), whereas that of flexors was decreased (Fig. 7C, bottom trace). Furthermore, dTC appeared to increase the frequency of background unit activity in extensors more than flexors (data not shown), suggesting that cholinergic connectivity to extensors and flexors differs.
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Figure 7. Blocking nicotinic receptors alters both the frequency and pattern of spontaneous bursting activity. A, Graph plotting intervals (in minutes) between bursting episodes before and after the addition of 5 µM dTC, a general blocker of nicotinic receptors. At both stages 25 (filled diamonds) and 28 (open diamonds), dTC caused the cessation of spontaneous activity for a period of time; however, after prolonged exposure, bursting returned at a lower frequency (arrow and dashed line mark the addition of dTC). B-D, Effects of dTC on structure of bursting. Low doses (0.5 µM) of dTC did not block spontaneous bursting (data not shown) but increased the number of bursts per episode and altered the burst duration and amplitude (C, compare to control in B). Extensor bursts tended to increase in duration (top trace), whereas flexors decreased in duration (bottom trace). D, At high doses (10 µM), dTC blocked spontaneous activity for over 20 min, but after activity resumed bursts were enhanced in both amplitude and duration in extensors (top trace) and reduced slightly in duration in flexors (bottom trace). Interestingly, at high doses, there was a reduction in the number of bursts, some episodes only containing one burst as opposed to the normal two in controls (B). B-D are from a single stage 28 embryo. Top traces, FEMORO nerves; bottom traces, SART nerves. FEMORO, Femorotibialis; SART, sartorius.
The response to dTC changed in a dose-dependent manner. At stage 28, low concentrations of dTC (5 × 10
To further test this hypothesis, we treated embryos with eserine, an acetylcholinesterase inhibitor, to block the degradation of endogenously released ACh. Eserine should thus enhance the action of any ACh released. Consistent with this, as shown in Figure 8, eserine had the opposite effect of cholinergic antagonists, such as dTC, on spontaneous bursting. First, eserine markedly increased the frequency of spontaneous bursting at both stages 25 and 28 (Fig. 8A). Second, whereas eserine did not affect burst structure at stage 25 (Fig. 8C), at stage 28, its affect on burst structure was quite profound (Fig. 8B). With each successive burst, presumably as ACh accumulated in the synapse, the two short bursts occurring at this stage were gradually replaced by a single burst of much longer duration (~20 sec compared with 1 sec in controls; see also Fig. 8B, bottom, traces on expanded time scale). It is interesting to note that the effect of eserine on burst shape and duration for flexors (sartorius; bottom trace of each pair) and extensors (femorotibialis; top trace of each pair) differed; flexor bursts tended to contain a long inhibitory period shortly after burst onset, whereas extensor bursts did not. These effects of eserine were blocked by pretreatment with dTC and atropine (data not shown), nicotinic and muscarinic receptor blockers, respectively, as would be expected if eserine were accentuating the effect of endogenously released ACh. The effect of enhancing the activity of endogenously released ACh (via eserine) on burst shape and duration was very similar to that produced by enhancing the effect of endogenously released GABA (via nipecotic acid) (compare Figs. 6F, 8B). This might arise if one effect of ACh was to facilitate the release of GABA, a possibility considered further in Discussion.
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Figure 8. The acetylcholinesterase inhibitor eserine increases the frequency of spontaneous episodes and alters burst structure. A, Graph showing that eserine (1 µM) increases the frequency of spontaneous episodes at both stages 25 (filled diamonds) and 28 (open diamonds). After prolonged exposure, episode frequency decreased in the presence of eserine, presumably as a result of ACh receptor desensitization. B, Eserine also altered the pattern of bursting in each episode. After eserine treatment, bursts became considerably elongated in duration (compare control bursting on left with bursting in eserine on right). In addition, flexors (bottom traces) were characterized by a period of quiescence shortly after onset of activity, whereas extensors (top traces) were not. Bottom panels in B show representative bursts on an expanded time scale. C, Although eserine increased bursting frequency in both stages 25 and 28 embryos (A), it did not alter bursting pattern at stage 25 (C, compare control on left with eserine on right). B, Top traces, FEMORO nerve recordings; bottom traces, SART nerve recordings. D, Top traces, Ischioflexorius nerve recordings; bottom traces, SART nerve recordings. FEMORO, Femorotibialis; SART, sartorius.
Acetylcholine acts primarily through non-
Nicotinic receptors blocked by dTC are known to be a heterogeneous population composed of different subunit types (for review, see Role and Berg, 1996
To distinguish between the effects of
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Figure 9. Acetylcholine acts primarily through non-
Low and high doses of nicotine affect spontaneous bursting differently
In other parts of the nervous system, ACh has been shown to act via many different receptor subtypes, which have different affinities for agonists, antagonists, and differing rates of desensitization (McGehe and Role, 1995
We found that high doses of nicotine (10 µM) induced a series of bursts, followed by an increase in spontaneous unit activity (Fig. 10A). As this unit activity returned to baseline, spontaneous bursting ceased for at least 1 hr (the duration of the drug application). This may have resulted from ACh receptor desensitization, which is common in many receptor subtypes especially at high agonist concentrations (Fenster et al., 1997
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Figure 10. Effect of nicotine on spontaneous bursting. A, At stage 28, high doses of nicotine (10 µM) induce a series of bursts (bottom traces, arrow marks addition of nicotine), followed by a transient increase in unit activity (asterisk). Top traces show bursting induced by high nicotine on an expanded time scale to demonstrate bursting characteristics. B, In contrast, low concentrations of nicotine (0.1 µM) cause the cessation of spontaneous bursting. Pretreatment of embryos for 30 min with DHBE (1 µM), but not
Early spontaneous bursting may be driven by either cholinergic or GABAergic circuits
Thus far, we have shown that both cholinergic and GABAergic, but not glutamatergic, neurons are important for early spontaneous motoneuron bursting in the chick. However, in all cases in which the action of a neurotransmitter was blocked by receptor antagonists, spontaneous bursting activity recovered in the continued presence of the drug(s), presumably now driven by a different transmitter system. At stage 36, for example, recovery of spontaneous activity can occur after blockade of excitatory transmission (Chub and O'Donovan, 1998
To determine which transmitter systems were capable of driving spontaneous activity at stage 25, we treated embryos with combinations of drugs. As shown for one embryo in Figure 11A, blockade of both GABA and glutamate receptors had no effect on spontaneous activity, in contrast to its effect at stage 36 (Chub and O'Donovan, 1998
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Figure 11. Spontaneous activity at stage 25 does not occur after blockade of both excitatory and inhibitory connections. Embryos were treated with a combination of receptor blockers to determine which transmitter systems were critical for spontaneous bursting. In each graph, arrows mark the addition of each receptor blocker. A, Blockade of GABA receptors with a combination of phaclofen (Ph, 50 µM) and bicuculline (Bic, 50 µM) has no effect on the frequency of spontaneous episodes. Addition of glutamate receptor blockers APV [50 µM and increasing to 100 µM (APV+)] and CNQX (20 µM) to the GABA receptor blockers also has no effect on activity. However, dTC (5 µM) causes a transient cessation of spontaneous activity for 25 min, followed by a recovery of bursting. Bursting ceases completely after atropine (1 µM) is added to the cocktail. Bursting resumes quickly (within 3 min) to near control frequency after washing in normal Tyrode's solution (arrow marks onset of wash at hour 6 of the experiment). The x symbols represent activity induced by electrical stimulation of the rostral cord, an example being shown in C. Such stimuli, which were used to determine whether descending input was able to elicit bursts under various drug applications, did not affect subsequent bursting or lack thereof. B, Shows that atropine (1 µM) has no effect on burst frequency when applied alone (Atr, arrow), but addition of dTC (5 µM) to atropine shuts off activity transiently, and bursting returns in the continued presence of both drugs. This resumed activity may be mediated by GABA, because the addition of bicuculline (50 µM) to the cocktail shuts off activity completely. One burst did occur after ~1 hr, but no other bursts occurred. C, Although no spontaneous activity occurred in the presence of GABAergic, glutamatergic, and cholinergic receptor blockers shown in A, bursting could be elicited by a single electrical stimulus to the rostral cord (C, arrow marks the stimulus artifact). Each stimulus evoked a single well formed burst.
To determine which of the transmitter systems was the most critical for spontaneous bursting, we next treated with a combination of cholinergic and GABAergic drugs. We found that spontaneous bursting activity was blocked completely in the presence of the cholinergic blockers atropine and dTC and the GABAA blocker bicuculline (Fig. 11B). These results indicate that spontaneous activity can be driven by either cholinergic or GABAergic circuits acting through GABAA receptors and that simultaneous blockade of both systems eliminates spontaneous activity.
Descending input
Despite our ability to block spontaneous activity with cocktails of receptor blockers, we were unable to block the ability of rostral cord stimulation to elicit a bursting episode. For example, in the experiment shown in Figure 11A, we were still able to evoke a burst by electrical stimulation of the rostral cord (x symbols indicate stimulated bursts), even when spontaneous activity was completely shut off (Fig. 11C, arrow marks stimulus). Two mechanisms could explain this finding. Descending input could use a different transmitter, such as norepinephrine, serotonin, or dopamine, to activate local circuits in the lumbosacral spinal cord that generate bursts. Alternatively, the drugs used might not fully block all GABA, glutamate, and ACh receptors, possibly because of different affinities for embryonic forms of the receptors. This finding also suggests the possibility that another transmitter is involved in the local pattern generator circuit, because blockade of GABA, glutamate, and ACh receptors did not eliminate the ability of descending input, to stimulate a well formed burst of motoneuron activity (Fig. 11C). Another possibility, that electrical coupling between networks of motoneurons could produce bursts when activated by descending input, will be considered next.
Potential role for electrical coupling in spontaneous burst generation
Our observations have clearly demonstrated the importance of chemical transmission in allowing patterned spontaneous bursting by early lumbosacral motoneuron pools. They do not, however, preclude a role for electrical coupling between motoneurons or interneurons in generating the spontaneous bursting episodes. Electrical coupling has been demonstrated between somatic motoneurons in Xenopus embryos (Perrins and Roberts 1995a
Octanol (3 mM), which blocks gap junction-mediated electrical coupling and which reduces the frequency of spontaneous Ca2+ transients in embryonic chick retina (Catsicas et al., 1998
DISCUSSION
Our major finding is that as early as E4 (stages 24-25), 1-1.5 d after being born (Hollyday and Hamburger, 1977
The circuit we have pharmacologically characterized has many similarities to the circuit that drives spontaneous waves of electrical activity in the developing retinas of both ferret (Meister et al., 1991
Similarities in early circuits that generate spontaneous activity
Unlike the local domains of spontaneous activity in developing visual cortex that are propagated by second messenger spread through gap junctions (Kandler and Katz, 1998
What is the source of the ACh that drives activity in early cord circuits? The chick circuit is contained entirely within the ventral cord (Ho and O'Donovan, 1993
